Increased transglutaminase activity during skin wound healing in rats

doi:10.1016/0304-4165(88)90014-1

Biochimica et Biophysica Acta (BBA) - General Subjects

Volume 967, Issue 2, 17 November 1988, Pages 234-240

https://doi.org/10.1016/0304-4165(88)90014-1 Get rights and content

Abstract

Outer, middle and inner layers from wounded or unwounded rat dorsal skin were separated and extracted first with buffer and then with Triton X-100 and dithiothreitol. The extracts and residues were assayed for transglutaminase activity and tissue transglutaminase antigen. Transglutaminase activities in all skin layers are increased in the period 1–5 days after wounding. Most of the increased activity is in the buffer-soluble fraction in the inner skin layer though there is no corresponding increase in antigen in this fraction. This suggests that there is production of activated soluble tissue transglutaminase in the wounded inner layer. In the 3–5-day wounded outer layer the largest fraction of both activity and antigen is associated with the insoluble residue remaining after extraction with Triton X-100. On DEAE-cellulose chromatography Triton X-100 extracts of the inner layer of wounded skin showed a single major peak of activity, corresponding approximately with rabbit liver transglutaminase; the outer layer showed the same peak plus a different one, eluting at lower salt concentration, which is thought to be epidermal transglutaminase.

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TG2 appears to promote net formation of ECM molecules such as fibronection and collagen by irreversible crosslinking of these molecules in tissue repair. TG2 expression and activity were increased following cutaneous wounding in animal models (Bowness et al. 1988; Upchurch et al. 1991; Raghunath et al. 1996). In addition, infiltrating macrophages in injured tissue express TG2 (Haroon et al. 1999).
Transglutaminase 2 (TG2) is a multifunctional protein that contributes to inflammatory disease when aberrantly expressed. Although macrophages express TG2, the factor stimulating TG2 expression remains poorly characterized in these cells. In the present study, we examined the effects of the stress-related catecholamines adrenaline and noradrenaline on macrophage expression of TG2 in RAW264.7 murine macrophages and murine bone marrow-derived macrophages. Treatment with adrenaline markedly increased TG2 mRNA expression and increased TG2 protein levels. While the β₂-adrenoceptor-selective antagonist ICI 118,551 completely blocked adrenaline-induced TG2 mRNA expression, the β₂-adrenoceptor specific agonist salmeterol increased TG2 expression. Noradrenaline also increased TG2 mRNA expression at higher doses than the effective doses of adrenaline. The effect of adrenaline on TG2 mRNA expression was mimicked by treatment with the membrane-permeable cAMP analog 8-Br-cAMP. Thus, increased intracellular cAMP following stimulation of β₂-adrenoceptors appeared to be responsible for adrenaline-induced TG2 expression. Because stress events activate the sympathetic nervous system and result in secretion of the catecholamines, adrenoceptor-mediated increase in macrophage TG2 expression might be associated with stress-related inflammatory disorders.
Complex formation between tissue transglutaminase II (tTG) and vascular endothelial growth factor receptor 2 (VEGFR-2): Proposed mechanism for modulation of endothelial cell response to VEGF
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We have recently demonstrated that thrombin-activated FXIII (FXIIIA-subunit), a plasma transglutaminase, activates VEGFR-2 by crosslinking it with the α_vβ₃ integrin on the surface of endothelial cells (EC), thereby stimulating angiogenesis. Tissue transglutaminase (tTG), which is functionally and structurally related to FXIIIA, is expressed by numerous cell types, among them EC. However, its role in EC function has not been fully characterized. In the present study, we investigated the potential involvement of tTG in angiogenesis. Using co-immunoprecipitation and immunofluorescent staining experiments, we observed that tTG forms a complex with VEGFR-2 on the cell surface and within the cytoplasm of EC. Stimulation of EC with VEGF resulted in translocation of the tTG–VEGFR-2 complex from the cytoplasm to the nucleus. In VEGF-treated cells, tTG–VEGFR-2 interaction resulted in incorporation of VEGFR-2 into high molecular weight crosslinked complex (es), as revealed by an antibody against γ-glutamyl-ε-lysine isopeptide bond. tTG –VEGFR-2 association was inhibited by a specific VEGFR-2 protein tyrosine