|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell Biology International (2010) 34, 325334 (Printed in Great Britain)
Recent advances in understanding the roles of transglutaminase 2 in alcoholic steatohepatitis
Hideki Tatsukawa and Soichi Kojima1
Molecular Ligand Biology Research Team, Chemical Genomics Research Group, Chemical Biology Department, RIKEN Advanced Science Institute, 21 Hirosawa, Wako, Saitama 3510198, Japan
Tissue TG (transglutaminase) or TG2 is the most ubiquitously expressed member of the large TG family that catalyses deamidation of a glutamine residue, formation of an Nε(γ-glutamyl)-lysine cross-linking between lysine and glutamine residues and/or covalent incorporation of polyamines into a glutamine residue, exerting a number of physiological and/or pathological functions. Extracellular TG2 contributes to wound healing and exacerbation of liver fibrosis through a role in extracellular matrix assembly and cell adhesion. Intracellular TG2 acts as a GTPase in normal cells when the intracellular Ca2+ concentration is as low as 10–20 nM, participating in the transmembrane signalling of phospholipase Cδ as a component of α1-adrenergic receptor complexes, and thereby supporting the growth of hepatic cells. When cells are injured and the intracellular Ca2+ concentration rises to more than 700–800 nM, TG2 dramatically alters its structure and transforms into a cross-linking enzyme. TG2 primarily exists in the cytosol in normal cells, but is distributed among multiple intracellular milieus during tissue injury or apoptosis. In particular, TG2 has been shown to be abundant in the nuclei of cells undergoing apoptosis, although its role in the nucleus and the underlying mechanisms remain unresolved. Recently, three findings in the study of alcoholic steatohepatitis have shed light on these issues. Omary’s group disclosed that TG2-mediated cross-linking of keratin 8 is essential for the formation of Mallory–Denk bodies. We have demonstrated that in both mouse models of alcoholic steatohepatitis and human patients with alcoholic steatohepatitis, TG2 translocates into the nucleus and provokes hepatocyte death via cross-linking and inactivation of a transcription factor, Sp1, leading to down-regulation of the hepatocyte growth factor receptor, c-Met. Furthermore, Giebeler et al. has reported that down-regulation of c-Met is associated with liver fibrosis. In the present review article, we introduce these recent advances in knowledge with regard to the the roles of TG2 in alcoholic steatohepatitis.
Key words: alcoholic steatohepatitis, apoptosis, c-Met, fibrosis, Sp1, transglutaminase
Abbreviations: ECM, extracellular matrix, HCV, hepatitis C virus, HGF, hepatocyte growth factor, IL, interleukin, MMP-9, matrix metalloproteinase 9, NASH, non-alcoholic steatohepatitis, NF-κB, nuclear factor κB, PLCδ1, phospholipase Cδ1, TG, transglutaminase, sTG2, short form of TG2, TGF-β, transforming growth factor-β, TNF-α, tumour necrosis factor-α
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Ethanol consumption represents a major global health burden, accounting for approx. 2 million deaths annually (World Health Organization, Global status report on alcohol 2004; http://www.who.int/substance_abuse/publications/global_status_report_2004_overview.pdf). Ethanol-induced liver injury follows a typical progression from its earliest stage of steatosis to more advanced injury, characterized by the development of hepatocyte apoptosis/necrosis, inflammation, fibrosis and finally cirrhosis accompanying the production of ROS (reactive oxygen species), and alteration of multiple signalling pathways (McVicker et al., 2007; Tsukamoto, 2007; Strnad and Omary, 2009). Much progress has been made over the past decade in the understanding of ethanol metabolism, the various enzymes and pathways involved, and of how ethanol, directly via its metabolism or indirectly via its solvent-like action affecting membranes, impacts on cellular functions. However, we still lack a complete understanding of the mechanisms by which alcohol causes cell injury and cell death.
TG (transglutaminase) was first described by Heinrich Waelsch more than 50 years ago as a liver enzyme incorporating amines into proteins (Sarkar et al., 1957; Lorand, 2002). TGs are now known to constitute a large family of enzymes working as ‘natural biological glues’. They catalyse a Ca2+-dependent acryl transfer reaction between the γ-carboxamide group of a polypeptide-bound glutamine residue and a hydroxy group in water, or primary amines in either a polypeptide-bound lysine residue or a polyamine. Such reactions result in the deamidation of glutamine residues in proteins to form glutamic acid, or the formation of intra- or inter-cross-linkings among proteins via Nε(γ-glutamyl)-lysine isopeptide bonds, or incorporation of polyamines into polypeptide-bound glutamine residues via (γ-glutamyl)-polyamine bonds (Figure 1A). TG2 catalyses a two-step double-displacement reaction (Figure 1A). TGs have been identified in organisms from immature micro-organisms such as Physarum polycephalum, an acellular slime mold, to humans (Wada et al., 2002; Figure 1B). In mammals, TGs are widely distributed throughout the body including the blood, extracellular spaces and intracellular compartments, and are involved in numerous biochemical processes such as blood coagulation, skin-barrier formation, hardening of the fertilization envelope, tissue remodelling, ECM (extracellular matrix) assembly and cell death or differentiation (Fesus and Piacentini, 2002; Griffin et al., 2002; Lorand and Graham, 2003). As summarized in Table 1, nine TGs have been identified in humans, among which Factor XIIIa and TG1–TG4 are relatively well characterized. Although TGs possess a variety of biological functions that are generally attributed to their protein-modifying activities, their function is in some instances due to specialized non-cross-linking actions, such as scaffolding of the cytoskeleton to maintain membrane integrity, cell adhesion, endocytosis and signal transduction through GTPase and/or kinase activities (Hasegawa et al., 2003; Lorand and Graham, 2003; Mishra et al., 2007).
