TRANSGLUTAMINASE REGULATION OF CELLFUNCTIONRichard L. Eckert, Mari T. Kaartinen, Maria Nurminskaya, Alexey M. Belkin, Gozde Colak,Gail V. W. Johnson, and Kapil Mehta
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore,Maryland; Division of Biomedical Sciences, Faculty of Dentistry, McGill University, Montreal, Quebec, Canada;Department of Anesthesiology, University of Rochester, Rochester, New York; and Department of ExperimentalTherapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas
LEckert RL, Kaartinen MT, Nurminskaya M, Belkin AM, Colak G, Johnson GVW,Mehta K. Transglutaminase Regulation of Cell Function. Physiol Rev 94: 383–417,2014.—Transglutaminases (TGs) are multifunctional proteins having enzymatic andscaffolding functions that participate in regulation of cell fate in a wide range of cellularsystems and are implicated to have roles in development of disease. This review
highlights the mechanism of action of these proteins with respect to their structure, impact on celldifferentiation and survival, role in cancer development and progression, and function in signaltransduction. We also discuss the mechanisms whereby TG level is controlled and how TGs controldownstream targets. The studies described herein begin to clarify the physiological roles of TGs inboth normal biology and disease states.
I. TRANSGLUTAMINASE: INTRODUCTION... 383II. REGULATION OF PROTEIN... 386III. TRANSGLUTAMINASE-REGULATED... 388IV. TGS IN CELL... 397V. ROLE OF TG2 IN CANCER 400VI. REGULATION OF TG2 EXPRESSION... 403VII. PERSPECTIVES AND FUTURE... 406
I. TRANSGLUTAMINASES: INTRODUCTIONAND OVERVIEW
Transglutaminases (TGs; EC 2.3.2.13) are a family of struc-turally and functionally related proteins that catalyze theCa2�-dependent posttranslational modification of proteinsby introducing covalent bonds between free amine groups(e.g., protein- or peptide-bound lysine) and �-carboxamidegroups of peptide-bound glutamines (FIGURE 1). Research-ers identified the first TG, now designated TG2, in 1959from guinea pig liver extracts based on its ability to catalyzeincorporation of low-molecular-weight primary aminesinto proteins (306). Since the discovery of TG2, additionalproteins with this activity have been identified from unicel-lular organisms, invertebrates, fish, mammals, and plants(122). Nine TG genes are present in humans. Eight arecatalytically active enzymes, and one is inactive (erythro-cyte membrane protein band 4.2) (122). These proteinsserve as scaffolds, maintain membrane integrity, regulatecell adhesion, and modulate signal transduction (TABLE 1)(308). Although the primary sequence of the TGs differ,with the exception of band 4.2, all share an identical aminoacid sequence at the active site (FIGURE 2). In addition to theprotein crosslinking and scaffolding functions, TGs cata-
lyze posttranslational modification of proteins via deami-dation and amine incorporation (FIGURE 1). For example,TG2-dependent deamidation of gliadin A, a component ofwheat and other cereals, is implicated in the pathogenesis ofceliac disease (189). Similarly, deamidation of Gln63 inRhoA activates this signaling protein (108). Moreover, TG-catalyzed incorporation of amines into proteins can modifythe function, stability, and immunogenicity of substrateproteins and contribute to autoimmune disease (220). Ofthe nine TGs identified in humans, TG2 is the most widelydistributed and most extensively studied. In this review, wedescribe the role of TGs in general, and TG2 in particular,and also explore the consequences of aberrant TG expres-sion and activation. TABLE 1 summarizes the general fea-tures of each member of the TG family.
A. Transglutaminase 1
Keratinocyte TG (TG1) is expressed in the stratified squa-mous epithelia of the skin and upper digestive tract and inthe lower female genital tract. The TGM1 gene promotercontains three activator protein AP2-like response elementslocated �0.5 kb from the transcription initiation site (238).Proteolytic cleavage, increased Ca2� level, and interactionwith tazarotene-induced gene 3 (TIG3) are known to acti-vate TG1 catalytic activity (98, 156, 331, 332). Phorbolesters induce and retinoic acid reduces TG1 mRNA andprotein expression (97). TG1 protein associates with theplasma membrane via fatty acyl linkage in the NH2-termi-nal cysteine residue and is released by proteolysis as 10-,33-, and 66-kDa fragments (183). Autosomal recessive la-mellar ichthyosis results from mutation of the TG1-encod-
ing gene (46, 71, 140, 141). Common mutations include aC-to-T change in the binding site for the transcription factorSp1 within the promoter region, a Gly143-to-Glu mutationin exon 3, and a Val382-to-Met mutation in exon 7. Lamel-lar ichthyosis is a rare keratinization disorder of the skincharacterized by abnormal cornification of the epidermis.Individuals with ichthyosis exhibit drastically reduced TG1activity and absence of detectable TG1 protein (46, 71, 140,141). TG1 knockout mice exhibit the lamellar ichthyosisphenotype (234).
B. Transglutaminase 2
Tissue TG (TG2), also referred to as TGc or Gh, is widelydistributed in tissues and cell types. TG2 is predominantly acytosolic protein but is also present in the nucleus and onthe plasma membrane (220). The TG2 gene promoter con-tains a retinoic acid response element (1.7 kb upstream ofthe initiation site), an interleukin (IL)-6 specific cis-regula-tory element (4 kb upstream of the promoter), a transform-ing growth factor-�1 (TGF-�1) response element (868 bp
OII
H2N-R
CH2-CH2-C – N- ROII
I+ NH3
CH2-CH2-CH2-CH2-H2N
CH2 CH2 C - OH + NH3
OII
P1-
OII
H2O
P2
(1)
(3)
Glutamine
+
Glutamate
P1-
Transamidation
Deamidation
Transamidation
IH
H
TGCa2+ Nε (γ-glutamyl) lysine bridgeε
ε P1- CH2- CH2- C – NH2 TG -Cys
+ NH3
P1-CH2-CH2-C-N-CH2-CH2-CH2-CH2- P2 (2)γ
γ
γ
γ
FIGURE 1. Enzymatic reactions catalyzed by transglutaminases (TGs). Transamidation crosslinking reactionsrequire the presence of Ca2� to covalently link primary amines including polyamines, monoamines, andprotein-bound amines (P2) to a glutamine residue of the acceptor protein (P1). These reactions form poly-amines or monoamine crosslinks with proteins (1) or protein-protein crosslinks to form an �-(�-glutamyl)lysineisopeptide bond (2). Under slightly acidic conditions, some TGs can utilize H2O to catalyze deamidation of theP1 protein (3).
Table 1. Properties of transglutaminase proteins
Gene ProteinChromosomal
LocationMolecularMass, kDa Main Function Tissue Distribution Alternate Names
upstream), and two AP2-like response elements (634 and183 bp upstream of the transcription initiation site). Reti-noic acid, vitamin D, TGF-�1, IL-6, tumor necrosis factor(TNF), NF-�B, epidermal growth factor (EGF), phorbolester, oxidative stress, and Hox-A7 induce TG2 expression.In addition to the transamidation reaction, TG2 displaysGTPase, ATPase, protein kinase, and protein disulfideisomerase (PDI) activity. It interacts with phopholipaseC�1, �-integrins, fibronectin, osteonectin, RhoA, multilin-eage kinases, retinoblastoma protein, PTEN, and I�B�.TG2 dysfunction contributes to celiac disease, neurodegen-erative disorders, and cataract formation. TG2 knockoutmice have no phenotype but display delayed wound healingand poor response to stress. Also, fibroblasts derived fromTG2 mice display altered attachment and motility (351).
C. Transglutaminase 3
Transglutaminase 3 (TG3) or epidermal TG is present inhair follicles, epidermis, and brain. The TG3 gene (TGM3)promoter contains Sp1- and Ets-motifs (128 and 91 bpupstream of the initiation site, respectively), and expressionof pro-transglutaminase 3 mRNA is increased by Ca2�.TG3 protein is encoded as two polypeptide chains derivedfrom a single precursor protein by proteolysis. Like TG2,TG3 binds to and hydrolyzes GTP. It catalyzes the cross-linking of trichohyalin and keratin intermediate filamentsto harden the inner root sheath of a hair follicle, which iscritical for hair fiber morphogenesis (133–136, 162). It alsoparticipates in cell envelope formation during the latterstages of differentiation (162). TG3 knockout mice showimpaired hair development and reduced skin barrier func-tion (36, 162).
D. Transglutaminase 4
Transglutaminase 4 (TG4) or prostate TG is present in theprostate gland, prostatic fluids, and seminal plasma (91,122, 160, 386). An Sp1-binding site, located �96 to �87
bp upstream of the transcription initiation site, is critical fortranscriptional regulation of the TG4 gene expression, andandrogen treatment increases TG4 mRNA level in the hu-man prostate cancer cells. In rats, the enzyme participates inthe formation of the copulatory plug in the female genitaltract, and in masking the antigenicity of the male gamete.TG4 knockout mice exhibit reduced fertility due to defectsin copulatory plug formation (84). The exact function ofTG4 in humans is not known, but some recent reports sug-gest a link between increased expression of TG4 and pro-motion of an aggressive prostate cancer phenotype (160).
E. Transglutaminase 5
Transglutaminase 5 (TG5) is mainly expressed in foreskin ker-atinocytes, epithelial barrier lining, and skeletal muscle (53).The TG5 gene (TGM5) has a TATA-less promoter but con-tains putative binding sites for several transcription factors,including C-Myb, AP-1, NF-�B, and NF-1. GTP and ATPinhibit the protein crosslinking activity of TG5, whereas Ca2�
reverses this inhibition. In addition to full-length TG5 protein,three alternatively spliced isoforms of TG5 have been de-scribed: delta3 (deletion of exon 3), delta11 (deletion of exon11), and delta3-delta11 (deletion of both exons). Full-lengthTG5 and the delta11 isoform are active, whereas delta3 anddelta3-delta11 have low activity. TG5 crosslinks loricrin, in-volucrin, and SPR3 in epidermis (49) and contributes to hy-perkeratosis in ichthyosis and psoriasis patients (48). TG5 in-activating mutations result in skin peeling syndrome (53).TG5 knockout mouse have not been generated.
F. Transglutaminase 6
Transglutaminase 6 (TG6) expression is localized in the hu-man testes and lungs, and in the brain of mice. Human carci-noma cells with neuronal characteristics also express TG6. Inaddition to full-length protein, alternative splicing produces ashort variant that lacks the second �-barrel domain (348). Thecatalytic function of TG6 is activated following proteolyticcleavage of the proenzyme; thus TG6 comprises two polypep-tide chains that are cleaved from a single precursor. TG6knockout mouse have not been generated.
G. Transglutaminase 7
Not much is known about TG7 gene regulation or function.Like TG6, expression is restricted to testes, lungs, andbrain. One report suggested that TG7 transcript levels areincreased in breast cancer cells of patients with poor prog-noses (159). TG7 knockout mice are not available.
H. Factor XIIIa
Plasma TG (FXIIIa) is an important component of theblood coagulation cascade. It is found in platelets, plasma,
FIGURE 2. The TG protein catalytic sites. Amino acid sequencesderived from the catalytic core of each of the nine known transglu-taminases. The catalytic cysteine residue (indicated by arrow) is partof the conserved motif that is required for the transamidation reac-tion. This residue is replaced with alanine in the only catalyticallyinactive member of TGs, band 4.2 protein.
TRANSGLUTAMINASES IN CELL FUNCTION
385Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
on April 4, 2014
Dow
nloaded from
astrocytes, macrophages, dermal dendritic cells, the pla-centa, chondrocytes, synovial fluid, the heart, the eyes, andin cells of osteoblast lineage. Expression of FXIIIa gene(F13A1) is regulated by a myeloid-enriched transcriptionfactor (MZF1-like protein) and two ubiquitous transcrip-tion factors (NF1 and Sp1). Also, two myeloid-enrichedfactors (GATA-1 and Ets-1) induce F13A1 expression.
FXIIIa is the last zymogen activated in the blood coagula-tion cascade (220, 221) and is a heterotetramer composedof two A and two B subunits. The catalytic site of FXIII islocalized in the A subunit, and the B subunit serves as acarrier protein. Upon activation by thrombin-dependentcleavage, the catalytic A subunit dissociates from the B sub-unit, yielding the active enzyme (FXIIIa). In the presence ofCa2�, the enzyme catalyzes crosslinking of fibrin moleculesto stabilize fibrin clots.
FXIIIa also plays a role in inflammation and bone syn-thesis. Crosslinking of the AT1 receptor, catalyzed byFXIIIa, results in enhanced signaling and promotesmonocyte adhesion in hypertensive patients, thereby ac-celerating atherogenesis. F13A1 deficiency is an auto-somal recessive disorder characterized by a lifelongbleeding tendency and impaired wound healing. FXIIIaknockout mice have a clotting defect, increased incidenceof miscarriage, decreased angiogenesis, and tissue re-modeling defects (77, 193, 254, 355).
I. Erythrocyte membrane protein band 4.2(Band 4.2)
Band 4.2 is a unique TG that lacks catalytic activity. ACys-Ala substitution within the active site of band 4.2 isresponsible for the lack of enzymatic activity (FIGURE 2).Band 4.2 is mainly present in erythrocytes, bone marrow,fetal liver, and spleen. Two isoforms of band 4.2 are pro-duced by alternative splicing of the EPB4.2 gene; theshorter isoform is more abundant. Band 4.2 is a majorcomponent of the erythrocyte membrane cytoskeleton andplays an important role in maintenance of membrane integ-rity and regulation of cell stability. Band 4.2 binds to thecytoplasmic domain of the erythrocyte anion transporter(308). Band 4.2 protein expression is partially or com-pletely absent in Japanese recessive spherocytic elliptocyto-sis patients. In these patients, the ankyrin protein is moreloosely associated with the membrane skeleton than in nor-mal individuals. Band 4.2 null mice show alterations in redblood cell function, including spherocytosis and altered iontransport (289).
Most tissues express multiple TG forms (www.ncbi.nlm.ni-h.gov/UniGene) and share common substrates (86). Thismay explain why TG family members can compensate forthe loss of an individual enzyme. Perhaps the best-studiedmodel for compensation is TGM2 gene knockout mouse.
Compensatory activation of the FXIIIa is observed inTG2�/� chondrocytes (266, 335), and TG1 and TG3 leveland activity are increased in TG2�/� joint tissue (86). How-ever, compensation is not observed in all tissues. For exam-ple, in skeletal muscle, loss of TG2 is not compensated (86).
II. REGULATION OF THE PROTEINCROSSLINKING FUNCTION OF TG2
A. Regulation of TG2 Conformation
TG2, also known as tissue transglutaminase, cytosolic typeII, or liver transglutaminase, is a unique member of thetransglutaminase family of enzymes. In addition to Ca2�-dependent posttranslational modification of proteins, it canalso bind and hydrolyze GTP and acts as a G protein (220).Therefore, from catalytic activity point of view, TG2 can bereferred to as bifunctional enzyme, owing to its ability tocatalyze Ca2�-dependent protein crosslinking activity andCa2�-independent GTP hydrolysis. Structurally TG2 iscomposed of four domains: an NH2-terminal �-sandwichthat contains integrin and fibronectin binding sites, a cata-lytic core domain which contains a catalytic triad (Cys277,His335, and Asp358) for acyl transfer reaction, and twoCOOH-terminal �-barrels. Although other members of TGfamily display a similar general structure, TG2 contains aunique guanine-binding site, located in the cleft between thecatalytic core and the first �-barrel. This sequence is codedby exon 10 of the TGM2 gene. The spatial arrangement ofthe four domains in TG2 is altered by interaction with co-factors (FIGURE 3). For example, the GTP/GDP bound formdisplays considerable interaction between the catalytic do-main and domains 3 and 4, which renders TG2 in a closedor compact conformation. This reduces accessibility andactivity of the Ca2�-dependent crosslinking site (217). Incontrast, Ca2� binding alters the conformation by movingdomains 3 and 4 further apart, allowing TG2 to acquire anopen/extended conformation and exposing the catalyticsite. This open configuration is associated with the acyltransfer “crosslinking” reaction (293) (FIGURE 3). Cross-linking activity requires Cys277, which attacks �-glutamylresidues on acyl donor substrates, on proteins and peptides,to drive formation of a thioester intermediate. The resultingacylated enzyme can then either react with an amine donor,typically an �-lysyl side chain of another protein/peptide,which associates with TG2 at a second substrate bindingsite. This results in isopeptide bond (crosslink) formation.Alternatively, in the absence of a suitable amine donor, thethioester is hydrolyzed to form glutamic acid, resulting in anet deamidation (FIGURE 1).
B. Allosteric Regulation of TransamidationActivity
TGs are present in intracellular and extracellular environ-ments, and activity is tightly controlled under physiological
conditions. For example, Ca2�, guanine nucleotides, andredox potential modulate TG2 crosslinking activity. Mu-tagenesis studies identify five potential Ca2�-binding sites(188), and structure studies show that some of these sitesare distorted when TG2 binds GTP/GDP (217). In essence,the protein can function as a G protein or as a transamida-tion enzyme. The transamidation catalytic activity of TG2is allosterically activated by Ca2� and inhibited by GTP,GDP, and GMP. One molecule of TG2 binds up to six Ca2�
with an apparent overall dissociation constant of 90 �M(32). In contrast, GTP and GDP bind TG2 with a dissocia-tion constant of 1.6 �M. GTP-bound TG2 cannot crosslinkproteins, and crosslinking activity is only observed at highcalcium Ca2� concentrations. Because TG2 inside livingcells is primarily GTP/GDP-bound, and calcium concentra-tions are low, it is believed that TG2 is predominantly pres-ent in a crosslinking-inactive form in cells. This may explainwhy overexpression of TG2 is not always associated withincreased intracellular crosslinking activity. In a recentstudy, using TG2 that is covalently conjugated to enhancedyellow (YFP) and cyan fluoresce proteins (CFP) at NH2 andCOOH terminus, respectively, Pavlyukov et al. (286) ob-served closed/inactive TG2 at a perinuclear location. In con-trast, crosslinking-active TG2 was present at the cell mem-brane. Using the fluoresce resonance energy transfer(FRET)-based approach, these authors observed that TG2changed from closed to open conformation in response toionophore-induced calcium influx (286). In addition, Ca-ron et al. (52) reported that an acrylamide-based TG2 in-hibitor induces the open conformation, and a cinnamoyltriazole inhibitor stabilizes the closed conformation (52).On balance, these observations support the contention thatintracellular TG2 is predominantly present as a catalytically
inactive form. Nevertheless, despite low intracellular cal-cium levels, multiple transamidation and crosslinking sub-strates of intracellular TG2 have been identified. This sug-gests that locally increased intracellular calcium and/or asyet uncharacterized interacting proteins may facilitate for-mation of open TG2. It should also be noted that someauthors have suggested that relatively low calcium concen-trations may be sufficient to activate TG2 crosslinking ac-tivity (173, 188). Finding additional TG2-binding proteinsinside the cell (e.g., using yeast-2-hybrid or proteomics ap-proach) and characterizing new TG2-interacting proteinsunder physiological conditions is expected to help addressthis issue.
A puzzling issue is why extracellular TG2 is inactive despitelow GTP levels and high calcium levels (319). A possibleexplanation is the response of the enzyme to oxidative con-ditions in the extracellular environment. TG2 forms intra-molecular disulfide bonds that are required for transamida-tion activity (37, 110), and a switch between the reduced(active) and oxidized (inactive) states of TG2 has been de-scribed (161, 327). This involves a triad of cysteine residues,including Cys370, Cys371, and Cys230, which have anunusually high redox potential (161). Mutation analysisand alkylation studies identified Cys230 as the key redoxsensor. Under oxidizing conditions, an interstrand disulfidebond between Cys230 and Cys370 forms which facilitatesformation of the more stable Cys370-Cys371 disulfidebond. These events inactivate the transamidation activity ofTG2 (293, 327). In contrast, reduction of TG2 results in anopen active conformation (327) (FIGURE 3). Thus the extra-cellular oxidative environment drives inactivation of itstransamidase activity (73, 319). Another factor that con-
GTP
Ca2+
Oxidation
SH
SH S
S
GTP
Closed
Catalytically inactive
Open
Catalytically active
Open
Catalytically inactive
Ca2+
Ca2+
Thioredoxin
FIGURE 3. Three TG2 conformations. Guanine nucleotide (GTP/GDP)-bound TG2 is compact (closed) andcatalytically inactive. Catalytic activity refers to ability of the enzyme to perform the transamidation reaction.The structure of TG2 in its Ca2�-bound form has not been resolved, but a putative Ca2�-binding site homolo-gous to FXIIIa is distorted by GTP/GDP binding to TG2. The binding of Ca2� to the catalytic domain of TG2 altersthe protein to move domains 3 and 4 away from the catalytic domain, thus making the active site accessible(open, catalytically active). Oxidation of the open/active protein results in loss of activity (open, catalyticallyinactive). The oxidized state can be prevented by treatment with thioredoxin. NH2-terminal domain is blue.COOH-terminal domain is red.
TRANSGLUTAMINASES IN CELL FUNCTION
387Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
on April 4, 2014
Dow
nloaded from
tributes to inactivation of extracellular TG2 is nitric oxide(NO), which drives nitrosylation of several Cys residues inTG2 (202). In vivo, a gradual decrease in NO bioavailabil-ity during aging increases TG2 transamidation activity inblood vessels and increases their stiffness due to accumula-tion of crosslinks in the vascular extracellular matrix(ECM) (170, 303). Furthermore, protein kinase A phos-phorylation of TG2(Ser216) stimulates TG2 kinase activ-ity, while inhibiting transamidase function (245, 246). Incontrast, thiol reductases (e.g., thioredoxin) activate extra-cellular TG2, thus antagonizing oxidative inactivation inthe extracellular environment (161). Acting together, thesefactors modulate the redox, nitrosylation, and phosphory-lation states of TG2 to control transamidase activity.
In summary, calcium, guanine nucleotides, and redox po-tential maintain mammalian TG2 activity in at least threedistinct states depending on local conditions (FIGURE 3).The two most common TG2 states, the GTP/GDP-boundform and the Ca2�-bound oxidized form, are catalyticallyinactive, whereas calcium binding activates the reducedform. Some thiol reductases, such as thioredoxin, are likelyto control the redox state of extracellular TG2. In addition,the TG2 crosslinking activity is inhibited through nitrosy-lation and phosphorylation. The physiological implicationsof these allosteric regulatory and posttranslational modifi-cation mechanisms are described in subsequent sections. Itis also important to note that protein-protein interactionsregulate TG2 crosslinking activity. For example, in theECM, TG2 interacts with a number of proteins, includingfibronectin, osteonectin, and integrins (396), and interac-tion with some of these proteins alters enzymatic activity(359).
III. TRANSGLUTAMINASE-REGULATEDCELL SIGNALING
Although investigators originally discovered TG2 as acrosslinking enzyme, new functions have been identified. Itis now appreciated that TG2 interacts with target proteinslocalized in the cytoplasm, membrane, ECM, nucleus, andmitochondria (151, 220, 278). This includes roles for TG2in transamidation and protein-protein crosslinking, as aGTPase/ATPase, as a nonenzymatic adapter, as a scaffoldprotein, and as a regulator of signal transduction.
A. TG2 and FXIIIa Signaling: The CellSurface and ECM
1. TG2 and FXIIIa crosslinking of ECM proteins
TG2 and FXIIIa are released into the extracellular environ-ment via a poorly understood nonclassical secretion path-way (69, 398) where they covalently modify ECM proteinsto form homo- and heteropolymers (3, 220) to enhanceECM stability (220). This crosslinking increases the rigidity
of fibronectin (262) and collagen fibrils (326). The resultingincrease in ECM stiffness enhances fibroblast and osteo-blast adhesion (56, 112) and enhances cell survival, growth,migration, and differentiation by impacting integrin-relatedmechanosensing pathways (34). Endothelial cell adherenceto the TG2-crosslinked fibrinogen �C increases integrinclustering and formation of focal adhesions, thereby elevat-ing outside-in activation of focal adhesion kinase (FAK) andextracellular signal-regulated kinase (ERK) 1/2 activity(30). In addition, ECM protein crosslinking may exposecryptic integrin receptor-binding sites. For example, TG2-mediated polymerization of osteopontin creates a bindingsite for integrin �9�1 binding, leading to enhanced chemot-actic migratory activity of neutrophils (264, 265). A similarmechanism influences vascular smooth muscle cell migra-tion into FXIIIa-crosslinked fibrin gels (255), and impactsangiogenesis (138, 168).
2. Extracellular TG2 regulates the TGF-� signalingpathway
TG2-induced modification can modify growth factor activ-ity in the extracellular environment (148, 220, 381). Forexample, TGF-�1 is a key regulator of ECM remodeling(387), and TGF-�1 activation involves integrins and pro-teases and is influenced by the oxidative environment andmechanical stress. TG2 covalently crosslinks latent TGF�1-binding protein and thereby controls TGF-�1 maturationand activity (191, 362, 380). In addition, in fibroblasts,TG2 increases TGF-� mRNA and protein expression via anuclear transcription factor (NF)-�B signaling mechanism(342). This results in a positive feedback loop in whichTGF-� and TG2 display reciprocal activation of expression(29). In cancer cells, the TGF-�-induced increase in TG2expression promotes epithelial-to-mesenchymal transition(EMT) (51, 196, 315).
3. Extracellular TG2 and FXIIIa enhanceintegrin-mediated signaling
Integrins are important transmembrane adhesion and sig-naling receptors that, although lacking intrinsic enzymaticactivity, regulate a host of intracellular signaling pathways.Integrins are activated by binding to ECM (147). TG2 in-teracts with ECM to enhance cell adhesion and integrin-mediated signaling via direct interaction with �1, �3 and �5integrin (FIGURE 4) (29, 396). TG2 also binds to the gelatin-binding region of fibronectin (297). The integrin-fibronec-tin binding is a weak interaction, while TG2 interactsstrongly with both fibronectin and integrin, and therebyenhances integrin/fibronectin interaction. This facilitatescell attachment to the matrix and activates integrin signal-ing (29). TG2 controls integrin function in cancer cells (229,309) and macrophages (29, 353). The interaction betweenintegrin-bound TG2 and fibronectin is important in variousdisease conditions, including mesenchymal stem cell (MSC)
interaction with infarcted myocardium (325), cancer cellmetastasis (309), and glial scarring (361).