kinase inhibitor (PTKI ), as well as by cystamine, inhibitor of the transglutaminase activity of tTG, but not by bacitracin which inhibits the protein-disulfide isomerase (PDI) activity of tTG. Furthermore, cystamine completely abolished the VEGF-induced nuclear translocation of the tTG–VEGFR-2 complex. Blockade of the crosslinking activity of tTG by cystamine enhanced VEGF-induced migration of EC in Boyden chamber by 31% (P < 0.02), and prolonged VEGF-induced signaling response, as demonstrated by sustained activation of the MAP kinase ERK. Taken together, our findings suggest that endothelial cell tTG might be involved in modulation of the cellular response to VEGF by forming an intracellular complex with VEGFR-2, and mediating its translocation into the nucleus upon VEGF stimulation.
Hypertrophic scar cells fail to undergo a form of apoptosis specific to contractile collagen - The role of tissue transglutaminase
2005, Journal of Investigative Dermatology
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The stabilization of newly formed ECM is thought to be largely accomplished through the actions of tissue transglutaminase (or transglutaminase II) (Ricard-Blum et al, 1996; Grenard et al, 2001). The activity of this enzyme is markedly increased during wound healing, substantially effecting both the breaking strength of wounds and the solubility of ECM proteins (Bowness et al, 1987b,1988; Dolynchuk et al, 1994; Haroon et al, 1999). Tissue transglutaminase cross-links proteins through the formation of isopeptide bonds between reactive γ-glutaminyl groups in certain specific proteins and the ε-amino group of lysine in other proteins.
Failure of apoptosis has been postulated to cause the hypercellularity and thus excess scar-tissue formation of hypertrophic scars (HTS). Here, we have examined the susceptibility of fibroblasts derived from normal or HTS to apoptosis induced during collagen-gel contraction, a wound-healing model. Normal scar (NS) fibroblasts underwent significant apoptosis (>40% total) in contractile collagen, whereas apoptosis was not detected in HTS cells. This inability was specific to apoptosis induced by contractile collagen because apoptosis could be induced using diverse modalities. Since chronic fibrotic tissue is known to be excessively cross-linked, we next examined whether collagen matrix that had been conditioned by HTS fibroblasts became refractory to enzymatic breakdown and indeed, found that it is resistant to breakdown by both collagenase D and matrix metalloproteinase-2. Newly formed extracellular matrix is stabilized by the enzyme, tissue transglutaminase, which we demonstrated to be overexpressed by HTS fibroblasts in vivo and in vitro. Reducing tissue transglutaminase activity in collagen gels containing HTS fibroblasts permitted induction of apoptosis on gel contraction, whereas increasing enzymic activity in NS cell-containing gels completely abrogated collagen-contraction-induced-apoptosis. Together, these observations show that HTS fibroblasts exhibit resistance to a specific form of apoptosis elicited by contraction of collagen gels, and that this phenomenon is dependent on excess activity of cell surface tissue transglutaminase.
Atorvastatin induces tissue transglutaminase in human endothelial cells
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Tissue transglutaminase (tTgase) contributes to the organisation of the basement membrane and is therefore thought to be important for the integrity and stability of the vessel wall. In the present study, we hypothesised that the HMG-CoA reductase inhibitor atorvastatin may up-regulate the tTgase expression in endothelial cells and thereby exert beneficial effects on endothelial function. Treatment of human umbilical vein endothelial cells (HUVEC) with atorvastatin (1–10 μM) caused a clear increased expression of tTgase in both permeabilised and non-permeabilised HUVEC. In contrast, stimulation of HUVEC with TNFα had no substantial effect on tTgase expression or localisation but inhibited the atorvastatin-induced up-regulation and externalisation of tTgase. Propidium iodide staining revealed that statin-induced apoptosis is not responsible for the enhanced expression. By inducing the expression of tTgase, statins may promote tTgase-mediated stabilisation of the basement membrane. This effect of atorvastatin may contribute to the beneficial role of statins on endothelial function.
Importance of tissue transglutaminase in repair of extracellular matrices and cell death of dermal fibroblasts after exposure to a solarium ultraviolet a source
