Research ArticleGremlin Activates the Smad Pathway Linked toEpithelial Mesenchymal Transdifferentiation inCultured Tubular Epithelial Cells
Raquel Rodrigues-Diez1 Rauacutel R Rodrigues-Diez1 Carolina Lavoz1
Gisselle Carvajal1 Alejandra Droguett2 Ana B Garcia-Redondo1 Isabel Rodriguez34
Alberto Ortiz45 Jesuacutes Egido46 Sergio Mezzano2 and Marta Ruiz-Ortega1
1 Cellular Biology in Renal Diseases Laboratory IIS-Fundacion Jimenez Dıaz Universidad Autonoma de MadridAvenida Reyes Catolicos 2 28040 Madrid Spain
2Division of Nephrology School of Medicine Universidad Austral Valdivia Chile3 Bone and Mineral Research Unit Hospital Universitario Central de Asturias 33006 Oviedo Spain4 Instituto Reina Sofıa de Investigacion Nefrologica 28003 Madrid Spain5 Division of Dialysis IIS-Fundacion Jimenez Dıaz Universidad Autonoma de Madrid Avenida Reyes Catolicos 228040 Madrid Spain
6Division of Nephrology and Hypertension IIS-Fundacion Jimenez Dıaz Universidad Autonoma de Madrid CIBERDEMAvenida Reyes Catolicos 2 28040 Madrid Spain
Correspondence should be addressed to Marta Ruiz-Ortega mruizofjdes
Received 27 February 2014 Revised 25 April 2014 Accepted 1 May 2014 Published 18 May 2014
Academic Editor Akito Maeshima
Copyright copy 2014 Raquel Rodrigues-Diez et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforminggrowth factor-120573 (TGF-120573) Epithelial mesenchymal transition (EMT) is one process involved in renal fibrosis In tubular epithelialcells we have recently described that Gremlin induces EMT and acts as a downstream TGF-120573mediator Our aim was to investigatewhether Gremlin participates in EMT by the regulation of the Smad pathway Stimulation of human tubular epithelial cells (HK2)with Gremlin caused an early activation of the Smad signaling pathway (Smad 23 phosphorylation nuclear translocation andSmad-dependent gene transcription) The blockade of TGF-120573 by a neutralizing antibody against active TGF-120573 did not modifyGremlin-induced early Smad activationThese data show thatGremlin directly by aTGF-120573 independent process activates the Smadpathway In tubular epithelial cells long-term incubation with Gremlin increased TGF-120573 production and caused a sustained Smadactivation and a phenotype conversion into myofibroblasts-like cells Smad 7 overexpression which blocks Smad 23 activationdiminished EMT changes observed in Gremlin-transfected tubuloepithelial cells TGF-120573 neutralization also diminished Gremlin-induced EMT changes In conclusion we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubularepithelial cells through activation of Smad pathway and induction of TGF-120573
1 Introduction
Many embryological expressed genes regulate morphogen-esis and then become quiescent in the normal adult kid-ney Recent studies have shown that some developmentalgenes are reactivated in the adult diseased kidneys [1] Thereemergence of these genes appears to be linked to tissue
repair but when an imprecise interaction of developmen-tal and inflammatory signals occurs complete healing isnot achieved Instead there is an excessive production ofmatrix proteins leading to a scar formation Gremlin wasidentified as one of the developmental genes induced incultured human mesangial cells exposed to high glucoseinitially known as induced in high glucose-2 (IHG-2) and
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 802841 11 pageshttpdxdoiorg1011552014802841
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also called downregulated by mos (Drm) [2] Gremlin isa member of cysteine knot superfamily [3] that includestransforming growth factor-120573 (TGF-120573) proteins and actsas a bone morphogenetic protein (BMP) antagonist [4]Analysis of the predicted amino acid sequence indicated thepresence of several significant features including potentialnuclear localization signals near the C-terminus potentialN-linked glycosylation sites and multiple potential sites forphosphorylation The signalling peptide and a predictedglycosylation site have been identified Gremlin is a glycosy-lated phosphorylated secreted protein present both on theexternal cell surface and within the ER-Golgi compartments[3] In many human renal diseases induction of Gremlinhas been described [5ndash8] Several experimental studies haveshown that Gremlin participates in renal damage [9 10]Therefore some authors have suggested that Gremlin couldbe considered as a mediator of renal injury
Chronic progressive fibrosis of the kidney remains anunresolved challenge Irrespective of the underlying causechronic kidney disease is linked to the development oftubulointerstitial fibrosis characterized by accumulation ofextracellularmatrix (ECM)Themechanisms of renal fibrosisare complex and our therapeutic armamentarium is limitedThe key cellular mediator of fibrosis is the myofibroblastThere are different sources of myofibroblasts including acti-vation of tissue fibroblasts and migration of circulating mes-enchymal progenitors or cell transitions including epithelial-mesenchymal transition (EMT) or endothelial mesenchymaltransition [11 12] The investigation of the mechanismsinvolved in renal fibrosis and the identification of novelmedi-ators with potential therapeutic application is an importantopen question in chronic kidney disease
TGF-1205731 signalingmainly through Smad proteins is a keyplayer in fibrosis and EMT [13ndash17] Because of its pleiotropicactions TGF-120573 blockade is not an ideal therapeutic tooltherefore novel targets are needed Among them Grem-lin may be an interesting candidate in progressive renaldiseases Recent in vitro studies developed by our grouphave shown that Gremlin gene silencing inhibited TGF-120573-mediated matrix production and EMT [18] Howeverthe involvement of TGF-120573 in Gremlin responses has notbeen investigated We have also reported the presence ofGremlin in glomerular crescents of human pauci-immuneglomerulonephritis and in the tubulointerstitium of chronicallograft nephropathy In these human diseases Gremlin cor-related with the degree of tubulointerstitial fibrosis and wasassociated with TGF-1205731 overexpression and Smad pathwayactivation [7 8] These studies suggest that Gremlin mayactivate the Smad pathway therefore the aim of this workwas to evaluate whether Gremlin could directly activate theSmad pathway in tubular epithelial cells evaluating whetherthis activation is linked to Gremlin-induced EMT the mainfibrotic effect observed in response to Gremlin stimulation inthese cells [18]
2 Materials and Methods
21 Cell Cultures Human renal proximal tubuloepithelialcells (HK2 cell line ATCC CRL-2190) were grown in RPMI
with 10 fetal bovine serum (FBS) 1 nonessential aminoacids 100UmL penicillin and 100120583gmL streptomycininsulin transferrin selenite (ITS) (5120583gmL) and hydrocorti-sone (36 ngmL) in 5CO
2at 37∘CAt 60ndash70of confluence
cells were growth-arrested in serum-free medium for 24hours before the experimentsThen cells were stimulated fordifferent timeswith recombinantGremlin (50 ngmL) (RampD)or human recombinant TGF-1205731 (1 ngmL Peprotech) Cellculture reagents were obtained from Life Technologies IncTGF-120573 was targeted by a pan-specific polyclonal anti-TGF-120573neutralizing antibody which recognizes bovine mouse andhuman TGF-1205731 and 1205732 isoforms (1 120583gmL) (RampD)
22 Transfection cDNA Constructs and Promoter StudiesHK2 cells were transiently transfected for 24ndash48 hourswith FuGENE (Roche) pCDNA3-Gremlin-myc-IRES2-eGFP plasmid (GREM-GFP) andor pCDNA-FLAG-Smad7expression vector (kindly donated by Dr MassagueMemorial Sloan-Keternig Cancer Center USA) or emptyvector (pCDNA) The GREM-GFP was generated as followsGREM1 cDNA was purchased from the Mammalian GeneNIH Collection (Bethesda Maryland USA) We addeda c-myc tag to the 31015840 portion of GREM1 using PCR withthe forward primer 51015840AGTGCGGCGGCTGAGGACCCGCCGCACTGACAT-31015840 and the reverse primer 51015840-ATAGCCGCCGCTTACAGATCCTCTTCTGAGATG-AGTTTTTGTTCATCCAAATCGATGGATATGC-31015840We also inserted an e-GFP sequence downstream ofhuman Gremlin as follows The IRES-eGFP sequencewas obtained by PCR using a pIRES2-EGFP plasmid(Clontech Mountain View CA USA) as the template withthe following primers IRES-eGFP-F (51015840-TACATTAAT-GGGCCCGGGATCCGCCCCTC-31015840) and IRES-eGFP-R(51015840-GGCCATATGCGCCTTAAGATACATTGATG-31015840) The GREM1-c-myc and IRES-eGFP fragments wereindependently cloned into a pGEMT-Easy vector andthen sequenced (Macrogen Seoul Korea) to confirm themodifications and absence of additional mutations Nextboth the GREM1-c-myc and IRES-eGFP fragments weresubcloned into a modified pCDNA3 vector using theEcoRI and NotI restriction sites respectively In Gremlin-transfected cells Gremlin production was confirmed byimmunofluorescence (not shown)
To demonstrate Smad 7 transfection efficacy an anti-FLAG antibody was used (not shown) Smad-dependent pro-moter activation was evaluated by transfection of Smadluc(kindly donated by Dr Volgestein Baltimore USA) and TK-renilla as internal control as described [19]
23 Protein Studies Total cellular protein extracts (10ndash50 120583glane) obtained in lysis buffer [50mM Tris-HCl pH 74150mMNaCl 2mMEDTA 2mMEGTA 02Triton X-10003 NP40 100 120583M phenylmethylsulphonylfluoride 1mMdithiothreitol 100 120583MNa
3VO4 and 1mM protease-inhibitor
cocktail (Sigma)] were separated on 8ndash12 polyacrylamide-SDS gels under reducing conditions Samples were thentransferred onto nitrocellulose membranes (Bio-Rad Her-cules CA) blocked with 5 nonfat dry milk in 50mM
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Tris-HCl pH 75 150mM NaCl with 005 Tween-20 andincubated overnight at 4∘C with the primary antibodiesand subsequently incubated with peroxidase-conjugated IgG(Amersham) and developed by ECL chemiluminescence(GE Healthcare Buckinghamshire UK)
Immunocytochemistry studies were performed in cellsgrowing on coverslips After the experiments cells werefixed in Merckofix (Merck) and permeabilized with 02Triton-X100 for 10min (except for E-cadherin staining)After blocking with 4 BSA and 8 serum for 1 hoursamples were incubated with primary antibodies overnightat 4∘C and then 1 hour at room temperature with fluoresceinisothiocyanate (FITC) [1200] or AlexaFluor 633 [1300]conjugated antibodies (Amersham) Nuclei were stainedwith 1 120583gmL propidium iodide (PI) or 410158406-Diamidino-2-phenyindole dilactate (DAPI) (Sigma-Aldrich) as control ofequal cell density Absence of primary antibody was used asnegative control Samples were mounted in Mowiol 40ndash88(Sigma-Aldrich) and examined by a Leica DM-IRB confocalmicroscope
TGF-1205731 protein was measured in the cell-conditionedmedium using a commercial enzyme-linked immunoassay(ELISA) (BD Sciences San Diego USA) following the man-ufacturerrsquos instructions TGF-1205731 levels were quantified bycomparison with a standard curve using increasing concen-trations of human TGF-1205731 Protein content was determinedby the BCA method (Pierce)
24 Gene Expression Total RNA was isolated from cells withTrizol (Invitrogen) according to the manufacturerrsquos protocolcDNA was synthesized from 2120583g of total RNA with randomhexamer primers using the High capacity cDNA Archive Kit(Applied) Real-time PCR was performed using human FAMTaqMan MGB probes designed by assay-on-demand geneexpression products (Applied) TGF-1205731 Hs99999918 m1connective tissue growth factor (CTGF) Hs00170014 m1plasminogen activator inhibitor 1 (PAI1) Hs 00167155 m1Data weres normalized to 18S eukaryotic ribosomal RNA4210893E (VIC) The mRNA copy numbers were calculatedfor each sample by the instrument software using Ct value(ldquoarithmetic fit point analysis for the lightcyclerrdquo) Resultswere expressed in copy numbers and calculated relative tounstimulated cells after normalization against 18S
25 Statistical Analysis Results throughout the text areexpressed as mean plusmn SEM Differences between agonist-treated groups and controls were assessed by one-way anal-ysis of variance followed by post hoc Bonferroni or Dunnetttest or Mann-Whitney test as appropriate 119875 lt 005 wasconsidered significant Statistical analysis was conductedusing the SPSS statistical software version 110 (SPSS)
3 Results
31 Gremlin Activates Smad Pathway in Human CulturedTubuloepithelial Cells Receptormediated activation of Smadproteins (R-Smads 2 and 3) occurs by direct C-terminalphosphorylation Smad 23 then form complexes with Smad4 and translocate into the nucleus where they associateand cooperate with DNA binding transcription factors toactivate or repress target gene transcription [17] In culturedHK2 cells stimulation with recombinant Gremlin increasedphosphorylation levels of Smad 3 as early as 5 minutes and itwas maintained until 15 minutes (Figure 1(a))
Although Smad is the main signaling mechanism ofTGF-120573 several factors involved in renal damage such asangiotensin II can directly activate the Smad pathway inde-pendent of endogenous TGF-120573 [17] Therefore to evaluatewhether early Smad activation caused by Gremlin was medi-ated or not by TGF-120573 cells were preincubated with a neu-tralizing antibody against active TGF-120573 Gremlin-inducedSmad activation (evaluated as p-Smad 3 levels) was notmodified in the presence of the TGF-120573 antibody (Figure 1(b))Similar lack of response was found in the presence of decorin(a proteoglycan that neutralizes active TGF-120573 not shown)These data indicates that Gremlin directly activates the Smadpathway
Some actions of Gremlin are due to its effect as BMPantagonist [4] To determine the contribution of BMPs inGremlin-induced Smad activation HK2 cells were prein-cubated with BMP-2 or BMP-4 and then stimulated withGremlin during 10 minutes Phosphorylation of Smad 3 wasnot modified in the presence of any of these BMPs (Fig-ure 1(c)) suggesting that Gremlin-induced Smad activationis independent of BMPs in tubular epithelial cells
By confocal microscopy we have confirmed that Gremlinrapidly increased Smad 3 translocation to the nucleus thelatter demonstrated by the yellow nuclear staining observedin the merge of Figure 2 while in untreated cells the nucleiare red Gremlin also increased nuclear localization of Smad2 and Smad 4 observed at 15 minutes (Figure 2)
To further confirm that Gremlin activates the Smadpathway cells were transfected with a Gremlin expressionvector (GREM-GFP) for 24 hours By confocal microscopywe observed that in positive Gremlin-transfected cells (GFP-green staining) there was a nuclear immunostaining showingthe translocation of Smad 3 and Smad 2 (characteristic ofSmad activation) compared to cells transfected with emptyvector (Figures 3(a) and 3(b)) In the merge of Figure 3a Gremlin expressing-cell marked by a yellow rectanglepresented a positive nuclear staining corresponding to thepresence of Smad 3 or 2 In contrast in cells transfectedwith empty vector there are no nuclear Smad 2 and Smad 3immunostaining (all cells present blue nuclei) as observed insome nontransfected cells
To investigatewhetherGremlin regulates Smad-mediatedgene expression cells were cotransfected with a Grem-lin expression vector (GREM-GFP) and a luciferase Smadreporter plasmid Gremlin transfected cells expressed higherSmad-dependent luciferase activity (SBE) than control cells(Figure 4) To demonstrate further the involvement of Smad
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Figure 1 (a) Stimulation with Gremlin rapidly increased Smad 3 phosphorylation in cultured human tubuloepithelial cells HK2 cells werestimulated with Gremlin (50 ngmL) for different times (b) Early Smad 3 phosphorylation induced by stimulation with Gremlin was notmediated by TGF-120573 TGF-120573 was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizing antibody andthen treated with Gremlin for 10 minutes (c) In some points HK2 cells were preincubated with BMP-2 or BMP-4 and then treated withGremlin for 10 minutes Total proteins were isolated and protein levels were evaluated by western blot GAPDH or Smad 3 were used asloading controls Figures show a representative western blot of phosphorylated levels of Smad 3 and data are expressed as n-fold over control(considered as 1) as the mean plusmn SEM of 3-4 independent experiments lowast119875 lt 005 versus control
pathway in Gremlin-induced responses a Smad 7 expressionvector that inhibits Smad-mediated transcriptional effects byinterfering with receptor-mediated activation of R-Smad wasused [17 19] In HK2 cells cotransfected with GREM-GFPand Smad 7 expression vectors the Smad-mediated luciferaseactivity was significantly lower than cells transfected withGREM-GFP alone showing the specific Smad 7 blockade ofGremlin-mediated Smad activation (Figure 4)
32 Gremlin-Induced EMT Is Mediated by Smad ActivationWe have previously demonstrated that in tubular epithe-lial cells long-term stimulation with recombinant Gremlininduced EMT [18] Now we have observed that transfec-tion of HK2 cells with GREM-GFP induced EMT-relatedphenotypic changes observed by confocal microscopy after48 hours (Figure 5) Cells transfected with empty-vectorshowed epithelial morphology including the presence of
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Figure 2 Stimulation with Gremlin induces a rapid activation of the Smad pathway in cultured human tubuloepithelial cells HK2 cells werestimulated with Gremlin (50 ngmL) for 15 minutes The localization of R-Smad 3 (a) and 2 (b) and Smad 4 (c) was evaluated by confocalmicroscopy with FITC-secondary antibodies (green staining) Nuclei were stained with propidium iodide (IP) (red) In themerge the yellowstaining indicates the nuclear localization of Smad proteinsThe results are representative of 3 independent confocal microscopy experiments
epithelial markers such as cytokeratin (red staining) andthere is no positive staining for mesenchymal marker 120572-SMA(Figure 5) In contrast overexpression of Gremlin causedchanges inmorphology to fibroblast-like shape and inductionof 120572-SMA (see the GREM-GFP positive cell that presentsyellow staining and elongated shape) Moreover in Gremlinexpressing cells cytokeratin staining was markedly dimin-ished (absence of red staining in an area with several GREM-GFPpositive green cells)Theblockade of Smad activation bycotransfection with Gremlin and Smad 7 diminished theseEMT changes (Figure 5) as shown by restoration of thecytokeratin immunostaining and the epithelial morphologyand diminution of 120572-SMA as observed in the green positivecell These data suggest that Gremlin regulates EMT throughthe Smad pathway
33 Role of Endogenous TGF-120573 on Gremlin-Induced EMTPreviously we have reported that Gremlin acts as a down-stream mediator of TGF-120573-induced fibrosis in cultured renal
cells and incubation with Gremlin for 24 hours induced asignificant upregulation of TGF-1205731 mRNA levels in culturedtubuloepithelial cells [18] We have further investigated therelation between GremlinTGF-120573 evaluating whether Grem-lin could regulate TGF-1205731 synthesis In HK2 cells activeTGF-1205731 protein increased in the supernatants of Gremlin-stimulated cells after 48 hours but not at 24 hours (Fig-ure 6(a)) suggesting that some of the profibrotic actions ofGremlin could bemediated by endogenousTGF-1205731 synthesisTherefore we blocked TGF-120573 before HK-2 stimulation withGremlin by adding a neutralizing antibody against activeTGF-120573 which is able to block angiotensin II-induced ECMproduction andEMT [19 20] TGF-120573neutralization inhibitedGremlin-induced gene upregulation of profibrotic factorsobserved after 24 hours including TGF-120573 CTGF and PAI-1 (Figure 6(b)) Moreover TGF-120573 blockade antagonizedseveral EMT-related changes induced by Gremlin after 48hours as shown by immunofluorescence (Figure 7(a)) Wealso observed by western blot that TGF-120573 neutralizationdiminished Vimentin and Slug induction caused by Gremlin
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(a)
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Figure 3 Gremlin overexpression causes a sustained Smad activation in cultured human tubuloepithelial cells HK2 cells were transientlytransfected with a Gremlin expression vector (GREM-GFP green) or empty vector for 24 hours The levels and localization of R-Smad 3(a) and R-Smad 2 (b) were evaluated by confocal microscopy with Alexa-633 secondary IgG (red) Nuclei were stained using 410158406-diamino-2-phenylindole dihydrochloride (DAPI blue) In Gremlin-transfected cells (green staining by GFP) the Smad 2 and Smad 3 were found inthe nuclei (white staining in the merge) Figures show representative images out of 3 independent observations
and restored E-cadherin levels decreased by Gremlin (Fig-ure 7(b))These data suggest that TGF-120573 is amediator of long-term responses of Gremlin in tubuloepithelial cells includingregulation of profibrotic factors and EMT changes
4 Discussion
Our in vitro studies in cultured tubuloepithelial cells showthat Gremlin directly activates the Smad pathway and par-ticipates in the EMT process via Smad signalling These datasuggest that Gremlin could be a mediator of renal fibrosis
Our study reveals that in cultured human tubuloepithelialcells Gremlin induces a rapid activation of the Smad path-way (observed after 5min of stimulation) characterized byincreased phosphorylation of the receptor-Smad (R-Smad)Smad 3 a critical downstream mediator of fibrosis [17] and
Smad 2 proteinsOnceR-Smad is phosphorylated it dimeriseswith Smad 4 and then shuttles to the nucleus to regulategene expression By confocal microscopy we have foundthat Gremlin caused a rapid translocation to the nucleusof R-SmadSmad 4 proteins In several cells types Gremlin-induced TGF-120573 production [18 21] as we have observedhere after 48 hours of incubationHowever Gremlin-inducedearly Smad activation is independent of endogenous TGF-120573as we have demonstrated using TGF-120573 blockers (Figure 8)Other important profibrotic factors such as angiotensin IIalso activates the Smad pathway rapidly and independent ofendogenous TGF-120573 [17]
Previous studies in tubular epithelial cells have shownthat the Smad route regulates EMT induced by key factorsinvolved in renal fibrosis such as TGF-120573 and angiotensinII [20] The activation of Smad pathway has been described
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SBE-
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Figure 4 Gremlin overexpression induces Smad-dependent genetranscription HK2 cells were transfected with GREM-GFP or emptyvector Smadluc promoter and TK-renilla for 24 hours In somepoints cells were cotransfected with Smad 7Then luciferaserenillaactivity was measured Data are expressed as increase in Smadbinding element (SBE) promoter-luciferase dependent expressionData are expressed as n-fold over control (considered as 1) as themean plusmn SEM of 5 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
in experimental renal fibrotic diseases including glomeru-losclerosis tubulointerstitial fibrosis hypertensive-inducedrenal damage and diabetic nephropathy [20ndash26] as well as inrenal tumor progression [27] In angiotensin II-induced renaldamage renal activation of the Smad pathway was associatedto EMT changes [20] Moreover Smad 7 overexpressionameliorates renal damage and fibrosis caused by unilateralureteral obstruction angiotensin II and diabetes [22ndash2428] We have observed that in tubuloepithelial cells Smad 7overexpression blocked Gremlin-induced EMT changes Theinvolvement of Smad pathway in Gremlin-mediated fibrosishas been also described in other cell types in vitro In opticnerve head astrocytes and lamina cribrosa cells recombinantGremlin stimulates ECM production through the activationof TGF-120573 receptor and Smad 3 phosphorylation suggestinga role for Gremlin in glaucoma [29] In healthy dermalfibroblasts IL-6 mediated induction of collagen is dependenton Gremlin production and activation of TGF-120573Smad sig-nalling [30] Besides the regulation of renal EMT and fibrosisGremlinSmad pathway could also be involved in the onset ofproteinuria by modulating podocyte injury and changing thedistribution of nephrin and synaptopodin [21]
Recent evidences suggest that Gremlin could be animportant promoter of fibrosis in different pathologiesincluding liver fibrosis lung diseases particularly pul-monary hypertension and idiopathic pulmonary fibrosis andmyocardial fibrosis [31ndash35] In several human renal diseasesGremlin overexpression was found mainly in areas of tubuleinterstitial fibrosis [5ndash8] Experimental studies in mice haveshown that Gremlin blockade diminished renal fibrosis asobserved in streptozotocin-induced diabetes in knockoutmice heterozygous for grem1 [9] and by Gremlin gene silenc-ing [10] Recent studies have demonstrated direct fibrogeniceffect of Gremlin in renal cells In mesangial cells Gremlin
increased cell proliferation and ECM accumulation via ERK[36] In renal fibroblasts Gremlin increased ECM production[18] including type I collagen In tubular epithelial cellsGremlin upregulates profibrotic genes such as TGF-120573 andCTGF and caused EMT changes [18] Gremlin also inducesEMT in airway epithelial cells [37] and in cancer cells [38]Although the contribution of EMT to renal fibrosis is amatterof intense debate [39 40] the lost of epithelial propertiesof the tubular epithelial cells including permeability andpolarity may result in decreased viability and contribute torenal injury [40 41] Therefore EMT-related changes arean initial step in renal damage and an important potentialtherapeutic target Our data demonstrate that Gremlin viaSmad pathway regulates EMT showing a novel mechanismof Gremlin action in renal cells
TGF-120573 is known as the major promoter of EMT duringembryogenesis cancer and fibrosis [13ndash17]In a mesothe-lioma cell line Gremlin-silencing inhibited cell proliferationassociated with downregulation of the transcription factorslug as well as mesenchymal proteins linked to cancerEMT [38] We have recently demonstrated that Gremlingene silencing blocked TGF-120573-induced EMT in tubularepithelial cells [18] Now we have observed that Gremlinincreased TGF-120573 production at 48 hours and this endoge-nous autocrine TGF-120573 acts as a downstream mediator ofGremlin-induced profibrotic and EMT related factors incultured human tubuloepithelial cells (Figure 8) All thesefindings reveal the complex relationship between Gremlinand TGF-120573 in the kidney disclosing a positive feedback loopconnection between them in promoting EMT and fibrosis
Gremlin exerts a potent inhibitory action via binding toand forming heterodimers with BMP-2 BMP-4 and BMP-7The binding of Gremlin to selective BMPs prevents ligand-receptor interaction and subsequent downstream signallingGremlin acting as a BMPs antagonist plays a critical role dur-ing the process of nephrogenesis [4] BMP-7 is the antagonistof TGF-1205731 signalling and has been found to inhibit TGF-1205731-induced renal fibrosis by reversing EMT process [42 43]In experimental lungs and pulmonary fibrosis upregulationof Gremlin was associated with downregulation of BMPsignalling [31 32] Gremlin overexpression has been foundto inhibit BMP-4 thus leading to enhance TGF-120573 signallingand ECM deposition in primary open angle glaucoma [44]However BMP-independent mechanismsmaymediate someactions of Gremlin Exogenous Gremlin may bind to andact directly on endothelial cells to modulate angiogenesisincluding endothelial cell migration [45 46] We have foundthat BMPs did no inhibit Gremlin-induced early Smad 3activation Thus a receptor-mediated mechanism of actionmay exist for GremlinTherefore future studies investigatingthe receptor involved in Gremlin responses in renal cells areneeded
5 Conclusion
Chronic progressive fibrosis of the kidney remains anunsolved challenge The investigation of the mediators andmechanisms involved in renal fibrosis could lead to better
8 BioMed Research InternationalG
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Figure 5 Gremlin-induced EMT via the Smad pathway HK2 cells were transiently transfected with empty Gremlin (GREM-GFP) alone orcotransfected with Smad 7 expression vectors EMT markers were evaluated after 48 hours Gremlin transfected cells express GFP (greenstaining) Confocal microscopy analysis of cytokeratin and 120572-SMA immunofluorescence was performed using specific primary antibodiesand Alexa-633 secondary IgG (red staining) Representative image out of 3 experiments
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Figure 6 (a) Gremlin increased TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 and 48 hours in serum-freemedium TGF-1205731 protein levels were measured in the cell-conditioned medium using a specific ELISA Data are expressed as mean plusmn SEMof 6 independent experiments lowast119875 lt 005 versus control (b) The late increase in gene expression of profibrotic factors caused by Gremlin ismediated by endogenous TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 hours in serum-free medium TGF-120573was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizing antibody Total cell RNAwas isolated to assessmRNA levels by real-time PCR Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005versus control 119875 lt 005 versus Gremlin
BioMed Research International 9
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Figure 7TGF-120573 is amediator of EMT-related changes following stimulationwith Gremlin HK2 cells were stimulatedwithGremlin (50 ngmL)for 48 hours in serum-free medium TGF-120573 was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizingantibody (a) EMT changes were evaluated by confocal microscopy E-cadherin pan-Cytokeratin and Vimentin were studied by indirectimmunofluorescence using FITC-secondary IgG (green) and confocal microscopy Nuclei are shown in blue Figure shows a representativeimage out of 3 independent observations (b)Total proteins were isolated and Vimentin E-cadherin and Slug levels were analyzed by westernblot Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
diagnostic tools and novel therapeutics approaches Manystudies have shown that renal expression of Gremlin isinduced in diabetic nephropathy and in other progressiverenal diseases associated with tubulointerstitial fibrosis andSmad activation [5ndash8] We show here that Gremlin activatesthe Smad signaling pathway and induces TGF-120573 and otherrelated factors involved in EMT and fibrotic events in renalcells All these data suggest that Gremlin could be a potential
novel molecular antifibrotic target and biomarker useful forprognostication disease monitoring and therapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
10 BioMed Research International
Gremlin
Smadactivation
Target genes
EMT-related changes
Early-response(min)
Long-response
TGF-120573 independent
TGF-120573 production
TGF-120573 mediated(gt24hours)
Induction of vimentin and 120572-SMAlost of E-cadherin and cytokeratin
Figure 8 Dual effects of Gremlin on Smad activation Gremlininduces an early (minutes) and direct TGF-120573-independent Smadpathway activation After 24 hours Gremlin increased severalprofibrotic genes including TGF-120573 and after 48 hours increasedTGF-120573 production and induced EMT features These long-termGremlin-induced profibrotic events require autocrine TGF-120573
Authorsrsquo Contribution
Raquel Rodrigues-Diez and Raul R Rodrigues-Diez equallycontributed to this work
Acknowledgments
This work was supported by grants from the Institutode Salud Carlos III (PI1101854 and REDINREN ISCIII-RETIC RD1200210002 and 0001) Sociedad Espanola deNefrologıa PCI Iberoamerica (A957107) CYTED IBER-ERC FONDECYT Chile 1080083 and 1120480 Comu-nidad de Madrid (Fibroteam S2010BMD-2321 S2010BMD-2378) Programa Intensificacion Actividad Investigadora(ISCIIIAgencia Laın-EntralgoCM) to AO Fundacion parael fomento en Asturias de la investigacion cientıfica aplicaday la tecnologıa (FICYT) The authors want to thank Ma MarGonzalezGarcia-Parreno for her technical help with confocalmicroscopy