The effect of TG2 on integrin function is evidenced by itsimpact on integrin clustering (155). The mechanismwhereby TG2 promotes integrin clustering is not known;however, the ability of TG2 to oligomerize and interactwith integrin-binding proteins, such as caveolin-1 and tet-raspanins, may promote clustering. Moreover, localizationof TG2 and �1-integrin in lipid rafts and caveolae (400)may enhance ECM interaction with these cholesterol-en-riched membrane microdomains. Therefore, TG2 is likelyto have a role in regulating membrane protein traffickingand compartmentalization during cell signaling.
TG2-induced integrin clustering potentiates integrin-de-pendent intracellular signaling (9, 155). This includes acti-vation of FAK, Src, and p190RhoGAP and increased ex-pression of active, GTP-bound RhoA and its downstreamtarget, ROCK. The net impact of these events is increasedfocal adhesion and actin stress fiber formation leading toenhanced actomyosin contractility (FIGURE 4).
TG2 and integrins are also important in macrophages.TG2�/� macrophages are deficient in phagocytosis owingto altered accumulation of �3-integrin at the engulfing por-tals (353). Efficient signaling via �3-integrin, which is re-quired for formation of the phagocytic cup and effectiveuptake of apoptotic cells, may require TG2 interaction with�3-integrin. TG2 activates downstream signaling targets of�3 integrin, including RhoG and Rac1, which are requiredfor efficient phagocytosis. Furthermore, overexpression of�3-integrin in TG2�/� macrophages partially restoresphagocytosis (354). Mechanistically, TG2 interacts with
the protein milk fat globule EGF factor 8, which is involvedin binding of �3-integrin to apoptotic cells, on the surface ofmacrophages. TG2-mediated stabilization of the �3-integ-rin/milk fat globule EGF factor 8 complex improves phago-cytic uptake of apoptotic cells, likely owing to upregulationof �3 integrin-mediated activation of RhoG and Rac1 sig-naling. Thus TG2 is an integrin coreceptor and signalingpartner.
FXIIIa, in contrast, is an integrin ligand and a covalentintegrin modifier. Platelet integrin �IIb�3 is the most com-mon binding site for plasma FXIII (70) and serves as atransamidation substrate for platelet-derived FXIIIa (66).Of note, extracellular platelet FXIIIa suppresses Ca2�-de-pendent activation of �IIb�3 integrin in cells that adhere tocollagen in a transamidation-dependent manner, implying arole for FXIIIa in preventing excessive platelet accumula-tion on thrombogenic surfaces (194). In addition, plasmaFXIII binds to integrin �v�3 on endothelial cells and medi-ates platelet/endothelial cell interaction by bridging endo-thelial cell �v�3 to platelet �IIb�3 integrins (79). FXIIIaalso stimulates endothelial cell, monocyte, and fibroblastproliferation and migration and inhibits apoptosis by inter-acting with �v�3 integrin on the cell surface to triggerdownstream signaling (76, 80). These effects of FXIIIa leadto increased vascularization and angiogenic actions of en-dothelial cells via activation of vascular endothelial growthfactor receptor (VEGFR) 2, leading to increased expressionof the Egr-1 and c-Jun transcription factors and downregu-lation of mRNA encoding the antiangiogenic ECM proteinthrombospondin-1 (76, 78, 80).
The mechanism of FXIIIa-mediated activation of VEGFR2in endothelial cells involves extracellular crosslinking of
Tcf/Lef
Transcription
PP
TG2
Integrin cluster
FAK PKCα β-cateninSrc
p190RhoGAP
RhoA
ROCK
Stress fiber andfocal adhesionformation
Reduced cellgrowth andmetastasis
Akt1FAKSrcERK1/2Shp2
GPR56TG2 TG2 TG2
TG2 TG2
PDGFRsyndecan-4
syndecan-2βα
PDGF
ECM
Fibronectin LPR5LPR6
dimer
clustering Gαq
Gβ
FIGURE 4. TG2-mediated adhesion/sig-naling at the cell surface. The solid blackarrows indicate TG2-mediated activation ofsignaling. The dotted black line indicatesbinding of activated PKC� to the integrincytoplasmic tails, causing their redistribu-tion on the cell surface. The dashed grayarrows outline activation of syndecan-2 byintracellular PKC� and syndecan-2-medi-ated activation of ROCK, which inducesstress fiber and focal adhesion formation.The dashed black arrow indicates nucleartranslocation of �-catenin, which leads tocomplex formation with Tcf/Lef and activa-tion of gene transcription. The dashed dou-ble black line indicates the unknown path-way of GPR56-induced G�q activation,which inhibits tumor cell growth and me-tastasis. The flat-headed arrows indicateinhibition of signaling.
TRANSGLUTAMINASES IN CELL FUNCTION
389Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
on April 4, 2014
Dow
nloaded from
this receptor to the �3 subunit of �v�3, an integrin thatfacilitates angiogenesis (75). Extracellular FXIIIa promoteshypertrophic differentiation of chondrocytes by enhancingTG2 secretion (164). This effect does not require transami-dating activity, but rather depends on FXIIIa interactionwith �1�1 integrin via a novel integrin-binding site at theFXIIIa NH2 terminus. Moreover, FXIIIa-dependent induc-tion of type X collagen synthesis, a hallmark of chondrocytedifferentiation, is mediated by �1�1-dependent activationof FAK and p38 MAPK signaling (165).
4. Modulation of syndecan-4 signaling byextracellular TG2
The heparan sulfate proteoglycan syndecan-4 localizes atpoints of cell-ECM contact, where it interacts via heparansulfate with the Hep-2 region of fibronectin. It collaborateswith integrins to enhance cell adhesion to fibronectin andfacilitate adhesion-dependent RhoA-mediated develop-ment of focal adhesions, stress fibers, and actomyosin con-tractility (389). Syndecan-4 is an important TG2-bindingpartner (311, 344). A heparan sulfate binding site,261LRRWK265, that may mediate this interaction, is presentin TG2 (366).
The high-affinity interaction of extracellular TG2 with syn-decan-4 activates protein kinase C-alpha (PKC�), which, inturn, binds directly to the cytoplasmic tail of �1 integrin(FIGURE 4). This interaction controls integrin level and dis-tribution on the cell surface as well as integrin stimulationof FAK and ERK1/2 (282, 311, 344, 379, 381). The abilityof activated PKC� to maintain adhesion of fibroblasts andosteoblasts, via formation of ECM-based TG2-fibronectincomplexes with cell surface syndecan-4, is mediated by syn-decan-2 (379, 381). Syndecan-2 does not bind to TG2 butacts as a downstream signaling effector in modulating cy-toskeletal organization via the ROCK pathway. These find-ings imply a major role for the TG2/fibronectin/syndecan-4complex as an adhesive and signaling platform (363). Theintegrin- and syndecan-4-based adhesion systems are likelyto physically interact with each other, as these two receptorsbind to nonadjacent regions of fibronectin, and functionallycollaborate by jointly regulating p190RhoGAP activity andlocalization during cell adhesion to the ECM (25, 343).Hence, this evidence indicates the existence of adhesion/signaling complexes composed of TG2, integrins, synde-can-4, and fibronectin. TG2 controls formation of thesecomplexes owing to its high affinity for syndecan-4 andfibronectin.
Syndecan-4 interaction with integrin-bound TG2 at the cellsurface and/or fibronectin-bound TG2 in the ECM may berequired for response to tissue damage and ECM degrada-tion. Thus increased TG2 expression during wound healingand tissue repair is likely to enhance cell adhesion and sig-naling to increase integrin-dependent adhesion and assem-bly of the fibronectin matrix (343, 367, 380). This may
promote clustering of TG2 binding partners on the cellsurface to enhance adhesion, prevent adhesion-mediatedapoptosis (anoikis), and facilitate cell survival.
5. Regulation of growth factor receptor signaling byextracellular TG2 and FXIIIa
Physical association between integrins and receptor ty-rosine kinases is required for the cell response to ECM andsoluble growth factors (392). A novel example of a role forTG2 in this context is the interaction between integrin andplatelet-derived growth factor receptor (PDGFR). TG2 in-teracts with PDGFR on the surface of fibroblasts and vas-cular smooth muscle cells, and enhances PDGFR interac-tion with integrins (397, 399) by bridging these receptorson the cell surface (FIGURE 4). The interaction with TG2promotes PDGFR clustering, PDGF- and adhesion-inducedPDGFR activation and downstream signaling, and PDGFRturnover. In particular, TG2 increases PDGF/PDGFR-me-diated activation of Akt1 and Shp2 in fibroblasts and vas-cular smooth muscle cells (397). Cell surface TG2 is re-quired for efficient PDGF-dependent fibroblast and vascu-lar smooth muscle cell proliferation and migration. TG2also enhances PDGF-induced vascular smooth muscle cellsurvival and suppresses differentiation. These studies re-vealed a novel function of cell surface TG2 in regulatingPDGFR/integrin signaling and PDGFR-dependent cell re-sponses, by coupling the adhesion-mediated and growthfactor-dependent signaling pathways. These findings alsosuggest that TG2 activity may have a proinflammatory rolein wound healing, tissue fibrosis, vascular restenosis, andtumor metastasis, diverse pathophysiological responsesthat often involve overactivation or dysregulation of thePDGF/PDGFR signaling axis (130).
The interaction of extracellular TG2 with growth factorreceptors may be a general phenomenon since, as notedearlier, TG2 also binds to VEGFR on the surface of endo-thelial cells and modulates VEGF signaling (75). In thiscase, TG2 covalently crosslinks VEGFR to form high-mo-lecular-weight complexes. In VEGF-treated cells, thesecomplexes shuttle to the nucleus to enhance VEGF-inducedERK activation. Extracellular FXIIIa also regulates VEGFRsignaling by enhancing noncovalent interaction betweenVEGFR and �v�3 integrin (75, 78). Future studies shouldidentify the molecular motifs required for association ofTG2 and FXIIIa with growth factor receptors, and addresswhether TG2 and FXIIIa interact with other structurallyrelated receptor tyrosine kinases.
6. Extracellular TG2 as an activator of LRP5/6-mediated �-catenin signaling
Extracellular TG2 binds to the LRP5 and LRP6 (low-den-sity lipoprotein receptor) transmembrane receptors on vas-cular smooth muscle cells (FIGURE 4) (102). Binding of TG2
to LRP5/6 triggers activation of the �-catenin pathway bydriving nuclear translocation of �-catenin, inducing Tcf/Leftranscription factors and decreasing p21Cip1 expression.TG2-mediated activation of �-catenin signaling promotescalcification of vascular smooth muscle cells (102). TG2synergizes with LRP6 in the activation of �-catenin-depen-dent gene expression in COS-7 cells. Interfering with theLRP5/6 receptor function attenuates TG2-induced activa-tion of �-catenin in these cells. Moreover, TG2 binds di-rectly to the extracellular domain of LRP6 which acts as asubstrate for TG2-mediated protein crosslinking (85). Fu-ture studies should assess the contribution of TG2-regu-lated LRP5/6 signaling to pathological conditions, such ascancer and calcification of blood vessels.
7. TG2 signaling via GPR56
GPR56 is an atypical G protein-coupled receptor (GPCR)that is reduced in level in metastatic melanoma cells, andinteracts with cell surface-localized TG2 in tumor stromacells (390). TG2 is proposed as a novel GPR56 ligand thatcooperates in the growth-inhibitory and tumor-suppressiveaction of GPR56; however, additional study will be neces-sary to understand the downstream signaling mechanismsinvolved in this activity.
B. Cytoplasmic TG2 and FXIIIa in CellSignaling
1. TG-mediated monoaminylation of cytoplasmicproteins regulates signaling, the cytoskeleton, andvesicular trafficking
Monoamines, including serotonin, histamine, dopamine,and norepinephrine, are competitive inhibitors of TG cross-linking activity. However, these amines also can be utilizedby TG to monoaminylation target proteins (FIGURE 5)(378). In this context, TG catalyzed serotonylation of theRhoA and Rab4A GTPases is required for cytoskeletal re-arrangement that leads to exocytosis of platelet �-granules,platelet activation, platelet adhesion, and platelet aggrega-tion (377). Given that TG2 and FXIIIa are both abundant inplatelets (220), knockout studies will be required to clarifywhich TG drives this reaction in vivo. In addition, seroto-nylation of Rab3A and Rab27A in pancreatic � cells isinvolved in the release of insulin (285). Although the TGthat is involved is not known, the presence of missensemutations of the TGM2 gene in patients with early-onsettype 2 diabetes mellitus is interesting (294). In one study,TG2 was identified to be the only TG significantly expressedin pancreatic � cells, and its deletion impaired glucose-stimulated insulin secretion (33). Thus, TG2 mutations(989T�G, 992T�A) that impair transamidation activityare linked with early onset of type 2 diabetes (294). How-ever, although these mutations are not found in normalpatients, heterozygous TGM2 mutations are not fully pen-
etrant and do not appear to cause diabetes in these families.Iismaa et al. (150) evaluated the role of TG2 in diabetes andconcluded that glucose homeostasis is TG2 independentand TG2 plays no role in pathophysiology of type 2 diabe-tes. Moreover, neither deletion nor activation of TG2transamidation activity in transgenic mouse models altersbasal or insulin-challenged glucose homeostasis. This isclearly an area that will require future study.
In vascular smooth muscle cells, TG2-mediated serotonyla-tion increases RhoA activity and degradation, which leadsto increased Akt1 activity and inhibition of muscle contrac-tion (124). TG2-mediated serotonylation of RhoA is alsoimplicated in pulmonary arterial remodeling and hyperten-sion (123). Moreover, TG2-mediated serotonylation of�-actin and other contractile apparatus proteins, in vascu-lar smooth muscle cells, increases arterial isometric contrac-tion (383). Similar TG2-mediated modification of smoothmuscle proteins is observed during vasoconstriction (166).Moreover, TG2-dependent serotonylation activates Rac1(another small GTPase) signaling in cortical neurons (74).In each of these examples, TG2-mediated incorporation ofprimary amines into cytoplasmic proteins influences activ-ity, to alter the cytoskeleton and vesicular trafficking (FIG-
Signalmediators
Monoaminereceptor
Cytosol
NH2
Monoaminetransporter
Monoaminehormone
Targetprotein
Monoaminylatedtarget protein
TG2
Hormone effects:Regulation of cytoskeleton and vesicular trafficking
FIGURE 5. Regulation of signaling by TG2-induced monoaminyla-tion. Monoamines (serotonin, norepinephrine, dopamine, etc.) inter-act with the monoamine receptor, but are also delivered to cells viamonoamine transporters. Intracellular monoamines are covalentlycrosslinked to cytoplasmic proteins by TG2. Target proteins includethe small regulatory GTPases (RhoA, Rac1, Rab3A, Rab4a, andRab27A) and cytoskeletal components such as �-actin. These TG2-induced posttranslational modifications alter target protein biologi-cal activity. The biological effects of these TG2-driven modificationsare important in diabetes, thrombosis, and arterial hypertension.
TRANSGLUTAMINASES IN CELL FUNCTION
391Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
on April 4, 2014
Dow
nloaded from
URE 5) (378). This mechanism is likely to be broadly impor-tant in cardiovascular and neurodegenerative disease.
2. TG2-dependent regulation of NF-�B signaling
TG2 crosslinking regulates NF-�B signaling (181, 240).NF-�B belongs to a family of transcription factors that areimportant in inflammatory disease and cancer (178). Undernormal cellular conditions, NF-�B is inactive in the cyto-plasm because of its association with I�B�. Exposure tostress stimuli activates pathways that ubiquitinate I�B� viaa mechanism that involves I�B kinase (IKK)-dependentI�B� phosphorylation (23). Proteasome-mediated degrada-tion of I�B� releases NF-�B, which translocates to the nu-cleus to activate gene expression (121) (FIGURE 6A). IKK-independent NF-�B activation, via a mechanism that in-volves TG2 crosslinking of I�B�, has recently beendescribed (FIGURE 6B) (205, 230). TG2-mediated polymer-ization of I�B� results in I�B� proteasomal degradation,leading to the NF-�B activation (205). TG2 also interacts
directly with I�B� to cause I�B� degradation via a nonpro-teasomal mechanism (198) (FIGURE 6B). These novel TG2-mediated, IKK-independent mechanisms of NF-�B activa-tion are important and suggest that targeting these eventsmay block inflammation (181, 370).
3. TG2-crosslinking of PPAR-� links oxidative stressand inflammation
Cystic fibrosis is caused by mutation of the cystic fibrosistransmembrane conductance regulator (CFTR) leading tochronic airway inflammation. Interestingly, human bron-chial epithelial cells that express functionally deficientCFTR express high levels of TG2, leading to increasedcrosslinking and sequestration of anti-inflammatoryPPAR-�. This suggests a role for TG2 in mediating theinflammatory response in cystic fibrosis patients (227). TG2crosslinking promotes accumulation of polymerized ubi-quitinated PPAR-� in perinuclear aggresomes and, as a re-sult, PPAR-� interaction with the N-CoR-histone deacety-
Inflammation (chronic)BA
p65 p50
p65 p50
IκBα
IκBα
IκBαIκBα
polymerization
IκBαdegradation
IκBα-Pdegradation
IκBαIκBα IκBα
Drug resistanceand metastasis
Promoter
TG2
TG2
TG2
TGFβ/ROS–
Proteasomeindependent
Cytosol
Nucleus
Proteasomedependent
HIF1αTG2
Promoter
HIF1β
HIF1αp65 p50
TG2IκBα
Promoter
p65 p50
Inflammation (acute)
p65 p50
p65 p50
IκBα
IκBα
IKKα/β
TNFα/IL1, etc.
P
FIGURE 6. TG2 expression results in constitutive activation of NF-�B via noncanonical pathway. Acuteinflammation is a tightly regulated physiological process in which NF-�B is transiently activated as a result ofIKK-complex mediated phosphorylation and degradation of the inhibitory protein I�B�. As I�B� is one of thedownstream targets of NF-�B, its expression results in feedback inhibition of NF-�B, which limits the inflam-matory response (A). In contrast, chronic inflammation is associated with constitutive activation of NF-�B owingto aberrant expression of TG2 (B). TG2 binds to I�B� resulting in its rapid degradation via a nonproteasomalpathway. Alternatively, TG2-mediated covalent crosslinking of I�B� may promote proteasomal degradation ofI�B� polymers (broken arrows). TG2-activated NF-�B regulates the expression of multiple target genes thatplay roles in cell survival, invasion, and drug resistance. One of the TG2/NF-�B target genes is HIF-1�, atranscription factor known to promote an aggressive phenotype in cancer cells.
lase 3 complex is reduced, thereby facilitating inflammatoryresponse gene expression (283). Moreover, inhibition ofTG2-mediated crosslinking restores normal PPAR-� leveland reduces inflammation in both cultured CFTR-defectivecells and cystic fibrosis tissue (227).
Oxidative stress in CFTR-defective cells also increasesSUMO ligase activity. SUMO ligase inhibits activatedSTAT-� leading to reduced TG2 SUMOylation which re-duces TG2 turnover and increases TG2 level and activity(224). This evidence is consistent with the finding of ele-vated reactive oxygen species and increased TG2SUMOylation in lung tissue in mutant �Phe508-CFTRmice, a model of cystic fibrosis, and suggests that control ofTG2 turnover serves as a central link between oxidativesignaling and inflammation in cystic fibrosis (223, 224).These findings established cytoplasmic TG2 as a novel me-diator that connects oxidative stress and inflammation.
4. TG2 crosslinking of beclin-1 and inhibition ofautophagy
In addition to an impact on protein aggregation, stress-induced accumulation of cytoplasmic TG2 and activationof TG2 crosslinking inhibits autophagy. Specifically, PKC�-mediated induction of TG2 expression in pancreatic carci-noma cells inhibits autophagy by crosslinking beclin-1 toinhibit its function (7, 277). This mechanism also operatesin CFTR-deficient lung cells where oxidative stress-relatedTG2-induced crosslinking of beclin-1 leads to sequestrationof beclin-1, and beclin-1 interacting proteins, in the ag-gresomes (223). These findings suggest a central role forTG2 in beclin-1 depletion, beclin-1 sequestration in ag-gresomes, and inhibition of autophagy, in patients withcystic fibrosis.
5. Cytoplasmic TG2 and EGF/EGFR signaling inepithelial cancer cells
EGFR activity, which is frequently increased in human ma-lignant cells, increases TG2 expression in cervical, breast,and lung epithelial cancer cells (15, 214). Moreover, induc-tion of TG2 expression and TG2-dependent transamida-tion are essential for EGF-mediated migration, invasion(15), and anchorage-independent cancer cell growth (213).The EGF-induced response is mediated by Ras- and Cdc42-induced activation of PI3K and NF-�B and requires TG2-mediated upregulation of Src expression (15). TG2-inducedSrc expression is associated with transamidation-dependentformation of cytoplasmic ternary complexes of Src, TG2,and keratin 19 (213). EGF signaling, via Ras and JNK,causes TG2 activity to accumulate at the leading edge ofcells. Accumulation of cytoplasmic TG2 at this location isnecessary for cell migration and requires interaction of TG2with heat shock protein 70 (Hsp70) (38). Similarly, EGF-induced upregulation of TG2 expression in TNF-related
apoptosis-inducing ligand (TRAIL)-resistant lung cancercells elevates MMP-9 expression, secretion, and activity,and this enhances the migration and invasiveness of thesecells (214). The mechanism of TG2 action in this contextremains to be defined; however, JNK/ERK signaling path-ways are implicated in this process (214). Thus cytoplasmicTG2 is a novel mediator of EGF/EGFR-induced signalingand oncogenesis in epithelial cancer cells that involves TG2transamidation-dependent and -independent actions.
6. Regulation of angiotensin signaling byFXIIIa-induced dimerization of AT1 receptors
GPCRs constitute a large family of cell surface receptors.GPCR homodimers and heterodimers influence many re-ceptor-related functions, including ligand binding, mem-brane localization, signaling, and desensitization (116).However, until recently, little was known about the patho-physiological importance of GPCR dimerization in vivo. Aninsightful study revealed a novel mechanism of FXIIIa-me-diated dimerization of the angiotensin II AT1 receptor inmonocytes (1). Crosslinking of AT1 dimers, via glutamineresidues, in the tail domain enhances receptor signaling.Moreover, FXIIIa-deficient individuals lack crosslinkedAT1 dimers, whereas patients with the common atherogenicrisk factor hypertension have elevated levels of thesedimers. The presence of these dimers correlates with en-hanced adhesion of angiotensin II-stimulated monocytes toendothelial cells (1). Importantly, in monocytes, these AT1
dimers promote atherogenesis, and inhibition of FXIIIacrosslinking activity reduces AT1 dimer formation and re-duces disease severity in atherosclerosis in mice. ThusFXIIIa, via an impact on AT1 receptors, appears to have arole in maintaining atherogenesis.
7. FXIIIa-mediated crosslinking of Glu-tubulin altersmicrotubule dynamics and controls osteoblast matrixdeposition
The transamidating activity of TG2 and FXIIIa, accompa-nied by collagen type I and fibronectin deposition into theECM, is associated with osteoblast differentiation. How-ever, the molecular mechanisms linking these events remainlargely unknown (266). A recent study showed that inhibi-tion of FXIIIa-mediated transamidation in osteoblasts re-sulted in microtubule destabilization as evidenced by re-duced Glu-tubulin levels and blocked formation of Glu-tubulin oligomers (11). In turn, blockage of this activityinhibited vesicle-based secretion and deposition of collagentype I and fibronectin. Thus this study provides potentialmechanistic clues regarding the role of transamidation andprotein crosslinking by FXIIIa in the regulation of ECMprotein secretion and deposition that leads to osteoblastdifferentiation.
TRANSGLUTAMINASES IN CELL FUNCTION
393Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
on April 4, 2014
Dow
nloaded from
8. Cytoplasmic TG2 as an atypical GTPase andmediator of GPCR-induced signaling
Although the discovery that TG2 can bind to and hydrolyzeGTP occurred in 1987 (2), researchers did not establish alink between this activity and GPCR function until 1994when a GTP-binding protein, termed Gh�, was isolatedwith the �1B adrenergic receptor. The study showed thatGH� was TG2 (FIGURE 7) (259). Other researchers ob-served a similar role for TG2 relative to the �1D adrenergic,thromboxane A2, oxytocin, and follicle-stimulating hor-mone receptors. In response to exposure to agonists of thesereceptors, PLC�1 activation leads to increased inositol1,4,5-trisphosphate level (19, 20, 104, 152, 153, 220, 241,279, 373).
The GTPase activity and associated signaling capacity ofTG2 is independent of its transamidating activity (58).Moreover, given the high intracellular GTP levels observedunder normal physiological conditions, the activity of TG2/Gh� as a GPCR-linked GTPase is physiologically relevant.TG2 binds to and hydrolyzes GTP with an affinity andcatalytic rate similar to that observed with the canonical �subunits of heterotrimeric and monomeric G proteins de-spite the absence of the four consensus GTP-binding motifscommon to the classical G proteins. Mutating Arg580 inTG2 results in a 100-fold reduction in GTP-binding affinityand eliminates GTP inhibition of TG2 transamidation ac-tivity (27, 28). The activation/deactivation GTPase cycle of
TG2 functions similarly to that of other heterotrimeric Gproteins (FIGURE 7) (220, 241). Agonist binding inducesexchange of GDP with GTP and dissociation of TG2/GTPfrom Gh�. Deactivation occurs when TG2 hydrolyzes GTPto GDP and reassociates with free Gh�. Two regions inTG2, R564-D581 and Q633-E646, are involved in TG2interaction with �1 adrenergic receptors and activation ofthe GTPase function (103).
