Transglutaminase 2 has opposing roles in the regulation of cellular functions as well as cell growth and death

Transglutaminase 2 (TG2) is primarily known as the most ubiquitously expressed member of the transglutaminase family with Ca2+-dependent protein crosslinking activity; however, this enzyme exhibits multiple additional functions through GTPase, cell adhesion, protein disulfide isomerase, kinase, and scaffold activities and is associated with cell growth, differentiation, and apoptosis. TG2 is found in the extracellular matrix, plasma membrane, cytosol, mitochondria, recycling endosomes, and nucleus, and its subcellular localization is an important determinant of its function. Depending upon the cell type and stimuli, TG2 changes its subcellular localization and biological activities, playing both anti- and pro-apoptotic roles. Increasing evidence indicates that the GTP-bound form of the enzyme (in its closed form) protects cells from apoptosis but that the transamidation activity of TG2 (in its open form) participates in both facilitating and inhibiting apoptosis. A difficulty in the study and understanding of this enigmatic protein is that opposing effects have been reported regarding its roles in the same physiological and/or pathological systems. These include neuroprotective or neurodegenerative effects, hepatic cell growth-promoting or hepatic cell death-inducing effects, exacerbating or having no effect on liver fibrosis, and anti- and pro-apoptotic effects on cancer cells. The reasons for these discrepancies have been ascribed to TG2's multifunctional activities, genetic variants, conformational changes induced by the immediate environment, and differences in the genetic background of the mice used in each of the experiments. In this article, we first report that TG2 has opposing roles like the protagonist in the novel Dr. Jekyll and Mr. Hyde, followed by a summary of the controversies reported, and finally discuss the possible reasons for these discrepancies.


Facts
Tissue transglutaminase (TG2) is a multifunctional enzyme that exhibits crosslinking, GTPase, cell adhesion, protein disulfide isomerase, kinase, and scaffold activities. By virtue of these multiple activities, TG2 is implicated in the regulation of cell growth, differentiation, and apoptosis. However, opposing effects have been reported regarding its roles even in the same physiological and pathological systems. To help understand these discrepancies, we summarize and discuss possible reasons for opposing effects in each case.

Open Questions
How can we measure the individual activity of multiple functions of TG2 in vivo? How are these multiple functions and genetic variants of TG2 regulated?
How is TG2's substrate specificity (other than its sublocalization) determined? Transglutaminase 2 (TG2) is a multifunctional enzyme and the most ubiquitously expressed member of the large TG family, a protein family of eight isozymes designated as blood coagulation factor XIII and TG1-7. TGs primarily deamidate γ-carboxamide groups of specific protein-bound glutamines while exchanging any primary amines, the ε-amino group of a lysine residue, or water, and form ammonia plus an N ɛ (γ-glutamyl)lysine crosslinking between glutamine and lysine residues or convert glutamine to glutamic acid. [1][2][3] TG2 exerts additional enzymatic activities that do not require Ca 2+ ; 1,3 that is, hydrolyzing ATP and GTP to mediate signal transduction through G-protein-coupled receptors, 4,5 protein disulfide isomerase and protein kinase, 6,7 interacting with several proteins as an adhesion or scaffold protein 8 (Figure 1).
TG2 is predominantly found in the cytosol. TG2 also appears in the nucleus, mitochondria, plasma membrane, and extracellular matrix (ECM). 9 In mammals, TG2 is widely present throughout the body including blood, extracellular spaces, and intracellular compartments of nearly all tissues, and induces tissue remodeling/wound healing and ECM assembly as well as cell growth, differentiation, and cell death. 10 TG2 is thus involved in the pathogenesis/treatment of cancer, diabetes, neurodegeneration, fibrosis, inflammatory, and autoimmune disorders 2 as well as liver diseases. [11][12][13] For the general functions and structure of TG2, including topics on genetically engineered mouse models and inherited disorders, refer to wellwritten review articles and/or a book recently released. 2,9,14 Opposing effects have been reported for the roles of TG2 in the same physiological and/or pathological systems due to differences in (1) The enzymatic and non-enzymatic activities or properties of TG2, that is, GTPase versus transamidation versus scaffold activities. 2,15,16 (2) Genetic variants including alternative splicing, that is, the short or truncated form (TG2-S) versus the long form (TG2-L) 17 and single-nucleotide polymorphisms. 18 (3) The conformational structure (open versus closed forms), which is affected by immediate cellular and tissue environments. 2,15 (4) The genetic background of mice. 2,13 The aim of this review is to summarize recently obtained knowledge of how TG2 plays Dr. Jekyll and Mr. Hyde (opposing roles) in the regulation of cell growth and death.