2003, Journal of Investigative Dermatology
Citation Excerpt :
From a more physiologic context we have also observed this increase in matrix associated tTgase activity without increase in enzyme expression in the tubulo-interstitium of rat kidney during renal scarring for hyperglycemia (Skill et al, 2001). A comparable observation was also demonstrated byBowness et al (1988) following the mechanical wounding of rat skin. Like factor XIIIa, tTgase may therefore have a defined physiologic function in the wound healing process through the stabilization of extracellular matrix proteins such as fibronectin and other extracellular matrix proteins known to act as substrates for the enzyme (Aeschlimann and Thomazy, 2000) following tissue damage.
Investigations were undertaken to study the role of the protein cross-linking enzyme tissue transglutaminase in changes associated with the extracellular matrix and in the cell death of human dermal fibroblasts following exposure to a solarium ultraviolet A source consisting of 98.8% ultraviolet A and 1.2% ultraviolet B. Exposure to nonlethal ultraviolet doses of 60 to 120 kJ per m² resulted in increased tissue transglutaminase activity when measured either in cell homogenates, “in situ” by incorporation of fluorescein-cadaverine into the extracellular matrix or by changes in the ε(γ-glutamyl) lysine cross-link. This increase in enzyme activity did not require de novo protein synthesis. Incorporation of fluorescein-cadaverine into matrix proteins was accompanied by the cross-linking of fibronectin and tissue transglutaminase into nonreducible high molecular weight polymers. Addition of exogenous tissue transglutaminase to cultured cells mimicking extensive cell leakage of the enzyme resulted in increased extracellular matrix deposition and a decreased rate of matrix turnover. Exposure of cells to 180 kJ per m² resulted in 40% to 50% cell death with dying cells showing extensive tissue transglutaminase cross-linking of intracellular proteins and increased cross-linking of the surrounding extracellular matrix, the latter probably occurring as a result of cell leakage of tissue transglutaminase. These cells demonstrated negligible caspase activation and DNA fragmentation but maintained their cell morphology. In contrast, exposure of cells to 240 kJ per m² resulted in increased cell death with caspase activation and some DNA fragmentation. These cells could be partially rescued from death by addition of caspase inhibitors. These data suggest that changes in cross-linking both in the intracellular and extracellular compartments elicited by tissue transglutaminase following exposure to ultraviolet provides a rapid tissue stabilization process following damage, but as such may be a contributory factor to the scarring process that results.
Transglutaminase-mediated gelatin matrices incorporating cell adhesion factors as a biomaterial for tissue engineering
2003, Journal of Bioscience and Bioengineering
The goal of this work was to develop a novel biomaterial to be used for either wound dressing or as a scaffold for tissue engineering. The biodegradable hydrogels were prepared through cross-linking of gelatin with transglutaminase (TGase) in an aqueous solution. We found that the concentrations of 5 wt% gelatin and 1 unit/ml TGase were optimum for the proliferation of NIH/3T3 fibroblasts. Then, we investigated whether the cell proliferation was enhanced by incorporation of cell adhesion factors into the gelatin hydrogels. Since vitronectin and fibronectin can bind with gelatin by the action of TGase, we added these cell adhesion proteins into the gelatin hydrogels. The hydrogels incorporating these cell adhesion proteins significantly enhanced cell proliferation compared with the gelatin hydrogels without these proteins (p<0.05). Two types of synthetic Arg-Gly-Asp (RGD) peptides, RGDLLQ and RGDLLG were also added to the gelatin solution where RGDLLQ is a substrate of TGase by virtue of a glutamine (Q) residue with an ε-amino group and RGDLLG is not. These two RGD peptides enhanced cell proliferation, but RGDLLQ significantly enhanced the proliferation compared with RGDLLG (p<0.05). These results suggest that T-Gase-mediated incorporation of cell adhesion factors into gelatin matrices enhanced cell proliferation and this novel biomaterial is a potent tool for wound dressing or tissue engineering.

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Increased transglutaminase activity during skin wound healing in rats

Abstract

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Connect. Tissue Res.

Annu. Rev. Biochem.