The role and specificity of TG2 in GPCR signaling is deter-mined not only by the range of receptors it interacts withbut also by the downstream effectors. PLC�1 is a key down-stream target of TG2/�1 adrenergic receptor coupling (21,81, 104). The Val665-Lys672 region in the COOH termi-nus of TG2 is involved in binding and activation of PLC�1
(146), which hydrolyzes phosphoinositide and increases in-tracellular free calcium level (104, 175). PLC�1 acts as botha guanine nucleotide exchange factor and a GTP hydrolysis-inhibitory factor for TG2 (21). TG2 also regulates othersignaling pathways via its GTPase activity. It participates inERK1/2 activation in cardiomyocytes (206). In fibroblastsand endothelial cells, overexpression of TG2, or transami-dation-inactive TG2, inhibits adenylyl cyclase activity,whereas knockdown of TG2 reverses this effect (119). TG2also increases adenylyl cyclase activity in neuroblastomacells, but this effect requires its transamidating activity(356), implying that TG2 can regulate signaling pathwaysdifferentially dependent upon cell type. Also, TG2 activatesthe large-conductance Ca2�-activated K� channels in vas-
GhβCRT
GhβCRT
GhβCRT PLCδ1
1
GPCR
Cytosol
Agonist
TG2Ca2+Ca2+
Ca2+
Ghα
TG2GDP
Ghα
TG2GTP
Ghα
TG2
Pi
PIP2 DAG
IP3Ghα
TG2GDP GTP
5
7
2 6
3
4
FIGURE 7. TG2 GTPase activity andTG2/Gh� signaling. The GDP-TG2/Gh�-CRT/Gh� complex is inactive. CRT is calre-ticulin. 1: Agonist stimulation of transmem-brane G protein-coupled receptors (GPCR)induces exchange of GDP with GTP anddissociation of GTP-bound TG2/Gh� fromCRT/Gh�. 2: GTP-bound TG2/Gh� acti-vates PLC�1. 3/4: Signal termination oc-curs with GTP hydrolysis and reassociationof GDP-bound TG2/Gh� with free CRT/Gh�. 5: PLC�1 promotes coupling effi-ciency by stabilizing GTP-TG2/Gh�. 6:PLC�1 catalyzes hydrolysis of phosphatidyl-inositol 4,5-bisphosphate to diacylglyceroland inositol 1,4,5-triphosphate, causingan increase in intracellular Ca2� level. 7:Switching off GTPase activity of TG2/Gh�is triggered by elevated intracellular Ca2�.
cular smooth muscle cells (207), and GTP-bound TG2binds to the cytoplasmic tail of �5 integrin to inhibit vascu-lar smooth muscle cell migration (177). In contrast, TG2promotes fibroblast migration via its GTP-binding activity(330). Finally, TG2 GTPase activity regulates cell-cycle pro-gression in fibrosarcoma cells (242) and mediates �1 adren-ergic receptor-induced proliferation of hepatocytes (388)and visceral smooth muscle cells (92).
Despite significant progress in understanding of the GTPasefunction of TG2, the pathophysiological role of the associ-ated intracellular signaling remains poorly understood. Forinstance, cardiac-specific overexpression of TG2 fails to al-ter PLC�1 activity, suggesting that TG2 acts as a TG ratherthan a GTPase in this context (323). Nonetheless, TG2GTPase activity is markedly reduced in ischemic heart, sug-gesting that loss of this activity may be an important factorin cardiac failure (146). TG2 GTPase activity may also beinvolved in liver regeneration owing to its involvement in �1
adrenergic receptor signaling (304, 388). Overall, TG2GTP-ase signaling is prosurvival and cytoprotective, as mutantsof TG2 defective in GTP binding appeared to induce apo-ptosis in NIH3T3 and HeLa cells independent of theirtransamidating activity (82). Moreover, the GTPase func-tion (FIGURE 3), and intracellular localization of TG2, areimportant in protecting cells from death caused by oxygenand glucose deprivation (67, 126).
C. Signaling Function of TG2 in the Nucleus
1. Nuclear TG2 transadmidation and regulation ofgene expression
TG2 nuclear localization was initially reported in hepato-cytes (128). Nuclear TG2 comprises �5% of the total TG2cellular pool (278). TG2 displays crosslinking and GTPaseactivity in this cellular compartment (321) and associateswith chromatin (209). The crosslinking activity of nuclearTG2 has a role in histone and transcription factor transami-dation which alters gene expression (236, 340). Perhaps thebest example of transamidation regulation of gene expres-sion is the TG2 impact on Sp1 function (FIGURE 8B) (317,339, 340). Sp1 is involved in alcohol-induced apoptosis, aprocess in which TG2 crosslinks Sp1 resulting in Sp1 inac-tivation (339). This leads to decreased expression of growthfactor receptors, such as c-Met, which results in cell death.TG2 regulation of Sp1 function is also observed in free fattyacid-treated hepatocytes and in patients with nonalcoholicsteatohepatitis (340).
2. TG2 as a transcriptional coregulator
Recent reports indicate that nuclear TG2 functions nonen-zymatically as a transcriptional coactivator (5, 106). TG2-dependent reduction in MMP-9 gene transcription in car-
diomyoblasts is mediated by direct noncovalent binding ofTG2 to c-Jun, thereby inhibiting c-Jun/c-Fos dimerizationand blocking interaction with the AP-1 site in the MMP-9gene promoter (FIGURE 8C) (5). A similar mechanism oper-ates in cortical neurons where nuclear TG2 interaction withHIF-1� prevents dimerizing with HIF-1� (FIGURE 8D)(106). This interaction attenuates expression of Bnip3 andother genes containing the hypoxia-response element(HRE) to reduce neuron death in patients with ischemia andreduce stroke. In addition, the protein kinase activity ofnuclear TG2 may be involved in the phosphorylation ofhistones H1 and H3 (248), p53 (247), and retinoblastomaprotein (245) (FIGURE 8A).
D. Signaling by Mitochondrial TG2
Although TG2 does not have a classical NH2-terminal mi-tochondrial targeting signal, the protein does associate withmitochondria in various cell types (291, 299). In the major-ity of cells, mitochondrial TG2 localizes at the outer mito-chondrial membrane and the inner mem-brane space; however, in 10% of cells, TG2 is present at theinner mitochondrial membrane and mitochondrial matrix(278, 299).
1. TG2 crosslinking of mitochondrial proteins isinvolved in the mitochondrial-driven apoptosis
The TG2 sequence includes 204LKNAGRDC211, which is70% homologous to the BH3 domain of Bcl-2 family pro-teins, suggesting that TG2 is a novel apoptotic BH3 protein(299). Mutation of the highly conserved Leu204 residue inthis motif attenuates TG2-mediated staurosporin-inducedneuroblastoma cell death, confirming earlier findings thatTG2-induced hyperpolarization of the mitochondrial mem-brane sensitizes cells to the intrinsic pathway of pro-grammed cell death (291, 299). Also, the TG2 BH3 peptideinteracts with proapoptotic Bax but not with anti-apoptoticBcl-2, and TG2-Bax interaction increases during cell death.In contrast, TG2 inhibits calcium-induced apoptosis inHEK293 cells by covalently crosslinking Bax and down-regulating Bax expression (61). Thus the proapoptotic orantiapoptotic function of mitochondrial TG2 may dependon the cell type and cell death inducer.
In addition, TG2-mediated transamidation of proteins inmitochondria has been reported (278, 305). Prohibitin is amembrane-bound chaperone essential for correct folding ofthe Hsp70 and Hsp90 respiratory chain components. Theorganizing protein Hsp60 cooperates with prohibitin andforms a membrane-tethered import motor complex in-volved in the unfolding of preprotein domains, whereas theATP synthase � chain is a key component of complex V ofthe respiratory chain. In apoptotic neural cells, these pro-teins are transamidated and crosslinked by TG2 (26, 275).The bifunctional adenine nucleotide translocator (ANT1),
TRANSGLUTAMINASES IN CELL FUNCTION
395Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
on April 4, 2014
Dow
nloaded from
a protein involved in ADP/ATP exchange and a core com-ponent of the permeability transition pore complex in theinternal mitochondrial membrane, is also crosslinked byTG2 (228). In general, TG2-mediated covalent modifica-tion of mitochondrial proteins does not occur in normaltissue; however, modification is likely in patients with “mi-tochondrial diseases,” including cardiovascular ischemia/reperfusion injury and neurodegenerative disorders such asHuntington disease. Accordingly, TG2-catalyzed crosslink-ing of mitochondrial matrix �-ketoglutarate dehydrogenase(68) and aconitase (186) is observed with decline in energymetabolism. Moreover, high-molecular-weight aggregatesof these enzymes are observed in Huntington disease pa-tients having elevated TG2 crosslinking activity. Additional
studies are needed to establish the precise role of TG2-mediated crosslinking in the mitochondrial apoptosis path-way.
2. TG2 PDI enzymatic activity and the mitochondria
TG2 also acts as a PDI to regulate mitochondrial function(228, 231, 305). Deletion of TG2 leads to defective disulfidebond formation in NADH-ubiquinone oxidoreductase(complex I), succinate-ubiquinone oxidoreductase (com-plex II), cytochrome c oxidase (complex IV), and ATP syn-thase (complex V). TG2 PDI activity may also control therespiratory chain by modulating protein complex forma-tion (231). Another target of TG2 PDI activity is ANT1
A
B
– – –
SP1
c-Jun c-Fos
PGC-1α promoter
c-Met promoter
Unaffected cells
HIF1β HIF1α
TG2
TG
– –
PGC-1α promoter
Huntington’s diseaseTG2
TG
Unmodifiedhistones
TG2
TG
Ad/Sc Ad/Sc
Aminylatedhistones
–
+ + +
SP1SP1
Inactivation
SP1 binding site
SP1
C
MMP9 promoter
TG2
AP1 binding site
D
Bnip3 promoter
TG2
Hypoxic response element
FIGURE 8. TG2 as a novel transcriptional regulator in the nucleus. A: TG2-mediated transcriptional regula-tion in patients with Huntington disease. Under normal conditions, the level of TG2 transamidation activity is lowand does not interfere with transcription of key genes involved in the regulation of mitochondrial and metabolicfunction (e.g., PGC-1�). In Huntington disease, TG2 activity increases, leading to aminylation of histones,thereby yielding an increased net positive charge that promotes tighter packing of DNA with histones. Thischromatin alteration represses target gene transcription. Reduced expression of PGC-1� and related genescontributes to the mitochondrial and metabolic dysfunction observed in Huntington disease. B: TG2-dependentenzymatic crosslinking of Sp1 transcription factor in the nucleus causes Sp1 inactivation and inhibits Sp1-mediated transcription of the prosurvival gene c-Met in hepatocytes. This transamidation-dependent mecha-nism mediated by nuclear TG2 is involved in liver steatohepatitis. C: TG2 binds noncovalently to c-Jun in thenucleus and prevents c-Jun/c-Fos dimerization, thereby decreasing AP1 transcription factor-dependent tran-scription of the MMP-9 gene in cardiomyoblasts. This nonenzymatic mechanism, mediated by nuclear TG2, isthought to be involved in ECM remodeling. D: TG2 interacts noncovalently with HIF-1� in the nucleus andprevents its dimerization with HIF-1�, thus inhibiting HIF-1 binding to the hypoxic response element (HRE) in thepromoter of the Bnip3 gene leading to reduced Bnip3 transcription in neuronal cells. This nonenzymaticnuclear TG2-driven mechanism plays a role in the prosurvival effect of TG2 in stroke patients. TG indicatestransamidating activity of TG2, and Ad/Sc indicates adapter/scaffolding nonenzymatic activity of TG2.
(228). ANT1 oligomerization is essential for activity, andTG2�/� mice have increased thiol-dependent ANT1 oli-gomer formation and elevated ANT1 ADP/ATP exchangeactivity in heart mitochondria. Thus the PDI activity of TG2reduces the level of oligomerized ANT1 and inhibits trans-porter activity by sequestering ANT1 monomers and pre-venting oligomer formation by direct binding to ANT1. Inaddition, the presence of TG2 is required for Bax/ANT1colocalization and interaction in mitochondria (228).Taken together, these findings reveal an important role forTG2 PDI enzymatic activity in vivo and indicate the exis-tence of a novel pathway that links this activity with regu-lation of mitochondrial physiology.
Future studies to understand TG2 trafficking and shuttlingamong various cellular compartments is important. Howdo cells regulate these trafficking events? What are the TG2molecular signature motifs (e.g., “barcodes”) for its tar-geted delivery to different organelles? What are the com-partment-specific binding partners of TG2? Which signal-ing pathways regulate distribution? Defining these issuesshould help to determine the compartment-specific func-tions of TG2. For example, the vexing lack of understand-ing of how intracellular TG2 moves to the cell exterior(398) is an important example of a trafficking mechanismthat needs to be clarified.
IV. TGS IN CELL DIFFERENTIATION
TG activity is fourfold higher in blastocysts than in two-cellembryos (225), suggesting that transglutaminases have animportant role in development. Little is known about whichTG isoforms are responsible for this activity. However,more is known about the role of transglutaminases in spe-cific tissues which will be discussed in this section.
A. Epidermis
The epidermis is a stratified epithelium formed by special-ized cells called keratinocytes (94, 95). It consists of thebasal, spinous, granular, and cornified layers. The prolifer-ating cells are in the basal layer, and the spinous and gran-ular and cornified layers display progressive differentiation(95). The cornified layer consists of completely differenti-ated cells that form the body surface. At the cellular level,the final stage of keratinocyte differentiation begins in theepidermal spinous and granular layers with accumulationof cornified envelope precursors on the inner surface of theplasma membrane (55, 95, 96, 99). These proteins arecrosslinked by TGs to form the rigid cornified cell envelopethat gives the differentiated keratinocyte its protectiveproperties (46, 329). TG crosslinks cornified envelope pre-cursors including involucrin, loricrin, filaggrin, and smallproline-rich proteins (50, 95, 97, 132).
At least four TGs are expressed in human epidermis: TG1,TG2, TG3, and TG5. TG2 is detected in basal keratino-
cytes, although the precise role of TG2 in skin cells is notknown (48, 184). TG1 and TG3 and TG5 are produced inthe differentiated cells of the spinous and granular layers(48, 184) where they act on substrates to assemble the cor-nified envelope (50, 95, 391). In addition, TG1 mediatescovalent crosslinking of ceramides to involucrin, whichcontributes to maintenance of the epidermal permeabilitybarrier (263).
TG1 and TG5 mutations produce defects in the cornifica-tion process. Mutation of TG1 in either the catalytic cys-teine, or surrounding region, is associated with lamellarichthyosis (140, 281, 302, 346). A similar phenotype isobserved in mice lacking the TG1 gene (234). These micehave defects in epidermal barrier formation and die shortlyafter birth. Interestingly, other TGs cannot functionally re-place TG1 action despite their ability to crosslink loricrin,involucrin, and other precursors (47). Similar to the criticalrole of TG1 activity in cornification, a loss-of-function mu-tation of TG5 is associated with acral peeling skin syn-drome (53). In contrast, a role for TG2 in keratinocytedifferentiation has yet to be demonstrated, and TG2-knock-out mice do not have any obvious skin defects (261). Like-wise, the role of TG3 in keratinocyte differentiation re-mains to be elucidated.
B. Nervous System
1. Neuronal differentiation
Dendritic extension and axonal branching are key featuresof neurogenesis and often used as markers of neuronal dif-ferentiation, and catalytically active TG2 may modulate therate and extent of neurite outgrowth. For example, an in-crease in total TG activity is associated with neurite out-growth in murine neuroblastoma cells (226), and forcedexpression of TG2 in SH-SY5Y neuroblastoma cells causesspontaneous neurite outgrowth and neuronal marker ex-pression following retinoic acid treatment (322). In con-trast, elevated expression of transamidating-inactive(C277S) TG2 represses neuroblastoma cell differentiation(341). These findings suggest a requirement for the TG2transamidating function in this process.
The specific molecular mechanisms underlying induction ofneuronal differentiation by the transamidating activity ofTG2 are emerging. TG2 protein is prominently localized atthe tips of outgrowing neuritis, suggesting that it has a rolein stabilizing extended structural projections (357). In ad-dition, a role for TG2-dependent activation of c-Jun NH2-terminal kinase (JNK) signaling in neurite outgrowth hasbeen proposed (322), as has activation of adenylyl cyclaseby the transamidating active form of TG2, leading to pro-tein kinase A-mediated CREB activation (356). This mech-anism seems to be specific to neuronal differentiation, be-cause TG2 inhibits adenylyl cyclase activity and decreases
TRANSGLUTAMINASES IN CELL FUNCTION
397Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
on April 4, 2014
Dow
nloaded from
cAMP levels in Balb-c 3T3 human fibroblasts and bovineaorta endothelial cells (119), and in MSCs undergoingchondrogenic differentiation (270).
In contrast to the proposed role of the transamidating ac-tivity of TG2 in neuronal differentiation, the role of thisenzyme in neurodegeneration is not clear. Neuronal TG2attenuates cell death in ischemic conditions (106, 107), butfacilitates cell death in excitoxic conditions (358). ThusTG2 accumulation in brains of Parkinson, Huntington, andAlzheimer’s patients may be a compensatory, protectiveresponse (158, 163, 185, 211).
2. Macroglia
Glial cells maintain homeostasis, form myelin, and supportand protect neurons. Similar to neurons, glial cells originatein the neural tube and neural crest of the developing em-bryo. The most common type of macroglial cell is the astro-cyte which has numerous projections that help anchor neu-rons to the blood supply. They also secrete regulatoryproteins that promote the myelinating activity of oligoden-drocytes, another abundant type of microglial cell. Oligo-dendrocytes form a specialized myelin membrane, whichcoats and protects axons in the central nervous system.Genetic ablation of TG2 results in delayed remyelination ininjured animals, suggesting a role for TG2 in glial differen-tiation. In addition, in vitro studies demonstrated that TG2regulates astrocyte migration, which is required for properremyelination in vivo and for differentiation of myelin-pro-ducing oligodendrocytes from precursor oligodendrocytes(361). Moreover, both of these processes are attenuated bythe TG2-specific inhibitor KCC009 {(S)-[3-(4-hydroxy-phenyl)-2-N-(phenylmethyloxycarbonyl)]aminopropanoicacid N’-(3=-bromo-4=,5=-dihydro-5=-isoxalyl)methylamide}(361). The precise mechanism of TG2 action remains to bedetermined, but reported data implicate a role for RhoA inTG2-dependent differentiation of oligodendrocytes (360).
C. Immune System
Accumulating evidence indicates an important role for TG2in cell-mediated immunity and suggests that TG2 contrib-utes to the immune response via regulation of differentia-tion in phagocytes, monocytes, neutrophils, and T cells.During monocyte differentiation, expression of FXIIIa andTG2 is induced and may contribute to neutrophilic andmonocytic differentiation (8, 208, 251, 252). Genetic andpharmacology studies identify TG2 as a positive regulatorof neutrophilic differentiation. For example, genetic abla-tion of TG2 in murine neutrophils results in diminishedsuperoxide anion production and impaired extravasation,indicative of delayed differentiation (22). Moreover, TG2silencing in the human promyelocytic leukemia cell lineNB-4 leads to a significant delay in the neutrophil differen-tiation process and downregulation of expression of genes
related to the innate immune system (72). Similar to itseffects on neutrophilic differentiation, TG2 activity playsan important role in monocytic differentiation to macro-phages and dendritic cells, the antigen-presenting cells thatact as messengers between innate and adaptive immunity.TG2 regulates cell adhesion and migration in differentiatingmacrophages (8, 251), whereas in dendritic cells, TG2 me-diates maturation of antigen-presenting cells in response tobacterial lipopolysaccharide (LPS) (233). Moreover, inhibi-tion of TG2 with KCC009 (62) reduces cytokine produc-tion and dendritic cell differentiation in vitro (233). Alteredresistance to LPS-induced septic shock in TG2-null micefurther confirms that TG2 has a significant role in dendriticcell maturation (233). In addition, FXIIIa may regulate ter-minal maturation in both macrophages and dendritic cells,and FXIIIa-deficient human monocytes demonstrate re-duced phagocytosis (307). In contrast, FXIIIa overexpres-sion results in enhanced dendritic cell motility (157).
D. Connective Tissues
Connective tissues, including cartilage, bone, adipose tis-sue, ligaments, and tendons, differentiate from MSCs (17,18, 325). TG2, but not FXIIIa, is expressed in MSCs (325);however, FXIIIa expression is induced in cells of severallineages during differentiation. Accumulating evidence sug-gests a role for TGs in regulation of MSC differentiation.TG2 can crosslink and stabilize cytoskeletal proteins in-cluding actin, tubulin, vimentin, and myosin, suggesting arole in establishing cellular phenotype (65, 87, 101). Inaddition, extracellular TG2 stabilizes the cytoskeleton bypromoting integrin clustering and regulating FAK, RhoAGTPase, ROCK, and mitogen-activated protein kinase(MAPK) activity (155). TG2-mediated crosslinking in-creases the stiffness of the ECM (262, 314, 326), and this issensed by integrin receptors (43, 180) and converted intosignaling output (316). MSC differentiation is controlled bymatrix stiffness (100, 137, 260), and TG2-modified colla-gen scaffolds promote chondrogenic differentiation of hu-man MSCs (314). Extracellular matrices with “high stiff-ness” favor osteoblast differentiation over neural, muscle,and chondrogenic differentiation (100, 260). Thus MSC-associated TG activity, both intracellular and extracellular,regulates MSC differentiation.
1. Cartilage and chondrocytes
Endochondral bone formation is a key event in skeletaldevelopment. This is a process in which cartilage is firstgenerated and then replaced by bone (219). The first step inendochondral bone formation is chondrocyte differentia-tion, which begins with MSC condensation and progressesthrough a series of maturation stages including resting, pro-liferative, prehypertrophic, and hypertrophic stages (219).Terminal differentiation of chondrocytes is characterizedby deposition of mineralized ECM that is associated with
cell death and replacement of cartilage with bone. TG2regulates the transition to the prehypertrophic stage, whichis characterized by cessation of ECM deposition and re-duced cell proliferation (270). Expression of FXIIIa andTG2 is markedly increased during this transition (267,270), and both enzymes are found in the intracellular andextracellular compartments during terminal differentiation(4, 268, 349). Premature expression of TG2 in culturedMSCs accelerates prehypertrophic differentiation and in-hibits deposition of the cartilaginous matrix, whereas treat-ment with the TG2 inhibitor ERW-1065 (382) enhances thematrix synthesis (270). In vivo, increased TG2 expressiondelays endochondral ossification in chicken embryos byblocking chondrocyte maturation during prehypertrophicdifferentiation. TG2-mediated inhibition of protein kinaseA activity is implicated as a major mechanisms underlyingthis regulation.
In contrast to endochondral cartilage, which is replaced bybone, articular cartilage remains cartilaginous throughoutlife. Articular chondrocytes are maintained in the restingstage and do not undergo proliferation or terminal differ-entiation (90). Both TG2 and FXIIIa are expressed byhealthy articular chondrocytes (301, 349) and may blockchondrocyte differentiation. In addition, a role for TG2 andFXIIIa in cell response to mechanical stress and abrasiveforce can be postulated based on the pattern of FXIIIa ex-pression and localization of TG activity to condylar regions(269).
Diseases and conditions such as osteoarthritis and aging areassociated with increased expression of both TG2 andFXIIIa. In vitro studies have shown that extracellular TG2enhances hypertrophic differentiation and induces matrixmineralization in cultured articular chondrocytes via �5�1-integrin-mediated activation of Rac1 and p38 in a fibronec-tin-independent manner (165, 335). Interestingly, these ef-fects require the GTP-bound form of TG2. Whether thismechanism is active in vivo where extracellular TG2 maynot be GTP-bound, remains to be determined. TG2 mobi-lization to the cell surface is regulated by FXIIIa interactionwith �1�1-integrin. Thus atypical maturation of articularchondrocytes may be regulated by a network of TGs andnot require transamidation activity (164). In addition,TG2-modified calgranulin/S100A11 may trigger abnormalmaturation of articular chondrocytes (54). Because hyper-trophic differentiation of articular chondrocytes is pro-posed as a mechanism that drives osteoarthritis, these find-ings implicate TGs in osteoarthritis. Indeed, severe kneeosteoarthritis in a guinea pig model is associated with in-creased synovial levels of TG2, but not FXIIIa (143), andcartilage degradation in the mouse model of injury-inducedosteroarthritis is reduced in the absence of TG2 (274).These proteins may also contribute to inflammatory osteo-arthritis by triggering illegitimate maturation of articularchondrocytes. These findings suggest that identification of
downstream mediators of TG-dependent chondrogenic dif-ferentiation may lead to development of novel therapeuticsto treat inflamed joints without affecting normal homeosta-sis in cartilaginous tissue.
2. Bone and osteoblast differentiation
Osteoblasts are bone cells responsible for production andsecretion of the collagen type I (COL I)-based mineralizedbone matrix. Osteoblast lineage differentiation is under themaster control of the transcription factors RUNX2 andOsterix, which control MSC commitment to the osteogeniclineage and the preosteoblast-to-osteoblast transition, re-spectively (192, 237, 324). Preosteoblasts form a transientprovisional fibronectin matrix (31), while mature osteo-blasts deposit the permanent COL I matrix (250, 336, 337).The advanced stages of osteoblast maturation are charac-terized by expression of alkaline phosphatase (114, 115),which is essential for matrix mineralization and a charac-teristic feature of fully differentiated osteoblasts (237). Thefinal stage of bone and osteoblast differentiation is osteo-blast transformation into osteocytes, which serves a mecha-nosensory function in the bone matrix and controls boneremodeling via regulation of osteoblast and osteoclast func-tion (237, 324). TG2 and FXIIIa are expressed during os-teoblast differentiation from early stages through osteocyteformation (11, 12, 171, 257, 266). Maturation and matrixmineralization in cultured osteoblasts are accelerated by theexpression of exogenous TGs, suggesting that TGs act aspositive autocrine regulators of osteoblast differentiation.Indeed, TG inhibitors arrest osteoblast differentiation at avery early stage, dramatically decrease COL I and fibronec-tin deposition and matrix accumulation as well as alkalinephosphatase expression and matrix mineralization (11, 12).These results suggest that TGs are essential for osteoblastdifferentiation. However, the precise molecular mechanismof this regulation is not known. During differentiation, TG2levels remain constant, and the TG2 protein is retained atthe cell surface in a high-molecular-weight complex that hasno detectable crosslinking activity but may possess signal-ing activity (11). It has been postulated that TG2 on the cellsurface promotes cell adhesion and spreading (56, 111,112, 129, 367, 381) and may regulate osteoblast differen-tiation via this mechanism. In addition, in cultures of ma-ture osteoblasts supplemented with ATP, TG2 may regulateECM mineralization by acting as an ATPase in the ECM(39, 256, 258, 374) and increasing phosphate levels forhydroxyapatite precipitation or via phosphate signaling topromote terminal differentiation of osteoblasts into osteo-cytes (402).
