Contents lists available at SciVerse ScienceDirect
Cellular Signalling
j ourna l homepage: www.e lsev ie r.com/ locate /ce l l s ig
Lysophosphatidic acid increases soluble ST2 expression in mouse lung and humanbronchial epithelial cells
Jing Zhao a, Qingyuan Chen a, Hong Li a,b, Michael Myerburg a, Ernst Wm. Spannhake c,Viswanathan Natarajan d,e,1, Yutong Zhao a,⁎,1
a Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USAb Department of Clinical Laboratory, Jiangxi Provincial Children's Hospital, Nanchang, Jiangxi, PR Chinac Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USAd Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, USAe Institute for Personalized Respiratory Medicine, University of Illinois at Chicago, Chicago, IL, USA
⁎ Corresponding author at: Department of Medicine, Uof Medicine, 3459 Fifth Ave, NW628MUH, Pittsburgh, PA9488.
E-mail address: [email protected] (Y. Zhao).1 Co-senior authors of this work.
Article history:Received 5 July 2011Received in revised form 8 August 2011Accepted 9 August 2011Available online 17 August 2011
Keywords:LysophospholipidSoluble ST2Gene expressionHistone acetylationEpithelial barrier function
Lysophosphatidic acid (LPA), a naturally occurring bioactive lysophospholipid increases the expression of bothpro-inflammatory and anti-inflammatory mediators in airway epithelial cells. Soluble ST2 (sST2), an anti-inflammatory mediator, has been known to function as a decoy receptor of interleukin (IL)-33 and attenuatesendotoxin-induced inflammatory responses. Here, we show that LPA increased sST2 mRNA expression andprotein release in a dose and timedependentmanner inhumanbronchial epithelial cells (HBEpCs). LPA receptorsantagonist and Gαi inhibitor, pertussis toxin, attenuated LPA-induced sST2 release. Inhibition of NF-κB or JNKpathway reduced LPA-induced sST2 release. LPA treatment decreased histone deacetylase 3 (HDAC3) expressionandenhancedacetylationof histoneH3at lysine9 thatbinds to the sST2promoter region. Furthermore, limitationof intracellular LPA generation by the down-regulation of acetyl glycerol kinase attenuated exogenous LPA-induced histone H3 acetylation on sST2 promoter region, as well as sST2 gene expression. Treatment of HBEpCswith recombinant sST2 protein or sST2-rich cell culturemedia attenuated endotoxin-induced phosphorylation ofPKC and airway epithelial barrier disruption. These results unravel a novel sST2mediated signaling pathway thathas physiological relevance to airway inflammation and remodeling.
niversity of Pittsburgh School, 15213, USA. Tel.: +1 412 648
Lysophosphatidic acid (LPA) is a bioactive phospholipid that playsan important role in several signaling pathways. It has been detectedin various biological fluids, including plasma [1] and bronchoalveolarlavage (BAL) fluids [2,3]. Elevated level of plasma LPA is considered asa biomarker for ovarian cancer [4]. So far, seven G protein-coupledLPA receptors (LPA1–7) have been cloned and characterized [5–7]. LPAtreatment induces activation of transcriptional factors (such as NF-κB,AP-1, and C/EBPβ) and signaling pathways (such as MAPKs and PKC)[5,7]. Increasing evidences show that LPA plays a dual role in airwayinflammation. Increases in LPA level are detected in BAL fluids fromchallenged asthmatic [2], idiopathic pulmonary fibrosis (IPF) patients[3], and murine models of asthma [8] and fibrosis [3]. LPA treatmentincreases IL-8 secretion in human bronchial epithelial cells (HBEpCs)
[9–11] that results in neutrophil influx into alveolar spaces [8,9],thereby suggesting a pro-inflammatory role for LPA. However, recentfindings show that LPA exhibits an anti-inflammatory effect on air-ways. For instance, LPA treatment of HBEpCs attenuates IL-13-inducedsignals through increasing expression of IL-13 decoy receptor, IL-13Rα[12]. LPA enhances airway epithelial barrier integrity by inducing theaccumulation of E-cadherin at cell–cell contacts [13]. Intratracheal orintravenous administration of LPA significantly protects against lipo-polysaccharide (LPS)-induced lung injury [13,14].
ST2, an IL-1 receptor relatedprotein, consists of four isoforms, solublesecreted form (sST2), transmembrane form (ST2L), ST2V, and ST2LV[15]. sST2 binds to IL-33, andnot to IL-1α, IL-1β, and IL-1 antagonist [16],and functions as a decoy receptor of IL-33[17]. In IL-33 receptor over-expressed murine thymoma cells (EL4), pretreatment with sST2suppressed IL-33-induced NF-κB activity and IL-4, IL-5, and IL-13expression [17]. sST2 has been implicated as an anti-inflammatorymediator in inflammatory responses [17–22]. Pretreatment withrecombinant sST2 protein attenuates LPS-induced expression of TNF,IL-6, and IL-12 inmacrophages [18,20]. Over-expression of sST2 reducesLPS-induced TNF, IL-6, and TLR4 levels in serum and myeloperoxidaseactivity in lung, and protests against LPS-induced pulmonary inflamma-tion in a murine model of lung injury [22].
78 J. Zhao et al. / Cellular Signalling 24 (2012) 77–85
The sST2 gene was first identified in fibroblasts [23,24]. Serum sST2levels are significantly elevated in patients with idiopathic pulmonaryfibrosis [25], heart failure [26–29], and other immune diseases [30]. Inhematopoietic cells,−26999G/A SNPpredominantly regulates ST2geneexpression [31]. The current study by using human bronchial epithelialcells, demonstrates that sST2 gene expression is up-regulated by thebioactive lysophospholipid, LPA. We show here that LPA induces bothsST2 mRNA expression and protein release through activation oftranscription factors NF-κB and AP-1, and histone acetylation at sST2promoter region. Further, sST2 has a protective effect against LPS-induced airway epithelial barrier dysfunction.