Tissue TG or TG2 (E.C. 188.8.131.52) is a multifunctional protein originally characterized as the most ubiquitous member of the TG family. It is expressed throughout the whole body, and not only displays cross-linking activity, but also functions as a GTPase (Fesus and Piacentini, 2002; Griffin et al., 2002; Lorand and Graham, 2003). Its cross-linking activity accounts for approx. 80% of the total TG activity in normal mouse liver (Strnad et al., 2007). TG2 has been implicated in a number of pathophysiological processes such as hepatic injury (Wu and Zern, 2004; Tatsukawa et al., 2009), degenerative disorders of neurons (Lesort et al., 2000), myocardial hypertrophy (Wettschureck et al., 2001) and fibrosis (Mirza et al., 1997; Grenard et al., 2001). Analyses of in vitro models overexpressing TG2 and animal-knockout models, as well as structural analyses of TG2 protein, have helped to delineate the molecular basis of its biochemical properties and have greatly facilitated our understanding of the roles of TG2 in a variety of pathological processes.
With regard to hepatic diseases, it is well known that extracellular TG2 contributes to wound healing and exacerbation of liver fibrosis by virtue of its role in ECM assembly and cell adhesion (Grenard et al., 2001; Wu and Zern, 2004). It is also well established that TG2 catalyses the deamidation of gliadin A (a component of wheat and other cereals), with a preference for Gln-X-Pro sequences (where X is any amino acid), which are the dominant epitopes for activating T-cells in coeliac disease (Shan et al., 2002). The intracellular functions of TG2 differ with the intracellular Ca2+ concentration, e.g. mainly acting either as a GTPase in normal hepatic cells, supporting their growth (Nakaoka et al., 1994; Wu et al., 2000; Lorand and Graham, 2003), or as a cross-linking enzyme, suppressing hepatic cell growth and inducing apoptosis (Wu et al., 2000; Begg et al., 2006). Furthermore, changes in the intracellular translocation of TG2 from the cytosol, where the enzyme is primarily located in normal cells, to various compartments including the plasma membrane, nucleus and extracellular space, are observed in injured or apoptotic cells (Lorand and Graham, 2003; Fesus and Szondy, 2005). In particular, the nuclear abundance of TG2 has been implicated in apoptosis, although the underlying mechanisms remain unresolved (Lorand and Graham, 2003; Fesus and Szondy, 2005). Recently, three findings in the study of alcoholic steatohepatitis have elucidated these mechanisms (Strnad and Omary, 2009): (i) a role of TG2 in the formation of Mallory–Denk bodies (Strnad et al., 2007); (ii) nuclear TG2 cross-linking, inactivation of Sp1 and down-regulation in c-Met, a novel apoptotic axis (Tatsukawa et al., 2009); and (iii) induction of liver fibrosis by down-regulation of c-Met (Giebeler et al., 2009). Below, we will introduce details of these topics to deepen our understanding of the roles of TG2 in alcohol-induced hepatic injury and fibrosis.
2. Structural switching of biochemical functions of TG2 whose expression is enhanced in liver-composing cells in the injured liver
Human TG2 is composed of 687 amino acid residues and has an approx. molecular mass of 76 kDa. The three-dimensional structure of TG2, crystallized in a dimer form in complex with GDP, has been reported (Liu et al., 2002), providing clues to understanding how its enzymatic transamidation (cross-linking) activity is regulated. TG2 has four distinct domains (Figure 1C), like another TG, Factor XIIIa: (i) an N-terminal β-sandwich domain consisting of residues Met1 to Phe139 (with fibronectin- and integrin-binding sites); (ii) a catalytic core domain consisting of Ala147 to Asn460; and (iii) two C-terminal β-barrel domains, which include Gly472 to Tyr583 and Ile591 to Ala687 [the second contains a PLCδ1 (phospholipase Cδ1)-binding sequence (Leu665 to Lys672)]. The catalytic core domain contains the catalytic triad for the acyl-transfer reaction, Cys277–His335–Asp358, conserved in all members of the TG family; Cys277, located in the middle of a groove within the catalytic domain, is the essential nucleophile for transamidation, and a conserved Trp241 is also essential for this catalytic activity (Murthy et al., 2002). The guanine nucleotide binds mainly to residues from the first and last strands of β-barrel 1 (Arg476–Ser482 and Arg580–Tyr583) and to two residues in the core domain (Lys173 and Phe174) that protrude on a loop to meet β-barrel 1 (Iismaa et al., 2000; Liu et al., 2002; Begg et al., 2006). When the intracellular Ca2+ concentration is as low as 10–20 nM, TG2 exists as the GDP-bound form, in which access to the transamidation active site is blocked by two loops, and the active site Cys277 is hydrogen-bonded to a tyrosine residue (Liu et al., 2002). Ca2+ exerts an activating signal for transamidation. As shown in Figure 1(D), when cells are injured and the intracellular Ca2+ concentration rises to more than 700–800 nM, the structure of TG2 dramatically alters and it transforms into a cross-linking enzyme (Begg et al., 2006). The putative Ca2+-binding site on TG2 is located near the end of the loop that connects the catalytic domain to the first β-barrel domain (Liu et al., 2002).
