Physiol Rev 94: 383– 417, 2014
doi:10.1152/physrev.00019.2013
TRANSGLUTAMINASE REGULATION OF CELL
FUNCTION
Richard 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 Experimental
Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas
Eckert 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 and
scaffolding functions that participate in regulation of cell fate in a wide range of cellular
systems 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 cell
differentiation and survival, role in cancer development and progression, and function in signal
transduction. We also discuss the mechanisms whereby TG level is controlled and how TGs control
downstream targets. The studies described herein begin to clarify the physiological roles of TGs in
both normal biology and disease states.
L
I.
II.
III.
IV.
V.
VI.
VII.
TRANSGLUTAMINASE: INTRODUCTION...
REGULATION OF PROTEIN...
TRANSGLUTAMINASE-REGULATED...
TGS IN CELL...
ROLE OF TG2 IN CANCER
REGULATION OF TG2 EXPRESSION...
PERSPECTIVES AND FUTURE...
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386
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397
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403
406
I. TRANSGLUTAMINASES: INTRODUCTION
AND OVERVIEW
Transglutaminases (TGs; EC 2.3.2.13) are a family of structurally and functionally related proteins that catalyze the
Ca2⫹-dependent posttranslational modification of proteins
by introducing covalent bonds between free amine groups
(e.g., protein- or peptide-bound lysine) and ␥-carboxamide
groups of peptide-bound glutamines (FIGURE 1). Researchers identified the first TG, now designated TG2, in 1959
from guinea pig liver extracts based on its ability to catalyze
incorporation of low-molecular-weight primary amines
into proteins (306). Since the discovery of TG2, additional
proteins with this activity have been identified from unicellular organisms, invertebrates, fish, mammals, and plants
(122). Nine TG genes are present in humans. Eight are
catalytically active enzymes, and one is inactive (erythrocyte membrane protein band 4.2) (122). These proteins
serve as scaffolds, maintain membrane integrity, regulate
cell 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 amino
acid sequence at the active site (FIGURE 2). In addition to the
protein crosslinking and scaffolding functions, TGs cata-
lyze posttranslational modification of proteins via deamidation and amine incorporation (FIGURE 1). For example,
TG2-dependent deamidation of gliadin A, a component of
wheat and other cereals, is implicated in the pathogenesis of
celiac disease (189). Similarly, deamidation of Gln63 in
RhoA activates this signaling protein (108). Moreover, TGcatalyzed incorporation of amines into proteins can modify
the function, stability, and immunogenicity of substrate
proteins and contribute to autoimmune disease (220). Of
the nine TGs identified in humans, TG2 is the most widely
distributed and most extensively studied. In this review, we
describe the role of TGs in general, and TG2 in particular,
and also explore the consequences of aberrant TG expression and activation. TABLE 1 summarizes the general features of each member of the TG family.
A. Transglutaminase 1
Keratinocyte TG (TG1) is expressed in the stratified squamous epithelia of the skin and upper digestive tract and in
the lower female genital tract. The TGM1 gene promoter
contains three activator protein AP2-like response elements
located ⬃0.5 kb from the transcription initiation site (238).
Proteolytic cleavage, increased Ca2⫹ level, and interaction
with tazarotene-induced gene 3 (TIG3) are known to activate TG1 catalytic activity (98, 156, 331, 332). Phorbol
esters induce and retinoic acid reduces TG1 mRNA and
protein expression (97). TG1 protein associates with the
plasma membrane via fatty acyl linkage in the NH2-terminal cysteine residue and is released by proteolysis as 10-,
33-, and 66-kDa fragments (183). Autosomal recessive lamellar ichthyosis results from mutation of the TG1-encod-
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ECKERT ET AL.
γ
O
II
P1- CH2-CH2-C – N- R + NH3 (1)
I
H
H2N-R
TG
Transamidation
Nε (γ-glutamyl) lysine bridge
ε
Ca2+
H2N CH2-CH2-CH2-CH2- P2
II
γ O
ε
Transamidation
II
P1- CH2- CH2- C – NH2 + TG -Cys
P1-CH2-CH2-C-N-CH2-CH2-CH2-CH2- P2 (2)
γ
O
I
H
Glutamine
+ NH3
Deamidation
H2O
γ
O
II
P1- CH2 CH2 C - OH + NH3 (3)
Glutamate
FIGURE 1. Enzymatic reactions catalyzed by transglutaminases (TGs). Transamidation crosslinking reactions
require the presence of Ca2⫹ to covalently link primary amines including polyamines, monoamines, and
protein-bound amines (P2) to a glutamine residue of the acceptor protein (P1). These reactions form polyamines or monoamine crosslinks with proteins (1) or protein-protein crosslinks to form an ⑀-(␥-glutamyl)lysine
isopeptide bond (2). Under slightly acidic conditions, some TGs can utilize H2O to catalyze deamidation of the
P1 protein (3).
ing gene (46, 71, 140, 141). Common mutations include a
C-to-T change in the binding site for the transcription factor
Sp1 within the promoter region, a Gly143-to-Glu mutation
in exon 3, and a Val382-to-Met mutation in exon 7. Lamellar ichthyosis is a rare keratinization disorder of the skin
characterized by abnormal cornification of the epidermis.
Individuals with ichthyosis exhibit drastically reduced TG1
activity and absence of detectable TG1 protein (46, 71, 140,
141). TG1 knockout mice exhibit the lamellar ichthyosis
phenotype (234).
B. Transglutaminase 2
Tissue TG (TG2), also referred to as TGc or Gh, is widely
distributed in tissues and cell types. TG2 is predominantly a
cytosolic protein but is also present in the nucleus and on
the plasma membrane (220). The TG2 gene promoter contains a retinoic acid response element (1.7 kb upstream of
the initiation site), an interleukin (IL)-6 specific cis-regulatory element (4 kb upstream of the promoter), a transforming growth factor-␤1 (TGF-␤1) response element (868 bp
Table 1. Properties of transglutaminase proteins
Gene
Protein
Chromosomal
Location
Molecular
Mass, kDa
Main Function
Tissue Distribution
Alternate Names
TGM1
TG1
14q11.2
90
Cell envelope formation
during keratinocyte
differentiation
Membrane-bound keratinocytes
TGk, keratinocyte TG, particulate TG
TGM2
TG2
20q11-12
80
Apoptosis, cell
adhesion, matrix
stabilization, signal
transduction
Many tissues: cytosolic, nuclear,
membrane, and extracellular
Tissue TG, TGc, liver TG, endothelial
TG, erythrocyte TG, Gh␣
TGM3
TG3
20q11-12
77
Cell envelope formation
during keratinocyte
differentiation
Hair follicle, epidermis, brain
TGE, callus TG, hair follicle TG,
bovine snout TG
TGM4
TG4
3q21-22
77
Reproduction, especially
in rodents as a result
of semen coagulation
Prostate
TGp androgen-regulated major
secretory protein, vesiculase,
dorsal prostate protein 1
TGM5
TG5
15q15.2
81
Cell envelope formation
in keratinocytes
Foreskin keratinocytes, epithelial
barrier lining, skeletal
muscular striatum
TGx
TGM6
TG6
20q11
78
Not known
Testis and lung
TGy
TGM7
TG7
15q15.2
81
Not known
Ubiquitous but predominately in
testis and lung
TGz
F13A1
FXIIIa
6q24-25
83
Blood clotting, wound
healing, bone
synthesis
Platelets, placenta, synovial fluid,
chondrocytes, astrocytes,
macrophages, osteoclasts
and osteoblasts
Fibrin-stabilizing factor, fibrinoligase,
plasma TG, Laki-Lorand factor
EPB42
Band4.2
15q15.2
72
Membrane integrity, cell
attachment, signal
transduction
Erythrocyte membranes, cone
marrow, spleen
B4.2, ATP-binding erythrocyte
membrane protein band 4.2
384
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TRANSGLUTAMINASES IN CELL FUNCTION
FIGURE 2. The TG protein catalytic sites. Amino acid sequences
derived from the catalytic core of each of the nine known transglutaminases. The catalytic cysteine residue (indicated by arrow) is part
of the conserved motif that is required for the transamidation reaction. This residue is replaced with alanine in the only catalytically
inactive member of TGs, band 4.2 protein.
upstream), and two AP2-like response elements (634 and
183 bp upstream of the transcription initiation site). Retinoic acid, vitamin D, TGF-␤1, IL-6, tumor necrosis factor
(TNF), NF-␬B, epidermal growth factor (EGF), phorbol
ester, oxidative stress, and Hox-A7 induce TG2 expression.
In addition to the transamidation reaction, TG2 displays
GTPase, ATPase, protein kinase, and protein disulfide
isomerase (PDI) activity. It interacts with phopholipase
C␦1, ␤-integrins, fibronectin, osteonectin, RhoA, multilineage kinases, retinoblastoma protein, PTEN, and I␬B␣.
TG2 dysfunction contributes to celiac disease, neurodegenerative disorders, and cataract formation. TG2 knockout
mice have no phenotype but display delayed wound healing
and poor response to stress. Also, fibroblasts derived from
TG2 mice display altered attachment and motility (351).
bp upstream of the transcription initiation site, is critical for
transcriptional regulation of the TG4 gene expression, and
androgen treatment increases TG4 mRNA level in the human prostate cancer cells. In rats, the enzyme participates in
the formation of the copulatory plug in the female genital
tract, and in masking the antigenicity of the male gamete.
TG4 knockout mice exhibit reduced fertility due to defects
in copulatory plug formation (84). The exact function of
TG4 in humans is not known, but some recent reports suggest a link between increased expression of TG4 and promotion of an aggressive prostate cancer phenotype (160).
E. Transglutaminase 5
Transglutaminase 5 (TG5) is mainly expressed in foreskin keratinocytes, epithelial barrier lining, and skeletal muscle (53).
The TG5 gene (TGM5) has a TATA-less promoter but contains putative binding sites for several transcription factors,
including C-Myb, AP-1, NF-␬B, and NF-1. GTP and ATP
inhibit 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 described: delta3 (deletion of exon 3), delta11 (deletion of exon
11), and delta3-delta11 (deletion of both exons). Full-length
TG5 and the delta11 isoform are active, whereas delta3 and
delta3-delta11 have low activity. TG5 crosslinks loricrin, involucrin, and SPR3 in epidermis (49) and contributes to hyperkeratosis in ichthyosis and psoriasis patients (48). TG5 inactivating mutations result in skin peeling syndrome (53).
TG5 knockout mouse have not been generated.
F. Transglutaminase 6
C. Transglutaminase 3
Transglutaminase 3 (TG3) or epidermal TG is present in
hair follicles, epidermis, and brain. The TG3 gene (TGM3)
promoter contains Sp1- and Ets-motifs (128 and 91 bp
upstream of the initiation site, respectively), and expression
of pro-transglutaminase 3 mRNA is increased by Ca2⫹.
TG3 protein is encoded as two polypeptide chains derived
from a single precursor protein by proteolysis. Like TG2,
TG3 binds to and hydrolyzes GTP. It catalyzes the crosslinking of trichohyalin and keratin intermediate filaments
to harden the inner root sheath of a hair follicle, which is
critical for hair fiber morphogenesis (133–136, 162). It also
participates in cell envelope formation during the latter
stages of differentiation (162). TG3 knockout mice show
impaired hair development and reduced skin barrier function (36, 162).
Transglutaminase 6 (TG6) expression is localized in the human testes and lungs, and in the brain of mice. Human carcinoma cells with neuronal characteristics also express TG6. In
addition to full-length protein, alternative splicing produces a
short variant that lacks the second ␤-barrel domain (348). The
catalytic function of TG6 is activated following proteolytic
cleavage of the proenzyme; thus TG6 comprises two polypeptide chains that are cleaved from a single precursor. TG6
knockout 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, and
brain. One report suggested that TG7 transcript levels are
increased in breast cancer cells of patients with poor prognoses (159). TG7 knockout mice are not available.
D. Transglutaminase 4
H. Factor XIIIa
Transglutaminase 4 (TG4) or prostate TG is present in the
prostate gland, prostatic fluids, and seminal plasma (91,
122, 160, 386). An Sp1-binding site, located ⫺96 to ⫺87
Plasma TG (FXIIIa) is an important component of the
blood coagulation cascade. It is found in platelets, plasma,
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ECKERT ET AL.
astrocytes, macrophages, dermal dendritic cells, the placenta, chondrocytes, synovial fluid, the heart, the eyes, and
in cells of osteoblast lineage. Expression of FXIIIa gene
(F13A1) is regulated by a myeloid-enriched transcription
factor (MZF1-like protein) and two ubiquitous transcription factors (NF1 and Sp1). Also, two myeloid-enriched
factors (GATA-1 and Ets-1) induce F13A1 expression.
FXIIIa is the last zymogen activated in the blood coagulation cascade (220, 221) and is a heterotetramer composed
of two A and two B subunits. The catalytic site of FXIII is
localized in the A subunit, and the B subunit serves as a
carrier protein. Upon activation by thrombin-dependent
cleavage, the catalytic A subunit dissociates from the B subunit, yielding the active enzyme (FXIIIa). In the presence of
Ca2⫹, the enzyme catalyzes crosslinking of fibrin molecules
to stabilize fibrin clots.
FXIIIa also plays a role in inflammation and bone synthesis. Crosslinking of the AT1 receptor, catalyzed by
FXIIIa, results in enhanced signaling and promotes
monocyte adhesion in hypertensive patients, thereby accelerating atherogenesis. F13A1 deficiency is an autosomal recessive disorder characterized by a lifelong
bleeding tendency and impaired wound healing. FXIIIa
knockout mice have a clotting defect, increased incidence
of miscarriage, decreased angiogenesis, and tissue remodeling 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. A
Cys-Ala substitution within the active site of band 4.2 is
responsible 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 produced by alternative splicing of the EPB4.2 gene; the
shorter isoform is more abundant. Band 4.2 is a major
component of the erythrocyte membrane cytoskeleton and
plays an important role in maintenance of membrane integrity and regulation of cell stability. Band 4.2 binds to the
cytoplasmic domain of the erythrocyte anion transporter
(308). Band 4.2 protein expression is partially or completely absent in Japanese recessive spherocytic elliptocytosis patients. In these patients, the ankyrin protein is more
loosely associated with the membrane skeleton than in normal individuals. Band 4.2 null mice show alterations in red
blood cell function, including spherocytosis and altered ion
transport (289).