2. Materials and methods
2.1. Materials
1-oleoyl (18:1) LPA and ki16425 were purchased from Sigma-Aldrich (St. Louis, MO). Pertusis toxin (PTx) and JNK inhibitor (JNKi II)were from Calbiochem (San Diego, CA). Bay11-7082 was purchasedfrom BioMol (Plymouth Meetings, PA). sST2, PKCα, and E-cadherin(K20) antibody were from Santa Cruz Biotechnology (Santa Cruz, CA).Antibodies to p-PKCα, HDAC1, HDAC2, HDAC3, acetylated lysine,histoneH3, and acetylated histone 3 at lysine 9were fromCell SignalingTechnology Inc. (Danvers, MA). Horseradish peroxidase-conjugatedgoat anti-rabbit and anti-mouse secondary antibodies were purchasedfromMolecular Probes (Eugene, OR). ECL kit for detection of proteins byWesternblottingwasobtained fromAmershamPharmacia (Piscataway,NJ). Human recombinant sST2-fc fusion protein was from R&D systems(Minneapolis, MN). Real-time PCR reagents were from Bio-RadLaboratories (Hercules, CA). Bronchial epithelial cell basal medium
sST2
0h 0.5h 1h 3h 6h 24h
0.0
5.0
10.0
15.0
20.0
25.0
30.0
0h 0.5h 1h 3h 6h 24h
sST
2 m
RN
A le
vels
(n
orm
aliz
ed t
o 0
h)
* *
*
*
0h 0.5h 1h 3h 6h 24h
* * *
0
10
20
30
40
50
60
Inte
nsi
ties
(n
orm
aliz
ed t
o 0
h)
A
C
E
LPA 1µM
LPA 1µM
Fig. 1. LPA increases sST2 gene expression and protein release in HBEpCs — A. HBEpCs grownfollowed. Total RNA was extracted and mRNA levels were determined by real-time RT-PCR wexperiments. *pb0.01 versus 0 h cells. B. HBEpCs grown to ~70–80% confluence were challengwere determined by real-time RT-PCR with primers for human sST2. Data represent mean±Ssupernatantswere collected, and thenwere subjected to 10% SDS–PAGEand analyzedwith specshown (mean±SD). *pb0.01 versus 0 h cells D. HBEpCswere treated as described in B, and themPAGE and immunoblotted with specific ant-ST2 antibody. F. Quantitative analysis from three i
(BEBM) and supplement kit were purchased from Lonza (Rockville,MD). MILLIPORE TM 10 kit was purchased from Millipore (Bedford,MA). All other reagents were of analytical grade.
2.2. Cell culture
HBEpCs were purchased from Lonza (Rockville, MD). The passage1 (P1) HBEpCs were cultured in serum free basal essential growthmedium (BEGM) and supplemented with growth factors. Cells wereincubated at 37 °C in 5% CO2 and 95% air to ~80% confluence andsubsequently propagated in 100-mm or 6-well collagen-coateddishes. All experiments were carried out between passages 1 and 4.
2.3. Preparation of cell lysates, media and western blotting
After the indicated treatments, media were collected and centrifugedat 500 ×g for 10 min, and supernatants were concentrated byMILLIPORETM10kit according tomanufacturer's instruction. Cellswere rinsed twicewith ice-cold PBS and lysed in 200 μl of buffer containing 20 mMTris–HCl(pH 7.4), 150 mM NaCl, 2 mM EGTA, 5 mM β-glycerophosphate, 1 mMMgCl2, 1% Triton X-100, 1 mM sodium orthovanadate, 10 μg/ml proteaseinhibitors, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin.Cell lysateswere incubatedat 4 °C for10 min, sonicatedon ice for10 s andcentrifuged at 500 ×g for 5 min at 4 °C in a microfuge. Proteinconcentrations were determined with a BCA protein assay kit (PierceChemical Co., Rockford, IL) using BSA as standard. Equal amounts ofcell lysates (20 μg) or concentrated media (30 μl) were subjected to 10%SDS–PAGE gels, transferred to polyvinylidene difluoride membranes,blocked with 5% (w/v) BSA in TBST (25 mM Tris–HCl, pH 7.4, 137 mMNaCl and 0.1% Tween-20) for 1 h, and incubatedwith primary antibodies
sST2
0.02.04.06.08.0
10.012.014.016.018.0
sST
2 m
RN
A le
vels
(n
orm
aliz
ed t
o C
on
t) * *
***
*
**
0
20
40
60
80
Inte
nsi
ties
(n
orm
aliz
ed t
o c
on
t)
B
D
F
LPA, µM 0 0.5 1.0 5.0 10.00.1
0 0.5 1.0 5.0 10.0LPA,3h
0 0.5 1.0 5.0 10.0LPA, µM
to ~70–80% confluence were challenged with LPA (1 μM) and the kinetic response wasith primers for human sST2. Data represent mean±SD generated from three different
ed with LPA at different concentrations for 3 h. Total RNA was extracted and mRNA levelsD of three values. *pb0.01 versus 0 μM. C. HBEpCs were treated as described in A, cultureific antibody to ST2. E. Quantitative analysis of blots from three independent experiments isediawere collected, centrifuged and concentrated. Theywere then subjected to 10% SDS–
ndependent experiments is shown (mean±SD). *pb0.01 versus 0 μM.
Control LPA 3h LPA18h
sST2
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
Control LPA 3h LPA 18h
Ch
ang
es o
f In
ten
siti
es
(no
rmal
ized
to
co
ntr
ol)
*
**
A
B
Fig. 2. Intratracheal administrationof LPA increases sST2 release inBALfluids—A.C57BL/6mice (3/group)were challengedwith LPA (5 μM) BAL fluids (at 3 h and 18 h) from controland LPA-challenged mice were concentrated with MILLIOPORE column and 30 μl ofconcentrated media were subjected to 10% SDS–PAGE gel and sST2 expression weredetermined by Western blotting with an antibody to sST2. B. Quantitative analysis ofWestern blot is shown. *pb0.01 versus control, **pb0.05 versus control.
79J. Zhao et al. / Cellular Signalling 24 (2012) 77–85
in 5% (w/v) BSA in TBST for 1–2 h. Themembranes were washed at leastthree timeswith TBST at 15 min intervals and then incubatedwith eithermouse or rabbit or goat horseradish peroxidase-conjugated secondaryantibody (1:3000) for 1 h. The membranes were developed withenhanced chemiluminescence detection system according to manufac-turer's instructions.