Multifunctional Activities of TG2 and Its Regulation
GTPase. Intracellular TG2 in its closed form acts as a Ca 2+ -independent GTPase in normal cells when the intracellular Ca 2+ concentration is as low as 10-20 nM, participating Figure 1 Diverse functions of TG2. TG2 regulates the post-translational modification of several proteins though several activities, including transamidation, GTPase, adhesion/scaffold, kinase, disulfide isomerase, and isopeptidase activities. Some functions of TG2 remain unclear. TG2 exerts different activities depending on the stimuli; these activities lead to several effects, including apoptosis, cell growth, and differentiation in the transmembrane signaling of phospholipase Cδ as a component of α 1 -adrenergic receptor complexes and supporting the growth of hepatic cells. 16,19,20 GTP binding with TG2 sustains the closed form and prevents its conformational change to the open form; the emergence of crosslinking activity is thus a result of binding to Ca 2+ , whereas the depletion of GTP causes conformational changes in the open form. In this scenario, the emergence of crosslinking activity induces apoptosis in islet β-cells 21 and other cell types. 22 Transamidase. When cells are injured (or receive certain stimuli) and the intracellular Ca 2+ concentration increases to more than 700-800 nM, TG2 markedly alters its structure and transforms into an open form that exerts crosslinking activity. 16,23 In the specific conditions such as tissue regeneration and remodeling, TG2 crosskinks ECM proteins, however, despite a high extracellular Ca 2+ concentration (in the mM range) outside of cells, TG2 usually does not exert transamidation but instead mediates multiple and complex regulatory interactions, particularly in the regulation of cell-ECM interactions and outside-in signaling via transmembrane receptors, indicating that Ca 2+ and/or GTP concentrations are not the sole determinants of transamidation activity.
Integrin-fibronectin interaction. TG2 has been identified as an important extracellular crosslinking enzyme involved in ECM turnover. [24][25][26][27] In contrast, TG2 is secreted into ECM and forms a hetero complex with its high-affinity binding partner fibronectin (FN) through its N-terminal 42-kD fragment in a crosslinking activity-independent manner. 28,29 The TG-FN complex promotes FN fibril deposition and RGDindependent cell adhesion via syndecan-4/2 and α5β1 integrin co-signaling, 29 and sustains cell survival in osteoblasts, 30 bone marrow-derived mesenchymal stem cells, 31 and many tumor cells. 32,33 Miscellaneous functions. Protein disulfide isomerase (PDI) activity 6 has been implicated in mitochondrial-dependent apoptosis, 34 whereas TG2 has been reported to exert an intrinsic serine/threonine kinase activity and to phosphorylate insulin-like growth factor (IGF)-binding protein-3 (IGFBP3), 7 p53 tumor suppressor protein, 35 H1-4 histones, 36 and Rb protein. 37 Moreover, TG2 affects the hypusination of eukaryotic initiation factor 5A (eIF5a) in BALB/c 3T3 cells; 38 this action may be related to the fact that eIF5a serves as a binding protein and/or substrate for TG2. 39,40 Regulation. The gene expression related to TG2 is modulated by endoplasmic reticulum (ER) stress, 11,41 tissue remodeling, 42 inflammation, 2,43 viral infection, 44 and apoptotic signals, 45,46 as well as cancers 2,47-49 and mediated by soluble factors such as transforming growth factor (TGF)-β/ bone morphogenetic protein 4, 50 interleukin (IL)-1, 51 IL-6, 52 tumor necrosis factor (TNF)-α, 53 and epidermal growth factor (EGF) 47 (Table 1). Retinoic acid (RA) induces TG2 expression; 54,55 in acute promyelocytic leukemia treated with RA, TG2 has an important role in the neutrophil-granulocyte differentiation and gene expression of neutrophil's cellular functions in addition to related adhesive, migratory, and phagocytic capacities. 56 The transcriptional activation of the TG2 gene, TGM2, is mediated by nuclear factor-kappa B (NF-κB), 41 RA receptor (RAR)/retinoid X receptor, liver X receptor/RAR, and Sp1. 