In the liver, both parenchymal and non-parenchymal cells, such as hepatic stellate cells, as well as sinusoidal endothelial and Kupffer cells, produce TG2, which appears to be released into the extracellular space through an unidentified mechanism(s) in which Tyr274 is important (Balklava et al., 2002; Tóth et al., 2009). TG2 expression is up-regulated when liver tissues or cells encounter an injurious process (Mirza et al., 1997). As summarized in Table 2, TG2 activity is also up-regulated in a tissue-specific manner by transcriptional activators such as retinoids (Nara et al., 1989; Nagy et al., 1997; Ou et al., 2000), vitamin D and steroids (Defacque et al., 1995), through retinoid-responsive elements and Sp1-binding sites (GC box) within the TG2 promoter (Shimada et al., 2001). There are also regions for regulation by TGF-β (transforming growth factor-β), ILs (interleukins), bone morphogenic protein 4 (Ritter and Davies, 1998) and NF-κB (nuclear factor κB) (Mirza et al., 1997). In hepatoma cell lines, IL-6 and TNF-α (tumour necrosis factor-α) modulate TG2 expression (Suto et al., 1993; Kuncio et al., 1998). It has been shown that enhancing α1-adrenergic receptor-coupled signalling via the G-protein activity of TG2 results in decreased TG2 cross-linking and enhanced hepatocyte proliferation; activation of TG2 cross-linking either by ethanol treatment or by Fas activation results in increased apoptosis and decreased hepatocyte proliferation (Wu et al., 2000). Hence, the alcohol-induced switch from the GTPase to the cross-linking function of TG2 may play an important role in ethanol-induced liver disease. In cells within an injured liver, especially in apoptotic cells, TG2 expression is stimulated by many soluble factors at the transcriptional level (Esposito and Caputo, 2005), and falling nucleotide levels and increasing Ca2+ levels activate the transamidation activity through structural alterations (Liu et al., 2002; Begg et al., 2006).
Table 2 List of factors reported to regulate TG2 expression
3. Growth-stimulating functions of TG2
As mentioned above, in a normal situation with low Ca2+ concentrations (10–20 nM), TG2 acts as a GTPase and supports hepatic cell proliferation. This unique characteristic of TG2, to bind and hydrolyse GTP with affinity and rates similar to those of other G-proteins, distinguishes it from other TGs, and suggests that, like other G-proteins, it participates in signalling pathways (Nakaoka et al., 1994). Among the studies implicating TG2 as a signal transducer in biological-response pathways, the best-documented describe its role in α1-adrenergic receptor-mediated stimulation of PLCδ1 activity (Feng et al., 1996). It was originally reported that an approx. 70–80 kDa GTP-binding protein (named Gh) was responsible for coupling α1-adrenergic agonists to the stimulation of phosphoinositide lipid metabolism (Baek et al., 1993), and it was subsequently demonstrated that Gh was identical with TG2; it is now called Gαh (Nakaoka et al., 1994). Its GTP-binding and GTPase activities are Ca2+-independent, and its link to downstream signal transduction pathways elicited a new array of research on its roles in intracellular Ca2+ homoeostasis, cell proliferation and other actions (Wu et al., 2000; Wu and Zern, 2004). In this regard, TG2 has been reported to be anti-apoptotic, an effect linked to both its GTP-binding and cross-linking activities (Fesus and Szondy, 2005; Sarang et al., 2005). TG2-null mice exhibited no obvious defect in the hepatic phenotype, but clearance of apoptotic cells by phagocytosis was impaired under stress conditions, and liver injury after carbon tetrachloride (CCl4) administration was more severe (Fesus and Piacentini, 2002; Nardacci et al., 2003).
4. Fibrogenic functions of TG2
In injured liver cells, TG2 transforms to a cross-linking enzyme, as noted above. One major role of TG2 cross-linking activity is its involvement in wound healing. Hepatic fibrosis is a healing process in chronic liver injury (Friedman, 2008). The cross-linking reaction results in the formation of an Nε(γ-glutamyl)-lysine isopeptide bound, which is one important step in the maturation or stabilization of ECM components, such as collagens in the extracellular space, exacerbating hepatic fibrosis (Grenard et al., 2001; Wu and Zern, 2004). The Nε(γ-glutamyl)-lysine cross-link, which is undetectable in normal liver tissue, was present extracellularly in the fibrotic livers of patients with a variety of chronic liver diseases, mostly in inflammatory areas where intense remodelling was occurring (Grenard et al., 2001). In addition, co-localization of osteonectin with Nε(γ-glutamyl)-lysine at the periphery of granulomas and in the ECM of inflammatory zones suggests that this protein was crosslinked by TG2 in fibrotic livers (Grenard et al., 2001). It was also observed that higher TG2 activity appeared at an early stage of HCV (hepatitis C virus) infection, and the enzyme was localized to hepatocytes facing the periportal infiltrate. TG2 was mostly located in ECM components during late stages of hepatic fibrosis after HCV infection (Nardacci et al., 2003).