2.4. RNA isolation
Total RNA was isolated from cultured HBEpCs using TRIzol®reagent (Life Technology, Rockville, MD) according to the manufac-turer's instructions. RNA was quantified spectrophotometrically andsamples with an absorbance ratio of ≥1.8 measured at 260/280 nmwere analyzed by real-time RT-PCR.
sST2
DMSO
LPA
Ki16425
0.0
1.0
2.0
3.0
4.0
Inte
nsi
ties
(n
orm
aliz
ed t
o
DM
SO
alo
ne)
A
DMSO
LPA
Ki16425
5.0*
**
C
B
D
Fig. 3. LPA induces sST2 release through Gαi-coupled LPA receptor — A. HBEpCs were pretrconcentrated culture supernatants were analyzed by 10% SDS–PAGE gel and immunoblottedLPA (1 μM, 3 h) treatment. Clarified and concentrated culture supernatants were subjectedanalysis from three independent experiments of A (mean±SD) is shown. *pb0.01 versuexperiments of B is shown (mean±SD). *pb0.01 versus vehicle cells; **pb0.05 versus LPA
2.5. Real-time RT-PCR and Reverse Transcription-PCR
1 μg of RNA was reverse transcripted using cDNA synthesis kit(Bio-Rad) and real-time PCR and regular PCR were performed toassess expression of the sST2 gene using primers designed basedon human mRNA sequences (NM_003856). sST2 forward primer: 5 -ACGTGAAGGAAGAGGATTTATTGCTGC-3 ; sST2 reverse primer: 5 -TCAGAAACACTCCTTAC-3 . For real-time RT-PCR, amplicon expres-sion in each sample was normalized to its 18S RNA content. Therelative abundance of target mRNA in each sample was calculated as2 raised to the negative of its threshold cycle value times 106 afterbeing normalized to the abundance of its corresponding 18S [e.g.,2−(sST2 Threshold Cycle) /2−(18S Threshold Cycle)×106].
2.6. E-cadherin immunofluorescence staining
HBEpCswere grown on glass chamber until ~90% confluence. Aftertreatment, cells were fixed with 3.7% formaldehyde for 20 minwithout permeabilization, then immunostained with E-cadherinantibody (K20) and fluorescence second antibody. Images werecaptured by Nikon ECLIPSE TE 300 inverted microscope.
2.7. Intratracheal administration of LPA
C57BL/6 mice (8–10-week) were intratracheally administered withLPA (5 μM in 50 μl PBS with 0.1% BSA). After 3 h and 18 h, lungs werelavaged by an intratracheal injection of 1 ml of PBS solution followed bygentle aspiration; the lavage was repeated twice to recover a totalvolume of 1.8–2.0 ml. The recovered BAL fluids were processed fordetermining sST2 levels by Western blotting. All procedures wereexecuted in accordance with approved protocols though the Universityof Pittsburgh Institutional Animal Care and Use Committee.
2.8. Chip experiments
After treatment, cells were cross-linked for 5 min with 0.3%formaldehyde and the reaction was terminated by adding glycine. Cell
sST2
Veh
LPA
PTx
*
0.0
5.0
10.0
15.0
20.0
25.0
Inte
nsi
ties
(n
orm
aliz
ed t
o V
eh)
**
Veh
LPA
PTx
eated with Ki16425 (10 μM) and challenged with LPA (1 μM) for 3 h. The clarified andwith anti-sST2 antibody. B. HBEpCs were treated with PTx (100 ng/ml) for 16 h prior toto 10% SDS–PAGE and analyzed with specific antibody to human ST2. C. Quantitatives veh; **pb0.01 versus LPA alone. D. Quantitative analysis from three independentalone.
Fig. 4. In silico analysis of ST2 proximal promoter region — DNA sequence of human ST2 proximal promoter region at exon 1 (810 bp, accession number: S74267) was analyzed byTRANSFAC 4.0 database. Two predicated NF-κB binding sites are shown in dashed boxes and five predicated AP-1 binding sites are shown in solid boxes. The sequences used for thedesign of real-time PCR primers for Chip analysis are underlined.
80 J. Zhao et al. / Cellular Signalling 24 (2012) 77–85
lysates were sonicated at 4 °C using a sonicator microtip at 50%–60%maximumpower. Immunoprecipitationwasperformedwith antibodiesagainst to acetylated lysine (KAc) or acetylated histone H3 at lysine 9(H3K9Ac) and chromatin DNA was extracted with phenol/chloroformfollowed by ethanol precipitation. sST2 proximal promoter region wasdetectedby regular PCRand real-timePCRwithprimers localizedwithinsST2 promoter. The sets of PCR primers used for the analysis of the
Bay11-7082
sST2
0.0
1.0
2.0
3.0
4.0
5.0
Inte
nsi
ties
(n
orm
aliz
ed t
o
DN
SO
alo
ne)
A
DMSO
LPA
**
*C
LPA
Bay11-7082DMSO
Fig. 5. LPA-induced the increase in release is through NF-κB and AP-1 pathways. HBEpCs groJNK inhibitor (JNKiII 10 μM) (B) for 1 h, and challenged with LPA (1 μM) for 3 h. Media wedetermined by Western blotting with an antibody to human sST2. C and D. Quantitative avehicle cells; **pb0.01 versus LPA alone.
human sST2 proximal promoter were: 5 -CCTCTCAAGGGATTACT-CAATG-3 and 5 -AGACACTCCTCCCATCCTGAAA-3 .
2.9. Measurement of transepithelial resistance (TER) by ECIS
HBEpCs were grown to confluence over gold microelectrodes. TERwas measured in an electrical cell-substrate impedance sensing
sST2
B
Inte
nsi
ties
(n
orm
aliz
ed
to D
MS
O a
lon
e)
DMSO
LPA
JNKiII
0.0
1.0
2.0
3.0
4.0 *
**
D
DMSOLPA
JNKiII
wn in 6-well plates were pretreated with the NF-κB inhibitor Bay11-7082 (5 μM) (A) orre collected, and then were subjected to 10% SDS–PAGE gel and sST2 expression werenalysis from three independent experiments is depicted (mean±SD). *pb0.01 versus
81J. Zhao et al. / Cellular Signalling 24 (2012) 77–85
system (Applied BioPhysics, Foster city, CA). The total TER measureddynamically across the epithelial monolayer was determined as thecombined resistance between the basal surface of the cell and theelectrode, reflecting alterations in cell–cell adhesion [7].