References
[1] S A Roxburgh M Murphy C A Pollock and D P BrazilldquoRecapitulation of embryological programmes in renalfibrosismdashthe importance of epithelial cell plasticity anddevelopmental genesrdquo Nephron Physiology vol 103 no 3 ppp139ndashp148 2006
[2] R McMahon MMurphy M Clarkson et al ldquoIHG-2 a mesan-gial cell gene induced by high glucose is human gremlin regu-lation by extracellular glucose concentration cyclic mechanicalstrain and transforming growth factor-1205731rdquo The Journal ofBiological Chemistry vol 275 no 14 pp 9901ndash9904 2000
[3] L Z Topol B Bardot Q Zhang et al ldquoBiosynthesis post-translation modification and functional characterization ofDrmGremlinrdquo The Journal of Biological Chemistry vol 275no 12 pp 8785ndash8793 2000
[4] O Michos L Panman K Vintersten K Beier R Zeller andA Zuniga ldquoGremlin-mediated BMP antagonism inducesthe epithelial-mesenchymal feedback signaling controllingmetanephric kidney and limb organogenesisrdquo Developmentvol 131 no 14 pp 3401ndash3410 2004
[5] V Dolan M Murphy D Sadlier et al ldquoExpression of gremlina bone morphogenetic protein antagonist in human diabeticnephropathyrdquo American Journal of Kidney Diseases vol 45 no6 pp 1034ndash1039 2005
[6] M Murphy J Crean D P Brazil D Sadlier F Martin andC Godson ldquoRegulation and consequences of differential geneexpression in diabetic kidney diseaserdquo Biochemical SocietyTransactions vol 36 no 5 pp 941ndash945 2008
[7] S Mezzano A Droguett M B Eugenia et al ldquoExpression ofgremlin a bonemorphogenetic protein antagonist in glomeru-lar crescents of pauci-immune glomerulonephritisrdquoNephrologyDialysis Transplantation vol 22 no 7 pp 1882ndash1890 2007
[8] G Carvajal A Droguett M E Burgos et al ldquoGremlin a novelmediator of epithelial mesenchymal transition and fibrosis inchronic allograft nephropathyrdquo Transplantation Proceedingsvol 40 no 3 pp 734ndash739 2008
[9] S A Roxburgh J J Kattla S P Curran et al ldquoAllelic depletionof grem1 attenuates diabetic kidney diseaserdquo Diabetes vol 58no 7 pp 1641ndash1650 2009
[10] Q Zhang Y Shi J Wada et al ldquoIn vivo delivery of gremlinsiRNA plasmid reveals therapeutic potential against diabeticnephropathy by recovering bone morphogenetic protein-7rdquoPLoS ONE vol 5 no 7 article e11709 2010
[11] M Zeisberg and E G Neilson ldquoMechanisms oftubulointerstitial fibrosisrdquo Journal of the American Societyof Nephrology vol 21 no 11 pp 1819ndash1834 2010
[12] Y Liu ldquoNew insights into epithelial-mesenchymal transition inkidney fibrosisrdquo Journal of the American Society of Nephrologyvol 21 no 2 pp 212ndash222 2010
[13] A Moustakas and C-H Heldin ldquoSignaling networks guidingepithelial-mesenchymal transitions during embryogenesisand cancer progressionrdquo Cancer Science vol 98 no 10 pp1512ndash1520 2007
[14] J-M Fan N G Yee-Yung P A Hill et al ldquoTransforminggrowth factor-120573 regulates tubular epithelial-myofibroblasttransdifferentiation in vitrordquo Kidney International vol 56 no4 pp 1455ndash1467 1999
[15] A B Roberts F Tian S D Byfield et al ldquoSmad3 is keyto TGF-120573-mediated epithelial-to-mesenchymal transitionfibrosis tumor suppression andmetastasisrdquo Cytokine amp GrowthFactor Reviews vol 17 no 1-2 pp 19ndash27 2006
BioMed Research International 11
[16] J Xu S Lamouille and R Derynck ldquoTGF-120573-induced epithelialto mesenchymal transitionrdquo Cell Research vol 19 no 2 pp156ndash172 2009
[17] M Ruiz-Ortega J Rodrıguez-Vita E Sanchez-Lopez GCarvajal and J Egido ldquoTGF-120573 signaling in vascular fibrosisrdquoCardiovascular Research vol 74 no 2 pp 196ndash206 2007
[18] R Rodrigues-Diez C Lavoz G Carvajal et al ldquoGremlin isa downstream profibrotic mediator of transforming growthfactor-beta in cultured renal cellsrdquo Nephron ExperimentalNephrology vol 122 no 1-2 pp 62ndash74 2012
[19] J Rodrıguez-Vita E Sanchez-Lopez V Esteban M Ruperez JEgido and M Ruiz-Ortega ldquoAngiotensin II activates the Smadpathway in vascular smooth muscle cells by a transforminggrowth factor-120573-independent mechanismrdquo Circulation vol 111no 19 pp 2509ndash2517 2005
[20] G Carvajal J Rodrıguez-Vita R Rodrigues-Dıez et alldquoAngiotensin II activates the Smad pathway during epithelialmesenchymal transdifferentiationrdquo Kidney International vol74 no 5 pp 585ndash595 2008
[21] G Li Y Li S Liu et al ldquoGremlin aggravates hyperglycemia-induced podocyte injury by a TGF120573smad dependent signalingpathwayrdquo Journal of Cellular Biochemistry vol 114 no 9 pp2101ndash2113 2013
[22] H Y Lan W Mu N Tomita et al ldquoInhibition of renalfibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in ratUUOmodelrdquo Journal of theAmericanSociety of Nephrology vol 14 no 6 pp 1535ndash1548 2003
[23] H Fukasawa T Yamamoto A Togawa et al ldquoDown-regulationof Smad7 expression by ubiquitin-dependent degradationcontributes to renal fibrosis in obstructive nephropathy inmicerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 23 pp 8687ndash8692 2004
[24] H Y Chen X R Huang WWang et al ldquoThe protective role ofSmad7 in diabetic kidney disease mechanism and therapeuticpotentialrdquo Diabetes vol 60 no 2 pp 590ndash601 2011
[25] X Wei Y Xia F Li et al ldquoKindlin-2 mediates activationof TGF-120573Smad signaling and renal fibrosisrdquo Journal of theAmerican Society Nephrology vol 24 no 9 pp 1387ndash1398 2013
[26] M Isono S Chen S W Hong M C Iglesias-de la Cruzand F N Ziyadeh ldquoSmad pathway is activated in the diabeticmouse kidney and smad3 mediates TGF-120573-induced fibronectinin mesangial cellsrdquo Biochemical and Biophysical ResearchCommunications vol 296 no 5 pp 1356ndash1365 2002
[27] J H Park C Lee J H Suh J Y Chae andK CMoon ldquoNuclearexpression of Smad proteins and its prognostic significance inclear cell renal cell carcinomardquo Human Pathology vol 44 no10 pp 2047ndash2054 2013
[28] G X Liu Y Q Li X R Huang et al ldquoSmad7 inhibitsAngII-mediated hypertensive nephropathy in amousemodel ofhypertensionrdquo Clinical Science vol 127 no 3 pp 195ndash208 2014
[29] G S Zode A F Clark and R J Wordinger ldquoBone morpho-genetic protein 4 inhibits TGF-1205732 stimulation of extracellularmatrix proteins in optic nerve head cells role of gremlin inECMmodulationrdquo Glia vol 57 no 7 pp 755ndash766 2009
[30] S OrsquoReilly M Ciechomska R Cant and J M van Laar ldquoIL-6trans signalling drives a STAT3 dependant pathway that leadsto hyperactive TGF-120573 signalling promoting SMAD3 activationand fibrosis via gremlinrdquo The Journal of Biological Chemistry2014
[31] M Myllarniemi P Lindholm M J Ryynanen et al ldquoGremlin-mediated decrease in bone morphogenetic protein signaling
promotes pulmonary fibrosisrdquo American Journal of Respiratoryand Critical Care Medicine vol 177 no 3 pp 321ndash329 2008
[32] E Cahill C M Costello S C Rowan et al ldquoGremlin playsa key role in the pathogenesis of pulmonary hypertensionrdquoCirculation vol 125 no 7 pp 920ndash930 2012
[33] W Boers S Aarrass C Linthorst M Pinzani R O Elferinkand P Bosma ldquoTranscriptional profiling reveals novel markersof liver fibrogenesis gremlin and insulin-like growth factor-binding proteinsrdquo The Journal of Biological Chemistry vol 281no 24 pp 16289ndash16295 2006
[34] CMCostello E Cahill FMartin S Gaine andPMcLoughlinldquoRole of gremlin in the lung development and diseaserdquoAmerican Journal of Respiratory Cell and Molecular Biologyvol 42 no 5 pp 517ndash523 2010
[35] K A Mueller E Tavlaki M Schneider et al ldquoGremlin-1identifies fibrosis and predicts adverse outcome in patientswith heart failure undergoing endomyocardial biopsyrdquo Journalof Cardiac Failure vol 19 no 10 pp 678ndash684 2013
[36] H Huang H Huang Y Li et al ldquoGremlin induces cellproliferation and extra cellular matrix accumulation in mousemesangial cells exposed to high glucose via the ERK12pathwayrdquo BMC Nephrology vol 14 article 33 2013
[37] N McCormack E L Molloy and S OrsquoDea ldquoBone morpho-genetic proteins enhance an epithelial-mesenchymal transitionin normal airway epithelial cells during restitution of a dis-rupted epitheliumrdquoRespiratory Research vol 14 article 36 2013
[38] J A Tamminen V Parviainen M Ronty et al ldquoGremlin-1associates with fibrillin microfibrils in vivo and regulatesmesothelioma cell survival through transcription factor slugrdquoOncogenesis vol 2 article e66 2013
[39] E M Zeisberg S E Potenta H Sugimoto M Zeisberg and RKalluri ldquoFibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transitionrdquo Journal of the American Society ofNephrology vol 19 no 12 pp 2282ndash2287 2008
[40] V S LeBleu G Taduri J OrsquoConnell et al ldquoOrigin and functionof myofibroblasts in kidney fibrosisrdquo Nature Medicine vol 19no 8 pp 1047ndash1053 2013
[41] W C Prozialeck and J R Edwards ldquoCell adhesion moleculesin chemically-induced renal injuryrdquo Pharmacology amp Thera-peutics vol 114 no 1 pp 74ndash93 2007
[42] M Zeisberg J Hanai H Sugimoto et al ldquoBMP-7 counteractsTGF-1205731-induced epithelial-to-mesenchymal transition andreverses chronic renal injuryrdquo Nature Medicine vol 9 no 7 pp964ndash968 2003
[43] Y-L Yang H-Z Ju S-F Liu et al ldquoBMP-2 suppresses renalinterstitial fibrosis by regulating epithelial-mesenchymaltransitionrdquo Journal of Cellular Biochemistry vol 112 no 9 pp2558ndash2565 2011
[44] R J Wordinger D L Fleenor P E Hellberg et al ldquoEffects ofTGF-1205732 BMP-4 and gremlin in the trabecular meshworkimplications for glaucomardquo Investigative Ophthalmology ampVisual Science vol 48 no 3 pp 1191ndash1200 2007
[45] B Chen M Athanasiou Q Gu and D G Blair ldquoDrmGremlintranscriptionally activates p21Cip1 via a novel mechanism andinhibits neoplastic transformationrdquoBiochemical andBiophysicalResearch Communications vol 295 no 5 pp 1135ndash1141 2002
[46] S Mitola C Ravelli E Moroni et al ldquoGremlin is a novelagonist of the major proangiogenic receptor VEGFR2rdquo Bloodvol 116 no 18 pp 3677ndash3680 2010
also called downregulated by mos (Drm) [2] Gremlin isa member of cysteine knot superfamily [3] that includestransforming growth factor-120573 (TGF-120573) proteins and actsas a bone morphogenetic protein (BMP) antagonist [4]Analysis of the predicted amino acid sequence indicated thepresence of several significant features including potentialnuclear localization signals near the C-terminus potentialN-linked glycosylation sites and multiple potential sites forphosphorylation The signalling peptide and a predictedglycosylation site have been identified Gremlin is a glycosy-lated phosphorylated secreted protein present both on theexternal cell surface and within the ER-Golgi compartments[3] In many human renal diseases induction of Gremlinhas been described [5ndash8] Several experimental studies haveshown that Gremlin participates in renal damage [9 10]Therefore some authors have suggested that Gremlin couldbe considered as a mediator of renal injury
Chronic progressive fibrosis of the kidney remains anunresolved challenge Irrespective of the underlying causechronic kidney disease is linked to the development oftubulointerstitial fibrosis characterized by accumulation ofextracellularmatrix (ECM)Themechanisms of renal fibrosisare complex and our therapeutic armamentarium is limitedThe key cellular mediator of fibrosis is the myofibroblastThere are different sources of myofibroblasts including acti-vation of tissue fibroblasts and migration of circulating mes-enchymal progenitors or cell transitions including epithelial-mesenchymal transition (EMT) or endothelial mesenchymaltransition [11 12] The investigation of the mechanismsinvolved in renal fibrosis and the identification of novelmedi-ators with potential therapeutic application is an importantopen question in chronic kidney disease
TGF-1205731 signalingmainly through Smad proteins is a keyplayer in fibrosis and EMT [13ndash17] Because of its pleiotropicactions TGF-120573 blockade is not an ideal therapeutic tooltherefore novel targets are needed Among them Grem-lin may be an interesting candidate in progressive renaldiseases Recent in vitro studies developed by our grouphave shown that Gremlin gene silencing inhibited TGF-120573-mediated matrix production and EMT [18] Howeverthe involvement of TGF-120573 in Gremlin responses has notbeen investigated We have also reported the presence ofGremlin in glomerular crescents of human pauci-immuneglomerulonephritis and in the tubulointerstitium of chronicallograft nephropathy In these human diseases Gremlin cor-related with the degree of tubulointerstitial fibrosis and wasassociated with TGF-1205731 overexpression and Smad pathwayactivation [7 8] These studies suggest that Gremlin mayactivate the Smad pathway therefore the aim of this workwas to evaluate whether Gremlin could directly activate theSmad pathway in tubular epithelial cells evaluating whetherthis activation is linked to Gremlin-induced EMT the mainfibrotic effect observed in response to Gremlin stimulation inthese cells [18]
2 Materials and Methods
21 Cell Cultures Human renal proximal tubuloepithelialcells (HK2 cell line ATCC CRL-2190) were grown in RPMI
with 10 fetal bovine serum (FBS) 1 nonessential aminoacids 100UmL penicillin and 100120583gmL streptomycininsulin transferrin selenite (ITS) (5120583gmL) and hydrocorti-sone (36 ngmL) in 5CO
2at 37∘CAt 60ndash70of confluence
cells were growth-arrested in serum-free medium for 24hours before the experimentsThen cells were stimulated fordifferent timeswith recombinantGremlin (50 ngmL) (RampD)or human recombinant TGF-1205731 (1 ngmL Peprotech) Cellculture reagents were obtained from Life Technologies IncTGF-120573 was targeted by a pan-specific polyclonal anti-TGF-120573neutralizing antibody which recognizes bovine mouse andhuman TGF-1205731 and 1205732 isoforms (1 120583gmL) (RampD)
22 Transfection cDNA Constructs and Promoter StudiesHK2 cells were transiently transfected for 24ndash48 hourswith FuGENE (Roche) pCDNA3-Gremlin-myc-IRES2-eGFP plasmid (GREM-GFP) andor pCDNA-FLAG-Smad7expression vector (kindly donated by Dr MassagueMemorial Sloan-Keternig Cancer Center USA) or emptyvector (pCDNA) The GREM-GFP was generated as followsGREM1 cDNA was purchased from the Mammalian GeneNIH Collection (Bethesda Maryland USA) We addeda c-myc tag to the 31015840 portion of GREM1 using PCR withthe forward primer 51015840AGTGCGGCGGCTGAGGACCCGCCGCACTGACAT-31015840 and the reverse primer 51015840-ATAGCCGCCGCTTACAGATCCTCTTCTGAGATG-AGTTTTTGTTCATCCAAATCGATGGATATGC-31015840We also inserted an e-GFP sequence downstream ofhuman Gremlin as follows The IRES-eGFP sequencewas obtained by PCR using a pIRES2-EGFP plasmid(Clontech Mountain View CA USA) as the template withthe following primers IRES-eGFP-F (51015840-TACATTAAT-GGGCCCGGGATCCGCCCCTC-31015840) and IRES-eGFP-R(51015840-GGCCATATGCGCCTTAAGATACATTGATG-31015840) The GREM1-c-myc and IRES-eGFP fragments wereindependently cloned into a pGEMT-Easy vector andthen sequenced (Macrogen Seoul Korea) to confirm themodifications and absence of additional mutations Nextboth the GREM1-c-myc and IRES-eGFP fragments weresubcloned into a modified pCDNA3 vector using theEcoRI and NotI restriction sites respectively In Gremlin-transfected cells Gremlin production was confirmed byimmunofluorescence (not shown)
To demonstrate Smad 7 transfection efficacy an anti-FLAG antibody was used (not shown) Smad-dependent pro-moter activation was evaluated by transfection of Smadluc(kindly donated by Dr Volgestein Baltimore USA) and TK-renilla as internal control as described [19]
23 Protein Studies Total cellular protein extracts (10ndash50 120583glane) obtained in lysis buffer [50mM Tris-HCl pH 74150mMNaCl 2mMEDTA 2mMEGTA 02Triton X-10003 NP40 100 120583M phenylmethylsulphonylfluoride 1mMdithiothreitol 100 120583MNa
3VO4 and 1mM protease-inhibitor
cocktail (Sigma)] were separated on 8ndash12 polyacrylamide-SDS gels under reducing conditions Samples were thentransferred onto nitrocellulose membranes (Bio-Rad Her-cules CA) blocked with 5 nonfat dry milk in 50mM
BioMed Research International 3
Tris-HCl pH 75 150mM NaCl with 005 Tween-20 andincubated overnight at 4∘C with the primary antibodiesand subsequently incubated with peroxidase-conjugated IgG(Amersham) and developed by ECL chemiluminescence(GE Healthcare Buckinghamshire UK)
Immunocytochemistry studies were performed in cellsgrowing on coverslips After the experiments cells werefixed in Merckofix (Merck) and permeabilized with 02Triton-X100 for 10min (except for E-cadherin staining)After blocking with 4 BSA and 8 serum for 1 hoursamples were incubated with primary antibodies overnightat 4∘C and then 1 hour at room temperature with fluoresceinisothiocyanate (FITC) [1200] or AlexaFluor 633 [1300]conjugated antibodies (Amersham) Nuclei were stainedwith 1 120583gmL propidium iodide (PI) or 410158406-Diamidino-2-phenyindole dilactate (DAPI) (Sigma-Aldrich) as control ofequal cell density Absence of primary antibody was used asnegative control Samples were mounted in Mowiol 40ndash88(Sigma-Aldrich) and examined by a Leica DM-IRB confocalmicroscope
TGF-1205731 protein was measured in the cell-conditionedmedium using a commercial enzyme-linked immunoassay(ELISA) (BD Sciences San Diego USA) following the man-ufacturerrsquos instructions TGF-1205731 levels were quantified bycomparison with a standard curve using increasing concen-trations of human TGF-1205731 Protein content was determinedby the BCA method (Pierce)
24 Gene Expression Total RNA was isolated from cells withTrizol (Invitrogen) according to the manufacturerrsquos protocolcDNA was synthesized from 2120583g of total RNA with randomhexamer primers using the High capacity cDNA Archive Kit(Applied) Real-time PCR was performed using human FAMTaqMan MGB probes designed by assay-on-demand geneexpression products (Applied) TGF-1205731 Hs99999918 m1connective tissue growth factor (CTGF) Hs00170014 m1plasminogen activator inhibitor 1 (PAI1) Hs 00167155 m1Data weres normalized to 18S eukaryotic ribosomal RNA4210893E (VIC) The mRNA copy numbers were calculatedfor each sample by the instrument software using Ct value(ldquoarithmetic fit point analysis for the lightcyclerrdquo) Resultswere expressed in copy numbers and calculated relative tounstimulated cells after normalization against 18S
25 Statistical Analysis Results throughout the text areexpressed as mean plusmn SEM Differences between agonist-treated groups and controls were assessed by one-way anal-ysis of variance followed by post hoc Bonferroni or Dunnetttest or Mann-Whitney test as appropriate 119875 lt 005 wasconsidered significant Statistical analysis was conductedusing the SPSS statistical software version 110 (SPSS)
3 Results
31 Gremlin Activates Smad Pathway in Human CulturedTubuloepithelial Cells Receptormediated activation of Smadproteins (R-Smads 2 and 3) occurs by direct C-terminalphosphorylation Smad 23 then form complexes with Smad4 and translocate into the nucleus where they associateand cooperate with DNA binding transcription factors toactivate or repress target gene transcription [17] In culturedHK2 cells stimulation with recombinant Gremlin increasedphosphorylation levels of Smad 3 as early as 5 minutes and itwas maintained until 15 minutes (Figure 1(a))
Although Smad is the main signaling mechanism ofTGF-120573 several factors involved in renal damage such asangiotensin II can directly activate the Smad pathway inde-pendent of endogenous TGF-120573 [17] Therefore to evaluatewhether early Smad activation caused by Gremlin was medi-ated or not by TGF-120573 cells were preincubated with a neu-tralizing antibody against active TGF-120573 Gremlin-inducedSmad activation (evaluated as p-Smad 3 levels) was notmodified in the presence of the TGF-120573 antibody (Figure 1(b))Similar lack of response was found in the presence of decorin(a proteoglycan that neutralizes active TGF-120573 not shown)These data indicates that Gremlin directly activates the Smadpathway
Some actions of Gremlin are due to its effect as BMPantagonist [4] To determine the contribution of BMPs inGremlin-induced Smad activation HK2 cells were prein-cubated with BMP-2 or BMP-4 and then stimulated withGremlin during 10 minutes Phosphorylation of Smad 3 wasnot modified in the presence of any of these BMPs (Fig-ure 1(c)) suggesting that Gremlin-induced Smad activationis independent of BMPs in tubular epithelial cells
By confocal microscopy we have confirmed that Gremlinrapidly increased Smad 3 translocation to the nucleus thelatter demonstrated by the yellow nuclear staining observedin the merge of Figure 2 while in untreated cells the nucleiare red Gremlin also increased nuclear localization of Smad2 and Smad 4 observed at 15 minutes (Figure 2)
To further confirm that Gremlin activates the Smadpathway cells were transfected with a Gremlin expressionvector (GREM-GFP) for 24 hours By confocal microscopywe observed that in positive Gremlin-transfected cells (GFP-green staining) there was a nuclear immunostaining showingthe translocation of Smad 3 and Smad 2 (characteristic ofSmad activation) compared to cells transfected with emptyvector (Figures 3(a) and 3(b)) In the merge of Figure 3a Gremlin expressing-cell marked by a yellow rectanglepresented a positive nuclear staining corresponding to thepresence of Smad 3 or 2 In contrast in cells transfectedwith empty vector there are no nuclear Smad 2 and Smad 3immunostaining (all cells present blue nuclei) as observed insome nontransfected cells
To investigatewhetherGremlin regulates Smad-mediatedgene expression cells were cotransfected with a Grem-lin expression vector (GREM-GFP) and a luciferase Smadreporter plasmid Gremlin transfected cells expressed higherSmad-dependent luciferase activity (SBE) than control cells(Figure 4) To demonstrate further the involvement of Smad
4 BioMed Research International
p-Smad 3
GAPDH
0
1
2
3
4
Control
Gremlin (min)
5 10 15 20
p-Sm
ad 3
expr
essio
n le
vels
(n-fo
ld)
lowast
lowast
lowast
(a)
p-Smad 3
p-Sm
ad 3
expr
essio
n le
vels
(n-fo
ld)
0
1
2
3
Control Gremlin
GAPDH
lowast
lowast
Gremlin
Anti-TGF-120573+
(b)
0
1
2
3
4
Control Gremlin BMP-2BMP-4Gremlin
p-Smad 3
GAPDH
p-Sm
ad 3
expr
essio
n le
vels
(n-fo
ld)
lowast
lowastlowast
(c)
Figure 1 (a) Stimulation with Gremlin rapidly increased Smad 3 phosphorylation in cultured human tubuloepithelial cells HK2 cells werestimulated with Gremlin (50 ngmL) for different times (b) Early Smad 3 phosphorylation induced by stimulation with Gremlin was notmediated by TGF-120573 TGF-120573 was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizing antibody andthen treated with Gremlin for 10 minutes (c) In some points HK2 cells were preincubated with BMP-2 or BMP-4 and then treated withGremlin for 10 minutes Total proteins were isolated and protein levels were evaluated by western blot GAPDH or Smad 3 were used asloading controls Figures show a representative western blot of phosphorylated levels of Smad 3 and data are expressed as n-fold over control(considered as 1) as the mean plusmn SEM of 3-4 independent experiments lowast119875 lt 005 versus control
pathway in Gremlin-induced responses a Smad 7 expressionvector that inhibits Smad-mediated transcriptional effects byinterfering with receptor-mediated activation of R-Smad wasused [17 19] In HK2 cells cotransfected with GREM-GFPand Smad 7 expression vectors the Smad-mediated luciferaseactivity was significantly lower than cells transfected withGREM-GFP alone showing the specific Smad 7 blockade ofGremlin-mediated Smad activation (Figure 4)
32 Gremlin-Induced EMT Is Mediated by Smad ActivationWe have previously demonstrated that in tubular epithe-lial cells long-term stimulation with recombinant Gremlininduced EMT [18] Now we have observed that transfec-tion of HK2 cells with GREM-GFP induced EMT-relatedphenotypic changes observed by confocal microscopy after48 hours (Figure 5) Cells transfected with empty-vectorshowed epithelial morphology including the presence of
BioMed Research International 5
Smad 3 IP MergeC
ontro
lG
rem
lin
(a)
Smad 2 IP Merge
Con
trol
Gre
mlin
(b)
Smad 4 IP Merge
Con
trol
Gre
mlin
(c)
Figure 2 Stimulation with Gremlin induces a rapid activation of the Smad pathway in cultured human tubuloepithelial cells HK2 cells werestimulated with Gremlin (50 ngmL) for 15 minutes The localization of R-Smad 3 (a) and 2 (b) and Smad 4 (c) was evaluated by confocalmicroscopy with FITC-secondary antibodies (green staining) Nuclei were stained with propidium iodide (IP) (red) In themerge the yellowstaining indicates the nuclear localization of Smad proteinsThe results are representative of 3 independent confocal microscopy experiments
epithelial markers such as cytokeratin (red staining) andthere is no positive staining for mesenchymal marker 120572-SMA(Figure 5) In contrast overexpression of Gremlin causedchanges inmorphology to fibroblast-like shape and inductionof 120572-SMA (see the GREM-GFP positive cell that presentsyellow staining and elongated shape) Moreover in Gremlinexpressing cells cytokeratin staining was markedly dimin-ished (absence of red staining in an area with several GREM-GFPpositive green cells)Theblockade of Smad activation bycotransfection with Gremlin and Smad 7 diminished theseEMT changes (Figure 5) as shown by restoration of thecytokeratin immunostaining and the epithelial morphologyand diminution of 120572-SMA as observed in the green positivecell These data suggest that Gremlin regulates EMT throughthe Smad pathway
33 Role of Endogenous TGF-120573 on Gremlin-Induced EMTPreviously we have reported that Gremlin acts as a down-stream mediator of TGF-120573-induced fibrosis in cultured renal
cells and incubation with Gremlin for 24 hours induced asignificant upregulation of TGF-1205731 mRNA levels in culturedtubuloepithelial cells [18] We have further investigated therelation between GremlinTGF-120573 evaluating whether Grem-lin could regulate TGF-1205731 synthesis In HK2 cells activeTGF-1205731 protein increased in the supernatants of Gremlin-stimulated cells after 48 hours but not at 24 hours (Fig-ure 6(a)) suggesting that some of the profibrotic actions ofGremlin could bemediated by endogenousTGF-1205731 synthesisTherefore we blocked TGF-120573 before HK-2 stimulation withGremlin by adding a neutralizing antibody against activeTGF-120573 which is able to block angiotensin II-induced ECMproduction andEMT [19 20] TGF-120573neutralization inhibitedGremlin-induced gene upregulation of profibrotic factorsobserved after 24 hours including TGF-120573 CTGF and PAI-1 (Figure 6(b)) Moreover TGF-120573 blockade antagonizedseveral EMT-related changes induced by Gremlin after 48hours as shown by immunofluorescence (Figure 7(a)) Wealso observed by western blot that TGF-120573 neutralizationdiminished Vimentin and Slug induction caused by Gremlin
6 BioMed Research International
Nuclei MergeSmad 3 GFP
Empt
y pC
DN
AG
REM
-GFP
(a)
Smad 2 Nuclei MergeGFP
Empt
y pC
DN
AG
REM
-GFP
(b)
Figure 3 Gremlin overexpression causes a sustained Smad activation in cultured human tubuloepithelial cells HK2 cells were transientlytransfected with a Gremlin expression vector (GREM-GFP green) or empty vector for 24 hours The levels and localization of R-Smad 3(a) and R-Smad 2 (b) were evaluated by confocal microscopy with Alexa-633 secondary IgG (red) Nuclei were stained using 410158406-diamino-2-phenylindole dihydrochloride (DAPI blue) In Gremlin-transfected cells (green staining by GFP) the Smad 2 and Smad 3 were found inthe nuclei (white staining in the merge) Figures show representative images out of 3 independent observations
and restored E-cadherin levels decreased by Gremlin (Fig-ure 7(b))These data suggest that TGF-120573 is amediator of long-term responses of Gremlin in tubuloepithelial cells includingregulation of profibrotic factors and EMT changes
4 Discussion
Our in vitro studies in cultured tubuloepithelial cells showthat Gremlin directly activates the Smad pathway and par-ticipates in the EMT process via Smad signalling These datasuggest that Gremlin could be a mediator of renal fibrosis
Our study reveals that in cultured human tubuloepithelialcells Gremlin induces a rapid activation of the Smad path-way (observed after 5min of stimulation) characterized byincreased phosphorylation of the receptor-Smad (R-Smad)Smad 3 a critical downstream mediator of fibrosis [17] and
Smad 2 proteinsOnceR-Smad is phosphorylated it dimeriseswith Smad 4 and then shuttles to the nucleus to regulategene expression By confocal microscopy we have foundthat Gremlin caused a rapid translocation to the nucleusof R-SmadSmad 4 proteins In several cells types Gremlin-induced TGF-120573 production [18 21] as we have observedhere after 48 hours of incubationHowever Gremlin-inducedearly Smad activation is independent of endogenous TGF-120573as we have demonstrated using TGF-120573 blockers (Figure 8)Other important profibrotic factors such as angiotensin IIalso activates the Smad pathway rapidly and independent ofendogenous TGF-120573 [17]
Previous studies in tubular epithelial cells have shownthat the Smad route regulates EMT induced by key factorsinvolved in renal fibrosis such as TGF-120573 and angiotensinII [20] The activation of Smad pathway has been described
BioMed Research International 7
0
1
2
3
4
Control GREM-GFP GREM-GFP+ Smad 7
SBE-
prom
otor
expr
essio
n (n
-fold
) lowast
Figure 4 Gremlin overexpression induces Smad-dependent genetranscription HK2 cells were transfected with GREM-GFP or emptyvector Smadluc promoter and TK-renilla for 24 hours In somepoints cells were cotransfected with Smad 7Then luciferaserenillaactivity was measured Data are expressed as increase in Smadbinding element (SBE) promoter-luciferase dependent expressionData are expressed as n-fold over control (considered as 1) as themean plusmn SEM of 5 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
in experimental renal fibrotic diseases including glomeru-losclerosis tubulointerstitial fibrosis hypertensive-inducedrenal damage and diabetic nephropathy [20ndash26] as well as inrenal tumor progression [27] In angiotensin II-induced renaldamage renal activation of the Smad pathway was associatedto EMT changes [20] Moreover Smad 7 overexpressionameliorates renal damage and fibrosis caused by unilateralureteral obstruction angiotensin II and diabetes [22ndash2428] We have observed that in tubuloepithelial cells Smad 7overexpression blocked Gremlin-induced EMT changes Theinvolvement of Smad pathway in Gremlin-mediated fibrosishas been also described in other cell types in vitro In opticnerve head astrocytes and lamina cribrosa cells recombinantGremlin stimulates ECM production through the activationof TGF-120573 receptor and Smad 3 phosphorylation suggestinga role for Gremlin in glaucoma [29] In healthy dermalfibroblasts IL-6 mediated induction of collagen is dependenton Gremlin production and activation of TGF-120573Smad sig-nalling [30] Besides the regulation of renal EMT and fibrosisGremlinSmad pathway could also be involved in the onset ofproteinuria by modulating podocyte injury and changing thedistribution of nephrin and synaptopodin [21]
Recent evidences suggest that Gremlin could be animportant promoter of fibrosis in different pathologiesincluding liver fibrosis lung diseases particularly pul-monary hypertension and idiopathic pulmonary fibrosis andmyocardial fibrosis [31ndash35] In several human renal diseasesGremlin overexpression was found mainly in areas of tubuleinterstitial fibrosis [5ndash8] Experimental studies in mice haveshown that Gremlin blockade diminished renal fibrosis asobserved in streptozotocin-induced diabetes in knockoutmice heterozygous for grem1 [9] and by Gremlin gene silenc-ing [10] Recent studies have demonstrated direct fibrogeniceffect of Gremlin in renal cells In mesangial cells Gremlin