In addition, this cross-linking ability of TG2 appears to be crucial for the fixation and activation of TGF-β (Kojima et al., 1993), the most fibrogenic cytokine (Friedman, 2008). TGF-β1 is released in a latent form (approx. 300 kDa), and must therefore be activated by removing the latency-associated protein and conversion into an active form of 25 kDa. Plasmin cleaves the latency-associated protein portion of the latent TGF-β molecule, leading to the activation of TGF-β1 (Rifkin, 2005). In many tissues, enhanced TG2 activity, which results from cell damage due to chronic intoxication, e.g. by ethanol or CCl4, is required for this activation of TGF-β via cross-linking of large latent complexes to the cell surface, or to fibronectin or other ECM components through the latent TGF-β binding protein portion (Kojima et al., 1993; Le et al., 2001; Gressner et al., 2002; Szondy et al., 2003; Shweke et al., 2008).
Increased TG2 activity is associated with ECM production and TGF-β, as shown after chronic CCl4 intoxication in rats (Mirza et al., 1997), directly through stabilizing the ECM in an insoluble form and indirectly through promoting the generation of active TGF-β, which strongly enhances hepatic fibrogenesis by increasing ECM production. The reaction is Ca2+-dependent, and reported classically to be the biochemical basis for TG2 involvement in hepatic fibrosis (Wu and Zern, 2004). Therefore amine substrates, such as putrescine and cystamine, competitive inhibitors of the cross-linking activity of the enzyme, have been shown to protect against ethanol-induced liver injury, as well as liver fibrosis induced by CCl4 (Diehl et al., 1990; Shibley et al., 1995; Qiu et al., 2007; Hsu et al., 2008).
5. Implication of TG2 in hepatic injury and cell death
The relationship between increased TG2 cross-linking activity and cell death has been established for nearly a decade. Accumulating evidence suggests a link between enhanced cross-linking by TG2 and neuronal cell death, e.g. enhanced expression of TG2 in Alzheimer’s disease patients (Citron et al., 2001) and enhanced TG activity in Huntington’s disease patients (Dunah et al., 2002; Karpuj et al., 2002; Li et al., 2002; Zhai et al., 2005). It has been demonstrated that TG2 cross-linking activity increases significantly with CCl4 or ethanol-induced liver injury in rats and in acute human liver injury (Mirza et al., 1997; Wu et al., 2000; Grenard et al., 2001; Strnad et al., 2007; Chen et al., 2008); however, its involvement in apoptosis remains incompletely defined and controversial. Generally, in TG2-transfected cells, elevated TG2 activity correlated with enhanced apoptosis in a neuroblastoma cell line (Melino et al., 1994), and treatment with antisense against TG2 reduced this activity (Oliverio et al., 1999). When TG2-overexpressing plasmid-transfected SH-SY5Y cells were treated with staurosporine or osmotic stress, there was activation of caspase 3, apoptotic nuclear changes and an increase in cross-linking activity. These changes did not occur in cells transfected with a control vector, or with a mutated TG2 plasmid lacking crosslinking activity (Tucholski and Johnson, 2002). Enhanced expression of a short form of TG2 (termed sTG2) has been reported in Alzheimer’s patients (Citron et al., 2001). This short form is expressed from an alternatively spliced transcript, lacks GTPase activity, has poor cross-linking activity and is pro-apoptotic. To date, expression of sTG2 has only been reported in human brain (Monsonego et al., 1998; Citron et al., 2001; Antonyak et al., 2006).
Mice with homozygous inactivation of Tgm2, the TG2 gene (TG2−/− mice) (De Laurenzi and Melino, 2001; Nanda et al., 2001), are more susceptible to toxin-induced hepatobiliary injury (Strnad et al., 2007) and CCl4-induced liver injury (Nardacci et al., 2003). On the other hand, elevated TG2 cross-linking activity caused by diminished levels of the TG2 inhibitor putrescine has been implicated in the ethanol-mediated inhibition of hepatocyte regeneration (Diehl et al., 1988, 1990). The implication that TG2 is involved in hepatic injury and cell death was further supported by the finding that ethanol exposure increases TG2 cross-linking activity, while decreasing hepatocyte proliferation (Wands et al., 1979; Diehl et al., 1988; Wu et al., 2000). Although no publication has reported that TG2 aggressively promotes hepatitis formation by stimulating inflammation, TG2 contributes to the clearance of apoptotic cells by promoting monocyte infiltration via dimerization of the monocyte chemotactic factor, S19 (Horino et al., 1998), followed by macrophage engulfment via integrin β3 (Tóth et al., 2009), leading to the suppression of inflammation. On the other hand, it has been reported that TG2 contributes to wound repair and tissue stabilization via cross-linking of various intracellular and extracellular proteins at sites of inflammation (Griffin et al., 2002; Nicholas et al., 2003). However, the mechanism that accounts for the pro-apoptotic function of TG2 at sites of inflammation remained unclear until our recent findings, which are described in detail below.
6. Recent mechanistic insights into the anti- and pro-apoptotic roles of TG2 during the pathogenesis of liver diseases
Recently, we and two other groups have added new insights into a role of TG2 in injured liver tissues and cells, where TG2 acts as a cross-linking enzyme (Figure 2A).
