FXIIIA (blood coagulation factor XIIIA)

expression through myosin heavy chain


Knockout interstitial fibrosis

Viable; impaired reproduction; reduced survival

Impaired clot stabilization; delayed bleeding arrest

mouse towards the wild-type phenotype. This finding suggested a role for matrix/cell surface TG2 in wound migration during tissue repair [Verderio, Telci, Li, Melino, Griffin, unpubl. data].

Although the genetic approach of targeting inactivation of TG genes by homologous recombination in embryonic stem cells has revealed the in vivo involvement of TG1 and TG2 in wound repair and confirmed the importance of FXIIIA, it cannot be ruled out that some of the TG functions may be hidden by compensatory pathways occurring in the knockout mice, including the increased expression of other members of the TG family.

General Role of TG2 in Cell-Matrix Interactions

Significant in Wound Repair

TG2 appears to be involved in the wound healing process at multiple levels and in many body compartments thus reflecting its wide expression profile. For this reason we have dedicated to it a separate section which reflects its involvement in general tissue repair.

Multiple Activities and Regulation of TG2

Unlike other TGs, TG2 is characterized by unique structural features, which leads to a wide range of biological activities and physio-pathological implications [2, 3, 7 and references therein]. Although some controversy and redundancy of information is present on the definitive functions of TG2, rapid advances have been made in the past few years and multiple biological activities have been ascribed to it which include: protein transamidation; GTPase; structural adhesive protein; protein disulphide isomerase and insulin-like growth factor-binding protein-3 kinase activities [2, 3, 7, 31].

Integrated with the Ca2+-regulated transamidase active site of TG2 is a GTP binding and hydrolysis site, which is responsible for the G-protein/ signal transduction function of TG2, and for this role is known as Gha. Binding of GTP negatively regulates the protein transamidation activity of TG2 by inducing a conformational change that blocks the access of substrates to the transamidating active site [32]. Reciprocally, the binding of Ca2+ inhibits the binding of GTP. The TG2-mediated protein transamidation is not only regulated by the Ca2+/GTP:GDP ratio but outside the cell also by matrix binding and the redox state of the Cys active site [33, 34]. Due to Ca2+ activation requirements it is generally believed that the transamidating activity is mostly latent intracellularly [35, 36]. More recently cross-linking-independent adhesive functions have also been ascribed to TG2, which would depend on and be regulated by its externalization and association with ECM FN [33, 37-39].

Externalization of TG2 during Cell Stress and TG2-Associated

Cell Death

TG2 is localized in three major cell compartments (cytosol, plasma membrane and nucleus) and it is also secreted into the ECM. The mechanism of secretion is unusual because TG2 lacks a signal peptide and is not secreted by a classical endoplasmic reticulum/golgi-dependent mechanism. It is known that TG2 secretion requires the active-state conformation of TG2 [39] and an intact N-terminal FN-binding site [40]. Due to its atypical secretion mechanism, TG2 is not efficiently released. However its externalization dramatically increases in situations of tissue damage and cellular stress [11,14, 18, 41-43] when it accumulates in the ECM in complex with FN [18, 41]. For this reason TG2 may be regarded as a novel cell/tissue response modulator [2, 16].