In early osteoblasts, cellular FXIIIa associates with theplasma membrane and appears to be required for microtu-bule stabilization and collagen trafficking and secretion(11), a function that likely involves TG-mediated crosslink-ing of detyrosinated tubulin to form homotypic or hetero-typic polymers. Tubulin has been described as a TG sub-
TRANSGLUTAMINASES IN CELL FUNCTION
399Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
strate in other systems (87). At the preosteoblastic stage,FXIIIa is secreted as a latent proenzyme that is incorporatedinto fibronectin and COL I matrix fibrils. Matrix-associatedFXIIIa seems to be activated to stabilize the permanentfibronectin/COL I matrix, which in turn promotes osteo-blast maturation (292). Expression, cellular localization,and secretion of FXIIIa are strongly stimulated by extracel-lular COL I and MAPK signaling (292).
A lack of any obvious skeletal phenotype in mouse modelslacking either TG2 or FXIIIa suggests that these two TGshave complementary functions and may act to compensatefor each other (266). In this respect, increased FXIIIa, TG1,and TG3 expression in bone and tendons in TG2-knockoutmice has been observed (86, 266, 273, 338).
3. Muscle and myoblasts
Formation of cardiac, smooth, and skeletal muscle involvesdifferentiation of myoblasts from MSCs. Expression of themuscle-specific contractile myofibrillar proteins (e.g., myo-sin, actin) is characteristic of myoblast differentiation, as ismyoblast fusion during skeletal muscle maturation (40).TG inhibitors interfere with proper myoblast fusion result-ing in anomalous elongation of the fused cells, which isindicative of defective cytoskeletal stabilization (35). TG-mediated crosslinking of the myosin chain occurs in differ-ent muscles, and this may contribute to myoblast differen-tiation and elongation (63, 139, 174). Therefore, stabiliza-tion of cytoskeletal elements via TG-mediated crosslinkingmight be the mechanism of TG-dependent regulation of celldifferentiation, which is especially pertinent in cells withreduced levels of ECM, such as muscle cells.
4. Tendons and ligaments
The role of TGs in differentiation of tenocytes has yet to beextensively investigated despite evidence of expression ofTG1 and TG2 in normal tendons (273). In mice, TG2-knockout tendons are anatomically normal. However, fur-ther biomechanical tests and injury models are needed totest a role for TG2 in tendon stabilization and response toinjury (273). Whether TG1 can compensate for loss of TG2expression in tendons also remains to be determined. TG3and TG5 have been found in tendons, and FXIIIa expres-sion is selectively increased in injured tendons. The sourceof FXIIIa in injured tendons remains to be determined, butmay be related to blood vessel injury. Interestingly, in-creased TG activity in tendocytes is associated with disease.TG2 and FXIIIa activity are increased in tendocytes ex-posed to carboxymethyl-lysine collagen and high glucoselevels, mimicking an AGE-rich environment and diabetes,respectively (284, 300). These findings suggest that in-creased TG crosslinking activity and crosslink formationmay contribute to the stiffening and pathological calcifica-tion of tendons in diabetic patients (10, 235). Similarly,
elevated TG2 level is observed in calcified ligamentum flavaof the spine (393), which provides additional evidence ofthe role of TG2 in pathology-related calcification of ten-dons.
V. ROLE OF TG2 IN CANCER
Cancer is a disease of dysregulated cell growth and differ-entiation. Genetic and epigenetic changes induced in re-sponse to chronic stressors (chemicals, infections, environ-mental) can constitutively activate survival signaling cas-cades and render them independent of growth factors. Inaddition to genetic and epigenetic alterations, cancer cellsrequire additional input (probably induced by tumorstroma) to gain metastatic competence and ability to sur-vive in host environment. In this context, chronic inflam-mation is considered to be an important factor in cancerinitiation and progression (364, 365). Because TG expres-sion, particularly TG2, is frequently increased in responseto inflammation (223, 224, 227), it is likely that TGs play arole in cancer. Several lines of evidence suggest the involve-ment of TGs during cancer development even though themolecular mechanisms remain controversial. TG2 expres-sion is low in primary tumors, but TG2 level is increased indrug-resistant and metastatic tumors (240, 280, 309, 372,394). These observations suggest that TG2 may supportcancer progression (6, 42, 229, 239, 271). In the followingsection, we discuss evidence that TG2 expression repro-grams inflammatory signaling pathways in epithelial cancercells to confer drug resistance and metastatic phenotypes.
A. TG2, Inflammation, and Cancer
A role for inflammation in cancer is well documented (151,364, 365). Moreover, many inflammatory cytokines includ-ing TGF-�, TNF-�, IL-1, and IL-6, which are secreted attumor cells, are known TG2 inducers (199, 296, 333). Ex-cessive deposition of collagen by fibroblasts is observed intumors in the form of a desmoplastic response, which isresponsible for the clinical presentation of the tumor as alump or dense stroma (16, 376) (FIGURE 9). The desmoplas-tic response of tumors appears to require TG2-catalyticactivity (FIGURE 9). Increased TG2 expression in responseto inflammatory cytokines, such as TGF-� in fibroblastsand epithelial cancer cells, accelerates the synthesis and de-position of fibronectin and collagen, whereas extracellularTG2 crosslinks them to stabilize the ECM. TG2-catalyzedcrosslinking of ECM proteins can result in adverse out-comes, including diabetic nephropathy, kidney scarring,and atherosclerosis (60, 167, 312), and probably plays asimilar role in the tumor desmoplastic response (FIGURE 9).The desmoplastic response involves an interplay betweenthe invading tumor cells and altered ECM (16), hence theTG2-mediated alteration of the ECM is likely to impacttumor cell behavior. For example, breast cancer progres-
sion requires collagen crosslinking, ECM stiffening, andincreased focal adhesion formation (212), and TG2-medi-ated crosslinking may alter the stiffness of the ECM andthereby promote a malignant phenotype.
B. TG2, Drug Resistance, and Metastasis
Resistance to therapy and metastasis are hallmarks of ad-vanced cancer. Identification of tumor-encoded genes thatpromote drug resistance and metastasis is an importantgoal, as these proteins may serve as cancer biomarkers andalso offer targets for therapy.
EMT is a major pathway that governs cell behavior duringcancer progression. EMT is an embryonic process that canbe reactivated in adult tissues in response to epigeneticchanges (347). EMT is considered to be an attempt of thehost to control inflammation and heal damaged tissue;however, in pathological contexts this response can causedamage. Epithelial tumor cells undergoing EMT lose epi-thelial tight junction proteins including E-cadherin and gainthe expression of mesenchymal markers such as SMA,
FSP1, vimentin, fibronectin, and desmin (172, 243, 347).These cells are involved in intravasation, movement of can-cer cells in the circulation, extravasation, and micrometas-tases formation.
TG2 activates FAK, Akt, and NF-�B signaling, which areknown to induce cancer cell EMT (315, 369, 371) (FIGURE9). Inhibition of TG2 expression suppresses and increasedTG2 expression enhances invasiveness and resistance tochemotherapy (131, 145, 280, 309, 368, 395). In addition,microvesicles, shed by TG2 overexpressing cancer cells, canconfer transformed characteristics (e.g., anchorage-inde-pendent growth and enhanced survival capability) on nor-mal fibroblasts and epithelial cells (14). Although TG2alone is not sufficient to transform fibroblasts, it is likelythat it collaborates with other proteins to mediate trans-forming action of the cancer cell-derived microvescles.
C. TG2, EMT, and Cancer Stem Cells
TG2 expression is associated with induction of EMT andacquisition of stem-cell phenotype (195–197, 315). For ex-
Inflammation
EMT
Myofibroblasts
Cell survivalinvasion
Drug resistanceMetastasis
Desmoplasticreaction
Transdifferentiation intomesenchymal state
Modified ECMECM
Active-TGF-β
Latent-TGF-β
Immunecell
Epithelial cells
TGF-β/TNF-α/IL-6/ROS–
Fibroblasts
Collagen, fibronectin
Fibrosis
TG2
TG2
NFκB
FIGURE 9. TG2 reprograms the inflammatory signaling circuitry. Inflammatory cytokines produced by infil-trating immune cells induce TG2 expression in cancer cells and host cells (tumor-associated fibroblasts). Dueto low Ca2� and high GTP levels, intracellular TG2 is predominantly present as a cryptic enzyme and serves asa scaffold protein. For example, it interacts with I�B� and results in its degradation via a proteasome-independent pathway. This results in increased activation of NF-�B. Activated NF-�B translocates to thenucleus and induces expression of multiple genes. TG2 is also a target gene for NF-�B, which results in anauto-induction loop between NF-�B and TG2. NF-�B regulates genes involved in promoting drug resistance andmetastasis. TG2 expression in fibroblasts induces the synthesis of ECM proteins (e.g., collagens, fibronectin).Extracellular TG2, in turn, crosslinks these and other proteins and stabilizes the ECM. Low turnover ofTG2-modified ECM, coupled with enhanced synthesis of ECM component proteins, results in fibrosis anddesmoplastic response, which further promote aggressive phenotype in cancer cells.
TRANSGLUTAMINASES IN CELL FUNCTION
401Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
ample, mammospheres generated from TG2-overexpress-ing nontransformed mammary epithelial cells (MCF10A)differentiate into mammary glandlike structures includingMuc1-positive luminal cells and integrin �6-positive basalcells in response to prolactin treatment (197). In addition,there is a correlation among TG2, EMT, and acquisition ofa stem cell-like phenotype in ovarian cancer cells (51). Can-cer stem cells exhibit intrinsic resistance to chemotherapyand treatment of MCF-7 breast cancer cells with doxorubi-cin selects a small subpopulation of cells with stem cellcharacteristics that express high levels of TG2 (44). Theseresults suggest that TG2 induces EMT and other stem celltraits in cancer cells.
Some information is available regarding TG2-regulated sig-naling pathways that drive these processes. TG2 expressionresults in constitutive activation of FAK, Akt, and NF-�Bsignaling (182, 369, 371), all of which are involved in can-cer progression (172, 243, 276, 347, 375). NF-�B plays aprominent role in cancer progression, stemming from itsability to activate pro-growth, pro-metastatis, and anti-ap-optotic genes (89, 328). Recently, a novel feedback loopwhere TG2 activates NF-�B and NF-�B, in turn, drives TG2expression was identified (41) (FIGURE 9). In cancer cells,this TG2/NF-�B feedback loop may be self-amplifying, be-cause high TG2 expression and elevated NF-�B activity arefrequently observed in late-stage cancers. Most efforts toinhibit NF-�B activation in cancer cells have focused onsmall molecules that block the IKK kinase activity. In viewof the observation that TG2-induced activation of NF-�B ismediated through IKK-independent mechanism (198),these inhibitors may not be effective. High-level NF-�Bwould, in turn, confer resistance to cell death and promoteEMT and metastasis (178, 187).
NF-�B collaborates with TG2 to regulate expression ofEMT transcriptional regulators including Snail, Twist,Zeb1, and Zeb2 (196, 315). TG2, in complex with the p65subunit of NF-�B, is recruited to the promoter of theSNAIL gene, to increase Snail levels (187). TG2 also inter-faces with TGF-�, a potent inducer of EMT. In breast can-cer cells, TGF-� induced EMT requires TG2 expression(196). These findings suggest that TG2 may be an importantregulator in the TGF� signaling pathway that is requiredfor EMT, and that crosstalk between TG2 and NF-�B maypromote metastatic competence.
HIF-1 is an important hypoxia-response transcription fac-tor that is regulated by NF-�B (142, 169, 318) and TG2(195, 196, 198, 315) and has an important role in cancercells. TG2-expressing cells display high basal levels ofHIF-1� expression even under normoxic conditions, andsuppression of either TG2 or NF-�B (p65/RelA) reducesHIF-1� level. Chromatin immunoprecipitation studies re-veal that TG2 forms a complex with p65/RelA and that thecomplex binds to the NF-�B binding site in the HIF-1�
promoter (FIGURE 6B). Like NF-�B and TG2, HIF-1� reg-ulates EMT-related signaling, including accumulation snail,twist, and Zeb1 (127). These results suggest that TG2-in-duced NF-�B activation regulates HIF-1� expression.
Taken together, these observations suggest that aberrantepigenetic regulation of TG2 expression in cancer cells canreprogram inflammatory signaling networks (NF-�B acti-vation, expression of HIF-1�, Snail, Twist, and Zeb) thatinfluence EMT and stemness to promote drug resistanceand the metastatic phenotype (FIGURE 10). Doxorubicin-resistant breast cancer cells express high levels of TG2 as-sociated with hypomethylation of TGM2 promoter,whereas in doxorubicin-sensitive cells, which show no de-tectable expression of TG2, the TGM2 promoter is hyper-methylated (6). In addition, treatment of drug-sensitive cellswith 5-azadC restores TG2 expression and reduces sensitiv-ity to doxorubicin (6). These findings suggest that TG2 maybe a promising anti-cancer therapeutic target, as in vivosilencing of TG2 by delivery of liposomal-siRNA inhibits
Hypomethylation ofTGM2 gene promoter
Drug resistance& Metastasis
Inflammation (chronic)TGF-β/TNF-α/IL-6/ROS–
TG2
NFκB
HIF1αVEGF
Angiogenesis
Zeb, Twist, Snail
EMTStem cell
traits
Warburg effect
Cell growthCell survival
FIGURE 10. TG2 regulates inflammatory signaling, drug resis-tance, and metastatic phenotype. Chronic exposure to inflammatorycytokines (produced by tumor-infiltrating immune cells) results inepigenetic regulation of TG2 and initiates the feedback cycle whereTG2 activates NF-�B, and NF-�B further increases TG2 expression.HIF-1� is a downstream target of TG2-induced NF-�B. Increasedexpression of HIF-1� (even under normal oxygen conditions) resultsin activation of multiple downstream target genes, which are in-volved in reprogramming of cancer cells for altered metabolism, andangiogenesis. Moreover, the level of key transcription repressors(e.g., Snail, Twist, Zeb) is upregulated in TG2/NF-�B/HIF-1�-ex-pressing cells, which leads to transdifferentiation of epithelial cells tothe mesenchymal state (EMT), an important step in tumor metas-tasis. TG2-induced EMT promotes stem-cell traits in cancer cellsand confers resistance and self-renewal ability for successful sur-vival and growth at metastatic sites.
the growth and dissemination of pancreatic and ovariancancer cells and renders them sensitive to chemotherapeuticdrugs in a nude mouse model (145, 368).
VI. REGULATION OF TG2 EXPRESSIONAND TG2-REGULATEDTRANSCRIPTIONAL PROCESSES
TG2 is gaining increasing attention for its role as a tran-scriptional regulator. In this section, we focus on how TG2expression is regulated and how TG2 regulates gene expres-sion.
A. Control of TG2 Gene Expression
Expression of TG2 is mainly controlled at the transcrip-tional level. Retinoids are well-known inducers of TG2 ex-pression (83, 253, 290), and induction of TG2 expressionby retinoids is controlled by the retinoic acid receptors(RARs) and retinoid X receptors (RXRs) (FIGURE 11).RARs and RXRs belong to a superfamily of nuclear recep-tors that bind to DNA as dimers. A study by Nagy et al.(253) showed that retinoid-dependent induction of murineTG2 expression is mediated by a tripartite response elementlocated in the TG2 gene promoter. This study demonstratedthat retinoic acid response elements are present at �1720bp (RRE-1) and �1731 bp (RRE-2) in the murine TG2promoter. Accordingly, RAR-RXR heterodimer and RXRhomodimer receptor complexes bind to the murine TG2promoter to regulate retinoic acid-dependent induction. Inaddition to RAR and RXR binding, the murine TG2 pro-moter requires an additional cooperating segment of thepromoter (HR-1) to mediate activation of TG2 expressionby retinoic acid (253). In the first characterization of theTG2 promoter, Lu et al. (222) reported that a 1.74-kb DNAregion at the 5=-flanking region of the human TG2 pro-moter did not contain retinoic acid response elements, asthe luciferase reporter they used failed to show induction inresponse to treatment with retinoic acid. However, subse-quent studies establish that human TG2 expression is in-creased by retinoids, and an unusual binding site present inthe TG2 promoter is likely to mediate induction (24, 357,401).
TG2 expression is essential for retinoic acid-induced differ-entiation of human neuroblastoma cells (322, 357). Themyc oncoproteins block differentiation and promote prolif-eration of malignant cancer cells (287), and TG2 expressionis repressed by N-myc in neuroblastoma cells and by c-Mycprotein in breast cancer cells, which may contribute to can-cer cell proliferation and migration owing to a lack of dif-ferentiation. N-myc appears to suppress TG2 expression byrecruiting histone deacetylase 1 to the Sp1 sites on the TG2gene promoter (218, 341). Also, TG2 expression is epige-netically regulated by hypomethylation of CpG islands in its
promoter in chemoresistant breast cancer (6), non-smallcell lung cancer (280), and glioma (93) cells.
Although TG2 expression is suppressed in some cancercells, it is increased in others, especially in cells resistant tochemotherapy or isolated from metastatic sites. TG2 ex-pression is elevated in pancreatic carcinoma (372), breastcarcinoma (239), malignant melanoma (109), ovarian car-cinoma (145), lung carcinoma (280), and glioblastoma(395). As outlined above, inflammation (201) and hypoxia(313) are important in cancer, and TG2 expression is up-regulated by these processes. Indeed, the TG2 promotercontains response elements involved in inflammatory anti-hypoxic signaling (118, 154, 199, 244).
Cytokines increase TG2 expression in neuronal cultures(216). Also, LPS stimulates TG2 expression in several mam-malian cell models, and metastatic tumor antigen 1 facili-tates LPS-induced TG2 expression (120). An IL-6 responseelement (�1190 bp) is present in the human TG2 promoter(83), and IL-6 treatment of HepG2 human hepatoblastomacells (333), human macrophages (117), and other cell types(232, 271) increases TG2. In addition, an NF-�B bindingsite is located at �1328 bp in the human TG2 promoter.NF-�B binding to this sequence in the TG2 promoter isobserved in CCl4-treated rats (244). TG2 expression alsocan be induced by treatment with TNF-� in HepG2 cells(199).
TGF-�1 increases TG2 expression in apoptotic hepatocy-toma cells (118), human dermal fibroblasts (296), and hu-man retinal pigment epithelial cells (295). The murine TG2promoter contains a TGF-� response element at �868 bpupstream of the transcription start site (298). TGF-�1 in-creases TG2 promoter activity in Mv1Lu cells, an epithelial-like cell line derived from normal mink lung. Interestingly,treatment with TGF-�1 inhibits TG2 promoter activity inMC3T3 murine pre-osteoblastic cell. Moreover, BMP2 andBMP4, which are members of the TGF-� superfamily, alsoreduce TG2 promoter activity (298), indicating that the roleof TGF-� family members in modulation of TG2 gene ex-pression is stimulus and cell type dependent.
HIF-1 increases TG2 expression (154), and promoter dele-tion analysis revealed a sequence at 367 bp upstream of thetranscription start site in the TG2 promoter that is requiredfor this action (154). A comparison of TG2 promoter se-quences shows that the response element is conserved in thehuman, murine, and guinea pig TG2 promoters. TG2 ex-pression is also increased in response to hypoxia or ischemiain several other models (107, 350). Indeed, TG2 mRNA andprotein level are elevated after stroke in rats, mice, andgerbils (107, 149, 350).
Lu et al. (222) characterized a TATA box (�29 bp) andCAAT box (�96 bp) in the human TG2 gene promoter
TRANSGLUTAMINASES IN CELL FUNCTION
403Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
on April 4, 2014
Dow
nloaded from
(FIGURE 11). A GC-rich region, located between the TATAbox and the CAAT box, encodes two Sp1 transcriptionfactor binding motifs at �54 and �43 bp, and additionalSp1-binding motifs are located in the 5=-untranslated regionof the TG2 gene at positions �59 and �65 bp. Four 3= halfsites of NF-1 are also present in the 5=-untranslated regionat �4 and �12 bp. TG2 expression can be upregulated inrat astroglial cells in response to various growth factors andsteroids (45), indicating that the putative glucocorticoidresponse element of the TG2 promoter is likely to be func-tional. In addition to the glucocorticoid response element,AP1 (�183 bp) and AP2 (�634 bp) response elements arelocated in the murine TG2 promoter (222). These findingsindicate that multiple signaling pathways regulate TG2gene expression. FIGURE 11 shows known and potentialregulatory elements in the human TG2 promoter. It will beimportant in future studies to confirm the functional role ofthese sites.
Variant forms of TG2 are also produced. The full-length687-amino acid form of TG2 is the most abundant; how-ever, other shorter TG2 variants are produced. Fraij et al.(113) isolated a truncated 548-amino acid form of TG2(TGase-H) in retinoic acid-treated human erythroleukemiacells that differs from full-length TG2 in the last 10 aminoacids (GKALCSWSIC versus EKSVPLCILY) (113). The 5=end of this short isoform is the same as that of the long formup to base 1747, but the sequence differs from base 1748 tothe termination codon. The divergent sequence containedan intron-exon boundary (CTGGTAA), which suggests al-ternative splicing (113). Another study described a similartruncated 548 amino acid isoform in an Alzheimer’s diseasebrain (64) along with a 555 amino acid isoform with aCOOH-terminal sequence of GKPCVTGAFVDRGLTTC
instead of QTQPITCQPSTQPGFIPR (64). Injured rat spi-nal cords express a 640-amino acid isoform of TG2, with29 different amino acids at the COOH terminus (105).Truncated forms are also present in cytokine-treated ratastrocytes (249), leukocytes, vascular smooth muscle cells,and endothelial cells (203). The common feature is the pres-ence of variable sequences at the COOH terminus. Becausethese short isoforms lack residues at the COOH terminus,GTP binding in TG2 is often impaired (203). Although themechanism by which these short isoforms are generated hasyet to be understood, some may result from intron read-through (64, 105, 113, 203, 249). Further studies areneeded to determine how these isoforms are generated andto identify their physiological function.
B. Transcriptional Regulation by TG2
TG2 transcriptionally regulates several important targets(FIGURE 12). For example, TG2 induces differentiation andneurite growth in SH-SY5Y cells via a mechanism that mayinvolve cAMP signaling, as TG2 induces adenylyl cyclase,increases cAMP level, and enhances CREB activity in SH-SY5Y cells (356). These responses require TG2 transami-dating activity (356). TG2 also increases CREB activity byinteracting with protein phosphatase 2, a protein that de-phosphorylates and inactivates CREB. The TG2-mediatedincrease in CREB activity leads to increased MMP-2 expres-sion (310). In addition, membrane recruitment of TG2 isincreased during erythroid differentiation of K562 cells inresponse to trans-retinoic acid and is associated with acti-vation of Akt. TG2 overexpression increases CREB phos-phorylation, an effect that is abrogated in the presence ofwortmannin, a phosphatidylinositol 3-kinase inhibitor, im-
FIGURE 11. Regulatory elements of human TG2 gene expression (TGM2). Glucocorticoid response element(�1399 bp), NF-�B (�1338 bp), IL-6 response element (�1190 bp), AP-2 (�634 bp), HRE (�367 bp), AP-1(�183 bp), CAAT box (�96 bp), GC box: Sp1-binding motifs (�54 bp, �43 bp, �59 bp, and �65 bp), TATAbox (�29 bp), and NF-1 (�4 bp and �12 bp). TG2 gene expression is upregulated in response to inflammationand hypoxia. Human TG2 expression is upregulated by treatment with retinoic acid, and potential retinoic acidresponse elements (RAR and RXR) are located in the human TG2 promoter. [From Gundemir et al. (125a),with permission from Elsevier.]
plying that TG2 activates Akt signaling leading to activa-tion of CREB (176). Similarly, cytosolic TG2, as discussedabove, can activate NF-�B, either by transamidating I�B�or facilitating its degradation. Given that NF-�B signalingcan facilitate neuritogenesis, this is another pathwaythrough which TG2 could facilitate neurite outgrowth(345).
TG2 signaling is also important in cancer. Elevated TG2expression is associated with acquisition of TRAIL resis-tance in lung cancer cells and increased MMP-9 expression,while TG2-siRNA reduces MMP-9 expression and restoresTRAIL sensitivity (214). TG2 knockdown also reduces cellattachment, migration and invasion, and secretion ofMMP-9 and MMP-1 in A431-III cells, an invasive cancercell line (59, 215). These studies indicated that increasedTG2 expression increases MMP-9 expression, perhaps byactivating NF-�B. This response is not uniformly observed,as other studies report that TG2 expression suppressesMMP-9 promoter activity and reduces MMP-9 level. Thiswas associated with reduced jun/fos recruitment to AP1transcription factor binding elements in the MMP-9 pro-moter (5). The reason for the different outcomes of thesestudies is not known.