6.1. Anti-apoptotic role of TG2 through formation of Mallory–Denk bodies
Mallory–Denk bodies are among the most prevalent protein deposits in humans and are characteristic of several liver disorders including alcoholic steatohepatitis and NASH (non-alcoholic steatohepatitis) (Jensen and Gluud, 1994; Denk et al., 2000). In addition to the effects on hepatocyte proliferation, TG2-mediated transamidation is essential for the formation of Mallory–Denk bodies via cross-linking of keratin 8 (Strnad et al., 2007, 2008; Zhong et al., 2009). Mallory–Denk bodies seem to be correlated with fibrosis and are thought to prevent cell death by trapping pro-apoptotic components such as keratin 8 into aggregates (Strnad and Omary, 2009).
6.2. Pro-apoptotic role of TG2 through cross-linking and silencing of Sp1 and reduced expression of c-Met
Using TG2+/+ and TG2−/− mice, we found evidence that TG2 has a primary role in hepatic injury-induced hepatocyte apoptosis in vivo, and this is mediated by accumulation of TG2 in the nucleus (Figure 2B; Tatsukawa et al., 2009). To address the role of nuclear TG2 in alcohol-mediated liver injury, we analysed gene expression in ethanol-treated and untreated hepatocytes isolated from TG2+/+ and TG2−/− mice. We found that c-Met is down-regulated in TG2+/+, but not TG2−/−, mice after exposure to ethanol (Tatsukawa et al., 2009). c-Met is a receptor for HGF (hepatocyte growth factor) and, importantly, the HGF–c-Met axis is indispensable for efficient liver regeneration (Huh et al., 2004). Given the involvement of the transcription factor Sp1 in c-Met activation (Liu, 1998) and the fact that Sp1 is a glutamine-rich protein, we demonstrated that the decreased c-Met levels are due to TG2-mediated cross-linking of Sp1 and consequent disruption of its activity (Tatsukawa et al., 2009). The human genome contains >12000 potential Sp1-binding sites. It is still unknown why Sp1 silencing predominantly decreases c-Met expression in ethanol-treated hepatocytes. From microarray data, there was no or only minimal (<2-fold) change in the expression of a number of major apoptotic and anti-apoptotic genes in this TG2-mediated apoptotic pathway, such as those for caspase family members and Bcl proteins, indicating that hepatocyte apoptosis induced by TG2-mediated cross-linking of Sp1 is a novel cell-death pathway basically independent of caspases. This was corroborated by the finding that the caspase inhibitors z-VAD and z-DEVD failed to prevent this apoptosis pathway. Pathway analysis of the genes that were up- or down-regulated by >2.5-fold in a TG2-dependent manner following ethanol treatment suggested highly significant activation (P<1×10−6) of both hepatic cell death and disease pathways, including TG2-dependent inhibition of c-Met expression.
In in vivo models of hepatic apoptosis, and in alcoholic steatohepatitis patients, we demonstrated that Sp1 is cross-linked, oligomerized and inactivated by nuclear TG2, leading to activation of a caspase-independent apoptotic process as a result of reduced expression of critical growth-factor-receptor genes such as c-Met (Tatsukawa et al., 2009). The TG2-induced decrease in c-Met might be involved in the impaired hepatocyte regeneration seen in patients with alcoholic liver disease (Diehl et al., 1988, 1990; Huh et al., 2004).
6.3. A novel mechanism for a pro-fibrogenic role of TG2 through reduced c-Met expression
We also observed nuclear accumulation of TG2 and cross-linking of Sp1 in the fibrotic area of patients with alcoholic steatohepatitis (Taksukawa, Nagatsuma, Matsuura and Kojima, unpublished data). It has been reported that decreased c-Met levels are implicated in the development of ethanol-induced fatty liver (Tomita et al., 2004) and that c-Met-deficient livers express significantly lower amounts of MMP-9 (matrix metalloproteinase 9), which causes decreased degradation of ECM in the liver, promoting increased formation of fibrosis (Ishikawa et al., 2008). Furthermore, Giebeler et al. (2009) recently showed that reduced c-Met levels may predispose to liver fibrosis probably via TNF-α, IL-6 and TGF-β1; and Ahn et al. (2008) showed that TG2 induced down-regulation of MMP-9 via blocking of Jun–Fos-complex binding to an AP-1 site. Also, in rats with acute hepatic failure, c-Met was down-regulated via inactivation of Sp1 (Mizuguchi et al., 2001). Combining these findings with our own strongly suggests that the down-regulation of c-Met observed in injured livers might be linked to fibrogenesis: an increase in the nuclear TG2-silencing Sp1 axis might provoke hepatic apoptosis and fibrosis via reductions in c-Met and MMP-9 levels respectively (Figure 2C).