increased cell proliferation and ECM accumulation via ERK[36] In renal fibroblasts Gremlin increased ECM production[18] including type I collagen In tubular epithelial cellsGremlin upregulates profibrotic genes such as TGF-120573 andCTGF and caused EMT changes [18] Gremlin also inducesEMT in airway epithelial cells [37] and in cancer cells [38]Although the contribution of EMT to renal fibrosis is amatterof intense debate [39 40] the lost of epithelial propertiesof the tubular epithelial cells including permeability andpolarity may result in decreased viability and contribute torenal injury [40 41] Therefore EMT-related changes arean initial step in renal damage and an important potentialtherapeutic target Our data demonstrate that Gremlin viaSmad pathway regulates EMT showing a novel mechanismof Gremlin action in renal cells
TGF-120573 is known as the major promoter of EMT duringembryogenesis cancer and fibrosis [13ndash17]In a mesothe-lioma cell line Gremlin-silencing inhibited cell proliferationassociated with downregulation of the transcription factorslug as well as mesenchymal proteins linked to cancerEMT [38] We have recently demonstrated that Gremlingene silencing blocked TGF-120573-induced EMT in tubularepithelial cells [18] Now we have observed that Gremlinincreased TGF-120573 production at 48 hours and this endoge-nous autocrine TGF-120573 acts as a downstream mediator ofGremlin-induced profibrotic and EMT related factors incultured human tubuloepithelial cells (Figure 8) All thesefindings reveal the complex relationship between Gremlinand TGF-120573 in the kidney disclosing a positive feedback loopconnection between them in promoting EMT and fibrosis
Gremlin exerts a potent inhibitory action via binding toand forming heterodimers with BMP-2 BMP-4 and BMP-7The binding of Gremlin to selective BMPs prevents ligand-receptor interaction and subsequent downstream signallingGremlin acting as a BMPs antagonist plays a critical role dur-ing the process of nephrogenesis [4] BMP-7 is the antagonistof TGF-1205731 signalling and has been found to inhibit TGF-1205731-induced renal fibrosis by reversing EMT process [42 43]In experimental lungs and pulmonary fibrosis upregulationof Gremlin was associated with downregulation of BMPsignalling [31 32] Gremlin overexpression has been foundto inhibit BMP-4 thus leading to enhance TGF-120573 signallingand ECM deposition in primary open angle glaucoma [44]However BMP-independent mechanismsmaymediate someactions of Gremlin Exogenous Gremlin may bind to andact directly on endothelial cells to modulate angiogenesisincluding endothelial cell migration [45 46] We have foundthat BMPs did no inhibit Gremlin-induced early Smad 3activation Thus a receptor-mediated mechanism of actionmay exist for GremlinTherefore future studies investigatingthe receptor involved in Gremlin responses in renal cells areneeded
5 Conclusion
Chronic progressive fibrosis of the kidney remains anunsolved challenge The investigation of the mediators andmechanisms involved in renal fibrosis could lead to better
8 BioMed Research InternationalG
REM
-GFP
GRE
M-G
FP S
mad
7Em
pty
pCD
NA
CytokeratinGFP Merge
(a)
GFP Merge
GRE
M-G
FPEm
pty
pCD
NA
120572-SMA
GRE
M-G
FP S
mad
7
(b)
Figure 5 Gremlin-induced EMT via the Smad pathway HK2 cells were transiently transfected with empty Gremlin (GREM-GFP) alone orcotransfected with Smad 7 expression vectors EMT markers were evaluated after 48 hours Gremlin transfected cells express GFP (greenstaining) Confocal microscopy analysis of cytokeratin and 120572-SMA immunofluorescence was performed using specific primary antibodiesand Alexa-633 secondary IgG (red staining) Representative image out of 3 experiments
Control Gremlin0
1
2
3
TGF-120573
expr
essio
n le
vels
(n-fo
ld)
24hours48hours
lowast
(a)
mRN
A le
vels
(n-fo
ld)
0
1
2
Control Gremlin
CTGFPAI-1
lowast lowast
lowast
Anti-TGF-120573+
GremlinTGF-120573
(b)
Figure 6 (a) Gremlin increased TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 and 48 hours in serum-freemedium TGF-1205731 protein levels were measured in the cell-conditioned medium using a specific ELISA Data are expressed as mean plusmn SEMof 6 independent experiments lowast119875 lt 005 versus control (b) The late increase in gene expression of profibrotic factors caused by Gremlin ismediated by endogenous TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 hours in serum-free medium TGF-120573was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizing antibody Total cell RNAwas isolated to assessmRNA levels by real-time PCR Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005versus control 119875 lt 005 versus Gremlin
BioMed Research International 9
Vim
entin
pan-
Cyto
kera
tinE-
cadh
erin
Control Gremlin
Gremlin+
anti-TGF-120573
(a)
0
1
2
Vimentin
GAPDH
Control Gremlin+
Gremlin
Vim
entin
GA
PDH
(n-fo
ld)
GAPDH
0
1
2
E-ca
dher
inG
APD
H (n
-fold
)
Control Gremlin+
Gremlin
Slug
GAPDH
0
1
2
Control Gremlin+
Gremlin
Slug
GA
PDH
(n-fo
ld)
E-cadherin
anti-TGF-120573 anti-TGF-120573 anti-TGF-120573
lowast
lowast
lowast
(b)
Figure 7TGF-120573 is amediator of EMT-related changes following stimulationwith Gremlin HK2 cells were stimulatedwithGremlin (50 ngmL)for 48 hours in serum-free medium TGF-120573 was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizingantibody (a) EMT changes were evaluated by confocal microscopy E-cadherin pan-Cytokeratin and Vimentin were studied by indirectimmunofluorescence using FITC-secondary IgG (green) and confocal microscopy Nuclei are shown in blue Figure shows a representativeimage out of 3 independent observations (b)Total proteins were isolated and Vimentin E-cadherin and Slug levels were analyzed by westernblot Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
diagnostic tools and novel therapeutics approaches Manystudies have shown that renal expression of Gremlin isinduced in diabetic nephropathy and in other progressiverenal diseases associated with tubulointerstitial fibrosis andSmad activation [5ndash8] We show here that Gremlin activatesthe Smad signaling pathway and induces TGF-120573 and otherrelated factors involved in EMT and fibrotic events in renalcells All these data suggest that Gremlin could be a potential
novel molecular antifibrotic target and biomarker useful forprognostication disease monitoring and therapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
10 BioMed Research International
Gremlin
Smadactivation
Target genes
EMT-related changes
Early-response(min)
Long-response
TGF-120573 independent
TGF-120573 production
TGF-120573 mediated(gt24hours)
Induction of vimentin and 120572-SMAlost of E-cadherin and cytokeratin
Figure 8 Dual effects of Gremlin on Smad activation Gremlininduces an early (minutes) and direct TGF-120573-independent Smadpathway activation After 24 hours Gremlin increased severalprofibrotic genes including TGF-120573 and after 48 hours increasedTGF-120573 production and induced EMT features These long-termGremlin-induced profibrotic events require autocrine TGF-120573
Authorsrsquo Contribution
Raquel Rodrigues-Diez and Raul R Rodrigues-Diez equallycontributed to this work
Acknowledgments
This work was supported by grants from the Institutode Salud Carlos III (PI1101854 and REDINREN ISCIII-RETIC RD1200210002 and 0001) Sociedad Espanola deNefrologıa PCI Iberoamerica (A957107) CYTED IBER-ERC FONDECYT Chile 1080083 and 1120480 Comu-nidad de Madrid (Fibroteam S2010BMD-2321 S2010BMD-2378) Programa Intensificacion Actividad Investigadora(ISCIIIAgencia Laın-EntralgoCM) to AO Fundacion parael fomento en Asturias de la investigacion cientıfica aplicaday la tecnologıa (FICYT) The authors want to thank Ma MarGonzalezGarcia-Parreno for her technical help with confocalmicroscopy
References
[1] S A Roxburgh M Murphy C A Pollock and D P BrazilldquoRecapitulation of embryological programmes in renalfibrosismdashthe importance of epithelial cell plasticity anddevelopmental genesrdquo Nephron Physiology vol 103 no 3 ppp139ndashp148 2006
[2] R McMahon MMurphy M Clarkson et al ldquoIHG-2 a mesan-gial cell gene induced by high glucose is human gremlin regu-lation by extracellular glucose concentration cyclic mechanicalstrain and transforming growth factor-1205731rdquo The Journal ofBiological Chemistry vol 275 no 14 pp 9901ndash9904 2000
[3] L Z Topol B Bardot Q Zhang et al ldquoBiosynthesis post-translation modification and functional characterization ofDrmGremlinrdquo The Journal of Biological Chemistry vol 275no 12 pp 8785ndash8793 2000
[4] O Michos L Panman K Vintersten K Beier R Zeller andA Zuniga ldquoGremlin-mediated BMP antagonism inducesthe epithelial-mesenchymal feedback signaling controllingmetanephric kidney and limb organogenesisrdquo Developmentvol 131 no 14 pp 3401ndash3410 2004
[5] V Dolan M Murphy D Sadlier et al ldquoExpression of gremlina bone morphogenetic protein antagonist in human diabeticnephropathyrdquo American Journal of Kidney Diseases vol 45 no6 pp 1034ndash1039 2005
[6] M Murphy J Crean D P Brazil D Sadlier F Martin andC Godson ldquoRegulation and consequences of differential geneexpression in diabetic kidney diseaserdquo Biochemical SocietyTransactions vol 36 no 5 pp 941ndash945 2008
[7] S Mezzano A Droguett M B Eugenia et al ldquoExpression ofgremlin a bonemorphogenetic protein antagonist in glomeru-lar crescents of pauci-immune glomerulonephritisrdquoNephrologyDialysis Transplantation vol 22 no 7 pp 1882ndash1890 2007
[8] G Carvajal A Droguett M E Burgos et al ldquoGremlin a novelmediator of epithelial mesenchymal transition and fibrosis inchronic allograft nephropathyrdquo Transplantation Proceedingsvol 40 no 3 pp 734ndash739 2008
[9] S A Roxburgh J J Kattla S P Curran et al ldquoAllelic depletionof grem1 attenuates diabetic kidney diseaserdquo Diabetes vol 58no 7 pp 1641ndash1650 2009
[10] Q Zhang Y Shi J Wada et al ldquoIn vivo delivery of gremlinsiRNA plasmid reveals therapeutic potential against diabeticnephropathy by recovering bone morphogenetic protein-7rdquoPLoS ONE vol 5 no 7 article e11709 2010
[11] M Zeisberg and E G Neilson ldquoMechanisms oftubulointerstitial fibrosisrdquo Journal of the American Societyof Nephrology vol 21 no 11 pp 1819ndash1834 2010
[12] Y Liu ldquoNew insights into epithelial-mesenchymal transition inkidney fibrosisrdquo Journal of the American Society of Nephrologyvol 21 no 2 pp 212ndash222 2010
[13] A Moustakas and C-H Heldin ldquoSignaling networks guidingepithelial-mesenchymal transitions during embryogenesisand cancer progressionrdquo Cancer Science vol 98 no 10 pp1512ndash1520 2007
[14] J-M Fan N G Yee-Yung P A Hill et al ldquoTransforminggrowth factor-120573 regulates tubular epithelial-myofibroblasttransdifferentiation in vitrordquo Kidney International vol 56 no4 pp 1455ndash1467 1999
[15] A B Roberts F Tian S D Byfield et al ldquoSmad3 is keyto TGF-120573-mediated epithelial-to-mesenchymal transitionfibrosis tumor suppression andmetastasisrdquo Cytokine amp GrowthFactor Reviews vol 17 no 1-2 pp 19ndash27 2006
BioMed Research International 11
[16] J Xu S Lamouille and R Derynck ldquoTGF-120573-induced epithelialto mesenchymal transitionrdquo Cell Research vol 19 no 2 pp156ndash172 2009
[17] M Ruiz-Ortega J Rodrıguez-Vita E Sanchez-Lopez GCarvajal and J Egido ldquoTGF-120573 signaling in vascular fibrosisrdquoCardiovascular Research vol 74 no 2 pp 196ndash206 2007
[18] R Rodrigues-Diez C Lavoz G Carvajal et al ldquoGremlin isa downstream profibrotic mediator of transforming growthfactor-beta in cultured renal cellsrdquo Nephron ExperimentalNephrology vol 122 no 1-2 pp 62ndash74 2012
[19] J Rodrıguez-Vita E Sanchez-Lopez V Esteban M Ruperez JEgido and M Ruiz-Ortega ldquoAngiotensin II activates the Smadpathway in vascular smooth muscle cells by a transforminggrowth factor-120573-independent mechanismrdquo Circulation vol 111no 19 pp 2509ndash2517 2005
[20] G Carvajal J Rodrıguez-Vita R Rodrigues-Dıez et alldquoAngiotensin II activates the Smad pathway during epithelialmesenchymal transdifferentiationrdquo Kidney International vol74 no 5 pp 585ndash595 2008
[21] G Li Y Li S Liu et al ldquoGremlin aggravates hyperglycemia-induced podocyte injury by a TGF120573smad dependent signalingpathwayrdquo Journal of Cellular Biochemistry vol 114 no 9 pp2101ndash2113 2013
[22] H Y Lan W Mu N Tomita et al ldquoInhibition of renalfibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in ratUUOmodelrdquo Journal of theAmericanSociety of Nephrology vol 14 no 6 pp 1535ndash1548 2003
[23] H Fukasawa T Yamamoto A Togawa et al ldquoDown-regulationof Smad7 expression by ubiquitin-dependent degradationcontributes to renal fibrosis in obstructive nephropathy inmicerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 23 pp 8687ndash8692 2004
[24] H Y Chen X R Huang WWang et al ldquoThe protective role ofSmad7 in diabetic kidney disease mechanism and therapeuticpotentialrdquo Diabetes vol 60 no 2 pp 590ndash601 2011
[25] X Wei Y Xia F Li et al ldquoKindlin-2 mediates activationof TGF-120573Smad signaling and renal fibrosisrdquo Journal of theAmerican Society Nephrology vol 24 no 9 pp 1387ndash1398 2013
[26] M Isono S Chen S W Hong M C Iglesias-de la Cruzand F N Ziyadeh ldquoSmad pathway is activated in the diabeticmouse kidney and smad3 mediates TGF-120573-induced fibronectinin mesangial cellsrdquo Biochemical and Biophysical ResearchCommunications vol 296 no 5 pp 1356ndash1365 2002
[27] J H Park C Lee J H Suh J Y Chae andK CMoon ldquoNuclearexpression of Smad proteins and its prognostic significance inclear cell renal cell carcinomardquo Human Pathology vol 44 no10 pp 2047ndash2054 2013
[28] G X Liu Y Q Li X R Huang et al ldquoSmad7 inhibitsAngII-mediated hypertensive nephropathy in amousemodel ofhypertensionrdquo Clinical Science vol 127 no 3 pp 195ndash208 2014
[29] G S Zode A F Clark and R J Wordinger ldquoBone morpho-genetic protein 4 inhibits TGF-1205732 stimulation of extracellularmatrix proteins in optic nerve head cells role of gremlin inECMmodulationrdquo Glia vol 57 no 7 pp 755ndash766 2009
[30] S OrsquoReilly M Ciechomska R Cant and J M van Laar ldquoIL-6trans signalling drives a STAT3 dependant pathway that leadsto hyperactive TGF-120573 signalling promoting SMAD3 activationand fibrosis via gremlinrdquo The Journal of Biological Chemistry2014
[31] M Myllarniemi P Lindholm M J Ryynanen et al ldquoGremlin-mediated decrease in bone morphogenetic protein signaling
promotes pulmonary fibrosisrdquo American Journal of Respiratoryand Critical Care Medicine vol 177 no 3 pp 321ndash329 2008
[32] E Cahill C M Costello S C Rowan et al ldquoGremlin playsa key role in the pathogenesis of pulmonary hypertensionrdquoCirculation vol 125 no 7 pp 920ndash930 2012
[33] W Boers S Aarrass C Linthorst M Pinzani R O Elferinkand P Bosma ldquoTranscriptional profiling reveals novel markersof liver fibrogenesis gremlin and insulin-like growth factor-binding proteinsrdquo The Journal of Biological Chemistry vol 281no 24 pp 16289ndash16295 2006
[34] CMCostello E Cahill FMartin S Gaine andPMcLoughlinldquoRole of gremlin in the lung development and diseaserdquoAmerican Journal of Respiratory Cell and Molecular Biologyvol 42 no 5 pp 517ndash523 2010
[35] K A Mueller E Tavlaki M Schneider et al ldquoGremlin-1identifies fibrosis and predicts adverse outcome in patientswith heart failure undergoing endomyocardial biopsyrdquo Journalof Cardiac Failure vol 19 no 10 pp 678ndash684 2013
[36] H Huang H Huang Y Li et al ldquoGremlin induces cellproliferation and extra cellular matrix accumulation in mousemesangial cells exposed to high glucose via the ERK12pathwayrdquo BMC Nephrology vol 14 article 33 2013
[37] N McCormack E L Molloy and S OrsquoDea ldquoBone morpho-genetic proteins enhance an epithelial-mesenchymal transitionin normal airway epithelial cells during restitution of a dis-rupted epitheliumrdquoRespiratory Research vol 14 article 36 2013
[38] J A Tamminen V Parviainen M Ronty et al ldquoGremlin-1associates with fibrillin microfibrils in vivo and regulatesmesothelioma cell survival through transcription factor slugrdquoOncogenesis vol 2 article e66 2013
[39] E M Zeisberg S E Potenta H Sugimoto M Zeisberg and RKalluri ldquoFibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transitionrdquo Journal of the American Society ofNephrology vol 19 no 12 pp 2282ndash2287 2008
[40] V S LeBleu G Taduri J OrsquoConnell et al ldquoOrigin and functionof myofibroblasts in kidney fibrosisrdquo Nature Medicine vol 19no 8 pp 1047ndash1053 2013
[41] W C Prozialeck and J R Edwards ldquoCell adhesion moleculesin chemically-induced renal injuryrdquo Pharmacology amp Thera-peutics vol 114 no 1 pp 74ndash93 2007
[42] M Zeisberg J Hanai H Sugimoto et al ldquoBMP-7 counteractsTGF-1205731-induced epithelial-to-mesenchymal transition andreverses chronic renal injuryrdquo Nature Medicine vol 9 no 7 pp964ndash968 2003
[43] Y-L Yang H-Z Ju S-F Liu et al ldquoBMP-2 suppresses renalinterstitial fibrosis by regulating epithelial-mesenchymaltransitionrdquo Journal of Cellular Biochemistry vol 112 no 9 pp2558ndash2565 2011
[44] R J Wordinger D L Fleenor P E Hellberg et al ldquoEffects ofTGF-1205732 BMP-4 and gremlin in the trabecular meshworkimplications for glaucomardquo Investigative Ophthalmology ampVisual Science vol 48 no 3 pp 1191ndash1200 2007
[45] B Chen M Athanasiou Q Gu and D G Blair ldquoDrmGremlintranscriptionally activates p21Cip1 via a novel mechanism andinhibits neoplastic transformationrdquoBiochemical andBiophysicalResearch Communications vol 295 no 5 pp 1135ndash1141 2002
[46] S Mitola C Ravelli E Moroni et al ldquoGremlin is a novelagonist of the major proangiogenic receptor VEGFR2rdquo Bloodvol 116 no 18 pp 3677ndash3680 2010
Tris-HCl pH 75 150mM NaCl with 005 Tween-20 andincubated overnight at 4∘C with the primary antibodiesand subsequently incubated with peroxidase-conjugated IgG(Amersham) and developed by ECL chemiluminescence(GE Healthcare Buckinghamshire UK)
Immunocytochemistry studies were performed in cellsgrowing on coverslips After the experiments cells werefixed in Merckofix (Merck) and permeabilized with 02Triton-X100 for 10min (except for E-cadherin staining)After blocking with 4 BSA and 8 serum for 1 hoursamples were incubated with primary antibodies overnightat 4∘C and then 1 hour at room temperature with fluoresceinisothiocyanate (FITC) [1200] or AlexaFluor 633 [1300]conjugated antibodies (Amersham) Nuclei were stainedwith 1 120583gmL propidium iodide (PI) or 410158406-Diamidino-2-phenyindole dilactate (DAPI) (Sigma-Aldrich) as control ofequal cell density Absence of primary antibody was used asnegative control Samples were mounted in Mowiol 40ndash88(Sigma-Aldrich) and examined by a Leica DM-IRB confocalmicroscope
TGF-1205731 protein was measured in the cell-conditionedmedium using a commercial enzyme-linked immunoassay(ELISA) (BD Sciences San Diego USA) following the man-ufacturerrsquos instructions TGF-1205731 levels were quantified bycomparison with a standard curve using increasing concen-trations of human TGF-1205731 Protein content was determinedby the BCA method (Pierce)
24 Gene Expression Total RNA was isolated from cells withTrizol (Invitrogen) according to the manufacturerrsquos protocolcDNA was synthesized from 2120583g of total RNA with randomhexamer primers using the High capacity cDNA Archive Kit(Applied) Real-time PCR was performed using human FAMTaqMan MGB probes designed by assay-on-demand geneexpression products (Applied) TGF-1205731 Hs99999918 m1connective tissue growth factor (CTGF) Hs00170014 m1plasminogen activator inhibitor 1 (PAI1) Hs 00167155 m1Data weres normalized to 18S eukaryotic ribosomal RNA4210893E (VIC) The mRNA copy numbers were calculatedfor each sample by the instrument software using Ct value(ldquoarithmetic fit point analysis for the lightcyclerrdquo) Resultswere expressed in copy numbers and calculated relative tounstimulated cells after normalization against 18S
25 Statistical Analysis Results throughout the text areexpressed as mean plusmn SEM Differences between agonist-treated groups and controls were assessed by one-way anal-ysis of variance followed by post hoc Bonferroni or Dunnetttest or Mann-Whitney test as appropriate 119875 lt 005 wasconsidered significant Statistical analysis was conductedusing the SPSS statistical software version 110 (SPSS)
3 Results
31 Gremlin Activates Smad Pathway in Human CulturedTubuloepithelial Cells Receptormediated activation of Smadproteins (R-Smads 2 and 3) occurs by direct C-terminalphosphorylation Smad 23 then form complexes with Smad4 and translocate into the nucleus where they associateand cooperate with DNA binding transcription factors toactivate or repress target gene transcription [17] In culturedHK2 cells stimulation with recombinant Gremlin increasedphosphorylation levels of Smad 3 as early as 5 minutes and itwas maintained until 15 minutes (Figure 1(a))
Although Smad is the main signaling mechanism ofTGF-120573 several factors involved in renal damage such asangiotensin II can directly activate the Smad pathway inde-pendent of endogenous TGF-120573 [17] Therefore to evaluatewhether early Smad activation caused by Gremlin was medi-ated or not by TGF-120573 cells were preincubated with a neu-tralizing antibody against active TGF-120573 Gremlin-inducedSmad activation (evaluated as p-Smad 3 levels) was notmodified in the presence of the TGF-120573 antibody (Figure 1(b))Similar lack of response was found in the presence of decorin(a proteoglycan that neutralizes active TGF-120573 not shown)These data indicates that Gremlin directly activates the Smadpathway
Some actions of Gremlin are due to its effect as BMPantagonist [4] To determine the contribution of BMPs inGremlin-induced Smad activation HK2 cells were prein-cubated with BMP-2 or BMP-4 and then stimulated withGremlin during 10 minutes Phosphorylation of Smad 3 wasnot modified in the presence of any of these BMPs (Fig-ure 1(c)) suggesting that Gremlin-induced Smad activationis independent of BMPs in tubular epithelial cells
By confocal microscopy we have confirmed that Gremlinrapidly increased Smad 3 translocation to the nucleus thelatter demonstrated by the yellow nuclear staining observedin the merge of Figure 2 while in untreated cells the nucleiare red Gremlin also increased nuclear localization of Smad2 and Smad 4 observed at 15 minutes (Figure 2)
To further confirm that Gremlin activates the Smadpathway cells were transfected with a Gremlin expressionvector (GREM-GFP) for 24 hours By confocal microscopywe observed that in positive Gremlin-transfected cells (GFP-green staining) there was a nuclear immunostaining showingthe translocation of Smad 3 and Smad 2 (characteristic ofSmad activation) compared to cells transfected with emptyvector (Figures 3(a) and 3(b)) In the merge of Figure 3a Gremlin expressing-cell marked by a yellow rectanglepresented a positive nuclear staining corresponding to thepresence of Smad 3 or 2 In contrast in cells transfectedwith empty vector there are no nuclear Smad 2 and Smad 3immunostaining (all cells present blue nuclei) as observed insome nontransfected cells
To investigatewhetherGremlin regulates Smad-mediatedgene expression cells were cotransfected with a Grem-lin expression vector (GREM-GFP) and a luciferase Smadreporter plasmid Gremlin transfected cells expressed higherSmad-dependent luciferase activity (SBE) than control cells(Figure 4) To demonstrate further the involvement of Smad
4 BioMed Research International
p-Smad 3
GAPDH
0
1
2
3
4
Control
Gremlin (min)
5 10 15 20
p-Sm
ad 3
expr
essio
n le
vels
(n-fo
ld)
lowast
lowast
lowast
(a)
p-Smad 3
p-Sm
ad 3
expr
essio
n le
vels
(n-fo
ld)
0
1
2
3
Control Gremlin
GAPDH
lowast
lowast
Gremlin
Anti-TGF-120573+
(b)
0
1
2
3
4
Control Gremlin BMP-2BMP-4Gremlin
p-Smad 3
GAPDH
p-Sm
ad 3
expr
essio
n le
vels
(n-fo
ld)
lowast
lowastlowast
(c)
Figure 1 (a) Stimulation with Gremlin rapidly increased Smad 3 phosphorylation in cultured human tubuloepithelial cells HK2 cells werestimulated with Gremlin (50 ngmL) for different times (b) Early Smad 3 phosphorylation induced by stimulation with Gremlin was notmediated by TGF-120573 TGF-120573 was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizing antibody andthen treated with Gremlin for 10 minutes (c) In some points HK2 cells were preincubated with BMP-2 or BMP-4 and then treated withGremlin for 10 minutes Total proteins were isolated and protein levels were evaluated by western blot GAPDH or Smad 3 were used asloading controls Figures show a representative western blot of phosphorylated levels of Smad 3 and data are expressed as n-fold over control(considered as 1) as the mean plusmn SEM of 3-4 independent experiments lowast119875 lt 005 versus control
pathway in Gremlin-induced responses a Smad 7 expressionvector that inhibits Smad-mediated transcriptional effects byinterfering with receptor-mediated activation of R-Smad wasused [17 19] In HK2 cells cotransfected with GREM-GFPand Smad 7 expression vectors the Smad-mediated luciferaseactivity was significantly lower than cells transfected withGREM-GFP alone showing the specific Smad 7 blockade ofGremlin-mediated Smad activation (Figure 4)
32 Gremlin-Induced EMT Is Mediated by Smad ActivationWe have previously demonstrated that in tubular epithe-lial cells long-term stimulation with recombinant Gremlininduced EMT [18] Now we have observed that transfec-tion of HK2 cells with GREM-GFP induced EMT-relatedphenotypic changes observed by confocal microscopy after48 hours (Figure 5) Cells transfected with empty-vectorshowed epithelial morphology including the presence of
BioMed Research International 5
Smad 3 IP MergeC
ontro
lG
rem
lin
(a)
Smad 2 IP Merge
Con
trol
Gre
mlin
(b)
Smad 4 IP Merge
Con
trol
Gre
mlin
(c)
Figure 2 Stimulation with Gremlin induces a rapid activation of the Smad pathway in cultured human tubuloepithelial cells HK2 cells werestimulated with Gremlin (50 ngmL) for 15 minutes The localization of R-Smad 3 (a) and 2 (b) and Smad 4 (c) was evaluated by confocalmicroscopy with FITC-secondary antibodies (green staining) Nuclei were stained with propidium iodide (IP) (red) In themerge the yellowstaining indicates the nuclear localization of Smad proteinsThe results are representative of 3 independent confocal microscopy experiments
epithelial markers such as cytokeratin (red staining) andthere is no positive staining for mesenchymal marker 120572-SMA(Figure 5) In contrast overexpression of Gremlin causedchanges inmorphology to fibroblast-like shape and inductionof 120572-SMA (see the GREM-GFP positive cell that presentsyellow staining and elongated shape) Moreover in Gremlinexpressing cells cytokeratin staining was markedly dimin-ished (absence of red staining in an area with several GREM-GFPpositive green cells)Theblockade of Smad activation bycotransfection with Gremlin and Smad 7 diminished theseEMT changes (Figure 5) as shown by restoration of thecytokeratin immunostaining and the epithelial morphologyand diminution of 120572-SMA as observed in the green positivecell These data suggest that Gremlin regulates EMT throughthe Smad pathway
33 Role of Endogenous TGF-120573 on Gremlin-Induced EMTPreviously we have reported that Gremlin acts as a down-stream mediator of TGF-120573-induced fibrosis in cultured renal
cells and incubation with Gremlin for 24 hours induced asignificant upregulation of TGF-1205731 mRNA levels in culturedtubuloepithelial cells [18] We have further investigated therelation between GremlinTGF-120573 evaluating whether Grem-lin could regulate TGF-1205731 synthesis In HK2 cells activeTGF-1205731 protein increased in the supernatants of Gremlin-stimulated cells after 48 hours but not at 24 hours (Fig-ure 6(a)) suggesting that some of the profibrotic actions ofGremlin could bemediated by endogenousTGF-1205731 synthesisTherefore we blocked TGF-120573 before HK-2 stimulation withGremlin by adding a neutralizing antibody against activeTGF-120573 which is able to block angiotensin II-induced ECMproduction andEMT [19 20] TGF-120573neutralization inhibitedGremlin-induced gene upregulation of profibrotic factorsobserved after 24 hours including TGF-120573 CTGF and PAI-1 (Figure 6(b)) Moreover TGF-120573 blockade antagonizedseveral EMT-related changes induced by Gremlin after 48hours as shown by immunofluorescence (Figure 7(a)) Wealso observed by western blot that TGF-120573 neutralizationdiminished Vimentin and Slug induction caused by Gremlin
6 BioMed Research International
Nuclei MergeSmad 3 GFP
Empt
y pC
DN
AG
REM
-GFP
(a)
Smad 2 Nuclei MergeGFP
Empt
y pC
DN
AG
REM
-GFP
(b)
Figure 3 Gremlin overexpression causes a sustained Smad activation in cultured human tubuloepithelial cells HK2 cells were transientlytransfected with a Gremlin expression vector (GREM-GFP green) or empty vector for 24 hours The levels and localization of R-Smad 3(a) and R-Smad 2 (b) were evaluated by confocal microscopy with Alexa-633 secondary IgG (red) Nuclei were stained using 410158406-diamino-2-phenylindole dihydrochloride (DAPI blue) In Gremlin-transfected cells (green staining by GFP) the Smad 2 and Smad 3 were found inthe nuclei (white staining in the merge) Figures show representative images out of 3 independent observations
and restored E-cadherin levels decreased by Gremlin (Fig-ure 7(b))These data suggest that TGF-120573 is amediator of long-term responses of Gremlin in tubuloepithelial cells includingregulation of profibrotic factors and EMT changes
4 Discussion
Our in vitro studies in cultured tubuloepithelial cells showthat Gremlin directly activates the Smad pathway and par-ticipates in the EMT process via Smad signalling These datasuggest that Gremlin could be a mediator of renal fibrosis
Our study reveals that in cultured human tubuloepithelialcells Gremlin induces a rapid activation of the Smad path-way (observed after 5min of stimulation) characterized byincreased phosphorylation of the receptor-Smad (R-Smad)Smad 3 a critical downstream mediator of fibrosis [17] and
Smad 2 proteinsOnceR-Smad is phosphorylated it dimeriseswith Smad 4 and then shuttles to the nucleus to regulategene expression By confocal microscopy we have foundthat Gremlin caused a rapid translocation to the nucleusof R-SmadSmad 4 proteins In several cells types Gremlin-induced TGF-120573 production [18 21] as we have observedhere after 48 hours of incubationHowever Gremlin-inducedearly Smad activation is independent of endogenous TGF-120573as we have demonstrated using TGF-120573 blockers (Figure 8)Other important profibrotic factors such as angiotensin IIalso activates the Smad pathway rapidly and independent ofendogenous TGF-120573 [17]
Previous studies in tubular epithelial cells have shownthat the Smad route regulates EMT induced by key factorsinvolved in renal fibrosis such as TGF-120573 and angiotensinII [20] The activation of Smad pathway has been described
BioMed Research International 7
0
1
2
3
4
Control GREM-GFP GREM-GFP+ Smad 7
SBE-
prom
otor
expr
essio
n (n
-fold
) lowast
Figure 4 Gremlin overexpression induces Smad-dependent genetranscription HK2 cells were transfected with GREM-GFP or emptyvector Smadluc promoter and TK-renilla for 24 hours In somepoints cells were cotransfected with Smad 7Then luciferaserenillaactivity was measured Data are expressed as increase in Smadbinding element (SBE) promoter-luciferase dependent expressionData are expressed as n-fold over control (considered as 1) as themean plusmn SEM of 5 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