The studies above focused on modulation of transcriptionby cytosolic TG2. However, nuclear TG2 also regulatestranscription. Although the levels of TG2 in the nucleus aresignificantly lower than in the cytoplasm, the presence ofnuclear TG2 is well-documented (67, 106, 126, 209, 320).The mechanism whereby TG2 translocates to the nucleus isnot known. TG2 has two putative nuclear localization sig-nals (NLSs) located at amino acids 259–263 and 597–602,respectively (236), and TG2 interacts with importin-�3(288), which may mediate nuclear translocation. However,study of this process is complicated, as mutation of the NLS
site at amino acids 259–263 also inactivates the transami-dating function of TG2 which complicates interpretation.However, mutation of the putative NLS site at amino acids597–602 does not prevent nuclear localization of TG2(236). It has also been suggested that TG2 contains a puta-tive nuclear export signal (LHMGLHKL) at the COOHterminus (amino acids 657–664) (200). A preliminarystudy showed that deleting part of the �-barrel domain ofTG2 (amino acids 591–687) resulted in TG2 accumulationin the nucleus, suggesting the presence of a nuclear exportsignal (200). Although these observations are intriguing,additional studies are needed to better understand this reg-ulation. TG2 nuclear accumulation is also stimulus depen-dent. Treatment of A375-S2 human melanocytic cells withsphingosine increases nuclear TG2 (334), and in astrocytes,growth factors cause nuclear TG2 accumulation (45). In-creased nuclear TG2 is also observed in differentiating NB4cells (22). Hypoxic stress is a critically important signal thatincreases nuclear TG2 in neurons (106), and nuclear TG2accumulation occurs in neurons in mice after stroke and instroke patients in the areas of infarction (107). Moreover,TG2 suppresses hypoxia-induced HIF-1� signaling in SH-SY5Y cells (106) and primary rat neurons (Gundemir andJohnson, unpublished data), and the suppression does notrequire TG2 transamidation activity (125, 126). Further-more, both wild-type and transamidation-inactive TG2forms attenuate ischemic cell death in both SH-SY5Y cellsand primary neurons (106). After stroke, infarcts in miceoverexpressing human TG2 selectively in neurons are muchsmaller than those in wild-type mice, and stroke-inducedproapoptotic gene expression is attenuated (107). Thus, inneurons and neuronal cell models, hypoxia causes accumu-lation of TG2 in the nucleus and increases cell survival.
In contrast, TG2 plays a different role in epithelial cells.Kumar and Mehta (198) demonstrated formation of a TG2
A
PGC-1αα
CREB
TG2
NFκB
Inflammation
TG2
cyt-c
E
BTG2
HRE
C D
Ethanol
IκB
HIF1βSP1
cAMP
Hypoxia Huntington’s disease
FIGURE 12. TG2 regulation of transcrip-tion. A: TG2 expression is upregulated byinflammation. TG2 transamidates I�B�leading to NF-�B activation in response toinflammation. B: increased TG2 expressionin hypoxia leads to HIF-1� dependent tran-scription of genes via hypoxia response ele-ment (HRE). C: TG2 suppresses PGC-1�and cyt c expression in mutant huntingtin-expressing cells. D: TG2 crosslinks Sp1 inresponse to ethanol treatment leading toreduced Sp1-dependent gene transcrip-tion. E: TG2 increases cAMP expression,leading to activation of CREB. [From Gun-demir et al. (125a), with permission fromElsevier.]
TRANSGLUTAMINASES IN CELL FUNCTION
405Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
on April 4, 2014
Dow
nloaded from
complex with the p65/RelA subunit of NF-�B and bindingof this complex to the HIF-1� promoter. This binding re-sults in increased transcription and accumulation of HIF-1�protein, even under normal oxygen conditions. The in-creased HIF-1, in turn, stimulates transcription of genesencoding glycolytic proteins including GLUT-1, hexokinaseII, and lactate dehydrogenase-A. The net impact is increasedglucose uptake, lactate production, and reduced mitochon-drial respiration (Kumar and Mehta, unpublished data).
There are also studies suggesting that TG2 is associatedwith neurodegeneration (13, 204, 385). TG2 expressionand activity are increased in patients with neurodegenera-tive disorders including Alzheimer’s (163, 185), Hunting-ton’s (179, 211), and Parkinson’s diseases (13). A recentstudy suggests that TG2 regulates transcription in Hunting-ton’s disease. PGC-1� and cyt c mRNA levels are lower inmutant huntingtin-expressing striatal cells compared withwild-type cells (236), and treatment of the mutant hunting-tin-expressing cells with a TG2 inhibitor or TG2 knock-down restores PGC-1� and cyt c mRNA level. Additionally,TG2-knockout mouse fibroblasts contain higher levels ofcyt c than wild-type mouse fibroblasts. Furthermore, in thepresence of mutant huntingtin, the interaction of TG2 withPGC-1� and cyt c promoters is increased, resulting in sup-pression of transcription. In these experiments, TG2transamidation activity is necessary for suppression of pro-moter activity. Considering the fact that TG2 expression isincreased in Huntington disease patient brains (211), sup-pression of PGC-1� and cyt c transcription by TG2 in mu-tant huntingtin expressing cells may contribute to diseaseprogression.
TG2 accumulates in the nucleus in ethanol exposed rathepatocytes (57). Gene expression analysis reveals that eth-anol treatment reduces c-Met expression to a greater extentin TG2 wild-type hepatocytes compared with TG2 knock-out mouse hepatocytes. c-Met is the receptor for hepatocytegrowth factor and is involved in liver regeneration (144).This is associated with TG2 crosslinking of Sp1 which isrequired for c-Met expression (339). This is consistent withpatient data, as TG2-mediated Sp1 crosslinking is observedin liver from patients with alcohol-related liver disease(384). In addition to increasing the expression of TG2,treatment with ethanol appears to increase TG2 transami-dation activity (339, 388). These findings indicate that nu-clear TG2 can modulate gene transcription in response toethanol. However, is it not known whether this effect re-quires TG2 transamidation activity.
VII. PERSPECTIVES AND FUTUREDIRECTIONS
Despite a growing understanding of the biological functionsof TGs and their mechanism of action, many questionsremain unanswered in a host of cell types. Several areas that
would benefit from further study are outlined below. Someof these areas are highly relevant to disease. For example,while the role of transglutaminases in differentiation is es-tablished, efforts are only beginning to address the role ofthese enzymes in the stem cell niche and the role of TGs instem and progenitor cells. This area has important implica-tions for tissue renewal and cancer cell survival. Diabetes isalso an important area that would benefit from additionalstudies. Knowledge is limited regarding the role of TG2 indiabetes and in adipocyte differentiation. In this context,FXIIIa was recently identified as a top obesity gene in agenome-wide screen for single nucleotide polymorphismlinked to basal metabolic index. The significant associationof FXIIIa with obesity was further confirmed in a largeEuropean ENGAGE consortium study of more than 21,000unrelated individuals as well as in the GenMets cohortstudy (261a). The interplay among various TG forms isanother area that is important in the context of disease. Thecompensatory response following loss of TG2 in specifictissues requires more detailed analysis. There is now sub-stantial evidence for compensation by other TG forms, butmore needs to be done. This analysis will advance our un-derstanding of the biological functions of TG-mediatedcrosslinking in different tissues and may also offer focusedapproaches to target TG in various diseases. The area ofinhibitors of transglutaminase is also an area for additionalinvestment, as it would be extremely useful to have com-pounds that specifically inhibit each of the TG forms.
Understanding the role of various TGs in tissues that ex-press multiple TG enzymes is also an important arena. Anexample is monocyte differentiation. TG2 and FXIIIa par-tially translocate to the nucleus in differentiating monocytes(22, 352), and both enzymes affect expression of a largenumber of genes (72, 352). Identification of TG2- andFXIIIa-responsive genes may be informative regarding therole of these proteins in monocyte differentiation. This isalso an issue in many other tissues. New knowledge wouldalso be useful regarding the role of transglutaminases inother immune cells. TGs may indirectly regulate inflamma-tory responses via activation of macrophages, natural killercells, and antigen-specific cytotoxic T lymphocytes. TG2,for example, causes epithelial cells to secrete IL-6 (272),which is likely to promote immune cell inversion.
Another important area is the role of TGs in cancer. It isinteresting that elevated expression of TG2 confers drugresistance. However, we have a long way to go before weunderstand the mechanism. A systematic study of a largecohort of patients should be performed to determinewhether TG2 is a valid prognostic marker. Similarly, TG2-induced activation of NF-�B and accumulation of HIF-1�represent interesting areas for future research. Constitutiveactivation of NF-�B is linked with etiology of variouschronic inflammatory conditions, including cancer. Under-standing of process whereby TG2-regulates NF-�B activa-
tion may yield ways to block this pathway and mitigatedisease. Similarly, inhibition of TG2-mediated increase inHIF-1� accumulation may be relevant in cancer therapy, asHIF-1� activation is a major pathway whereby cancer cellsinfluence glucose metabolism and aerobic glycolysis (88).
A remarkable feature of these studies is that no more thantwo decades ago transglutaminases were thought to func-tion solely as enzymes that covalently crosslinked proteinsto assemble barriers. The scope of this review points to thevast amount of new knowledge that has accumulated in thepast two decades which point to a pivotal role for theseproteins in regulating cell homeostasis.
ACKNOWLEDGMENTS
We thank Kathleen Reinecke for persistent, patient, andexpert assistance in the preparation of this manuscript. Wealso thank Donald R. Norwood for critical reading of andeditorial help with this review and Dr. Soner Gundemir forthe helpful discussion. We also thank Drs. Candace Kerrand Ellen Rorke for critically reading the manuscript.
Address for reprint requests and other correspondence: R.Eckert, Dept. of Biochemistry and Molecular Biology, Univ.of Maryland School of Medicine, 108 N. Greene St., Rm.103, Baltimore, MD 21201 (e-mail: [email protected]).
GRANTS
This work was supported by National Institutes of HealthGrants AR053851 and CA131974 (to R. L. Eckert);AR057126, HL093305, and DK071920 (to M. Nurmins-kaya); and NS0065825 (to G. Johnson); a grant from theBayer Healthcare System (Grants4Targets) and SK Agarwaldonor funds (to K. Mehta); Canadian Institutes of HealthResearch Grants MOP-119103, NHG-107768, and IMH-89827 (to M. T. Kaartinen); and a grant from AmericanHeart Association (to A. M. Belkin).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declaredby the authors.
REFERENCES
1. AbdAlla S, Lother H, Langer A, el FY, Quitterer U. Factor XIIIA transglutaminasecrosslinks AT1 receptor dimers of monocytes at the onset of atherosclerosis. Cell 119:343–354, 2004.
2. Achyuthan KE, Greenberg CS. Identification of a guanosine triphosphate-binding siteon guinea pig liver transglutaminase. Role of GTP and calcium ions in modulatingactivity. J Biol Chem 262: 1901–1906, 1987.
3. Aeschlimann D, Thomazy V. Protein crosslinking in assembly and remodelling ofextracellular matrices: the role of transglutaminases. Connect Tissue Res 41: 1–27,2000.
4. Aeschlimann D, Wetterwald A, Fleisch H, Paulsson M. Expression of tissue transglu-taminase in skeletal tissues correlates with events of terminal differentiation of chon-drocytes. J Cell Biol 120: 1461–1470, 1993.
5. Ahn JS, Kim MK, Hahn JH, Park JH, Park KH, Cho BR, Park SB, Kim DJ. Tissuetransglutaminase-induced down-regulation of matrix metalloproteinase-9. BiochemBiophys Res Commun 376: 743–747, 2008.
6. Ai L, Kim WJ, Demircan B, Dyer LM, Bray KJ, Skehan RR, Massoll NA, Brown KD. Thetransglutaminase 2 gene (TGM2), a potential molecular marker for chemotherapeuticdrug sensitivity, is epigenetically silenced in breast cancer. Carcinogenesis 29: 510–518, 2008.
7. Akar U, Ozpolat B, Mehta K, Fok J, Kondo Y, Lopez-Berestein G. Tissue transglutami-nase inhibits autophagy in pancreatic cancer cells. Mol Cancer Res 5: 241–249, 2007.
8. Akimov SS, Belkin AM. Cell surface tissue transglutaminase is involved in adhesion andmigration of monocytic cells on fibronectin. Blood 98: 1567–1576, 2001.
9. Akimov SS, Krylov D, Fleischman LF, Belkin AM. Tissue transglutaminase is an integ-rin-binding adhesion coreceptor for fibronectin. J Cell Biol 148: 825–838, 2000.
10. Akturk M, Karaahmetoglu S, Kacar M, Muftuoglu O. Thickness of the supraspinatusand biceps tendons in diabetic patients. Diabetes Care 25: 408, 2002.
11. Al-Jallad HF, Myneni VD, Piercy-Kotb SA, Chabot N, Mulani A, Keillor JW, Kaartinen MT.Plasma membrane factor XIIIA transglutaminase activity regulates osteoblast matrixsecretion and deposition by affecting microtubule dynamics. PLoS One 6: e15893,2011.
12. Al-Jallad HF, Nakano Y, Chen JL, McMillan E, Lefebvre C, Kaartinen MT. Transglu-taminase activity regulates osteoblast differentiation and matrix mineralization inMC3T3–E1 osteoblast cultures. Matrix Biol 25: 135–148, 2006.
13. Andringa G, Lam KY, Chegary M, Wang X, Chase TN, Bennett MC. Tissue transglu-taminase catalyzes the formation of alpha-synuclein crosslinks in Parkinson’s disease.FASEB J 18: 932–934, 2004.
14. Antonyak MA, Li B, Boroughs LK, Johnson JL, Druso JE, Bryant KL, Holowka DA,Cerione RA. Cancer cell-derived microvesicles induce transformation by transferringtissue transglutaminase and fibronectin to recipient cells. Proc Natl Acad Sci USA 108:4852–4857, 2011.
15. Antonyak MA, Li B, Regan AD, Feng Q, Dusaban SS, Cerione RA. Tissue transglutami-nase is an essential participant in the epidermal growth factor-stimulated signalingpathway leading to cancer cell migration and invasion. J Biol Chem 284: 17914–17925,2009.
17. Augello A, De BC. The regulation of differentiation in mesenchymal stem cells. HumGene Ther 21: 1226–1238, 2010.
18. Augello A, Kurth TB, De BC. Mesenchymal stem cells: a perspective from in vitrocultures to in vivo migration and niches. Eur Cell Mater 20: 121–133, 2010.
19. Baek KJ, Das T, Gray C, Antar S, Murugesan G, Im MJ. Evidence that the Gh proteinis a signal mediator from alpha 1-adrenoceptor to a phospholipase C. I. Identificationof alpha 1-adrenoceptor-coupled Gh family and purification of Gh7 from bovine heart.J Biol Chem 268: 27390–27397, 1993.
20. Baek KJ, Das T, Gray CD, Desai S, Hwang KC, Gacchui R, Ludwig M, Im MJ. A 50 KDaprotein modulates guanine nucleotide binding of transglutaminase II. Biochemistry 35:2651–2657, 1996.
21. Baek KJ, Kang S, Damron D, Im M. Phospholipase Cdelta1 is a guanine nucleotideexchanging factor for transglutaminase II (Galpha h) and promotes alpha 1B-adreno-receptor-mediated GTP binding and intracellular calcium release. J Biol Chem 276:5591–5597, 2001.
22. Balajthy Z, Csomos K, Vamosi G, Szanto A, Lanotte M, Fesus L. Tissue-transglutami-nase contributes to neutrophil granulocyte differentiation and functions. Blood 108:2045–2054, 2006.
TRANSGLUTAMINASES IN CELL FUNCTION
407Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
23. Baldwin AS Jr. The NF-kappa B and I kappa B proteins: new discoveries and insights.Annu Rev Immunol 14: 649–683, 1996.
24. Balmer JE, Blomhoff R. Gene expression regulation by retinoic acid. J Lipid Res 43:1773–1808, 2002.
25. Bass MD, Morgan MR, Roach KA, Settleman J, Goryachev AB, Humphries MJ.p190RhoGAP is the convergence point of adhesion signals from alpha 5 beta 1 integrinand syndecan-4. J Cell Biol 181: 1013–1026, 2008.
26. Battaglia G, Farrace MG, Mastroberardino PG, Viti I, Fimia GM, Van BJ, Devreese B,Melino G, Molinaro G, Busceti CL, Biagioni F, Nicoletti F, Piacentini M. Transglutami-nase 2 ablation leads to defective function of mitochondrial respiratory complex Iaffecting neuronal vulnerability in experimental models of extrapyramidal disorders. JNeurochem 100: 36–49, 2007.
27. Begg GE, Carrington L, Stokes PH, Matthews JM, Wouters MA, Husain A, Lorand L,Iismaa SE, Graham RM. Mechanism of allosteric regulation of transglutaminase 2 byGTP. Proc Natl Acad Sci USA 103: 19683–19688, 2006.
28. Begg GE, Holman SR, Stokes PH, Matthews JM, Graham RM, Iismaa SE. Mutation ofa critical arginine in the GTP-binding site of transglutaminase 2 disinhibits intracellularcross-linking activity. J Biol Chem 281: 12603–12609, 2006.
30. Belkin AM, Tsurupa G, Zemskov E, Veklich Y, Weisel JW, Medved L. Transglutami-nase-mediated oligomerization of the fibrin(ogen) alphaC domains promotes integ-rin-dependent cell adhesion and signaling. Blood 105: 3561–3568, 2005.
31. Bentmann A, Kawelke N, Moss D, Zentgraf H, Bala Y, Berger I, Gasser JA, Nakch-bandi IA. Circulating fibronectin affects bone matrix, whereas osteoblast fibronectinmodulates osteoblast function. J Bone Miner Res 25: 706–715, 2010.
32. Bergamini CM. GTP modulates calcium binding and cation-induced conformationalchanges in erythrocyte transglutaminase. FEBS Lett 239: 255–258, 1988.
33. Bernassola F, Federici M, Corazzari M, Terrinoni A, Hribal ML, De LV, Ranalli M,Massa O, Sesti G, McLean WH, Citro G, Barbetti F, Melino G. Role of transglutami-nase 2 in glucose tolerance: knockout mice studies and a putative mutation in a MODYpatient. FASEB J 16: 1371–1378, 2002.
34. Bershadsky A, Kozlov M, Geiger B. Adhesion-mediated mechanosensitivity: a time toexperiment, and a time to theorize. Curr Opin Cell Biol 18: 472–481, 2006.
35. Bersten AM, Ahkong QF, Hallinan T, Nelson SJ, Lucy JA. Inhibition of the formation ofmyotubes in vitro by inhibitors of transglutaminase. Biochim Biophys Acta 762: 429–436, 1983.
36. Bognar P, Nemeth I, Mayer B, Haluszka D, Wikonkal N, Ostorhazi E, John S, PaulssonM, Smyth N, Pasztoi M, Buzas EI, Szipocs R, Kolonics A, Temesvari E, Karpati S.Reduced inflammatory threshold indicates skin barrier defect in transglutaminase 3knockout mice. J Invest Dermatol 2013, doi: 10.1038/jid.2013.307.
37. Boothe RL, Folk JE. A reversible, calcium-dependent, copper-catalyzed inactivation ofguinea pig liver transglutaminase. J Biol Chem 244: 399–405, 1969.
38. Boroughs LK, Antonyak MA, Johnson JL, Cerione RA. A unique role for heat shockprotein 70 and its binding partner tissue transglutaminase in cancer cell migration. JBiol Chem 286: 37094–37107, 2011.
39. Boskey AL, Doty SB, Binderman I. Adenosine 5=-triphosphate promotes mineraliza-tion in differentiating chick limb-bud mesenchymal cell cultures. Microsc Res Tech 28:492–504, 1994.
41. Brown KD. Transglutaminase 2 and NF-kappaB: an odd couple that shapes breastcancer phenotype. Breast Cancer Res Treat 137: 329–336, 2013.
42. Budillon A, Carbone C, Di Gennaro E. Tissue transglutaminase: a new target toreverse cancer drug resistance. Amino Acids 44: 63–72, 2013.
43. Buxboim A, Ivanovska IL, Discher DE. Matrix elasticity, cytoskeletal forces and phys-ics of the nucleus: how deeply do cells “feel” outside and in? J Cell Sci 123: 297–308,2010.
44. Calcagno AM, Salcido CD, Gillet JP, Wu CP, Fostel JM, Mumau MD, Gottesman MM,Varticovski L, Ambudkar SV. Prolonged drug selection of breast cancer cells andenrichment of cancer stem cell characteristics. J Natl Cancer Inst 102: 1637–1652,2010.
45. Campisi A, Bramanti V, Caccamo D, Li VG, Cannavo G, Curro M, Raciti G, Galvano F,Amenta F, Vanella A, Ientile R, Avola R. Effect of growth factors and steroids ontransglutaminase activity and expression in primary astroglial cell cultures. J NeurosciRes 86: 1297–1305, 2008.
46. Candi E, Melino G, Lahm A, Ceci R, Rossi A, Kim IG, Ciani B, Steinert PM. Transglu-taminase 1 mutations in lamellar ichthyosis. Loss of activity due to failure of activationby proteolytic processing. J Biol Chem 273: 13693–13702, 1998.
47. Candi E, Melino G, Lahm A, Ceci R, Rossi A, Kim IG, Ciani B, Steinert PM. Transglu-taminase 1 mutations in lamellar ichthyosis. Loss of activity due to failure of activationby proteolytic processing. J Biol Chem 273: 13693–13702, 1998.
48. Candi E, Oddi S, Paradisi A, Terrinoni A, Ranalli M, Teofoli P, Citro G, Scarpato S,Puddu P, Melino G. Expression of transglutaminase 5 in normal and pathologic humanepidermis. J Invest Dermatol 119: 670–677, 2002.
49. Candi E, Oddi S, Terrinoni A, Paradisi A, Ranalli M, Finazzi-Agro A, Melino G. Trans-glutaminase 5 cross-links loricrin, involucrin, and small proline-rich proteins in vitro. JBiol Chem 276: 35014–35023, 2001.
50. Candi E, Schmidt R, Melino G. The cornified envelope: a model of cell death in theskin. Nat Rev Mol Cell Biol 6: 328–340, 2005.
51. Cao L, Shao M, Schilder J, Guise T, Mohammad KS, Matei D. Tissue transglutaminaselinks TGF-beta, epithelial to mesenchymal transition and a stem cell phenotype inovarian cancer. Oncogene 31: 2521–2534, 2012.
52. Caron NS, Munsie LN, Keillor JW, Truant R. Using FLIM-FRET to measure confor-mational changes of transglutaminase type 2 in live cells. PLoS One 7: e44159, 2012.
53. Cassidy AJ, van Steensel MA, Steijlen PM, van Geel M, van der Velden J, Morley SM,Terrinoni A, Melino G, Candi E, McLean WH. A homozygous missense mutation inTGM5 abolishes epidermal transglutaminase 5 activity and causes acral peeling skinsyndrome. Am J Hum Genet 77: 909–917, 2005.
54. Cecil DL, Terkeltaub R. Transamidation by transglutaminase 2 transforms S100A11calgranulin into a procatabolic cytokine for chondrocytes. J Immunol 180: 8378–8385,2008.
55. Chakravarty R, Rong XH, Rice RH. Phorbol ester-stimulated phosphorylation of ker-atinocyte transglutaminase in the membrane anchorage region. Biochem J 271: 25–30,1990.
56. Chau DY, Collighan RJ, Verderio EA, Addy VL, Griffin M. The cellular response totransglutaminase-cross-linked collagen. Biomaterials 26: 6518–6529, 2005.
57. Chen CS, Wu CH, Lai YC, Lee WS, Chen HM, Chen RJ, Chen LC, Ho YS, Wang YJ.NF-kappaB-activated tissue transglutaminase is involved in ethanol-induced hepaticinjury and the possible role of propolis in preventing fibrogenesis. Toxicology 246:148–157, 2008.
58. Chen S, Lin F, Iismaa S, Lee KN, Birckbichler PJ, Graham RM. Alpha1-adrenergicreceptor signaling via Gh is subtype specific and independent of its transglutaminaseactivity. J Biol Chem 271: 32385–32391, 1996.
59. Chen SH, Lin CY, Lee LT, Chang GD, Lee PP, Hung CC, Kao WT, Tsai PH, Schally AV,Hwang JJ, Lee MT. Up-regulation of fibronectin and tissue transglutaminase promotescell invasion involving increased association with integrin and MMP expression in A431cells. Anticancer Res 30: 4177–4186, 2010.
60. Cho BR, Kim MK, Suh DH, Hahn JH, Lee BG, Choi YC, Kwon TJ, Kim SY, Kim DJ.Increased tissue transglutaminase expression in human atherosclerotic coronary ar-teries. Coron Artery Dis 19: 459–468, 2008.
61. Cho SY, Lee JH, Bae HD, Jeong EM, Jang GY, Kim CW, Shin DM, Jeon JH, Kim IG.Transglutaminase 2 inhibits apoptosis induced by calcium-overload through down-regulation of Bax. Exp Mol Med 42: 639–650, 2010.
62. Choi K, Siegel M, Piper JL, Yuan L, Cho E, Strnad P, Omary B, Rich KM, Khosla C.Chemistry and biology of dihydroisoxazole derivatives: selective inhibitors of humantransglutaminase 2. Chem Biol 12: 469–475, 2005.
63. Chowdhury ZA, Barsigian C, Chalupowicz GD, Bach TL, Garcia-Manero G, MartinezJ. Colocalization of tissue transglutaminase and stress fibers in human vascular smoothmuscle cells and human umbilical vein endothelial cells. Exp Cell Res 231: 38–49, 1997.
64. Citron BA, SantaCruz KS, Davies PJ, Festoff BW. Intron-exon swapping of transglu-taminase mRNA and neuronal Tau aggregation in Alzheimer’s disease. J Biol Chem 276:3295–3301, 2001.
65. Clement S, Velasco PT, Murthy SN, Wilson JH, Lukas TJ, Goldman RD, Lorand L. Theintermediate filament protein, vimentin, in the lens is a target for cross-linking bytransglutaminase. J Biol Chem 273: 7604–7609, 1998.
66. Cohen I, Glaser T, Veis A, Bruner-Lorand J. Ca2�-dependent cross-linking processesin human platelets. Biochim Biophys Acta 676: 137–147, 1981.
67. Colak G, Keillor JW, Johnson GV. Cytosolic guanine nucledotide binding deficientform of transglutaminase 2 (R580a) potentiates cell death in oxygen glucose depriva-tion. PLoS One 6: e16665, 2011.
68. Cooper AJ, Jeitner TM, Blass JP. The role of transglutaminases in neurodegenerativediseases: overview. Neurochem Int 40: 1–5, 2002.
69. Cordell PA, Kile BT, Standeven KF, Josefsson EC, Pease RJ, Grant PJ. Association ofcoagulation factor XIII-A with Golgi proteins within monocyte-macrophages: impli-cations for subcellular trafficking and secretion. Blood 115: 2674–2681, 2010.
70. Cox AD, Devine DV. Factor XIIIa binding to activated platelets is mediated throughactivation of glycoprotein IIb-IIIa. Blood 83: 1006–1016, 1994.
