Tissue transglutaminase, a key enzyme involved in liver diseases

Hepatology Research 29 (2004) 1–8

Tissue transglutaminase, a key enzyme involved in liver diseases Jian Wu, Mark A. Zern∗ Transplant Research Institute, University of California, Davis Medical Center, 4635 2nd Ave. Suite 1001, Sacramento, CA 95817, USA Received 25 November 2003; received in revised form 26 January 2004; accepted 4 February 2004

Abstract Ubiquitous tissue transglutaminase (tTG) is one member of the large transglutaminase (TG) family, which catalyze posttranslational modification of proteins by establishing ⑀(␥-glutamyl)lysine cross-linking and/or covalent incorporation of polyamines. The unique characteristics of tTG include: (1) possessing both cross-linking activity and GTPase activity; (2) functioning as a G protein; and (3) participating in the signal transduction of ␣1-adrenergic receptor coupling. A growing body of literature suggests that increased tTG levels in the cytosolic or nuclear compartments contribute to the apoptotic process, and lines of evidence exist that nuclear translocation and cross-linking of transcriptional factor Sp1 may represent the underlying mechanisms of these proapoptotic effects of tTG. Our studies indicate that tTG GTPase activation may be responsible for enhanced hepatocyte proliferation, whereas, its tTGase activity may cause increased apoptosis. Moreover, it appears that tTG cross-linking activity contributes to hepatic fibrogenesis in animal models and in human liver disease. Understanding the roles of tTG in the pathogenesis of liver disease could facilitate the development of new treatment regimens. © 2004 Elsevier B.V. All rights reserved. Keywords: Apoptosis; Fibrosis; Liver; Regeneration; Tissue transglutaminase

1. Introduction Transglutaminases (TGs) are a large family of enzymes that catalyze a calcium-dependent acryl transfer reaction between the ␥-carboxamide group of a polypeptide bound lysine residue to form an ε(␥-glutamyl)lysine isopeptide bond. TGs can also catalyze the incorporation of a polyamine into a polypeptide-bound glutamine leading to the formation of a (␥-glutamyl)polyamine bond. TGs, known as “natural biological glues”, are widely localized in blood, extracellular space and intracellular compartments, and are involved in numerous biochemical processes, such as blood clotting, wound healing, tissue remodeling, matrix stabilization, and cell death or differentiation [1]. As summarized in Table 1, there are eight human TGs, and five

Abbreviations: CCl4 , carbon tetrachloride; ECM, extracellular matrix; GTP, guanosine 5 -triphosphate; PLC, phospholipase C; TGF-␤, transforming growth factor-␤; TG, transglutaminase; tTG, tissue transglutaminase; TNF-␣, tumor necrosis factor-␣ ∗ Corresponding author. Tel.: +1-916-734-8063; fax: +1-916-734-8097. E-mail address: [email protected] (M.A. Zern). 1386-6346/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.hepres.2004.02.007

of them are well characterized. In addition, factor XIIIa is a well characterized molecule with transglutaminase activity. Although TGs possess a variety of biological functions that are generally attributed to their protein-modifying activity, in some instances, their biological function is due to specialized non-catalytic actions, such as scaffolding of the cytoskeleton to maintain membrane integrity, cell adhesion, endocytosis, and signal transduction [2]. Thus, TGs are essential isozymes of cell components and function in multiple facets of biological processes. Tissue transglutaminase (tTG or TG2, E.C. 2.3.2.13) not only displays cross-linking activity, but also functions as a GTPase, and it has been shown to be involved in a number of pathophysiologic processes, such as degenerative disorders of neurons [3], myocardial hypertrophy [4], hepatic injury [5,6], and fibrosis [7,8]. Employing overexpression of tTG in in vitro models or examining knock-out animal models and the three dimension crystal structure of the protein configuration has helped to delineate the molecular bases for its biochemical properties, and has greatly facilitated our understanding of the enzyme’s role in a variety of pathologic processes. The present review summarizes recent studies of tTG’s biochemical features, and its involvement in

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Table 1 Human transglutaminases (TGs), biological functions and relevant diseases (Lorand and Graham 2003; Griffin et al. 2002) Synonyms

Residues (kDa)

Gene

Gene map locus

Tissue expression

Localization

Function

Possible link to disease

Factor XIIIa

Fibrin-stabilizing factor, pro-fibrinoligase, or plasma pro-TG

732 (83)