in experimental renal fibrotic diseases including glomeru-losclerosis tubulointerstitial fibrosis hypertensive-inducedrenal damage and diabetic nephropathy [20ndash26] as well as inrenal tumor progression [27] In angiotensin II-induced renaldamage renal activation of the Smad pathway was associatedto EMT changes [20] Moreover Smad 7 overexpressionameliorates renal damage and fibrosis caused by unilateralureteral obstruction angiotensin II and diabetes [22ndash2428] We have observed that in tubuloepithelial cells Smad 7overexpression blocked Gremlin-induced EMT changes Theinvolvement of Smad pathway in Gremlin-mediated fibrosishas been also described in other cell types in vitro In opticnerve head astrocytes and lamina cribrosa cells recombinantGremlin stimulates ECM production through the activationof TGF-120573 receptor and Smad 3 phosphorylation suggestinga role for Gremlin in glaucoma [29] In healthy dermalfibroblasts IL-6 mediated induction of collagen is dependenton Gremlin production and activation of TGF-120573Smad sig-nalling [30] Besides the regulation of renal EMT and fibrosisGremlinSmad pathway could also be involved in the onset ofproteinuria by modulating podocyte injury and changing thedistribution of nephrin and synaptopodin [21]
Recent evidences suggest that Gremlin could be animportant promoter of fibrosis in different pathologiesincluding liver fibrosis lung diseases particularly pul-monary hypertension and idiopathic pulmonary fibrosis andmyocardial fibrosis [31ndash35] In several human renal diseasesGremlin overexpression was found mainly in areas of tubuleinterstitial fibrosis [5ndash8] Experimental studies in mice haveshown that Gremlin blockade diminished renal fibrosis asobserved in streptozotocin-induced diabetes in knockoutmice heterozygous for grem1 [9] and by Gremlin gene silenc-ing [10] Recent studies have demonstrated direct fibrogeniceffect of Gremlin in renal cells In mesangial cells Gremlin
increased cell proliferation and ECM accumulation via ERK[36] In renal fibroblasts Gremlin increased ECM production[18] including type I collagen In tubular epithelial cellsGremlin upregulates profibrotic genes such as TGF-120573 andCTGF and caused EMT changes [18] Gremlin also inducesEMT in airway epithelial cells [37] and in cancer cells [38]Although the contribution of EMT to renal fibrosis is amatterof intense debate [39 40] the lost of epithelial propertiesof the tubular epithelial cells including permeability andpolarity may result in decreased viability and contribute torenal injury [40 41] Therefore EMT-related changes arean initial step in renal damage and an important potentialtherapeutic target Our data demonstrate that Gremlin viaSmad pathway regulates EMT showing a novel mechanismof Gremlin action in renal cells
TGF-120573 is known as the major promoter of EMT duringembryogenesis cancer and fibrosis [13ndash17]In a mesothe-lioma cell line Gremlin-silencing inhibited cell proliferationassociated with downregulation of the transcription factorslug as well as mesenchymal proteins linked to cancerEMT [38] We have recently demonstrated that Gremlingene silencing blocked TGF-120573-induced EMT in tubularepithelial cells [18] Now we have observed that Gremlinincreased TGF-120573 production at 48 hours and this endoge-nous autocrine TGF-120573 acts as a downstream mediator ofGremlin-induced profibrotic and EMT related factors incultured human tubuloepithelial cells (Figure 8) All thesefindings reveal the complex relationship between Gremlinand TGF-120573 in the kidney disclosing a positive feedback loopconnection between them in promoting EMT and fibrosis
Gremlin exerts a potent inhibitory action via binding toand forming heterodimers with BMP-2 BMP-4 and BMP-7The binding of Gremlin to selective BMPs prevents ligand-receptor interaction and subsequent downstream signallingGremlin acting as a BMPs antagonist plays a critical role dur-ing the process of nephrogenesis [4] BMP-7 is the antagonistof TGF-1205731 signalling and has been found to inhibit TGF-1205731-induced renal fibrosis by reversing EMT process [42 43]In experimental lungs and pulmonary fibrosis upregulationof Gremlin was associated with downregulation of BMPsignalling [31 32] Gremlin overexpression has been foundto inhibit BMP-4 thus leading to enhance TGF-120573 signallingand ECM deposition in primary open angle glaucoma [44]However BMP-independent mechanismsmaymediate someactions of Gremlin Exogenous Gremlin may bind to andact directly on endothelial cells to modulate angiogenesisincluding endothelial cell migration [45 46] We have foundthat BMPs did no inhibit Gremlin-induced early Smad 3activation Thus a receptor-mediated mechanism of actionmay exist for GremlinTherefore future studies investigatingthe receptor involved in Gremlin responses in renal cells areneeded
5 Conclusion
Chronic progressive fibrosis of the kidney remains anunsolved challenge The investigation of the mediators andmechanisms involved in renal fibrosis could lead to better
8 BioMed Research InternationalG
REM
-GFP
GRE
M-G
FP S
mad
7Em
pty
pCD
NA
CytokeratinGFP Merge
(a)
GFP Merge
GRE
M-G
FPEm
pty
pCD
NA
120572-SMA
GRE
M-G
FP S
mad
7
(b)
Figure 5 Gremlin-induced EMT via the Smad pathway HK2 cells were transiently transfected with empty Gremlin (GREM-GFP) alone orcotransfected with Smad 7 expression vectors EMT markers were evaluated after 48 hours Gremlin transfected cells express GFP (greenstaining) Confocal microscopy analysis of cytokeratin and 120572-SMA immunofluorescence was performed using specific primary antibodiesand Alexa-633 secondary IgG (red staining) Representative image out of 3 experiments
Control Gremlin0
1
2
3
TGF-120573
expr
essio
n le
vels
(n-fo
ld)
24hours48hours
lowast
(a)
mRN
A le
vels
(n-fo
ld)
0
1
2
Control Gremlin
CTGFPAI-1
lowast lowast
lowast
Anti-TGF-120573+
GremlinTGF-120573
(b)
Figure 6 (a) Gremlin increased TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 and 48 hours in serum-freemedium TGF-1205731 protein levels were measured in the cell-conditioned medium using a specific ELISA Data are expressed as mean plusmn SEMof 6 independent experiments lowast119875 lt 005 versus control (b) The late increase in gene expression of profibrotic factors caused by Gremlin ismediated by endogenous TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 hours in serum-free medium TGF-120573was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizing antibody Total cell RNAwas isolated to assessmRNA levels by real-time PCR Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005versus control 119875 lt 005 versus Gremlin
BioMed Research International 9
Vim
entin
pan-
Cyto
kera
tinE-
cadh
erin
Control Gremlin
Gremlin+
anti-TGF-120573
(a)
0
1
2
Vimentin
GAPDH
Control Gremlin+
Gremlin
Vim
entin
GA
PDH
(n-fo
ld)
GAPDH
0
1
2
E-ca
dher
inG
APD
H (n
-fold
)
Control Gremlin+
Gremlin
Slug
GAPDH
0
1
2
Control Gremlin+
Gremlin
Slug
GA
PDH
(n-fo
ld)
E-cadherin
anti-TGF-120573 anti-TGF-120573 anti-TGF-120573
lowast
lowast
lowast
(b)
Figure 7TGF-120573 is amediator of EMT-related changes following stimulationwith Gremlin HK2 cells were stimulatedwithGremlin (50 ngmL)for 48 hours in serum-free medium TGF-120573 was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizingantibody (a) EMT changes were evaluated by confocal microscopy E-cadherin pan-Cytokeratin and Vimentin were studied by indirectimmunofluorescence using FITC-secondary IgG (green) and confocal microscopy Nuclei are shown in blue Figure shows a representativeimage out of 3 independent observations (b)Total proteins were isolated and Vimentin E-cadherin and Slug levels were analyzed by westernblot Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
diagnostic tools and novel therapeutics approaches Manystudies have shown that renal expression of Gremlin isinduced in diabetic nephropathy and in other progressiverenal diseases associated with tubulointerstitial fibrosis andSmad activation [5ndash8] We show here that Gremlin activatesthe Smad signaling pathway and induces TGF-120573 and otherrelated factors involved in EMT and fibrotic events in renalcells All these data suggest that Gremlin could be a potential
novel molecular antifibrotic target and biomarker useful forprognostication disease monitoring and therapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
10 BioMed Research International
Gremlin
Smadactivation
Target genes
EMT-related changes
Early-response(min)
Long-response
TGF-120573 independent
TGF-120573 production
TGF-120573 mediated(gt24hours)
Induction of vimentin and 120572-SMAlost of E-cadherin and cytokeratin
Figure 8 Dual effects of Gremlin on Smad activation Gremlininduces an early (minutes) and direct TGF-120573-independent Smadpathway activation After 24 hours Gremlin increased severalprofibrotic genes including TGF-120573 and after 48 hours increasedTGF-120573 production and induced EMT features These long-termGremlin-induced profibrotic events require autocrine TGF-120573
Authorsrsquo Contribution
Raquel Rodrigues-Diez and Raul R Rodrigues-Diez equallycontributed to this work
Acknowledgments
This work was supported by grants from the Institutode Salud Carlos III (PI1101854 and REDINREN ISCIII-RETIC RD1200210002 and 0001) Sociedad Espanola deNefrologıa PCI Iberoamerica (A957107) CYTED IBER-ERC FONDECYT Chile 1080083 and 1120480 Comu-nidad de Madrid (Fibroteam S2010BMD-2321 S2010BMD-2378) Programa Intensificacion Actividad Investigadora(ISCIIIAgencia Laın-EntralgoCM) to AO Fundacion parael fomento en Asturias de la investigacion cientıfica aplicaday la tecnologıa (FICYT) The authors want to thank Ma MarGonzalezGarcia-Parreno for her technical help with confocalmicroscopy
References
[1] S A Roxburgh M Murphy C A Pollock and D P BrazilldquoRecapitulation of embryological programmes in renalfibrosismdashthe importance of epithelial cell plasticity anddevelopmental genesrdquo Nephron Physiology vol 103 no 3 ppp139ndashp148 2006
[2] R McMahon MMurphy M Clarkson et al ldquoIHG-2 a mesan-gial cell gene induced by high glucose is human gremlin regu-lation by extracellular glucose concentration cyclic mechanicalstrain and transforming growth factor-1205731rdquo The Journal ofBiological Chemistry vol 275 no 14 pp 9901ndash9904 2000
[3] L Z Topol B Bardot Q Zhang et al ldquoBiosynthesis post-translation modification and functional characterization ofDrmGremlinrdquo The Journal of Biological Chemistry vol 275no 12 pp 8785ndash8793 2000
[4] O Michos L Panman K Vintersten K Beier R Zeller andA Zuniga ldquoGremlin-mediated BMP antagonism inducesthe epithelial-mesenchymal feedback signaling controllingmetanephric kidney and limb organogenesisrdquo Developmentvol 131 no 14 pp 3401ndash3410 2004
[5] V Dolan M Murphy D Sadlier et al ldquoExpression of gremlina bone morphogenetic protein antagonist in human diabeticnephropathyrdquo American Journal of Kidney Diseases vol 45 no6 pp 1034ndash1039 2005
[6] M Murphy J Crean D P Brazil D Sadlier F Martin andC Godson ldquoRegulation and consequences of differential geneexpression in diabetic kidney diseaserdquo Biochemical SocietyTransactions vol 36 no 5 pp 941ndash945 2008
[7] S Mezzano A Droguett M B Eugenia et al ldquoExpression ofgremlin a bonemorphogenetic protein antagonist in glomeru-lar crescents of pauci-immune glomerulonephritisrdquoNephrologyDialysis Transplantation vol 22 no 7 pp 1882ndash1890 2007
[8] G Carvajal A Droguett M E Burgos et al ldquoGremlin a novelmediator of epithelial mesenchymal transition and fibrosis inchronic allograft nephropathyrdquo Transplantation Proceedingsvol 40 no 3 pp 734ndash739 2008
[9] S A Roxburgh J J Kattla S P Curran et al ldquoAllelic depletionof grem1 attenuates diabetic kidney diseaserdquo Diabetes vol 58no 7 pp 1641ndash1650 2009
[10] Q Zhang Y Shi J Wada et al ldquoIn vivo delivery of gremlinsiRNA plasmid reveals therapeutic potential against diabeticnephropathy by recovering bone morphogenetic protein-7rdquoPLoS ONE vol 5 no 7 article e11709 2010
[11] M Zeisberg and E G Neilson ldquoMechanisms oftubulointerstitial fibrosisrdquo Journal of the American Societyof Nephrology vol 21 no 11 pp 1819ndash1834 2010
[12] Y Liu ldquoNew insights into epithelial-mesenchymal transition inkidney fibrosisrdquo Journal of the American Society of Nephrologyvol 21 no 2 pp 212ndash222 2010
[13] A Moustakas and C-H Heldin ldquoSignaling networks guidingepithelial-mesenchymal transitions during embryogenesisand cancer progressionrdquo Cancer Science vol 98 no 10 pp1512ndash1520 2007
[14] J-M Fan N G Yee-Yung P A Hill et al ldquoTransforminggrowth factor-120573 regulates tubular epithelial-myofibroblasttransdifferentiation in vitrordquo Kidney International vol 56 no4 pp 1455ndash1467 1999
[15] A B Roberts F Tian S D Byfield et al ldquoSmad3 is keyto TGF-120573-mediated epithelial-to-mesenchymal transitionfibrosis tumor suppression andmetastasisrdquo Cytokine amp GrowthFactor Reviews vol 17 no 1-2 pp 19ndash27 2006
BioMed Research International 11
[16] J Xu S Lamouille and R Derynck ldquoTGF-120573-induced epithelialto mesenchymal transitionrdquo Cell Research vol 19 no 2 pp156ndash172 2009
[17] M Ruiz-Ortega J Rodrıguez-Vita E Sanchez-Lopez GCarvajal and J Egido ldquoTGF-120573 signaling in vascular fibrosisrdquoCardiovascular Research vol 74 no 2 pp 196ndash206 2007
[18] R Rodrigues-Diez C Lavoz G Carvajal et al ldquoGremlin isa downstream profibrotic mediator of transforming growthfactor-beta in cultured renal cellsrdquo Nephron ExperimentalNephrology vol 122 no 1-2 pp 62ndash74 2012
[19] J Rodrıguez-Vita E Sanchez-Lopez V Esteban M Ruperez JEgido and M Ruiz-Ortega ldquoAngiotensin II activates the Smadpathway in vascular smooth muscle cells by a transforminggrowth factor-120573-independent mechanismrdquo Circulation vol 111no 19 pp 2509ndash2517 2005
[20] G Carvajal J Rodrıguez-Vita R Rodrigues-Dıez et alldquoAngiotensin II activates the Smad pathway during epithelialmesenchymal transdifferentiationrdquo Kidney International vol74 no 5 pp 585ndash595 2008
[21] G Li Y Li S Liu et al ldquoGremlin aggravates hyperglycemia-induced podocyte injury by a TGF120573smad dependent signalingpathwayrdquo Journal of Cellular Biochemistry vol 114 no 9 pp2101ndash2113 2013
[22] H Y Lan W Mu N Tomita et al ldquoInhibition of renalfibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in ratUUOmodelrdquo Journal of theAmericanSociety of Nephrology vol 14 no 6 pp 1535ndash1548 2003
[23] H Fukasawa T Yamamoto A Togawa et al ldquoDown-regulationof Smad7 expression by ubiquitin-dependent degradationcontributes to renal fibrosis in obstructive nephropathy inmicerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 23 pp 8687ndash8692 2004
[24] H Y Chen X R Huang WWang et al ldquoThe protective role ofSmad7 in diabetic kidney disease mechanism and therapeuticpotentialrdquo Diabetes vol 60 no 2 pp 590ndash601 2011
[25] X Wei Y Xia F Li et al ldquoKindlin-2 mediates activationof TGF-120573Smad signaling and renal fibrosisrdquo Journal of theAmerican Society Nephrology vol 24 no 9 pp 1387ndash1398 2013
[26] M Isono S Chen S W Hong M C Iglesias-de la Cruzand F N Ziyadeh ldquoSmad pathway is activated in the diabeticmouse kidney and smad3 mediates TGF-120573-induced fibronectinin mesangial cellsrdquo Biochemical and Biophysical ResearchCommunications vol 296 no 5 pp 1356ndash1365 2002
[27] J H Park C Lee J H Suh J Y Chae andK CMoon ldquoNuclearexpression of Smad proteins and its prognostic significance inclear cell renal cell carcinomardquo Human Pathology vol 44 no10 pp 2047ndash2054 2013
[28] G X Liu Y Q Li X R Huang et al ldquoSmad7 inhibitsAngII-mediated hypertensive nephropathy in amousemodel ofhypertensionrdquo Clinical Science vol 127 no 3 pp 195ndash208 2014
[29] G S Zode A F Clark and R J Wordinger ldquoBone morpho-genetic protein 4 inhibits TGF-1205732 stimulation of extracellularmatrix proteins in optic nerve head cells role of gremlin inECMmodulationrdquo Glia vol 57 no 7 pp 755ndash766 2009
[30] S OrsquoReilly M Ciechomska R Cant and J M van Laar ldquoIL-6trans signalling drives a STAT3 dependant pathway that leadsto hyperactive TGF-120573 signalling promoting SMAD3 activationand fibrosis via gremlinrdquo The Journal of Biological Chemistry2014
[31] M Myllarniemi P Lindholm M J Ryynanen et al ldquoGremlin-mediated decrease in bone morphogenetic protein signaling
promotes pulmonary fibrosisrdquo American Journal of Respiratoryand Critical Care Medicine vol 177 no 3 pp 321ndash329 2008
[32] E Cahill C M Costello S C Rowan et al ldquoGremlin playsa key role in the pathogenesis of pulmonary hypertensionrdquoCirculation vol 125 no 7 pp 920ndash930 2012
[33] W Boers S Aarrass C Linthorst M Pinzani R O Elferinkand P Bosma ldquoTranscriptional profiling reveals novel markersof liver fibrogenesis gremlin and insulin-like growth factor-binding proteinsrdquo The Journal of Biological Chemistry vol 281no 24 pp 16289ndash16295 2006
[34] CMCostello E Cahill FMartin S Gaine andPMcLoughlinldquoRole of gremlin in the lung development and diseaserdquoAmerican Journal of Respiratory Cell and Molecular Biologyvol 42 no 5 pp 517ndash523 2010
[35] K A Mueller E Tavlaki M Schneider et al ldquoGremlin-1identifies fibrosis and predicts adverse outcome in patientswith heart failure undergoing endomyocardial biopsyrdquo Journalof Cardiac Failure vol 19 no 10 pp 678ndash684 2013
[36] H Huang H Huang Y Li et al ldquoGremlin induces cellproliferation and extra cellular matrix accumulation in mousemesangial cells exposed to high glucose via the ERK12pathwayrdquo BMC Nephrology vol 14 article 33 2013
[37] N McCormack E L Molloy and S OrsquoDea ldquoBone morpho-genetic proteins enhance an epithelial-mesenchymal transitionin normal airway epithelial cells during restitution of a dis-rupted epitheliumrdquoRespiratory Research vol 14 article 36 2013
[38] J A Tamminen V Parviainen M Ronty et al ldquoGremlin-1associates with fibrillin microfibrils in vivo and regulatesmesothelioma cell survival through transcription factor slugrdquoOncogenesis vol 2 article e66 2013
[39] E M Zeisberg S E Potenta H Sugimoto M Zeisberg and RKalluri ldquoFibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transitionrdquo Journal of the American Society ofNephrology vol 19 no 12 pp 2282ndash2287 2008
[40] V S LeBleu G Taduri J OrsquoConnell et al ldquoOrigin and functionof myofibroblasts in kidney fibrosisrdquo Nature Medicine vol 19no 8 pp 1047ndash1053 2013
[41] W C Prozialeck and J R Edwards ldquoCell adhesion moleculesin chemically-induced renal injuryrdquo Pharmacology amp Thera-peutics vol 114 no 1 pp 74ndash93 2007
[42] M Zeisberg J Hanai H Sugimoto et al ldquoBMP-7 counteractsTGF-1205731-induced epithelial-to-mesenchymal transition andreverses chronic renal injuryrdquo Nature Medicine vol 9 no 7 pp964ndash968 2003
[43] Y-L Yang H-Z Ju S-F Liu et al ldquoBMP-2 suppresses renalinterstitial fibrosis by regulating epithelial-mesenchymaltransitionrdquo Journal of Cellular Biochemistry vol 112 no 9 pp2558ndash2565 2011
[44] R J Wordinger D L Fleenor P E Hellberg et al ldquoEffects ofTGF-1205732 BMP-4 and gremlin in the trabecular meshworkimplications for glaucomardquo Investigative Ophthalmology ampVisual Science vol 48 no 3 pp 1191ndash1200 2007
[45] B Chen M Athanasiou Q Gu and D G Blair ldquoDrmGremlintranscriptionally activates p21Cip1 via a novel mechanism andinhibits neoplastic transformationrdquoBiochemical andBiophysicalResearch Communications vol 295 no 5 pp 1135ndash1141 2002
[46] S Mitola C Ravelli E Moroni et al ldquoGremlin is a novelagonist of the major proangiogenic receptor VEGFR2rdquo Bloodvol 116 no 18 pp 3677ndash3680 2010
Figure 1 (a) Stimulation with Gremlin rapidly increased Smad 3 phosphorylation in cultured human tubuloepithelial cells HK2 cells werestimulated with Gremlin (50 ngmL) for different times (b) Early Smad 3 phosphorylation induced by stimulation with Gremlin was notmediated by TGF-120573 TGF-120573 was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizing antibody andthen treated with Gremlin for 10 minutes (c) In some points HK2 cells were preincubated with BMP-2 or BMP-4 and then treated withGremlin for 10 minutes Total proteins were isolated and protein levels were evaluated by western blot GAPDH or Smad 3 were used asloading controls Figures show a representative western blot of phosphorylated levels of Smad 3 and data are expressed as n-fold over control(considered as 1) as the mean plusmn SEM of 3-4 independent experiments lowast119875 lt 005 versus control
pathway in Gremlin-induced responses a Smad 7 expressionvector that inhibits Smad-mediated transcriptional effects byinterfering with receptor-mediated activation of R-Smad wasused [17 19] In HK2 cells cotransfected with GREM-GFPand Smad 7 expression vectors the Smad-mediated luciferaseactivity was significantly lower than cells transfected withGREM-GFP alone showing the specific Smad 7 blockade ofGremlin-mediated Smad activation (Figure 4)
32 Gremlin-Induced EMT Is Mediated by Smad ActivationWe have previously demonstrated that in tubular epithe-lial cells long-term stimulation with recombinant Gremlininduced EMT [18] Now we have observed that transfec-tion of HK2 cells with GREM-GFP induced EMT-relatedphenotypic changes observed by confocal microscopy after48 hours (Figure 5) Cells transfected with empty-vectorshowed epithelial morphology including the presence of
BioMed Research International 5
Smad 3 IP MergeC
ontro
lG
rem
lin
(a)
Smad 2 IP Merge
Con
trol
Gre
mlin
(b)
Smad 4 IP Merge
Con
trol
Gre
mlin
(c)
Figure 2 Stimulation with Gremlin induces a rapid activation of the Smad pathway in cultured human tubuloepithelial cells HK2 cells werestimulated with Gremlin (50 ngmL) for 15 minutes The localization of R-Smad 3 (a) and 2 (b) and Smad 4 (c) was evaluated by confocalmicroscopy with FITC-secondary antibodies (green staining) Nuclei were stained with propidium iodide (IP) (red) In themerge the yellowstaining indicates the nuclear localization of Smad proteinsThe results are representative of 3 independent confocal microscopy experiments
epithelial markers such as cytokeratin (red staining) andthere is no positive staining for mesenchymal marker 120572-SMA(Figure 5) In contrast overexpression of Gremlin causedchanges inmorphology to fibroblast-like shape and inductionof 120572-SMA (see the GREM-GFP positive cell that presentsyellow staining and elongated shape) Moreover in Gremlinexpressing cells cytokeratin staining was markedly dimin-ished (absence of red staining in an area with several GREM-GFPpositive green cells)Theblockade of Smad activation bycotransfection with Gremlin and Smad 7 diminished theseEMT changes (Figure 5) as shown by restoration of thecytokeratin immunostaining and the epithelial morphologyand diminution of 120572-SMA as observed in the green positivecell These data suggest that Gremlin regulates EMT throughthe Smad pathway
33 Role of Endogenous TGF-120573 on Gremlin-Induced EMTPreviously we have reported that Gremlin acts as a down-stream mediator of TGF-120573-induced fibrosis in cultured renal
cells and incubation with Gremlin for 24 hours induced asignificant upregulation of TGF-1205731 mRNA levels in culturedtubuloepithelial cells [18] We have further investigated therelation between GremlinTGF-120573 evaluating whether Grem-lin could regulate TGF-1205731 synthesis In HK2 cells activeTGF-1205731 protein increased in the supernatants of Gremlin-stimulated cells after 48 hours but not at 24 hours (Fig-ure 6(a)) suggesting that some of the profibrotic actions ofGremlin could bemediated by endogenousTGF-1205731 synthesisTherefore we blocked TGF-120573 before HK-2 stimulation withGremlin by adding a neutralizing antibody against activeTGF-120573 which is able to block angiotensin II-induced ECMproduction andEMT [19 20] TGF-120573neutralization inhibitedGremlin-induced gene upregulation of profibrotic factorsobserved after 24 hours including TGF-120573 CTGF and PAI-1 (Figure 6(b)) Moreover TGF-120573 blockade antagonizedseveral EMT-related changes induced by Gremlin after 48hours as shown by immunofluorescence (Figure 7(a)) Wealso observed by western blot that TGF-120573 neutralizationdiminished Vimentin and Slug induction caused by Gremlin
6 BioMed Research International
Nuclei MergeSmad 3 GFP
Empt
y pC
DN
AG
REM
-GFP
(a)
Smad 2 Nuclei MergeGFP
Empt
y pC
DN
AG
REM
-GFP
(b)
Figure 3 Gremlin overexpression causes a sustained Smad activation in cultured human tubuloepithelial cells HK2 cells were transientlytransfected with a Gremlin expression vector (GREM-GFP green) or empty vector for 24 hours The levels and localization of R-Smad 3(a) and R-Smad 2 (b) were evaluated by confocal microscopy with Alexa-633 secondary IgG (red) Nuclei were stained using 410158406-diamino-2-phenylindole dihydrochloride (DAPI blue) In Gremlin-transfected cells (green staining by GFP) the Smad 2 and Smad 3 were found inthe nuclei (white staining in the merge) Figures show representative images out of 3 independent observations
and restored E-cadherin levels decreased by Gremlin (Fig-ure 7(b))These data suggest that TGF-120573 is amediator of long-term responses of Gremlin in tubuloepithelial cells includingregulation of profibrotic factors and EMT changes
4 Discussion
Our in vitro studies in cultured tubuloepithelial cells showthat Gremlin directly activates the Smad pathway and par-ticipates in the EMT process via Smad signalling These datasuggest that Gremlin could be a mediator of renal fibrosis
Our study reveals that in cultured human tubuloepithelialcells Gremlin induces a rapid activation of the Smad path-way (observed after 5min of stimulation) characterized byincreased phosphorylation of the receptor-Smad (R-Smad)Smad 3 a critical downstream mediator of fibrosis [17] and
Smad 2 proteinsOnceR-Smad is phosphorylated it dimeriseswith Smad 4 and then shuttles to the nucleus to regulategene expression By confocal microscopy we have foundthat Gremlin caused a rapid translocation to the nucleusof R-SmadSmad 4 proteins In several cells types Gremlin-induced TGF-120573 production [18 21] as we have observedhere after 48 hours of incubationHowever Gremlin-inducedearly Smad activation is independent of endogenous TGF-120573as we have demonstrated using TGF-120573 blockers (Figure 8)Other important profibrotic factors such as angiotensin IIalso activates the Smad pathway rapidly and independent ofendogenous TGF-120573 [17]
Previous studies in tubular epithelial cells have shownthat the Smad route regulates EMT induced by key factorsinvolved in renal fibrosis such as TGF-120573 and angiotensinII [20] The activation of Smad pathway has been described
BioMed Research International 7
0
1
2
3
4
Control GREM-GFP GREM-GFP+ Smad 7
SBE-
prom
otor
expr
essio
n (n
-fold
) lowast
Figure 4 Gremlin overexpression induces Smad-dependent genetranscription HK2 cells were transfected with GREM-GFP or emptyvector Smadluc promoter and TK-renilla for 24 hours In somepoints cells were cotransfected with Smad 7Then luciferaserenillaactivity was measured Data are expressed as increase in Smadbinding element (SBE) promoter-luciferase dependent expressionData are expressed as n-fold over control (considered as 1) as themean plusmn SEM of 5 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
in experimental renal fibrotic diseases including glomeru-losclerosis tubulointerstitial fibrosis hypertensive-inducedrenal damage and diabetic nephropathy [20ndash26] as well as inrenal tumor progression [27] In angiotensin II-induced renaldamage renal activation of the Smad pathway was associatedto EMT changes [20] Moreover Smad 7 overexpressionameliorates renal damage and fibrosis caused by unilateralureteral obstruction angiotensin II and diabetes [22ndash2428] We have observed that in tubuloepithelial cells Smad 7overexpression blocked Gremlin-induced EMT changes Theinvolvement of Smad pathway in Gremlin-mediated fibrosishas been also described in other cell types in vitro In opticnerve head astrocytes and lamina cribrosa cells recombinantGremlin stimulates ECM production through the activationof TGF-120573 receptor and Smad 3 phosphorylation suggestinga role for Gremlin in glaucoma [29] In healthy dermalfibroblasts IL-6 mediated induction of collagen is dependenton Gremlin production and activation of TGF-120573Smad sig-nalling [30] Besides the regulation of renal EMT and fibrosisGremlinSmad pathway could also be involved in the onset ofproteinuria by modulating podocyte injury and changing thedistribution of nephrin and synaptopodin [21]
Recent evidences suggest that Gremlin could be animportant promoter of fibrosis in different pathologiesincluding liver fibrosis lung diseases particularly pul-monary hypertension and idiopathic pulmonary fibrosis andmyocardial fibrosis [31ndash35] In several human renal diseasesGremlin overexpression was found mainly in areas of tubuleinterstitial fibrosis [5ndash8] Experimental studies in mice haveshown that Gremlin blockade diminished renal fibrosis asobserved in streptozotocin-induced diabetes in knockoutmice heterozygous for grem1 [9] and by Gremlin gene silenc-ing [10] Recent studies have demonstrated direct fibrogeniceffect of Gremlin in renal cells In mesangial cells Gremlin
increased cell proliferation and ECM accumulation via ERK[36] In renal fibroblasts Gremlin increased ECM production[18] including type I collagen In tubular epithelial cellsGremlin upregulates profibrotic genes such as TGF-120573 andCTGF and caused EMT changes [18] Gremlin also inducesEMT in airway epithelial cells [37] and in cancer cells [38]Although the contribution of EMT to renal fibrosis is amatterof intense debate [39 40] the lost of epithelial propertiesof the tubular epithelial cells including permeability andpolarity may result in decreased viability and contribute torenal injury [40 41] Therefore EMT-related changes arean initial step in renal damage and an important potentialtherapeutic target Our data demonstrate that Gremlin viaSmad pathway regulates EMT showing a novel mechanismof Gremlin action in renal cells
TGF-120573 is known as the major promoter of EMT duringembryogenesis cancer and fibrosis [13ndash17]In a mesothe-lioma cell line Gremlin-silencing inhibited cell proliferationassociated with downregulation of the transcription factorslug as well as mesenchymal proteins linked to cancerEMT [38] We have recently demonstrated that Gremlingene silencing blocked TGF-120573-induced EMT in tubularepithelial cells [18] Now we have observed that Gremlinincreased TGF-120573 production at 48 hours and this endoge-nous autocrine TGF-120573 acts as a downstream mediator ofGremlin-induced profibrotic and EMT related factors incultured human tubuloepithelial cells (Figure 8) All thesefindings reveal the complex relationship between Gremlinand TGF-120573 in the kidney disclosing a positive feedback loopconnection between them in promoting EMT and fibrosis
Gremlin exerts a potent inhibitory action via binding toand forming heterodimers with BMP-2 BMP-4 and BMP-7The binding of Gremlin to selective BMPs prevents ligand-receptor interaction and subsequent downstream signallingGremlin acting as a BMPs antagonist plays a critical role dur-ing the process of nephrogenesis [4] BMP-7 is the antagonistof TGF-1205731 signalling and has been found to inhibit TGF-1205731-induced renal fibrosis by reversing EMT process [42 43]In experimental lungs and pulmonary fibrosis upregulationof Gremlin was associated with downregulation of BMPsignalling [31 32] Gremlin overexpression has been foundto inhibit BMP-4 thus leading to enhance TGF-120573 signallingand ECM deposition in primary open angle glaucoma [44]However BMP-independent mechanismsmaymediate someactions of Gremlin Exogenous Gremlin may bind to andact directly on endothelial cells to modulate angiogenesisincluding endothelial cell migration [45 46] We have foundthat BMPs did no inhibit Gremlin-induced early Smad 3activation Thus a receptor-mediated mechanism of actionmay exist for GremlinTherefore future studies investigatingthe receptor involved in Gremlin responses in renal cells areneeded
5 Conclusion
Chronic progressive fibrosis of the kidney remains anunsolved challenge The investigation of the mediators andmechanisms involved in renal fibrosis could lead to better
8 BioMed Research InternationalG
REM
-GFP
GRE
M-G
FP S
mad
7Em
pty
pCD
NA
CytokeratinGFP Merge
(a)
GFP Merge
GRE
M-G
FPEm
pty
pCD
NA
120572-SMA
GRE
M-G
FP S
mad
7