55 Two examples of a positive feedback loop are known to act in the regulation of TG2 expression. TG2 is indispensable for latent TGF-β activation in many tissues, [57][58][59][60][61][62][63] and the generation of TGF-β may stimulate TG2 gene expression. TG2 activates NF-κB via the depletion of inhibitor of κB (I-κB)α via polymerization in the absence of I-κBα kinase activation. This activity results in the dissociation of NF-κB and its translocation to the nucleus, where it may upregulate TG2 in cancer cells. [64][65][66] Ca 2+ and nucleotides (GTP/ATP), respectively, act as the activator and suppressors of the transamidase activity of TG2. Nitric oxide (NO) is also a potent inhibitor by promoting S-nitrosylation on the active site C277. 67 The decreased S-nitrosylation of TG2 contributes to age-related increases in vascular stiffness. 68 Micro-RNA (miR-19) directly downregulates TG2 expression and enhances the invasion of colorectal cancer cells. 18 Finally, the SUMOylation of TG2 enhances its

Opposing Roles of TG2 In Vitro
The accumulation of TG2 in various cell and tissue types undergoing apoptosis has been demonstrated; 70,71 elevated TG2 activity is correlated with enhanced apoptosis in a neuroblastoma cell line, 72 whereas treatment with antisense against TG2 reduces apoptosis. 73 When neuroblastoma cells are transfected with plasmid overexpressing TG2 but not mutated TG2 lacking crosslinking activity and then treated with staurosporine or osmotic stress, an increased crosslinking activity accompanies the activation of caspase-3 and apoptotic nuclear changes. 74 In mitochondria, interaction with Bax through TG2's BH3 domain plus the predicted crosslinking of Bax causes mitochondrial depolarization, the release of cytochrome c, and cell death. 45 Nuclear TG2 causes caspaseindependent cell death via the crosslinking and inactivation of the general transcription factor Sp1, resulting in the reduced expression of growth factor receptors, such as c-Met and EGF receptors, that are essential for cell survival. 12,75 In contrast, numerous reports have found a protective effect of TG2 in cell death induced by TNF-α, 76 RA, 77 and stressors such as thapsigargin, hyperosmotic stress, and oxygen/glucose deprivation. 78 Below are examples in which TG2 has Dr. Jekyll and Mr. Hyde (opposing functions; Tables 2,3,4,5,6).

Neuroprotective Versus Neurodegenerative Roles by Modulating HIF1 and Huntingtin Functions, Respectively
Increased activity and expression of TG2 are observed in the ischemic hippocampus after reperfusion (Table 2). 79,80 TG2 expression is induced by oxidative stress, and the induction of inducible NO synthase and NO production, which contribute to neurodegeneration, are closely associated with TG2 expression in the lipopolysaccharide-stimulated activation of astrocytes. 81 The infarction volume was smaller in both TG2 knockout mice and mice treated with cystamine than in control mice. 82 In contrast, both SH-SY5Y human neuroblastoma cells 83 and a permanent middle cerebral artery ligation stroke model 84 demonstrated that hypoxic conditions increased nuclear TG2, TG2 binding to HIF1β independently of transamidase activity, and the prevention of the upregulation of proapoptotic Bnip3 85 and Noxa, 86 thereby protecting neuronal cells from hypoxia-induced death in ischemia and stroke. 83,84 Increased TG2 levels and/or activity, especially the nvolvement of nuclear TG2, have been observed in many neurodegenerative diseases such as Alzheimer's disease (AD), Huntington's disease (HD), and Parkinson's disease (PD). 87 AD is characterized by the formation of extracellular neurotoxic aggregates consisting of amyloid-β protein or intracellular neurotoxic aggregates consisting of hyperphosphorylated tau. TG2 mediates the crosslinking of both amyloid-β and tau in vitro 88,89 and the polyamination of the tau protein, resulting in an increased resistance of tau to proteolytic degradation by calpain. This change leads to higher levels of non-degradable tau within the neuron, 90 which indicates that TG2 may accelerate the aggregation process of amyloid-β and tau in AD patients. Furthermore, the enhanced expression of a short form of TG2 (sTG2), an alternatively spliced transcript lacking GTPase activity that maintains poor crosslinking activity and thus has a pro-apoptotic property, has been reported in AD patients, 22,91,92 whereas TG2 is not a biochemical marker for AD disease because no colocalization of TG2 with tau or amyloid-β deposits is found in neocortex sections. 