F13A1

6q24-25

Cytosolic, extracellular

Blood coagulation, bone growth

Factor XIII deficiency

TG1

TGk, keratinocyte TG, particulate TG

814 (90)

TGM1

14q11.2

Platelets, astrocytes, dermal dendritic cells, placenta, plasma, and synovial fluid Keratinocytes, brain

Membrane, cytosolic

Lamellar ichthyosis

TG2

TGc, tissue TG (tTG), endothelial TG, erythrocyte TG, Gh␣ TGe, epidermal TG, hair follicle TG

686 (80)

TGM2

20q11-12

Ubiquitous

Cytosolic, membrane, and nuclear, extracellular

692 (77)

TGM3

20q11-12

Squamous epithelium, brain

cytosolic

TG4

TGp, prostate TG

683 (77)

TGM4

3q21-22

Prostate

Unknown

TG5

TGx

791 (81)

TGM5

15q15.2

Unknown

TG6 TG7 Band 4.2

TGy TGz ATP-binding erythrocyte membrane protein band 4.2

Unknown 710 690 (72)

TGM6 TGM7 EPB4.2

20q11 15q15.2 15q15.2

Ubiquitous except the CNS and lymphatic tissue Unknown Ubiquitous Red blood cells, bone marrow, fetal liver, and spleen

Cell envelope formation in the differentiation of keratinocytes Cell death, differentiation, matrix stabilization, adhesion protein Cell envelope formation during terminal differentiation of keratinoytes Reproductive function involving semen coagulation, especially in rodents Epidermal differentiation

TG3

Unknown Unknown Membrane

Not characterized Not characterized Membrane skeletal components

Hepatic fibrogenesis?

Not defined

Unknown

Unknown Unknown Unknown Hereditary spherocytosis

J. Wu, M.A. Zern / Hepatology Research 29 (2004) 1–8

Protein

J. Wu, M.A. Zern / Hepatology Research 29 (2004) 1–8

hepatic injury and fibrosis, as well as hepatocyte proliferation.

2. Three dimensional structure and gene regulation of tTG tTG is constitutively expressed in endothelial, mesangial and smooth muscle cells, and localized in the cytosol, plasma membrane, and nucleus of cells in addition to the extracellular space. It is composed of 686 amino acid residues with an approximate molecular weight of 80 kDa. The overall X-ray crystallographic human tTG structure demonstrates a dimer [9,10] (Fig. 1). Each monomer has four distinct domains: the amino-terminal ␤-sandwich domain consisting of residues Met-1 to Phe-139, the transamidation catalytic core domain consisting of Ala-147 to Asn-460, and two carboxyl-terminal ␤-barrel domains, which include Gly-472 to Tyr-583, and Ile-591 to Ala-687, respectively. The guanine nucleotide-binding site is located in a segment of 15 amino acid residues (159 YVLTQQGFIYQGSVK173 ) [9], which is a cleft between the catalytic core and the first ␤-barrel domain, close to the dimerization interface. The catalytic domain contains two residues interacting with the guanine base, but does not reveal any bound Mg2+ . tTG can bind GTP-␥-S, GTP or GDP with high affinity in the absence of Mg2+ , which differs markedly from the nucleotide-binding domain conserved among the ␣ subunits of large heterotrimeric G proteins. The tTG structure also provides clues regarding the regulation of its enzymatic transamidation activity. It has been well established that

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Cys-277 is the essential nucleophile for transamidation. In the tTG structure, Cys-277 is located in the middle of a groove within the catalytic domain and participates in a catalytic triad, Cys-277–His-335–Asp-358. These three catalytic residues are conserved in all members of the TG family. Calcium ions exert an activating signal for transamidation. The putative Ca2+ -binding site on TG is located near the end of the loop that connects the catalytic transamidation domain to the first ␤-barrel domain [10]. In the liver, both parenchymal and non-parenchyma cells produce tTG, and tTG appears to be released into the extracellular space. The tTG expression is up-regulated when there is any injuring process [7], and by tumor necrosis factor-␣ (TNF-␣) in a hepatoma cell line [11]. tTG activity is also regulated by transcriptional activators, such as retinoids [12], vitamin D and steroids in a tissue-specific manner [13], or by transcriptional factors, such as SP-1 at the promoter region which contains retinoid-responsive elements, sites for SP-1 binding, as well as regions for regulation by TGF-␤, interleukins, morphogenic protein 4, and possible steroid receptors. In an apoptotic cell, falling nucleotide levels and increasing Ca2+ levels seems to activate tTG’s transamidation activity (10).