Most tissues express multiple TG forms (www.ncbi.nlm.nih.gov/UniGene) and share common substrates (86). This
may explain why TG family members can compensate for
the loss of an individual enzyme. Perhaps the best-studied
model for compensation is TGM2 gene knockout mouse.
386
Compensatory activation of the FXIIIa is observed in
TG2⫺/⫺ chondrocytes (266, 335), and TG1 and TG3 level
and activity are increased in TG2⫺/⫺ joint tissue (86). However, compensation is not observed in all tissues. For example, in skeletal muscle, loss of TG2 is not compensated (86).
II. REGULATION OF THE PROTEIN
CROSSLINKING FUNCTION OF TG2
A. Regulation of TG2 Conformation
TG2, also known as tissue transglutaminase, cytosolic type
II, or liver transglutaminase, is a unique member of the
transglutaminase family of enzymes. In addition to Ca2⫹dependent posttranslational modification of proteins, it can
also bind and hydrolyze GTP and acts as a G protein (220).
Therefore, from catalytic activity point of view, TG2 can be
referred to as bifunctional enzyme, owing to its ability to
catalyze Ca2⫹-dependent protein crosslinking activity and
Ca2⫹-independent GTP hydrolysis. Structurally TG2 is
composed of four domains: an NH2-terminal ␤-sandwich
that contains integrin and fibronectin binding sites, a catalytic core domain which contains a catalytic triad (Cys277,
His335, and Asp358) for acyl transfer reaction, and two
COOH-terminal ␤-barrels. Although other members of TG
family display a similar general structure, TG2 contains a
unique guanine-binding site, located in the cleft between the
catalytic core and the first ␤-barrel. This sequence is coded
by exon 10 of the TGM2 gene. The spatial arrangement of
the four domains in TG2 is altered by interaction with cofactors (FIGURE 3). For example, the GTP/GDP bound form
displays considerable interaction between the catalytic domain and domains 3 and 4, which renders TG2 in a closed
or compact conformation. This reduces accessibility and
activity of the Ca2⫹-dependent crosslinking site (217). In
contrast, Ca2⫹ binding alters the conformation by moving
domains 3 and 4 further apart, allowing TG2 to acquire an
open/extended conformation and exposing the catalytic
site. This open configuration is associated with the acyl
transfer “crosslinking” reaction (293) (FIGURE 3). Crosslinking activity requires Cys277, which attacks ␥-glutamyl
residues on acyl donor substrates, on proteins and peptides,
to drive formation of a thioester intermediate. The resulting
acylated 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 binding
site. This results in isopeptide bond (crosslink) formation.
Alternatively, in the absence of a suitable amine donor, the
thioester is hydrolyzed to form glutamic acid, resulting in a
net deamidation (FIGURE 1).
B. Allosteric Regulation of Transamidation
Activity
TGs are present in intracellular and extracellular environments, and activity is tightly controlled under physiological
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TRANSGLUTAMINASES IN CELL FUNCTION
Ca2+
Thioredoxin
GTP
GTP
S
SH
Ca2+
Closed
Ca2+
Oxidation
SH
Open
Catalytically inactive
S
Open
Catalytically active
Catalytically inactive
FIGURE 3. Three TG2 conformations. Guanine nucleotide (GTP/GDP)-bound TG2 is compact (closed) and
catalytically 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 homologous to FXIIIa is distorted by GTP/GDP binding to TG2. The binding of Ca2⫹ to the catalytic domain of TG2 alters
the 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, catalytically
inactive). The oxidized state can be prevented by treatment with thioredoxin. NH2-terminal domain is blue.
COOH-terminal domain is red.
conditions. For example, Ca2⫹, guanine nucleotides, and
redox potential modulate TG2 crosslinking activity. Mutagenesis studies identify five potential Ca2⫹-binding sites
(188), and structure studies show that some of these sites
are distorted when TG2 binds GTP/GDP (217). In essence,
the protein can function as a G protein or as a transamidation enzyme. The transamidation catalytic activity of TG2
is 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 dissociation constant of 1.6 ␮M. GTP-bound TG2 cannot crosslink
proteins, and crosslinking activity is only observed at high
calcium Ca2⫹ concentrations. Because TG2 inside living
cells is primarily GTP/GDP-bound, and calcium concentrations are low, it is believed that TG2 is predominantly present in a crosslinking-inactive form in cells. This may explain
why overexpression of TG2 is not always associated with
increased intracellular crosslinking activity. In a recent
study, using TG2 that is covalently conjugated to enhanced
yellow (YFP) and cyan fluoresce proteins (CFP) at NH2 and
COOH terminus, respectively, Pavlyukov et al. (286) observed closed/inactive TG2 at a perinuclear location. In contrast, crosslinking-active TG2 was present at the cell membrane. Using the fluoresce resonance energy transfer
(FRET)-based approach, these authors observed that TG2
changed from closed to open conformation in response to
ionophore-induced calcium influx (286). In addition, Caron et al. (52) reported that an acrylamide-based TG2 inhibitor induces the open conformation, and a cinnamoyl
triazole inhibitor stabilizes the closed conformation (52).
On balance, these observations support the contention that
intracellular TG2 is predominantly present as a catalytically
inactive form. Nevertheless, despite low intracellular calcium levels, multiple transamidation and crosslinking substrates of intracellular TG2 have been identified. This suggests that locally increased intracellular calcium and/or as
yet uncharacterized interacting proteins may facilitate formation of open TG2. It should also be noted that some
authors have suggested that relatively low calcium concentrations may be sufficient to activate TG2 crosslinking activity (173, 188). Finding additional TG2-binding proteins
inside the cell (e.g., using yeast-2-hybrid or proteomics approach) and characterizing new TG2-interacting proteins
under physiological conditions is expected to help address
this issue.
A puzzling issue is why extracellular TG2 is inactive despite
low GTP levels and high calcium levels (319). A possible
explanation is the response of the enzyme to oxidative conditions in the extracellular environment. TG2 forms intramolecular disulfide bonds that are required for transamidation activity (37, 110), and a switch between the reduced
(active) and oxidized (inactive) states of TG2 has been described (161, 327). This involves a triad of cysteine residues,
including Cys370, Cys371, and Cys230, which have an
unusually high redox potential (161). Mutation analysis
and alkylation studies identified Cys230 as the key redox
sensor. Under oxidizing conditions, an interstrand disulfide
bond between Cys230 and Cys370 forms which facilitates
formation of the more stable Cys370-Cys371 disulfide
bond. These events inactivate the transamidation activity of
TG2 (293, 327). In contrast, reduction of TG2 results in an
open active conformation (327) (FIGURE 3). Thus the extracellular oxidative environment drives inactivation of its
transamidase activity (73, 319). Another factor that con-
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387
ECKERT ET AL.
tributes to inactivation of extracellular TG2 is nitric oxide
(NO), which drives nitrosylation of several Cys residues in
TG2 (202). In vivo, a gradual decrease in NO bioavailability during aging increases TG2 transamidation activity in
blood vessels and increases their stiffness due to accumulation of crosslinks in the vascular extracellular matrix
(ECM) (170, 303). Furthermore, protein kinase A phosphorylation of TG2(Ser216) stimulates TG2 kinase activity, while inhibiting transamidase function (245, 246). In
contrast, thiol reductases (e.g., thioredoxin) activate extracellular TG2, thus antagonizing oxidative inactivation in
the extracellular environment (161). Acting together, these
factors modulate the redox, nitrosylation, and phosphorylation states of TG2 to control transamidase activity.
In summary, calcium, guanine nucleotides, and redox potential maintain mammalian TG2 activity in at least three
distinct states depending on local conditions (FIGURE 3).
The two most common TG2 states, the GTP/GDP-bound
form and the Ca2⫹-bound oxidized form, are catalytically
inactive, whereas calcium binding activates the reduced
form. Some thiol reductases, such as thioredoxin, are likely
to control the redox state of extracellular TG2. In addition,
the TG2 crosslinking activity is inhibited through nitrosylation and phosphorylation. The physiological implications
of these allosteric regulatory and posttranslational modification mechanisms are described in subsequent sections. It
is also important to note that protein-protein interactions
regulate TG2 crosslinking activity. For example, in the
ECM, TG2 interacts with a number of proteins, including
fibronectin, osteonectin, and integrins (396), and interaction with some of these proteins alters enzymatic activity
(359).
III. TRANSGLUTAMINASE-REGULATED
CELL SIGNALING
Although investigators originally discovered TG2 as a
crosslinking enzyme, new functions have been identified. It
is now appreciated that TG2 interacts with target proteins
localized in the cytoplasm, membrane, ECM, nucleus, and
mitochondria (151, 220, 278). This includes roles for TG2
in transamidation and protein-protein crosslinking, as a
GTPase/ATPase, as a nonenzymatic adapter, as a scaffold
protein, and as a regulator of signal transduction.
A. TG2 and FXIIIa Signaling: The Cell
Surface and ECM
1. TG2 and FXIIIa crosslinking of ECM proteins
TG2 and FXIIIa are released into the extracellular environment via a poorly understood nonclassical secretion pathway (69, 398) where they covalently modify ECM proteins
to form homo- and heteropolymers (3, 220) to enhance
ECM stability (220). This crosslinking increases the rigidity
388
of fibronectin (262) and collagen fibrils (326). The resulting
increase in ECM stiffness enhances fibroblast and osteoblast adhesion (56, 112) and enhances cell survival, growth,
migration, and differentiation by impacting integrin-related
mechanosensing pathways (34). Endothelial cell adherence
to the TG2-crosslinked fibrinogen ␣C increases integrin
clustering and formation of focal adhesions, thereby elevating outside-in activation of focal adhesion kinase (FAK) and
extracellular signal-regulated kinase (ERK) 1/2 activity
(30). In addition, ECM protein crosslinking may expose
cryptic integrin receptor-binding sites. For example, TG2mediated polymerization of osteopontin creates a binding
site for integrin ␣9␤1 binding, leading to enhanced chemotactic migratory activity of neutrophils (264, 265). A similar
mechanism influences vascular smooth muscle cell migration into FXIIIa-crosslinked fibrin gels (255), and impacts
angiogenesis (138, 168).
2. Extracellular TG2 regulates the TGF-␤ signaling
pathway
TG2-induced modification can modify growth factor activity in the extracellular environment (148, 220, 381). For
example, TGF-␤1 is a key regulator of ECM remodeling
(387), and TGF-␤1 activation involves integrins and proteases and is influenced by the oxidative environment and
mechanical stress. TG2 covalently crosslinks latent TGF␤1binding protein and thereby controls TGF-␤1 maturation
and activity (191, 362, 380). In addition, in fibroblasts,
TG2 increases TGF-␤ mRNA and protein expression via a
nuclear transcription factor (NF)-␬B signaling mechanism
(342). This results in a positive feedback loop in which
TGF-␤ and TG2 display reciprocal activation of expression
(29). In cancer cells, the TGF-␤-induced increase in TG2
expression promotes epithelial-to-mesenchymal transition
(EMT) (51, 196, 315).
3. Extracellular TG2 and FXIIIa enhance
integrin-mediated signaling
Integrins are important transmembrane adhesion and signaling receptors that, although lacking intrinsic enzymatic
activity, regulate a host of intracellular signaling pathways.
Integrins are activated by binding to ECM (147). TG2 interacts with ECM to enhance cell adhesion and integrinmediated signaling via direct interaction with ␤1, ␤3 and ␤5
integrin (FIGURE 4) (29, 396). TG2 also binds to the gelatinbinding region of fibronectin (297). The integrin-fibronectin binding is a weak interaction, while TG2 interacts
strongly with both fibronectin and integrin, and thereby
enhances integrin/fibronectin interaction. This facilitates
cell attachment to the matrix and activates integrin signaling (29). TG2 controls integrin function in cancer cells (229,
309) and macrophages (29, 353). The interaction between
integrin-bound TG2 and fibronectin is important in various
disease conditions, including mesenchymal stem cell (MSC)
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TRANSGLUTAMINASES IN CELL FUNCTION
ECM
PDGF
Fibronectin
LPR5
LPR6
Integrin cluster
TG2
TG2TG2
dimer
α
β
TG2
syndecan-2
p190
RhoGAP
FAK
PKCα
RhoA
GPR56
PDGFR
syndecan-4
clustering
Src
TG2
TG2
P
P
Gαq
β-catenin
Akt1
FAK
Src
ERK1/2
Shp2
Gβ
Tcf/Lef
ROCK
Transcription
Stress fiber and
focal adhesion
formation
Reduced cell
growth and
metastasis
interaction with infarcted myocardium (325), cancer cell
metastasis (309), and glial scarring (361).
The effect of TG2 on integrin function is evidenced by its
impact on integrin clustering (155). The mechanism
whereby TG2 promotes integrin clustering is not known;
however, the ability of TG2 to oligomerize and interact
with integrin-binding proteins, such as caveolin-1 and tetraspanins, may promote clustering. Moreover, localization
of TG2 and ␤1-integrin in lipid rafts and caveolae (400)
may enhance ECM interaction with these cholesterol-enriched membrane microdomains. Therefore, TG2 is likely
to have a role in regulating membrane protein trafficking
and compartmentalization during cell signaling.
TG2-induced integrin clustering potentiates integrin-dependent intracellular signaling (9, 155). This includes activation of FAK, Src, and p190RhoGAP and increased expression of active, GTP-bound RhoA and its downstream
target, ROCK. The net impact of these events is increased
focal adhesion and actin stress fiber formation leading to
enhanced actomyosin contractility (FIGURE 4).
TG2 and integrins are also important in macrophages.