93 In the frontal cortex of postmortem HD brain tissues, 99% colocalization is observed between ε-(γ-glutamyl)lysine crosslinks and huntingtin aggregates in the nucleus, 94 indicating an involvement of nuclear TG2 in HD. Furthermore, the TG inhibitors cystamine and monodansylcadaverine partially suppress aggregate formation and apoptosis in cells expressing truncated dentatorubral-pallidoluysian atrophy protein with an expanded polyglutamine stretch. 95 Although, (i) the in vivo ablation of the TG2 gene ameliorates HD symptoms and leads to unaltered or even increased numbers of neuronal intranuclear inclusions, 96,97 which has recently been ascribed at least in part to a loss of TG2's regulatory effect on autophagy; 98 (ii) cystamine inhibits not only TG2 but also caspase-3; 99 (iii) neuronal intranuclear inclusions are likely to be formed at a late stage of aggregation and do not directly affect the progression of pathogenesis; 100 and (iv) the genetic deletion or enzymatic inhibition of TG2 alleviates the degenerative process and improves survival as well as life span, 97,101  isolated polyglutamine forms aggregates through a β-sheetbased nucleation mechanism, indicating a causative role for nuclear TG2 in HD pathogenesis via mechanisms other than the formation of neuronal intranuclear inclusions. 102 Transcription dysregulation and impaired energy homeostasis have important roles. The introduction of ZDON, a peptide inhibitor of TG2, ameliorates HD symptoms. 101,103 The mutant huntingtin binds to and inactivates other polyglutamine-enriched proteins such as transcription factors, including the general transcription factor Sp1 or its coactivator TAFII130, [104][105][106] which may repress the Sp1-dependent transcription of BDNF, 107 dopamine D2 receptor, 104,105 preproenkephalin, 104 the mitochondrial proteins PGC-1α, and cytochrome c. 108,109 These results imply that although nuclear TG2 is not essential for inducing HD, it may be an important factor in exaggerating HD symptoms through the transcriptional dysregulation of these survival factors and key metabolic genes. Defective nuclear actin remodeling causes faster cell death in correlation with disease progression. 110 Cofilin is an actin binding protein that is required for actin treadmilling. 111 The formation of the dynamic cytoskeleton, referred to as 'actin-cofilin rods', is a self-protecting reaction against cellular stress and is sustained by an association with normal huntingtin upon its release from the ER and localization to the nucleus under stress conditions. In response to cellular stress, delayed and aberrant actin-cofilin rods are formed in neurodegenerative disease by (i) mutant huntingtin via the impairment of the normal function of huntingtin and (ii) an excessive crosslinking of actin-cofilin complexes by stress-activated nuclear TG2. 110 As stated above, TG2 binds Promotive effect CCl 4 -induced liver injury and fibrosis Different animal backgrounds 121 Liver damage and injury in ASH/alcohol, NASH/FFAs 11,12,136,137 Liver damage in NZB/W F1 mice (a well-known lupus-prone strain) 139 No effect CCl 4 -or thioacetamide-induced liver fibrosis 141 Chemoprevention by retinoid Different chemopreventive agents and cell types 75 Growth arrest/apoptosis in RA-treated U937 cells Lost activity of Rb protein 149 Phosphorylation of Rb protein in MCF-7 37 No effect Cell growth/apoptosis in TG2-transfected fibrosarcoma cell Different cell types 146

FN through its N-terminal domain and interacts with many