2.10. Statistical analyses
All results were subjected to statistical analysis using one-wayANOVA and, wherever appropriate, the data was also analyzed byStudent–Newman–Keuls test and expressed as mean±S.D. Data wascollected from at least three independent experiments and Pb0.05was considered significant.
3. Results
3.1. LPA induces sST2 expression and release
In order to determine the role of IL-1R related protein sST2 inhuman lungs, we first investigated sST2 gene expression and proteinrelease in response to LPA challenge in HBEpCs. The basal sST2 mRNAlevels were relatively low in HBEpCs, however LPA significantly
TSA, µ
sST
2 m
RN
A le
vels
(no
rmal
ized
to
co
nt)
E
H3K9Ac
H3
C
**
0.0
2.0
4.0
6.0
Inte
nsi
ties
(no
rmal
ized
to
0 h
)
* * **
LPA, h 0 0.5 1.0 2.0 3.0
LPA, h 0 0.5 1.0 2.0 3.0
Veh
HDAC1
HDAC2
HDAC3
LPA
Inte
nsi
ties
(%
of
Veh
)BA
D
Fig. 6. LPA induces sST2 expression through histone H3 acetylation— A. HBEpCs were treatedto HDAC1, HDAC2, and HDAC3. B. Quantitative analysis from three independent experimendifferent time points and cell lysates were analyzed by immunoblotting with antibodies to athree independent experiments is shown (mean±SD). *pb0.05 versus 0 h; **pb0.01 versustreatment. Total RNA was extracted and sST2 mRNA levels were detected by real-time RT-Pveh; **pb0.05 versus LPA alone; ***pb0.01 versus LPA alone.
induced sST2 mRNA levels in a dose and time dependent manner(Fig. 1A and B). The peak of sST2 mRNA expression was at 6 h and themaximum response was by 1 μM of LPA. A similar increase in sST2protein release in cell culture media was also observed, however thepeak levels were observed at 24 h and the maximum response was by10 μM of LPA (Fig. 1C–F).
Further, we investigated the effect of LPA on sST2 release in vivo.C57BL/6 mice (8–10 weeks) were challenged with LPA intratracheallyand BAL fluids were collected. As shown in Fig. 2A and B, LPAchallenge increased sST2 levels in BAL fluids after 3 h (~9.1 fold) and18 h (~5.5 fold). These data indicated that LPA induces sST2 geneexpression in pulmonary epithelial cells and that it is also secretedinto the extracellular pulmonary fluids.
3.2. Role of LPA receptor-mediatedNF-κBand JNKpathways in LPA-inducedsST2 expression
LPA receptors (LPA1–3) have been detected in HBEpCs [32]. Inorder to determine whether LPA-induced the increase in sST2 releaseis mediated through the canonical LPA1–3-mediated signaling, HBEpCswere treated with a LPA1 and LPA3 antagonist (Ki16425, 10 μM) or
**
*
***
0
20
40
60
80
100
120
0
LPA
M 30010050300 0
0
20
40
60
80
100
Veh LPA
HDAC1 HDAC2 HDAC3
*
with LPA (1 μM) for 3 h. Cell lysates were analyzed byWestern blotting with antibodiests is shown (mean±SD). *pb0.01 versus veh. C. Cells were treated with LPA (1 μM) atcetylated histone H3 at lysine 9 (H3K9Ac) and histone H3. D. Quantitative analysis from0 h. E. Cells were pretreated with TSA (50, 100, and 300 μM) prior to LPA (1 μM, 3 h)
CR with sST2 primers. Data points represent mean±SD of three values. *pb0.01 versus
ST2
proximal
promoter
InputIgG
H3K9AcKAc
InputIgG
H3K9AcKAc
Cont LPA
*
0.0
2.0
4.0
6.0
sST
2 p
rom
ote
r D
NA
(no
rmal
ized
to
IgG
)
*
LPA
Veh
B
A
H3K9AcKAc
Fig. 7. LPA induces histone H3 acetylation on the ST2 promoter region — HBEpCs weretreated with LPA (1 μM) for 3 h and then subjected to Chip assay. IgG antibodywas usedas negative control. ST2 proximal promoter was detected by regular PCR (32 cycles)with primers designed based on human ST2 proximal promoter sequence (accessionnumber: S S74267). Amplified DNAwas separated on 1.5% agarose gel (A). B. After DNAwas extracted from chromatin immunoprecipitated complex, levels of ST2 proximalpromoter region were determined by real-time PCR with same primers used in A. Datawas normalized to IgG control. Data are the mean±SD of three values is shown.*pb0.01 versus veh.
82 J. Zhao et al. / Cellular Signalling 24 (2012) 77–85
pertussis toxin (PTx) prior to LPA challenge. Ki16425 blocked LPA(1 μM, 3 h)-induced sST2 release (Fig. 3A and C), suggesting LPA-induced sST2 release through cell surface LPA receptors, at least LPA1
and LPA3. Further, pretreatment with PTx (100 ng/ml, 4 h) attenuatedLPA-induced sST2 secretion, indicating that LPA-induced sST2 releasewas mediated through Gαi-coupled LPA receptors. Ki16425 is solvedin DMSO. DMSO itself affected LPA-induced sST2 release (Fig. 3A).Transcription factors NF-κB and JNK/AP-1 mediate LPA-induced geneexpression of IL-8 [9–11], COX-2 [33], and IL-13Rα2 [12] in HBEpCs. Insilico analysis of transcriptional factor binding sites identified at leasttwo NF-κB and five AP-1 binding sites within human ST2 proximalpromoter region in exon 1 (accession number: S74267, Fig. 4).Inhibition of NF-κB by Bay11-7082 (Fig. 5A and C) or inhibition of JNKby JNK inhibitor II (JNKiII) (Fig. 5B and D) completely blocked LPA-induced sST2 expression, suggesting that activation of these twotranscriptional factors by LPA treatment is essential for LPA-inducedsST2 expression in HBEpCs.