3. Calcium-dependent cross-linking activity and calcium-independent GTPase activity Transamidation is a basic function of cross-linking activity of tTG, and peptide-bound ␥-glutaminyl residues serve as substrates for tTG. tTG catalyzes a two-step double dis-

Fig. 1. Overall structure of a human tissue transglutaminase (TG) dimer with bound GDP. tTG is shown in ribbon drawing with the ␤-sandwich domain, the catalytic core domain, and the first and second ␤-barrel domain shown in green, red, cyan, and yellow, respectively. The loops connecting the first ␤-barrel domain to the catalytic core and the second ␤-barrel are shown in purple. GDP is shown as a ball-and-stick model between the catalytic core and the first ␤-barrel (from [10] with reprint permission from Proceedings of the National Academy of Science).

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O

tTG +Ca2+ H2N-(CH2)4Lysine

NH4+

-(CH2)-C-NH-(CH2)4-

+ O

Cross-linking

-(CH2)2-C-NH Glutamyl residue O

tTG +Ca2+ -(CH2)-C-NH-(CH2)4-NH3+

+ NH4+ +

H2N-(CH2)4-NH3+ Putrescine

Polyamine incorporation

Fig. 2. tTG catalyzes a calcium-dependent acryl transfer reaction between the ␥-carboxyamide group of polypeptide bound glutamine and the primary amino group of either a polypeptide bound lysine to form an isopeptide bond between or within proteins, or a polyamine (e.g. putrescine) yielding a (␥-glutamyl)polyamine bond. (modified from [3]).

placement reaction, as illustrated in Fig. 2. The cross-linking reaction results in the formation of an ε(␥-glutamyl)lysine isopeptide bound, which is one important step of maturation or stabilization of extracellular matrixes, such as collagens in the extracellular space. In addition, this cross-linking appears to play a crucial role in the secretion and fixation of TGF-␤ in the extracellular matrix (ECM) by linkage of latent TGF-␤ binding protein (LTBP) to fibronectin or other ECM components [14,15]. The reaction is Ca2+ -dependent, and is the biochemical basis for tTG involvement in hepatic fibrosis. Since the enzyme recognizes putrescine as a substrate, putrescine may serve as a competitive inhibitor for the cross-linking activity of the enzyme, and has been shown to be protective in ethanol-induced liver injury, in which enhanced tTG cross-linking activity was observed in in vitro and animal models [16–18]. As mentioned before, GDP or GTP binding to its binding sites reduces the cross-linking activity due to the conformational change in the catalytic core of the enzyme. The binding inhibition can be overcome by high concentrations of Ca2+ [10]. One characteristic of tTG is to bind and to hydrolyze GTP with affinity and rates similar to those of other G proteins; this distinguishes tTG from other transglutaminases, and suggests that tTG, like other G proteins, participates in signaling pathways. Among the studies implicating tTG as a signal transducer in biological response pathways, the best documented is its role in ␣1-adrenergic receptor-mediated stimulation of phospholipase C (PLC) ␦ activity [19]. It was originally reported that an approximately 70–80 kDa GTP-binding protein (named Gh) was responsible for coupling ␣1-adrenergic agonists to the stimulation of phosphoinositide lipid metabolism [20], and it was subsequently demonstrated that Gh was identical to tTG, and it is now called G␣h [21,22]. The GTP binding and GTPase activity is calcium-independent, and its linking with the downstream signal transduction pathways elicited a new array of

research on its roles in intracellular calcium homeostasis (oscillations), cell proliferation, and other actions [5].