7. Conclusions and prospects
The unique multifunctional characteristics of TG2 elicit extensive interest in exploring its roles in several processes in liver disease, such as hepatic injury, fibrosis and regeneration. Although some findings with regard to the roles of TG2 in apoptosis are contradictory, data obtained from a variety of settings indicate that TG2 is certainly associated with apoptosis, and may represent an early component of the apoptosis cascade through its transformation (activation) to the cross-linking enzyme and nuclear localization. Using loss- and gain-of-function approaches for TG2 and Sp1 in in vitro and in vivo models, we have shown that TG2 is causative in hepatocyte apoptosis induced by ethanol and Fas. As an underlying molecular mechanism, we show that TG2-mediated impairment of Sp1 and its related c-Met signalling cascade may be a culprit in hepatocyte death. These effects define a novel pro-apoptotic pathway that results in caspase-independent apoptosis accompanied by chromatin condensation; responses that can be rescued by Sp1 overexpression or by growth-factor-receptor activation. Moreover, we found very recently that a similar TG2-dependent hepatic apoptosis pathway appears relevant to the pathogenesis of NASH (Kuo, Taksukawa, Tsukamoto and Kojima, unpublished data). However, we cannot explain why Sp1 cross-linking selectively down-regulates c-Met expression, whereas other Sp1-dependent genes are much less affected. We speculate that differences in Sp1-dependence may depend on the cell type and culture conditions used, and that other member(s) of the Sp/Krüppel-like factor family may compensate for the function of Sp1 in the transactivation of less-affected genes. Indeed, we recently found that nuclear TG2 and cross-linked Sp1 are associated with apoptosis induced by acyclic retinoid (a novel anti-cancer drug) in hepatocellular carcinoma and that, in this case, the putative target is EGFR (epidermal growth factor receptor) rather than c-Met (Tatsukawa and Kojima, unpublished data). In addition to its role in an early stage of apoptosis, it has been suggested that TG2 is essential for the phagocytosis of apoptotic cells in experiments using peritoneal macrophages (Tóth et al., 2009). It will be intriguing to see whether TG2 also plays a similar role in the liver.
Elevated TG2 activity leads to activation of the most potent fibrogenic cytokine, TGF-β, and the stabilization of a large number of ECM components. Conflicting results have been obtained using TG2−/− mice with different backgrounds, i.e. TG2 has a promotive (Qiu et al., 2007; Shweke et al., 2008) or protective (Fesus and Piacentini, 2002; Nardacci et al., 2003) effect on liver injury and fibrosis. The complexity of this issue is highlighted by the fact that TG2 is multifunctional, exerting mainly G-protein or cross-linking activities, and the enzyme may function differently in animals with different backgrounds. In addition, it remains to be determined whether the effects of TG2 (or other TGs) will be synergistic or antagonistic given the heterogeneous hepatocyte/cholangiocyte/endothelial/stellate and other mesenchymal cell components of the liver.
In summary, we have increased our understanding of the role of TG2 in alcoholic steatohepatitis through identifying it as a novel potential link to ethanol-mediated liver injury (Tatsukawa et al., 2009). Although our own studies have focused on the silencing of the Sp1–c-Met axis, future work will focus on (i) the identification of additional TG2 substrates, which might be equally important in the pathogenesis of alcoholic liver diseases, (ii) the pathological specificity of TG2-mediated liver injury among liver diseases, and (iii) the nuclear localization of TG2, which might be the important step in promoting alcoholic liver injury. Alternatively, making and analysing the agents that directly influence the TG2–Sp1–c-Met axis might also be an important study for treating and preventing alcoholic liver disease.
We thank Dr H. Senoo (Akita University, Akita, Japan) for providing the opportunity of writing the review and his critical reading of the draft.
The authors’ experimental work referred to in this review was supported partly by Grant-in-Aids from the
Ahn, JS, Kim, MK, Hahn, JH, Park, JH, Park, KH and Cho, BR (2008) Tissue transglutaminase-induced down-regulation of matrix metalloproteinase-9. Biochem Biophys Res Commun 376, 743-7
Antonyak, MA, Jansen, JM, Miller, AM, Ly, TK, Endo, M and Cerione, RA (2006) Two isoforms of tissue transglutaminase mediate opposing cellular fates. Proc Natl Acad Sci USA 103, 18609-14
Baek, KJ, Das, T, Gray, C, Antar, S, Murugesan, G and Im, MJ (1993) Evidence that the Gh protein is a signal mediator from α1-adrenoceptor to a phospholipase C. I. Identification of α1-adrenoceptor-coupled Gh family and purification of Gh7 from bovine heart. J Biol Chem 268, 27390-7
Balklava, Z, Verderio, E, Collighan, R, Gross, S, Adams, J and Griffin, M (2002) Analysis of tissue transglutaminase function in the migration of Swiss 3T3 fibroblasts: the active-state conformation of the enzyme does not affect cell motility but is important for its secretion. J Biol Chem 277, 16567-75