3.3. Role of histone acetylation in LPA-induced sST2 gene expression
Histones are acetylated and deacetylated in lysine sites and thedegree of histone acetylation is known to regulate transcriptionalactivity. Histone deacetylases (HDACs) catalyze the removal of acetylgroups on the lysine residues of core nucleosomal histones, andinhibition of HDACs increase histone acetylation and gene expressions[34]. LPA treatment of HBEpCs for 3 h had no effects on expression ofHDAC1 and 2; however, there was a dramatic reduction in HDAC3expression (Fig. 6A and B). To investigate the effect of LPA on histoneacetylation, we determined histone H3 acetylation by Westernblotting after LPA treatment at different times. As shown in Fig. 6Cand D, LPA treatment significantly induced acetylation of histone H3at lysine 9 at 0.5 h and thereafter increased through 3 h. To furtherinvestigate the effect of histone acetylation on LPA-induced sST2 geneexpression, trichostatin A (TSA) was used to inhibit HDACs andenhance acetylation of histones. Pretreatment with TSA alonesignificantly increased sST2 gene expression, while TSA plus LPAfurther enhanced sST2 gene expression (Fig. 6E). These data suggestthat LPA increased the sST2 gene expression through the reduction ofHDAC3 expression and the increase in histone H3 acetylation.
3.4. LPA induces histone H3 acetylation at sST2 promoter region
To further investigate the epigenetic regulation of sST2 geneexpression, we performed Chip experiments to determine whetherhistone H3 is acetylated on sST2 promoter region in response to LPAtreatment. Antibodies to acetylated lysine (KAc) and acetylatedhistone H3 at K9 (H3K9Ac) were used to immunoprecipitate activatedtranscriptional nucleosomal regions and sST2 promoter region wasamplified by real-time PCR. The primers for sST2 promoter weredesigned based on sST2 proximal promoter region. As shown inFig. 7A and B, LPA treatment induced histone acetylation in generaland histone H3 at K9 in particular, on sST2 promoter region,suggesting that LPA increased sST2 gene expression through histoneH3 acetylation on sST2 promoter region.
3.5. AGK regulates LPS-induced histone acetylation and sST2 gene expression
Our previous study had shown that reduction of intracellular LPAlevels by down-regulation of acyl glycerol kinase (AGK) attenuatedexogenous LPA-induced NF-κB activation and IL-8 expression inHBEpCs [35]. Here, by using Chip experiments, we found that down-regulation of AGK by AGK siRNA transfection (50 nM, 72 h)attenuated LPA-induced histone H3 acetylation on sST2 promoterregion (Fig. 8A), suggesting a role of intracellular LPA in regulation ofhistone H3 acetylation and chromatin modification. The effect of AGKsiRNA on abrogating AGK expression using real-time RT-PCR with
human AGK primers has been previously reported [35]. Further, theeffect of AGK siRNA on LPA-induced sST2 gene expression wasexamined. AGK siRNA transfection attenuated LPA-induced sST2 geneexpression (Fig. 8C). This result is consistent with our previous findingthat intracellular LPA regulates exogenous LPA-induced gene expres-sion through activation of NF-κB and AP-1 pathways [35].
3.6. sST2 protects HBEpCs from LPS-induced airway epithelial barrierdisruption
In order to ascertain the physiological role of sST2 in regulation oflung epithelial integrity, we determined the effect of LPA-releasedsST2 on barrier integrity. HBEpC monolayers were challenged withconditional supernatants from control cell culture or LPA (1 μM,24 h)-treated cell culture and the transepithelial resistance (TER) wasmeasured [13]. Control cell culture supernatant alone had no effect onbasal TER (Fig. 9A). LPS (10 μg/ml) reduced TER in the presence ofcontrol conditional supernatant, but not in the presence of LPA-treated conditional supernatant (Fig. 9A). Our previous study hasshown that LPA treatment rapidly increased TER [13], however, thedata here shows LPA (1 μM, 24 h)-treated conditional supernatanthad no effect on changes of TER, suggesting that exogenous LPA hasbeen degraded after 24 h in LPA (1 μM, 24 h)-treated conditionalsupernatant. LPA half-life in HBEpCs cell culture supernatant is around~20 min [36]. In order to demonstrate the direct protective effect ofsST2 on airway epithelial barrier dysfunction, the cells were exposedto increasing concentrations of recombinant human sST2-fc fusionprotein following LPS (10 μg/ml) challenge. Recombinant sST2-fcfusion protein alone had no discernable effect on TER, however, itattenuated LPS-induced decreases in TER in a dose-dependentmanner (Fig. 9B). Immunofluorescence staining showed that LPS
0
5
10
15
20
25
30
Cont siRNA AGK siRNA
sST
2 m
RN
A e
xpre
ssio
n
(no
rmal
ized
to
co
nt
siR
NA
)
Veh
LPA
AG
K m
RN
A e
xpre
ssio
n
0
20
30
40
50
Cont siRNA AGK siRNA
10
0.0
1.0
2.0
3.0
4.0
Cont siRNA+LPA
AGKsiRNA+LPA
sST
2 P
rom
ote
r D
NA
(n
orm
aliz
ed t
o in
pu
t)
**
*
H3K9Ac in promoterA
C
B
Cont siRNA AGK siRNA
*
*
Fig. 8. Down-regulation of AGK attenuates LPA-induced acetylation of histone H3 and sST2 gene expression—A.HBEpCs (~40% confluence)were transfectedwith AGK siRNA (50 nM) for72 h, and then treated with LPA (1 μM) for 3 h. Cell lysates were then subjected to Chip assay with an antibody to H3K9Ac. Levels of ST2 proximal promoter region in acetylated H3K9Accomplexeswere determined by real-time PCR. Datawas normalized to IgG control. Data are themean±SDof three values. *pb0.01 versus control siRNA+LPA. B. After 72 h of AGK siRNA(50 nM) transfection, total RNAwas extracted andAGKmRNA levelswere examinedby real-timeRT-PCRwith humanAGKprimers. Datawasnormalized to18S.Data are themean±SDofthree values. *pb0.01 versus control siRNA. C. HBEpCs (~40% confluence) were transfected with AGK siRNA (50 nM) for 72 h, and then treated with LPA (1 μM) for 3 h. Total RNA wasextractedand sST2mRNA levelswereexaminedby real-timeRT-PCRwith humansST2primers. Datawasnormalized to 18S.Data are themean±SDof three values. *pb0.01 versus controlsiRNA; **pb0.05 versus cont siRNA+LPA.