other scaffold proteins such as integrins and lamins A and C. 28,112,113 In PD patients' brains, TG2 expression is increased in the substantia nigra, and TG2-catalyzed crosslinking has been shown to colocalize with α-synuclein, a substrate for TG2 in vivo, 114 in dementia with Lewy bodies. 115 Hepatic Protective Versus Insult Roles Through Sp1 Crosslinking TG2 crosslinking activity significantly increases with carbon tetrachloride (CCl 4 ) or ethanol-induced liver injury in rats and in acute human liver injury (Table 3). [116][117][118][119][120] Cystamine and garlic extract prevent CCl 4 -induced liver injury and fibrosis via the inhibition of TG2. 121 Sp1 is a general transcription factor that is rich in lysine and glutamine residues, 122 thereby serving as a good substrate for TG2 both in vitro and in vivo. 12 An upregulation of Sp1's transcriptional activation activity upon crosslinking with nuclear TG2 in human 293T cells likely occurred because Sp1 exerts higher transcriptional activation activity as a dimer or trimer, 123,124 whereas in alcohol or free fatty acid (FFA)-treated hepatic cells, highly crosslinked, oligomerized Sp1 loses its transcriptional activation activity. A defect in Sp1 activity causes the decreased expression of c-Met, the major receptor for hepatocyte growth factor, leading to caspase-independent hepatic cell death in culture systems and animal models as well as in patients with both alcoholic steatohepatitis (ASH) and non-ASH (NASH). 11,12,75 FFAs increase ER stress, NF-κB activation, and nuclear TG2 through a pancreatic ER kinase (PERK)-dependent pathway, whereas ethanol-induced nuclear TG2 is dependent at least in part on retinoid signaling. 11 RA enhances the transcription of the TG2 gene via GC box motifs in its promoter region through a physical interaction between newly synthesized RARs and preexisting Sp1. 55 Therefore, the crosslinking and silencing of Sp1 by TG2 may be a feedback mechanism to control the excessive expression of TG2 and prevent it from playing Mr. Hyde. Furthermore, TG2 contributes to the clearance of apoptotic cells by promoting monocyte infiltration via the dimerization of the monocyte chemotactic factor S19 125 followed by macrophage engulfment via integrin β3, 126 leading to inflammation suppression. In addition, TG2 contributes to wound repair and tissue stabilization via the crosslinking of various intracellular and extracellular proteins at inflammation sites. 42 A peptide with anti-TG2 activity decreases lung inflammation accompanied by reduced neutrophil infiltration and cytokines expression. 127 However, TG2 has been reported to be anti-apoptotic, and the effect is linked to both its GTP binding and crosslinking activity. 15,20 TG2-null mice do not exhibit an obvious hepatic phenotype defect but do exhibit an impaired clearance of apoptotic cells by phagocytosis under stress conditions and experience a more severe liver injury after CCl 4 or Fas administration. 20,128 This discrepancy can be explained as follows. Sarang et al. 20 reported that TG2 protects against high-dose (1 μg/g body weight) Jo2 (anti-Fas antibody)induced liver injury; TG2 − / − mice were more sensitive to Jo2-mediated necrosis than TG2 +/+ animals. In those studies, Sarang et al. 20 used FVB mice as wild-type controls. We used wild-type littermates from heterozygous TG2 +/ − crosses and applied a high dose of Jo2 (used by Sarang et al. 20 ); this treatment resulted in massive hepatic necrosis both in TG2 +/+ and TG2 − / − mice. Thus, the varying genetic backgrounds of the TG2 +/+ versus TG2 − / − mice used by Sarang et al. 20 might have contributed to the discrepancy between their findings and ours. However, we reproduced the protective effect of TG2 silencing on hepatic injury with a low dose (0.1 μg/g body weight) of Jo2 using the exact same TG2 − / − mouse line used by Sarang et al. 20 Thus, TG2 may promote the hepatic apoptosis caused by relatively low doses (but not higher doses) of Jo2.
Using the same TG2 − / − mouse line used by Sarang et al., 20 Nardacci et al. 128 reported that TG2 is protective in CCl 4mediated liver injury and speculated that the observed increase in TG2 expression during the initial stages of liver fibrosis in HCV-infected patients may protect against liver injury. However, another explanation may be that high levels of TG2 contribute to liver injury in these patients; as we have shown in an animal model, 12 TG2 inhibitors may be a useful treatment for the prevention of hepatic apoptosis.