4. tTGase activity and its effects on liver injury and hepatocyte proliferation The relationship between increased tTG cross-linking activity (also called tTGase or transamidating activity) and cell death has been established for nearly a decade. However, whether the increase in tTG activity causes cell death in various pathologic processes, or whether the two phenomena have only a loose relationship is still uncertain. In tTG-transfected cells, elevated tTG activity was correlated with enhanced apoptosis [23]. Impaired mitochondrial function results in increased tTG activity in neuroblastoma SH-SY5Y cells [24]. Treatment of tTG plasmid-transfected SH-SY5Y cells with staurosporine or osmotic stress led to activation of caspase-3, apoptotic nuclear changes, and an increase in transamidating activity, but these changes did not occur in cells transfected with a control vector, or with a mutated tTG plasmid lacking transamidating activity [25]. Treatment of antisense against tTG reduced apoptosis in dopaminergic neurons [26]. Treatment of cultured rat hepatocytes with ethanol led to apoptosis, an increase in cytosolic cytochrome C levels, and enhanced tTGase activity in cytosol and nuclear compartments [27]. Hepatocytes isolated from tTG knock-out mice displayed less apoptosis than those from wild-type mice under the same treatment. Much lower serum alanine aminotransferase levels were detected in tTG knock-out mice after Fas ligand antibody (Jo2) injection compared to levels in wild-type mice [28]. In situ apoptosis staining showed that many fewer apoptotic cells were visualized in the livers of tTG knock-out mice than in those from wild-type mice. These findings suggest the concept that tTG is a crucial factor in the apoptotic process

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caused by a variety of factors. However, an Italian group showed conflicting results, and claimed that tTG is a protective factor against apoptosis based on their observation that more tTG knock-out mice died after 5 weeks of CCl4 intoxication than did wild-type animals [6], and more apoptotic cells were found in the liver of the mice after PbNO3 injection due to defective clearance of apoptotic cells in the liver [29]. The explanation for these divergent findings is unknown, but these two groups used two independent strains of tTG knock-out mice [29,30]. The speculation is that the genetic background difference in the mouse strains may lead to the different results. It is well established that ethanol exposure inhibits hepatocyte mitogenesis and proliferation in both in vitro [31] and in vivo [32] systems. Ethanol has been shown to decrease putrescine levels in the liver, and the administration of putrescine or its precursors, alanine or glutamine, abrogates this inhibition of liver regeneration by ethanol administration [16–18]. The mechanism by which putrescine abolishes this effect of ethanol on regeneration may be that putrescine acts as a competitive substrate inhibitor of tTGase activity [33]. This action of putrescine is important because tTG has been shown to inhibit cell proliferation in other systems. This is thought to occur by delaying the progression of the cells from S-phase to G2 /M [34,35]. Our speculation is that ethanol or other profibrogenic agents may enhance tTG expression and tTGase cross-linking activity, leading in turn to an inhibition of the proliferation cascade, and that putrescine may enhance hepatocyte proliferation by inhibiting tTGase activity. Our results indicate that ethanol administration enhances tTGase activity at concentrations that inhibit hepatocyte proliferation in vitro. Moreover, three inhibitors of tTGase activity, putrescine, guanosine 5 -O-(3 thiotriphosphate) (GTP-␥-S), and phenylephrine (each acting by a different mechanism to inhibit tTGase activity) abrogated the ethanol inhibition of hepatocyte proliferation at the same time that they inhibited the tTGase cross-linking activity [5]. These findings suggest that ethanol-induced inhibition of hepatocyte proliferation may be caused, at least in part, by enhanced tTGase activity. It appears that liver cell death secondary to ethanol administration is occurring by apoptosis, not by liver cell necrosis [36,37]. Recent reports suggest that the Fas ligand system may be involved in drug and ethanol-induced apoptosis [38,39]. Our findings suggest that the well-known association between tTG and apoptosis may also be a factor in apoptosis induced by ethanol. However, others may argue for a predominant role of tTG in the downstream stage of apoptosis, with its cross-linking activity important only for some of the morphologic events in the late stages of the process. The activation of tTG leads to the assembly of intracellular cross-linked protein polymers which irreversibly modify cell structure, thus contributing to the ultrasturctural changes occurring in cells undergoing apoptosis [40,41]. Whether tTG is an active mediator at an early stage in the process of programmed cell death is still controversial.