TG2⫺/⫺ macrophages are deficient in phagocytosis owing
to altered accumulation of ␤3-integrin at the engulfing portals (353). Efficient signaling via ␤3-integrin, which is required for formation of the phagocytic cup and effective
uptake of apoptotic cells, may require TG2 interaction with
␤3-integrin. TG2 activates downstream signaling targets of
␤3 integrin, including RhoG and Rac1, which are required
for efficient phagocytosis. Furthermore, overexpression of
␤3-integrin in TG2⫺/⫺ macrophages partially restores
phagocytosis (354). Mechanistically, TG2 interacts with
FIGURE 4. TG2-mediated adhesion/signaling at the cell surface. The solid black
arrows indicate TG2-mediated activation of
signaling. The dotted black line indicates
binding of activated PKC␣ to the integrin
cytoplasmic tails, causing their redistribution on the cell surface. The dashed gray
arrows outline activation of syndecan-2 by
intracellular PKC␣ and syndecan-2-mediated activation of ROCK, which induces
stress fiber and focal adhesion formation.
The dashed black arrow indicates nuclear
translocation of ␤-catenin, which leads to
complex formation with Tcf/Lef and activation of gene transcription. The dashed double black line indicates the unknown pathway of GPR56-induced G␣q activation,
which inhibits tumor cell growth and metastasis. The flat-headed arrows indicate
inhibition of signaling.
the protein milk fat globule EGF factor 8, which is involved
in binding of ␤3-integrin to apoptotic cells, on the surface of
macrophages. TG2-mediated stabilization of the ␤3-integrin/milk fat globule EGF factor 8 complex improves phagocytic uptake of apoptotic cells, likely owing to upregulation
of ␤3 integrin-mediated activation of RhoG and Rac1 signaling. Thus TG2 is an integrin coreceptor and signaling
partner.
FXIIIa, in contrast, is an integrin ligand and a covalent
integrin modifier. Platelet integrin ␣IIb␤3 is the most common binding site for plasma FXIII (70) and serves as a
transamidation substrate for platelet-derived FXIIIa (66).
Of note, extracellular platelet FXIIIa suppresses Ca2⫹-dependent activation of ␣IIb␤3 integrin in cells that adhere to
collagen in a transamidation-dependent manner, implying a
role for FXIIIa in preventing excessive platelet accumulation on thrombogenic surfaces (194). In addition, plasma
FXIII binds to integrin ␣v␤3 on endothelial cells and mediates platelet/endothelial cell interaction by bridging endothelial cell ␣v␤3 to platelet ␣IIb␤3 integrins (79). FXIIIa
also stimulates endothelial cell, monocyte, and fibroblast
proliferation and migration and inhibits apoptosis by interacting with ␣v␤3 integrin on the cell surface to trigger
downstream signaling (76, 80). These effects of FXIIIa lead
to increased vascularization and angiogenic actions of endothelial cells via activation of vascular endothelial growth
factor receptor (VEGFR) 2, leading to increased expression
of the Egr-1 and c-Jun transcription factors and downregulation of mRNA encoding the antiangiogenic ECM protein
thrombospondin-1 (76, 78, 80).
The mechanism of FXIIIa-mediated activation of VEGFR2
in endothelial cells involves extracellular crosslinking of
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ECKERT ET AL.
this receptor to the ␤3 subunit of ␣v␤3, an integrin that
facilitates angiogenesis (75). Extracellular FXIIIa promotes
hypertrophic differentiation of chondrocytes by enhancing
TG2 secretion (164). This effect does not require transamidating activity, but rather depends on FXIIIa interaction
with ␣1␤1 integrin via a novel integrin-binding site at the
FXIIIa NH2 terminus. Moreover, FXIIIa-dependent induction of type X collagen synthesis, a hallmark of chondrocyte
differentiation, is mediated by ␣1␤1-dependent activation
of FAK and p38 MAPK signaling (165).
4. Modulation of syndecan-4 signaling by
extracellular TG2
The heparan sulfate proteoglycan syndecan-4 localizes at
points of cell-ECM contact, where it interacts via heparan
sulfate with the Hep-2 region of fibronectin. It collaborates
with integrins to enhance cell adhesion to fibronectin and
facilitate adhesion-dependent RhoA-mediated development of focal adhesions, stress fibers, and actomyosin contractility (389). Syndecan-4 is an important TG2-binding
partner (311, 344). A heparan sulfate binding site,
261
LRRWK265, that may mediate this interaction, is present
in TG2 (366).
The high-affinity interaction of extracellular TG2 with syndecan-4 activates protein kinase C-alpha (PKC␣), which, in
turn, binds directly to the cytoplasmic tail of ␤1 integrin
(FIGURE 4). This interaction controls integrin level and distribution on the cell surface as well as integrin stimulation
of FAK and ERK1/2 (282, 311, 344, 379, 381). The ability
of activated PKC␣ to maintain adhesion of fibroblasts and
osteoblasts, via formation of ECM-based TG2-fibronectin
complexes with cell surface syndecan-4, is mediated by syndecan-2 (379, 381). Syndecan-2 does not bind to TG2 but
acts as a downstream signaling effector in modulating cytoskeletal organization via the ROCK pathway. These findings imply a major role for the TG2/fibronectin/syndecan-4
complex as an adhesive and signaling platform (363). The
integrin- and syndecan-4-based adhesion systems are likely
to physically interact with each other, as these two receptors
bind to nonadjacent regions of fibronectin, and functionally
collaborate by jointly regulating p190RhoGAP activity and
localization during cell adhesion to the ECM (25, 343).
Hence, this evidence indicates the existence of adhesion/
signaling complexes composed of TG2, integrins, syndecan-4, and fibronectin. TG2 controls formation of these
complexes owing to its high affinity for syndecan-4 and
fibronectin.
Syndecan-4 interaction with integrin-bound TG2 at the cell
surface and/or fibronectin-bound TG2 in the ECM may be
required for response to tissue damage and ECM degradation. Thus increased TG2 expression during wound healing
and tissue repair is likely to enhance cell adhesion and signaling to increase integrin-dependent adhesion and assembly of the fibronectin matrix (343, 367, 380). This may
390
promote clustering of TG2 binding partners on the cell
surface to enhance adhesion, prevent adhesion-mediated
apoptosis (anoikis), and facilitate cell survival.
5. Regulation of growth factor receptor signaling by
extracellular TG2 and FXIIIa
Physical association between integrins and receptor tyrosine kinases is required for the cell response to ECM and
soluble growth factors (392). A novel example of a role for
TG2 in this context is the interaction between integrin and
platelet-derived growth factor receptor (PDGFR). TG2 interacts with PDGFR on the surface of fibroblasts and vascular smooth muscle cells, and enhances PDGFR interaction with integrins (397, 399) by bridging these receptors
on the cell surface (FIGURE 4). The interaction with TG2
promotes PDGFR clustering, PDGF- and adhesion-induced
PDGFR activation and downstream signaling, and PDGFR
turnover. In particular, TG2 increases PDGF/PDGFR-mediated activation of Akt1 and Shp2 in fibroblasts and vascular smooth muscle cells (397). Cell surface TG2 is required for efficient PDGF-dependent fibroblast and vascular smooth muscle cell proliferation and migration. TG2
also enhances PDGF-induced vascular smooth muscle cell
survival and suppresses differentiation. These studies revealed a novel function of cell surface TG2 in regulating
PDGFR/integrin signaling and PDGFR-dependent cell responses, by coupling the adhesion-mediated and growth
factor-dependent signaling pathways. These findings also
suggest that TG2 activity may have a proinflammatory role
in wound healing, tissue fibrosis, vascular restenosis, and
tumor metastasis, diverse pathophysiological responses
that often involve overactivation or dysregulation of the
PDGF/PDGFR signaling axis (130).
The interaction of extracellular TG2 with growth factor
receptors may be a general phenomenon since, as noted
earlier, TG2 also binds to VEGFR on the surface of endothelial cells and modulates VEGF signaling (75). In this
case, TG2 covalently crosslinks VEGFR to form high-molecular-weight complexes. In VEGF-treated cells, these
complexes shuttle to the nucleus to enhance VEGF-induced
ERK activation. Extracellular FXIIIa also regulates VEGFR
signaling by enhancing noncovalent interaction between
VEGFR and ␣v␤3 integrin (75, 78). Future studies should
identify the molecular motifs required for association of
TG2 and FXIIIa with growth factor receptors, and address
whether TG2 and FXIIIa interact with other structurally
related receptor tyrosine kinases.
6. Extracellular TG2 as an activator of LRP5/
6-mediated ␤-catenin signaling
Extracellular TG2 binds to the LRP5 and LRP6 (low-density lipoprotein receptor) transmembrane receptors on vascular smooth muscle cells (FIGURE 4) (102). Binding of TG2
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TRANSGLUTAMINASES IN CELL FUNCTION
to LRP5/6 triggers activation of the ␤-catenin pathway by
driving nuclear translocation of ␤-catenin, inducing Tcf/Lef
transcription factors and decreasing p21Cip1 expression.
TG2-mediated activation of ␤-catenin signaling promotes
calcification of vascular smooth muscle cells (102). TG2
synergizes with LRP6 in the activation of ␤-catenin-dependent gene expression in COS-7 cells. Interfering with the
LRP5/6 receptor function attenuates TG2-induced activation of ␤-catenin in these cells. Moreover, TG2 binds directly to the extracellular domain of LRP6 which acts as a
substrate for TG2-mediated protein crosslinking (85). Future studies should assess the contribution of TG2-regulated LRP5/6 signaling to pathological conditions, such as
cancer and calcification of blood vessels.
NH2
Monoamine
hormone
Monoamine
receptor
Cytosol
TG2
Signal
mediators
Target
protein
7. TG2 signaling via GPR56
GPR56 is an atypical G protein-coupled receptor (GPCR)
that is reduced in level in metastatic melanoma cells, and
interacts with cell surface-localized TG2 in tumor stroma
cells (390). TG2 is proposed as a novel GPR56 ligand that
cooperates in the growth-inhibitory and tumor-suppressive
action of GPR56; however, additional study will be necessary to understand the downstream signaling mechanisms
involved in this activity.
B. Cytoplasmic TG2 and FXIIIa in Cell
Signaling
1. TG-mediated monoaminylation of cytoplasmic
proteins regulates signaling, the cytoskeleton, and
vesicular trafficking
Monoamines, including serotonin, histamine, dopamine,
and norepinephrine, are competitive inhibitors of TG crosslinking activity. However, these amines also can be utilized
by TG to monoaminylation target proteins (FIGURE 5)
(378). In this context, TG catalyzed serotonylation of the
RhoA and Rab4A GTPases is required for cytoskeletal rearrangement that leads to exocytosis of platelet ␣-granules,
platelet activation, platelet adhesion, and platelet aggregation (377). Given that TG2 and FXIIIa are both abundant in
platelets (220), knockout studies will be required to clarify
which TG drives this reaction in vivo. In addition, serotonylation of Rab3A and Rab27A in pancreatic ␤ cells is
involved in the release of insulin (285). Although the TG
that is involved is not known, the presence of missense
mutations of the TGM2 gene in patients with early-onset
type 2 diabetes mellitus is interesting (294). In one study,
TG2 was identified to be the only TG significantly expressed
in pancreatic ␤ cells, and its deletion impaired glucosestimulated insulin secretion (33). Thus, TG2 mutations
(989T⬎G, 992T⬎A) that impair transamidation activity
are linked with early onset of type 2 diabetes (294). However, although these mutations are not found in normal
patients, heterozygous TGM2 mutations are not fully pen-
Monoamine
transporter
Monoaminylated
target protein
Hormone effects:
Regulation of cytoskeleton and vesicular trafficking
FIGURE 5. Regulation of signaling by TG2-induced monoaminylation. Monoamines (serotonin, norepinephrine, dopamine, etc.) interact with the monoamine receptor, but are also delivered to cells via
monoamine transporters. Intracellular monoamines are covalently
crosslinked to cytoplasmic proteins by TG2. Target proteins include
the small regulatory GTPases (RhoA, Rac1, Rab3A, Rab4a, and
Rab27A) and cytoskeletal components such as ␣-actin. These TG2induced posttranslational modifications alter target protein biological activity. The biological effects of these TG2-driven modifications
are important in diabetes, thrombosis, and arterial hypertension.
etrant and do not appear to cause diabetes in these families.
Iismaa et al. (150) evaluated the role of TG2 in diabetes and
concluded that glucose homeostasis is TG2 independent
and TG2 plays no role in pathophysiology of type 2 diabetes. Moreover, neither deletion nor activation of TG2
transamidation activity in transgenic mouse models alters
basal or insulin-challenged glucose homeostasis. This is
clearly an area that will require future study.
In vascular smooth muscle cells, TG2-mediated serotonylation increases RhoA activity and degradation, which leads
to increased Akt1 activity and inhibition of muscle contraction (124). TG2-mediated serotonylation of RhoA is also
implicated in pulmonary arterial remodeling and hypertension (123). Moreover, TG2-mediated serotonylation of
␣-actin and other contractile apparatus proteins, in vascular smooth muscle cells, increases arterial isometric contraction (383). Similar TG2-mediated modification of smooth
muscle 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 of
primary amines into cytoplasmic proteins influences activity, to alter the cytoskeleton and vesicular trafficking (FIG-
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ECKERT ET AL.
URE 5) (378). This mechanism is likely to be broadly important 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 are
important in inflammatory disease and cancer (178). Under
normal cellular conditions, NF-␬B is inactive in the cytoplasm because of its association with I␬B␣. Exposure to
stress stimuli activates pathways that ubiquitinate I␬B␣ via
a mechanism that involves I␬B kinase (IKK)-dependent
I␬B␣ phosphorylation (23). Proteasome-mediated degradation of I␬B␣ releases NF-␬B, which translocates to the nucleus to activate gene expression (121) (FIGURE 6A). IKKindependent NF-␬B activation, via a mechanism that involves TG2 crosslinking of I␬B␣, has recently been
described (FIGURE 6B) (205, 230). TG2-mediated polymerization of I␬B␣ results in I␬B␣ proteasomal degradation,
leading to the NF-␬B activation (205). TG2 also interacts
A
Inflammation (acute)
B
directly with I␬B␣ to cause I␬B␣ degradation via a nonproteasomal mechanism (198) (FIGURE 6B). These novel TG2mediated, IKK-independent mechanisms of NF-␬B activation are important and suggest that targeting these events
may block inflammation (181, 370).