(b)
Figure 5 Gremlin-induced EMT via the Smad pathway HK2 cells were transiently transfected with empty Gremlin (GREM-GFP) alone orcotransfected with Smad 7 expression vectors EMT markers were evaluated after 48 hours Gremlin transfected cells express GFP (greenstaining) Confocal microscopy analysis of cytokeratin and 120572-SMA immunofluorescence was performed using specific primary antibodiesand Alexa-633 secondary IgG (red staining) Representative image out of 3 experiments
Control Gremlin0
1
2
3
TGF-120573
expr
essio
n le
vels
(n-fo
ld)
24hours48hours
lowast
(a)
mRN
A le
vels
(n-fo
ld)
0
1
2
Control Gremlin
CTGFPAI-1
lowast lowast
lowast
Anti-TGF-120573+
GremlinTGF-120573
(b)
Figure 6 (a) Gremlin increased TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 and 48 hours in serum-freemedium TGF-1205731 protein levels were measured in the cell-conditioned medium using a specific ELISA Data are expressed as mean plusmn SEMof 6 independent experiments lowast119875 lt 005 versus control (b) The late increase in gene expression of profibrotic factors caused by Gremlin ismediated by endogenous TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 hours in serum-free medium TGF-120573was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizing antibody Total cell RNAwas isolated to assessmRNA levels by real-time PCR Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005versus control 119875 lt 005 versus Gremlin
BioMed Research International 9
Vim
entin
pan-
Cyto
kera
tinE-
cadh
erin
Control Gremlin
Gremlin+
anti-TGF-120573
(a)
0
1
2
Vimentin
GAPDH
Control Gremlin+
Gremlin
Vim
entin
GA
PDH
(n-fo
ld)
GAPDH
0
1
2
E-ca
dher
inG
APD
H (n
-fold
)
Control Gremlin+
Gremlin
Slug
GAPDH
0
1
2
Control Gremlin+
Gremlin
Slug
GA
PDH
(n-fo
ld)
E-cadherin
anti-TGF-120573 anti-TGF-120573 anti-TGF-120573
lowast
lowast
lowast
(b)
Figure 7TGF-120573 is amediator of EMT-related changes following stimulationwith Gremlin HK2 cells were stimulatedwithGremlin (50 ngmL)for 48 hours in serum-free medium TGF-120573 was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizingantibody (a) EMT changes were evaluated by confocal microscopy E-cadherin pan-Cytokeratin and Vimentin were studied by indirectimmunofluorescence using FITC-secondary IgG (green) and confocal microscopy Nuclei are shown in blue Figure shows a representativeimage out of 3 independent observations (b)Total proteins were isolated and Vimentin E-cadherin and Slug levels were analyzed by westernblot Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
diagnostic tools and novel therapeutics approaches Manystudies have shown that renal expression of Gremlin isinduced in diabetic nephropathy and in other progressiverenal diseases associated with tubulointerstitial fibrosis andSmad activation [5ndash8] We show here that Gremlin activatesthe Smad signaling pathway and induces TGF-120573 and otherrelated factors involved in EMT and fibrotic events in renalcells All these data suggest that Gremlin could be a potential
novel molecular antifibrotic target and biomarker useful forprognostication disease monitoring and therapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
10 BioMed Research International
Gremlin
Smadactivation
Target genes
EMT-related changes
Early-response(min)
Long-response
TGF-120573 independent
TGF-120573 production
TGF-120573 mediated(gt24hours)
Induction of vimentin and 120572-SMAlost of E-cadherin and cytokeratin
Figure 8 Dual effects of Gremlin on Smad activation Gremlininduces an early (minutes) and direct TGF-120573-independent Smadpathway activation After 24 hours Gremlin increased severalprofibrotic genes including TGF-120573 and after 48 hours increasedTGF-120573 production and induced EMT features These long-termGremlin-induced profibrotic events require autocrine TGF-120573
Authorsrsquo Contribution
Raquel Rodrigues-Diez and Raul R Rodrigues-Diez equallycontributed to this work
Acknowledgments
This work was supported by grants from the Institutode Salud Carlos III (PI1101854 and REDINREN ISCIII-RETIC RD1200210002 and 0001) Sociedad Espanola deNefrologıa PCI Iberoamerica (A957107) CYTED IBER-ERC FONDECYT Chile 1080083 and 1120480 Comu-nidad de Madrid (Fibroteam S2010BMD-2321 S2010BMD-2378) Programa Intensificacion Actividad Investigadora(ISCIIIAgencia Laın-EntralgoCM) to AO Fundacion parael fomento en Asturias de la investigacion cientıfica aplicaday la tecnologıa (FICYT) The authors want to thank Ma MarGonzalezGarcia-Parreno for her technical help with confocalmicroscopy
References
[1] S A Roxburgh M Murphy C A Pollock and D P BrazilldquoRecapitulation of embryological programmes in renalfibrosismdashthe importance of epithelial cell plasticity anddevelopmental genesrdquo Nephron Physiology vol 103 no 3 ppp139ndashp148 2006
[2] R McMahon MMurphy M Clarkson et al ldquoIHG-2 a mesan-gial cell gene induced by high glucose is human gremlin regu-lation by extracellular glucose concentration cyclic mechanicalstrain and transforming growth factor-1205731rdquo The Journal ofBiological Chemistry vol 275 no 14 pp 9901ndash9904 2000
[3] L Z Topol B Bardot Q Zhang et al ldquoBiosynthesis post-translation modification and functional characterization ofDrmGremlinrdquo The Journal of Biological Chemistry vol 275no 12 pp 8785ndash8793 2000
[4] O Michos L Panman K Vintersten K Beier R Zeller andA Zuniga ldquoGremlin-mediated BMP antagonism inducesthe epithelial-mesenchymal feedback signaling controllingmetanephric kidney and limb organogenesisrdquo Developmentvol 131 no 14 pp 3401ndash3410 2004
[5] V Dolan M Murphy D Sadlier et al ldquoExpression of gremlina bone morphogenetic protein antagonist in human diabeticnephropathyrdquo American Journal of Kidney Diseases vol 45 no6 pp 1034ndash1039 2005
[6] M Murphy J Crean D P Brazil D Sadlier F Martin andC Godson ldquoRegulation and consequences of differential geneexpression in diabetic kidney diseaserdquo Biochemical SocietyTransactions vol 36 no 5 pp 941ndash945 2008
[7] S Mezzano A Droguett M B Eugenia et al ldquoExpression ofgremlin a bonemorphogenetic protein antagonist in glomeru-lar crescents of pauci-immune glomerulonephritisrdquoNephrologyDialysis Transplantation vol 22 no 7 pp 1882ndash1890 2007
[8] G Carvajal A Droguett M E Burgos et al ldquoGremlin a novelmediator of epithelial mesenchymal transition and fibrosis inchronic allograft nephropathyrdquo Transplantation Proceedingsvol 40 no 3 pp 734ndash739 2008
[9] S A Roxburgh J J Kattla S P Curran et al ldquoAllelic depletionof grem1 attenuates diabetic kidney diseaserdquo Diabetes vol 58no 7 pp 1641ndash1650 2009
[10] Q Zhang Y Shi J Wada et al ldquoIn vivo delivery of gremlinsiRNA plasmid reveals therapeutic potential against diabeticnephropathy by recovering bone morphogenetic protein-7rdquoPLoS ONE vol 5 no 7 article e11709 2010
[11] M Zeisberg and E G Neilson ldquoMechanisms oftubulointerstitial fibrosisrdquo Journal of the American Societyof Nephrology vol 21 no 11 pp 1819ndash1834 2010
[12] Y Liu ldquoNew insights into epithelial-mesenchymal transition inkidney fibrosisrdquo Journal of the American Society of Nephrologyvol 21 no 2 pp 212ndash222 2010
[13] A Moustakas and C-H Heldin ldquoSignaling networks guidingepithelial-mesenchymal transitions during embryogenesisand cancer progressionrdquo Cancer Science vol 98 no 10 pp1512ndash1520 2007
[14] J-M Fan N G Yee-Yung P A Hill et al ldquoTransforminggrowth factor-120573 regulates tubular epithelial-myofibroblasttransdifferentiation in vitrordquo Kidney International vol 56 no4 pp 1455ndash1467 1999
[15] A B Roberts F Tian S D Byfield et al ldquoSmad3 is keyto TGF-120573-mediated epithelial-to-mesenchymal transitionfibrosis tumor suppression andmetastasisrdquo Cytokine amp GrowthFactor Reviews vol 17 no 1-2 pp 19ndash27 2006
BioMed Research International 11
[16] J Xu S Lamouille and R Derynck ldquoTGF-120573-induced epithelialto mesenchymal transitionrdquo Cell Research vol 19 no 2 pp156ndash172 2009
[17] M Ruiz-Ortega J Rodrıguez-Vita E Sanchez-Lopez GCarvajal and J Egido ldquoTGF-120573 signaling in vascular fibrosisrdquoCardiovascular Research vol 74 no 2 pp 196ndash206 2007
[18] R Rodrigues-Diez C Lavoz G Carvajal et al ldquoGremlin isa downstream profibrotic mediator of transforming growthfactor-beta in cultured renal cellsrdquo Nephron ExperimentalNephrology vol 122 no 1-2 pp 62ndash74 2012
[19] J Rodrıguez-Vita E Sanchez-Lopez V Esteban M Ruperez JEgido and M Ruiz-Ortega ldquoAngiotensin II activates the Smadpathway in vascular smooth muscle cells by a transforminggrowth factor-120573-independent mechanismrdquo Circulation vol 111no 19 pp 2509ndash2517 2005
[20] G Carvajal J Rodrıguez-Vita R Rodrigues-Dıez et alldquoAngiotensin II activates the Smad pathway during epithelialmesenchymal transdifferentiationrdquo Kidney International vol74 no 5 pp 585ndash595 2008
[21] G Li Y Li S Liu et al ldquoGremlin aggravates hyperglycemia-induced podocyte injury by a TGF120573smad dependent signalingpathwayrdquo Journal of Cellular Biochemistry vol 114 no 9 pp2101ndash2113 2013
[22] H Y Lan W Mu N Tomita et al ldquoInhibition of renalfibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in ratUUOmodelrdquo Journal of theAmericanSociety of Nephrology vol 14 no 6 pp 1535ndash1548 2003
[23] H Fukasawa T Yamamoto A Togawa et al ldquoDown-regulationof Smad7 expression by ubiquitin-dependent degradationcontributes to renal fibrosis in obstructive nephropathy inmicerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 23 pp 8687ndash8692 2004
[24] H Y Chen X R Huang WWang et al ldquoThe protective role ofSmad7 in diabetic kidney disease mechanism and therapeuticpotentialrdquo Diabetes vol 60 no 2 pp 590ndash601 2011
[25] X Wei Y Xia F Li et al ldquoKindlin-2 mediates activationof TGF-120573Smad signaling and renal fibrosisrdquo Journal of theAmerican Society Nephrology vol 24 no 9 pp 1387ndash1398 2013
[26] M Isono S Chen S W Hong M C Iglesias-de la Cruzand F N Ziyadeh ldquoSmad pathway is activated in the diabeticmouse kidney and smad3 mediates TGF-120573-induced fibronectinin mesangial cellsrdquo Biochemical and Biophysical ResearchCommunications vol 296 no 5 pp 1356ndash1365 2002
[27] J H Park C Lee J H Suh J Y Chae andK CMoon ldquoNuclearexpression of Smad proteins and its prognostic significance inclear cell renal cell carcinomardquo Human Pathology vol 44 no10 pp 2047ndash2054 2013
[28] G X Liu Y Q Li X R Huang et al ldquoSmad7 inhibitsAngII-mediated hypertensive nephropathy in amousemodel ofhypertensionrdquo Clinical Science vol 127 no 3 pp 195ndash208 2014
[29] G S Zode A F Clark and R J Wordinger ldquoBone morpho-genetic protein 4 inhibits TGF-1205732 stimulation of extracellularmatrix proteins in optic nerve head cells role of gremlin inECMmodulationrdquo Glia vol 57 no 7 pp 755ndash766 2009
[30] S OrsquoReilly M Ciechomska R Cant and J M van Laar ldquoIL-6trans signalling drives a STAT3 dependant pathway that leadsto hyperactive TGF-120573 signalling promoting SMAD3 activationand fibrosis via gremlinrdquo The Journal of Biological Chemistry2014
[31] M Myllarniemi P Lindholm M J Ryynanen et al ldquoGremlin-mediated decrease in bone morphogenetic protein signaling
promotes pulmonary fibrosisrdquo American Journal of Respiratoryand Critical Care Medicine vol 177 no 3 pp 321ndash329 2008
[32] E Cahill C M Costello S C Rowan et al ldquoGremlin playsa key role in the pathogenesis of pulmonary hypertensionrdquoCirculation vol 125 no 7 pp 920ndash930 2012
[33] W Boers S Aarrass C Linthorst M Pinzani R O Elferinkand P Bosma ldquoTranscriptional profiling reveals novel markersof liver fibrogenesis gremlin and insulin-like growth factor-binding proteinsrdquo The Journal of Biological Chemistry vol 281no 24 pp 16289ndash16295 2006
[34] CMCostello E Cahill FMartin S Gaine andPMcLoughlinldquoRole of gremlin in the lung development and diseaserdquoAmerican Journal of Respiratory Cell and Molecular Biologyvol 42 no 5 pp 517ndash523 2010
[35] K A Mueller E Tavlaki M Schneider et al ldquoGremlin-1identifies fibrosis and predicts adverse outcome in patientswith heart failure undergoing endomyocardial biopsyrdquo Journalof Cardiac Failure vol 19 no 10 pp 678ndash684 2013
[36] H Huang H Huang Y Li et al ldquoGremlin induces cellproliferation and extra cellular matrix accumulation in mousemesangial cells exposed to high glucose via the ERK12pathwayrdquo BMC Nephrology vol 14 article 33 2013
[37] N McCormack E L Molloy and S OrsquoDea ldquoBone morpho-genetic proteins enhance an epithelial-mesenchymal transitionin normal airway epithelial cells during restitution of a dis-rupted epitheliumrdquoRespiratory Research vol 14 article 36 2013
[38] J A Tamminen V Parviainen M Ronty et al ldquoGremlin-1associates with fibrillin microfibrils in vivo and regulatesmesothelioma cell survival through transcription factor slugrdquoOncogenesis vol 2 article e66 2013
[39] E M Zeisberg S E Potenta H Sugimoto M Zeisberg and RKalluri ldquoFibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transitionrdquo Journal of the American Society ofNephrology vol 19 no 12 pp 2282ndash2287 2008
[40] V S LeBleu G Taduri J OrsquoConnell et al ldquoOrigin and functionof myofibroblasts in kidney fibrosisrdquo Nature Medicine vol 19no 8 pp 1047ndash1053 2013
[41] W C Prozialeck and J R Edwards ldquoCell adhesion moleculesin chemically-induced renal injuryrdquo Pharmacology amp Thera-peutics vol 114 no 1 pp 74ndash93 2007
[42] M Zeisberg J Hanai H Sugimoto et al ldquoBMP-7 counteractsTGF-1205731-induced epithelial-to-mesenchymal transition andreverses chronic renal injuryrdquo Nature Medicine vol 9 no 7 pp964ndash968 2003
[43] Y-L Yang H-Z Ju S-F Liu et al ldquoBMP-2 suppresses renalinterstitial fibrosis by regulating epithelial-mesenchymaltransitionrdquo Journal of Cellular Biochemistry vol 112 no 9 pp2558ndash2565 2011
[44] R J Wordinger D L Fleenor P E Hellberg et al ldquoEffects ofTGF-1205732 BMP-4 and gremlin in the trabecular meshworkimplications for glaucomardquo Investigative Ophthalmology ampVisual Science vol 48 no 3 pp 1191ndash1200 2007
[45] B Chen M Athanasiou Q Gu and D G Blair ldquoDrmGremlintranscriptionally activates p21Cip1 via a novel mechanism andinhibits neoplastic transformationrdquoBiochemical andBiophysicalResearch Communications vol 295 no 5 pp 1135ndash1141 2002
[46] S Mitola C Ravelli E Moroni et al ldquoGremlin is a novelagonist of the major proangiogenic receptor VEGFR2rdquo Bloodvol 116 no 18 pp 3677ndash3680 2010
Figure 2 Stimulation with Gremlin induces a rapid activation of the Smad pathway in cultured human tubuloepithelial cells HK2 cells werestimulated with Gremlin (50 ngmL) for 15 minutes The localization of R-Smad 3 (a) and 2 (b) and Smad 4 (c) was evaluated by confocalmicroscopy with FITC-secondary antibodies (green staining) Nuclei were stained with propidium iodide (IP) (red) In themerge the yellowstaining indicates the nuclear localization of Smad proteinsThe results are representative of 3 independent confocal microscopy experiments
epithelial markers such as cytokeratin (red staining) andthere is no positive staining for mesenchymal marker 120572-SMA(Figure 5) In contrast overexpression of Gremlin causedchanges inmorphology to fibroblast-like shape and inductionof 120572-SMA (see the GREM-GFP positive cell that presentsyellow staining and elongated shape) Moreover in Gremlinexpressing cells cytokeratin staining was markedly dimin-ished (absence of red staining in an area with several GREM-GFPpositive green cells)Theblockade of Smad activation bycotransfection with Gremlin and Smad 7 diminished theseEMT changes (Figure 5) as shown by restoration of thecytokeratin immunostaining and the epithelial morphologyand diminution of 120572-SMA as observed in the green positivecell These data suggest that Gremlin regulates EMT throughthe Smad pathway
33 Role of Endogenous TGF-120573 on Gremlin-Induced EMTPreviously we have reported that Gremlin acts as a down-stream mediator of TGF-120573-induced fibrosis in cultured renal
cells and incubation with Gremlin for 24 hours induced asignificant upregulation of TGF-1205731 mRNA levels in culturedtubuloepithelial cells [18] We have further investigated therelation between GremlinTGF-120573 evaluating whether Grem-lin could regulate TGF-1205731 synthesis In HK2 cells activeTGF-1205731 protein increased in the supernatants of Gremlin-stimulated cells after 48 hours but not at 24 hours (Fig-ure 6(a)) suggesting that some of the profibrotic actions ofGremlin could bemediated by endogenousTGF-1205731 synthesisTherefore we blocked TGF-120573 before HK-2 stimulation withGremlin by adding a neutralizing antibody against activeTGF-120573 which is able to block angiotensin II-induced ECMproduction andEMT [19 20] TGF-120573neutralization inhibitedGremlin-induced gene upregulation of profibrotic factorsobserved after 24 hours including TGF-120573 CTGF and PAI-1 (Figure 6(b)) Moreover TGF-120573 blockade antagonizedseveral EMT-related changes induced by Gremlin after 48hours as shown by immunofluorescence (Figure 7(a)) Wealso observed by western blot that TGF-120573 neutralizationdiminished Vimentin and Slug induction caused by Gremlin
6 BioMed Research International
Nuclei MergeSmad 3 GFP
Empt
y pC
DN
AG
REM
-GFP
(a)
Smad 2 Nuclei MergeGFP
Empt
y pC
DN
AG
REM
-GFP
(b)
Figure 3 Gremlin overexpression causes a sustained Smad activation in cultured human tubuloepithelial cells HK2 cells were transientlytransfected with a Gremlin expression vector (GREM-GFP green) or empty vector for 24 hours The levels and localization of R-Smad 3(a) and R-Smad 2 (b) were evaluated by confocal microscopy with Alexa-633 secondary IgG (red) Nuclei were stained using 410158406-diamino-2-phenylindole dihydrochloride (DAPI blue) In Gremlin-transfected cells (green staining by GFP) the Smad 2 and Smad 3 were found inthe nuclei (white staining in the merge) Figures show representative images out of 3 independent observations
and restored E-cadherin levels decreased by Gremlin (Fig-ure 7(b))These data suggest that TGF-120573 is amediator of long-term responses of Gremlin in tubuloepithelial cells includingregulation of profibrotic factors and EMT changes
4 Discussion
Our in vitro studies in cultured tubuloepithelial cells showthat Gremlin directly activates the Smad pathway and par-ticipates in the EMT process via Smad signalling These datasuggest that Gremlin could be a mediator of renal fibrosis
Our study reveals that in cultured human tubuloepithelialcells Gremlin induces a rapid activation of the Smad path-way (observed after 5min of stimulation) characterized byincreased phosphorylation of the receptor-Smad (R-Smad)Smad 3 a critical downstream mediator of fibrosis [17] and
Smad 2 proteinsOnceR-Smad is phosphorylated it dimeriseswith Smad 4 and then shuttles to the nucleus to regulategene expression By confocal microscopy we have foundthat Gremlin caused a rapid translocation to the nucleusof R-SmadSmad 4 proteins In several cells types Gremlin-induced TGF-120573 production [18 21] as we have observedhere after 48 hours of incubationHowever Gremlin-inducedearly Smad activation is independent of endogenous TGF-120573as we have demonstrated using TGF-120573 blockers (Figure 8)Other important profibrotic factors such as angiotensin IIalso activates the Smad pathway rapidly and independent ofendogenous TGF-120573 [17]
Previous studies in tubular epithelial cells have shownthat the Smad route regulates EMT induced by key factorsinvolved in renal fibrosis such as TGF-120573 and angiotensinII [20] The activation of Smad pathway has been described
BioMed Research International 7
0
1
2
3
4
Control GREM-GFP GREM-GFP+ Smad 7
SBE-
prom
otor
expr
essio
n (n
-fold
) lowast
Figure 4 Gremlin overexpression induces Smad-dependent genetranscription HK2 cells were transfected with GREM-GFP or emptyvector Smadluc promoter and TK-renilla for 24 hours In somepoints cells were cotransfected with Smad 7Then luciferaserenillaactivity was measured Data are expressed as increase in Smadbinding element (SBE) promoter-luciferase dependent expressionData are expressed as n-fold over control (considered as 1) as themean plusmn SEM of 5 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
in experimental renal fibrotic diseases including glomeru-losclerosis tubulointerstitial fibrosis hypertensive-inducedrenal damage and diabetic nephropathy [20ndash26] as well as inrenal tumor progression [27] In angiotensin II-induced renaldamage renal activation of the Smad pathway was associatedto EMT changes [20] Moreover Smad 7 overexpressionameliorates renal damage and fibrosis caused by unilateralureteral obstruction angiotensin II and diabetes [22ndash2428] We have observed that in tubuloepithelial cells Smad 7overexpression blocked Gremlin-induced EMT changes Theinvolvement of Smad pathway in Gremlin-mediated fibrosishas been also described in other cell types in vitro In opticnerve head astrocytes and lamina cribrosa cells recombinantGremlin stimulates ECM production through the activationof TGF-120573 receptor and Smad 3 phosphorylation suggestinga role for Gremlin in glaucoma [29] In healthy dermalfibroblasts IL-6 mediated induction of collagen is dependenton Gremlin production and activation of TGF-120573Smad sig-nalling [30] Besides the regulation of renal EMT and fibrosisGremlinSmad pathway could also be involved in the onset ofproteinuria by modulating podocyte injury and changing thedistribution of nephrin and synaptopodin [21]
Recent evidences suggest that Gremlin could be animportant promoter of fibrosis in different pathologiesincluding liver fibrosis lung diseases particularly pul-monary hypertension and idiopathic pulmonary fibrosis andmyocardial fibrosis [31ndash35] In several human renal diseasesGremlin overexpression was found mainly in areas of tubuleinterstitial fibrosis [5ndash8] Experimental studies in mice haveshown that Gremlin blockade diminished renal fibrosis asobserved in streptozotocin-induced diabetes in knockoutmice heterozygous for grem1 [9] and by Gremlin gene silenc-ing [10] Recent studies have demonstrated direct fibrogeniceffect of Gremlin in renal cells In mesangial cells Gremlin
increased cell proliferation and ECM accumulation via ERK[36] In renal fibroblasts Gremlin increased ECM production[18] including type I collagen In tubular epithelial cellsGremlin upregulates profibrotic genes such as TGF-120573 andCTGF and caused EMT changes [18] Gremlin also inducesEMT in airway epithelial cells [37] and in cancer cells [38]Although the contribution of EMT to renal fibrosis is amatterof intense debate [39 40] the lost of epithelial propertiesof the tubular epithelial cells including permeability andpolarity may result in decreased viability and contribute torenal injury [40 41] Therefore EMT-related changes arean initial step in renal damage and an important potentialtherapeutic target Our data demonstrate that Gremlin viaSmad pathway regulates EMT showing a novel mechanismof Gremlin action in renal cells
TGF-120573 is known as the major promoter of EMT duringembryogenesis cancer and fibrosis [13ndash17]In a mesothe-lioma cell line Gremlin-silencing inhibited cell proliferationassociated with downregulation of the transcription factorslug as well as mesenchymal proteins linked to cancerEMT [38] We have recently demonstrated that Gremlingene silencing blocked TGF-120573-induced EMT in tubularepithelial cells [18] Now we have observed that Gremlinincreased TGF-120573 production at 48 hours and this endoge-nous autocrine TGF-120573 acts as a downstream mediator ofGremlin-induced profibrotic and EMT related factors incultured human tubuloepithelial cells (Figure 8) All thesefindings reveal the complex relationship between Gremlinand TGF-120573 in the kidney disclosing a positive feedback loopconnection between them in promoting EMT and fibrosis
Gremlin exerts a potent inhibitory action via binding toand forming heterodimers with BMP-2 BMP-4 and BMP-7The binding of Gremlin to selective BMPs prevents ligand-receptor interaction and subsequent downstream signallingGremlin acting as a BMPs antagonist plays a critical role dur-ing the process of nephrogenesis [4] BMP-7 is the antagonistof TGF-1205731 signalling and has been found to inhibit TGF-1205731-induced renal fibrosis by reversing EMT process [42 43]In experimental lungs and pulmonary fibrosis upregulationof Gremlin was associated with downregulation of BMPsignalling [31 32] Gremlin overexpression has been foundto inhibit BMP-4 thus leading to enhance TGF-120573 signallingand ECM deposition in primary open angle glaucoma [44]However BMP-independent mechanismsmaymediate someactions of Gremlin Exogenous Gremlin may bind to andact directly on endothelial cells to modulate angiogenesisincluding endothelial cell migration [45 46] We have foundthat BMPs did no inhibit Gremlin-induced early Smad 3activation Thus a receptor-mediated mechanism of actionmay exist for GremlinTherefore future studies investigatingthe receptor involved in Gremlin responses in renal cells areneeded
5 Conclusion
Chronic progressive fibrosis of the kidney remains anunsolved challenge The investigation of the mediators andmechanisms involved in renal fibrosis could lead to better
8 BioMed Research InternationalG
REM
-GFP
GRE
M-G
FP S
mad
7Em
pty
pCD
NA
CytokeratinGFP Merge
(a)
GFP Merge
GRE
M-G
FPEm
pty
pCD
NA
120572-SMA
GRE
M-G
FP S
mad
7
(b)
Figure 5 Gremlin-induced EMT via the Smad pathway HK2 cells were transiently transfected with empty Gremlin (GREM-GFP) alone orcotransfected with Smad 7 expression vectors EMT markers were evaluated after 48 hours Gremlin transfected cells express GFP (greenstaining) Confocal microscopy analysis of cytokeratin and 120572-SMA immunofluorescence was performed using specific primary antibodiesand Alexa-633 secondary IgG (red staining) Representative image out of 3 experiments
Control Gremlin0
1
2
3
TGF-120573
expr
essio
n le
vels
(n-fo
ld)
24hours48hours
lowast
(a)
mRN
A le
vels
(n-fo
ld)
0
1
2
Control Gremlin
CTGFPAI-1
lowast lowast
lowast
Anti-TGF-120573+
GremlinTGF-120573
(b)
Figure 6 (a) Gremlin increased TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 and 48 hours in serum-freemedium TGF-1205731 protein levels were measured in the cell-conditioned medium using a specific ELISA Data are expressed as mean plusmn SEMof 6 independent experiments lowast119875 lt 005 versus control (b) The late increase in gene expression of profibrotic factors caused by Gremlin ismediated by endogenous TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 hours in serum-free medium TGF-120573was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizing antibody Total cell RNAwas isolated to assessmRNA levels by real-time PCR Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005versus control 119875 lt 005 versus Gremlin
BioMed Research International 9
Vim
entin
pan-
Cyto
kera
tinE-
cadh
erin
Control Gremlin
Gremlin+
anti-TGF-120573
(a)
0
1
2
Vimentin
GAPDH
Control Gremlin+
Gremlin
Vim
entin
GA
PDH
(n-fo
ld)
GAPDH
0
1
2
E-ca
dher
inG
APD
H (n
-fold
)
Control Gremlin+
Gremlin
Slug
GAPDH
0
1
2
Control Gremlin+
Gremlin
Slug
GA
PDH
(n-fo
ld)
E-cadherin
anti-TGF-120573 anti-TGF-120573 anti-TGF-120573
lowast
lowast
lowast
(b)
Figure 7TGF-120573 is amediator of EMT-related changes following stimulationwith Gremlin HK2 cells were stimulatedwithGremlin (50 ngmL)for 48 hours in serum-free medium TGF-120573 was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizingantibody (a) EMT changes were evaluated by confocal microscopy E-cadherin pan-Cytokeratin and Vimentin were studied by indirectimmunofluorescence using FITC-secondary IgG (green) and confocal microscopy Nuclei are shown in blue Figure shows a representativeimage out of 3 independent observations (b)Total proteins were isolated and Vimentin E-cadherin and Slug levels were analyzed by westernblot Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
diagnostic tools and novel therapeutics approaches Manystudies have shown that renal expression of Gremlin isinduced in diabetic nephropathy and in other progressiverenal diseases associated with tubulointerstitial fibrosis andSmad activation [5ndash8] We show here that Gremlin activatesthe Smad signaling pathway and induces TGF-120573 and otherrelated factors involved in EMT and fibrotic events in renalcells All these data suggest that Gremlin could be a potential
novel molecular antifibrotic target and biomarker useful forprognostication disease monitoring and therapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
10 BioMed Research International
Gremlin
Smadactivation
Target genes
EMT-related changes
Early-response(min)
Long-response
TGF-120573 independent
TGF-120573 production
TGF-120573 mediated(gt24hours)
Induction of vimentin and 120572-SMAlost of E-cadherin and cytokeratin
Figure 8 Dual effects of Gremlin on Smad activation Gremlininduces an early (minutes) and direct TGF-120573-independent Smadpathway activation After 24 hours Gremlin increased severalprofibrotic genes including TGF-120573 and after 48 hours increasedTGF-120573 production and induced EMT features These long-termGremlin-induced profibrotic events require autocrine TGF-120573
Authorsrsquo Contribution
Raquel Rodrigues-Diez and Raul R Rodrigues-Diez equallycontributed to this work
Acknowledgments
This work was supported by grants from the Institutode Salud Carlos III (PI1101854 and REDINREN ISCIII-RETIC RD1200210002 and 0001) Sociedad Espanola deNefrologıa PCI Iberoamerica (A957107) CYTED IBER-ERC FONDECYT Chile 1080083 and 1120480 Comu-nidad de Madrid (Fibroteam S2010BMD-2321 S2010BMD-2378) Programa Intensificacion Actividad Investigadora(ISCIIIAgencia Laın-EntralgoCM) to AO Fundacion parael fomento en Asturias de la investigacion cientıfica aplicaday la tecnologıa (FICYT) The authors want to thank Ma MarGonzalezGarcia-Parreno for her technical help with confocalmicroscopy
References
[1] S A Roxburgh M Murphy C A Pollock and D P BrazilldquoRecapitulation of embryological programmes in renalfibrosismdashthe importance of epithelial cell plasticity anddevelopmental genesrdquo Nephron Physiology vol 103 no 3 ppp139ndashp148 2006
[2] R McMahon MMurphy M Clarkson et al ldquoIHG-2 a mesan-gial cell gene induced by high glucose is human gremlin regu-lation by extracellular glucose concentration cyclic mechanicalstrain and transforming growth factor-1205731rdquo The Journal ofBiological Chemistry vol 275 no 14 pp 9901ndash9904 2000
[3] L Z Topol B Bardot Q Zhang et al ldquoBiosynthesis post-translation modification and functional characterization ofDrmGremlinrdquo The Journal of Biological Chemistry vol 275no 12 pp 8785ndash8793 2000
[4] O Michos L Panman K Vintersten K Beier R Zeller andA Zuniga ldquoGremlin-mediated BMP antagonism inducesthe epithelial-mesenchymal feedback signaling controllingmetanephric kidney and limb organogenesisrdquo Developmentvol 131 no 14 pp 3401ndash3410 2004
[5] V Dolan M Murphy D Sadlier et al ldquoExpression of gremlina bone morphogenetic protein antagonist in human diabeticnephropathyrdquo American Journal of Kidney Diseases vol 45 no6 pp 1034ndash1039 2005
[6] M Murphy J Crean D P Brazil D Sadlier F Martin andC Godson ldquoRegulation and consequences of differential geneexpression in diabetic kidney diseaserdquo Biochemical SocietyTransactions vol 36 no 5 pp 941ndash945 2008
[7] S Mezzano A Droguett M B Eugenia et al ldquoExpression ofgremlin a bonemorphogenetic protein antagonist in glomeru-lar crescents of pauci-immune glomerulonephritisrdquoNephrologyDialysis Transplantation vol 22 no 7 pp 1882ndash1890 2007
[8] G Carvajal A Droguett M E Burgos et al ldquoGremlin a novelmediator of epithelial mesenchymal transition and fibrosis inchronic allograft nephropathyrdquo Transplantation Proceedingsvol 40 no 3 pp 734ndash739 2008
[9] S A Roxburgh J J Kattla S P Curran et al ldquoAllelic depletionof grem1 attenuates diabetic kidney diseaserdquo Diabetes vol 58no 7 pp 1641ndash1650 2009