Role of TG2 in Matrix Synthesis and Degradation

Successful tissue repair much depends on an optimum balance between ECM synthesis (formation of granulation tissue and deposition of collagen) and matrix degradation. This is controlled by matrix metalloproteinases (MMPs) and plasminogen activators and further regulated by tissue inhibitors of proteases. Matrix synthesis is essential to allow cell migration, while degradation is important to create a migration path in the deposited ECM, which together with wound contraction stimulates wound closure. TG2 actively participates in the remodeling of the ECM and a variety of mechanisms have been proposed. TG2 may facilitate the stabilization of the ECM after synthesis. In this respect, the ability of TG2 to form nonreducible multimers of ECM FN leading to its stabilization is well demonstrated [18, 36, 44, 45]. Apart from FN, TG2 has also been shown to cross-link a number of different types of collagen (II, III V, VII and XI) [46 and references therein] and more recently collagen type I [47]. We have recently shown that TG2-mediated modification of type I and III collagen leads to an increased rate of fibril formation and to the formation of an improved substratum for cell adhesion of fibroblasts and osteoblasts in culture [48]. Collagen type I when cross-linked by TG2 also becomes increasingly stable to MMP-1, thus facilitating its accumulation [42]. Recent work has shown that TG2 deficiency in fibroblasts transfected with antisense TG2 cDNA leads to decreased collagen matrix tension and decreased activation of gelatinase (MMP-2), which correlated with reduced levels of MT1-MMP activity [49]. Collagen remodeling by TG2 may therefore lead to the formation of large bundles with the creation of new intermolecular cross-links, which give tensile strength to the scar.

TG2 however may also affect matrix deposition indirectly by participating in the activation mechanism of latent TGF-p1 [50, 51]. It is indeed well documented that TGF-p1 plays a primary role in regulating proliferation and synthesis of ECM in fibroblasts, namely collagen synthesis. The involvement of TG2 with TGF-p1 activation is further demonstrated by the recent finding that lack of TG2 leads to a deficiency in activation of TGF-p1, which is related to a reduced rate of apoptotic thymocytes clearance by TG2-/- macrophages [26]. In turn, TGF-^1 can stimulate TG2 expression [52], possibly through a TGF-p1/BMP4 response element in the TG2 gene [53], leading to a positive amplification loop. This finding is supported by the recent observation that neutralizing anti-TGF-^ antibodies significantly reduce TG2 expression in mice [26].

Matrix Structural Role of TG2

Our studies and those of other groups have confirmed that TG2 is a FN-binding protein in cultured cells, which tightly associates with cell surface and ECM FN [36, 37 40, 51]. TG2 is able to modulate and stabilize the FN matrix by forming nonreducible Ca2+-dependent e(7-glutamyl)lysine cross-links [45, 36]. Reciprocally, FN binding protects TG2 from proteolytic degradation [54] and controls its transamidating activity [55], which otherwise would be unsus-tainably high and eventually detrimental to cells in the high Ca2+ containing extracellular environment. It is likely that once externalized and bound to FN, TG2 becomes gradually inactive as a protein cross-linker and assumes instead a structural role in complex with FN. In a recent study, we developed a model of TG2-rich FN matrix, which would form in response to tissue injury, when TG2 is upregulated and exported to the ECM. TG2 would either directly bind to FN fibrils or bind plasma FN, which is then deposited in the damaged area [36, 40, 55]. We demonstrated that matrices of FN in complex with TG2 have a distinctive adhesive role. In response to TG2-FN, various cell types could largely restore loss of cell adhesion following inhibition of the classical FN ArgGlyAsp (RGD)-dependent adhesion pathway mediated by a5p1 integrin receptors [33]. This process however was not linked to the intrinsic TG2 ability to modify the FN matrix by Ca2+-dependent transamidation, but is consistent with the previously described transamidating-independent role for TG2 in cell-matrix interactions [37, 39]. This matrix complex was sufficient to support the formation of focal contacts in the presence of RGD peptide, the assembly of associated actin stress fibers and activation of focal adhesion kinase. A PKC-a inhibitor (GO6976) negatively affected RGD-independent cell adhesion to TG2-FN but not normal cell adhesion to FN, suggesting the involvement of PKC-a [33].

At this stage, it is still not entirely clear how signals are conveyed from the extracellular TG2-FN. However, we demonstrated that treatment of human osteoblasts cells with heparitinase, an enzyme that catalyses the eliminative cleavage of heparin and heparan sulfate, greatly diminished the RGD-indepen-dent adhesion in response to TG2-FN, suggesting that cell-surface heparan sulphate proteoglycans may mediate RGD-independent cell adhesion to TG2-FN (see also fig. 4).