71. Cserhalmi-Friedman PB, Milstone LM, Christiano AM. Diagnosis of autosomal reces-sive lamellar ichthyosis with mutations in the TGM1 gene. Br J Dermatol 144: 726–730,2001.
72. Csomos K, Nemet I, Fesus L, Balajthy Z. Tissue transglutaminase contributes to theall-trans-retinoic acid-induced differentiation syndrome phenotype in the NB4 modelof acute promyelocytic leukemia. Blood 116: 3933–3943, 2010.
73. Dafik L, Albertelli M, Stamnaes J, Sollid LM, Khosla C. Activation and inhibition oftransglutaminase 2 in mice. PLoS One 7: e30642, 2012.
74. Dai Y, Dudek NL, Patel TB, Muma NA. Transglutaminase-catalyzed transamidation: anovel mechanism for Rac1 activation by 5-hydroxytryptamine2A receptor stimula-tion. J Pharmacol Exp Ther 326: 153–162, 2008.
75. Dardik R, Inbal A. Complex formation between tissue transglutaminase II (tTG) andvascular endothelial growth factor receptor 2 (VEGFR-2): proposed mechanism formodulation of endothelial cell response to VEGF. Exp Cell Res 312: 2973–2982, 2006.
76. Dardik R, Krapp T, Rosenthal E, Loscalzo J, Inbal A. Effect of FXIII on monocyte andfibroblast function. Cell Physiol Biochem 19: 113–120, 2007.
77. Dardik R, Leor J, Skutelsky E, Castel D, Holbova R, Schiby G, Shaish A, Dickneite G,Loscalzo J, Inbal A. Evaluation of the pro-angiogenic effect of factor XIII in heterotopicmouse heart allografts and FXIII-deficient mice. Thromb Haemost 95: 546–550, 2006.
78. Dardik R, Loscalzo J, Eskaraev R, Inbal A. Molecular mechanisms underlying theproangiogenic effect of factor XIII. Arterioscler Thromb Vasc Biol 25: 526–532, 2005.
79. Dardik R, Shenkman B, Tamarin I, Eskaraev R, Harsfalvi J, Varon D, Inbal A. Factor XIIImediates adhesion of platelets to endothelial cells through alpha(v)beta(3) and glyco-protein IIb/IIIa integrins. Thromb Res 105: 317–323, 2002.
80. Dardik R, Solomon A, Loscalzo J, Eskaraev R, Bialik A, Goldberg I, Schiby G, Inbal A.Novel proangiogenic effect of factor XIII associated with suppression of thrombos-pondin 1 expression. Arterioscler Thromb Vasc Biol 23: 1472–1477, 2003.
81. Das T, Baek KJ, Gray C, Im MJ. Evidence that the Gh protein is a signal mediator fromalpha 1-adrenoceptor to a phospholipase C. II. Purification and characterization of aGh-coupled 69-kDa phospholipase C and reconstitution of alpha 1-adrenoceptor, Ghfamily, and phospholipase C. J Biol Chem 268: 27398–27405, 1993.
82. Datta S, Antonyak MA, Cerione RA. GTP-binding-defective forms of tissue transglu-taminase trigger cell death. Biochemistry 46: 14819–14829, 2007.
83. Davies PJ, Murtaugh MP, Moore WT Jr, Johnson GS, Lucas D. Retinoic acid-inducedexpression of tissue transglutaminase in human promyelocytic leukemia (HL-60) cells.J Biol Chem 260: 5166–5174, 1985.
84. Dean MD. Genetic disruption of the copulatory plug in mice leads to severely reducedfertility. PLoS Genet 9: e1003185, 2013.
85. Deasey S, Nurminsky D, Shanmugasundaram S, Lima F, Nurminskaya M. Transglu-taminase 2 as a novel activator of LRP6/beta-catenin signaling. Cell Signal 25: 2646–2651, 2013.
86. Deasey S, Shanmugasundaram S, Nurminskaya M. Tissue-specific responses to loss oftransglutaminase 2. Amino Acids 44: 179–187, 2013.
87. Del DS, Serafini-Fracassini D, Bonner P, Cresti M, Cai G. Effects of post-translationalmodifications catalysed by pollen transglutaminase on the functional properties ofmicrotubules and actin filaments. Biochem J 418: 651–664, 2009.
88. Denko NC. Hypoxia, HIF1 and glucose metabolism in the solid tumour. Nat RevCancer 8: 705–713, 2008.
89. DiDonato JA, Mercurio F, Karin M. NF-kappaB and the link between inflammationand cancer. Immunol Rev 246: 379–400, 2012.
90. Dreier R. Hypertrophic differentiation of chondrocytes in osteoarthritis: the devel-opmental aspect of degenerative joint disorders. Arthritis Res Ther 12: 216, 2010.
91. Dubbink HJ, Cleutjens KB, van der Korput HA, Trapman J, Romijn JC. An Sp1 bindingsite is essential for basal activity of the human prostate-specific transglutaminase gene(TGM4) promoter. Gene 240: 261–267, 1999.
92. Dupuis M, Levy A, Mhaouty-Kodja S. Functional coupling of rat myometrial alpha1-adrenergic receptors to Gh alpha/tissue transglutaminase 2 during pregnancy. J BiolChem 279: 19257–19263, 2004.
93. Dyer LM, Schooler KP, Ai L, Klop C, Qiu J, Robertson KD, Brown KD. The transglu-taminase 2 gene is aberrantly hypermethylated in glioma. J Neurooncol 101: 429–440,2011.
94. Eckert RL. Structure, function, and differentiation of the keratinocyte. Physiol Rev 69:1316–1346, 1989.
95. Eckert RL, Crish JF, Robinson NA. The epidermal keratinocyte as a model for thestudy of gene regulation and cell differentiation. Physiol Rev 77: 397–424, 1997.
96. Eckert RL, Green H. Structure and evolution of the human involucrin gene. Cell 46:583–589, 1986.
100. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineagespecification. Cell 126: 677–689, 2006.
101. Esposito C, Caputo I. Mammalian transglutaminases. Identification of substrates as akey to physiological function and physiopathological relevance. FEBS J 272: 615–631,2005.
103. Feng JF, Gray CD, Im MJ. Alpha 1B-adrenoceptor interacts with multiple sites oftransglutaminase II: characteristics of the interaction in binding and activation. Bio-chemistry 38: 2224–2232, 1999.
104. Feng JF, Rhee SG, Im MJ. Evidence that phospholipase delta1 is the effector in the Gh(transglutaminase II)-mediated signaling. J Biol Chem 271: 16451–16454, 1996.
105. Festoff BW, SantaCruz K, Arnold PM, Sebastian CT, Davies PJ, Citron BA. Injury-induced “switch” from GTP-regulated to novel GTP-independent isoform of tissuetransglutaminase in the rat spinal cord. J Neurochem 81: 708–718, 2002.
106. Filiano AJ, Bailey CD, Tucholski J, Gundemir S, Johnson GV. Transglutaminase 2protects against ischemic insult, interacts with HIF1beta, and attenuates HIF1 signal-ing. FASEB J 22: 2662–2675, 2008.
108. Flatau G, Landraud L, Boquet P, Bruzzone M, Munro P. Deamidation of RhoA glu-tamine 63 by the Escherichia coli CNF1 toxin requires a short sequence of the GTPaseswitch 2 domain. Biochem Biophys Res Commun 267: 588–592, 2000.
109. Fok JY, Ekmekcioglu S, Mehta K. Implications of tissue transglutaminase expression inmalignant melanoma. Mol Cancer Ther 5: 1493–1503, 2006.
110. Folk JE, Cole PW. Mechanism of action of guinea pig liver transglutaminase. I. Purifi-cation and properties of the enzyme: identification of a functional cysteine essentialfor activity. J Biol Chem 241: 5518–5525, 1966.
111. Forsprecher J, Wang Z, Goldberg HA, Kaartinen MT. Transglutaminase-mediatedoligomerization promotes osteoblast adhesive properties of osteopontin and bonesialoprotein. Cell Adh Migr 5: 65–72, 2011.
113. Fraij BM, Birckbichler PJ, Patterson MK Jr, Lee KN, Gonzales RA. A retinoic acid-inducible mRNA from human erythroleukemia cells encodes a novel tissue transglu-taminase homologue. J Biol Chem 267: 22616–22623, 1992.
114. Franceschi RT, Iyer BS. Relationship between collagen synthesis and expression of theosteoblast phenotype in MC3T3–E1 cells. J Bone Miner Res 7: 235–246, 1992.
115. Franceschi RT, Iyer BS, Cui Y. Effects of ascorbic acid on collagen matrix formationand osteoblast differentiation in murine MC3T3–E1 cells. J Bone Miner Res 9: 843–854, 1994.
116. Franco R, Casado V, Cortes A, Ferrada C, Mallol J, Woods A, Lluis C, Canela EI, FerreS. Basic concepts in G-protein-coupled receptor homo- and heterodimerization. Sci-entific World J 7: 48–57, 2007.
117. Frisdal E, Lesnik P, Olivier M, Robillard P, Chapman MJ, Huby T, Guerin M, Le GW.Interleukin-6 protects human macrophages from cellular cholesterol accumulationand attenuates the proinflammatory response. J Biol Chem 286: 30926–30936, 2011.
118. Fukuda K, Kojiro M, Chiu JF. Differential regulation of tissue transglutaminase in rathepatoma cell lines McA-RH7777 and McA-RH8994: relation to growth rate and celldeath. J Cell Biochem 54: 67–77, 1994.
119. Gentile V, Porta R, Chiosi E, Spina A, Valente F, Pezone R, Davies PJ, Alaadik A, IllianoG. tTGase/G alpha h protein expression inhibits adenylate cyclase activity in Balb-C3T3 fibroblasts membranes. Biochim Biophys Acta 1357: 115–122, 1997.
120. Ghanta KS, Pakala SB, Reddy SD, Li DQ, Nair SS, Kumar R. MTA1 coregulation oftransglutaminase 2 expression and function during inflammatory response. J Biol Chem286: 7132–7138, 2011.
121. Ghosh S, Baltimore D. Activation in vitro of NF-kappa B by phosphorylation of itsinhibitor I kappa B. Nature 344: 678–682, 1990.
122. Grenard P, Bates MK, Aeschlimann D. Evolution of transglutaminase genes: identifi-cation of a transglutaminase gene cluster on human chromosome 15q15. Structure ofthe gene encoding transglutaminase X and a novel gene family member, transglutami-nase Z. J Biol Chem 276: 33066–33078, 2001.
123. Guilluy C, Eddahibi S, Agard C, Guignabert C, Izikki M, Tu L, Savale L, Humbert M,Fadel E, Adnot S, Loirand G, Pacaud P. RhoA and Rho kinase activation in humanpulmonary hypertension: role of 5-HT signaling. Am J Respir Crit Care Med 179: 1151–1158, 2009.
124. Guilluy C, Rolli-Derkinderen M, Tharaux PL, Melino G, Pacaud P, Loirand G. Trans-glutaminase-dependent RhoA activation and depletion by serotonin in vascularsmooth muscle cells. J Biol Chem 282: 2918–2928, 2007.
125. Gundemir S, Colak G, Feola J, Blouin R, Johnson GV. Transglutaminase 2 facilitates orameliorates HIF signaling and ischemic cell death depending on its conformation andlocalization. Biochim Biophys Acta 1833: 1–10, 2013.
125a. Gundemir S, Colak G, Tucholski J, Johnson GVW. Transglutaminase 2: a molecularSwiss army knife. Biochim Biophys Acta 1823: 406–419, 2012.
126. Gundemir S, Johnson GV. Intracellular localization and conformational state of trans-glutaminase 2: implications for cell death. PLoS One 4: e6123, 2009.
127. Haase VH. Oxygen regulates epithelial-to-mesenchymal transition: insights into mo-lecular mechanisms and relevance to disease. Kidney Int 76: 492–499, 2009.
128. Haddox MK, Russell DH. Increased nuclear conjugated polyamines and transglutami-nase during liver regeneration. Proc Natl Acad Sci USA 78: 1712–1716, 1981.
129. Heath DJ, Downes S, Verderio E, Griffin M. Characterization of tissue transglutami-nase in human osteoblast-like cells. J Bone Miner Res 16: 1477–1485, 2001.
130. Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derivedgrowth factor. Physiol Rev 79: 1283–1316, 1999.
131. Herman JF, Mangala LS, Mehta K. Implications of increased tissue transglutaminase(TG2) expression in drug-resistant breast cancer (MCF-7) cells. Oncogene 25: 3049–3058, 2006.
132. Hitomi K. Transglutaminases in skin epidermis. Eur J Dermatol 15: 313–319, 2005.
133. Hitomi K, Horio Y, Ikura K, Yamanishi K, Maki M. Analysis of epidermal-type trans-glutaminase (TGase 3) expression in mouse tissues and cell lines. Int J Biochem Cell Biol33: 491–498, 2001.
134. Hitomi K, Ikeda N, Maki M. Immunological detection of proteolytically activatedepidermal-type transglutaminase (TGase 3) using cleavage-site-specific antibody. Bio-sci Biotechnol Biochem 67: 2492–2494, 2003.
135. Hitomi K, Kanehiro S, Ikura K, Maki M. Characterization of recombinant mouseepidermal-type transglutaminase (TGase 3): regulation of its activity by proteolysisand guanine nucleotides. J Biochem 125: 1048–1054, 1999.
136. Hitomi K, Presland RB, Nakayama T, Fleckman P, Dale BA, Maki M. Analysis ofepidermal-type transglutaminase (transglutaminase 3) in human stratified epitheliaand cultured keratinocytes using monoclonal antibodies. J Dermatol Sci 32: 95–103,2003.
137. Hsiong SX, Carampin P, Kong HJ, Lee KY, Mooney DJ. Differentiation stage altersmatrix control of stem cells. J Biomed Mater Res A 85: 145–156, 2008.
138. Huang L, Haylor JL, Hau Z, Jones RA, Vickers ME, Wagner B, Griffin M, Saint RE,Coutts IG, El Nahas AM, Johnson TS. Transglutaminase inhibition ameliorates exper-imental diabetic nephropathy. Kidney Int 76: 383–394, 2009.
139. Huang YP, Seguro K, Motoki M, Tawada K. Cross-linking of contractile proteins fromskeletal muscle by treatment with microbial transglutaminase. J Biochem 112: 229–234, 1992.
140. Huber M, Rettler I, Bernasconi K, Frenk E, Lavrijsen SP, Ponec M, Bon A, Lauten-schlager S, Schorderet DF, Hohl D. Mutations of keratinocyte transglutaminase inlamellar ichthyosis. Science 267: 525–528, 1995.
141. Huber M, Rettler I, Bernasconi K, Wyss M, Hohl D. Lamellar ichthyosis is geneticallyheterogeneous–cases with normal keratinocyte transglutaminase. J Invest Dermatol105: 653–654, 1995.
143. Huebner JL, Johnson KA, Kraus VB, Terkeltaub RA. Transglutaminase 2 is a marker ofchondrocyte hypertrophy and osteoarthritis severity in the Hartley guinea pig modelof knee OA. Osteoarthritis Cartilage 17: 1056–1064, 2009.
144. Huh CG, Factor VM, Sanchez A, Uchida K, Conner EA, Thorgeirsson SS. Hepatocytegrowth factor/c-met signaling pathway is required for efficient liver regeneration andrepair. Proc Natl Acad Sci USA 101: 4477–4482, 2004.
145. Hwang JY, Mangala LS, Fok JY, Lin YG, Merritt WM, Spannuth WA, Nick AM, Fiter-man DJ, Vivas-Mejia PE, Deavers MT, Coleman RL, Lopez-Berestein G, Mehta K,Sood AK. Clinical and biological significance of tissue transglutaminase in ovariancarcinoma. Cancer Res 68: 5849–5858, 2008.
146. Hwang KC, Gray CD, Sivasubramanian N, Im MJ. Interaction site of GTP binding Gh(transglutaminase II) with phospholipase C. J Biol Chem 270: 27058–27062, 1995.
150. Iismaa SE, Aplin M, Holman S, Yiu TW, Jackson K, Burchfield JG, Mitchell CJ, O’ReillyL, Davenport A, Cantley J, Schmitz-Peiffer C, Biden TJ, Cooney GJ, Graham RM.Glucose homeostasis in mice is transglutaminase 2 independent. PLoS One 8: e63346,2013.
151. Iismaa SE, Mearns BM, Lorand L, Graham RM. Transglutaminases and disease: lessonsfrom genetically engineered mouse models and inherited disorders. Physiol Rev 89:991–1023, 2009.
152. Im MJ, Graham RM. A novel guanine nucleotide-binding protein coupled to the alpha1-adrenergic receptor. I. Identification by photolabeling or membrane and ternarycomplex preparation. J Biol Chem 265: 18944–18951, 1990.
153. Im MJ, Riek RP, Graham RM. A novel guanine nucleotide-binding protein coupled tothe alpha 1-adrenergic receptor. II. Purification, characterization, and reconstitution.J Biol Chem 265: 18952–18960, 1990.
154. Jang GY, Jeon JH, Cho SY, Shin DM, Kim CW, Jeong EM, Bae HC, Kim TW, Lee SH,Choi Y, Lee DS, Park SC, Kim IG. Transglutaminase 2 suppresses apoptosis by mod-ulating caspase 3 and NF-kappaB activity in hypoxic tumor cells. Oncogene 29: 356–367, 2010.
155. Janiak A, Zemskov EA, Belkin AM. Cell surface transglutaminase promotes RhoAactivation via integrin clustering and suppression of the Src-p190RhoGAP signalingpathway. Mol Biol Cell 17: 1606–1619, 2006.
156. Jans R, Sturniolo MT, Eckert RL. Localization of the TIG3 transglutaminase interactiondomain and demonstration that the amino-terminal region is required for TIG3 func-tion as a keratinocyte differentiation regulator. J Invest Dermatol 128: 517–529, 2008.
157. Jayo A, Conde I, Lastres P, Jimenez-Yuste V, Gonzalez-Manchon C. Possible role forcellular FXIII in monocyte-derived dendritic cell motility. Eur J Cell Biol 88: 423–431,2009.
158. Jeitner TM, Muma NA, Battaile KP, Cooper AJ. Transglutaminase activation in neu-rodegenerative diseases. Future Neurol 4: 449–467, 2009.
159. Jiang WG, Ablin R, Douglas-Jones A, Mansel RE. Expression of transglutaminases inhuman breast cancer and their possible clinical significance. Oncol Rep 10: 2039–2044,2003.
160. Jiang WG, Ablin RJ. Prostate transglutaminase: a unique transglutaminase and its rolein prostate cancer. Biomark Med 5: 285–291, 2011.
161. Jin X, Stamnaes J, Klock C, DiRaimondo TR, Sollid LM, Khosla C. Activation of extra-cellular transglutaminase 2 by thioredoxin. J Biol Chem 286: 37866–37873, 2011.
162. John S, Thiebach L, Frie C, Mokkapati S, Bechtel M, Nischt R, Rosser-Davies S,Paulsson M, Smyth N. Epidermal transglutaminase (TGase 3) is required for properhair development, but not the formation of the epidermal barrier. PLoS One 7:e34252, 2012.
163. Johnson GV, Cox TM, Lockhart JP, Zinnerman MD, Miller ML, Powers RE. Transglu-taminase activity is increased in Alzheimer’s disease brain. Brain Res 751: 323–329,1997.
164. Johnson KA, Rose DM, Terkeltaub RA. Factor XIIIA mobilizes transglutaminase 2 toinduce chondrocyte hypertrophic differentiation. J Cell Sci 121: 2256–2264, 2008.
165. Johnson KA, Terkeltaub RA. External GTP-bound transglutaminase 2 is a molecularswitch for chondrocyte hypertrophic differentiation and calcification. J Biol Chem 280:15004–15012, 2005.
166. Johnson KB, Thompson JM, Watts SW. Modification of proteins by norepinephrine isimportant for vascular contraction. Front Physiol 1: 131, 2010.
167. Johnson TS, El-Koraie AF, Skill NJ, Baddour NM, El Nahas AM, Njloma M, Adam AG,Griffin M. Tissue transglutaminase and the progression of human renal scarring. J AmSoc Nephrol 14: 2052–2062, 2003.
168. Jones RA, Kotsakis P, Johnson TS, Chau DY, Ali S, Melino G, Griffin M. Matrix changesinduced by transglutaminase 2 lead to inhibition of angiogenesis and tumor growth.Cell Death Differ 13: 1442–1453, 2006.
169. Julien S, Puig I, Caretti E, Bonaventure J, Nelles L, van RF, Dargemont C, de HerrerosAG, Bellacosa A, Larue L. Activation of NF-kappaB by Akt upregulates Snail expres-sion and induces epithelium mesenchyme transition. Oncogene 26: 7445–7456, 2007.
170. Jung SM, Jandu S, Steppan J, Belkin A, An SS, Pak A, Choi EY, Nyhan D, Butlin M, ViegasK, Avolio A, Berkowitz DE, Santhanam L. Increased tissue transglutaminase activity
contributes to central vascular stiffness in eNOS knockout mice. Am J Physiol HeartCirc Physiol 305: H803–H810, 2013.
171. Kaartinen MT, El-Maadawy S, Rasanen NH, McKee MD. Tissue transglutaminase andits substrates in bone. J Bone Miner Res 17: 2161–2173, 2002.
172. Kalluri R. EMT: when epithelial cells decide to become mesenchymal-like cells. J ClinInvest 119: 1417–1419, 2009.
173. Kanchan K, Ergulen E, Kiraly R, Simon-Vecsei Z, Fuxreiter M, Fesus L. Identification ofa specific one amino acid change in recombinant human transglutaminase 2 thatregulates its activity and calcium sensitivity. Biochem J 455: 261–272, 2013.
174. Kang SJ, Shin KS, Song WK, Ha DB, Chung CH, Kang MS. Involvement of transglu-taminase in myofibril assembly of chick embryonic myoblasts in culture. J Cell Biol 130:1127–1136, 1995.
175. Kang SK, Kim DK, Damron DS, Baek KJ, Im MJ. Modulation of intracellular Ca2� viaalpha(1B)-adrenoreceptor signaling molecules, G alpha(h) (transglutaminase II) andphospholipase C-delta 1. Biochem Biophys Res Commun 293: 383–390, 2002.
176. Kang SK, Lee JY, Chung TW, Kim CH. Overexpression of transglutaminase 2 accel-erates the erythroid differentiation of human chronic myelogenous leukemia K562cell line through PI3K/Akt signaling pathway. FEBS Lett 577: 361–366, 2004.
177. Kang SK, Yi KS, Kwon NS, Park KH, Kim UH, Baek KJ, Im MJ. Alpha1B-adrenoceptorsignaling and cell motility: GTPase function of Gh/transglutaminase 2 inhibits cellmigration through interaction with cytoplasmic tail of integrin alpha subunits. J BiolChem 279: 36593–36600, 2004.
178. Karin M, Greten FR. NF-kappaB: linking inflammation and immunity to cancer devel-opment and progression. Nat Rev Immunol 5: 749–759, 2005.
179. Karpuj MV, Garren H, Slunt H, Price DL, Gusella J, Becher MW, Steinman L. Trans-glutaminase aggregates huntingtin into nonamyloidogenic polymers, and its enzymaticactivity increases in Huntington’s disease brain nuclei. Proc Natl Acad Sci USA 96:7388–7393, 1999.
180. Khatiwala CB, Kim PD, Peyton SR, Putnam AJ. ECM compliance regulates osteogen-esis by influencing MAPK signaling downstream of RhoA and ROCK. J Bone Miner Res24: 886–898, 2009.
181. Kim SY. Transglutaminase 2 in inflammation. Front Biosci 11: 3026–3035, 2006.
182. Kim SY. Transglutaminase 2: a new paradigm for NF-kappaB involvement in disease.Adv Enzymol Relat Areas Mol Biol 78: 161–195, 2011.
183. Kim SY, Chung SI, Steinert PM. Highly active soluble processed forms of the trans-glutaminase 1 enzyme in epidermal keratinocytes. J Biol Chem 270: 18026–18035,1995.
184. Kim SY, Chung SI, Yoneda K, Steinert PM. Expression of transglutaminase 1 in humanepidermis. J Invest Dermatol 104: 211–217, 1995.
185. Kim SY, Grant P, Lee JH, Pant HC, Steinert PM. Differential expression of multipletransglutaminases in human brain. Increased expression and cross-linking by transglu-taminases 1 and 2 in Alzheimer’s disease. J Biol Chem 274: 30715–30721, 1999.
186. Kim SY, Marekov L, Bubber P, Browne SE, Stavrovskaya I, Lee J, Steinert PM, Blass JP,Beal MF, Gibson GE, Cooper AJ. Mitochondrial aconitase is a transglutaminase 2substrate: transglutamination is a probable mechanism contributing to high-molecu-lar-weight aggregates of aconitase and loss of aconitase activity in Huntington diseasebrain. Neurochem Res 30: 1245–1255, 2005.
187. Kim Y, Eom S, Kim K, Lee YS, Choe J, Hahn JH, Lee H, Kim YM, Ha KS, Ro JY, JeoungD. Transglutaminase II interacts with rac1, regulates production of reactive oxygenspecies, expression of snail, secretion of Th2 cytokines and mediates in vitro and invivo allergic inflammation. Mol Immunol 47: 1010–1022, 2010.
188. Kiraly R, Csosz E, Kurtan T, Antus S, Szigeti K, Simon-Vecsei Z, Korponay-Szabo IR,Keresztessy Z, Fesus L. Functional significance of five noncanonical Ca2�-binding sitesof human transglutaminase 2 characterized by site-directed mutagenesis. FEBS J 276:7083–7096, 2009.
189. Klock C, DiRaimondo TR, Khosla C. Role of transglutaminase 2 in celiac diseasepathogenesis. Semin Immunopathol 34: 513–522, 2012.
190. Kojima S, Kuo TF, Tatsukawa H. Regulation of transglutaminase-mediated hepatic celldeath in alcoholic steatohepatitis and non-alcoholic steatohepatitis. J GastroenterolHepatol 27 Suppl 2: 52–57, 2012.
TRANSGLUTAMINASES IN CELL FUNCTION
411Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
191. Kojima S, Nara K, Rifkin DB. Requirement for transglutaminase in the activation oflatent transforming growth factor-beta in bovine endothelial cells. J Cell Biol 121:439–448, 1993.