[10] Q Zhang Y Shi J Wada et al ldquoIn vivo delivery of gremlinsiRNA plasmid reveals therapeutic potential against diabeticnephropathy by recovering bone morphogenetic protein-7rdquoPLoS ONE vol 5 no 7 article e11709 2010
[11] M Zeisberg and E G Neilson ldquoMechanisms oftubulointerstitial fibrosisrdquo Journal of the American Societyof Nephrology vol 21 no 11 pp 1819ndash1834 2010
[12] Y Liu ldquoNew insights into epithelial-mesenchymal transition inkidney fibrosisrdquo Journal of the American Society of Nephrologyvol 21 no 2 pp 212ndash222 2010
[13] A Moustakas and C-H Heldin ldquoSignaling networks guidingepithelial-mesenchymal transitions during embryogenesisand cancer progressionrdquo Cancer Science vol 98 no 10 pp1512ndash1520 2007
[14] J-M Fan N G Yee-Yung P A Hill et al ldquoTransforminggrowth factor-120573 regulates tubular epithelial-myofibroblasttransdifferentiation in vitrordquo Kidney International vol 56 no4 pp 1455ndash1467 1999
[15] A B Roberts F Tian S D Byfield et al ldquoSmad3 is keyto TGF-120573-mediated epithelial-to-mesenchymal transitionfibrosis tumor suppression andmetastasisrdquo Cytokine amp GrowthFactor Reviews vol 17 no 1-2 pp 19ndash27 2006
BioMed Research International 11
[16] J Xu S Lamouille and R Derynck ldquoTGF-120573-induced epithelialto mesenchymal transitionrdquo Cell Research vol 19 no 2 pp156ndash172 2009
[17] M Ruiz-Ortega J Rodrıguez-Vita E Sanchez-Lopez GCarvajal and J Egido ldquoTGF-120573 signaling in vascular fibrosisrdquoCardiovascular Research vol 74 no 2 pp 196ndash206 2007
[18] R Rodrigues-Diez C Lavoz G Carvajal et al ldquoGremlin isa downstream profibrotic mediator of transforming growthfactor-beta in cultured renal cellsrdquo Nephron ExperimentalNephrology vol 122 no 1-2 pp 62ndash74 2012
[19] J Rodrıguez-Vita E Sanchez-Lopez V Esteban M Ruperez JEgido and M Ruiz-Ortega ldquoAngiotensin II activates the Smadpathway in vascular smooth muscle cells by a transforminggrowth factor-120573-independent mechanismrdquo Circulation vol 111no 19 pp 2509ndash2517 2005
[20] G Carvajal J Rodrıguez-Vita R Rodrigues-Dıez et alldquoAngiotensin II activates the Smad pathway during epithelialmesenchymal transdifferentiationrdquo Kidney International vol74 no 5 pp 585ndash595 2008
[21] G Li Y Li S Liu et al ldquoGremlin aggravates hyperglycemia-induced podocyte injury by a TGF120573smad dependent signalingpathwayrdquo Journal of Cellular Biochemistry vol 114 no 9 pp2101ndash2113 2013
[22] H Y Lan W Mu N Tomita et al ldquoInhibition of renalfibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in ratUUOmodelrdquo Journal of theAmericanSociety of Nephrology vol 14 no 6 pp 1535ndash1548 2003
[23] H Fukasawa T Yamamoto A Togawa et al ldquoDown-regulationof Smad7 expression by ubiquitin-dependent degradationcontributes to renal fibrosis in obstructive nephropathy inmicerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 23 pp 8687ndash8692 2004
[24] H Y Chen X R Huang WWang et al ldquoThe protective role ofSmad7 in diabetic kidney disease mechanism and therapeuticpotentialrdquo Diabetes vol 60 no 2 pp 590ndash601 2011
[25] X Wei Y Xia F Li et al ldquoKindlin-2 mediates activationof TGF-120573Smad signaling and renal fibrosisrdquo Journal of theAmerican Society Nephrology vol 24 no 9 pp 1387ndash1398 2013
[26] M Isono S Chen S W Hong M C Iglesias-de la Cruzand F N Ziyadeh ldquoSmad pathway is activated in the diabeticmouse kidney and smad3 mediates TGF-120573-induced fibronectinin mesangial cellsrdquo Biochemical and Biophysical ResearchCommunications vol 296 no 5 pp 1356ndash1365 2002
[27] J H Park C Lee J H Suh J Y Chae andK CMoon ldquoNuclearexpression of Smad proteins and its prognostic significance inclear cell renal cell carcinomardquo Human Pathology vol 44 no10 pp 2047ndash2054 2013
[28] G X Liu Y Q Li X R Huang et al ldquoSmad7 inhibitsAngII-mediated hypertensive nephropathy in amousemodel ofhypertensionrdquo Clinical Science vol 127 no 3 pp 195ndash208 2014
[29] G S Zode A F Clark and R J Wordinger ldquoBone morpho-genetic protein 4 inhibits TGF-1205732 stimulation of extracellularmatrix proteins in optic nerve head cells role of gremlin inECMmodulationrdquo Glia vol 57 no 7 pp 755ndash766 2009
[30] S OrsquoReilly M Ciechomska R Cant and J M van Laar ldquoIL-6trans signalling drives a STAT3 dependant pathway that leadsto hyperactive TGF-120573 signalling promoting SMAD3 activationand fibrosis via gremlinrdquo The Journal of Biological Chemistry2014
[31] M Myllarniemi P Lindholm M J Ryynanen et al ldquoGremlin-mediated decrease in bone morphogenetic protein signaling
promotes pulmonary fibrosisrdquo American Journal of Respiratoryand Critical Care Medicine vol 177 no 3 pp 321ndash329 2008
[32] E Cahill C M Costello S C Rowan et al ldquoGremlin playsa key role in the pathogenesis of pulmonary hypertensionrdquoCirculation vol 125 no 7 pp 920ndash930 2012
[33] W Boers S Aarrass C Linthorst M Pinzani R O Elferinkand P Bosma ldquoTranscriptional profiling reveals novel markersof liver fibrogenesis gremlin and insulin-like growth factor-binding proteinsrdquo The Journal of Biological Chemistry vol 281no 24 pp 16289ndash16295 2006
[34] CMCostello E Cahill FMartin S Gaine andPMcLoughlinldquoRole of gremlin in the lung development and diseaserdquoAmerican Journal of Respiratory Cell and Molecular Biologyvol 42 no 5 pp 517ndash523 2010
[35] K A Mueller E Tavlaki M Schneider et al ldquoGremlin-1identifies fibrosis and predicts adverse outcome in patientswith heart failure undergoing endomyocardial biopsyrdquo Journalof Cardiac Failure vol 19 no 10 pp 678ndash684 2013
[36] H Huang H Huang Y Li et al ldquoGremlin induces cellproliferation and extra cellular matrix accumulation in mousemesangial cells exposed to high glucose via the ERK12pathwayrdquo BMC Nephrology vol 14 article 33 2013
[37] N McCormack E L Molloy and S OrsquoDea ldquoBone morpho-genetic proteins enhance an epithelial-mesenchymal transitionin normal airway epithelial cells during restitution of a dis-rupted epitheliumrdquoRespiratory Research vol 14 article 36 2013
[38] J A Tamminen V Parviainen M Ronty et al ldquoGremlin-1associates with fibrillin microfibrils in vivo and regulatesmesothelioma cell survival through transcription factor slugrdquoOncogenesis vol 2 article e66 2013
[39] E M Zeisberg S E Potenta H Sugimoto M Zeisberg and RKalluri ldquoFibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transitionrdquo Journal of the American Society ofNephrology vol 19 no 12 pp 2282ndash2287 2008
[40] V S LeBleu G Taduri J OrsquoConnell et al ldquoOrigin and functionof myofibroblasts in kidney fibrosisrdquo Nature Medicine vol 19no 8 pp 1047ndash1053 2013
[41] W C Prozialeck and J R Edwards ldquoCell adhesion moleculesin chemically-induced renal injuryrdquo Pharmacology amp Thera-peutics vol 114 no 1 pp 74ndash93 2007
[42] M Zeisberg J Hanai H Sugimoto et al ldquoBMP-7 counteractsTGF-1205731-induced epithelial-to-mesenchymal transition andreverses chronic renal injuryrdquo Nature Medicine vol 9 no 7 pp964ndash968 2003
[43] Y-L Yang H-Z Ju S-F Liu et al ldquoBMP-2 suppresses renalinterstitial fibrosis by regulating epithelial-mesenchymaltransitionrdquo Journal of Cellular Biochemistry vol 112 no 9 pp2558ndash2565 2011
[44] R J Wordinger D L Fleenor P E Hellberg et al ldquoEffects ofTGF-1205732 BMP-4 and gremlin in the trabecular meshworkimplications for glaucomardquo Investigative Ophthalmology ampVisual Science vol 48 no 3 pp 1191ndash1200 2007
[45] B Chen M Athanasiou Q Gu and D G Blair ldquoDrmGremlintranscriptionally activates p21Cip1 via a novel mechanism andinhibits neoplastic transformationrdquoBiochemical andBiophysicalResearch Communications vol 295 no 5 pp 1135ndash1141 2002
[46] S Mitola C Ravelli E Moroni et al ldquoGremlin is a novelagonist of the major proangiogenic receptor VEGFR2rdquo Bloodvol 116 no 18 pp 3677ndash3680 2010
Figure 3 Gremlin overexpression causes a sustained Smad activation in cultured human tubuloepithelial cells HK2 cells were transientlytransfected with a Gremlin expression vector (GREM-GFP green) or empty vector for 24 hours The levels and localization of R-Smad 3(a) and R-Smad 2 (b) were evaluated by confocal microscopy with Alexa-633 secondary IgG (red) Nuclei were stained using 410158406-diamino-2-phenylindole dihydrochloride (DAPI blue) In Gremlin-transfected cells (green staining by GFP) the Smad 2 and Smad 3 were found inthe nuclei (white staining in the merge) Figures show representative images out of 3 independent observations
and restored E-cadherin levels decreased by Gremlin (Fig-ure 7(b))These data suggest that TGF-120573 is amediator of long-term responses of Gremlin in tubuloepithelial cells includingregulation of profibrotic factors and EMT changes
4 Discussion
Our in vitro studies in cultured tubuloepithelial cells showthat Gremlin directly activates the Smad pathway and par-ticipates in the EMT process via Smad signalling These datasuggest that Gremlin could be a mediator of renal fibrosis
Our study reveals that in cultured human tubuloepithelialcells Gremlin induces a rapid activation of the Smad path-way (observed after 5min of stimulation) characterized byincreased phosphorylation of the receptor-Smad (R-Smad)Smad 3 a critical downstream mediator of fibrosis [17] and
Smad 2 proteinsOnceR-Smad is phosphorylated it dimeriseswith Smad 4 and then shuttles to the nucleus to regulategene expression By confocal microscopy we have foundthat Gremlin caused a rapid translocation to the nucleusof R-SmadSmad 4 proteins In several cells types Gremlin-induced TGF-120573 production [18 21] as we have observedhere after 48 hours of incubationHowever Gremlin-inducedearly Smad activation is independent of endogenous TGF-120573as we have demonstrated using TGF-120573 blockers (Figure 8)Other important profibrotic factors such as angiotensin IIalso activates the Smad pathway rapidly and independent ofendogenous TGF-120573 [17]
Previous studies in tubular epithelial cells have shownthat the Smad route regulates EMT induced by key factorsinvolved in renal fibrosis such as TGF-120573 and angiotensinII [20] The activation of Smad pathway has been described
BioMed Research International 7
0
1
2
3
4
Control GREM-GFP GREM-GFP+ Smad 7
SBE-
prom
otor
expr
essio
n (n
-fold
) lowast
Figure 4 Gremlin overexpression induces Smad-dependent genetranscription HK2 cells were transfected with GREM-GFP or emptyvector Smadluc promoter and TK-renilla for 24 hours In somepoints cells were cotransfected with Smad 7Then luciferaserenillaactivity was measured Data are expressed as increase in Smadbinding element (SBE) promoter-luciferase dependent expressionData are expressed as n-fold over control (considered as 1) as themean plusmn SEM of 5 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
in experimental renal fibrotic diseases including glomeru-losclerosis tubulointerstitial fibrosis hypertensive-inducedrenal damage and diabetic nephropathy [20ndash26] as well as inrenal tumor progression [27] In angiotensin II-induced renaldamage renal activation of the Smad pathway was associatedto EMT changes [20] Moreover Smad 7 overexpressionameliorates renal damage and fibrosis caused by unilateralureteral obstruction angiotensin II and diabetes [22ndash2428] We have observed that in tubuloepithelial cells Smad 7overexpression blocked Gremlin-induced EMT changes Theinvolvement of Smad pathway in Gremlin-mediated fibrosishas been also described in other cell types in vitro In opticnerve head astrocytes and lamina cribrosa cells recombinantGremlin stimulates ECM production through the activationof TGF-120573 receptor and Smad 3 phosphorylation suggestinga role for Gremlin in glaucoma [29] In healthy dermalfibroblasts IL-6 mediated induction of collagen is dependenton Gremlin production and activation of TGF-120573Smad sig-nalling [30] Besides the regulation of renal EMT and fibrosisGremlinSmad pathway could also be involved in the onset ofproteinuria by modulating podocyte injury and changing thedistribution of nephrin and synaptopodin [21]
Recent evidences suggest that Gremlin could be animportant promoter of fibrosis in different pathologiesincluding liver fibrosis lung diseases particularly pul-monary hypertension and idiopathic pulmonary fibrosis andmyocardial fibrosis [31ndash35] In several human renal diseasesGremlin overexpression was found mainly in areas of tubuleinterstitial fibrosis [5ndash8] Experimental studies in mice haveshown that Gremlin blockade diminished renal fibrosis asobserved in streptozotocin-induced diabetes in knockoutmice heterozygous for grem1 [9] and by Gremlin gene silenc-ing [10] Recent studies have demonstrated direct fibrogeniceffect of Gremlin in renal cells In mesangial cells Gremlin
increased cell proliferation and ECM accumulation via ERK[36] In renal fibroblasts Gremlin increased ECM production[18] including type I collagen In tubular epithelial cellsGremlin upregulates profibrotic genes such as TGF-120573 andCTGF and caused EMT changes [18] Gremlin also inducesEMT in airway epithelial cells [37] and in cancer cells [38]Although the contribution of EMT to renal fibrosis is amatterof intense debate [39 40] the lost of epithelial propertiesof the tubular epithelial cells including permeability andpolarity may result in decreased viability and contribute torenal injury [40 41] Therefore EMT-related changes arean initial step in renal damage and an important potentialtherapeutic target Our data demonstrate that Gremlin viaSmad pathway regulates EMT showing a novel mechanismof Gremlin action in renal cells
TGF-120573 is known as the major promoter of EMT duringembryogenesis cancer and fibrosis [13ndash17]In a mesothe-lioma cell line Gremlin-silencing inhibited cell proliferationassociated with downregulation of the transcription factorslug as well as mesenchymal proteins linked to cancerEMT [38] We have recently demonstrated that Gremlingene silencing blocked TGF-120573-induced EMT in tubularepithelial cells [18] Now we have observed that Gremlinincreased TGF-120573 production at 48 hours and this endoge-nous autocrine TGF-120573 acts as a downstream mediator ofGremlin-induced profibrotic and EMT related factors incultured human tubuloepithelial cells (Figure 8) All thesefindings reveal the complex relationship between Gremlinand TGF-120573 in the kidney disclosing a positive feedback loopconnection between them in promoting EMT and fibrosis
Gremlin exerts a potent inhibitory action via binding toand forming heterodimers with BMP-2 BMP-4 and BMP-7The binding of Gremlin to selective BMPs prevents ligand-receptor interaction and subsequent downstream signallingGremlin acting as a BMPs antagonist plays a critical role dur-ing the process of nephrogenesis [4] BMP-7 is the antagonistof TGF-1205731 signalling and has been found to inhibit TGF-1205731-induced renal fibrosis by reversing EMT process [42 43]In experimental lungs and pulmonary fibrosis upregulationof Gremlin was associated with downregulation of BMPsignalling [31 32] Gremlin overexpression has been foundto inhibit BMP-4 thus leading to enhance TGF-120573 signallingand ECM deposition in primary open angle glaucoma [44]However BMP-independent mechanismsmaymediate someactions of Gremlin Exogenous Gremlin may bind to andact directly on endothelial cells to modulate angiogenesisincluding endothelial cell migration [45 46] We have foundthat BMPs did no inhibit Gremlin-induced early Smad 3activation Thus a receptor-mediated mechanism of actionmay exist for GremlinTherefore future studies investigatingthe receptor involved in Gremlin responses in renal cells areneeded
5 Conclusion
Chronic progressive fibrosis of the kidney remains anunsolved challenge The investigation of the mediators andmechanisms involved in renal fibrosis could lead to better
8 BioMed Research InternationalG
REM
-GFP
GRE
M-G
FP S
mad
7Em
pty
pCD
NA
CytokeratinGFP Merge
(a)
GFP Merge
GRE
M-G
FPEm
pty
pCD
NA
120572-SMA
GRE
M-G
FP S
mad
7
(b)
Figure 5 Gremlin-induced EMT via the Smad pathway HK2 cells were transiently transfected with empty Gremlin (GREM-GFP) alone orcotransfected with Smad 7 expression vectors EMT markers were evaluated after 48 hours Gremlin transfected cells express GFP (greenstaining) Confocal microscopy analysis of cytokeratin and 120572-SMA immunofluorescence was performed using specific primary antibodiesand Alexa-633 secondary IgG (red staining) Representative image out of 3 experiments
Control Gremlin0
1
2
3
TGF-120573
expr
essio
n le
vels
(n-fo
ld)
24hours48hours
lowast
(a)
mRN
A le
vels
(n-fo
ld)
0
1
2
Control Gremlin
CTGFPAI-1
lowast lowast
lowast
Anti-TGF-120573+
GremlinTGF-120573
(b)
Figure 6 (a) Gremlin increased TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 and 48 hours in serum-freemedium TGF-1205731 protein levels were measured in the cell-conditioned medium using a specific ELISA Data are expressed as mean plusmn SEMof 6 independent experiments lowast119875 lt 005 versus control (b) The late increase in gene expression of profibrotic factors caused by Gremlin ismediated by endogenous TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 hours in serum-free medium TGF-120573was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizing antibody Total cell RNAwas isolated to assessmRNA levels by real-time PCR Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005versus control 119875 lt 005 versus Gremlin
BioMed Research International 9
Vim
entin
pan-
Cyto
kera
tinE-
cadh
erin
Control Gremlin
Gremlin+
anti-TGF-120573
(a)
0
1
2
Vimentin
GAPDH
Control Gremlin+
Gremlin
Vim
entin
GA
PDH
(n-fo
ld)
GAPDH
0
1
2
E-ca
dher
inG
APD
H (n
-fold
)
Control Gremlin+
Gremlin
Slug
GAPDH
0
1
2
Control Gremlin+
Gremlin
Slug
GA
PDH
(n-fo
ld)
E-cadherin
anti-TGF-120573 anti-TGF-120573 anti-TGF-120573
lowast
lowast
lowast
(b)
Figure 7TGF-120573 is amediator of EMT-related changes following stimulationwith Gremlin HK2 cells were stimulatedwithGremlin (50 ngmL)for 48 hours in serum-free medium TGF-120573 was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizingantibody (a) EMT changes were evaluated by confocal microscopy E-cadherin pan-Cytokeratin and Vimentin were studied by indirectimmunofluorescence using FITC-secondary IgG (green) and confocal microscopy Nuclei are shown in blue Figure shows a representativeimage out of 3 independent observations (b)Total proteins were isolated and Vimentin E-cadherin and Slug levels were analyzed by westernblot Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
diagnostic tools and novel therapeutics approaches Manystudies have shown that renal expression of Gremlin isinduced in diabetic nephropathy and in other progressiverenal diseases associated with tubulointerstitial fibrosis andSmad activation [5ndash8] We show here that Gremlin activatesthe Smad signaling pathway and induces TGF-120573 and otherrelated factors involved in EMT and fibrotic events in renalcells All these data suggest that Gremlin could be a potential
novel molecular antifibrotic target and biomarker useful forprognostication disease monitoring and therapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
10 BioMed Research International
Gremlin
Smadactivation
Target genes
EMT-related changes
Early-response(min)
Long-response
TGF-120573 independent
TGF-120573 production
TGF-120573 mediated(gt24hours)
Induction of vimentin and 120572-SMAlost of E-cadherin and cytokeratin
Figure 8 Dual effects of Gremlin on Smad activation Gremlininduces an early (minutes) and direct TGF-120573-independent Smadpathway activation After 24 hours Gremlin increased severalprofibrotic genes including TGF-120573 and after 48 hours increasedTGF-120573 production and induced EMT features These long-termGremlin-induced profibrotic events require autocrine TGF-120573
Authorsrsquo Contribution
Raquel Rodrigues-Diez and Raul R Rodrigues-Diez equallycontributed to this work
Acknowledgments
This work was supported by grants from the Institutode Salud Carlos III (PI1101854 and REDINREN ISCIII-RETIC RD1200210002 and 0001) Sociedad Espanola deNefrologıa PCI Iberoamerica (A957107) CYTED IBER-ERC FONDECYT Chile 1080083 and 1120480 Comu-nidad de Madrid (Fibroteam S2010BMD-2321 S2010BMD-2378) Programa Intensificacion Actividad Investigadora(ISCIIIAgencia Laın-EntralgoCM) to AO Fundacion parael fomento en Asturias de la investigacion cientıfica aplicaday la tecnologıa (FICYT) The authors want to thank Ma MarGonzalezGarcia-Parreno for her technical help with confocalmicroscopy
References
[1] S A Roxburgh M Murphy C A Pollock and D P BrazilldquoRecapitulation of embryological programmes in renalfibrosismdashthe importance of epithelial cell plasticity anddevelopmental genesrdquo Nephron Physiology vol 103 no 3 ppp139ndashp148 2006
[2] R McMahon MMurphy M Clarkson et al ldquoIHG-2 a mesan-gial cell gene induced by high glucose is human gremlin regu-lation by extracellular glucose concentration cyclic mechanicalstrain and transforming growth factor-1205731rdquo The Journal ofBiological Chemistry vol 275 no 14 pp 9901ndash9904 2000
[3] L Z Topol B Bardot Q Zhang et al ldquoBiosynthesis post-translation modification and functional characterization ofDrmGremlinrdquo The Journal of Biological Chemistry vol 275no 12 pp 8785ndash8793 2000
[4] O Michos L Panman K Vintersten K Beier R Zeller andA Zuniga ldquoGremlin-mediated BMP antagonism inducesthe epithelial-mesenchymal feedback signaling controllingmetanephric kidney and limb organogenesisrdquo Developmentvol 131 no 14 pp 3401ndash3410 2004
[5] V Dolan M Murphy D Sadlier et al ldquoExpression of gremlina bone morphogenetic protein antagonist in human diabeticnephropathyrdquo American Journal of Kidney Diseases vol 45 no6 pp 1034ndash1039 2005
[6] M Murphy J Crean D P Brazil D Sadlier F Martin andC Godson ldquoRegulation and consequences of differential geneexpression in diabetic kidney diseaserdquo Biochemical SocietyTransactions vol 36 no 5 pp 941ndash945 2008
[7] S Mezzano A Droguett M B Eugenia et al ldquoExpression ofgremlin a bonemorphogenetic protein antagonist in glomeru-lar crescents of pauci-immune glomerulonephritisrdquoNephrologyDialysis Transplantation vol 22 no 7 pp 1882ndash1890 2007
[8] G Carvajal A Droguett M E Burgos et al ldquoGremlin a novelmediator of epithelial mesenchymal transition and fibrosis inchronic allograft nephropathyrdquo Transplantation Proceedingsvol 40 no 3 pp 734ndash739 2008
[9] S A Roxburgh J J Kattla S P Curran et al ldquoAllelic depletionof grem1 attenuates diabetic kidney diseaserdquo Diabetes vol 58no 7 pp 1641ndash1650 2009
[10] Q Zhang Y Shi J Wada et al ldquoIn vivo delivery of gremlinsiRNA plasmid reveals therapeutic potential against diabeticnephropathy by recovering bone morphogenetic protein-7rdquoPLoS ONE vol 5 no 7 article e11709 2010
[11] M Zeisberg and E G Neilson ldquoMechanisms oftubulointerstitial fibrosisrdquo Journal of the American Societyof Nephrology vol 21 no 11 pp 1819ndash1834 2010
[12] Y Liu ldquoNew insights into epithelial-mesenchymal transition inkidney fibrosisrdquo Journal of the American Society of Nephrologyvol 21 no 2 pp 212ndash222 2010
[13] A Moustakas and C-H Heldin ldquoSignaling networks guidingepithelial-mesenchymal transitions during embryogenesisand cancer progressionrdquo Cancer Science vol 98 no 10 pp1512ndash1520 2007
[14] J-M Fan N G Yee-Yung P A Hill et al ldquoTransforminggrowth factor-120573 regulates tubular epithelial-myofibroblasttransdifferentiation in vitrordquo Kidney International vol 56 no4 pp 1455ndash1467 1999
[15] A B Roberts F Tian S D Byfield et al ldquoSmad3 is keyto TGF-120573-mediated epithelial-to-mesenchymal transitionfibrosis tumor suppression andmetastasisrdquo Cytokine amp GrowthFactor Reviews vol 17 no 1-2 pp 19ndash27 2006
BioMed Research International 11
[16] J Xu S Lamouille and R Derynck ldquoTGF-120573-induced epithelialto mesenchymal transitionrdquo Cell Research vol 19 no 2 pp156ndash172 2009
[17] M Ruiz-Ortega J Rodrıguez-Vita E Sanchez-Lopez GCarvajal and J Egido ldquoTGF-120573 signaling in vascular fibrosisrdquoCardiovascular Research vol 74 no 2 pp 196ndash206 2007
[18] R Rodrigues-Diez C Lavoz G Carvajal et al ldquoGremlin isa downstream profibrotic mediator of transforming growthfactor-beta in cultured renal cellsrdquo Nephron ExperimentalNephrology vol 122 no 1-2 pp 62ndash74 2012
[19] J Rodrıguez-Vita E Sanchez-Lopez V Esteban M Ruperez JEgido and M Ruiz-Ortega ldquoAngiotensin II activates the Smadpathway in vascular smooth muscle cells by a transforminggrowth factor-120573-independent mechanismrdquo Circulation vol 111no 19 pp 2509ndash2517 2005
[20] G Carvajal J Rodrıguez-Vita R Rodrigues-Dıez et alldquoAngiotensin II activates the Smad pathway during epithelialmesenchymal transdifferentiationrdquo Kidney International vol74 no 5 pp 585ndash595 2008
[21] G Li Y Li S Liu et al ldquoGremlin aggravates hyperglycemia-induced podocyte injury by a TGF120573smad dependent signalingpathwayrdquo Journal of Cellular Biochemistry vol 114 no 9 pp2101ndash2113 2013
[22] H Y Lan W Mu N Tomita et al ldquoInhibition of renalfibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in ratUUOmodelrdquo Journal of theAmericanSociety of Nephrology vol 14 no 6 pp 1535ndash1548 2003
[23] H Fukasawa T Yamamoto A Togawa et al ldquoDown-regulationof Smad7 expression by ubiquitin-dependent degradationcontributes to renal fibrosis in obstructive nephropathy inmicerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 23 pp 8687ndash8692 2004
[24] H Y Chen X R Huang WWang et al ldquoThe protective role ofSmad7 in diabetic kidney disease mechanism and therapeuticpotentialrdquo Diabetes vol 60 no 2 pp 590ndash601 2011
[25] X Wei Y Xia F Li et al ldquoKindlin-2 mediates activationof TGF-120573Smad signaling and renal fibrosisrdquo Journal of theAmerican Society Nephrology vol 24 no 9 pp 1387ndash1398 2013
[26] M Isono S Chen S W Hong M C Iglesias-de la Cruzand F N Ziyadeh ldquoSmad pathway is activated in the diabeticmouse kidney and smad3 mediates TGF-120573-induced fibronectinin mesangial cellsrdquo Biochemical and Biophysical ResearchCommunications vol 296 no 5 pp 1356ndash1365 2002
[27] J H Park C Lee J H Suh J Y Chae andK CMoon ldquoNuclearexpression of Smad proteins and its prognostic significance inclear cell renal cell carcinomardquo Human Pathology vol 44 no10 pp 2047ndash2054 2013
[28] G X Liu Y Q Li X R Huang et al ldquoSmad7 inhibitsAngII-mediated hypertensive nephropathy in amousemodel ofhypertensionrdquo Clinical Science vol 127 no 3 pp 195ndash208 2014
[29] G S Zode A F Clark and R J Wordinger ldquoBone morpho-genetic protein 4 inhibits TGF-1205732 stimulation of extracellularmatrix proteins in optic nerve head cells role of gremlin inECMmodulationrdquo Glia vol 57 no 7 pp 755ndash766 2009
[30] S OrsquoReilly M Ciechomska R Cant and J M van Laar ldquoIL-6trans signalling drives a STAT3 dependant pathway that leadsto hyperactive TGF-120573 signalling promoting SMAD3 activationand fibrosis via gremlinrdquo The Journal of Biological Chemistry2014
[31] M Myllarniemi P Lindholm M J Ryynanen et al ldquoGremlin-mediated decrease in bone morphogenetic protein signaling
promotes pulmonary fibrosisrdquo American Journal of Respiratoryand Critical Care Medicine vol 177 no 3 pp 321ndash329 2008
[32] E Cahill C M Costello S C Rowan et al ldquoGremlin playsa key role in the pathogenesis of pulmonary hypertensionrdquoCirculation vol 125 no 7 pp 920ndash930 2012
[33] W Boers S Aarrass C Linthorst M Pinzani R O Elferinkand P Bosma ldquoTranscriptional profiling reveals novel markersof liver fibrogenesis gremlin and insulin-like growth factor-binding proteinsrdquo The Journal of Biological Chemistry vol 281no 24 pp 16289ndash16295 2006
[34] CMCostello E Cahill FMartin S Gaine andPMcLoughlinldquoRole of gremlin in the lung development and diseaserdquoAmerican Journal of Respiratory Cell and Molecular Biologyvol 42 no 5 pp 517ndash523 2010
[35] K A Mueller E Tavlaki M Schneider et al ldquoGremlin-1identifies fibrosis and predicts adverse outcome in patientswith heart failure undergoing endomyocardial biopsyrdquo Journalof Cardiac Failure vol 19 no 10 pp 678ndash684 2013
[36] H Huang H Huang Y Li et al ldquoGremlin induces cellproliferation and extra cellular matrix accumulation in mousemesangial cells exposed to high glucose via the ERK12pathwayrdquo BMC Nephrology vol 14 article 33 2013
[37] N McCormack E L Molloy and S OrsquoDea ldquoBone morpho-genetic proteins enhance an epithelial-mesenchymal transitionin normal airway epithelial cells during restitution of a dis-rupted epitheliumrdquoRespiratory Research vol 14 article 36 2013
[38] J A Tamminen V Parviainen M Ronty et al ldquoGremlin-1associates with fibrillin microfibrils in vivo and regulatesmesothelioma cell survival through transcription factor slugrdquoOncogenesis vol 2 article e66 2013
[39] E M Zeisberg S E Potenta H Sugimoto M Zeisberg and RKalluri ldquoFibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transitionrdquo Journal of the American Society ofNephrology vol 19 no 12 pp 2282ndash2287 2008
[40] V S LeBleu G Taduri J OrsquoConnell et al ldquoOrigin and functionof myofibroblasts in kidney fibrosisrdquo Nature Medicine vol 19no 8 pp 1047ndash1053 2013
[41] W C Prozialeck and J R Edwards ldquoCell adhesion moleculesin chemically-induced renal injuryrdquo Pharmacology amp Thera-peutics vol 114 no 1 pp 74ndash93 2007
[42] M Zeisberg J Hanai H Sugimoto et al ldquoBMP-7 counteractsTGF-1205731-induced epithelial-to-mesenchymal transition andreverses chronic renal injuryrdquo Nature Medicine vol 9 no 7 pp964ndash968 2003
[43] Y-L Yang H-Z Ju S-F Liu et al ldquoBMP-2 suppresses renalinterstitial fibrosis by regulating epithelial-mesenchymaltransitionrdquo Journal of Cellular Biochemistry vol 112 no 9 pp2558ndash2565 2011
[44] R J Wordinger D L Fleenor P E Hellberg et al ldquoEffects ofTGF-1205732 BMP-4 and gremlin in the trabecular meshworkimplications for glaucomardquo Investigative Ophthalmology ampVisual Science vol 48 no 3 pp 1191ndash1200 2007
[45] B Chen M Athanasiou Q Gu and D G Blair ldquoDrmGremlintranscriptionally activates p21Cip1 via a novel mechanism andinhibits neoplastic transformationrdquoBiochemical andBiophysicalResearch Communications vol 295 no 5 pp 1135ndash1141 2002
[46] S Mitola C Ravelli E Moroni et al ldquoGremlin is a novelagonist of the major proangiogenic receptor VEGFR2rdquo Bloodvol 116 no 18 pp 3677ndash3680 2010
Figure 4 Gremlin overexpression induces Smad-dependent genetranscription HK2 cells were transfected with GREM-GFP or emptyvector Smadluc promoter and TK-renilla for 24 hours In somepoints cells were cotransfected with Smad 7Then luciferaserenillaactivity was measured Data are expressed as increase in Smadbinding element (SBE) promoter-luciferase dependent expressionData are expressed as n-fold over control (considered as 1) as themean plusmn SEM of 5 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
in experimental renal fibrotic diseases including glomeru-losclerosis tubulointerstitial fibrosis hypertensive-inducedrenal damage and diabetic nephropathy [20ndash26] as well as inrenal tumor progression [27] In angiotensin II-induced renaldamage renal activation of the Smad pathway was associatedto EMT changes [20] Moreover Smad 7 overexpressionameliorates renal damage and fibrosis caused by unilateralureteral obstruction angiotensin II and diabetes [22ndash2428] We have observed that in tubuloepithelial cells Smad 7overexpression blocked Gremlin-induced EMT changes Theinvolvement of Smad pathway in Gremlin-mediated fibrosishas been also described in other cell types in vitro In opticnerve head astrocytes and lamina cribrosa cells recombinantGremlin stimulates ECM production through the activationof TGF-120573 receptor and Smad 3 phosphorylation suggestinga role for Gremlin in glaucoma [29] In healthy dermalfibroblasts IL-6 mediated induction of collagen is dependenton Gremlin production and activation of TGF-120573Smad sig-nalling [30] Besides the regulation of renal EMT and fibrosisGremlinSmad pathway could also be involved in the onset ofproteinuria by modulating podocyte injury and changing thedistribution of nephrin and synaptopodin [21]