Such a complex of TG2 and FN may be necessary to ensure adhesion-mediated cell survival in situations of cell wounding or cell stress, where the increased expression of matrix-degrading metalloproteinases, would lead to fragmentation of the ECM and formation of matrix peptides which act as competitive inhibitors of the classic RGD-dependent cell adhesion [33].

Role of TG2 in Cell Migration

Participation of TG2 in cellular processes which are relevant to wound healing, such as cell migration, has been well described [39, 56, 49] although, controversy still exists on whether these activities depend on the TG-mediated protein modification as opposed to a TG matrix structural role [37] or both or an intracellular G-protein function [49]. Cell migration on FN has been shown to depend on the expression of TG2 and does not seem to require TG2 transamidating activity [39]. The ability of anti-TG2 monoclonal antibody Cub7402 to reduce either cell adhesion or migration on FN in a dose-dependent manner in many cell types [36, 39, 45, 57] is reminiscent of the modulation of cell-matrix interactions by antibodies directed against cell surface integrin receptors ( and a5 and indicates that cell-surface TG2 is an important component in the migration of cells. Akimov and Belkin [56] have shown that TG2 is expressed on the surface of monocytic cells and it is also involved in the adhesion and migration of monocytic cells on a FN matrix. These authors also showed that TG2 might act as a coreceptor of ( and (3 integrins by mediating cell adhesion to the gelatin-binding site of FN [37]. In contrast, migration of fibroblasts on collagen does not seem to be related to its role as a structural protein since unlike migration studies on FN matrices incubation of cells with antibody to TG2 does not modulate cell migration [49].

Role of TG2 in the Inflammatory Response

Soon after tissue damage the inflammatory phase starts with neutrophils and monocytes migrating into the wound tissue to destroy tissue debris and pathogenic microorganisms (fig. 1a). An increase in TG2 activity has also been associated with the inflammatory phase, with the majority of attention focused on macrophages, which mediate the transition between inflammation and repair [1]. The TG2 antigen was found to be particularly expressed in macrophages, adjacent to the re-epithelialization zone and in the provisional fibrin matrix during rat dermal wound healing [11]. It was also reported that inflammatory cytokines such as IFN-7 in rat small intestinal cells, IL-6 or TGF-(1 might induce TG2 expression, which in turn could also affect the adhesion/motility of white blood cells during inflammation [58 and references therein].

TG2 and Activation of Phospholipase A2

Some reports have clearly linked TG2 with the inflammatory response by demonstrating that TG2 enhances the activity of sPLA2s (secretory isoforms of phospholipase A2). This would occur through the formation of an isopeptide bond within sPLA2 either via its crosslinking or the incorporation of polyamines into sPLA2s [59, 60]. The activated sPLA2 would then enhance the release of arachidonic acid from the cell membrane during inflammation, which is the rate limiting step in the biosynthesis of eicosanoids by cyclooxygenase. Sohn et al. [59] have recently shown that new chimeric peptides which are derived from proelafin, a cornified cell envelope component discovered in hyperproliferative epidermis, and antiflammins, peptides originating from natural PLA2-inhibitory proteins, can inhibit sPLA2 and TG2 activity including TG2-mediated modification of sPLA2 and display strong in vivo anti-inflammatory activity. In vivo studies conducted in a transgenic mouse model where TG2 is specifically over-expressed in ventricular myocytes (table 1) [27, 28], have confirmed the in vitro prediction of a link between TG2 upregulation and cyclooxygenase-2. The consequent cardiomyocyte overexpression of thromboxane synthase (TxS) and the receptor for thromboxane (TxA2) results in cardiac failure [27].

TG2 and Inflammatory Conditions

The implication of TG2 in inflammation is further supported by many reports showing the association of TG2 with various inflammatory conditions that are characterized by mucosal inflammation, such as celiac disease [61-63], other intestinal diseases such as Crohn's disease and other autoimmune diseases affecting various tissues and associated with chronic inflammation (e.g. sporadic inclusion body myositis or SIBM) [3, 64 and reference therein].