Begg, GE, Carrington, L, Stokes, PH, Matthews, JM, Wouters, MA and Husain, A (2006) Mechanism of allosteric regulation of transglutaminase 2 by GTP. Proc Natl Acad Sci USA 103, 19683-8
Chen, CS, Wu, CH, Lai, YC, Lee, WS, Chen, HM and Chen, RJ (2008) NF-κB-activated tissue transglutaminase is involved in ethanol-induced hepatic injury and the possible role of propolis in preventing fibrogenesis. Toxicology 246, 148-57
Citron, BA, SantaCruz, KS, Davies, PJ and Festoff, BW (2001) Intron-exon swapping of transglutaminase mRNA and neuronal Tau aggregation in Alzheimer’s disease. J Biol Chem 276, 3295-301
Defacque, H, Commes, T, Contet, V, Sevilla, C and Marti, J (1995) Differentiation of U937 myelomonocytic cell line by all-trans retinoic acid and 1,25-dihydroxyvitamin D3: synergistic effects on tissue transglutaminase. Leukemia 9, 1762-7
Dunah, AW, Jeong, H, Griffin, A, Kim, YM, Standaert, DG and Hersch, SM (2002) Sp1 and TAFII130 transcriptional activity disrupted in early Huntington’s disease. Science 296, 2238-43
Esposito, C and Caputo, I (2005) Mammalian transglutaminases. Identification of substrates as a key to physiological function and physiopathological relevance. FEBS J 272, 615-31
Giebeler, A, Boekschoten, MV, Klein, C, Borowiak, M, Birchmeier, C and Gassler, N (2009) c-Met confers protection against chronic liver tissue damage and fibrosis progression after bile duct ligation in mice. Gastroenterology 137, 297-308
Grenard, P, Bresson-Hadni, S, El Alaoui, S, Chevallier, M, Vuitton, DA and Ricard-Blum, S (2001) Transglutaminase-mediated cross-linking is involved in the stabilization of extracellular matrix in human liver fibrosis. J Hepatol 35, 367-75
Hasegawa, G, Suwa, M, Ichikawa, Y, Ohtsuka, T, Kumagai, S and Kikuchi, M (2003) A novel function of tissue-type transglutaminase: protein disulphide isomerase. Biochem J 373, 793-803
Horino, K, Nishiura, H, Ohsako, T, Shibuya, Y, Hiraoka, T and Kitamura, N (1998) A monocyte chemotactic factor, S19 ribosomal protein dimer, in phagocytic clearance of apoptotic cells. Lab Invest 78, 603-17
Hsu, TC, Huang, CY, Chiang, SY, Lai, WX, Tsai, CH and Tzang, BS (2008) Transglutaminase inhibitor cystamine alleviates the abnormality in liver from NZB/W F1 mice. Eur J Pharmacol 579, 382-9
Huh, CG, Factor, VM, Sanchez, A, Uchida, K, Conner, EA and Thorgeirsson, SS (2004) Hepatocyte growth factor/c-met signaling pathway is required for efficient liver regeneration and repair. Proc Natl Acad Sci USA 101, 4477-82
Iismaa, SE, Wu, MJ, Nanda, N, Church, WB and Graham, RM (2000) GTP binding and signaling by Gh/transglutaminase II involves distinct residues in a unique GTP-binding pocket. J Biol Chem 275, 18259-65
Ishikawa, T, Conner, EA, Hoang, T, Factor, VM and Thorgeirsson, SS (2008) c-Met signaling is essential for stem cell mediated liver regeneration Hepatology. 48, 341A
Karpuj, MV, Becher, MW, Springer, JE, Chabas, D, Youssef, S and Pedotti, R (2002) Prolonged survival and decreased abnormal movements in transgenic model of Huntington disease, with administration of the transglutaminase inhibitor cystamine. Nat Med 8, 143-9
Kojima, S, Nara, K and Rifkin, DB (1993) Requirement for transglutaminase in the activation of latent transforming growth factor-β in bovine endothelial cells. J Cell Biol 121, 439-48
Le, M, Gohr, CM and Rosenthal, AK (2001) Transglutaminase participates in the incorporation of latent TGFβ into the extracellular matrix of aging articular chondrocytes. Connect Tissue Res 42, 245-53
Liu, S, Cerione, RA and Clardy, J (2002) Structural basis for the guanine nucleotide-binding activity of tissue transglutaminase and its regulation of transamidation activity. Proc Natl Acad Sci USA 99, 2743-7
Melino, G, Annicchiarico-Petruzzelli, M, Piredda, L, Candi, E, Gentile, V and Davies, PJ (1994) Tissue transglutaminase and apoptosis: sense and antisense transfection studies with human neuroblastoma cells. Mol Cell Biol 14, 6584-96
Mirza, A, Liu, SL, Frizell, E, Zhu, J, Maddukuri, S and Martinez, J (1997) A role for tissue transglutaminase in hepatic injury and fibrogenesis, and its regulation by NF-κB. Am J Physiol 272, G281-8
Mishra, S, Melino, G and Murphy, LJ (2007) Transglutaminase 2 kinase activity facilitates protein kinase A-induced phosphorylation of retinoblastoma protein. J Biol Chem 282, 18108-15
Monsonego, A, Friedmann, I, Shani, Y, Eisenstein, M and Schwartz, M (1998) GTP-dependent conformational changes associated with the functional switch between Gα and cross-linking activities in brain-derived tissue transglutaminase. J Mol Biol 282, 713-20
Murthy, SN, Iismaa, S, Begg, G, Freymann, DM, Graham, RM and Lorand, L (2002) Conserved tryptophan in the core domain of transglutaminase is essential for catalytic activity. Proc Natl Acad Sci USA 99, 2738-42
Nagy, L, Thomazy, VA, Saydak, MM, Stein, JP and Davies, PJ (1997) The promoter of the mouse tissue transglutaminase gene directs tissue-specific, retinoid-regulated and apoptosis-linked expression. Cell Death Differ 4, 534-47