3. TG2-crosslinking of PPAR-␥ links oxidative stress
and inflammation
Cystic fibrosis is caused by mutation of the cystic fibrosis
transmembrane conductance regulator (CFTR) leading to
chronic airway inflammation. Interestingly, human bronchial epithelial cells that express functionally deficient
CFTR express high levels of TG2, leading to increased
crosslinking and sequestration of anti-inflammatory
PPAR-␥. This suggests a role for TG2 in mediating the
inflammatory response in cystic fibrosis patients (227). TG2
crosslinking promotes accumulation of polymerized ubiquitinated PPAR-␥ in perinuclear aggresomes and, as a result, PPAR-␥ interaction with the N-CoR-histone deacety-
Inflammation (chronic)
TGFβ/ROS–
IκBα
IκBα
TNFα/IL1, etc.
IκBα
TG2
IκBα
IκBα
p65
IκBα
polymerization
p50
p65
IκBα
Proteasome
dependent
IκBα
IKKα/β
p50
P
TG2
IκBα
Proteasome
independent
IκBα
degradation
TG2
IκBα-P
degradation
p65
p50
p65
p50
Cytosol
Nucleus
IκBα
p65
p50
Promoter
TG2
TG2
p65
HIF1α
HIF1β
p50
HIF1α
Promoter
Promoter
Drug resistance
and metastasis
FIGURE 6. TG2 expression results in constitutive activation of NF-␬B via noncanonical pathway. Acute
inflammation is a tightly regulated physiological process in which NF-␬B is transiently activated as a result of
IKK-complex mediated phosphorylation and degradation of the inhibitory protein I␬B␣. As I␬B␣ is one of the
downstream targets of NF-␬B, its expression results in feedback inhibition of NF-␬B, which limits the inflammatory response (A). In contrast, chronic inflammation is associated with constitutive activation of NF-␬B owing
to aberrant expression of TG2 (B). TG2 binds to I␬B␣ resulting in its rapid degradation via a nonproteasomal
pathway. Alternatively, TG2-mediated covalent crosslinking of I␬B␣ may promote proteasomal degradation of
I␬B␣ polymers (broken arrows). TG2-activated NF-␬B regulates the expression of multiple target genes that
play roles in cell survival, invasion, and drug resistance. One of the TG2/NF-␬B target genes is HIF-1␣, a
transcription factor known to promote an aggressive phenotype in cancer cells.
392
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TRANSGLUTAMINASES IN CELL FUNCTION
lase 3 complex is reduced, thereby facilitating inflammatory
response gene expression (283). Moreover, inhibition of
TG2-mediated crosslinking restores normal PPAR-␥ level
and reduces inflammation in both cultured CFTR-defective
cells and cystic fibrosis tissue (227).
Oxidative stress in CFTR-defective cells also increases
SUMO ligase activity. SUMO ligase inhibits activated
STAT-␥ leading to reduced TG2 SUMOylation which reduces TG2 turnover and increases TG2 level and activity
(224). This evidence is consistent with the finding of elevated reactive oxygen species and increased TG2
SUMOylation in lung tissue in mutant ⌬Phe508-CFTR
mice, a model of cystic fibrosis, and suggests that control of
TG2 turnover serves as a central link between oxidative
signaling and inflammation in cystic fibrosis (223, 224).
These findings established cytoplasmic TG2 as a novel mediator that connects oxidative stress and inflammation.
4. TG2 crosslinking of beclin-1 and inhibition of
autophagy
In addition to an impact on protein aggregation, stressinduced accumulation of cytoplasmic TG2 and activation
of TG2 crosslinking inhibits autophagy. Specifically, PKC␦mediated induction of TG2 expression in pancreatic carcinoma cells inhibits autophagy by crosslinking beclin-1 to
inhibit its function (7, 277). This mechanism also operates
in CFTR-deficient lung cells where oxidative stress-related
TG2-induced crosslinking of beclin-1 leads to sequestration
of beclin-1, and beclin-1 interacting proteins, in the aggresomes (223). These findings suggest a central role for
TG2 in beclin-1 depletion, beclin-1 sequestration in aggresomes, and inhibition of autophagy, in patients with
cystic fibrosis.
5. Cytoplasmic TG2 and EGF/EGFR signaling in
epithelial cancer cells
EGFR activity, which is frequently increased in human malignant cells, increases TG2 expression in cervical, breast,
and lung epithelial cancer cells (15, 214). Moreover, induction of TG2 expression and TG2-dependent transamidation are essential for EGF-mediated migration, invasion
(15), and anchorage-independent cancer cell growth (213).
The EGF-induced response is mediated by Ras- and Cdc42induced activation of PI3K and NF-␬B and requires TG2mediated upregulation of Src expression (15). TG2-induced
Src expression is associated with transamidation-dependent
formation 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 of
cells. Accumulation of cytoplasmic TG2 at this location is
necessary for cell migration and requires interaction of TG2
with heat shock protein 70 (Hsp70) (38). Similarly, EGFinduced upregulation of TG2 expression in TNF-related
apoptosis-inducing ligand (TRAIL)-resistant lung cancer
cells elevates MMP-9 expression, secretion, and activity,
and this enhances the migration and invasiveness of these
cells (214). The mechanism of TG2 action in this context
remains to be defined; however, JNK/ERK signaling pathways are implicated in this process (214). Thus cytoplasmic
TG2 is a novel mediator of EGF/EGFR-induced signaling
and oncogenesis in epithelial cancer cells that involves TG2
transamidation-dependent and -independent actions.
6. Regulation of angiotensin signaling by
FXIIIa-induced dimerization of AT1 receptors
GPCRs constitute a large family of cell surface receptors.
GPCR homodimers and heterodimers influence many receptor-related functions, including ligand binding, membrane localization, signaling, and desensitization (116).
However, until recently, little was known about the pathophysiological importance of GPCR dimerization in vivo. An
insightful study revealed a novel mechanism of FXIIIa-mediated dimerization of the angiotensin II AT1 receptor in
monocytes (1). Crosslinking of AT1 dimers, via glutamine
residues, in the tail domain enhances receptor signaling.
Moreover, FXIIIa-deficient individuals lack crosslinked
AT1 dimers, whereas patients with the common atherogenic
risk factor hypertension have elevated levels of these
dimers. The presence of these dimers correlates with enhanced adhesion of angiotensin II-stimulated monocytes to
endothelial cells (1). Importantly, in monocytes, these AT1
dimers promote atherogenesis, and inhibition of FXIIIa
crosslinking activity reduces AT1 dimer formation and reduces disease severity in atherosclerosis in mice. Thus
FXIIIa, via an impact on AT1 receptors, appears to have a
role in maintaining atherogenesis.
7. FXIIIa-mediated crosslinking of Glu-tubulin alters
microtubule dynamics and controls osteoblast matrix
deposition
The transamidating activity of TG2 and FXIIIa, accompanied by collagen type I and fibronectin deposition into the
ECM, is associated with osteoblast differentiation. However, the molecular mechanisms linking these events remain
largely unknown (266). A recent study showed that inhibition of FXIIIa-mediated transamidation in osteoblasts resulted in microtubule destabilization as evidenced by reduced Glu-tubulin levels and blocked formation of Glutubulin oligomers (11). In turn, blockage of this activity
inhibited vesicle-based secretion and deposition of collagen
type I and fibronectin. Thus this study provides potential
mechanistic clues regarding the role of transamidation and
protein crosslinking by FXIIIa in the regulation of ECM
protein secretion and deposition that leads to osteoblast
differentiation.
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8. Cytoplasmic TG2 as an atypical GTPase and
mediator of GPCR-induced signaling
Although the discovery that TG2 can bind to and hydrolyze
GTP occurred in 1987 (2), researchers did not establish a
link between this activity and GPCR function until 1994
when a GTP-binding protein, termed Gh␣, was isolated
with the ␣1B adrenergic receptor. The study showed that
GH␣ was TG2 (FIGURE 7) (259). Other researchers observed a similar role for TG2 relative to the ␣1D adrenergic,
thromboxane A2, oxytocin, and follicle-stimulating hormone receptors. In response to exposure to agonists of these
receptors, PLC␦1 activation leads to increased inositol
1,4,5-trisphosphate level (19, 20, 104, 152, 153, 220, 241,
279, 373).
The GTPase activity and associated signaling capacity of
TG2 is independent of its transamidating activity (58).
Moreover, given the high intracellular GTP levels observed
under 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 and
catalytic rate similar to that observed with the canonical ␣
subunits of heterotrimeric and monomeric G proteins despite the absence of the four consensus GTP-binding motifs
common to the classical G proteins. Mutating Arg580 in
TG2 results in a 100-fold reduction in GTP-binding affinity
and eliminates GTP inhibition of TG2 transamidation activity (27, 28). The activation/deactivation GTPase cycle of
TG2 functions similarly to that of other heterotrimeric G
proteins (FIGURE 7) (220, 241). Agonist binding induces
exchange of GDP with GTP and dissociation of TG2/GTP
from Gh␤. Deactivation occurs when TG2 hydrolyzes GTP
to GDP and reassociates with free Gh␤. Two regions in
TG2, R564-D581 and Q633-E646, are involved in TG2
interaction with ␣1 adrenergic receptors and activation of
the GTPase function (103).
The role and specificity of TG2 in GPCR signaling is determined not only by the range of receptors it interacts with
but also by the downstream effectors. PLC␦1 is a key downstream target of TG2/␣1 adrenergic receptor coupling (21,
81, 104). The Val665-Lys672 region in the COOH terminus of TG2 is involved in binding and activation of PLC␦1
(146), which hydrolyzes phosphoinositide and increases intracellular free calcium level (104, 175). PLC␦1 acts as both
a guanine nucleotide exchange factor and a GTP hydrolysisinhibitory factor for TG2 (21). TG2 also regulates other
signaling pathways via its GTPase activity. It participates in
ERK1/2 activation in cardiomyocytes (206). In fibroblasts
and endothelial cells, overexpression of TG2, or transamidation-inactive TG2, inhibits adenylyl cyclase activity,
whereas knockdown of TG2 reverses this effect (119). TG2
also increases adenylyl cyclase activity in neuroblastoma
cells, but this effect requires its transamidating activity
(356), implying that TG2 can regulate signaling pathways
differentially dependent upon cell type. Also, TG2 activates
the large-conductance Ca2⫹-activated K⫹ channels in vas-
Agonist
GPCR
Cytosol
Ca2+
Ca2+ TG2
Ghβ
CRT
1
7
Ghα
Ghβ
CRT
Ghα
5
GDP
GTP
TG2
TG2
2
6
PIP2
4
Ghβ
CRT
DAG
PLCδ1
Ghα
IP3
Ghα
GDP
GTP
TG2
TG2
Ca2+
FIGURE 7. TG2 GTPase activity and
TG2/Gh␣ signaling. The GDP-TG2/Gh␣CRT/Gh␤ complex is inactive. CRT is calreticulin. 1: Agonist stimulation of transmembrane G protein-coupled receptors (GPCR)
induces exchange of GDP with GTP and
dissociation of GTP-bound TG2/Gh␣ from
CRT/Gh␤. 2: GTP-bound TG2/Gh␣ activates PLC␦1. 3/4: Signal termination occurs with GTP hydrolysis and reassociation
of GDP-bound TG2/Gh␣ with free CRT/
Gh␤. 5: PLC␦1 promotes coupling efficiency by stabilizing GTP-TG2/Gh␣. 6:
PLC␦1 catalyzes hydrolysis of phosphatidylinositol 4,5-bisphosphate to diacylglycerol
and inositol 1,4,5-triphosphate, causing
an increase in intracellular Ca2⫹ level. 7:
Switching off GTPase activity of TG2/Gh␣
is triggered by elevated intracellular Ca2⫹.
3
Pi
394
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TRANSGLUTAMINASES IN CELL FUNCTION
cular smooth muscle cells (207), and GTP-bound TG2
binds to the cytoplasmic tail of ␣5 integrin to inhibit vascular smooth muscle cell migration (177). In contrast, TG2
promotes fibroblast migration via its GTP-binding activity
(330). Finally, TG2 GTPase activity regulates cell-cycle progression in fibrosarcoma cells (242) and mediates ␣1 adrenergic receptor-induced proliferation of hepatocytes (388)
and visceral smooth muscle cells (92).
Despite significant progress in understanding of the GTPase
function of TG2, the pathophysiological role of the associated intracellular signaling remains poorly understood. For
instance, cardiac-specific overexpression of TG2 fails to alter PLC␦1 activity, suggesting that TG2 acts as a TG rather
than a GTPase in this context (323). Nonetheless, TG2
GTPase activity is markedly reduced in ischemic heart, suggesting that loss of this activity may be an important factor
in cardiac failure (146). TG2 GTPase activity may also be
involved in liver regeneration owing to its involvement in ␣1
adrenergic receptor signaling (304, 388). Overall, TG2
GTPase signaling is prosurvival and cytoprotective, as mutants
of TG2 defective in GTP binding appeared to induce apoptosis in NIH3T3 and HeLa cells independent of their
transamidating activity (82). Moreover, the GTPase function (FIGURE 3), and intracellular localization of TG2, are
important in protecting cells from death caused by oxygen
and glucose deprivation (67, 126).
C. Signaling Function of TG2 in the Nucleus
1. Nuclear TG2 transadmidation and regulation of
gene expression
TG2 nuclear localization was initially reported in hepatocytes (128). Nuclear TG2 comprises ⬃5% of the total TG2
cellular pool (278). TG2 displays crosslinking and GTPase
activity in this cellular compartment (321) and associates
with chromatin (209). The crosslinking activity of nuclear
TG2 has a role in histone and transcription factor transamidation which alters gene expression (236, 340). Perhaps the
best example of transamidation regulation of gene expression is the TG2 impact on Sp1 function (FIGURE 8B) (317,
339, 340). Sp1 is involved in alcohol-induced apoptosis, a
process in which TG2 crosslinks Sp1 resulting in Sp1 inactivation (339). This leads to decreased expression of growth
factor receptors, such as c-Met, which results in cell death.