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Several groups are now suggesting that cross-linking of specific proteins may be crucial in some of the initial events of apoptosis. This includes a role for the cross-linking of pRB and polyglutamine proteins [42,43]. In addition, Dr. Kojima and colleagues have suggested that nuclear tTG may induce apoptosis through cross-linking of nuclear proteins [23]. In support of this hypothesis, tTGase has been found in the nucleus by a series of investigators [44,45], and some have suggested that its transport to the nucleus may be due to its interaction with the nuclear transport protein, importin-␣ [46]. Kojima and colleagues have demonstrated that cross-linking of Sp1 by tTGase may play a significant role in the process of apoptosis [23]. They have shown that tTGase induces the formation of a highly cross-linked complex of Sp1 in a cell-free system and in cells transfected with tTGase [45]. When this cross-linking occurs, Sp1 loses its ability to bind to the GC box motif and its capacity to transactivate GC box-containing genes. Moreover, this Sp1 inactivation coincides with chromatin fragmentation and an increase in caspase 3 activity, as well as the appearance of TUNEL-positive cells. When the caspase activity is blocked with caspase inhibitors, the transfected cells still undergo apoptosis. On the other hand, restoration of Sp1 activity by transfection of an Sp1 expression plasmid prevents the tTGase-induced apoptosis without affecting tTGase levels. These results suggest that Sp1 is cross-linked, aggregated, and inactivated by nuclear tTGase, and that this leads to caspase-independent apoptosis.

5. tTG GTPase activity and enhanced hepatocyte proliferation It has been demonstrated that ␣1-adrenergic agonists such as epinephrine and phenylephrine enhance hepatocyte proliferation [47,48]. Adrenergic signaling occurs through activation of phospholipase C (PLC), which in turn produces the two intracellular messengers, diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3 ). These intermediate messengers mediate the activation of protein kinase C (PKC) and increases in intracellular [Ca2+ ], resulting in calcium oscillations in the cells. The downstream events include cell proliferation. A variety of hormones and other agonists act through specific receptors coupled to different G proteins to stimulate distinct classes of PLC isozymes. For example, our experiments support the notion that phenlyephrine (PNLP) activates PLC-␦1 through coupling to the tTG G protein, G␣h [5]. Hepatocytes are also rich in vasopressin V1␣ receptors that activate the PLC-␤1 isoform through G␣q ; whereas growth factors, such as EGF or HGF activate the PLC-␥1 isoform through receptor tyrosine kinases [49]. All these PLC isoforms hydrolyze phosphatidylinositol-4, 5-bisphosphate (PIP2 ) to generate diacylglycerol and the second messenger, inositol 1,4,5-triphosphate (IP3 ), which causes the release of intracellular Ca2+ stores by binding

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to the IP3 receptor in the endoplasmic reticulum. Yet, for reasons that have not been elucidated, different agonists give rise to substantially distinct Ca2+ oscillation patterns. This may reflect specific features of control or subcellular localization of the different PLC isoforms. The functional consequences of these different PLC signaling branches are not known. However, previous studies established that the enhancement of cell proliferation by G protein-coupled receptor agonists is much more potent for PNLP than for vasopressin or other hormones acting through different receptors [50]. Thus, it is likely that the different PLC isoforms activate distinct downstream signaling pathways leading to different functional consequences for the cell. Our data suggest that the G␣h -dependent activation of PLC-␦1 by phenylephrine is preferentially involved in the enhancement of cell proliferation [5]. It is well known that multiple receptors can converge on a single G protein, and that a single receptor can activate more than one G protein. On the other hand, there is frequently a high degree of specificity with paired receptor-G proteineffector interactions [51]. Thus, an unanswered question is the specificity of the ␣1-adrenergic receptor/G␣h /PLC␦1 signaling. Delineating some of the downstream effects initiated by phenylephrine activation of the ␣1-adrenergic receptor-G␣h complex is an active area of research investigation.

•Viral infection •Drug toxicity •Alcoholism •Autoimmune reaction n tio va i t Ac

Our results clearly indicate that tTG has two different functions that lead to either proliferation or apoptosis. For example, alternative mRNA splicing or post-translational modifications of the same gene can have such effects in the Ced-9/Bcl-2 family [52]. Cytochrome C is involved in mitochondrial electron transport or in the activation of caspase 9, depending on its intracellular localization [53]. It would be instructive to explain the apparent “switch” that occurs between tTGase cross-linking activity located in the cytosol and the GTPase activity residing in the membrane fraction. It is not clear whether this switch represents a translocation of the protein from a cytosolic to a plasma membrane location, or a change in configuration that occurs in the receptor/G protein complex, or some other mechanism.