Blood vessel Reports Possible causation References
Positive role Formation and stability of blood vessels Promotion of angiogenic process via VEGF signals in endothelium 161,162 Migration and tubule formation of HUVEC TG2 inhibition results in reduced FN deposition, matrix-bound VEGFA, phosphorylation of VEGF receptor 2, and then suppresses migration and tubule formation 30 Endothelial sprouting, migration of both endothelial and vascular mesenchymal cells, and organization of actin cytoskeleton Promotion of the angiogenic process in endothelial and mesenchymal cells 164 Cell spreading and adhesion in the human endothelial cell line ECV304 Adhesion activity with integrin or FN 113,166 Colocalization and binding with endostatin Promotion of angiogenesis via endostatin-TG2 binding 167,168 Negative role Inhibition of angiogenesis and tumor growth via matrix changes through an intratumoral injection of TG2 into mice In injured liver cells, TG2 transforms into a crosslinking enzyme (open form) and contributes to the wound healing process and fibrosis (Table 3). First, the crosslinking reaction results in the formation of an N ɛ (γ-glutamyl)lysine isopeptide bond, which is one important step in the maturation or stabilization of ECMs (such as collagens) in the extracellular space, exacerbating hepatic fibrosis. 117,129 The N ɛ (γ-glutamyl)lysine crosslink, which is undetectable in normal liver tissue, is present extracellularly in the fibrotic livers of patients with a variety of chronic liver diseases, primarily in inflammatory areas in which an intense remodeling is occurring. 117 Second, this crosslinking ability of TG2 appears to have a crucial role in the fixation and activation of TGF-β, 57 the most fibrogenic cytokine. 130 TGF-β1 is released in a latent form (~300 kD) and converted to an active form of 25 kD. Enhanced TG2 activity is required in many tissues for this activation of TGF-β via its crosslinking of large latent complexes to the cell surface or to FN and other ECM components through a latent TGF-β-binding protein portion. 57,61,62,131,132 In addition to the liver, similar fibrogenic roles via the generation of TGF-β have been reported in articular cartilage, 60 kidney, 61 lung, 59 and pancreas. 133 Third, in in vivo models of hepatic apoptosis and in ASH patients, Sp1 is crosslinked, oligomerized, and inactivated by nuclear TG2, leading to the activation of a caspaseindependent apoptotic process resulting from the reduced expression of c-Met. 12 The TG2-induced decrease in c-Met may be involved in the impaired hepatocyte regeneration observed in patients with alcoholic liver diseases. 13,134,135 Furthermore, Giebeler et al. 136 reported that the downregulation of c-Met is associated with liver fibrosis, indicating a novel apoptotic axis accompanied by liver fibrosis, namely, a nuclear TG2/crosslinked Sp1/decline in c-Met. Supporting this hypothesis, the nuclear accumulation of TG2 and crosslinking of Sp1 are observed in the fibrotic area of patients with ASH. 137 In summary, increased TG2 activity is associated with ECM production and TGF-β, as shown after chronic CCl 4 intoxication in rats 118 directly by stabilizing the ECM in an insoluble form and indirectly by promoting the generation of active TGF-β, which strongly enhances hepatic fibrogenesis by increasing ECM production. The reaction is Ca 2+ dependent and is classically reported to be the biochemical basis for TG2 involvement in hepatic fibrosis. 129 Therefore, amine substrates, such as putrescine and cystamine (competitive inhibitors of the crosslinking activity of the enzyme), have been shown to be protective in ethanol-induced liver injury as well as liver fibrosis induced by CCl 4 . [138][139][140] In contrast, Popov et al. 141 demonstrated no change in the extent of liver fibrosis after the treatment of TG2 − / − mice with CCl 4 or thioacetamide. Again, potential explanations for this discrepancy include differences in mouse background, the method used for TG2 gene targeting, and broad biological and non-specific activities of TG inhibitors.