83J. Zhao et al. / Cellular Signalling 24 (2012) 77–85
(10 μg/ml, 18 h) reduced E-cadherin expression on HBEpC cell surfaceand increased cell–cell gaps, while pretreatment with sST2-fc fusionprotein (10 ng/ml) attenuated these effects of LPS (Fig. 9C). Wehave shown that PKCα was downstream of LPS and inhibition ofPKCα attenuated LPS-induced E-cadherin relocalization from cellsurface to cytoplasm [13]. Here we found that pretreatment withLPA (1 μM, 24 h) or sST2-fc fusion protein (10 ng/ml, 1 h) atten-uated LPS (10 μg/ml, 1 h)-induced phosphorylation of PKCα(Fig. 9D–G). These results suggest that sST2 protects against LPS-induced airway epithelial barrier dysfunction.
4. Discussion
sST2 is a soluble form of IL-1R related protein [15,21]. Elevatedplasma levels of sST2 have been detected in various diseases, includingcardiovascular [27–29], asthma [37], and autoimmune diseases [38].sST2 functions as a decoy receptor for IL-33 and blocks IL-33-inducedsignals and cellular responses [17]. Recent studies suggest that sST2attenuates LPS-induced production of cytokines in macrophages [20].However, the regulation of sST2 expression and its function in airwayepithelial cells are still unknown. Here, we report that a bioactivelysophospholipid mediator, LPA increases sST2 gene expression and
protein release into extracellular fluid in HBEpCs. LPA-induced sST2gene expression is principally mediated by activation of transcriptionfactors (NF-κB and AP-1) and acetylation of histone H3 on sST2promoter region. Further, our studies revealed that intracellular LPAcontributes to LPA-induced modification of histone and sST2 geneexpression. The data demonstrating the ability of sST2 fusion protein tosuppress LPS-induced airway epithelial barrier dysfunction reveal apotential ameliorating role for sST2 in lung injury.
LPA is a bioactive lysophospholipid that induces a wide range ofcellular responses through its G-protein-coupled receptors [6,7,39].Our previous studies have shown that LPA is an agonist of humanbronchial epithelial cells and induced gene expression of severalinflammatory mediators, including IL-8 [9–11] and COX-2 [33]. Thecurrent study, on the contrary reveals a potential anti-inflammatoryrole of LPA by its ability to induce sST2 gene expression and release.The underlying mechanisms involved previously shown mediators ofLPA activity such as the Gαi-coupled receptor and its downstreamtranscription factors such as NF-κB and AP-1 [9,10,12,33,35].
Gene expression is regulated by nuclear histone acetylation. Forinstance, histone acetylation weakens histone–DNA interaction andthus plays a critical role in transcriptional activation [40]. Two enzymesystems regulate histone acetylation: histone acetyl transferases (HATs)
BA
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Cont media LPA media
TE
R (
no
rmal
ized
to
co
nt
med
ia-t
reat
ed c
ells
)
Veh
LPS
*
**
0.5
0.6
0.7
0.8
0.9
1.0
1.1Veh
LPS
sST2 ng/ml 0 50 0 1 10 50
TE
R (
no
rmal
ized
to
u
ntr
eate
d c
ells
)
******
***
P-PKCα
sST2 LPS sST2+LPSVeh
PKCα
sST2 LPS sST2+LPSVeh
C
0.0
1.0
2.0
3.0
Inte
nsi
ties
(n
orm
aliz
ed t
o v
eh)
D F
E G
P-PKCα
PKCα
Cont media
+LPS
LPA media
+LPS
Cont media
0.0
2.0
4.0
6.0
Inte
nsi
ties
(n
orm
aliz
ed t
o Ig
G)
LPA media
* *
****
Fig. 9. sST2 offers protection to LPS-induced airway epithelial barrier disruption— A. HBEpCs grown on ECIS gold electrodes were incubated with conditional media (from control orLPA (1 μM, 24 h)-treated cells) for 1 h, and then challenged with LPS (10 μg/ml) for 6 h. TER measurements were carried out with the ECIS system. Data were normalized to 0 h.*pb0.01, versus veh; **pb0.01, versus control media+LPS treatment. B. HBEpCs grown on ECIS gold electrodes were treated with recombinant sST2-fc fusion protein (1, 10, and50 ng/ml) for 1 h, then challenged with LPS (10 μg/ml) for 6 h. TER values were measuredwith ECIS system. Data were normalized to 0 h. *pb0.01, compared to veh; **pb0.05, versusLPS alone; ***pb0.01, versus LPS alone. C. HBEpCs grown on glass chamber to ~90–100% confluencewere pretreated with sST2-fc fusion protein (10 ng/ml, 1 h) prior to LPS treatment(10 μg/ml, 18 h). Localization of E-cadherin was examined by immunofluorescence staining. Cell–cell gaps were shown by arrows. Shown are represent images. D. HBEpCS wereincubated in control conditional media or LPA (1 μM, 24 h)-treated conditional media for 1 h prior to LPS treatment (10 μg/ml, 1 h). Cell lysates were analyzed by Western blottingwith antibodies to p-PKCα and PKCα. E. Quantitative analysis from three independent experiments are depicted (mean±SD). *pb0.01 versus control media-incubated cells;**pb0.01 versus control media+LPS. F. HBEpCs were treated with sST2-fc fusion protein (10 ng/ml, 1 h) prior to LPS treatment (10 μg/ml, 1 h). Cell lysates were analyzed byWesternblotting with antibodies to p-PKCα and PKCα. G. Quantitative analysis from three independent experiments are depicted (mean±SD). *pb0.01 versus vehicle cells; **pb0.01 versusLPS alone.
84 J. Zhao et al. / Cellular Signalling 24 (2012) 77–85
and HDACs [34]. LPA-induced sST2 gene expression is dependent onchromatinmodification, especially, acetylation of histone H3 in HBEpCs(Fig. 6). Since HDACs can negatively regulate histone acetylation, wedetermined the levels of HDAC1, HDAC2 and HDAC3 that are abundantHBEpCs. Our data clearly supports a role for HDAC3 in histoneacetylation as its level significantly reduced in LPA-treated HBEpCs(Fig. 6A). Further support is gained from theobservation that inHBEpCs,LPA challenge induces histone 3 acetylation at Lysine 9 (Fig. 6C). Also,we verified the above by demonstrating that pretreatment of HBEpCs
with TSA, an inhibitor of class I and II HDACs also resulted in significantdose-dependent increase in LPA-induced sST2 transcripts (Fig. 6D).However, Ishdorj et al. showed that LPA reduced histone acetylationthrough inhibition of HDAC1 activity with a concomitant increase inHAT activity in transformed cell lines (CaOv-3 and I-83) [41]. Theseopposite effects of LPAonhistone acetylation suggest that LPA elicits celltype-specific responses. For instance, LPA had limited effect onphosphorylation of AKT in HBEpCs (data not shown), while LPA-induced activation of AKT in I-83 and CaVO-3 cells [42].