TG2 regulation of Sp1 function is also observed in free fatty
acid-treated hepatocytes and in patients with nonalcoholic
steatohepatitis (340).
2. TG2 as a transcriptional coregulator
Recent reports indicate that nuclear TG2 functions nonenzymatically as a transcriptional coactivator (5, 106). TG2dependent reduction in MMP-9 gene transcription in car-
diomyoblasts is mediated by direct noncovalent binding of
TG2 to c-Jun, thereby inhibiting c-Jun/c-Fos dimerization
and blocking interaction with the AP-1 site in the MMP-9
gene promoter (FIGURE 8C) (5). A similar mechanism operates in cortical neurons where nuclear TG2 interaction with
HIF-1␤ prevents dimerizing with HIF-1␣ (FIGURE 8D)
(106). This interaction attenuates expression of Bnip3 and
other genes containing the hypoxia-response element
(HRE) to reduce neuron death in patients with ischemia and
reduce stroke. In addition, the protein kinase activity of
nuclear TG2 may be involved in the phosphorylation of
histones H1 and H3 (248), p53 (247), and retinoblastoma
protein (245) (FIGURE 8A).
D. Signaling by Mitochondrial TG2
Although TG2 does not have a classical NH2-terminal mitochondrial targeting signal, the protein does associate with
mitochondria in various cell types (291, 299). In the majority of cells, mitochondrial TG2 localizes at the outer mitochondrial
membrane
and
the
inner
membrane space; however, in 10% of cells, TG2 is present at the
inner mitochondrial membrane and mitochondrial matrix
(278, 299).
1. TG2 crosslinking of mitochondrial proteins is
involved in the mitochondrial-driven apoptosis
The TG2 sequence includes 204LKNAGRDC211, which is
70% homologous to the BH3 domain of Bcl-2 family proteins, suggesting that TG2 is a novel apoptotic BH3 protein
(299). Mutation of the highly conserved Leu204 residue in
this motif attenuates TG2-mediated staurosporin-induced
neuroblastoma cell death, confirming earlier findings that
TG2-induced hyperpolarization of the mitochondrial membrane sensitizes cells to the intrinsic pathway of programmed cell death (291, 299). Also, the TG2 BH3 peptide
interacts with proapoptotic Bax but not with anti-apoptotic
Bcl-2, and TG2-Bax interaction increases during cell death.
In contrast, TG2 inhibits calcium-induced apoptosis in
HEK293 cells by covalently crosslinking Bax and downregulating Bax expression (61). Thus the proapoptotic or
antiapoptotic function of mitochondrial TG2 may depend
on the cell type and cell death inducer.
In addition, TG2-mediated transamidation of proteins in
mitochondria has been reported (278, 305). Prohibitin is a
membrane-bound chaperone essential for correct folding of
the Hsp70 and Hsp90 respiratory chain components. The
organizing protein Hsp60 cooperates with prohibitin and
forms a membrane-tethered import motor complex involved in the unfolding of preprotein domains, whereas the
ATP synthase ␤ chain is a key component of complex V of
the respiratory chain. In apoptotic neural cells, these proteins are transamidated and crosslinked by TG2 (26, 275).
The bifunctional adenine nucleotide translocator (ANT1),
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ECKERT ET AL.
A
Unaffected cells
TG2
TG
TG
Unmodified
histones
–
–
+
–
B
–
Aminylated
histones
–
D
TG2
TG2
Ad/Sc
c-Jun
TG
+
PGC-1α promoter
C
TG2
+
–
PGC-1α promoter
SP1 SP1 SP1
Huntington’s disease
TG2
c-Fos
Ad/Sc
HIF1β
HIF1α
SP1
Inactivation
SP1 binding site
c-Met promoter
AP1 binding site
Hypoxic response element
MMP9 promoter
Bnip3 promoter
FIGURE 8. TG2 as a novel transcriptional regulator in the nucleus. A: TG2-mediated transcriptional regulation in patients with Huntington disease. Under normal conditions, the level of TG2 transamidation activity is low
and does not interfere with transcription of key genes involved in the regulation of mitochondrial and metabolic
function (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. This
chromatin alteration represses target gene transcription. Reduced expression of PGC-1␣ and related genes
contributes to the mitochondrial and metabolic dysfunction observed in Huntington disease. B: TG2-dependent
enzymatic crosslinking of Sp1 transcription factor in the nucleus causes Sp1 inactivation and inhibits Sp1mediated transcription of the prosurvival gene c-Met in hepatocytes. This transamidation-dependent mechanism mediated by nuclear TG2 is involved in liver steatohepatitis. C: TG2 binds noncovalently to c-Jun in the
nucleus and prevents c-Jun/c-Fos dimerization, thereby decreasing AP1 transcription factor-dependent transcription of the MMP-9 gene in cardiomyoblasts. This nonenzymatic mechanism, mediated by nuclear TG2, is
thought to be involved in ECM remodeling. D: TG2 interacts noncovalently with HIF-1␤ in the nucleus and
prevents its dimerization with HIF-1␣, thus inhibiting HIF-1 binding to the hypoxic response element (HRE) in the
promoter of the Bnip3 gene leading to reduced Bnip3 transcription in neuronal cells. This nonenzymatic
nuclear TG2-driven mechanism plays a role in the prosurvival effect of TG2 in stroke patients. TG indicates
transamidating activity of TG2, and Ad/Sc indicates adapter/scaffolding nonenzymatic activity of TG2.
a protein involved in ADP/ATP exchange and a core component of the permeability transition pore complex in the
internal mitochondrial membrane, is also crosslinked by
TG2 (228). In general, TG2-mediated covalent modification of mitochondrial proteins does not occur in normal
tissue; however, modification is likely in patients with “mitochondrial diseases,” including cardiovascular ischemia/
reperfusion injury and neurodegenerative disorders such as
Huntington disease. Accordingly, TG2-catalyzed crosslinking of mitochondrial matrix ␣-ketoglutarate dehydrogenase
(68) and aconitase (186) is observed with decline in energy
metabolism. Moreover, high-molecular-weight aggregates
of these enzymes are observed in Huntington disease patients having elevated TG2 crosslinking activity. Additional
396
studies are needed to establish the precise role of TG2mediated crosslinking in the mitochondrial apoptosis pathway.
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 disulfide
bond formation in NADH-ubiquinone oxidoreductase
(complex I), succinate-ubiquinone oxidoreductase (complex II), cytochrome c oxidase (complex IV), and ATP synthase (complex V). TG2 PDI activity may also control the
respiratory chain by modulating protein complex formation (231). Another target of TG2 PDI activity is ANT1
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TRANSGLUTAMINASES IN CELL FUNCTION
(228). ANT1 oligomerization is essential for activity, and
TG2⫺/⫺ mice have increased thiol-dependent ANT1 oligomer formation and elevated ANT1 ADP/ATP exchange
activity in heart mitochondria. Thus the PDI activity of TG2
reduces the level of oligomerized ANT1 and inhibits transporter activity by sequestering ANT1 monomers and preventing oligomer formation by direct binding to ANT1. In
addition, the presence of TG2 is required for Bax/ANT1
colocalization and interaction in mitochondria (228).
Taken together, these findings reveal an important role for
TG2 PDI enzymatic activity in vivo and indicate the existence of a novel pathway that links this activity with regulation of mitochondrial physiology.
Future studies to understand TG2 trafficking and shuttling
among various cellular compartments is important. How
do cells regulate these trafficking events? What are the TG2
molecular signature motifs (e.g., “barcodes”) for its targeted delivery to different organelles? What are the compartment-specific binding partners of TG2? Which signaling pathways regulate distribution? Defining these issues
should help to determine the compartment-specific functions of TG2. For example, the vexing lack of understanding of how intracellular TG2 moves to the cell exterior
(398) is an important example of a trafficking mechanism
that needs to be clarified.
IV. TGS IN CELL DIFFERENTIATION
TG activity is fourfold higher in blastocysts than in two-cell
embryos (225), suggesting that transglutaminases have an
important role in development. Little is known about which
TG isoforms are responsible for this activity. However,
more is known about the role of transglutaminases in specific tissues which will be discussed in this section.
A. Epidermis
The epidermis is a stratified epithelium formed by specialized cells called keratinocytes (94, 95). It consists of the
basal, spinous, granular, and cornified layers. The proliferating cells are in the basal layer, and the spinous and granular and cornified layers display progressive differentiation
(95). The cornified layer consists of completely differentiated cells that form the body surface. At the cellular level,
the final stage of keratinocyte differentiation begins in the
epidermal spinous and granular layers with accumulation
of cornified envelope precursors on the inner surface of the
plasma membrane (55, 95, 96, 99). These proteins are
crosslinked by TGs to form the rigid cornified cell envelope
that gives the differentiated keratinocyte its protective
properties (46, 329). TG crosslinks cornified envelope precursors including involucrin, loricrin, filaggrin, and small
proline-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 not
known (48, 184). TG1 and TG3 and TG5 are produced in
the differentiated cells of the spinous and granular layers
(48, 184) where they act on substrates to assemble the cornified envelope (50, 95, 391). In addition, TG1 mediates
covalent crosslinking of ceramides to involucrin, which
contributes to maintenance of the epidermal permeability
barrier (263).
TG1 and TG5 mutations produce defects in the cornification process. Mutation of TG1 in either the catalytic cysteine, or surrounding region, is associated with lamellar
ichthyosis (140, 281, 302, 346). A similar phenotype is
observed in mice lacking the TG1 gene (234). These mice
have defects in epidermal barrier formation and die shortly
after birth. Interestingly, other TGs cannot functionally replace TG1 action despite their ability to crosslink loricrin,
involucrin, and other precursors (47). Similar to the critical
role of TG1 activity in cornification, a loss-of-function mutation of TG5 is associated with acral peeling skin syndrome (53). In contrast, a role for TG2 in keratinocyte
differentiation has yet to be demonstrated, and TG2-knockout mice do not have any obvious skin defects (261). Likewise, the role of TG3 in keratinocyte differentiation remains to be elucidated.
B. Nervous System
1. Neuronal differentiation
Dendritic extension and axonal branching are key features
of neurogenesis and often used as markers of neuronal differentiation, and catalytically active TG2 may modulate the
rate and extent of neurite outgrowth. For example, an increase in total TG activity is associated with neurite outgrowth in murine neuroblastoma cells (226), and forced
expression of TG2 in SH-SY5Y neuroblastoma cells causes
spontaneous neurite outgrowth and neuronal marker expression following retinoic acid treatment (322). In contrast, elevated expression of transamidating-inactive
(C277S) TG2 represses neuroblastoma cell differentiation
(341). These findings suggest a requirement for the TG2
transamidating function in this process.
The specific molecular mechanisms underlying induction of
neuronal differentiation by the transamidating activity of
TG2 are emerging. TG2 protein is prominently localized at
the tips of outgrowing neuritis, suggesting that it has a role
in stabilizing extended structural projections (357). In addition, a role for TG2-dependent activation of c-Jun NH2terminal kinase (JNK) signaling in neurite outgrowth has
been proposed (322), as has activation of adenylyl cyclase
by the transamidating active form of TG2, leading to protein kinase A-mediated CREB activation (356). This mechanism seems to be specific to neuronal differentiation, because TG2 inhibits adenylyl cyclase activity and decreases
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ECKERT ET AL.
cAMP levels in Balb-c 3T3 human fibroblasts and bovine
aorta endothelial cells (119), and in MSCs undergoing
chondrogenic differentiation (270).
In contrast to the proposed role of the transamidating activity of TG2 in neuronal differentiation, the role of this
enzyme in neurodegeneration is not clear. Neuronal TG2
attenuates cell death in ischemic conditions (106, 107), but
facilitates cell death in excitoxic conditions (358). Thus
TG2 accumulation in brains of Parkinson, Huntington, and
Alzheimer’s patients may be a compensatory, protective
response (158, 163, 185, 211).
2. Macroglia
Glial cells maintain homeostasis, form myelin, and support
and protect neurons. Similar to neurons, glial cells originate
in the neural tube and neural crest of the developing embryo. The most common type of macroglial cell is the astrocyte which has numerous projections that help anchor neurons to the blood supply. They also secrete regulatory
proteins that promote the myelinating activity of oligodendrocytes, another abundant type of microglial cell. Oligodendrocytes form a specialized myelin membrane, which
coats and protects axons in the central nervous system.
Genetic ablation of TG2 results in delayed remyelination in
injured animals, suggesting a role for TG2 in glial differentiation. In addition, in vitro studies demonstrated that TG2
regulates astrocyte migration, which is required for proper
remyelination in vivo and for differentiation of myelin-producing oligodendrocytes from precursor oligodendrocytes
(361). Moreover, both of these processes are attenuated by
the TG2-specific inhibitor KCC009 {(S)-[3-(4-hydroxyphenyl)-2-N-(phenylmethyloxycarbonyl)]aminopropanoic
acid N’-(3=-bromo-4=,5=-dihydro-5=-isoxalyl)methylamide}
(361). The precise mechanism of TG2 action remains to be
determined, but reported data implicate a role for RhoA in
TG2-dependent differentiation of oligodendrocytes (360).
C. Immune System
Accumulating evidence indicates an important role for TG2
in cell-mediated immunity and suggests that TG2 contributes to the immune response via regulation of differentiation in phagocytes, monocytes, neutrophils, and T cells.
During monocyte differentiation, expression of FXIIIa and
TG2 is induced and may contribute to neutrophilic and
monocytic differentiation (8, 208, 251, 252). Genetic and
pharmacology studies identify TG2 as a positive regulator
of neutrophilic differentiation. For example, genetic ablation of TG2 in murine neutrophils results in diminished
superoxide anion production and impaired extravasation,
indicative of delayed differentiation (22). Moreover, TG2
silencing in the human promyelocytic leukemia cell line
NB-4 leads to a significant delay in the neutrophil differentiation process and downregulation of expression of genes
398
related to the innate immune system (72). Similar to its
effects on neutrophilic differentiation, TG2 activity plays
an important role in monocytic differentiation to macrophages and dendritic cells, the antigen-presenting cells that
act as messengers between innate and adaptive immunity.