Wound Healing and Fibrosis in the Kidney

As noted earlier in the introduction to cutaneous wound healing, there are a number of events in the wound healing process that are common to all tissues. For example the kidney, like all other tissues and organs is susceptible to frequent insult and wounding encountered in daily living, e.g. dietary components, drugs, environmental factors and disease. This triggers a programmed and controlled repair process that is not that dissimilar to that found in skin [65], which ultimately terminates and resolves. However, since the kidney is an internal organ both the damage and repair process go virtually unnoticed. In fact, the only time it becomes apparent that these processes are occurring is when the insult becomes chronic or severe, or if the repair process becomes aberrant.

Aberrant Wound Healing in the Kidney Leading to Progressive

Renal Scarring

The highly efficient wound resolution within the kidney (as in all other major internal organs) remains largely unnoticed, however it is aberrations in this process that lead to greater than 99% of all cases of end-stage kidney failure requiring either dialysis or transplantation. The problems arise when the




Early scarring

Advanced scarring

End-stage kidney failure


Early scarring

Advanced scarring

Falling kidney function

End-stage kidney failure

Fig. 2. Progressive scarring in the kidney - aberrant wound healing. The images are stained with Masson's Trichrome which stains collagenous material blue/green, cell cytoplasm pink and nuclei purple. In normal glomeruli there is a thin discrete glomerular basement membrane (GBM), a clearly defined Bowman's Space, the blood capillaries have good spatial orientation each supported by podocytes and mesangial cells. In progressive scarring the GBM progressively thickens, the Bowman's space fills with collagenous material and mesangial cell and fibroblast proliferation becomes deregulated. This ultimately leads to podocyte death, capillary collapse and a remodeled glomerulus completely filled with ECM. In the normal tubulointerstitium there is intricate tight packing of tubules, each having a fine bore for optimal reabsorption. There is a small interstitial space with minimal fibroblasts detectable. Disease development is seen in both hypertrophy and flattening of the tubular epithelium leading to a distended lumen and tubular atrophy to epithelial cell loss. Interstitial fibroblasts proliferate and inflammatory cells invade the interstitium. The interstitial space widens as thickening of the tubular basement membrane (TBM) occurs due to excessive ECM accumulation. At end-stage kidney failure the tubulointerstitium is little more than a sea of collagenous material packed with fibroblasts with a few remaining tubules.

Fig. 3. a-d Tissue transglutaminase (TG2) staining (red) in normal human cortical tubulointerstitium (a) and glomeruli (b) compared to that in renal scarring resulting from focal segmental glomerulosclerosis (c, d). Arrow 1 indicates increased staining in the expanded tubulointerstitium, arrow 2 the mesangial matrix/glomerular basement membrane and arrow 3 periglomerular. e-h e(7-glutamyl)lysine staining (red) in biopsies from implanted human allografts [cortical tubulointerstitium (e) and Glomeruli (f)] compared to that in scarred grafts resulting from chronic rejection in the glomeruli (g) and cortical tubulointerstitium (h). Arrows indicate as in panels a-d. i-l Transglutaminase in situ activity (red)

Fig. 3. a-d Tissue transglutaminase (TG2) staining (red) in normal human cortical tubulointerstitium (a) and glomeruli (b) compared to that in renal scarring resulting from focal segmental glomerulosclerosis (c, d). Arrow 1 indicates increased staining in the expanded tubulointerstitium, arrow 2 the mesangial matrix/glomerular basement membrane and arrow 3 periglomerular. e-h e(7-glutamyl)lysine staining (red) in biopsies from implanted human allografts [cortical tubulointerstitium (e) and Glomeruli (f)] compared to that in scarred grafts resulting from chronic rejection in the glomeruli (g) and cortical tubulointerstitium (h). Arrows indicate as in panels a-d. i-l Transglutaminase in situ activity (red)