Recent evidences suggest that Gremlin could be animportant promoter of fibrosis in different pathologiesincluding liver fibrosis lung diseases particularly pul-monary hypertension and idiopathic pulmonary fibrosis andmyocardial fibrosis [31ndash35] In several human renal diseasesGremlin overexpression was found mainly in areas of tubuleinterstitial fibrosis [5ndash8] Experimental studies in mice haveshown that Gremlin blockade diminished renal fibrosis asobserved in streptozotocin-induced diabetes in knockoutmice heterozygous for grem1 [9] and by Gremlin gene silenc-ing [10] Recent studies have demonstrated direct fibrogeniceffect of Gremlin in renal cells In mesangial cells Gremlin
increased cell proliferation and ECM accumulation via ERK[36] In renal fibroblasts Gremlin increased ECM production[18] including type I collagen In tubular epithelial cellsGremlin upregulates profibrotic genes such as TGF-120573 andCTGF and caused EMT changes [18] Gremlin also inducesEMT in airway epithelial cells [37] and in cancer cells [38]Although the contribution of EMT to renal fibrosis is amatterof intense debate [39 40] the lost of epithelial propertiesof the tubular epithelial cells including permeability andpolarity may result in decreased viability and contribute torenal injury [40 41] Therefore EMT-related changes arean initial step in renal damage and an important potentialtherapeutic target Our data demonstrate that Gremlin viaSmad pathway regulates EMT showing a novel mechanismof Gremlin action in renal cells
TGF-120573 is known as the major promoter of EMT duringembryogenesis cancer and fibrosis [13ndash17]In a mesothe-lioma cell line Gremlin-silencing inhibited cell proliferationassociated with downregulation of the transcription factorslug as well as mesenchymal proteins linked to cancerEMT [38] We have recently demonstrated that Gremlingene silencing blocked TGF-120573-induced EMT in tubularepithelial cells [18] Now we have observed that Gremlinincreased TGF-120573 production at 48 hours and this endoge-nous autocrine TGF-120573 acts as a downstream mediator ofGremlin-induced profibrotic and EMT related factors incultured human tubuloepithelial cells (Figure 8) All thesefindings reveal the complex relationship between Gremlinand TGF-120573 in the kidney disclosing a positive feedback loopconnection between them in promoting EMT and fibrosis
Gremlin exerts a potent inhibitory action via binding toand forming heterodimers with BMP-2 BMP-4 and BMP-7The binding of Gremlin to selective BMPs prevents ligand-receptor interaction and subsequent downstream signallingGremlin acting as a BMPs antagonist plays a critical role dur-ing the process of nephrogenesis [4] BMP-7 is the antagonistof TGF-1205731 signalling and has been found to inhibit TGF-1205731-induced renal fibrosis by reversing EMT process [42 43]In experimental lungs and pulmonary fibrosis upregulationof Gremlin was associated with downregulation of BMPsignalling [31 32] Gremlin overexpression has been foundto inhibit BMP-4 thus leading to enhance TGF-120573 signallingand ECM deposition in primary open angle glaucoma [44]However BMP-independent mechanismsmaymediate someactions of Gremlin Exogenous Gremlin may bind to andact directly on endothelial cells to modulate angiogenesisincluding endothelial cell migration [45 46] We have foundthat BMPs did no inhibit Gremlin-induced early Smad 3activation Thus a receptor-mediated mechanism of actionmay exist for GremlinTherefore future studies investigatingthe receptor involved in Gremlin responses in renal cells areneeded
5 Conclusion
Chronic progressive fibrosis of the kidney remains anunsolved challenge The investigation of the mediators andmechanisms involved in renal fibrosis could lead to better
8 BioMed Research InternationalG
REM
-GFP
GRE
M-G
FP S
mad
7Em
pty
pCD
NA
CytokeratinGFP Merge
(a)
GFP Merge
GRE
M-G
FPEm
pty
pCD
NA
120572-SMA
GRE
M-G
FP S
mad
7
(b)
Figure 5 Gremlin-induced EMT via the Smad pathway HK2 cells were transiently transfected with empty Gremlin (GREM-GFP) alone orcotransfected with Smad 7 expression vectors EMT markers were evaluated after 48 hours Gremlin transfected cells express GFP (greenstaining) Confocal microscopy analysis of cytokeratin and 120572-SMA immunofluorescence was performed using specific primary antibodiesand Alexa-633 secondary IgG (red staining) Representative image out of 3 experiments
Control Gremlin0
1
2
3
TGF-120573
expr
essio
n le
vels
(n-fo
ld)
24hours48hours
lowast
(a)
mRN
A le
vels
(n-fo
ld)
0
1
2
Control Gremlin
CTGFPAI-1
lowast lowast
lowast
Anti-TGF-120573+
GremlinTGF-120573
(b)
Figure 6 (a) Gremlin increased TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 and 48 hours in serum-freemedium TGF-1205731 protein levels were measured in the cell-conditioned medium using a specific ELISA Data are expressed as mean plusmn SEMof 6 independent experiments lowast119875 lt 005 versus control (b) The late increase in gene expression of profibrotic factors caused by Gremlin ismediated by endogenous TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 hours in serum-free medium TGF-120573was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizing antibody Total cell RNAwas isolated to assessmRNA levels by real-time PCR Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005versus control 119875 lt 005 versus Gremlin
BioMed Research International 9
Vim
entin
pan-
Cyto
kera
tinE-
cadh
erin
Control Gremlin
Gremlin+
anti-TGF-120573
(a)
0
1
2
Vimentin
GAPDH
Control Gremlin+
Gremlin
Vim
entin
GA
PDH
(n-fo
ld)
GAPDH
0
1
2
E-ca
dher
inG
APD
H (n
-fold
)
Control Gremlin+
Gremlin
Slug
GAPDH
0
1
2
Control Gremlin+
Gremlin
Slug
GA
PDH
(n-fo
ld)
E-cadherin
anti-TGF-120573 anti-TGF-120573 anti-TGF-120573
lowast
lowast
lowast
(b)
Figure 7TGF-120573 is amediator of EMT-related changes following stimulationwith Gremlin HK2 cells were stimulatedwithGremlin (50 ngmL)for 48 hours in serum-free medium TGF-120573 was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizingantibody (a) EMT changes were evaluated by confocal microscopy E-cadherin pan-Cytokeratin and Vimentin were studied by indirectimmunofluorescence using FITC-secondary IgG (green) and confocal microscopy Nuclei are shown in blue Figure shows a representativeimage out of 3 independent observations (b)Total proteins were isolated and Vimentin E-cadherin and Slug levels were analyzed by westernblot Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
diagnostic tools and novel therapeutics approaches Manystudies have shown that renal expression of Gremlin isinduced in diabetic nephropathy and in other progressiverenal diseases associated with tubulointerstitial fibrosis andSmad activation [5ndash8] We show here that Gremlin activatesthe Smad signaling pathway and induces TGF-120573 and otherrelated factors involved in EMT and fibrotic events in renalcells All these data suggest that Gremlin could be a potential
novel molecular antifibrotic target and biomarker useful forprognostication disease monitoring and therapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
10 BioMed Research International
Gremlin
Smadactivation
Target genes
EMT-related changes
Early-response(min)
Long-response
TGF-120573 independent
TGF-120573 production
TGF-120573 mediated(gt24hours)
Induction of vimentin and 120572-SMAlost of E-cadherin and cytokeratin
Figure 8 Dual effects of Gremlin on Smad activation Gremlininduces an early (minutes) and direct TGF-120573-independent Smadpathway activation After 24 hours Gremlin increased severalprofibrotic genes including TGF-120573 and after 48 hours increasedTGF-120573 production and induced EMT features These long-termGremlin-induced profibrotic events require autocrine TGF-120573
Authorsrsquo Contribution
Raquel Rodrigues-Diez and Raul R Rodrigues-Diez equallycontributed to this work
Acknowledgments
This work was supported by grants from the Institutode Salud Carlos III (PI1101854 and REDINREN ISCIII-RETIC RD1200210002 and 0001) Sociedad Espanola deNefrologıa PCI Iberoamerica (A957107) CYTED IBER-ERC FONDECYT Chile 1080083 and 1120480 Comu-nidad de Madrid (Fibroteam S2010BMD-2321 S2010BMD-2378) Programa Intensificacion Actividad Investigadora(ISCIIIAgencia Laın-EntralgoCM) to AO Fundacion parael fomento en Asturias de la investigacion cientıfica aplicaday la tecnologıa (FICYT) The authors want to thank Ma MarGonzalezGarcia-Parreno for her technical help with confocalmicroscopy
References
[1] S A Roxburgh M Murphy C A Pollock and D P BrazilldquoRecapitulation of embryological programmes in renalfibrosismdashthe importance of epithelial cell plasticity anddevelopmental genesrdquo Nephron Physiology vol 103 no 3 ppp139ndashp148 2006
[2] R McMahon MMurphy M Clarkson et al ldquoIHG-2 a mesan-gial cell gene induced by high glucose is human gremlin regu-lation by extracellular glucose concentration cyclic mechanicalstrain and transforming growth factor-1205731rdquo The Journal ofBiological Chemistry vol 275 no 14 pp 9901ndash9904 2000
[3] L Z Topol B Bardot Q Zhang et al ldquoBiosynthesis post-translation modification and functional characterization ofDrmGremlinrdquo The Journal of Biological Chemistry vol 275no 12 pp 8785ndash8793 2000
[4] O Michos L Panman K Vintersten K Beier R Zeller andA Zuniga ldquoGremlin-mediated BMP antagonism inducesthe epithelial-mesenchymal feedback signaling controllingmetanephric kidney and limb organogenesisrdquo Developmentvol 131 no 14 pp 3401ndash3410 2004
[5] V Dolan M Murphy D Sadlier et al ldquoExpression of gremlina bone morphogenetic protein antagonist in human diabeticnephropathyrdquo American Journal of Kidney Diseases vol 45 no6 pp 1034ndash1039 2005
[6] M Murphy J Crean D P Brazil D Sadlier F Martin andC Godson ldquoRegulation and consequences of differential geneexpression in diabetic kidney diseaserdquo Biochemical SocietyTransactions vol 36 no 5 pp 941ndash945 2008
[7] S Mezzano A Droguett M B Eugenia et al ldquoExpression ofgremlin a bonemorphogenetic protein antagonist in glomeru-lar crescents of pauci-immune glomerulonephritisrdquoNephrologyDialysis Transplantation vol 22 no 7 pp 1882ndash1890 2007
[8] G Carvajal A Droguett M E Burgos et al ldquoGremlin a novelmediator of epithelial mesenchymal transition and fibrosis inchronic allograft nephropathyrdquo Transplantation Proceedingsvol 40 no 3 pp 734ndash739 2008
[9] S A Roxburgh J J Kattla S P Curran et al ldquoAllelic depletionof grem1 attenuates diabetic kidney diseaserdquo Diabetes vol 58no 7 pp 1641ndash1650 2009
[10] Q Zhang Y Shi J Wada et al ldquoIn vivo delivery of gremlinsiRNA plasmid reveals therapeutic potential against diabeticnephropathy by recovering bone morphogenetic protein-7rdquoPLoS ONE vol 5 no 7 article e11709 2010
[11] M Zeisberg and E G Neilson ldquoMechanisms oftubulointerstitial fibrosisrdquo Journal of the American Societyof Nephrology vol 21 no 11 pp 1819ndash1834 2010
[12] Y Liu ldquoNew insights into epithelial-mesenchymal transition inkidney fibrosisrdquo Journal of the American Society of Nephrologyvol 21 no 2 pp 212ndash222 2010
[13] A Moustakas and C-H Heldin ldquoSignaling networks guidingepithelial-mesenchymal transitions during embryogenesisand cancer progressionrdquo Cancer Science vol 98 no 10 pp1512ndash1520 2007
[14] J-M Fan N G Yee-Yung P A Hill et al ldquoTransforminggrowth factor-120573 regulates tubular epithelial-myofibroblasttransdifferentiation in vitrordquo Kidney International vol 56 no4 pp 1455ndash1467 1999
[15] A B Roberts F Tian S D Byfield et al ldquoSmad3 is keyto TGF-120573-mediated epithelial-to-mesenchymal transitionfibrosis tumor suppression andmetastasisrdquo Cytokine amp GrowthFactor Reviews vol 17 no 1-2 pp 19ndash27 2006
BioMed Research International 11
[16] J Xu S Lamouille and R Derynck ldquoTGF-120573-induced epithelialto mesenchymal transitionrdquo Cell Research vol 19 no 2 pp156ndash172 2009
[17] M Ruiz-Ortega J Rodrıguez-Vita E Sanchez-Lopez GCarvajal and J Egido ldquoTGF-120573 signaling in vascular fibrosisrdquoCardiovascular Research vol 74 no 2 pp 196ndash206 2007
[18] R Rodrigues-Diez C Lavoz G Carvajal et al ldquoGremlin isa downstream profibrotic mediator of transforming growthfactor-beta in cultured renal cellsrdquo Nephron ExperimentalNephrology vol 122 no 1-2 pp 62ndash74 2012
[19] J Rodrıguez-Vita E Sanchez-Lopez V Esteban M Ruperez JEgido and M Ruiz-Ortega ldquoAngiotensin II activates the Smadpathway in vascular smooth muscle cells by a transforminggrowth factor-120573-independent mechanismrdquo Circulation vol 111no 19 pp 2509ndash2517 2005
[20] G Carvajal J Rodrıguez-Vita R Rodrigues-Dıez et alldquoAngiotensin II activates the Smad pathway during epithelialmesenchymal transdifferentiationrdquo Kidney International vol74 no 5 pp 585ndash595 2008
[21] G Li Y Li S Liu et al ldquoGremlin aggravates hyperglycemia-induced podocyte injury by a TGF120573smad dependent signalingpathwayrdquo Journal of Cellular Biochemistry vol 114 no 9 pp2101ndash2113 2013
[22] H Y Lan W Mu N Tomita et al ldquoInhibition of renalfibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in ratUUOmodelrdquo Journal of theAmericanSociety of Nephrology vol 14 no 6 pp 1535ndash1548 2003
[23] H Fukasawa T Yamamoto A Togawa et al ldquoDown-regulationof Smad7 expression by ubiquitin-dependent degradationcontributes to renal fibrosis in obstructive nephropathy inmicerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 23 pp 8687ndash8692 2004
[24] H Y Chen X R Huang WWang et al ldquoThe protective role ofSmad7 in diabetic kidney disease mechanism and therapeuticpotentialrdquo Diabetes vol 60 no 2 pp 590ndash601 2011
[25] X Wei Y Xia F Li et al ldquoKindlin-2 mediates activationof TGF-120573Smad signaling and renal fibrosisrdquo Journal of theAmerican Society Nephrology vol 24 no 9 pp 1387ndash1398 2013
[26] M Isono S Chen S W Hong M C Iglesias-de la Cruzand F N Ziyadeh ldquoSmad pathway is activated in the diabeticmouse kidney and smad3 mediates TGF-120573-induced fibronectinin mesangial cellsrdquo Biochemical and Biophysical ResearchCommunications vol 296 no 5 pp 1356ndash1365 2002
[27] J H Park C Lee J H Suh J Y Chae andK CMoon ldquoNuclearexpression of Smad proteins and its prognostic significance inclear cell renal cell carcinomardquo Human Pathology vol 44 no10 pp 2047ndash2054 2013
[28] G X Liu Y Q Li X R Huang et al ldquoSmad7 inhibitsAngII-mediated hypertensive nephropathy in amousemodel ofhypertensionrdquo Clinical Science vol 127 no 3 pp 195ndash208 2014
[29] G S Zode A F Clark and R J Wordinger ldquoBone morpho-genetic protein 4 inhibits TGF-1205732 stimulation of extracellularmatrix proteins in optic nerve head cells role of gremlin inECMmodulationrdquo Glia vol 57 no 7 pp 755ndash766 2009
[30] S OrsquoReilly M Ciechomska R Cant and J M van Laar ldquoIL-6trans signalling drives a STAT3 dependant pathway that leadsto hyperactive TGF-120573 signalling promoting SMAD3 activationand fibrosis via gremlinrdquo The Journal of Biological Chemistry2014
[31] M Myllarniemi P Lindholm M J Ryynanen et al ldquoGremlin-mediated decrease in bone morphogenetic protein signaling
promotes pulmonary fibrosisrdquo American Journal of Respiratoryand Critical Care Medicine vol 177 no 3 pp 321ndash329 2008
[32] E Cahill C M Costello S C Rowan et al ldquoGremlin playsa key role in the pathogenesis of pulmonary hypertensionrdquoCirculation vol 125 no 7 pp 920ndash930 2012
[33] W Boers S Aarrass C Linthorst M Pinzani R O Elferinkand P Bosma ldquoTranscriptional profiling reveals novel markersof liver fibrogenesis gremlin and insulin-like growth factor-binding proteinsrdquo The Journal of Biological Chemistry vol 281no 24 pp 16289ndash16295 2006
[34] CMCostello E Cahill FMartin S Gaine andPMcLoughlinldquoRole of gremlin in the lung development and diseaserdquoAmerican Journal of Respiratory Cell and Molecular Biologyvol 42 no 5 pp 517ndash523 2010
[35] K A Mueller E Tavlaki M Schneider et al ldquoGremlin-1identifies fibrosis and predicts adverse outcome in patientswith heart failure undergoing endomyocardial biopsyrdquo Journalof Cardiac Failure vol 19 no 10 pp 678ndash684 2013
[36] H Huang H Huang Y Li et al ldquoGremlin induces cellproliferation and extra cellular matrix accumulation in mousemesangial cells exposed to high glucose via the ERK12pathwayrdquo BMC Nephrology vol 14 article 33 2013
[37] N McCormack E L Molloy and S OrsquoDea ldquoBone morpho-genetic proteins enhance an epithelial-mesenchymal transitionin normal airway epithelial cells during restitution of a dis-rupted epitheliumrdquoRespiratory Research vol 14 article 36 2013
[38] J A Tamminen V Parviainen M Ronty et al ldquoGremlin-1associates with fibrillin microfibrils in vivo and regulatesmesothelioma cell survival through transcription factor slugrdquoOncogenesis vol 2 article e66 2013
[39] E M Zeisberg S E Potenta H Sugimoto M Zeisberg and RKalluri ldquoFibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transitionrdquo Journal of the American Society ofNephrology vol 19 no 12 pp 2282ndash2287 2008
[40] V S LeBleu G Taduri J OrsquoConnell et al ldquoOrigin and functionof myofibroblasts in kidney fibrosisrdquo Nature Medicine vol 19no 8 pp 1047ndash1053 2013
[41] W C Prozialeck and J R Edwards ldquoCell adhesion moleculesin chemically-induced renal injuryrdquo Pharmacology amp Thera-peutics vol 114 no 1 pp 74ndash93 2007
[42] M Zeisberg J Hanai H Sugimoto et al ldquoBMP-7 counteractsTGF-1205731-induced epithelial-to-mesenchymal transition andreverses chronic renal injuryrdquo Nature Medicine vol 9 no 7 pp964ndash968 2003
[43] Y-L Yang H-Z Ju S-F Liu et al ldquoBMP-2 suppresses renalinterstitial fibrosis by regulating epithelial-mesenchymaltransitionrdquo Journal of Cellular Biochemistry vol 112 no 9 pp2558ndash2565 2011
[44] R J Wordinger D L Fleenor P E Hellberg et al ldquoEffects ofTGF-1205732 BMP-4 and gremlin in the trabecular meshworkimplications for glaucomardquo Investigative Ophthalmology ampVisual Science vol 48 no 3 pp 1191ndash1200 2007
[45] B Chen M Athanasiou Q Gu and D G Blair ldquoDrmGremlintranscriptionally activates p21Cip1 via a novel mechanism andinhibits neoplastic transformationrdquoBiochemical andBiophysicalResearch Communications vol 295 no 5 pp 1135ndash1141 2002
[46] S Mitola C Ravelli E Moroni et al ldquoGremlin is a novelagonist of the major proangiogenic receptor VEGFR2rdquo Bloodvol 116 no 18 pp 3677ndash3680 2010
Figure 5 Gremlin-induced EMT via the Smad pathway HK2 cells were transiently transfected with empty Gremlin (GREM-GFP) alone orcotransfected with Smad 7 expression vectors EMT markers were evaluated after 48 hours Gremlin transfected cells express GFP (greenstaining) Confocal microscopy analysis of cytokeratin and 120572-SMA immunofluorescence was performed using specific primary antibodiesand Alexa-633 secondary IgG (red staining) Representative image out of 3 experiments
Control Gremlin0
1
2
3
TGF-120573
expr
essio
n le
vels
(n-fo
ld)
24hours48hours
lowast
(a)
mRN
A le
vels
(n-fo
ld)
0
1
2
Control Gremlin
CTGFPAI-1
lowast lowast
lowast
Anti-TGF-120573+
GremlinTGF-120573
(b)
Figure 6 (a) Gremlin increased TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 and 48 hours in serum-freemedium TGF-1205731 protein levels were measured in the cell-conditioned medium using a specific ELISA Data are expressed as mean plusmn SEMof 6 independent experiments lowast119875 lt 005 versus control (b) The late increase in gene expression of profibrotic factors caused by Gremlin ismediated by endogenous TGF-120573 production HK2 cells were stimulated with Gremlin (50 ngmL) for 24 hours in serum-free medium TGF-120573was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizing antibody Total cell RNAwas isolated to assessmRNA levels by real-time PCR Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005versus control 119875 lt 005 versus Gremlin
BioMed Research International 9
Vim
entin
pan-
Cyto
kera
tinE-
cadh
erin
Control Gremlin
Gremlin+
anti-TGF-120573
(a)
0
1
2
Vimentin
GAPDH
Control Gremlin+
Gremlin
Vim
entin
GA
PDH
(n-fo
ld)
GAPDH
0
1
2
E-ca
dher
inG
APD
H (n
-fold
)
Control Gremlin+
Gremlin
Slug
GAPDH
0
1
2
Control Gremlin+
Gremlin
Slug
GA
PDH
(n-fo
ld)
E-cadherin
anti-TGF-120573 anti-TGF-120573 anti-TGF-120573
lowast
lowast
lowast
(b)
Figure 7TGF-120573 is amediator of EMT-related changes following stimulationwith Gremlin HK2 cells were stimulatedwithGremlin (50 ngmL)for 48 hours in serum-free medium TGF-120573 was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizingantibody (a) EMT changes were evaluated by confocal microscopy E-cadherin pan-Cytokeratin and Vimentin were studied by indirectimmunofluorescence using FITC-secondary IgG (green) and confocal microscopy Nuclei are shown in blue Figure shows a representativeimage out of 3 independent observations (b)Total proteins were isolated and Vimentin E-cadherin and Slug levels were analyzed by westernblot Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
diagnostic tools and novel therapeutics approaches Manystudies have shown that renal expression of Gremlin isinduced in diabetic nephropathy and in other progressiverenal diseases associated with tubulointerstitial fibrosis andSmad activation [5ndash8] We show here that Gremlin activatesthe Smad signaling pathway and induces TGF-120573 and otherrelated factors involved in EMT and fibrotic events in renalcells All these data suggest that Gremlin could be a potential
novel molecular antifibrotic target and biomarker useful forprognostication disease monitoring and therapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
10 BioMed Research International
Gremlin
Smadactivation
Target genes
EMT-related changes
Early-response(min)
Long-response
TGF-120573 independent
TGF-120573 production
TGF-120573 mediated(gt24hours)
Induction of vimentin and 120572-SMAlost of E-cadherin and cytokeratin
Figure 8 Dual effects of Gremlin on Smad activation Gremlininduces an early (minutes) and direct TGF-120573-independent Smadpathway activation After 24 hours Gremlin increased severalprofibrotic genes including TGF-120573 and after 48 hours increasedTGF-120573 production and induced EMT features These long-termGremlin-induced profibrotic events require autocrine TGF-120573
Authorsrsquo Contribution
Raquel Rodrigues-Diez and Raul R Rodrigues-Diez equallycontributed to this work
Acknowledgments
This work was supported by grants from the Institutode Salud Carlos III (PI1101854 and REDINREN ISCIII-RETIC RD1200210002 and 0001) Sociedad Espanola deNefrologıa PCI Iberoamerica (A957107) CYTED IBER-ERC FONDECYT Chile 1080083 and 1120480 Comu-nidad de Madrid (Fibroteam S2010BMD-2321 S2010BMD-2378) Programa Intensificacion Actividad Investigadora(ISCIIIAgencia Laın-EntralgoCM) to AO Fundacion parael fomento en Asturias de la investigacion cientıfica aplicaday la tecnologıa (FICYT) The authors want to thank Ma MarGonzalezGarcia-Parreno for her technical help with confocalmicroscopy
References
[1] S A Roxburgh M Murphy C A Pollock and D P BrazilldquoRecapitulation of embryological programmes in renalfibrosismdashthe importance of epithelial cell plasticity anddevelopmental genesrdquo Nephron Physiology vol 103 no 3 ppp139ndashp148 2006
[2] R McMahon MMurphy M Clarkson et al ldquoIHG-2 a mesan-gial cell gene induced by high glucose is human gremlin regu-lation by extracellular glucose concentration cyclic mechanicalstrain and transforming growth factor-1205731rdquo The Journal ofBiological Chemistry vol 275 no 14 pp 9901ndash9904 2000
[3] L Z Topol B Bardot Q Zhang et al ldquoBiosynthesis post-translation modification and functional characterization ofDrmGremlinrdquo The Journal of Biological Chemistry vol 275no 12 pp 8785ndash8793 2000
[4] O Michos L Panman K Vintersten K Beier R Zeller andA Zuniga ldquoGremlin-mediated BMP antagonism inducesthe epithelial-mesenchymal feedback signaling controllingmetanephric kidney and limb organogenesisrdquo Developmentvol 131 no 14 pp 3401ndash3410 2004
[5] V Dolan M Murphy D Sadlier et al ldquoExpression of gremlina bone morphogenetic protein antagonist in human diabeticnephropathyrdquo American Journal of Kidney Diseases vol 45 no6 pp 1034ndash1039 2005
[6] M Murphy J Crean D P Brazil D Sadlier F Martin andC Godson ldquoRegulation and consequences of differential geneexpression in diabetic kidney diseaserdquo Biochemical SocietyTransactions vol 36 no 5 pp 941ndash945 2008
[7] S Mezzano A Droguett M B Eugenia et al ldquoExpression ofgremlin a bonemorphogenetic protein antagonist in glomeru-lar crescents of pauci-immune glomerulonephritisrdquoNephrologyDialysis Transplantation vol 22 no 7 pp 1882ndash1890 2007
[8] G Carvajal A Droguett M E Burgos et al ldquoGremlin a novelmediator of epithelial mesenchymal transition and fibrosis inchronic allograft nephropathyrdquo Transplantation Proceedingsvol 40 no 3 pp 734ndash739 2008
[9] S A Roxburgh J J Kattla S P Curran et al ldquoAllelic depletionof grem1 attenuates diabetic kidney diseaserdquo Diabetes vol 58no 7 pp 1641ndash1650 2009
[10] Q Zhang Y Shi J Wada et al ldquoIn vivo delivery of gremlinsiRNA plasmid reveals therapeutic potential against diabeticnephropathy by recovering bone morphogenetic protein-7rdquoPLoS ONE vol 5 no 7 article e11709 2010
[11] M Zeisberg and E G Neilson ldquoMechanisms oftubulointerstitial fibrosisrdquo Journal of the American Societyof Nephrology vol 21 no 11 pp 1819ndash1834 2010
[12] Y Liu ldquoNew insights into epithelial-mesenchymal transition inkidney fibrosisrdquo Journal of the American Society of Nephrologyvol 21 no 2 pp 212ndash222 2010
[13] A Moustakas and C-H Heldin ldquoSignaling networks guidingepithelial-mesenchymal transitions during embryogenesisand cancer progressionrdquo Cancer Science vol 98 no 10 pp1512ndash1520 2007
[14] J-M Fan N G Yee-Yung P A Hill et al ldquoTransforminggrowth factor-120573 regulates tubular epithelial-myofibroblasttransdifferentiation in vitrordquo Kidney International vol 56 no4 pp 1455ndash1467 1999
[15] A B Roberts F Tian S D Byfield et al ldquoSmad3 is keyto TGF-120573-mediated epithelial-to-mesenchymal transitionfibrosis tumor suppression andmetastasisrdquo Cytokine amp GrowthFactor Reviews vol 17 no 1-2 pp 19ndash27 2006
BioMed Research International 11
[16] J Xu S Lamouille and R Derynck ldquoTGF-120573-induced epithelialto mesenchymal transitionrdquo Cell Research vol 19 no 2 pp156ndash172 2009
[17] M Ruiz-Ortega J Rodrıguez-Vita E Sanchez-Lopez GCarvajal and J Egido ldquoTGF-120573 signaling in vascular fibrosisrdquoCardiovascular Research vol 74 no 2 pp 196ndash206 2007
[18] R Rodrigues-Diez C Lavoz G Carvajal et al ldquoGremlin isa downstream profibrotic mediator of transforming growthfactor-beta in cultured renal cellsrdquo Nephron ExperimentalNephrology vol 122 no 1-2 pp 62ndash74 2012
[19] J Rodrıguez-Vita E Sanchez-Lopez V Esteban M Ruperez JEgido and M Ruiz-Ortega ldquoAngiotensin II activates the Smadpathway in vascular smooth muscle cells by a transforminggrowth factor-120573-independent mechanismrdquo Circulation vol 111no 19 pp 2509ndash2517 2005
[20] G Carvajal J Rodrıguez-Vita R Rodrigues-Dıez et alldquoAngiotensin II activates the Smad pathway during epithelialmesenchymal transdifferentiationrdquo Kidney International vol74 no 5 pp 585ndash595 2008
[21] G Li Y Li S Liu et al ldquoGremlin aggravates hyperglycemia-induced podocyte injury by a TGF120573smad dependent signalingpathwayrdquo Journal of Cellular Biochemistry vol 114 no 9 pp2101ndash2113 2013
[22] H Y Lan W Mu N Tomita et al ldquoInhibition of renalfibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in ratUUOmodelrdquo Journal of theAmericanSociety of Nephrology vol 14 no 6 pp 1535ndash1548 2003
[23] H Fukasawa T Yamamoto A Togawa et al ldquoDown-regulationof Smad7 expression by ubiquitin-dependent degradationcontributes to renal fibrosis in obstructive nephropathy inmicerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 23 pp 8687ndash8692 2004
[24] H Y Chen X R Huang WWang et al ldquoThe protective role ofSmad7 in diabetic kidney disease mechanism and therapeuticpotentialrdquo Diabetes vol 60 no 2 pp 590ndash601 2011
[25] X Wei Y Xia F Li et al ldquoKindlin-2 mediates activationof TGF-120573Smad signaling and renal fibrosisrdquo Journal of theAmerican Society Nephrology vol 24 no 9 pp 1387ndash1398 2013
[26] M Isono S Chen S W Hong M C Iglesias-de la Cruzand F N Ziyadeh ldquoSmad pathway is activated in the diabeticmouse kidney and smad3 mediates TGF-120573-induced fibronectinin mesangial cellsrdquo Biochemical and Biophysical ResearchCommunications vol 296 no 5 pp 1356ndash1365 2002
[27] J H Park C Lee J H Suh J Y Chae andK CMoon ldquoNuclearexpression of Smad proteins and its prognostic significance inclear cell renal cell carcinomardquo Human Pathology vol 44 no10 pp 2047ndash2054 2013
[28] G X Liu Y Q Li X R Huang et al ldquoSmad7 inhibitsAngII-mediated hypertensive nephropathy in amousemodel ofhypertensionrdquo Clinical Science vol 127 no 3 pp 195ndash208 2014
[29] G S Zode A F Clark and R J Wordinger ldquoBone morpho-genetic protein 4 inhibits TGF-1205732 stimulation of extracellularmatrix proteins in optic nerve head cells role of gremlin inECMmodulationrdquo Glia vol 57 no 7 pp 755ndash766 2009
[30] S OrsquoReilly M Ciechomska R Cant and J M van Laar ldquoIL-6trans signalling drives a STAT3 dependant pathway that leadsto hyperactive TGF-120573 signalling promoting SMAD3 activationand fibrosis via gremlinrdquo The Journal of Biological Chemistry2014
[31] M Myllarniemi P Lindholm M J Ryynanen et al ldquoGremlin-mediated decrease in bone morphogenetic protein signaling
promotes pulmonary fibrosisrdquo American Journal of Respiratoryand Critical Care Medicine vol 177 no 3 pp 321ndash329 2008
[32] E Cahill C M Costello S C Rowan et al ldquoGremlin playsa key role in the pathogenesis of pulmonary hypertensionrdquoCirculation vol 125 no 7 pp 920ndash930 2012
[33] W Boers S Aarrass C Linthorst M Pinzani R O Elferinkand P Bosma ldquoTranscriptional profiling reveals novel markersof liver fibrogenesis gremlin and insulin-like growth factor-binding proteinsrdquo The Journal of Biological Chemistry vol 281no 24 pp 16289ndash16295 2006
[34] CMCostello E Cahill FMartin S Gaine andPMcLoughlinldquoRole of gremlin in the lung development and diseaserdquoAmerican Journal of Respiratory Cell and Molecular Biologyvol 42 no 5 pp 517ndash523 2010
[35] K A Mueller E Tavlaki M Schneider et al ldquoGremlin-1identifies fibrosis and predicts adverse outcome in patientswith heart failure undergoing endomyocardial biopsyrdquo Journalof Cardiac Failure vol 19 no 10 pp 678ndash684 2013
[36] H Huang H Huang Y Li et al ldquoGremlin induces cellproliferation and extra cellular matrix accumulation in mousemesangial cells exposed to high glucose via the ERK12pathwayrdquo BMC Nephrology vol 14 article 33 2013
[37] N McCormack E L Molloy and S OrsquoDea ldquoBone morpho-genetic proteins enhance an epithelial-mesenchymal transitionin normal airway epithelial cells during restitution of a dis-rupted epitheliumrdquoRespiratory Research vol 14 article 36 2013
[38] J A Tamminen V Parviainen M Ronty et al ldquoGremlin-1associates with fibrillin microfibrils in vivo and regulatesmesothelioma cell survival through transcription factor slugrdquoOncogenesis vol 2 article e66 2013
[39] E M Zeisberg S E Potenta H Sugimoto M Zeisberg and RKalluri ldquoFibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transitionrdquo Journal of the American Society ofNephrology vol 19 no 12 pp 2282ndash2287 2008
[40] V S LeBleu G Taduri J OrsquoConnell et al ldquoOrigin and functionof myofibroblasts in kidney fibrosisrdquo Nature Medicine vol 19no 8 pp 1047ndash1053 2013