TG2 regulates cell adhesion and migration in differentiating
macrophages (8, 251), whereas in dendritic cells, TG2 mediates maturation of antigen-presenting cells in response to
bacterial lipopolysaccharide (LPS) (233). Moreover, inhibition of TG2 with KCC009 (62) reduces cytokine production and dendritic cell differentiation in vitro (233). Altered
resistance to LPS-induced septic shock in TG2-null mice
further confirms that TG2 has a significant role in dendritic
cell maturation (233). In addition, FXIIIa may regulate terminal maturation in both macrophages and dendritic cells,
and FXIIIa-deficient human monocytes demonstrate reduced phagocytosis (307). In contrast, FXIIIa overexpression results in enhanced dendritic cell motility (157).
D. Connective Tissues
Connective tissues, including cartilage, bone, adipose tissue, 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 several
lineages during differentiation. Accumulating evidence suggests a role for TGs in regulation of MSC differentiation.
TG2 can crosslink and stabilize cytoskeletal proteins including actin, tubulin, vimentin, and myosin, suggesting a
role in establishing cellular phenotype (65, 87, 101). In
addition, extracellular TG2 stabilizes the cytoskeleton by
promoting integrin clustering and regulating FAK, RhoA
GTPase, ROCK, and mitogen-activated protein kinase
(MAPK) activity (155). TG2-mediated crosslinking increases the stiffness of the ECM (262, 314, 326), and this is
sensed by integrin receptors (43, 180) and converted into
signaling output (316). MSC differentiation is controlled by
matrix stiffness (100, 137, 260), and TG2-modified collagen scaffolds promote chondrogenic differentiation of human MSCs (314). Extracellular matrices with “high stiffness” favor osteoblast differentiation over neural, muscle,
and chondrogenic differentiation (100, 260). Thus MSCassociated TG activity, both intracellular and extracellular,
regulates MSC differentiation.
1. Cartilage and chondrocytes
Endochondral bone formation is a key event in skeletal
development. This is a process in which cartilage is first
generated and then replaced by bone (219). The first step in
endochondral bone formation is chondrocyte differentiation, which begins with MSC condensation and progresses
through a series of maturation stages including resting, proliferative, prehypertrophic, and hypertrophic stages (219).
Terminal differentiation of chondrocytes is characterized
by deposition of mineralized ECM that is associated with
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cell death and replacement of cartilage with bone. TG2
regulates the transition to the prehypertrophic stage, which
is characterized by cessation of ECM deposition and reduced cell proliferation (270). Expression of FXIIIa and
TG2 is markedly increased during this transition (267,
270), and both enzymes are found in the intracellular and
extracellular compartments during terminal differentiation
(4, 268, 349). Premature expression of TG2 in cultured
MSCs accelerates prehypertrophic differentiation and inhibits deposition of the cartilaginous matrix, whereas treatment with the TG2 inhibitor ERW-1065 (382) enhances the
matrix synthesis (270). In vivo, increased TG2 expression
delays endochondral ossification in chicken embryos by
blocking chondrocyte maturation during prehypertrophic
differentiation. TG2-mediated inhibition of protein kinase
A activity is implicated as a major mechanisms underlying
this regulation.
In contrast to endochondral cartilage, which is replaced by
bone, articular cartilage remains cartilaginous throughout
life. Articular chondrocytes are maintained in the resting
stage and do not undergo proliferation or terminal differentiation (90). Both TG2 and FXIIIa are expressed by
healthy articular chondrocytes (301, 349) and may block
chondrocyte differentiation. In addition, a role for TG2 and
FXIIIa in cell response to mechanical stress and abrasive
force can be postulated based on the pattern of FXIIIa expression and localization of TG activity to condylar regions
(269).
Diseases and conditions such as osteoarthritis and aging are
associated with increased expression of both TG2 and
FXIIIa. In vitro studies have shown that extracellular TG2
enhances hypertrophic differentiation and induces matrix
mineralization in cultured articular chondrocytes via ␣5␤1integrin-mediated activation of Rac1 and p38 in a fibronectin-independent manner (165, 335). Interestingly, these effects require the GTP-bound form of TG2. Whether this
mechanism is active in vivo where extracellular TG2 may
not be GTP-bound, remains to be determined. TG2 mobilization to the cell surface is regulated by FXIIIa interaction
with ␣1␤1-integrin. Thus atypical maturation of articular
chondrocytes may be regulated by a network of TGs and
not require transamidation activity (164). In addition,
TG2-modified calgranulin/S100A11 may trigger abnormal
maturation of articular chondrocytes (54). Because hypertrophic differentiation of articular chondrocytes is proposed as a mechanism that drives osteoarthritis, these findings implicate TGs in osteoarthritis. Indeed, severe knee
osteoarthritis in a guinea pig model is associated with increased synovial levels of TG2, but not FXIIIa (143), and
cartilage degradation in the mouse model of injury-induced
osteroarthritis is reduced in the absence of TG2 (274).
These proteins may also contribute to inflammatory osteoarthritis by triggering illegitimate maturation of articular
chondrocytes. These findings suggest that identification of
downstream mediators of TG-dependent chondrogenic differentiation may lead to development of novel therapeutics
to treat inflamed joints without affecting normal homeostasis in cartilaginous tissue.
2. Bone and osteoblast differentiation
Osteoblasts are bone cells responsible for production and
secretion of the collagen type I (COL I)-based mineralized
bone matrix. Osteoblast lineage differentiation is under the
master control of the transcription factors RUNX2 and
Osterix, which control MSC commitment to the osteogenic
lineage and the preosteoblast-to-osteoblast transition, respectively (192, 237, 324). Preosteoblasts form a transient
provisional fibronectin matrix (31), while mature osteoblasts deposit the permanent COL I matrix (250, 336, 337).
The advanced stages of osteoblast maturation are characterized by expression of alkaline phosphatase (114, 115),
which is essential for matrix mineralization and a characteristic feature of fully differentiated osteoblasts (237). The
final stage of bone and osteoblast differentiation is osteoblast transformation into osteocytes, which serves a mechanosensory function in the bone matrix and controls bone
remodeling via regulation of osteoblast and osteoclast function (237, 324). TG2 and FXIIIa are expressed during osteoblast differentiation from early stages through osteocyte
formation (11, 12, 171, 257, 266). Maturation and matrix
mineralization in cultured osteoblasts are accelerated by the
expression of exogenous TGs, suggesting that TGs act as
positive autocrine regulators of osteoblast differentiation.
Indeed, TG inhibitors arrest osteoblast differentiation at a
very early stage, dramatically decrease COL I and fibronectin deposition and matrix accumulation as well as alkaline
phosphatase expression and matrix mineralization (11, 12).
These results suggest that TGs are essential for osteoblast
differentiation. However, the precise molecular mechanism
of this regulation is not known. During differentiation, TG2
levels remain constant, and the TG2 protein is retained at
the cell surface in a high-molecular-weight complex that has
no detectable crosslinking activity but may possess signaling activity (11). It has been postulated that TG2 on the cell
surface promotes cell adhesion and spreading (56, 111,
112, 129, 367, 381) and may regulate osteoblast differentiation via this mechanism. In addition, in cultures of mature osteoblasts supplemented with ATP, TG2 may regulate
ECM mineralization by acting as an ATPase in the ECM
(39, 256, 258, 374) and increasing phosphate levels for
hydroxyapatite precipitation or via phosphate signaling to
promote terminal differentiation of osteoblasts into osteocytes (402).
In early osteoblasts, cellular FXIIIa associates with the
plasma membrane and appears to be required for microtubule stabilization and collagen trafficking and secretion
(11), a function that likely involves TG-mediated crosslinking of detyrosinated tubulin to form homotypic or heterotypic polymers. Tubulin has been described as a TG sub-
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ECKERT ET AL.
strate in other systems (87). At the preosteoblastic stage,
FXIIIa is secreted as a latent proenzyme that is incorporated
into fibronectin and COL I matrix fibrils. Matrix-associated
FXIIIa seems to be activated to stabilize the permanent
fibronectin/COL I matrix, which in turn promotes osteoblast maturation (292). Expression, cellular localization,
and secretion of FXIIIa are strongly stimulated by extracellular COL I and MAPK signaling (292).
A lack of any obvious skeletal phenotype in mouse models
lacking either TG2 or FXIIIa suggests that these two TGs
have complementary functions and may act to compensate
for each other (266). In this respect, increased FXIIIa, TG1,
and TG3 expression in bone and tendons in TG2-knockout
mice has been observed (86, 266, 273, 338).
3. Muscle and myoblasts
Formation of cardiac, smooth, and skeletal muscle involves
differentiation of myoblasts from MSCs. Expression of the
muscle-specific contractile myofibrillar proteins (e.g., myosin, actin) is characteristic of myoblast differentiation, as is
myoblast fusion during skeletal muscle maturation (40).
TG inhibitors interfere with proper myoblast fusion resulting in anomalous elongation of the fused cells, which is
indicative of defective cytoskeletal stabilization (35). TGmediated crosslinking of the myosin chain occurs in different muscles, and this may contribute to myoblast differentiation and elongation (63, 139, 174). Therefore, stabilization of cytoskeletal elements via TG-mediated crosslinking
might be the mechanism of TG-dependent regulation of cell
differentiation, which is especially pertinent in cells with
reduced levels of ECM, such as muscle cells.
4. Tendons and ligaments
The role of TGs in differentiation of tenocytes has yet to be
extensively investigated despite evidence of expression of
TG1 and TG2 in normal tendons (273). In mice, TG2knockout tendons are anatomically normal. However, further biomechanical tests and injury models are needed to
test a role for TG2 in tendon stabilization and response to
injury (273). Whether TG1 can compensate for loss of TG2
expression in tendons also remains to be determined. TG3
and TG5 have been found in tendons, and FXIIIa expression is selectively increased in injured tendons. The source
of FXIIIa in injured tendons remains to be determined, but
may be related to blood vessel injury. Interestingly, increased TG activity in tendocytes is associated with disease.
TG2 and FXIIIa activity are increased in tendocytes exposed to carboxymethyl-lysine collagen and high glucose
levels, mimicking an AGE-rich environment and diabetes,
respectively (284, 300). These findings suggest that increased TG crosslinking activity and crosslink formation
may contribute to the stiffening and pathological calcification of tendons in diabetic patients (10, 235). Similarly,
400
elevated TG2 level is observed in calcified ligamentum flava
of the spine (393), which provides additional evidence of
the role of TG2 in pathology-related calcification of tendons.
V. ROLE OF TG2 IN CANCER
Cancer is a disease of dysregulated cell growth and differentiation. Genetic and epigenetic changes induced in response to chronic stressors (chemicals, infections, environmental) can constitutively activate survival signaling cascades and render them independent of growth factors. In
addition to genetic and epigenetic alterations, cancer cells
require additional input (probably induced by tumor
stroma) to gain metastatic competence and ability to survive in host environment. In this context, chronic inflammation is considered to be an important factor in cancer
initiation and progression (364, 365). Because TG expression, particularly TG2, is frequently increased in response
to inflammation (223, 224, 227), it is likely that TGs play a
role in cancer. Several lines of evidence suggest the involvement of TGs during cancer development even though the
molecular mechanisms remain controversial. TG2 expression is low in primary tumors, but TG2 level is increased in
drug-resistant and metastatic tumors (240, 280, 309, 372,
394). These observations suggest that TG2 may support
cancer progression (6, 42, 229, 239, 271). In the following
section, we discuss evidence that TG2 expression reprograms inflammatory signaling pathways in epithelial cancer
cells 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 including TGF-␤, TNF-␣, IL-1, and IL-6, which are secreted at
tumor cells, are known TG2 inducers (199, 296, 333). Excessive deposition of collagen by fibroblasts is observed in
tumors in the form of a desmoplastic response, which is
responsible for the clinical presentation of the tumor as a
lump or dense stroma (16, 376) (FIGURE 9). The desmoplastic response of tumors appears to require TG2-catalytic
activity (FIGURE 9). Increased TG2 expression in response
to inflammatory cytokines, such as TGF-␤ in fibroblasts
and epithelial cancer cells, accelerates the synthesis and deposition of fibronectin and collagen, whereas extracellular
TG2 crosslinks them to stabilize the ECM. TG2-catalyzed
crosslinking of ECM proteins can result in adverse outcomes, including diabetic nephropathy, kidney scarring,
and atherosclerosis (60, 167, 312), and probably plays a
similar role in the tumor desmoplastic response (FIGURE 9).
The desmoplastic response involves an interplay between
the invading tumor cells and altered ECM (16), hence the
TG2-mediated alteration of the ECM is likely to impact
tumor cell behavior. For example, breast cancer progres-
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TRANSGLUTAMINASES IN CELL FUNCTION
Inflammation
Immune
cell
TGF-β/TNF-α/IL-6/ROS–
Epithelial cells
Transdifferentiation into
mesenchymal state
NFκB
EMT
Cell survival
invasion
TG2
ECM
Modified ECM
Drug resistance
Metastasis
Fibroblasts
Collagen, fibronectin
Myofibroblasts
Active-TGF-β
Desmoplastic
reaction
TG2
Latent-TGF-β
Fibrosis
FIGURE 9. TG2 reprograms the inflammatory signaling circuitry. Inflammatory cytokines produced by infiltrating immune cells induce TG2 expression in cancer cells and host cells (tumor-associated fibroblasts). Due
to low Ca2⫹ and high GTP levels, intracellular TG2 is predominantly present as a cryptic enzyme and serves as
a scaffold protein. For example, it interacts with I␬B␣ and results in its degradation via a proteasomeindependent pathway. This results in increased activation of NF-␬B. Activated NF-␬B translocates to the
nucleus and induces expression of multiple genes. TG2 is also a target gene for NF-␬B, which results in an
auto-induction loop between NF-␬B and TG2. NF-␬B regulates genes involved in promoting drug resistance and
metastasis. 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 of
TG2-modified ECM, coupled with enhanced synthesis of ECM component proteins, results in fibrosis and
desmoplastic response, which further promote aggressive phenotype in cancer cells.
sion requires collagen crosslinking, ECM stiffening, and
increased focal adhesion formation (212), and TG2-mediated crosslinking may alter the stiffness of the ECM and
thereby promote a malignant phenotype.