192. Komori T. Regulation of osteoblast differentiation by transcription factors. J CellBiochem 99: 1233–1239, 2006.
193. Koseki-Kuno S, Yamakawa M, Dickneite G, Ichinose A. Factor XIII A subunit-deficientmice developed severe uterine bleeding events and subsequent spontaneous miscar-riages. Blood 102: 4410–4412, 2003.
194. Kulkarni S, Jackson SP. Platelet factor XIII and calpain negatively regulate integrinalphaIIbbeta3 adhesive function and thrombus growth. J Biol Chem 279: 30697–30706, 2004.
195. Kumar A, Gao H, Xu J, Reuben J, Yu D, Mehta K. Evidence that aberrant expressionof tissue transglutaminase promotes stem cell characteristics in mammary epithelialcells. PLoS One 6: e20701, 2011.
196. Kumar A, Xu J, Brady S, Gao H, Yu D, Reuben J, Mehta K. Tissue transglutaminasepromotes drug resistance and invasion by inducing mesenchymal transition in mam-mary epithelial cells. PLoS One 5: e13390, 2010.
197. Kumar A, Xu J, Sung B, Kumar S, Yu D, Aggarwal BB, Mehta K. Evidence that GTP-binding domain but not catalytic domain of transglutaminase 2 is essential for epithe-lial-to-mesenchymal transition in mammary epithelial cells. Breast Cancer Res 14: R4,2012.
198. Kumar S, Mehta K. Tissue transglutaminase constitutively activates HIF-1alpha pro-moter and nuclear factor-kappaB via a non-canonical pathway. PLoS One 7: e49321,2012.
199. Kuncio GS, Tsyganskaya M, Zhu J, Liu SL, Nagy L, Thomazy V, Davies PJ, Zern MA.TNF-alpha modulates expression of the tissue transglutaminase gene in liver cells. AmJ Physiol Gastrointest Liver Physiol 274: G240–G245, 1998.
200. Kuo TF, Tatsukawa H, Kojima S. New insights into the functions and localization ofnuclear transglutaminase 2. FEBS J 278: 4756–4767, 2011.
201. Kuraishy A, Karin M, Grivennikov SI. Tumor promotion via injury- and death-inducedinflammation. Immunity 35: 467–477, 2011.
202. Lai TS, Hausladen A, Slaughter TF, Eu JP, Stamler JS, Greenberg CS. Calcium regulatesS-nitrosylation, denitrosylation, and activity of tissue transglutaminase. Biochemistry40: 4904–4910, 2001.
203. Lai TS, Liu Y, Li W, Greenberg CS. Identification of two GTP-independent alterna-tively spliced forms of tissue transglutaminase in human leukocytes, vascular smoothmuscle, and endothelial cells. FASEB J 21: 4131–4143, 2007.
204. Lai TS, Tucker T, Burke JR, Strittmatter WJ, Greenberg CS. Effect of tissue transglu-taminase on the solubility of proteins containing expanded polyglutamine repeats. JNeurochem 88: 1253–1260, 2004.
205. Lee J, Kim YS, Choi DH, Bang MS, Han TR, Joh TH, Kim SY. Transglutaminase 2induces nuclear factor-kappaB activation via a novel pathway in BV-2 microglia. J BiolChem 279: 53725–53735, 2004.
206. Lee KH, Lee N, Lim S, Jung H, Ko YG, Park HY, Jang Y, Lee H, Hwang KC. Calreticulininhibits the MEK1,2-ERK1,2 pathway in alpha 1-adrenergic receptor/Gh-stimulatedhypertrophy of neonatal rat cardiomyocytes. J Steroid Biochem Mol Biol 84: 101–107,2003.
207. Lee MY, Chung S, Bang HW, Baek KJ, Uhm D. Modulation of large conductanceCa2�-activated K� channel by Galphah (transglutaminase II) in the vascular smoothmuscle cell. Pflügers Arch 433: 671–673, 1997.
208. Leroy P, Berto F, Bourget I, Rossi B. Down-regulation of Hox A7 is required for celladhesion and migration on fibronectin during early HL-60 monocytic differentiation. JLeukoc Biol 75: 680–688, 2004.
209. Lesort M, Attanavanich K, Zhang J, Johnson GV. Distinct nuclear localization andactivity of tissue transglutaminase. J Biol Chem 273: 11991–11994, 1998.
211. Lesort M, Chun W, Johnson GV, Ferrante RJ. Tissue transglutaminase is increased inHuntington’s disease brain. J Neurochem 73: 2018–2027, 1999.
213. Li B, Antonyak MA, Druso JE, Cheng L, Nikitin AY, Cerione RA. EGF potentiatedoncogenesis requires a tissue transglutaminase-dependent signaling pathway leadingto Src activation. Proc Natl Acad Sci USA 107: 1408–1413, 2010.
214. Li Z, Xu X, Bai L, Chen W, Lin Y. Epidermal growth factor receptor-mediated tissuetransglutaminase overexpression couples acquired tumor necrosis factor-related ap-optosis-inducing ligand resistance and migration through c-FLIP and MMP-9 proteinsin lung cancer cells. J Biol Chem 286: 21164–21172, 2011.
215. Lin CY, Tsai PH, Kandaswami CC, Chang GD, Cheng CH, Huang CJ, Lee PP, HwangJJ, Lee MT. Role of tissue transglutaminase 2 in the acquisition of a mesenchymal-likephenotype in highly invasive A431 tumor cells. Mol Cancer 10: 87, 2011.
217. Liu S, Cerione RA, Clardy J. Structural basis for the guanine nucleotide-binding activityof tissue transglutaminase and its regulation of transamidation activity. Proc Natl AcadSci USA 99: 2743–2747, 2002.
218. Liu T, Tee AE, Porro A, Smith SA, Dwarte T, Liu PY, Iraci N, Sekyere E, Haber M,Norris MD, Diolaiti D, Della VG, Perini G, Marshall GM. Activation of tissue transglu-taminase transcription by histone deacetylase inhibition as a therapeutic approach forMyc oncogenesis. Proc Natl Acad Sci USA 104: 18682–18687, 2007.
219. Long F, Ornitz DM. Development of the endochondral skeleton. Cold Spring HarbPerspect Biol 5: a008334, 2013.
220. Lorand L, Graham RM. Transglutaminases: crosslinking enzymes with pleiotropicfunctions. Nat Rev Mol Cell Biol 4: 140–156, 2003.
221. Lorand L, Jeong JM, Radek JT, Wilson J. Human plasma factor XIII: subunit interactionsand activation of zymogen. Methods Enzymol 222: 22–35, 1993.
222. Lu S, Saydak M, Gentile V, Stein JP, Davies PJ. Isolation and characterization of thehuman tissue transglutaminase gene promoter. J Biol Chem 270: 9748–9756, 1995.
223. Luciani A, Villella VR, Esposito S, Brunetti-Pierri N, Medina D, Settembre C, Gavina M,Pulze L, Giardino I, Pettoello-Mantovani M, D’Apolito M, Guido S, Masliah E, SpencerB, Quaratino S, Raia V, Ballabio A, Maiuri L. Defective CFTR induces aggresomeformation and lung inflammation in cystic fibrosis through ROS-mediated autophagyinhibition. Nat Cell Biol 12: 863–875, 2010.
224. Luciani A, Villella VR, Vasaturo A, Giardino I, Raia V, Pettoello-Mantovani M, D’ApolitoM, Guido S, Leal T, Quaratino S, Maiuri L. SUMOylation of tissue transglutaminase aslink between oxidative stress and inflammation. J Immunol 183: 2775–2784, 2009.
225. Maccioni RB, Arechaga J. Transglutaminase (TG) involvement in early embryogenesis.Exp Cell Res 167: 266–270, 1986.
227. Maiuri L, Luciani A, Giardino I, Raia V, Villella VR, D’Apolito M, Pettoello-MantovaniM, Guido S, Ciacci C, Cimmino M, Cexus ON, Londei M, Quaratino S. Tissue trans-glutaminase activation modulates inflammation in cystic fibrosis via PPARgammadown-regulation. J Immunol 180: 7697–7705, 2008.
228. Malorni W, Farrace MG, Matarrese P, Tinari A, Ciarlo L, Mousavi-Shafaei P, D’ElettoM, Di GG, Melino G, Palmieri L, Rodolfo C, Piacentini M. The adenine nucleotidetranslocator 1 acts as a type 2 transglutaminase substrate: implications for mitochon-drial-dependent apoptosis. Cell Death Differ 16: 1480–1492, 2009.
229. Mangala LS, Fok JY, Zorrilla-Calancha IR, Verma A, Mehta K. Tissue transglutaminaseexpression promotes cell attachment, invasion and survival in breast cancer cells.Oncogene 26: 2459–2470, 2007.
230. Mann AP, Verma A, Sethi G, Manavathi B, Wang H, Fok JY, Kunnumakkara AB, KumarR, Aggarwal BB, Mehta K. Overexpression of tissue transglutaminase leads to consti-tutive activation of nuclear factor-kappaB in cancer cells: delineation of a novel path-way. Cancer Res 66: 8788–8795, 2006.
231. Mastroberardino PG, Farrace MG, Viti I, Pavone F, Fimia GM, Melino G, Rodolfo C,Piacentini M. “Tissue” transglutaminase contributes to the formation of disulphidebridges in proteins of mitochondrial respiratory complexes. Biochim Biophys Acta1757: 1357–1365, 2006.
232. Matarese G, Picerno I, Caccamo D, Spataro P, Cordasco G, Ientile R. Increasedtransglutaminase activity was associated with IL-6 release in cultured human gingivalfibroblasts exposed to dental cast alloys. Amino Acids 30: 267–271, 2006.
233. Matic I, Sacchi A, Rinaldi A, Melino G, Khosla C, Falasca L, Piacentini M. Character-ization of transglutaminase type II role in dendritic cell differentiation and function. JLeukoc Biol 88: 181–188, 2010.
234. Matsuki M, Yamashita F, Ishida Yamamoto A, Yamada K, Kinoshita C, Fushiki S, UedaE, Morishima Y, Tabata K, Yasuno H, Hashida M, Iizuka H, Ikawa M, Okabe M,Kondoh G, Kinoshita T, Takeda J, Yamanishi K. Defective stratum corneum and earlyneonatal death in mice lacking the gene for transglutaminase 1 (keratinocyte transglu-taminase). Prod Natl Acad Sci USA 95: 1044–1049, 1998.
235. Mavrikakis ME, Drimis S, Kontoyannis DA, Rasidakis A, Moulopoulou ES, KontoyannisS. Calcific shoulder periarthritis (tendinitis) in adult onset diabetes mellitus: a con-trolled study. Ann Rheum Dis 48: 211–214, 1989.
236. McConoughey SJ, Basso M, Niatsetskaya ZV, Sleiman SF, Smirnova NA, Langley BC,Mahishi L, Cooper AJ, Antonyak MA, Cerione RA, Li B, Starkov A, Chaturvedi RK, BealMF, Coppola G, Geschwind DH, Ryu H, Xia L, Iismaa SE, Pallos J, Pasternack R, HilsM, Fan J, Raymond LA, Marsh JL, Thompson LM, Ratan RR. Inhibition of transglutami-nase 2 mitigates transcriptional dysregulation in models of Huntington disease. EMBOMol Med 2: 349–370, 2010.
237. McKee MD, Addison WN, Kaartinen MT. Hierarchies of extracellular matrix andmineral organization in bone of the craniofacial complex and skeleton. Cells TissuesOrgans 181: 176–188, 2005.
238. Mehta K. Mammalian transglutaminases: a family portrait. Prog Exp Tumor Res 38:1–18, 2005.
239. Mehta K, Fok J, Miller FR, Koul D, Sahin AA. Prognostic significance of tissue trans-glutaminase in drug resistant and metastatic breast cancer. Clin Cancer Res 10: 8068–8076, 2004.
240. Mehta K, Kumar A, Kim HI. Transglutaminase 2: a multi-tasking protein in the com-plex circuitry of inflammation and cancer. Biochem Pharmacol 80: 1921–1929, 2010.
241. Mhaouty-Kodja S. Ghalpha/tissue transglutaminase 2: an emerging G protein in signaltransduction. Biol Cell 96: 363–367, 2004.
242. Mian S, el AS, Lawry J, Gentile V, Davies PJ, Griffin M. The importance of the GTP-binding protein tissue transglutaminase in the regulation of cell cycle progression.FEBS Lett 370: 27–31, 1995.
243. Micalizzi DS, Farabaugh SM, Ford HL. Epithelial-mesenchymal transition in cancer:parallels between normal development and tumor progression. J Mammary Gland BiolNeoplasia 15: 117–134, 2010.
244. Mirza A, Liu SL, Frizell E, Zhu J, Maddukuri S, Martinez J, Davies P, Schwarting R,Norton P, Zern MA. A role for tissue transglutaminase in hepatic injury and fibrogen-esis, and its regulation by NF-kappaB. Am J Physiol Gastrointest Liver Physiol 272:G281–G288, 1997.
246. Mishra S, Murphy LJ. Phosphorylation of transglutaminase 2 by PKA at Ser216 creates14–3-3 binding sites. Biochem Biophys Res Commun 347: 1166–1170, 2006.
247. Mishra S, Murphy LJ. The p53 oncoprotein is a substrate for tissue transglutaminasekinase activity. Biochem Biophys Res Commun 339: 726–730, 2006.
249. Monsonego A, Shani Y, Friedmann I, Paas Y, Eizenberg O, Schwartz M. Expression ofGTP-dependent and GTP-independent tissue-type transglutaminase in cytokine-treated rat brain astrocytes. J Biol Chem 272: 3724–3732, 1997.
251. Murtaugh MP, Mehta K, Johnson J, Myers M, Juliano RL, Davies PJ. Induction of tissuetransglutaminase in mouse peritoneal macrophages. J Biol Chem 258: 11074–11081,1983.
252. Muszbek L, Bereczky Z, Bagoly Z, Komaromi I, Katona E. Factor XIII: a coagulationfactor with multiple plasmatic and cellular functions. Physiol Rev 91: 931–972, 2011.
253. Nagy L, Saydak M, Shipley N, Lu S, Basilion JP, Yan ZH, Syka P, Chandraratna RA, SteinJP, Heyman RA, Davies PJ. Identification and characterization of a versatile retinoidresponse element (retinoic acid receptor response element-retinoid X receptor re-sponse element) in the mouse tissue transglutaminase gene promoter. J Biol Chem271: 4355–4365, 1996.
254. Nahrendorf M, Hu K, Frantz S, Jaffer FA, Tung CH, Hiller KH, Voll S, Nordbeck P,Sosnovik D, Gattenlohner S, Novikov M, Dickneite G, Reed GL, Jakob P, RosenzweigA, Bauer WR, Weissleder R, Ertl G. Factor XIII deficiency causes cardiac rupture,impairs wound healing, and aggravates cardiac remodeling in mice with myocardialinfarction. Circulation 113: 1196–1202, 2006.
255. Naito M, Nomura H, Iguchi A, Thompson WD, Smith EB. Effect of crosslinking byfactor XIIIa on the migration of vascular smooth muscle cells into fibrin gels. ThrombRes 90: 111–116, 1998.
256. Nakano Y, Addison WN, Kaartinen MT. ATP-mediated mineralization of MC3T3–E1osteoblast cultures. Bone 41: 549–561, 2007.
257. Nakano Y, Al-Jallad HF, Mousa A, Kaartinen MT. Expression and localization of plasmatransglutaminase factor XIIIA in bone. J Histochem Cytochem 55: 675–685, 2007.
258. Nakano Y, Forsprecher J, Kaartinen MT. Regulation of ATPase activity of transglu-taminase 2 by MT1-MMP: implications for mineralization of MC3T3–E1 osteoblastcultures. J Cell Physiol 223: 260–269, 2010.
259. Nakaoka H, Perez DM, Baek KJ, Das T, Husain A, Misono K, Im MJ, Graham RM. Gh:a GTP-binding protein with transglutaminase activity and receptor signaling function.Science 264: 1593–1596, 1994.
260. Nam J, Johnson J, Lannutti JJ, Agarwal S. Modulation of embryonic mesenchymalprogenitor cell differentiation via control over pure mechanical modulus in electro-spun nanofibers. Acta Biomater 7: 1516–1524, 2011.
261. Nanda N, Iismaa SE, Owens WA, Husain A, Mackay F, Graham RM. Targeted inacti-vation of Gh/tissue transglutaminase II. J Biol Chem 276: 20673–20678, 2001.
261a.Naukkarinen J, Surakka I, Pietilainen KH, Rissanen A, Salomaa V, Ripatti S, Yki-JarvinenH, van Duijn CM, Wichmann HE, Kaprio T, Taskinen MR, Peltonen L. ENGAGEConsortium. Use of genome-wide expression data to mine the “Gray Zone” of GWAstudies leads to novel candidate obesity genes. PLOS Genet 3: e1000976, 2010.
262. Nelea V, Nakano Y, Kaartinen MT. Size distribution and molecular associations ofplasma fibronectin and fibronectin crosslinked by transglutaminase 2. Protein J 27:223–233, 2008.
263. Nemes Z, Marekov LN, Fesus L, Steinert PM. A novel function for transglutaminase 1:attachment of long-chain omega-hydroxyceramides to involucrin by ester bond for-mation. Proc Natl Acad Sci USA 96: 8402–8407, 1999.
264. Nishimichi N, Hayashita-Kinoh H, Chen C, Matsuda H, Sheppard D, Yokosaki Y.Osteopontin undergoes polymerization in vivo and gains chemotactic activity forneutrophils mediated by integrin alpha9beta1. J Biol Chem 286: 11170–11178, 2011.
265. Nishimichi N, Higashikawa F, Kinoh HH, Tateishi Y, Matsuda H, Yokosaki Y. Poly-meric osteopontin employs integrin alpha9beta1 as a receptor and attracts neutro-phils by presenting a de novo binding site. J Biol Chem 284: 14769–14776, 2009.
266. Nurminskaya M, Kaartinen MT. Transglutaminases in mineralized tissues. Front Biosci11: 1591–1606, 2006.
267. Nurminskaya M, Magee C, Nurminsky D, Linsenmayer TF. Plasma transglutaminasein hypertrophic chondrocytes: expression and cell-specific intracellular activation pro-duce cell death and externalization. J Cell Biol 142: 1135–1144, 1998.
268. Nurminskaya MV, Linsenmayer TF. Immunohistological analysis of transglutaminasefactor XIIIA expression in mouse embryonic growth plate. J Orthop Res 20: 575–578,2002.
269. Nurminskaya MV, Recheis B, Nimpf J, Magee C, Linsenmayer TF. Transglutaminasefactor XIIIA in the cartilage of developing avian long bones. Dev Dyn 223: 24–32, 2002.
270. Nurminsky D, Shanmugasundaram S, Deasey S, Michaud C, Allen S, Hendig D,Dastjerdi A, Francis-West P, Nurminskaya M. Transglutaminase 2 regulates earlychondrogenesis and glycosaminoglycan synthesis. Mech Dev 128: 234–245, 2011.
TRANSGLUTAMINASES IN CELL FUNCTION
413Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
271. Oh K, Ko E, Kim HS, Park AK, Moon HG, Noh DY, Lee DS. Transglutaminase 2facilitates the distant hematogenous metastasis of breast cancer by modulating inter-leukin-6 in cancer cells. Breast Cancer Res 13: R96, 2011.
272. Oh K, Park HB, Byoun OJ, Shin DM, Jeong EM, Kim YW, Kim YS, Melino G, Kim IG,Lee DS. Epithelial transglutaminase 2 is needed for T cell interleukin-17 productionand subsequent pulmonary inflammation and fibrosis in bleomycin-treated mice. J ExpMed 208: 1707–1719, 2011.
273. Oliva F, Zocchi L, Codispoti A, Candi E, Celi M, Melino G, Maffulli N, Tarantino U.Transglutaminases expression in human supraspinatus tendon ruptures and in mousetendons. Biochem Biophys Res Commun 379: 887–891, 2009.
274. Orlandi A, Oliva F, Taurisano G, Candi E, Di LA, Melino G, Spagnoli LG, Tarantino U.Transglutaminase-2 differently regulates cartilage destruction and osteophyte forma-tion in a surgical model of osteoarthritis. Amino Acids 36: 755–763, 2009.
275. Orru S, Caputo I, D’Amato A, Ruoppolo M, Esposito C. Proteomics identification ofacyl-acceptor and acyl-donor substrates for transglutaminase in a human intestinalepithelial cell line. Implications for celiac disease. J Biol Chem 278: 31766–31773,2003.
276. Ouyang G, Wang Z, Fang X, Liu J, Yang CJ. Molecular signaling of the epithelial tomesenchymal transition in generating and maintaining cancer stem cells. Cell Mol LifeSci 67: 2605–2618, 2010.
277. Ozpolat B, Akar U, Mehta K, Lopez-Berestein G. PKC delta and tissue transglutami-nase are novel inhibitors of autophagy in pancreatic cancer cells. Autophagy 3: 480–483, 2007.
278. Park D, Choi SS, Ha KS. Transglutaminase 2: a multi-functional protein in multiplesubcellular compartments. Amino Acids 39: 619–631, 2010.
279. Park ES, Won JH, Han KJ, Suh PG, Ryu SH, Lee HS, Yun HY, Kwon NS, Baek KJ.Phospholipase C-delta1 and oxytocin receptor signalling: evidence of its role as aneffector. Biochem J 331: 283–289, 1998.
280. Park KS, Kim HK, Lee JH, Choi YB, Park SY, Yang SH, Kim SY, Hong KM. Transglu-taminase 2 as a cisplatin resistance marker in non-small cell lung cancer. J Cancer ResClin Oncol 136: 493–502, 2010.
281. Parmentier L, Blanchet-Bardon C, Nguyen S, Prud’homme JF, Dubertret L, Weissen-bach J. Autosomal recessive lamellar ichthyosis: identification of a new mutation intransglutaminase 1 and evidence for genetic heterogeneity. Hum Mol Genet 4: 1391–1395, 1995.
282. Parsons M, Keppler MD, Kline A, Messent A, Humphries MJ, Gilchrist R, Hart IR,Quittau-Prevostel C, Hughes WE, Parker PJ, Ng T. Site-directed perturbation ofprotein kinase C-integrin interaction blocks carcinoma cell chemotaxis. Mol Cell Biol22: 5897–5911, 2002.
283. Pascual G, Fong AL, Ogawa S, Gamliel A, Li AC, Perissi V, Rose DW, Willson TM,Rosenfeld MG, Glass CK. A SUMOylation-dependent pathway mediates transrepres-sion of inflammatory response genes by PPAR-gamma. Nature 437: 759–763, 2005.
284. Paul RG, Bailey AJ. Glycation of collagen: the basis of its central role in the latecomplications of ageing and diabetes. Int J Biochem Cell Biol 28: 1297–1310, 1996.
285. Paulmann N, Grohmann M, Voigt JP, Bert B, Vowinckel J, Bader M, Skelin M, JevsekM, Fink H, Rupnik M, Walther DJ. Intracellular serotonin modulates insulin secretionfrom pancreatic beta-cells by protein serotonylation. PLoS Biol 7: e1000229, 2009.
286. Pavlyukov MS, Antipova NV, Balashova MV, Shakhparonov MI. Detection of transglu-taminase 2 conformational changes in living cell. Biochem Biophys Res Commun 421:773–779, 2012.
287. Pelengaris S, Khan M, Evan G. c-MYC: more than just a matter of life and death. NatRev Cancer 2: 764–776, 2002.
288. Peng X, Zhang Y, Zhang H, Graner S, Williams JF, Levitt ML, Lokshin A. Interaction oftissue transglutaminase with nuclear transport protein importin-alpha3. FEBS Lett446: 35–39, 1999.
289. Peters LL, Jindel HK, Gwynn B, Korsgren C, John KM, Lux SE, Mohandas N, CohenCM, Cho MR, Golan DE, Brugnara C. Mild spherocytosis and altered red cell iontransport in protein 4.2-null mice. J Clin Invest 103: 1527–1537, 1999.
290. Piacentini M, Ceru MP, Dini L, Di RM, Piredda L, Thomazy V, Davies PJ, Fesus L. Invivo and in vitro induction of “tissue” transglutaminase in rat hepatocytes by retinoicacid. Biochim Biophys Acta 1135: 171–179, 1992.
291. Piacentini M, Farrace MG, Piredda L, Matarrese P, Ciccosanti F, Falasca L, Rodolfo C,Giammarioli AM, Verderio E, Griffin M, Malorni W. Transglutaminase overexpressionsensitizes neuronal cell lines to apoptosis by increasing mitochondrial membranepotential and cellular oxidative stress. J Neurochem 81: 1061–1072, 2002.
292. Piercy-Kotb SA, Mousa A, Al-Jallad HF, Myneni VD, Chicatun F, Nazhat SN, KaartinenMT. Factor XIIIA transglutaminase expression and secretion by osteoblasts is regu-lated by extracellular matrix collagen and the MAP kinase signaling pathway. J CellPhysiol 227: 2936–2946, 2012.
293. Pinkas DM, Strop P, Brunger AT, Khosla C. Transglutaminase 2 undergoes a largeconformational change upon activation. PLoS Biol 5: e327, 2007.
294. Porzio O, Massa O, Cunsolo V, Colombo C, Malaponti M, Bertuzzi F, Hansen T,Johansen A, Pedersen O, Meschi F, Terrinoni A, Melino G, Federici M, Decarlo N,Menicagli M, Campani D, Marchetti P, Ferdaoussi M, Froguel P, Federici G, VaxillaireM, Barbetti F. Missense mutations in the TGM2 gene encoding transglutaminase 2 arefound in patients with early-onset type 2 diabetes. Mutation in brief no 982 online.Hum Mutat 28: 1150, 2007.
295. Priglinger SG, Alge CS, Neubauer AS, Kristin N, Hirneiss C, Eibl K, Kampik A, Welge-Lussen U. TGF-beta2-induced cell surface tissue transglutaminase increases adhesionand migration of RPE cells on fibronectin through the gelatin-binding domain. InvestOphthalmol Vis Sci 45: 955–963, 2004.