Anti-Versus Pro-Apoptotic Roles of TG2 in Cancer Cells Partially Through the Regulation of Rb and E2F1 Activities
Multiple studies have shown elevated TG2 expression in many types of cancer cells 9 to be associated with increased drug resistance, metastasis, and poor patient survival (Table 4). 33,142 For example, an analysis of more than 30,000 genes from tumor samples revealed that TG2 is a highly expressed gene in pancreatic adenocarcinoma. 143 An important property of the highly malignant tumor cells is their ability to survive in hostile host environments as they passes through the lymphatic system or the bloodstream in their attempt to colonize distant sites. TG2 stabilized contact points of tumor cells with the subendothelial matrix in free-floating melanoma cells isolated from arterioles. 144 In contrast, the downregulation of TG2 expression in melanoma cancer cells promoted their ability to metastasize. 145 The ectopic expression of TG2 in a highly malignant hamster fibrosarcoma cell line significantly reduces tumor incidence despite the fact that TG2-transfected clones exhibit no significant differences in growth rates, cell morphology, or levels of spontaneous apoptosis in vitro. 146 This finding indicates a suppressive effect of TG2 on tumor growth and confirms the importance of TG2 in the phenotypic changes associated with cancer. Similar nuclear TG2-mediated cell death (as observed in ASH and NASH) has been found in the chemoprevention of cancer. Acyclic retinoid, a synthetic retinoid, stimulates the nuclear localization and activation of TG2, 147 resulting in the crosslinking and inactivation of Sp1, thereby causing cell death in hepatocellular carcinoma cell lines through the downregulation of EGF receptors. 75 In contrast, TG2 enhances EGF receptor expression in glioblastomas and then induces cell transformation. 148 Therefore, a precise understanding of the expression and function of TG2 in the context of cancer stages and types is important for the implementation of TG2-based interventions to disrupt malignant invasion, growth, and survival.
In U937 human leukemic monocyte lymphoma cells undergoing apoptosis, nuclear TG2 polymerizes Rb protein, which culminates in the loss of anti-apoptotic action by Rb protein due to its interaction with E2F1 and the prevention of Rb degradation, which accelerates the degradation of E2F1 and leads to cell growth arrest/apoptosis. 149 In contrast, in some cell lines (NIH3T3 mouse embryonic fibroblast cells, HL-60 human promyelocytic leukemia cells, and mouse embryonic lung fibroblasts) treated with RA or N-(4-hydroxyphenyl) retinamide, TG2 prolongs the anti-apoptotic action of Rb protein, 150 protecting it from degradation by caspase-7 due to transamidation and/or the GTP-binding activities instead of undergoing polymerization. This conclusion has been drawn from the finding that monodansylcadaverine, a transamidation inhibitor, blocks the protective effect, whereas a transamidation-defective [C277V] TG2 mutant also exerts a protective effect. 150 The latter is corroborated in HEK293 human embryonic kidney cells in which nuclear localization of the [C277S] TG2 mutant attenuates apoptosis due to its GTP binding or scaffold protein characteristics upon complexing with Rb protein. 151 Furthermore, the phosphorylation of the Rb protein at Ser780 by TG2 destabilizes the Rb-E2F1 complex, ameliorating apoptosis in MCF-7 human breast carcinoma cells, which is further stimulated by the phosphorylation of TG2 with protein kinase A and abrogated by high Ca 2+ concentrations. 37 These observations indicate that in general, although the outcome differs depending on cell types and treatments, nuclear TG2 heavily polymerizes Rb protein under TG2 has Dr. Jekyll and Mr. Hyde (opposing roles) H Tatsukawa et al extremely high Ca 2+ concentrations, and low ATP concentrations, resulting in apoptosis through accelerating E2F1 degradation; otherwise, nuclear TG2 stabilizes Rb protein through the phosphorylation of Ser780 and the transamidation of certain glutamine residue(s) (but not through crosslinking) and preventing Rb degradation as a scaffold protein. This behavior indicates that intranuclear Ca 2+ and GTP concentrations are important in determining whether TG2 acts as Dr. Jekyll or Mr. Hyde (opposing functions). 16 Diabetes TG2 is involved in the membrane-mediated events required for glucose-stimulated insulin release from pancreatic β cells (Table 5). [152][153][154] Vitamin A also induces insulin secretion from islets via TG2, 155 and steroid hormone-induced TG2 facilitates the crosslinking of IGFBP1 and increases IGF-I actions. 156 As a controversial effect, inhibitors of TG2, such as monodansylcadaverine and glycine methylester, do not prevent insulin secretion induced by either cAMP or phorbol ester at basal levels (10 nM) of Ca 2+ ; however, these inhibitors prevent insulin secretion induced by Ca 2+ . 157 These data indicate that the transamidation activity of TG2 has a critical role in insulin secretion.