insult becomes chronic rather than acute [65]. In the kidney, this can occur for many reasons. Worldwide, the largest cause of chronic kidney disease is diabetes (both type 1 and 2) leading to diabetic nephropathy in about 30% of diabetic patients [United States Renal Database System 2003 (USRDS)]. Other causes of chronic kidney damage can result from autoimmunity to the basement membrane resulting in glomerulonephritis, high blood pressure or drug toxicity. Once the insult becomes chronic, then the wound healing also becomes chronic leading to a nonresolving inflammatory response, fibroblast proliferation [66] and ultimately a massive overexpression and accumulation of ECM components leading to cell deletion and destruction of the kidney [67]. This remodeling once progressive leads to scarring and fibrosis of the kidney and eventually the subsequent deletion of specialized renal cells and the ultimate destruction of the kidney (fig. 2).

The Role of TG2 in Wound Healing and Scarring in the Kidney

Recent studies on progressive kidney scarring both in animal models and using human biopsy material have shown that there are considerable increases in the levels of TG2 and most importantly the e(7-glutamyl)lysine isodipeptide cross-link during the scarring process suggesting that TG2 may have a role in progressive renal scarring (fig. 3) [42, 68, 69, 70, 72]. However, there is also a significant level of TG2 protein in a normal kidney [68]. In localized areas of damage, the cells e.g. tubular, epithelial, mesangial, within and surrounding the lesion stain far more strongly for TG2, but more importantly there is infinitely stronger staining for TG2 outside the cell in a pattern that would be consistent with TG2 localized within the ECM that forms the basement membranes; either glomerular or tubular depending on the location. These observations are highly significant in that they confirm first, that following stress/damage TG2 can move to the extracellular environment where the high Ca2+ and low nucleotide levels would lead to the activation of TG2 [32, 35]. Secondly, like the observations for cutaneous wound healing, that TG2 also has a normal role in the wound response as outlined in the earlier sections of this chapter.

in normal rat kidney (i) and that from the 5/6th subtotal nephrectomy model of renel scarring (/-/). k excludes autofluorescent emissions (green) showing renal morphology. Arrow 4 shows activity is strongest peritubular in the tubular basement membrane and that TG2 in the expanded ECM loses most of its activity. m-q TG2 in situ hybridization (black) in normal rat kidney (m) and that from a 5/6th subtotal nephrectomy model of renal scarring (n, o). Arrow 5 shows that proximal tubular cells are the predominant source of TG2 in renal scarring; although arrow 6 indicates isolated patches of interstitial cells (macrophages or myofibroblasts) are also able to synthesize TG2. p shows a glomeruli from a patient with crescentic nephritis with TG2 synthesis within the scarring crescent (arrow 7) (myofibroblasts) and q shows mesangial cell synthesis in a patient with mesangial proliferative glomerulonephritis.

TG2 in Progressive Renal Scarring

In some renal conditions, most notably diabetic nephropathy, there is an initial (i.e. preclinical presentation) increase in e(7-glutamyl)lysine levels independent of changes in overall renal TG levels as measured by the activation of total renal TG and in situ enzyme activity [69]. The e(7-glutamyl)lysine dipeptide bonds are located within an expanding ECM and seem to result initially from export of existing levels of TG2, although the contribution of other isoforms cannot be excluded [69]. As the disease develops and moves into a progressive scarring phase, there is an increase of TG2 at the mRNA and protein level predominantly in tubular epithelial cells (proximal and loop of Henle cells mainly) and mesangial cells (fig. 3), irrespective of the disease type [70-72]. Most importantly, most of this increased production of TG2 in epithelial cells is passed straight out of the cell leading to a massive pool of TG2 in the extracellular environment where high Ca2+ and low nucleotide levels lead to its activation, an event that can be recreated in cell culture [73]. With increasing levels of enzyme secreted from cells, modulation of its activity either through the binding of ECM FN or through proteolytic degradation [54] becomes increasingly ineffective resulting in the cross-linking of any appropriate substrate. This results in both quantitative and qualitative changes to the local ECM affecting both its deposition and proteolytic clearance [18, 48, 74]. Analysis of diseased kidneys clearly shows that the TG2 antigen, TG activity and increased levels of e(7-glutamyl)lysine are associated with expanding tubular and glomerular basement membranes indicating ECM components as the major target for the enzyme (fig. 3) [42, 65, 71, 72].