Nakaoka, H, Perez, DM, Baek, KJ, Das, T, Husain, A and Misono, K (1994) Gh: a GTP-binding protein with transglutaminase activity and receptor signaling function. Science 264, 1593-6
Nara, K, Nakanishi, K, Hagiwara, H, Wakita, K, Kojima, S and Hirose, S (1989) Retinol-induced morphological changes of cultured bovine endothelial cells are accompanied by a marked increase in transglutaminase. J Biol Chem 264, 19308-12
Nardacci, R, Lo Iacono, O, Ciccosanti, F, Falasca, L, Addesso, M and Amendola, A (2003) Transglutaminase type II plays a protective role in hepatic injury. Am J Pathol 162, 1293-303
Nicholas, B, Smethurst, P, Verderio, E, Jones, R and Griffin, M (2003) Cross-linking of cellular proteins by tissue transglutaminase during necrotic cell death: a mechanism for maintaining tissue integrity. Biochem J 371, 413-22
Oliverio, S, Amendola, A, Rodolfo, C, Spinedi, A and Piacentini, M (1999) Inhibition of ‘tissue’ transglutaminase increases cell survival by preventing apoptosis. J Biol Chem 274, 34123-8
Ou, H, Haendeler, J, Aebly, MR, Kelly, LA, Cholewa, BC and Koike, G (2000) Retinoic acid-induced tissue transglutaminase and apoptosis in vascular smooth muscle cells. Circ Res 87, 881-7
Qiu, JF, Zhang, ZQ, Chen, W and Wu, ZY (2007) Cystamine ameliorates liver fibrosis induced by carbon tetrachloride via inhibition of tissue transglutaminase. World J Gastroenterol 13, 4328-32
Ritter, SJ and Davies, PJ (1998) Identification of a transforming growth factor-beta1/bone morphogenetic protein 4 (TGF-β1/BMP4) response element within the mouse tissue transglutaminase gene promoter. J Biol Chem 273, 12798-806
Sarang, Z, Molnar, P, Nemeth, T, Gomba, S, Kardon, T and Melino, G (2005) Tissue transglutaminase (TG2) acting as G protein protects hepatocytes against Fas-mediated cell death in mice. Hepatology 42, 578-87
Shibley, IA Jr, Gavigan, MD and Pennington, SN (1995) Ethanol’s effect on tissue polyamines and ornithine decarboxylase activity: a concise review. Alcohol Clin Exp Res 19, 209-15
Shimada, J, Suzuki, Y, Kim, SJ, Wang, PC, Matsumura, M and Kojima, S (2001) Transactivation via RAR/RXR-Sp1 interaction: characterization of binding between Sp1 and GC box motif. Mol Endocrinol 15, 1677-92
Shweke, N, Boulos, N, Jouanneau, C, Vandermeersch, S, Melino, G and Dussaule, JC (2008) Tissue transglutaminase contributes to interstitial renal fibrosis by favoring accumulation of fibrillar collagen through TGF-β activation and cell infiltration. Am J Pathol 173, 631-42
Strnad, P, Harada, M, Siegel, M, Terkeltaub, RA, Graham, RM and Khosla, C (2007) Transglutaminase 2 regulates mallory body inclusion formation and injury-associated liver enlargement. Gastroenterology 132, 1515-26
Strnad, P, Tao, GZ, Zhou, Q, Harada, M, Toivola, DM and Brunt, EM (2008) Keratin mutation predisposes to mouse liver fibrosis and unmasks differential effects of the carbon tetrachloride and thioacetamide models. Gastroenterology 134, 1169-79
Szondy, Z, Sarang, Z, Molnar, P, Nemeth, T, Piacentini, M and Mastroberardino, PG (2003) Transglutaminase 2−/− mice reveal a phagocytosis-associated crosstalk between macrophages and apoptotic cells. Proc Natl Acad Sci USA 100, 7812-7
Tatsukawa, H, Fukaya, Y, Frampton, G, Martinez-Fuentes, A, Suzuki, K and Kuo, TF (2009) Role of transglutaminase 2 in liver injury via cross-linking and silencing of transcription factor Sp1. Gastroenterology 136, 1783-95
Tomita, K, Azuma, T, Kitamura, N, Nishida, J, Tamiya, G and Oka, A (2004) Pioglitazone prevents alcohol-induced fatty liver in rats through up-regulation of c-Met. Gastroenterology 126, 873-85
Tóth, B, Garabuczi, E, Sarang, Z, Vereb, G, Vámosi, G and Aeschlimann, D (2009) Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophages engulfing apoptotic cells. J Immunol 182, 2084-92
Wada, F, Nakamura, A, Masutani, T, Ikura, K, Maki, M and Hitomi, K (2002) Identification of mammalian-type transglutaminase in Physarum polycephalum. Evidence from the cDNA sequence and involvement of GTP in the regulation of transamidating activity. Eur J Biochem 269, 3451-60
Wettschureck, N, Rutten, H, Zywietz, A, Gehring, D, Wilkie, TM and Chen, J (2001) Absence of pressure overload induced myocardial hypertrophy after conditional inactivation of Gαq/Gα11 in cardiomyocytes. Nat Med 7, 1236-40
Wu, J, Liu, SL, Zhu, JL, Norton, PA, Nojiri, S and Hoek, JB (2000) Roles of tissue transglutaminase in ethanol-induced inhibition of hepatocyte proliferation and α1-adrenergic signal transduction. J Biol Chem 275, 22213-9
Zhai, W, Jeong, H, Cui, L, Krainc, D and Tjian, R (2005) In vitro analysis of huntingtin-mediated transcriptional repression reveals multiple transcription factor targets. Cell 123, 1241-53
Zhong, B, Strnad, P, Selmi, C, Invernizzi, P, Tao, GZ and Caleffi, A (2009) Keratin variants are overrepresented in primary biliary cirrhosis and associate with disease severity. Hepatology 50, 546-54
Received 2 September 2009/9 December 2009; accepted 5 January 2010
Published online 22 February 2010, doi:10.1042/CBI20090130
© The Author(s) Journal compilation © 2010 Portland Press Ltd
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)