TG2 − / − mice have impaired glucose-stimulated insulin secretion and show glucose intolerance after intraperitoneal glucose loading. 158 The TG2 − / − mouse phenotype resembles that of maturity-onset diabetes in young patients who have several missense mutations located near the catalytic site of TG2 and impaired transamidation activity. 158,159 However, TG2-disrupted mice and the constitutive transamidation active form of TG2-expressing mice show no significant differences in responses to a glucose or insulin challenge, suggesting that glucose homeostasis is TG2 independent. 160

Blood Vessel Formation
TG2 has a positive role in the formation and stability of blood vessels (Table 6). 161,162 Coeliac disease-specific autoantibodies targeting TG2 disturb several steps of angiogenesis, including endothelial sprouting, the migration of both endothelial, and vascular mesenchymal cells, ECM degradation, the organization of the actin cytoskeleton in capillary cell types, alterations of cell-ECM interactions to thereby affect endothelial cell adhesion, polarization, and motility. [163][164][165] At the endothelium, TG2 co-localizes with integrin and has an important role in cell spreading and adhesion. 113,166 Moreover, TG2 is co-localized with endostatin in the ECM secreted by endothelial cells under hypoxia, which stimulates angiogenesis and tumorigenesis. 167,168 TG2 inhibition leads to a reduction in FN deposition and matrix-bound VEGFA in HUVECs; TG2 thus positively regulates angiogenesis in a VEGF-dependent manner. 169 Microarray data show that TG2 is specifically upregulated by turbulent shear stress in coronary artery endothelial cells. 170 Given the important role of shear stress in the development of atherosclerosis, we assert that TG2 relates the progression of this disease. In an opposite effect, matrix changes induced by TG2 lead to the inhibition of angiogenesis and tumor growth, 171 indicating that endothelial TG2 primarily promotes blood vessel formation via enhanced stabilization and migration of neovascularization; however, a part of extracellular TG2 reduces organized vasculature via the excess stabilization and accumulation of ECM following their crosslinking. In cultured endothelial cells, TG2 suppressed cell migration via the formation of TGF-β. 57 In vascular smooth muscle cells, TG2 is involved in the all-trans RA-mediated apoptosis and reduction of neointimal mass in balloon-injured blood vessels. 54 More recently, TG2 was shown to block extracellular VEGF's binding to heparan sulfate proteoglycans and inhibit an early stage of angiogenesis. 172 Thus, the effect of TG2 on angiogenesis appears to differ depending on its stage and TG2's subcellular localization.

Conclusions and Prospects
The crosslinking activity of TG2 appears to represent the dominant function of TG2 during apoptotic cell death accompanied by an elevation of intracellular Ca 2+ . In addition to crosslinking activity, at normal intracellular Ca 2+ concentrations, the diverse cellular functions of TG2 appear to be attributed to G h , PDI, and kinase activities. These various biochemical activities of TG2 are differentially regulated depending on its subcellular localization; they also determine whether TG2 has Dr. Jekyll or Mr. Hyde. In the outer membrane, TG2 is released from cells by an unknown mechanism and constitutively exhibits crosslinking activity for ECM and anchoring protein. In the intracellular membrane and cytosol, TG2 typically adopts a closed conformation with the binding of GTP but not that of Ca 2+ , which contributes to intracellular Ca 2+ homeostasis, cell proliferation, and other actions. Both growth-stimulating (anti-apoptotic) and proapoptotic functions of TG2 may depend on the cell type, the type of death stimuli, the intracellular localization of the enzyme and which of its activities are switched on. 15 Thus, the use of an existing inhibitor, such as cystamine, is not effective for the regulation of TG2-related pathogenesis. It is important to develop specific regulatory compounds against crosslinking, GTPase, FN-binding, PDI, the phosphorylation activities of TG2, and its cofactors. To control cell death and survival, we are now establishing and screening inhibitors that regulate the nuclear localization of TG2.

Conflict of Interest
The authors declare no conflict of interest.