6. tTG cross-linking activity and hepatic fibrosis One major role of tTG cross-linking activity is its involvement in the wound healing process. Hepatic fibrosis is a healing process of chronic liver injury. For example, tTGase activity increased associated with TGF-␤ and EMC production after chronic CCl4 intoxication in rats [7]. The Nε (␥-glutamyl)lysine cross-link, which is undetectable in normal liver tissue, was present extracellularly in the fibrotic

Kupffer cells

Plasminogen Latent TGF−β

na sig

ls

tTG↑ TNF-α

tTG

Plasmin

PDGF

Active TGF-β tTG↑ Damaging signals

Hepatocytes Necrosis/Apoptosis

Activation of HSC tTG↑ Production of extracellular matrix (Cross-linking activity for mature ECM)

Fig. 3. Schematic illustration of the involvement of tTG in hepatic injury and fibrogenesis. Hepatocytes are damaged by various etiologies, and the damaging process leads to elevated levels of tTG in the cells. Elevated tTG is released into the extracellular space, and contributes to the activation of TGF-␤ and cross-linking of EMC components. Activated Kupffer cells during the injuring process releases TNF-␣ and other cytokines, which may up-regulate the expression of tTG by both parenchymal (hepatocytes) and non-parenchymal cells (Kupffer cells, sinusoidal endothelial cells, and stellate cells). The details of tTG’s involvement in the injury process have not been elucidated yet and the relationship of increased tTG cross-linking activity and apoptosis remains to be established. PDGF is platelet-derived growth factor; HSC, hepatic stellate cells, TNF-␣, tumor necrosis factor-␣; and ECM the extracellular matrix.

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livers of patients with a variety of chronic liver diseases, mostly in inflammatory areas where an intense remodeling was occurring [8]. In addition, co-localization of osteonectin with Nε (␥-glutamyl)lysine at the periphery of granulomas and in the extracellular matrix of inflammatory zones, suggests that this protein was cross-linked by tTG in fibrotic livers [8]. It was also observed that higher levels of tTGase activity appeared at an early stage of HCV infection, and that the enzyme was localized to hepatocytes facing the periportal infiltrate. tTG was mostly localized in the ECM components during late stages of hepatic fibrosis after HCV infection [6]. It is speculated that enhanced tTGase activity, which results from cell damage due to chronic intoxication, such as ethanol or CCl4 , contributes to the binding of tTG to the ECM and the activation of latent TGF-␤ to its active form [13]. TGF-␤1 is released in a latent form (about 300 kDa), and it is activated by removing the latency-associated protein, and converted to an active form of 25 kDa. Plasmin cleaves the latency-associated protein which leads to the activation of TGF-␤1. Plasminogen is converted by a plasminogen activator to plasmin before it displays this proteolytic activity [54]. tTG is thought to be required for this activation of TGF-␤ via its cross-linking of large latent complexes to the cell surface or matrix [14]. Under these circumstances, the activated TGF-␤ will enhance hepatic fibrogenesis by both increasing ECM production and by stabilizing the ECM in an insoluble form, since many ECM components serve as substrates of tTGase activity. However, the recent study by an Italian group contradicts the prevailing hypothesis. Findings from their tTG knock-out model did not support a promoting role for tTG in CCl4 -induced injury and fibrosis, and greater mortality and more severe hepatic fibrosis were observed in the knock-out mice when compared to wild-type mice [6]. Thus, more studies are needed to clarify the importance of tTG during hepatic fibrosis.

7. Conclusions and prospects The unique bi-functional characteristics of tTG elicit extensive interest to explore its roles in several processes in liver disease, such as hepatic injury, fibrosis, and regeneration. Although contradictory findings exist regarding the roles of tTG in apoptosis, the data generated from a variety of settings indicate that tTG is certainly associated with apoptosis, and may represent an early component of the apoptosis cascade. The involvement of tTG in hepatic fibrogenesis appears to be more certain. It is currently thought that elevated tTGase activity leads to the activation of the most potent fibrogenic cytokine, TGF-␤, and the stabilization of a large number of ECM components. The GTP binding and GTPase activity of tTG appears to be a component in ␣1-adrenergic signaling, and thus may play a role in hepatocyte proliferation. The better understanding of the actions of tTG in various forms of liver injury, as illus-

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trated in Fig. 3, may well lead to new therapeutic strategies, such as the use of putrescine and cystamine, both of which have been shown to be beneficial in acute and chronic liver injury models in animals [55–57].

Acknowledgements The studies discussed in the present work were supported by an NIH grant (AA06386 to MAZ) and by a Liver Scholar Award from the American Liver Foundation (JW).

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