B. TG2, Drug Resistance, and Metastasis
Resistance to therapy and metastasis are hallmarks of advanced cancer. Identification of tumor-encoded genes that
promote drug resistance and metastasis is an important
goal, as these proteins may serve as cancer biomarkers and
also offer targets for therapy.
EMT is a major pathway that governs cell behavior during
cancer progression. EMT is an embryonic process that can
be reactivated in adult tissues in response to epigenetic
changes (347). EMT is considered to be an attempt of the
host to control inflammation and heal damaged tissue;
however, in pathological contexts this response can cause
damage. Epithelial tumor cells undergoing EMT lose epithelial tight junction proteins including E-cadherin and gain
the expression of mesenchymal markers such as SMA,
FSP1, vimentin, fibronectin, and desmin (172, 243, 347).
These cells are involved in intravasation, movement of cancer cells in the circulation, extravasation, and micrometastases formation.
TG2 activates FAK, Akt, and NF-␬B signaling, which are
known to induce cancer cell EMT (315, 369, 371) (FIGURE
9). Inhibition of TG2 expression suppresses and increased
TG2 expression enhances invasiveness and resistance to
chemotherapy (131, 145, 280, 309, 368, 395). In addition,
microvesicles, shed by TG2 overexpressing cancer cells, can
confer transformed characteristics (e.g., anchorage-independent growth and enhanced survival capability) on normal fibroblasts and epithelial cells (14). Although TG2
alone is not sufficient to transform fibroblasts, it is likely
that it collaborates with other proteins to mediate transforming action of the cancer cell-derived microvescles.
C. TG2, EMT, and Cancer Stem Cells
TG2 expression is associated with induction of EMT and
acquisition of stem-cell phenotype (195–197, 315). For ex-
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ECKERT ET AL.
ample, mammospheres generated from TG2-overexpressing nontransformed mammary epithelial cells (MCF10A)
differentiate into mammary glandlike structures including
Muc1-positive luminal cells and integrin ␣6-positive basal
cells in response to prolactin treatment (197). In addition,
there is a correlation among TG2, EMT, and acquisition of
a stem cell-like phenotype in ovarian cancer cells (51). Cancer stem cells exhibit intrinsic resistance to chemotherapy
and treatment of MCF-7 breast cancer cells with doxorubicin selects a small subpopulation of cells with stem cell
characteristics that express high levels of TG2 (44). These
results suggest that TG2 induces EMT and other stem cell
traits in cancer cells.
Some information is available regarding TG2-regulated signaling pathways that drive these processes. TG2 expression
results in constitutive activation of FAK, Akt, and NF-␬B
signaling (182, 369, 371), all of which are involved in cancer progression (172, 243, 276, 347, 375). NF-␬B plays a
prominent role in cancer progression, stemming from its
ability to activate pro-growth, pro-metastatis, and anti-apoptotic genes (89, 328). Recently, a novel feedback loop
where TG2 activates NF-␬B and NF-␬B, in turn, drives TG2
expression was identified (41) (FIGURE 9). In cancer cells,
this TG2/NF-␬B feedback loop may be self-amplifying, because high TG2 expression and elevated NF-␬B activity are
frequently observed in late-stage cancers. Most efforts to
inhibit NF-␬B activation in cancer cells have focused on
small molecules that block the IKK kinase activity. In view
of the observation that TG2-induced activation of NF-␬B is
mediated through IKK-independent mechanism (198),
these inhibitors may not be effective. High-level NF-␬B
would, in turn, confer resistance to cell death and promote
EMT and metastasis (178, 187).
promoter (FIGURE 6B). Like NF-␬B and TG2, HIF-1␣ regulates EMT-related signaling, including accumulation snail,
twist, and Zeb1 (127). These results suggest that TG2-induced NF-␬B activation regulates HIF-1␣ expression.
Taken together, these observations suggest that aberrant
epigenetic regulation of TG2 expression in cancer cells can
reprogram inflammatory signaling networks (NF-␬B activation, expression of HIF-1␣, Snail, Twist, and Zeb) that
influence EMT and stemness to promote drug resistance
and the metastatic phenotype (FIGURE 10). Doxorubicinresistant breast cancer cells express high levels of TG2 associated with hypomethylation of TGM2 promoter,
whereas in doxorubicin-sensitive cells, which show no detectable expression of TG2, the TGM2 promoter is hypermethylated (6). In addition, treatment of drug-sensitive cells
with 5-azadC restores TG2 expression and reduces sensitivity to doxorubicin (6). These findings suggest that TG2 may
be a promising anti-cancer therapeutic target, as in vivo
silencing of TG2 by delivery of liposomal-siRNA inhibits
Inflammation (chronic)
TGF-β/TNF-α/IL-6/ROS–
Hypomethylation of
TGM2 gene promoter
TG2
NFκB
VEGF
NF-␬B collaborates with TG2 to regulate expression of
EMT transcriptional regulators including Snail, Twist,
Zeb1, and Zeb2 (196, 315). TG2, in complex with the p65
subunit of NF-␬B, is recruited to the promoter of the
SNAIL gene, to increase Snail levels (187). TG2 also interfaces with TGF-␤, a potent inducer of EMT. In breast cancer cells, TGF-␤ induced EMT requires TG2 expression
(196). These findings suggest that TG2 may be an important
regulator in the TGF␤ signaling pathway that is required
for EMT, and that crosstalk between TG2 and NF-␬B may
promote metastatic competence.
HIF-1 is an important hypoxia-response transcription factor that is regulated by NF-␬B (142, 169, 318) and TG2
(195, 196, 198, 315) and has an important role in cancer
cells. TG2-expressing cells display high basal levels of
HIF-1␣ expression even under normoxic conditions, and
suppression of either TG2 or NF-␬B (p65/RelA) reduces
HIF-1␣ level. Chromatin immunoprecipitation studies reveal that TG2 forms a complex with p65/RelA and that the
complex binds to the NF-␬B binding site in the HIF-1␣
402
HIF1α
Angiogenesis
Zeb, Twist, Snail
EMT
Warburg effect
Cell growth
Cell survival
Stem cell
traits
Drug resistance
& Metastasis
FIGURE 10. TG2 regulates inflammatory signaling, drug resistance, and metastatic phenotype. Chronic exposure to inflammatory
cytokines (produced by tumor-infiltrating immune cells) results in
epigenetic regulation of TG2 and initiates the feedback cycle where
TG2 activates NF-␬B, and NF-␬B further increases TG2 expression.
HIF-1␣ is a downstream target of TG2-induced NF-␬B. Increased
expression of HIF-1␣ (even under normal oxygen conditions) results
in activation of multiple downstream target genes, which are involved in reprogramming of cancer cells for altered metabolism, and
angiogenesis. Moreover, the level of key transcription repressors
(e.g., Snail, Twist, Zeb) is upregulated in TG2/NF-␬B/HIF-1␣-expressing cells, which leads to transdifferentiation of epithelial cells to
the mesenchymal state (EMT), an important step in tumor metastasis. TG2-induced EMT promotes stem-cell traits in cancer cells
and confers resistance and self-renewal ability for successful survival and growth at metastatic sites.
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TRANSGLUTAMINASES IN CELL FUNCTION
the growth and dissemination of pancreatic and ovarian
cancer cells and renders them sensitive to chemotherapeutic
drugs in a nude mouse model (145, 368).
VI. REGULATION OF TG2 EXPRESSION
AND TG2-REGULATED
TRANSCRIPTIONAL PROCESSES
TG2 is gaining increasing attention for its role as a transcriptional regulator. In this section, we focus on how TG2
expression is regulated and how TG2 regulates gene expression.
A. Control of TG2 Gene Expression
Expression of TG2 is mainly controlled at the transcriptional level. Retinoids are well-known inducers of TG2 expression (83, 253, 290), and induction of TG2 expression
by 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 receptors that bind to DNA as dimers. A study by Nagy et al.
(253) showed that retinoid-dependent induction of murine
TG2 expression is mediated by a tripartite response element
located in the TG2 gene promoter. This study demonstrated
that retinoic acid response elements are present at ⫺1720
bp (RRE-1) and ⫺1731 bp (RRE-2) in the murine TG2
promoter. Accordingly, RAR-RXR heterodimer and RXR
homodimer receptor complexes bind to the murine TG2
promoter to regulate retinoic acid-dependent induction. In
addition to RAR and RXR binding, the murine TG2 promoter requires an additional cooperating segment of the
promoter (HR-1) to mediate activation of TG2 expression
by retinoic acid (253). In the first characterization of the
TG2 promoter, Lu et al. (222) reported that a 1.74-kb DNA
region at the 5=-flanking region of the human TG2 promoter did not contain retinoic acid response elements, as
the luciferase reporter they used failed to show induction in
response to treatment with retinoic acid. However, subsequent studies establish that human TG2 expression is increased by retinoids, and an unusual binding site present in
the TG2 promoter is likely to mediate induction (24, 357,
401).
TG2 expression is essential for retinoic acid-induced differentiation of human neuroblastoma cells (322, 357). The
myc oncoproteins block differentiation and promote proliferation of malignant cancer cells (287), and TG2 expression
is repressed by N-myc in neuroblastoma cells and by c-Myc
protein in breast cancer cells, which may contribute to cancer cell proliferation and migration owing to a lack of differentiation. N-myc appears to suppress TG2 expression by
recruiting histone deacetylase 1 to the Sp1 sites on the TG2
gene promoter (218, 341). Also, TG2 expression is epigenetically regulated by hypomethylation of CpG islands in its
promoter in chemoresistant breast cancer (6), non-small
cell lung cancer (280), and glioma (93) cells.
Although TG2 expression is suppressed in some cancer
cells, it is increased in others, especially in cells resistant to
chemotherapy or isolated from metastatic sites. TG2 expression is elevated in pancreatic carcinoma (372), breast
carcinoma (239), malignant melanoma (109), ovarian carcinoma (145), lung carcinoma (280), and glioblastoma
(395). As outlined above, inflammation (201) and hypoxia
(313) are important in cancer, and TG2 expression is upregulated by these processes. Indeed, the TG2 promoter
contains response elements involved in inflammatory antihypoxic signaling (118, 154, 199, 244).
Cytokines increase TG2 expression in neuronal cultures
(216). Also, LPS stimulates TG2 expression in several mammalian cell models, and metastatic tumor antigen 1 facilitates LPS-induced TG2 expression (120). An IL-6 response
element (⫺1190 bp) is present in the human TG2 promoter
(83), and IL-6 treatment of HepG2 human hepatoblastoma
cells (333), human macrophages (117), and other cell types
(232, 271) increases TG2. In addition, an NF-␬B binding
site is located at ⫺1328 bp in the human TG2 promoter.
NF-␬B binding to this sequence in the TG2 promoter is
observed in CCl4-treated rats (244). TG2 expression also
can be induced by treatment with TNF-␣ in HepG2 cells
(199).
TGF-␤1 increases TG2 expression in apoptotic hepatocytoma cells (118), human dermal fibroblasts (296), and human retinal pigment epithelial cells (295). The murine TG2
promoter contains a TGF-␤ response element at ⬃868 bp
upstream of the transcription start site (298). TGF-␤1 increases TG2 promoter activity in Mv1Lu cells, an epitheliallike cell line derived from normal mink lung. Interestingly,
treatment with TGF-␤1 inhibits TG2 promoter activity in
MC3T3 murine pre-osteoblastic cell. Moreover, BMP2 and
BMP4, which are members of the TGF-␤ superfamily, also
reduce TG2 promoter activity (298), indicating that the role
of TGF-␤ family members in modulation of TG2 gene expression is stimulus and cell type dependent.
HIF-1 increases TG2 expression (154), and promoter deletion analysis revealed a sequence at 367 bp upstream of the
transcription start site in the TG2 promoter that is required
for this action (154). A comparison of TG2 promoter sequences shows that the response element is conserved in the
human, murine, and guinea pig TG2 promoters. TG2 expression is also increased in response to hypoxia or ischemia
in several other models (107, 350). Indeed, TG2 mRNA and
protein level are elevated after stroke in rats, mice, and
gerbils (107, 149, 350).
Lu et al. (222) characterized a TATA box (⫺29 bp) and
CAAT box (⫺96 bp) in the human TG2 gene promoter
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ECKERT ET AL.
5’
RAR
RRE-2 RRE-1
–1740
–1399
–1338
–1190
–634
GRE
NFκB
IL-6
–367
HRE
–183
–96
–54 –43
–29
AP-1
RXR
AP-2
SP1
CAAT
GC GC
TATA
+1 +4
+12
NF-1 NF-1
+59 +65
GC GC
3’
TGM2
Possible retinoic acid
response elements
TG2
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), TATA
box (⫺29 bp), and NF-1 (⫹4 bp and ⫹12 bp). TG2 gene expression is upregulated in response to inflammation
and hypoxia. Human TG2 expression is upregulated by treatment with retinoic acid, and potential retinoic acid
response elements (RAR and RXR) are located in the human TG2 promoter. [From Gundemir et al. (125a),
with permission from Elsevier.]
(FIGURE 11). A GC-rich region, located between the TATA
box and the CAAT box, encodes two Sp1 transcription
factor binding motifs at ⫺54 and ⫺43 bp, and additional
Sp1-binding motifs are located in the 5=-untranslated region
of the TG2 gene at positions ⫹59 and ⫹65 bp. Four 3= half
sites of NF-1 are also present in the 5=-untranslated region
at ⫹4 and ⫹12 bp. TG2 expression can be upregulated in
rat astroglial cells in response to various growth factors and
steroids (45), indicating that the putative glucocorticoid
response element of the TG2 promoter is likely to be functional. In addition to the glucocorticoid response element,
AP1 (⫺183 bp) and AP2 (⫺634 bp) response elements are
located in the murine TG2 promoter (222). These findings
indicate that multiple signaling pathways regulate TG2
gene expression. FIGURE 11 shows known and potential
regulatory elements in the human TG2 promoter. It will be
important in future studies to confirm the functional role of
these sites.