In vitro, the effect of growing renal cells (mesangial and tubular epithelial cells) in a high glucose environment is associated with an increased synthesis and accumulation of ECM and represents a basic model of diabetic nephropathy [75, 76]. These changes are thought to be driven through the production of various growth factors, most notably TGF-^1 [77] through a PDGF loop [78]. Recently, using OK proximal tubular epithelial cells we have been able to demonstrate that increases in ECM and particularly collagen are associated with the increases in glucose levels [93] and that these changes lead to increases in TG2 synthesis and subsequent changes in TG2 and e(7-glutamyl)lysine in the ECM [48]. To test if TG2 had any role in the accumulation of ECM or it is concomitant to this, similar studies have been performed in the presence of a specific site-directed TG inhibitor 1,3-dimethyl-2[(oxopropyl)thio] imidazolium [79, 80]. These studies categorically show that the level of ECM accumulated can be reduced if TG2 is blocked thus confirming a causative role in the accumulation of ECM associated with hyperglycemia [48]. Further, they clearly show that TG2 has a direct action on the ECM, as the effects of these inhibitors are independent of TGF-p1 synthesis or activity in this cell line over the time frame used.

In support of these results is the finding that ECM generated from proximal tubular epithelial cells transfected to give increased expression of TG2 has a higher collagen content with increased resistance to trypsin, MMP-1 and MMP-2 [74, and unpubl. data]. To determine the importance of TG2 in the aberrant wound healing leading to scarring, fibrosis and loss of renal function in the kidney, we have used mini osmotic pump technology to deliver a number of site-directed TG inhibitors to the kidney. As well as the well established site-directed inhibitor 1,3-dimethyl-2[(oxopropyl)thio] imidazolium [79, 80], an inhibitor based on a synthetic CBZ-glutaminyl glycine analogue molecule that has poor transfer across the cell membrane (thus effectively limiting its action to the extracellular compartment) [48] has been used. Using the 5/6th subtotal nephrectomy model of focal segmental glomerulosclerosis [81] and the streptozotocin model of type 1 diabetes mellitus and diabetic nephropathy (unpubl. data), we have been able to reduce the elevated levels of extracellular TG2 activity in excess of 50% for up to 8 months which results in an almost total prevention of the build up in e(7-glutamyl)lysine that is associated with kidney scarring. Most importantly the consequence of the prevention of e(7-glutamyl)lysine cross-linking is to dramatically reduce the degree of scarring that occurs in both these models of renal failure [81]. Anti-TG compounds therefore offer great potential not only in the treatment of kidney scarring, but the modulation of all types of tissue scarring due to the similarity between different tissues.

Dual Approach to Transglutaminase-Based Wound Repair: Transglutaminase as a Novel Molecular Target and as a Biocatalyst for Tissue Engineering

In general, wounds can be managed by a variety of approaches depending on the severity of the injury but treatments for severe and chronic wounds (e.g. skin burns, diabetic ulcers, critical-size bone fractures) and conditions leading to abnormal repair (e.g. fibroproliferative conditions) are still difficult to manage. Healing promotion by modulation of cytokines or other wound mediators is an attractive strategy e.g. neutralizing antibodies towards growth factors can be used to inhibit their function in cutaneous wounds and wound fluids [82]. However, their clinical usage has been limited by the complexity and range of cytokines and other wound mediators involved in cutaneous healing whose role in vivo is only partly understood. The use of tissue-engineered products both acellular, such as matrices that stimulate cell migration, angiogenesis and induce growth factor function, and cellular-based products has demonstrated to be a valid approach for wound repair and healing of chronic wounds [83, 84].

Table 2. Examples of transglutaminase-based wound repair: Transglutaminase as a novel molecular target and as a biocatalyst for tissue engineering
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