85J. Zhao et al. / Cellular Signalling 24 (2012) 77–85
Recent studies suggest that in addition to the effects mediated bytheir cell surface receptors, intracellular lysophospholipids alsoinduce signals and cellular responses [35,43,44]. Intracellular sphin-gosine-1-phosphate (S1P) regulates intracellular calcium concentra-tion [45], phosphorylation of I-κB, p38MAPK, and activation of Rac1[43]. Hait et al. reported that intracellular S1P interacted with HDAC1and 2 and associated with acetylation of histone [46]. Here, wedemonstrated that intracellular LPA regulates gene expressionthrough histone acetylation (Fig. 6). However, the mechanisms bywhich intracellular LPA-mediated histone acetylation are not wellunderstood. Hait et al. reported that intracellular LPA had no ability tointeract with HDAC1 and 2, while our previous study showed thatintracellular LPA, regulated by AGK, mediated phosphorylation of I-κBand p38MAPK in HBEpCs [35]. PPARγ is known as an intracellularreceptor for LPA [47]. The effect of LPA on histone acetylation may bethrough its binding to the nuclear factor, PPARγ, a possibility we arecurrently investigating. Recent study from Zou et al. shows a role oflipid modification of histone in regulating cellular mRNA expressionin lung epithelial cells [48]. The effect of intracellular LPA on histonelipid modification will be investigated in the future.
sST2 gene expression is known to be dependent on GATA-3binding on ST2 proximal promoter region [49]. We provided hereevidence that the transcription factors NF-κB and AP-1 and histoneacetylation on ST2 proximal promoter region regulate sST2 geneexpression in airway epithelial cells. sST2 is known to reduce IL-33-and LPS-mediated inflammatory responses [17–22]; however, theeffect of sST2 on airway epithelial barrier function has not beenstudied. Our present study is the first report to demonstrate that sST2has a protective effect against LPS-induced airway epithelial barrierdysfunction.
Our study revealed a novel molecular mechanism underlying LPA-induced sST2 expression and release through activation of transcrip-tional factors and acetylation of histone on sST2 promoter region. Thisstudy identified potential molecular targets for developing futuretherapeutic approaches to combat lung inflammatory diseases.
This work was supported by the National Institutes of HealthR01 grant HL091916 (to YZ), R37 079396 (to VN), P30 DK072506(to M.M.), and by Cystic Fibrosis Foundation RDP (to M.M.). Theauthors declare no conflict interest related to this work.
References
[1] J. Aoki, A. Taira, Y. Takanezawa, Y. Kishi, K. Hama, T. Kishimoto, K. Mizuno, K. Saku,R. Taguchi, H. Arai, J. Biol. Chem. 277 (2002) 48737–48744.
[2] S.N. Georas, E. Berdyshev, W. Hubbard, I.A. Gorshkova, P.V. Usatyuk, B. Saatian,A.C. Myers, M.A. Williams, H.Q. Xiao, M. Liu, V. Natarajan, Clin. Exp. Allergy 37(2007) 311–322.
[3] A.M. Tager, P. LaCamera, B.S. Shea, G.S. Campanella, M. Selman, Z. Zhao, V.Polosukhin, J. Wain, B.A. Karimi-Shah, N.D. Kim,W.K. Hart, A. Pardo, T.S. Blackwell,Y. Xu, J. Chun, A.D. Luster, Nat. Med. 14 (2008) 45–54.
[4] Y. Xu, Z. Shen, D.W.Wiper, M.Wu, R.E. Morton, P. Elson, A.W. Kennedy, J. Belinson,M. Markman, G. Casey, JAMA 280 (1998) 719–723.
[5] N. Fukushima, J. Chun, Prostaglandins 64 (2001) 21–32.[6] T. Mutoh, J. Chun, Subcell. Biochem. 49 (2008) 269–297.[7] Y. Zhao, V. Natarajan, Cell. Signal. 21 (2009) 367–377.[8] Y. Zhao, J. Tong, D. He, S. Pendyala, B. Evgeny, J. Chun, A.I. Sperling, V. Natarajan,
Respir. Res. 10 (2009) 114.[9] R. Cummings, Y. Zhao, D. Jacoby, E.W. Spannhake, M. Ohba, J.G. Garcia, T. Watkins,
D. He, B. Saatian, V. Natarajan, J. Biol. Chem. 279 (2004) 41085–41094.[10] B. Saatian, Y. Zhao, D. He, S.N. Georas, T. Watkins, E.W. Spannhake, V. Natarajan,
Biochem. J. 393 (2006) 657–668.[11] Y. Zhao, D. He, B. Saatian, T. Watkins, E.W. Spannhake, N.J. Pyne, V. Natarajan,
J. Biol. Chem. 281 (2006) 19501–19511.[12] Y. Zhao, D. He, J. Zhao, L. Wang, A.R. Leff, E.W. Spannhake, S. Georas, V. Natarajan,
J. Biol. Chem. 282 (2007) 10172–10179.[13] D. He, Y. Su, P.V. Usatyuk, E.W. Spannhake, P. Kogut, J. Solway, V. Natarajan, Y.
Zhao, J. Biol. Chem. 284 (2009) 24123–24132.[14] H. Fan, B. Zingarelli, V. Harris, G.E. Tempel, P.V. Halushka, J.A. Cook, Mol. Med. 14
(2008) 422–428.[15] M.A. Gayle, J.L. Slack, T.P. Bonnert, B.R. Renshaw, G. Sonoda, T. Taguchi, J.R. Testa,
S.K. Dower, J.E. Sims, J. Biol. Chem. 271 (1996) 5784–5789.[16] S. Kumar, M.D. Minnich, P.R. Young, J. Biol. Chem. 270 (1995) 27905–27913.[17] H. Hayakawa, M. Hayakawa, A. Kume, S. Tominaga, J. Biol. Chem. 282 (2007)
26369–26380.[18] K. Oshikawa, K. Yanagisawa, S. Tominaga, Y. Sugiyama, Biochem. Biophys. Res.