296. Quan G, Choi JY, Lee DS, Lee SC. TGF-beta1 up-regulates transglutaminase two andfibronectin in dermal fibroblasts: a possible mechanism for the stabilization of tissueinflammation. Arch Dermatol Res 297: 84–90, 2005.
297. Radek JT, Jeong JM, Murthy SN, Ingham KC, Lorand L. Affinity of human erythrocytetransglutaminase for a 42-kDa gelatin-binding fragment of human plasma fibronectin.Proc Natl Acad Sci USA 90: 3152–3156, 1993.
298. Ritter SJ, Davies PJ. Identification of a transforming growth factor-beta1/bone mor-phogenetic protein 4 (TGF-beta1/BMP4) response element within the mouse tissuetransglutaminase gene promoter. J Biol Chem 273: 12798–12806, 1998.
299. Rodolfo C, Mormone E, Matarrese P, Ciccosanti F, Farrace MG, Garofano E, PireddaL, Fimia GM, Malorni W, Piacentini M. Tissue transglutaminase is a multifunctionalBH3-only protein. J Biol Chem 279: 54783–54792, 2004.
300. Rosenthal AK, Gohr CM, Mitton E, Monnier V, Burner T. Advanced glycation endproducts increase transglutaminase activity in primary porcine tenocytes. J Invest Med57: 460–466, 2009.
301. Rosenthal AK, Masuda I, Gohr CM, Derfus BA, Le M. The transglutaminase, FactorXIIIA, is present in articular chondrocytes. Osteoarthritis Cartilage 9: 578–581, 2001.
302. Russell LJ, DiGiovanna JJ, Rogers GR, Steinert PM, Hashem N, Compton JG, Bale SJ.Mutations in the gene for transglutaminase 1 in autosomal recessive lamellar ichthyo-sis. Nat Genet 9: 279–283, 1995.
303. Santhanam L, Tuday EC, Webb AK, Dowzicky P, Kim JH, Oh YJ, Sikka G, Kuo M,Halushka MK, Macgregor AM, Dunn J, Gutbrod S, Yin D, Shoukas A, Nyhan D,Flavahan NA, Belkin AM, Berkowitz DE. Decreased S-nitrosylation of tissue transglu-taminase contributes to age-related increases in vascular stiffness. Circ Res 107: 117–125, 2010.
304. Sarang Z, Molnar P, Nemeth T, Gomba S, Kardon T, Melino G, Cotecchia S, Fesus L,Szondy Z. Tissue transglutaminase (TG2) acting as G protein protects hepatocytesagainst Fas-mediated cell death in mice. Hepatology 42: 578–587, 2005.
305. Sarang Z, Toth B, Balajthy Z, Koroskenyi K, Garabuczi E, Fesus L, Szondy Z. Somelessons from the tissue transglutaminase knockout mouse. Amino Acids 36: 625–631,2009.
306. Sarkar NK, Clarke DD, Waelsch H. An enzymically catalyzed incorporation of aminesinto proteins. Biochim Biophys Acta 25: 451–452, 1957.
307. Sarvary A, Szucs S, Balogh I, Becsky A, Bardos H, Kavai M, Seligsohn U, Egbring R,Lopaciuk S, Muszbek L, Adany R. Possible role of factor XIII subunit A in Fcgamma andcomplement receptor-mediated phagocytosis. Cell Immunol 228: 81–90, 2004.
308. Satchwell TJ, Shoemark DK, Sessions RB, Toye AM. Protein 4.2: a complex linker.Blood Cells Mol Dis 42: 201–210, 2009.
309. Satpathy M, Cao L, Pincheira R, Emerson R, Bigsby R, Nakshatri H, Matei D. Enhancedperitoneal ovarian tumor dissemination by tissue transglutaminase. Cancer Res 67:7194–7202, 2007.
310. Satpathy M, Shao M, Emerson R, Donner DB, Matei D. Tissue transglutaminaseregulates matrix metalloproteinase-2 in ovarian cancer by modulating cAMP-re-sponse element-binding protein activity. J Biol Chem 284: 15390–15399, 2009.
311. Scarpellini A, Germack R, Lortat-Jacob H, Muramatsu T, Billett E, Johnson T, VerderioEA. Heparan sulfate proteoglycans are receptors for the cell-surface trafficking andbiological activity of transglutaminase-2. J Biol Chem 284: 18411–18423, 2009.
312. Schelling JR. Tissue transglutaminase inhibition as treatment for diabetic glomerularscarring: it’s good to be glueless. Kidney Int 76: 363–365, 2009.
313. Semenza GL. Oxygen sensing, homeostasis, disease. N Engl J Med 365: 537–547,2011.
314. Shanmugasundaram S, Logan-Mauney S, Burgos K, Nurminskaya M. Tissue transglu-taminase regulates chondrogenesis in mesenchymal stem cells on collagen type XImatrices. Amino Acids 42: 1045–1053, 2012.
315. Shao M, Cao L, Shen C, Satpathy M, Chelladurai B, Bigsby RM, Nakshatri H, Matei D.Epithelial-to-mesenchymal transition and ovarian tumor progression induced by tis-sue transglutaminase. Cancer Res 69: 9192–9201, 2009.
316. Shekaran A, Garcia AJ. Extracellular matrix-mimetic adhesive biomaterials for bonerepair. J Biomed Mater Res A 96: 261–272, 2011.
317. Shimada J, Suzuki Y, Kim SJ, Wang PC, Matsumura M, Kojima S. Transactivation viaRAR/RXR-Sp1 interaction: characterization of binding between Sp1 and GC box mo-tif. Mol Endocrinol 15: 1677–1692, 2001.
318. Shostak K, Chariot A. NF-kappaB, stem cells and breast cancer: the links get stronger.Breast Cancer Res 13: 214, 2011.
319. Siegel M, Strnad P, Watts RE, Choi K, Jabri B, Omary MB, Khosla C. Extracellulartransglutaminase 2 is catalytically inactive, but is transiently activated upon tissueinjury. PLoS One 3: e1861, 2008.
320. Singh US, Cerione RA. Biochemical effects of retinoic acid on GTP-binding Protein/transglutaminases in HeLa cells. Stimulation of GTP-binding and transglutaminaseactivity, membrane association, and phosphatidylinositol lipid turnover. J Biol Chem271: 27292–27298, 1996.
321. Singh US, Erickson JW, Cerione RA. Identification and biochemical characterization ofan 80 kilodalton GTP-binding/transglutaminase from rabbit liver nuclei. Biochemistry34: 15863–15871, 1995.
322. Singh US, Pan J, Kao YL, Joshi S, Young KL, Baker KM. Tissue transglutaminasemediates activation of RhoA and MAP kinase pathways during retinoic acid-inducedneuronal differentiation of SH-SY5Y cells. J Biol Chem 278: 391–399, 2003.
323. Small K, Feng JF, Lorenz J, Donnelly ET, Yu A, Im MJ, Dorn GW, Liggett SB. Cardiacspecific overexpression of transglutaminase II [G(h)] results in a unique hypertrophyphenotype independent of phospholipase C activation. J Biol Chem 274: 21291–21296, 1999.
324. Sommerfeldt DW, Rubin CT. Biology of bone and how it orchestrates the form andfunction of the skeleton. Eur Spine J 10 Suppl 2: S86–S95, 2001.
325. Song H, Chang W, Lim S, Seo HS, Shim CY, Park S, Yoo KJ, Kim BS, Min BH, Lee H,Jang Y, Chung N, Hwang KC. Tissue transglutaminase is essential for integrin-medi-ated survival of bone marrow-derived mesenchymal stem cells. Stem Cells 25: 1431–1438, 2007.
326. Spurlin TA, Bhadriraju K, Chung KH, Tona A, Plant AL. The treatment of collagenfibrils by tissue transglutaminase to promote vascular smooth muscle cell contractilesignaling. Biomaterials 30: 5486–5496, 2009.
328. Staudt LM. Oncogenic activation of NF-kappaB. Cold Spring Harb Perspect Biol 2:a000109, 2010.
329. Steinert PM. A model for the hierarchical structure of the human epidermal cornifiedcell envelope. Cell Death Differ 2: 33–40, 1995.
330. Stephens P, Grenard P, Aeschlimann P, Langley M, Blain E, Errington R, Kipling D,Thomas D, Aeschlimann D. Crosslinking and G-protein functions of transglutaminase2 contribute differentially to fibroblast wound healing responses. J Cell Sci 117: 3389–3403, 2004.
331. Sturniolo MT, Chandraratna RA, Eckert RL. A novel transglutaminase activator formsa complex with type 1 transglutaminase. Oncogene 24: 2963–2972, 2005.
332. Sturniolo MT, Dashti SR, Deucher A, Rorke EA, Broome AM, Chandraratna RA,Keepers T, Eckert RL. A novel tumor suppressor protein promotes keratinocyteterminal differentiation via activation of type I transglutaminase. J Biol Chem 278:48066–48073, 2003.
333. Suto N, Ikura K, Sasaki R. Expression induced by interleukin-6 of tissue-type transglu-taminase in human hepatoblastoma HepG2 cells. J Biol Chem 268: 7469–7473, 1993.
334. Takeuchi Y, Ohashi H, Birckbichler PJ, Ikejima T. Nuclear translocation of tissue typetransglutaminase during sphingosine-induced cell death: a novel aspect of the enzymewith DNA hydrolytic activity. Z Naturforsch C 53: 352–358, 1998.
335. Tanaka K, Yokosaki Y, Higashikawa F, Saito Y, Eboshida A, Ochi M. The integrinalpha5beta1 regulates chondrocyte hypertrophic differentiation induced by GTP-bound transglutaminase 2. Matrix Biol 26: 409–418, 2007.
336. Tang CH, Yang RS, Huang TH, Liu SH, Fu WM. Enhancement of fibronectin fibrillo-genesis and bone formation by basic fibroblast growth factor via protein kinase C-de-pendent pathway in rat osteoblasts. Mol Pharmacol 66: 440–449, 2004.
337. Tang CH, Yang RS, Liou HC, Fu WM. Enhancement of fibronectin synthesis and fibrillo-genesis by BMP-4 in cultured rat osteoblast. J Bone Miner Res 18: 502–511, 2003.
338. Tarantino U, Oliva F, Taurisano G, Orlandi A, Pietroni V, Candi E, Melino G, MaffulliN. FXIIIA and TGF-beta over-expression produces normal musculo-skeletal pheno-type in TG2�/� mice. Amino Acids 36: 679–684, 2009.
339. Tatsukawa H, Fukaya Y, Frampton G, Martinez-Fuentes A, Suzuki K, Kuo TF, Nagat-suma K, Shimokado K, Okuno M, Wu J, Iismaa S, Matsuura T, Tsukamoto H, Zern MA,Graham RM, Kojima S. Role of transglutaminase 2 in liver injury via cross-linking andsilencing of transcription factor Sp1. Gastroenterology 136: 1783–1795, 2009.
340. Tatsukawa H, Kojima S. Recent advances in understanding the roles of transglutami-nase 2 in alcoholic steatohepatitis. Cell Biol Int 34: 325–334, 2010.
341. Tee AE, Marshall GM, Liu PY, Xu N, Haber M, Norris MD, Iismaa SE, Liu T. Opposingeffects of two tissue transglutaminase protein isoforms in neuroblastoma cell differ-entiation. J Biol Chem 285: 3561–3567, 2010.
342. Telci D, Collighan RJ, Basaga H, Griffin M. Increased TG2 expression can result ininduction of transforming growth factor beta1, causing increased synthesis and depo-sition of matrix proteins, which can be regulated by nitric oxide. J Biol Chem 284:29547–29558, 2009.
344. Telci D, Wang Z, Li X, Verderio EA, Humphries MJ, Baccarini M, Basaga H, Griffin M. Fi-bronectin-tissue transglutaminase matrix rescues RGD-impaired cell adhesionthrough syndecan-4 and beta1 integrin co-signaling. J Biol Chem 283: 20937–20947,2008.
345. Teng FY, Tang BL. NF-kappaB signaling in neurite growth and neuronal survival. RevNeurosci 21: 299–313, 2010.
346. Terrinoni A, Serra V, Codispoti A, Talamonti E, Bui L, Palombo R, Sette M, CampioneE, Didona B, Annicchiarico-Petruzzelli M, Zambruno G, Melino G, Candi E. Noveltransglutaminase 1 mutations in patients affected by lamellar ichthyosis. Cell Death Dis3: e416, 2012.
348. Thomas H, Beck K, Adamczyk M, Aeschlimann P, Langley M, Oita RC, Thiebach L,Hils M, Aeschlimann D. Transglutaminase 6: a protein associated with central nervoussystem development and motor function. Amino Acids 44: 161–177, 2013.
349. Thomazy VA, Davies PJ. Expression of tissue transglutaminase in the developingchicken limb is associated both with apoptosis and endochondral ossification. CellDeath Differ 6: 146–154, 1999.
350. Tolentino PJ, Waghray A, Wang KK, Hayes RL. Increased expression of tissue-typetransglutaminase following middle cerebral artery occlusion in rats. J Neurochem 89:1301–1307, 2004.
TRANSGLUTAMINASES IN CELL FUNCTION
415Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
351. Tong L, Png E, Aihua H, Yong SS, Yeo HL, Riau A, Mendoz E, Chaurasia SS, Lim CT,Yiu TW, Iismaa SE. Molecular mechanism of transglutaminase-2 in corneal epithelialmigration and adhesion. Biochim Biophys Acta 1833: 1304–1315, 2013.
352. Torocsik D, Szeles L, Paragh G Jr, Rakosy Z, Bardos H, Nagy L, Balazs M, Inbal A,Adany R. Factor XIII-A is involved in the regulation of gene expression in alternativelyactivated human macrophages. Thromb Haemost 104: 709–717, 2010.
353. Toth B, Garabuczi E, Sarang Z, Vereb G, Vamosi G, Aeschlimann D, Blasko B, Becsi B,Erdodi F, Lacy-Hulbert A, Zhang A, Falasca L, Birge RB, Balajthy Z, Melino G, Fesus L,Szondy Z. Transglutaminase 2 is needed for the formation of an efficient phagocyteportal in macrophages engulfing apoptotic cells. J Immunol 182: 2084–2092, 2009.
354. Toth B, Sarang Z, Vereb G, Zhang A, Tanaka S, Melino G, Fesus L, Szondy Z. Over-expression of integrin beta3 can partially overcome the defect of integrin beta3 sig-naling in transglutaminase 2 null macrophages. Immunol Lett 126: 22–28, 2009.
355. Tsujimoto I, Moriya K, Sakai K, Dickneite G, Sakai T. Critical role of factor XIII in theinitial stages of carbon tetrachloride-induced adult liver remodeling. Am J Pathol 179:3011–3019, 2011.
356. Tucholski J, Johnson GV. Tissue transglutaminase directly regulates adenylyl cyclaseresulting in enhanced cAMP-response element-binding protein (CREB) activation. JBiol Chem 278: 26838–26843, 2003.
357. Tucholski J, Lesort M, Johnson GV. Tissue transglutaminase is essential for neurite out-growth in human neuroblastoma SH-SY5Y cells. Neuroscience 102: 481–491, 2001.
358. Tucholski J, Roth KA, Johnson GV. Tissue transglutaminase overexpression in the brainpotentiates calcium-induced hippocampal damage. J Neurochem 97: 582–594, 2006.
359. Turner PM, Lorand L. Complexation of fibronectin with tissue transglutaminase.Biochemistry 28: 628–635, 1989.
360. Van Strien ME, Baron W, Bakker EN, Bauer J, Bol JG, Breve JJ, Binnekade R, VanDer Laarse WJ, Drukarch B, Van Dam AM. Tissue transglutaminase activity isinvolved in the differentiation of oligodendrocyte precursor cells into myelin-formingoligodendrocytes during CNS remyelination. Glia 59: 1622–1634, 2011.
361. Van Strien ME, Breve JJ, Fratantoni S, Schreurs MW, Bol JG, Jongenelen CA, DrukarchB, Van Dam AM. Astrocyte-derived tissue transglutaminase interacts with fibronectin:a role in astrocyte adhesion and migration? PLoS One 6: e25037, 2011.
362. Verderio E, Gaudry C, Gross S, Smith C, Downes S, Griffin M. Regulation of cellsurface tissue transglutaminase: effects on matrix storage of latent transforminggrowth factor-beta binding protein-1. J Histochem Cytochem 47: 1417–1432, 1999.
363. Verderio E, Scarpellini A. Significance of the syndecan-4-transglutaminase-2 interac-tion. Sci World J 10: 1073–1077, 2010.
364. Verderio EA, Johnson T, Griffin M. Tissue transglutaminase in normal and abnormalwound healing: review article. Amino Acids 26: 387–404, 2004.
365. Verderio EA, Johnson TS, Griffin M. Transglutaminases in wound healing and inflam-mation. Prog Exp Tumor Res 38: 89–114, 2005.
366. Verderio EA, Scarpellini A, Johnson TS. Novel interactions of TG2 with heparansulfate proteoglycans: reflection on physiological implications. Amino Acids 36: 671–677, 2009.
367. Verderio EA, Telci D, Okoye A, Melino G, Griffin M. A novel RGD-independent celadhesion pathway mediated by fibronectin-bound tissue transglutaminase rescuescells from anoikis. J Biol Chem 278: 42604–42614, 2003.
368. Verma A, Guha S, Diagaradjane P, Kunnumakkara AB, Sanguino AM, Lopez-BeresteinG, Sood AK, Aggarwal BB, Krishnan S, Gelovani JG, Mehta K. Therapeutic significanceof elevated tissue transglutaminase expression in pancreatic cancer. Clin Cancer Res14: 2476–2483, 2008.
369. Verma A, Guha S, Wang H, Fok JY, Koul D, Abbruzzese J, Mehta K. Tissue transglu-taminase regulates focal adhesion kinase/AKT activation by modulating PTEN expres-sion in pancreatic cancer cells. Clin Cancer Res 14: 1997–2005, 2008.
370. Verma A, Mehta K. Tissue transglutaminase-mediated chemoresistance in cancercells. Drug Resist Update 10: 144–151, 2007.
371. Verma A, Mehta K. Transglutaminase-mediated activation of nuclear transcriptionfactor-kappaB in cancer cells: a new therapeutic opportunity. Curr Cancer Drug Targets7: 559–565, 2007.
372. Verma A, Wang H, Manavathi B, Fok JY, Mann AP, Kumar R, Mehta K. Increasedexpression of tissue transglutaminase in pancreatic ductal adenocarcinoma and itsimplications in drug resistance and metastasis. Cancer Res 66: 10525–10533, 2006.
373. Vezza R, Habib A, FitzGerald GA. Differential signaling by the thromboxanereceptor isoforms via the novel GTP-binding protein, Gh. J Biol Chem 274: 12774–12779, 1999.
374. Villa-Bellosta R, Wang X, Millan JL, Dubyak GR, O’Neill WC. Extracellular pyrophos-phate metabolism and calcification in vascular smooth muscle. Am J Physiol Heart CircPhysiol 301: H61–H68, 2011.
375. Voulgari A, Pintzas A. Epithelial-mesenchymal transition in cancer metastasis: mech-anisms, markers and strategies to overcome drug resistance in the clinic. BiochimBiophys Acta 1796: 75–90, 2009.
376. Walker RA. The complexities of breast cancer desmoplasia. Breast Cancer Res 3:143–145, 2001.
377. Walther DJ, Peter JU, Winter S, Holtje M, Paulmann N, Grohmann M, Vowinckel J,Alamo-Bethencourt V, Wilhelm CS, Ahnert-Hilger G, Bader M. Serotonylation ofsmall GTPases is a signal transduction pathway that triggers platelet alpha-granulerelease. Cell 115: 851–862, 2003.
378. Walther DJ, Stahlberg S, Vowinckel J. Novel roles for biogenic monoamines: frommonoamines in transglutaminase-mediated post-translational protein modification tomonoaminylation deregulation diseases. FEBS J 278: 4740–4755, 2011.
379. Wang Z, Collighan RJ, Gross SR, Danen EH, Orend G, Telci D, Griffin M. RGD-independent cell adhesion via a tissue transglutaminase-fibronectin matrix promotesfibronectin fibril deposition and requires syndecan-4/2 and �5�1 integrin co-signaling.J Biol Chem 285: 40212–40229, 2010.
380. Wang Z, Griffin M. TG2, a novel extracellular protein with multiple functions. AminoAcids 42: 939–949, 2012.
381. Wang Z, Telci D, Griffin M. Importance of syndecan-4 and syndecan-2 in osteoblastcell adhesion and survival mediated by a tissue transglutaminase-fibronectin complex.Exp Cell Res 317: 367–381, 2011.
382. Watts RE, Siegel M, Khosla C. Structure-activity relationship analysis of the selectiveinhibition of transglutaminase 2 by dihydroisoxazoles. J Med Chem 49: 7493–7501,2006.
383. Watts SW, Priestley JR, Thompson JM. Serotonylation of vascular proteins importantto contraction. PLoS One 4: e5682, 2009.
384. Wierstra I. Sp1: emerging roles–beyond constitutive activation of TATA-less house-keeping genes. Biochem Biophys Res Commun 372: 1–13, 2008.
385. Wilhelmus MM, Grunberg SC, Bol JG, Van Dam AM, Hoozemans JJ, Rozemuller AJ, DrukarchB. Transglutaminases and transglutaminase-catalyzed cross-links colocalize with thepathological lesions in Alzheimer’s disease brain. Brain Pathol 19: 612–622, 2009.
386. Williams-Ashman HG, Wilson J, Beil RE, Lorand L. Transglutaminase reactions asso-ciated with the rat semen clotting system: modulation by macromolecular polyanions.Biochem Biophys Res Commun 79: 1192–1198, 1977.
387. Worthington JJ, Klementowicz JE, Travis MA. TGFbeta: a sleeping giant awoken byintegrins. Trends Biochem Sci 36: 47–54, 2011.
388. Wu J, Liu SL, Zhu JL, Norton PA, Nojiri S, Hoek JB, Zern MA. Roles of tissue trans-glutaminase in ethanol-induced inhibition of hepatocyte proliferation and alpha 1-ad-renergic signal transduction. J Biol Chem 275: 22213–22219, 2000.
389. Xian X, Gopal S, Couchman JR. Syndecans as receptors and organizers of the extra-cellular matrix. Cell Tissue Res 339: 31–46, 2010.
390. Xu L, Begum S, Hearn JD, Hynes RO. GPR56, an atypical G protein-coupled receptor,binds tissue transglutaminase, TG2, and inhibits melanoma tumor growth and metas-tasis. Proc Natl Acad Sci USA 103: 9023–9028, 2006.
391. Yamada K, Matsuki M, Morishima Y, Ueda E, Tabata K, Yasuno H, Suzuki M, Yamani-shi K. Activation of the human transglutaminase 1 promoter in transgenic mice: ter-minal differentiation-specific expression of the TGM1-lacZ transgene in keratinizedstratified squamous epithelia. Hum Mol Genet 6: 2223–2231, 1997.
392. Yamada KM, Even-Ram S. Integrin regulation of growth factor receptors. Nat Cell Biol4: E75–E76, 2002.
393. Yin X, Chen Z, Guo Z, Liu X, Yu H. Tissue transglutaminase expression and activity inhuman ligamentum flavum cells derived from thoracic ossification of ligamentumflavum. Spine 35: E1018–E1024, 2010.
394. Yuan L, Behdad A, Siegel M, Khosla C, Higashikubo R, Rich KM. Tissue transgluami-nase 2 expression in meningiomas. J Neurooncol 90: 125–132, 2008.
395. Yuan L, Siegel M, Choi K, Khosla C, Miller CR, Jackson EN, Piwnica-Worms D, RichKM. Transglutaminase 2 inhibitor, KCC009, disrupts fibronectin assembly in the ex-tracellular matrix and sensitizes orthotopic glioblastomas to chemotherapy. Oncogene26: 2563–2573, 2007.
396. Zemskov EA, Janiak A, Hang J, Waghray A, Belkin AM. The role of tissue transglu-taminase in cell-matrix interactions. Front Biosci 11: 1057–1076, 2006.
397. Zemskov EA, Loukinova E, Mikhailenko I, Coleman RA, Strickland DK, Belkin AM.Regulation of platelet-derived growth factor receptor function by integrin-associatedcell surface transglutaminase. J Biol Chem 284: 16693–16703, 2009.
398. Zemskov EA, Mikhailenko I, Hsia RC, Zaritskaya L, Belkin AM. Unconventional se-cretion of tissue transglutaminase involves phospholipid-dependent delivery into re-cycling endosomes. PLoS One 6: e19414, 2011.
399. Zemskov EA, Mikhailenko I, Smith EP, Belkin AM. Tissue transglutaminase promotesPDGF/PDGFR-mediated signaling and responses in vascular smooth muscle cells. JCell Physiol 227: 2089–2096, 2012.
400. Zemskov EA, Mikhailenko I, Strickland DK, Belkin AM. Cell-surface transglutaminaseundergoes internalization and lysosomal degradation: an essential role for LRP1. J CellSci 120: 3188–3199, 2007.
401. Zhang J, Lesort M, Guttmann RP, Johnson GV. Modulation of the in situ activity oftissue transglutaminase by calcium and GTP. J Biol Chem 273: 2288–2295, 1998.
402. Zhang R, Lu Y, Ye L, Yuan B, Yu S, Qin C, Xie Y, Gao T, Drezner MK, Bonewald LF,Feng JQ. Unique roles of phosphorus in endochondral bone formation and osteocytematuration. J Bone Miner Res 26: 1047–1056, 2011.
TRANSGLUTAMINASES IN CELL FUNCTION
417Physiol Rev • VOL 94 • APRIL 2014 • www.prv.org
doi:10.1152/physrev.00019.2013 94:383-417, 2014.Physiol RevGozde Colak, Gail V. W. Johnson and Kapil MehtaRichard L. Eckert, Mari T. Kaartinen, Maria Nurminskaya, Alexey M. Belkin,Transglutaminase Regulation of Cell Function
You might find this additional info useful...
403 articles, 152 of which can be accessed free at:This article cites /content/94/2/383.full.html#ref-list-1
including high resolution figures, can be found at:Updated information and services /content/94/2/383.full.html
can be found at:Physiological Reviewsabout Additional material and information http://www.the-aps.org/publications/prv