Variant forms of TG2 are also produced. The full-length
687-amino acid form of TG2 is the most abundant; however, 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 erythroleukemia
cells that differs from full-length TG2 in the last 10 amino
acids (GKALCSWSIC versus EKSVPLCILY) (113). The 5=
end of this short isoform is the same as that of the long form
up to base 1747, but the sequence differs from base 1748 to
the termination codon. The divergent sequence contained
an intron-exon boundary (CTGGTAA), which suggests alternative splicing (113). Another study described a similar
truncated 548 amino acid isoform in an Alzheimer’s disease
brain (64) along with a 555 amino acid isoform with a
COOH-terminal sequence of GKPCVTGAFVDRGLTTC
404
instead of QTQPITCQPSTQPGFIPR (64). Injured rat spinal cords express a 640-amino acid isoform of TG2, with
29 different amino acids at the COOH terminus (105).
Truncated forms are also present in cytokine-treated rat
astrocytes (249), leukocytes, vascular smooth muscle cells,
and endothelial cells (203). The common feature is the presence of variable sequences at the COOH terminus. Because
these short isoforms lack residues at the COOH terminus,
GTP binding in TG2 is often impaired (203). Although the
mechanism by which these short isoforms are generated has
yet to be understood, some may result from intron readthrough (64, 105, 113, 203, 249). Further studies are
needed to determine how these isoforms are generated and
to identify their physiological function.
B. Transcriptional Regulation by TG2
TG2 transcriptionally regulates several important targets
(FIGURE 12). For example, TG2 induces differentiation and
neurite growth in SH-SY5Y cells via a mechanism that may
involve cAMP signaling, as TG2 induces adenylyl cyclase,
increases cAMP level, and enhances CREB activity in SHSY5Y cells (356). These responses require TG2 transamidating activity (356). TG2 also increases CREB activity by
interacting with protein phosphatase 2, a protein that dephosphorylates and inactivates CREB. The TG2-mediated
increase in CREB activity leads to increased MMP-2 expression (310). In addition, membrane recruitment of TG2 is
increased during erythroid differentiation of K562 cells in
response to trans-retinoic acid and is associated with activation of Akt. TG2 overexpression increases CREB phosphorylation, an effect that is abrogated in the presence of
wortmannin, a phosphatidylinositol 3-kinase inhibitor, im-
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TRANSGLUTAMINASES IN CELL FUNCTION
TG2
Inflammation
A
E
IκB
cAMP
TG2
Ethanol
NFκB
Hypoxia
B
Huntington’s disease
C
TG2
D
PGC-1α
α
cyt-c
HIF1β
SP1
CREB
FIGURE 12. TG2 regulation of transcription. A: TG2 expression is upregulated by
inflammation. TG2 transamidates I␬B␣
leading to NF-␬B activation in response to
inflammation. B: increased TG2 expression
in hypoxia leads to HIF-1␤ dependent transcription of genes via hypoxia response element (HRE). C: TG2 suppresses PGC-1␣
and cyt c expression in mutant huntingtinexpressing cells. D: TG2 crosslinks Sp1 in
response to ethanol treatment leading to
reduced Sp1-dependent gene transcription. E: TG2 increases cAMP expression,
leading to activation of CREB. [From Gundemir et al. (125a), with permission from
Elsevier.]
HRE
plying that TG2 activates Akt signaling leading to activation of CREB (176). Similarly, cytosolic TG2, as discussed
above, can activate NF-␬B, either by transamidating I␬B␣
or facilitating its degradation. Given that NF-␬B signaling
can facilitate neuritogenesis, this is another pathway
through which TG2 could facilitate neurite outgrowth
(345).
TG2 signaling is also important in cancer. Elevated TG2
expression is associated with acquisition of TRAIL resistance in lung cancer cells and increased MMP-9 expression,
while TG2-siRNA reduces MMP-9 expression and restores
TRAIL sensitivity (214). TG2 knockdown also reduces cell
attachment, migration and invasion, and secretion of
MMP-9 and MMP-1 in A431-III cells, an invasive cancer
cell line (59, 215). These studies indicated that increased
TG2 expression increases MMP-9 expression, perhaps by
activating NF-␬B. This response is not uniformly observed,
as other studies report that TG2 expression suppresses
MMP-9 promoter activity and reduces MMP-9 level. This
was associated with reduced jun/fos recruitment to AP1
transcription factor binding elements in the MMP-9 promoter (5). The reason for the different outcomes of these
studies is not known.
The studies above focused on modulation of transcription
by cytosolic TG2. However, nuclear TG2 also regulates
transcription. Although the levels of TG2 in the nucleus are
significantly lower than in the cytoplasm, the presence of
nuclear TG2 is well-documented (67, 106, 126, 209, 320).
The mechanism whereby TG2 translocates to the nucleus is
not known. TG2 has two putative nuclear localization signals (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 transamidating function of TG2 which complicates interpretation.
However, mutation of the putative NLS site at amino acids
597– 602 does not prevent nuclear localization of TG2
(236). It has also been suggested that TG2 contains a putative nuclear export signal (LHMGLHKL) at the COOH
terminus (amino acids 657– 664) (200). A preliminary
study showed that deleting part of the ␤-barrel domain of
TG2 (amino acids 591– 687) resulted in TG2 accumulation
in the nucleus, suggesting the presence of a nuclear export
signal (200). Although these observations are intriguing,
additional studies are needed to better understand this regulation. TG2 nuclear accumulation is also stimulus dependent. Treatment of A375-S2 human melanocytic cells with
sphingosine increases nuclear TG2 (334), and in astrocytes,
growth factors cause nuclear TG2 accumulation (45). Increased nuclear TG2 is also observed in differentiating NB4
cells (22). Hypoxic stress is a critically important signal that
increases nuclear TG2 in neurons (106), and nuclear TG2
accumulation occurs in neurons in mice after stroke and in
stroke patients in the areas of infarction (107). Moreover,
TG2 suppresses hypoxia-induced HIF-1␣ signaling in SHSY5Y cells (106) and primary rat neurons (Gundemir and
Johnson, unpublished data), and the suppression does not
require TG2 transamidation activity (125, 126). Furthermore, both wild-type and transamidation-inactive TG2
forms attenuate ischemic cell death in both SH-SY5Y cells
and primary neurons (106). After stroke, infarcts in mice
overexpressing human TG2 selectively in neurons are much
smaller than those in wild-type mice, and stroke-induced
proapoptotic gene expression is attenuated (107). Thus, in
neurons and neuronal cell models, hypoxia causes accumulation 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
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ECKERT ET AL.
complex with the p65/RelA subunit of NF-␬B and binding
of this complex to the HIF-1␣ promoter. This binding results in increased transcription and accumulation of HIF-1␣
protein, even under normal oxygen conditions. The increased HIF-1, in turn, stimulates transcription of genes
encoding glycolytic proteins including GLUT-1, hexokinase
II, and lactate dehydrogenase-A. The net impact is increased
glucose uptake, lactate production, and reduced mitochondrial respiration (Kumar and Mehta, unpublished data).
There are also studies suggesting that TG2 is associated
with neurodegeneration (13, 204, 385). TG2 expression
and activity are increased in patients with neurodegenerative disorders including Alzheimer’s (163, 185), Huntington’s (179, 211), and Parkinson’s diseases (13). A recent
study suggests that TG2 regulates transcription in Huntington’s disease. PGC-1␣ and cyt c mRNA levels are lower in
mutant huntingtin-expressing striatal cells compared with
wild-type cells (236), and treatment of the mutant huntingtin-expressing cells with a TG2 inhibitor or TG2 knockdown restores PGC-1␣ and cyt c mRNA level. Additionally,
TG2-knockout mouse fibroblasts contain higher levels of
cyt c than wild-type mouse fibroblasts. Furthermore, in the
presence of mutant huntingtin, the interaction of TG2 with
PGC-1␣ and cyt c promoters is increased, resulting in suppression of transcription. In these experiments, TG2
transamidation activity is necessary for suppression of promoter activity. Considering the fact that TG2 expression is
increased in Huntington disease patient brains (211), suppression of PGC-1␣ and cyt c transcription by TG2 in mutant huntingtin expressing cells may contribute to disease
progression.
TG2 accumulates in the nucleus in ethanol exposed rat
hepatocytes (57). Gene expression analysis reveals that ethanol treatment reduces c-Met expression to a greater extent
in TG2 wild-type hepatocytes compared with TG2 knockout mouse hepatocytes. c-Met is the receptor for hepatocyte
growth factor and is involved in liver regeneration (144).
This is associated with TG2 crosslinking of Sp1 which is
required for c-Met expression (339). This is consistent with
patient data, as TG2-mediated Sp1 crosslinking is observed
in liver from patients with alcohol-related liver disease
(384). In addition to increasing the expression of TG2,
treatment with ethanol appears to increase TG2 transamidation activity (339, 388). These findings indicate that nuclear TG2 can modulate gene transcription in response to
ethanol. However, is it not known whether this effect requires TG2 transamidation activity.
VII. PERSPECTIVES AND FUTURE
DIRECTIONS
Despite a growing understanding of the biological functions
of TGs and their mechanism of action, many questions
remain unanswered in a host of cell types. Several areas that
406
would benefit from further study are outlined below. Some
of these areas are highly relevant to disease. For example,
while the role of transglutaminases in differentiation is established, efforts are only beginning to address the role of
these enzymes in the stem cell niche and the role of TGs in
stem and progenitor cells. This area has important implications for tissue renewal and cancer cell survival. Diabetes is
also an important area that would benefit from additional
studies. Knowledge is limited regarding the role of TG2 in
diabetes and in adipocyte differentiation. In this context,
FXIIIa was recently identified as a top obesity gene in a
genome-wide screen for single nucleotide polymorphism
linked to basal metabolic index. The significant association
of FXIIIa with obesity was further confirmed in a large
European ENGAGE consortium study of more than 21,000
unrelated individuals as well as in the GenMets cohort
study (261a). The interplay among various TG forms is
another area that is important in the context of disease. The
compensatory response following loss of TG2 in specific
tissues requires more detailed analysis. There is now substantial evidence for compensation by other TG forms, but
more needs to be done. This analysis will advance our understanding of the biological functions of TG-mediated
crosslinking in different tissues and may also offer focused
approaches to target TG in various diseases. The area of
inhibitors of transglutaminase is also an area for additional
investment, as it would be extremely useful to have compounds that specifically inhibit each of the TG forms.
Understanding the role of various TGs in tissues that express multiple TG enzymes is also an important arena. An
example is monocyte differentiation. TG2 and FXIIIa partially translocate to the nucleus in differentiating monocytes
(22, 352), and both enzymes affect expression of a large
number of genes (72, 352). Identification of TG2- and
FXIIIa-responsive genes may be informative regarding the
role of these proteins in monocyte differentiation. This is
also an issue in many other tissues. New knowledge would
also be useful regarding the role of transglutaminases in
other immune cells. TGs may indirectly regulate inflammatory responses via activation of macrophages, natural killer
cells, 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 is
interesting that elevated expression of TG2 confers drug
resistance. However, we have a long way to go before we
understand the mechanism. A systematic study of a large
cohort of patients should be performed to determine
whether TG2 is a valid prognostic marker. Similarly, TG2induced activation of NF-␬B and accumulation of HIF-1␣
represent interesting areas for future research. Constitutive
activation of NF-␬B is linked with etiology of various
chronic inflammatory conditions, including cancer. Understanding of process whereby TG2-regulates NF-␬B activa-
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TRANSGLUTAMINASES IN CELL FUNCTION
tion may yield ways to block this pathway and mitigate
disease. Similarly, inhibition of TG2-mediated increase in
HIF-1␣ accumulation may be relevant in cancer therapy, as
HIF-1␣ activation is a major pathway whereby cancer cells
influence glucose metabolism and aerobic glycolysis (88).
3. Aeschlimann D, Thomazy V. Protein crosslinking in assembly and remodelling of
extracellular matrices: the role of transglutaminases. Connect Tissue Res 41: 1–27,
2000.
A remarkable feature of these studies is that no more than
two decades ago transglutaminases were thought to function solely as enzymes that covalently crosslinked proteins
to assemble barriers. The scope of this review points to the
vast amount of new knowledge that has accumulated in the
past two decades which point to a pivotal role for these
proteins in regulating cell homeostasis.
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transglutaminase-induced down-regulation of matrix metalloproteinase-9. Biochem
Biophys Res Commun 376: 743–747, 2008.
ACKNOWLEDGMENTS
We thank Kathleen Reinecke for persistent, patient, and
expert assistance in the preparation of this manuscript. We
also thank Donald R. Norwood for critical reading of and
editorial help with this review and Dr. Soner Gundemir for
the helpful discussion. We also thank Drs. Candace Kerr
and Ellen Rorke for critically reading the manuscript.
Present e-mail address of K. Mehta: kapilmehta@icloud.
com.
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: reckert@umaryland.
edu).
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14. Antonyak MA, Li B, Boroughs LK, Johnson JL, Druso JE, Bryant KL, Holowka DA,
Cerione RA. Cancer cell-derived microvesicles induce transformation by transferring
tissue transglutaminase and fibronectin to recipient cells. Proc Natl Acad Sci USA 108:
4852– 4857, 2011.
GRANTS
This work was supported by National Institutes of Health
Grants AR053851 and CA131974 (to R. L. Eckert);
AR057126, HL093305, and DK071920 (to M. Nurminskaya); and NS0065825 (to G. Johnson); a grant from the
Bayer Healthcare System (Grants4Targets) and SK Agarwal
donor funds (to K. Mehta); Canadian Institutes of Health
Research Grants MOP-119103, NHG-107768, and IMH89827 (to M. T. Kaartinen); and a grant from American
Heart Association (to A. M. Belkin).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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