[41] W C Prozialeck and J R Edwards ldquoCell adhesion moleculesin chemically-induced renal injuryrdquo Pharmacology amp Thera-peutics vol 114 no 1 pp 74ndash93 2007
[42] M Zeisberg J Hanai H Sugimoto et al ldquoBMP-7 counteractsTGF-1205731-induced epithelial-to-mesenchymal transition andreverses chronic renal injuryrdquo Nature Medicine vol 9 no 7 pp964ndash968 2003
[43] Y-L Yang H-Z Ju S-F Liu et al ldquoBMP-2 suppresses renalinterstitial fibrosis by regulating epithelial-mesenchymaltransitionrdquo Journal of Cellular Biochemistry vol 112 no 9 pp2558ndash2565 2011
[44] R J Wordinger D L Fleenor P E Hellberg et al ldquoEffects ofTGF-1205732 BMP-4 and gremlin in the trabecular meshworkimplications for glaucomardquo Investigative Ophthalmology ampVisual Science vol 48 no 3 pp 1191ndash1200 2007
[45] B Chen M Athanasiou Q Gu and D G Blair ldquoDrmGremlintranscriptionally activates p21Cip1 via a novel mechanism andinhibits neoplastic transformationrdquoBiochemical andBiophysicalResearch Communications vol 295 no 5 pp 1135ndash1141 2002
[46] S Mitola C Ravelli E Moroni et al ldquoGremlin is a novelagonist of the major proangiogenic receptor VEGFR2rdquo Bloodvol 116 no 18 pp 3677ndash3680 2010
Figure 7TGF-120573 is amediator of EMT-related changes following stimulationwith Gremlin HK2 cells were stimulatedwithGremlin (50 ngmL)for 48 hours in serum-free medium TGF-120573 was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-120573 neutralizingantibody (a) EMT changes were evaluated by confocal microscopy E-cadherin pan-Cytokeratin and Vimentin were studied by indirectimmunofluorescence using FITC-secondary IgG (green) and confocal microscopy Nuclei are shown in blue Figure shows a representativeimage out of 3 independent observations (b)Total proteins were isolated and Vimentin E-cadherin and Slug levels were analyzed by westernblot Data are expressed as n-fold over control (considered as 1) as the mean plusmn SEM of 3 experiments lowast119875 lt 005 versus control 119875 lt 005versus Gremlin
diagnostic tools and novel therapeutics approaches Manystudies have shown that renal expression of Gremlin isinduced in diabetic nephropathy and in other progressiverenal diseases associated with tubulointerstitial fibrosis andSmad activation [5ndash8] We show here that Gremlin activatesthe Smad signaling pathway and induces TGF-120573 and otherrelated factors involved in EMT and fibrotic events in renalcells All these data suggest that Gremlin could be a potential
novel molecular antifibrotic target and biomarker useful forprognostication disease monitoring and therapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
10 BioMed Research International
Gremlin
Smadactivation
Target genes
EMT-related changes
Early-response(min)
Long-response
TGF-120573 independent
TGF-120573 production
TGF-120573 mediated(gt24hours)
Induction of vimentin and 120572-SMAlost of E-cadherin and cytokeratin
Figure 8 Dual effects of Gremlin on Smad activation Gremlininduces an early (minutes) and direct TGF-120573-independent Smadpathway activation After 24 hours Gremlin increased severalprofibrotic genes including TGF-120573 and after 48 hours increasedTGF-120573 production and induced EMT features These long-termGremlin-induced profibrotic events require autocrine TGF-120573
Authorsrsquo Contribution
Raquel Rodrigues-Diez and Raul R Rodrigues-Diez equallycontributed to this work
Acknowledgments
This work was supported by grants from the Institutode Salud Carlos III (PI1101854 and REDINREN ISCIII-RETIC RD1200210002 and 0001) Sociedad Espanola deNefrologıa PCI Iberoamerica (A957107) CYTED IBER-ERC FONDECYT Chile 1080083 and 1120480 Comu-nidad de Madrid (Fibroteam S2010BMD-2321 S2010BMD-2378) Programa Intensificacion Actividad Investigadora(ISCIIIAgencia Laın-EntralgoCM) to AO Fundacion parael fomento en Asturias de la investigacion cientıfica aplicaday la tecnologıa (FICYT) The authors want to thank Ma MarGonzalezGarcia-Parreno for her technical help with confocalmicroscopy
References
[1] S A Roxburgh M Murphy C A Pollock and D P BrazilldquoRecapitulation of embryological programmes in renalfibrosismdashthe importance of epithelial cell plasticity anddevelopmental genesrdquo Nephron Physiology vol 103 no 3 ppp139ndashp148 2006
[2] R McMahon MMurphy M Clarkson et al ldquoIHG-2 a mesan-gial cell gene induced by high glucose is human gremlin regu-lation by extracellular glucose concentration cyclic mechanicalstrain and transforming growth factor-1205731rdquo The Journal ofBiological Chemistry vol 275 no 14 pp 9901ndash9904 2000
[3] L Z Topol B Bardot Q Zhang et al ldquoBiosynthesis post-translation modification and functional characterization ofDrmGremlinrdquo The Journal of Biological Chemistry vol 275no 12 pp 8785ndash8793 2000
[4] O Michos L Panman K Vintersten K Beier R Zeller andA Zuniga ldquoGremlin-mediated BMP antagonism inducesthe epithelial-mesenchymal feedback signaling controllingmetanephric kidney and limb organogenesisrdquo Developmentvol 131 no 14 pp 3401ndash3410 2004
[5] V Dolan M Murphy D Sadlier et al ldquoExpression of gremlina bone morphogenetic protein antagonist in human diabeticnephropathyrdquo American Journal of Kidney Diseases vol 45 no6 pp 1034ndash1039 2005
[6] M Murphy J Crean D P Brazil D Sadlier F Martin andC Godson ldquoRegulation and consequences of differential geneexpression in diabetic kidney diseaserdquo Biochemical SocietyTransactions vol 36 no 5 pp 941ndash945 2008
[7] S Mezzano A Droguett M B Eugenia et al ldquoExpression ofgremlin a bonemorphogenetic protein antagonist in glomeru-lar crescents of pauci-immune glomerulonephritisrdquoNephrologyDialysis Transplantation vol 22 no 7 pp 1882ndash1890 2007
[8] G Carvajal A Droguett M E Burgos et al ldquoGremlin a novelmediator of epithelial mesenchymal transition and fibrosis inchronic allograft nephropathyrdquo Transplantation Proceedingsvol 40 no 3 pp 734ndash739 2008
[9] S A Roxburgh J J Kattla S P Curran et al ldquoAllelic depletionof grem1 attenuates diabetic kidney diseaserdquo Diabetes vol 58no 7 pp 1641ndash1650 2009
[10] Q Zhang Y Shi J Wada et al ldquoIn vivo delivery of gremlinsiRNA plasmid reveals therapeutic potential against diabeticnephropathy by recovering bone morphogenetic protein-7rdquoPLoS ONE vol 5 no 7 article e11709 2010
[11] M Zeisberg and E G Neilson ldquoMechanisms oftubulointerstitial fibrosisrdquo Journal of the American Societyof Nephrology vol 21 no 11 pp 1819ndash1834 2010
[12] Y Liu ldquoNew insights into epithelial-mesenchymal transition inkidney fibrosisrdquo Journal of the American Society of Nephrologyvol 21 no 2 pp 212ndash222 2010
[13] A Moustakas and C-H Heldin ldquoSignaling networks guidingepithelial-mesenchymal transitions during embryogenesisand cancer progressionrdquo Cancer Science vol 98 no 10 pp1512ndash1520 2007
[14] J-M Fan N G Yee-Yung P A Hill et al ldquoTransforminggrowth factor-120573 regulates tubular epithelial-myofibroblasttransdifferentiation in vitrordquo Kidney International vol 56 no4 pp 1455ndash1467 1999
[15] A B Roberts F Tian S D Byfield et al ldquoSmad3 is keyto TGF-120573-mediated epithelial-to-mesenchymal transitionfibrosis tumor suppression andmetastasisrdquo Cytokine amp GrowthFactor Reviews vol 17 no 1-2 pp 19ndash27 2006
BioMed Research International 11
[16] J Xu S Lamouille and R Derynck ldquoTGF-120573-induced epithelialto mesenchymal transitionrdquo Cell Research vol 19 no 2 pp156ndash172 2009
[17] M Ruiz-Ortega J Rodrıguez-Vita E Sanchez-Lopez GCarvajal and J Egido ldquoTGF-120573 signaling in vascular fibrosisrdquoCardiovascular Research vol 74 no 2 pp 196ndash206 2007
[18] R Rodrigues-Diez C Lavoz G Carvajal et al ldquoGremlin isa downstream profibrotic mediator of transforming growthfactor-beta in cultured renal cellsrdquo Nephron ExperimentalNephrology vol 122 no 1-2 pp 62ndash74 2012
[19] J Rodrıguez-Vita E Sanchez-Lopez V Esteban M Ruperez JEgido and M Ruiz-Ortega ldquoAngiotensin II activates the Smadpathway in vascular smooth muscle cells by a transforminggrowth factor-120573-independent mechanismrdquo Circulation vol 111no 19 pp 2509ndash2517 2005
[20] G Carvajal J Rodrıguez-Vita R Rodrigues-Dıez et alldquoAngiotensin II activates the Smad pathway during epithelialmesenchymal transdifferentiationrdquo Kidney International vol74 no 5 pp 585ndash595 2008
[21] G Li Y Li S Liu et al ldquoGremlin aggravates hyperglycemia-induced podocyte injury by a TGF120573smad dependent signalingpathwayrdquo Journal of Cellular Biochemistry vol 114 no 9 pp2101ndash2113 2013
[22] H Y Lan W Mu N Tomita et al ldquoInhibition of renalfibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in ratUUOmodelrdquo Journal of theAmericanSociety of Nephrology vol 14 no 6 pp 1535ndash1548 2003
[23] H Fukasawa T Yamamoto A Togawa et al ldquoDown-regulationof Smad7 expression by ubiquitin-dependent degradationcontributes to renal fibrosis in obstructive nephropathy inmicerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 23 pp 8687ndash8692 2004
[24] H Y Chen X R Huang WWang et al ldquoThe protective role ofSmad7 in diabetic kidney disease mechanism and therapeuticpotentialrdquo Diabetes vol 60 no 2 pp 590ndash601 2011
[25] X Wei Y Xia F Li et al ldquoKindlin-2 mediates activationof TGF-120573Smad signaling and renal fibrosisrdquo Journal of theAmerican Society Nephrology vol 24 no 9 pp 1387ndash1398 2013
[26] M Isono S Chen S W Hong M C Iglesias-de la Cruzand F N Ziyadeh ldquoSmad pathway is activated in the diabeticmouse kidney and smad3 mediates TGF-120573-induced fibronectinin mesangial cellsrdquo Biochemical and Biophysical ResearchCommunications vol 296 no 5 pp 1356ndash1365 2002
[27] J H Park C Lee J H Suh J Y Chae andK CMoon ldquoNuclearexpression of Smad proteins and its prognostic significance inclear cell renal cell carcinomardquo Human Pathology vol 44 no10 pp 2047ndash2054 2013
[28] G X Liu Y Q Li X R Huang et al ldquoSmad7 inhibitsAngII-mediated hypertensive nephropathy in amousemodel ofhypertensionrdquo Clinical Science vol 127 no 3 pp 195ndash208 2014
[29] G S Zode A F Clark and R J Wordinger ldquoBone morpho-genetic protein 4 inhibits TGF-1205732 stimulation of extracellularmatrix proteins in optic nerve head cells role of gremlin inECMmodulationrdquo Glia vol 57 no 7 pp 755ndash766 2009
[30] S OrsquoReilly M Ciechomska R Cant and J M van Laar ldquoIL-6trans signalling drives a STAT3 dependant pathway that leadsto hyperactive TGF-120573 signalling promoting SMAD3 activationand fibrosis via gremlinrdquo The Journal of Biological Chemistry2014
[31] M Myllarniemi P Lindholm M J Ryynanen et al ldquoGremlin-mediated decrease in bone morphogenetic protein signaling
promotes pulmonary fibrosisrdquo American Journal of Respiratoryand Critical Care Medicine vol 177 no 3 pp 321ndash329 2008
[32] E Cahill C M Costello S C Rowan et al ldquoGremlin playsa key role in the pathogenesis of pulmonary hypertensionrdquoCirculation vol 125 no 7 pp 920ndash930 2012
[33] W Boers S Aarrass C Linthorst M Pinzani R O Elferinkand P Bosma ldquoTranscriptional profiling reveals novel markersof liver fibrogenesis gremlin and insulin-like growth factor-binding proteinsrdquo The Journal of Biological Chemistry vol 281no 24 pp 16289ndash16295 2006
[34] CMCostello E Cahill FMartin S Gaine andPMcLoughlinldquoRole of gremlin in the lung development and diseaserdquoAmerican Journal of Respiratory Cell and Molecular Biologyvol 42 no 5 pp 517ndash523 2010
[35] K A Mueller E Tavlaki M Schneider et al ldquoGremlin-1identifies fibrosis and predicts adverse outcome in patientswith heart failure undergoing endomyocardial biopsyrdquo Journalof Cardiac Failure vol 19 no 10 pp 678ndash684 2013
[36] H Huang H Huang Y Li et al ldquoGremlin induces cellproliferation and extra cellular matrix accumulation in mousemesangial cells exposed to high glucose via the ERK12pathwayrdquo BMC Nephrology vol 14 article 33 2013
[37] N McCormack E L Molloy and S OrsquoDea ldquoBone morpho-genetic proteins enhance an epithelial-mesenchymal transitionin normal airway epithelial cells during restitution of a dis-rupted epitheliumrdquoRespiratory Research vol 14 article 36 2013
[38] J A Tamminen V Parviainen M Ronty et al ldquoGremlin-1associates with fibrillin microfibrils in vivo and regulatesmesothelioma cell survival through transcription factor slugrdquoOncogenesis vol 2 article e66 2013
[39] E M Zeisberg S E Potenta H Sugimoto M Zeisberg and RKalluri ldquoFibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transitionrdquo Journal of the American Society ofNephrology vol 19 no 12 pp 2282ndash2287 2008
[40] V S LeBleu G Taduri J OrsquoConnell et al ldquoOrigin and functionof myofibroblasts in kidney fibrosisrdquo Nature Medicine vol 19no 8 pp 1047ndash1053 2013
[41] W C Prozialeck and J R Edwards ldquoCell adhesion moleculesin chemically-induced renal injuryrdquo Pharmacology amp Thera-peutics vol 114 no 1 pp 74ndash93 2007
[42] M Zeisberg J Hanai H Sugimoto et al ldquoBMP-7 counteractsTGF-1205731-induced epithelial-to-mesenchymal transition andreverses chronic renal injuryrdquo Nature Medicine vol 9 no 7 pp964ndash968 2003
[43] Y-L Yang H-Z Ju S-F Liu et al ldquoBMP-2 suppresses renalinterstitial fibrosis by regulating epithelial-mesenchymaltransitionrdquo Journal of Cellular Biochemistry vol 112 no 9 pp2558ndash2565 2011
[44] R J Wordinger D L Fleenor P E Hellberg et al ldquoEffects ofTGF-1205732 BMP-4 and gremlin in the trabecular meshworkimplications for glaucomardquo Investigative Ophthalmology ampVisual Science vol 48 no 3 pp 1191ndash1200 2007
[45] B Chen M Athanasiou Q Gu and D G Blair ldquoDrmGremlintranscriptionally activates p21Cip1 via a novel mechanism andinhibits neoplastic transformationrdquoBiochemical andBiophysicalResearch Communications vol 295 no 5 pp 1135ndash1141 2002
[46] S Mitola C Ravelli E Moroni et al ldquoGremlin is a novelagonist of the major proangiogenic receptor VEGFR2rdquo Bloodvol 116 no 18 pp 3677ndash3680 2010
Induction of vimentin and 120572-SMAlost of E-cadherin and cytokeratin
Figure 8 Dual effects of Gremlin on Smad activation Gremlininduces an early (minutes) and direct TGF-120573-independent Smadpathway activation After 24 hours Gremlin increased severalprofibrotic genes including TGF-120573 and after 48 hours increasedTGF-120573 production and induced EMT features These long-termGremlin-induced profibrotic events require autocrine TGF-120573
Authorsrsquo Contribution
Raquel Rodrigues-Diez and Raul R Rodrigues-Diez equallycontributed to this work
Acknowledgments
This work was supported by grants from the Institutode Salud Carlos III (PI1101854 and REDINREN ISCIII-RETIC RD1200210002 and 0001) Sociedad Espanola deNefrologıa PCI Iberoamerica (A957107) CYTED IBER-ERC FONDECYT Chile 1080083 and 1120480 Comu-nidad de Madrid (Fibroteam S2010BMD-2321 S2010BMD-2378) Programa Intensificacion Actividad Investigadora(ISCIIIAgencia Laın-EntralgoCM) to AO Fundacion parael fomento en Asturias de la investigacion cientıfica aplicaday la tecnologıa (FICYT) The authors want to thank Ma MarGonzalezGarcia-Parreno for her technical help with confocalmicroscopy
References
[1] S A Roxburgh M Murphy C A Pollock and D P BrazilldquoRecapitulation of embryological programmes in renalfibrosismdashthe importance of epithelial cell plasticity anddevelopmental genesrdquo Nephron Physiology vol 103 no 3 ppp139ndashp148 2006
[2] R McMahon MMurphy M Clarkson et al ldquoIHG-2 a mesan-gial cell gene induced by high glucose is human gremlin regu-lation by extracellular glucose concentration cyclic mechanicalstrain and transforming growth factor-1205731rdquo The Journal ofBiological Chemistry vol 275 no 14 pp 9901ndash9904 2000
[3] L Z Topol B Bardot Q Zhang et al ldquoBiosynthesis post-translation modification and functional characterization ofDrmGremlinrdquo The Journal of Biological Chemistry vol 275no 12 pp 8785ndash8793 2000
[4] O Michos L Panman K Vintersten K Beier R Zeller andA Zuniga ldquoGremlin-mediated BMP antagonism inducesthe epithelial-mesenchymal feedback signaling controllingmetanephric kidney and limb organogenesisrdquo Developmentvol 131 no 14 pp 3401ndash3410 2004
[5] V Dolan M Murphy D Sadlier et al ldquoExpression of gremlina bone morphogenetic protein antagonist in human diabeticnephropathyrdquo American Journal of Kidney Diseases vol 45 no6 pp 1034ndash1039 2005
[6] M Murphy J Crean D P Brazil D Sadlier F Martin andC Godson ldquoRegulation and consequences of differential geneexpression in diabetic kidney diseaserdquo Biochemical SocietyTransactions vol 36 no 5 pp 941ndash945 2008
[7] S Mezzano A Droguett M B Eugenia et al ldquoExpression ofgremlin a bonemorphogenetic protein antagonist in glomeru-lar crescents of pauci-immune glomerulonephritisrdquoNephrologyDialysis Transplantation vol 22 no 7 pp 1882ndash1890 2007
[8] G Carvajal A Droguett M E Burgos et al ldquoGremlin a novelmediator of epithelial mesenchymal transition and fibrosis inchronic allograft nephropathyrdquo Transplantation Proceedingsvol 40 no 3 pp 734ndash739 2008
[9] S A Roxburgh J J Kattla S P Curran et al ldquoAllelic depletionof grem1 attenuates diabetic kidney diseaserdquo Diabetes vol 58no 7 pp 1641ndash1650 2009
[10] Q Zhang Y Shi J Wada et al ldquoIn vivo delivery of gremlinsiRNA plasmid reveals therapeutic potential against diabeticnephropathy by recovering bone morphogenetic protein-7rdquoPLoS ONE vol 5 no 7 article e11709 2010
[11] M Zeisberg and E G Neilson ldquoMechanisms oftubulointerstitial fibrosisrdquo Journal of the American Societyof Nephrology vol 21 no 11 pp 1819ndash1834 2010
[12] Y Liu ldquoNew insights into epithelial-mesenchymal transition inkidney fibrosisrdquo Journal of the American Society of Nephrologyvol 21 no 2 pp 212ndash222 2010
[13] A Moustakas and C-H Heldin ldquoSignaling networks guidingepithelial-mesenchymal transitions during embryogenesisand cancer progressionrdquo Cancer Science vol 98 no 10 pp1512ndash1520 2007
[14] J-M Fan N G Yee-Yung P A Hill et al ldquoTransforminggrowth factor-120573 regulates tubular epithelial-myofibroblasttransdifferentiation in vitrordquo Kidney International vol 56 no4 pp 1455ndash1467 1999
[15] A B Roberts F Tian S D Byfield et al ldquoSmad3 is keyto TGF-120573-mediated epithelial-to-mesenchymal transitionfibrosis tumor suppression andmetastasisrdquo Cytokine amp GrowthFactor Reviews vol 17 no 1-2 pp 19ndash27 2006
BioMed Research International 11
[16] J Xu S Lamouille and R Derynck ldquoTGF-120573-induced epithelialto mesenchymal transitionrdquo Cell Research vol 19 no 2 pp156ndash172 2009
[17] M Ruiz-Ortega J Rodrıguez-Vita E Sanchez-Lopez GCarvajal and J Egido ldquoTGF-120573 signaling in vascular fibrosisrdquoCardiovascular Research vol 74 no 2 pp 196ndash206 2007
[18] R Rodrigues-Diez C Lavoz G Carvajal et al ldquoGremlin isa downstream profibrotic mediator of transforming growthfactor-beta in cultured renal cellsrdquo Nephron ExperimentalNephrology vol 122 no 1-2 pp 62ndash74 2012
[19] J Rodrıguez-Vita E Sanchez-Lopez V Esteban M Ruperez JEgido and M Ruiz-Ortega ldquoAngiotensin II activates the Smadpathway in vascular smooth muscle cells by a transforminggrowth factor-120573-independent mechanismrdquo Circulation vol 111no 19 pp 2509ndash2517 2005
[20] G Carvajal J Rodrıguez-Vita R Rodrigues-Dıez et alldquoAngiotensin II activates the Smad pathway during epithelialmesenchymal transdifferentiationrdquo Kidney International vol74 no 5 pp 585ndash595 2008
[21] G Li Y Li S Liu et al ldquoGremlin aggravates hyperglycemia-induced podocyte injury by a TGF120573smad dependent signalingpathwayrdquo Journal of Cellular Biochemistry vol 114 no 9 pp2101ndash2113 2013
[22] H Y Lan W Mu N Tomita et al ldquoInhibition of renalfibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in ratUUOmodelrdquo Journal of theAmericanSociety of Nephrology vol 14 no 6 pp 1535ndash1548 2003
[23] H Fukasawa T Yamamoto A Togawa et al ldquoDown-regulationof Smad7 expression by ubiquitin-dependent degradationcontributes to renal fibrosis in obstructive nephropathy inmicerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 23 pp 8687ndash8692 2004
[24] H Y Chen X R Huang WWang et al ldquoThe protective role ofSmad7 in diabetic kidney disease mechanism and therapeuticpotentialrdquo Diabetes vol 60 no 2 pp 590ndash601 2011
[25] X Wei Y Xia F Li et al ldquoKindlin-2 mediates activationof TGF-120573Smad signaling and renal fibrosisrdquo Journal of theAmerican Society Nephrology vol 24 no 9 pp 1387ndash1398 2013
[26] M Isono S Chen S W Hong M C Iglesias-de la Cruzand F N Ziyadeh ldquoSmad pathway is activated in the diabeticmouse kidney and smad3 mediates TGF-120573-induced fibronectinin mesangial cellsrdquo Biochemical and Biophysical ResearchCommunications vol 296 no 5 pp 1356ndash1365 2002
[27] J H Park C Lee J H Suh J Y Chae andK CMoon ldquoNuclearexpression of Smad proteins and its prognostic significance inclear cell renal cell carcinomardquo Human Pathology vol 44 no10 pp 2047ndash2054 2013
[28] G X Liu Y Q Li X R Huang et al ldquoSmad7 inhibitsAngII-mediated hypertensive nephropathy in amousemodel ofhypertensionrdquo Clinical Science vol 127 no 3 pp 195ndash208 2014
[29] G S Zode A F Clark and R J Wordinger ldquoBone morpho-genetic protein 4 inhibits TGF-1205732 stimulation of extracellularmatrix proteins in optic nerve head cells role of gremlin inECMmodulationrdquo Glia vol 57 no 7 pp 755ndash766 2009
[30] S OrsquoReilly M Ciechomska R Cant and J M van Laar ldquoIL-6trans signalling drives a STAT3 dependant pathway that leadsto hyperactive TGF-120573 signalling promoting SMAD3 activationand fibrosis via gremlinrdquo The Journal of Biological Chemistry2014
[31] M Myllarniemi P Lindholm M J Ryynanen et al ldquoGremlin-mediated decrease in bone morphogenetic protein signaling
promotes pulmonary fibrosisrdquo American Journal of Respiratoryand Critical Care Medicine vol 177 no 3 pp 321ndash329 2008
[32] E Cahill C M Costello S C Rowan et al ldquoGremlin playsa key role in the pathogenesis of pulmonary hypertensionrdquoCirculation vol 125 no 7 pp 920ndash930 2012
[33] W Boers S Aarrass C Linthorst M Pinzani R O Elferinkand P Bosma ldquoTranscriptional profiling reveals novel markersof liver fibrogenesis gremlin and insulin-like growth factor-binding proteinsrdquo The Journal of Biological Chemistry vol 281no 24 pp 16289ndash16295 2006
[34] CMCostello E Cahill FMartin S Gaine andPMcLoughlinldquoRole of gremlin in the lung development and diseaserdquoAmerican Journal of Respiratory Cell and Molecular Biologyvol 42 no 5 pp 517ndash523 2010
[35] K A Mueller E Tavlaki M Schneider et al ldquoGremlin-1identifies fibrosis and predicts adverse outcome in patientswith heart failure undergoing endomyocardial biopsyrdquo Journalof Cardiac Failure vol 19 no 10 pp 678ndash684 2013
[36] H Huang H Huang Y Li et al ldquoGremlin induces cellproliferation and extra cellular matrix accumulation in mousemesangial cells exposed to high glucose via the ERK12pathwayrdquo BMC Nephrology vol 14 article 33 2013
[37] N McCormack E L Molloy and S OrsquoDea ldquoBone morpho-genetic proteins enhance an epithelial-mesenchymal transitionin normal airway epithelial cells during restitution of a dis-rupted epitheliumrdquoRespiratory Research vol 14 article 36 2013
[38] J A Tamminen V Parviainen M Ronty et al ldquoGremlin-1associates with fibrillin microfibrils in vivo and regulatesmesothelioma cell survival through transcription factor slugrdquoOncogenesis vol 2 article e66 2013
[39] E M Zeisberg S E Potenta H Sugimoto M Zeisberg and RKalluri ldquoFibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transitionrdquo Journal of the American Society ofNephrology vol 19 no 12 pp 2282ndash2287 2008
[40] V S LeBleu G Taduri J OrsquoConnell et al ldquoOrigin and functionof myofibroblasts in kidney fibrosisrdquo Nature Medicine vol 19no 8 pp 1047ndash1053 2013
[41] W C Prozialeck and J R Edwards ldquoCell adhesion moleculesin chemically-induced renal injuryrdquo Pharmacology amp Thera-peutics vol 114 no 1 pp 74ndash93 2007
[42] M Zeisberg J Hanai H Sugimoto et al ldquoBMP-7 counteractsTGF-1205731-induced epithelial-to-mesenchymal transition andreverses chronic renal injuryrdquo Nature Medicine vol 9 no 7 pp964ndash968 2003
[43] Y-L Yang H-Z Ju S-F Liu et al ldquoBMP-2 suppresses renalinterstitial fibrosis by regulating epithelial-mesenchymaltransitionrdquo Journal of Cellular Biochemistry vol 112 no 9 pp2558ndash2565 2011
[44] R J Wordinger D L Fleenor P E Hellberg et al ldquoEffects ofTGF-1205732 BMP-4 and gremlin in the trabecular meshworkimplications for glaucomardquo Investigative Ophthalmology ampVisual Science vol 48 no 3 pp 1191ndash1200 2007
[45] B Chen M Athanasiou Q Gu and D G Blair ldquoDrmGremlintranscriptionally activates p21Cip1 via a novel mechanism andinhibits neoplastic transformationrdquoBiochemical andBiophysicalResearch Communications vol 295 no 5 pp 1135ndash1141 2002
[46] S Mitola C Ravelli E Moroni et al ldquoGremlin is a novelagonist of the major proangiogenic receptor VEGFR2rdquo Bloodvol 116 no 18 pp 3677ndash3680 2010
[16] J Xu S Lamouille and R Derynck ldquoTGF-120573-induced epithelialto mesenchymal transitionrdquo Cell Research vol 19 no 2 pp156ndash172 2009
[17] M Ruiz-Ortega J Rodrıguez-Vita E Sanchez-Lopez GCarvajal and J Egido ldquoTGF-120573 signaling in vascular fibrosisrdquoCardiovascular Research vol 74 no 2 pp 196ndash206 2007
[18] R Rodrigues-Diez C Lavoz G Carvajal et al ldquoGremlin isa downstream profibrotic mediator of transforming growthfactor-beta in cultured renal cellsrdquo Nephron ExperimentalNephrology vol 122 no 1-2 pp 62ndash74 2012
[19] J Rodrıguez-Vita E Sanchez-Lopez V Esteban M Ruperez JEgido and M Ruiz-Ortega ldquoAngiotensin II activates the Smadpathway in vascular smooth muscle cells by a transforminggrowth factor-120573-independent mechanismrdquo Circulation vol 111no 19 pp 2509ndash2517 2005
[20] G Carvajal J Rodrıguez-Vita R Rodrigues-Dıez et alldquoAngiotensin II activates the Smad pathway during epithelialmesenchymal transdifferentiationrdquo Kidney International vol74 no 5 pp 585ndash595 2008
[21] G Li Y Li S Liu et al ldquoGremlin aggravates hyperglycemia-induced podocyte injury by a TGF120573smad dependent signalingpathwayrdquo Journal of Cellular Biochemistry vol 114 no 9 pp2101ndash2113 2013
[22] H Y Lan W Mu N Tomita et al ldquoInhibition of renalfibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in ratUUOmodelrdquo Journal of theAmericanSociety of Nephrology vol 14 no 6 pp 1535ndash1548 2003
[23] H Fukasawa T Yamamoto A Togawa et al ldquoDown-regulationof Smad7 expression by ubiquitin-dependent degradationcontributes to renal fibrosis in obstructive nephropathy inmicerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 23 pp 8687ndash8692 2004
[24] H Y Chen X R Huang WWang et al ldquoThe protective role ofSmad7 in diabetic kidney disease mechanism and therapeuticpotentialrdquo Diabetes vol 60 no 2 pp 590ndash601 2011
[25] X Wei Y Xia F Li et al ldquoKindlin-2 mediates activationof TGF-120573Smad signaling and renal fibrosisrdquo Journal of theAmerican Society Nephrology vol 24 no 9 pp 1387ndash1398 2013
[26] M Isono S Chen S W Hong M C Iglesias-de la Cruzand F N Ziyadeh ldquoSmad pathway is activated in the diabeticmouse kidney and smad3 mediates TGF-120573-induced fibronectinin mesangial cellsrdquo Biochemical and Biophysical ResearchCommunications vol 296 no 5 pp 1356ndash1365 2002
[27] J H Park C Lee J H Suh J Y Chae andK CMoon ldquoNuclearexpression of Smad proteins and its prognostic significance inclear cell renal cell carcinomardquo Human Pathology vol 44 no10 pp 2047ndash2054 2013
[28] G X Liu Y Q Li X R Huang et al ldquoSmad7 inhibitsAngII-mediated hypertensive nephropathy in amousemodel ofhypertensionrdquo Clinical Science vol 127 no 3 pp 195ndash208 2014
[29] G S Zode A F Clark and R J Wordinger ldquoBone morpho-genetic protein 4 inhibits TGF-1205732 stimulation of extracellularmatrix proteins in optic nerve head cells role of gremlin inECMmodulationrdquo Glia vol 57 no 7 pp 755ndash766 2009
[30] S OrsquoReilly M Ciechomska R Cant and J M van Laar ldquoIL-6trans signalling drives a STAT3 dependant pathway that leadsto hyperactive TGF-120573 signalling promoting SMAD3 activationand fibrosis via gremlinrdquo The Journal of Biological Chemistry2014
[31] M Myllarniemi P Lindholm M J Ryynanen et al ldquoGremlin-mediated decrease in bone morphogenetic protein signaling
promotes pulmonary fibrosisrdquo American Journal of Respiratoryand Critical Care Medicine vol 177 no 3 pp 321ndash329 2008
[32] E Cahill C M Costello S C Rowan et al ldquoGremlin playsa key role in the pathogenesis of pulmonary hypertensionrdquoCirculation vol 125 no 7 pp 920ndash930 2012
[33] W Boers S Aarrass C Linthorst M Pinzani R O Elferinkand P Bosma ldquoTranscriptional profiling reveals novel markersof liver fibrogenesis gremlin and insulin-like growth factor-binding proteinsrdquo The Journal of Biological Chemistry vol 281no 24 pp 16289ndash16295 2006
[34] CMCostello E Cahill FMartin S Gaine andPMcLoughlinldquoRole of gremlin in the lung development and diseaserdquoAmerican Journal of Respiratory Cell and Molecular Biologyvol 42 no 5 pp 517ndash523 2010
[35] K A Mueller E Tavlaki M Schneider et al ldquoGremlin-1identifies fibrosis and predicts adverse outcome in patientswith heart failure undergoing endomyocardial biopsyrdquo Journalof Cardiac Failure vol 19 no 10 pp 678ndash684 2013
[36] H Huang H Huang Y Li et al ldquoGremlin induces cellproliferation and extra cellular matrix accumulation in mousemesangial cells exposed to high glucose via the ERK12pathwayrdquo BMC Nephrology vol 14 article 33 2013
[37] N McCormack E L Molloy and S OrsquoDea ldquoBone morpho-genetic proteins enhance an epithelial-mesenchymal transitionin normal airway epithelial cells during restitution of a dis-rupted epitheliumrdquoRespiratory Research vol 14 article 36 2013
[38] J A Tamminen V Parviainen M Ronty et al ldquoGremlin-1associates with fibrillin microfibrils in vivo and regulatesmesothelioma cell survival through transcription factor slugrdquoOncogenesis vol 2 article e66 2013
[39] E M Zeisberg S E Potenta H Sugimoto M Zeisberg and RKalluri ldquoFibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transitionrdquo Journal of the American Society ofNephrology vol 19 no 12 pp 2282ndash2287 2008
[40] V S LeBleu G Taduri J OrsquoConnell et al ldquoOrigin and functionof myofibroblasts in kidney fibrosisrdquo Nature Medicine vol 19no 8 pp 1047ndash1053 2013
[41] W C Prozialeck and J R Edwards ldquoCell adhesion moleculesin chemically-induced renal injuryrdquo Pharmacology amp Thera-peutics vol 114 no 1 pp 74ndash93 2007
[42] M Zeisberg J Hanai H Sugimoto et al ldquoBMP-7 counteractsTGF-1205731-induced epithelial-to-mesenchymal transition andreverses chronic renal injuryrdquo Nature Medicine vol 9 no 7 pp964ndash968 2003
[43] Y-L Yang H-Z Ju S-F Liu et al ldquoBMP-2 suppresses renalinterstitial fibrosis by regulating epithelial-mesenchymaltransitionrdquo Journal of Cellular Biochemistry vol 112 no 9 pp2558ndash2565 2011
[44] R J Wordinger D L Fleenor P E Hellberg et al ldquoEffects ofTGF-1205732 BMP-4 and gremlin in the trabecular meshworkimplications for glaucomardquo Investigative Ophthalmology ampVisual Science vol 48 no 3 pp 1191ndash1200 2007
[45] B Chen M Athanasiou Q Gu and D G Blair ldquoDrmGremlintranscriptionally activates p21Cip1 via a novel mechanism andinhibits neoplastic transformationrdquoBiochemical andBiophysicalResearch Communications vol 295 no 5 pp 1135ndash1141 2002
[46] S Mitola C Ravelli E Moroni et al ldquoGremlin is a novelagonist of the major proangiogenic receptor VEGFR2rdquo Bloodvol 116 no 18 pp 3677ndash3680 2010
![Untitled-1 [] Company... · 2018. 1. 16. · Smad Construction Smad Sm Smad Construction mad Construction nstruction MAD Smad Construction MAD' Smad Construction MAD Smad Construction](https://static.documents.pub/doc/80x56/60b16e4aa21c90011033e8c0/untitled-1-company-2018-1-16-smad-construction-smad-sm-smad-construction.jpg)