Immunol. 164 (2011) 248–255.[23] S. Tominaga, FEBS Lett. 258 (1989) 301–304.[24] A.K. Werenskiold, S. Hoffmann, R. Klemenz, Mol. Cell. Biol. 9 (1989) 5207–5214.[25] S. Tajima, K. Oshikawa, S. Tominaga, Y. Sugiyama, Chest 124 (2003) 1206–1214.[26] J. Bartunek, L. Delrue, F. Van Durme, O. Muller, F. Casselman, B. De Wiest, R. Croes,
S. Verstreken, M. Goethals, H. de Raedt, J. Sarma, L. Joseph, M. Vanderheyden, E.O.Weinberg, J. Am. Coll. Cardiol. 52 (2008) 2166–2174.
[27] M. Shimpo, D.A. Morrow, E.O. Weinberg, M.S. Sabatine, S.A. Murphy, E.M. Antman,R.T. Lee, Circulation 109 (2004) 2186–2190.
[28] E.O. Weinberg, M. Shimpo, G.W. De Keulenaer, C. MacGillivray, S. Tominaga, S.D.Solomon, J.L. Rouleau, R.T. Lee, Circulation 106 (2002) 2961–2966.
[29] E.O. Weinberg, M. Shimpo, S. Hurwitz, S. Tominaga, J.L. Rouleau, R.T. Lee,Circulation 107 (2003) 721–726.
[30] M. Brunner, C. Krenn, G. Roth, B. Moser, M. Dworschak, E. Jensen-Jarolim, A.Spittler, T. Sautner, N. Bonaros, E. Wolner, G. Boltz-Nitulescu, H.J. Ankersmit,Intensive Care Med. 30 (2004) 1468–1473.
[31] M. Shimizu, A. Matsuda, K. Yanagisawa, T. Hirota, M. Akahoshi, N. Inomata, K. Ebe,K. Tanaka, H. Sugiura, K. Nakashima, M. Tamari, N. Takahashi, K. Obara, T.Enomoto, Y. Okayama, P.S. Gao, S.K. Huang, S. Tominaga, Z. Ikezawa, T. Shirakawa,Hum. Mol. Genet. 14 (2005) 2919–2927.
[32] L. Wang, R. Cummings, Y. Zhao, A. Kazlauskas, J.K. Sham, A. Morris, S. Georas, D.N.Brindley, V. Natarajan, J. Biol. Chem. 278 (2003) 39931–39940.
[33] D. He, V. Natarajan, R. Stern, I.A. Gorshkova, J. Solway, E.W. Spannhake, Y. Zhao,Biochem. J. 412 (2008) 153–162.
[34] M.H. Kuo, C.D. Allis, Bioessays 20 (1998) 615–626.[35] S. Kalari, Y. Zhao, E.W. Spannhake, E.V. Berdyshev, V. Natarajan, Am. J. Physiol.
Lung Cell. Mol. Physiol. 296 (2009) L328–336.[36] Y. Zhao, P.V. Usatyuk, R. Cummings, B. Saatian, D. He, T. Watkins, A. Morris, E.W.
Spannhake, D.N. Brindley, V. Natarajan, Biochem. J. 385 (2005) 493–502.[37] K. Oshikawa, K. Kuroiwa, K. Tago, H. Iwahana, K. Yanagisawa, S. Ohno, S.I.
Tominaga, Y. Sugiyama, Am. J. Respir. Crit. Care Med. 164 (2001) 277–281.[38] K. Kuroiwa, T. Arai, H. Okazaki, S. Minota, S. Tominaga, Biochem. Biophys. Res.
Commun. 284 (2001) 1104–1108.[39] Y. Takuwa, N. Takuwa, N. Sugimoto, J. Biochem. 131 (2002) 767–771.[40] A. Izzo, R. Schneider, Brief Funct, Genomics 9 (2010) 429–443.[41] G. Ishdorj, B.A. Graham, X. Hu, J. Chen, J.B. Johnston, X. Fang, S.B. Gibson, J. Biol.
Chem. 283 (2008) 16818–16829.[42] X. Hu, N. Haney, D. Kropp, A.F. Kabore, J.B. Johnston, S.B. Gibson, J. Biol. Chem. 280
(2005) 9498–9508.[43] Y. Zhao, S.K. Kalari, P.V. Usatyuk, I. Gorshkova, D. He, T. Watkins, D.N. Brindley, C.
Sun, R. Bittman, J.G. Garcia, E.V. Berdyshev, V. Natarajan, J. Biol. Chem. 282 (2007)14165–14177.
[44] E.V. Berdyshev, I.A. Gorshkova, P. Usatyuk, Y. Zhao, B. Saatian, W. Hubbard, V.Natarajan, Cell. Signal. 18 (2006) 1779–1792.
[45] D. Meyer zu Heringdorf, K. Liliom, M. Schaefer, K. Danneberg, J.H. Jaggar, G. Tigyi,K.H. Jakobs, FEBS Lett. 554 (2003) 443–449.
[46] N.C. Hait, J. Allegood, M. Maceyka, G.M. Strub, K.B. Harikumar, S.K. Singh, C. Luo, R.Marmorstein, T. Kordula, S. Milstien, S. Spiegel, Science 325 (2009) 1254–1257.
[47] T.M. McIntyre, A.V. Pontsler, A.R. Silva, A. St Hilaire, Y. Xu, J.C. Hinshaw, G.A.Zimmerman, K. Hama, J. Aoki, H. Arai, G.D. Prestwich, Proc. Natl. Acad. Sci. U.S.A.100 (2003) 131–136.
[48] C. Zou, B.M. Ellis, R.M. Smith, B.B. Chen, Y. Zhao, R.K. Mallampalli, J. Biol. Chem. 286(2011) 28019–28025.
[49] M. Hayakawa, K. Yanagisawa, S. Aoki, H. Hayakawa, N. Takezako, S. Tominaga,Biochim. Biophys. Acta 1728 (2005) 53–64.
