Proteolysis of Cell-Surface Tissue Transglutaminase by Matrix Metalloproteinase-2 Contributes to the Adhesive Defect and Matrix Abnormalities in Thrombospondin-2-Null Fibroblasts and Mice
2005; Elsevier BV; Volume: 167; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)62955-0
ISSN1525-2191
AutoresAzin Agah, Themis R. Kyriakides, Paul Börnstein,
Tópico(s)Tendon Structure and Treatment
ResumoThrombospondin (TSP)-2-null dermal fibroblasts display an attachment defect that results from increased matrix metalloproteinase (MMP)-2 levels in their conditioned media. To investigate the molecular mechanisms responsible for this defect, we analyzed the activity of tissue transglutaminase (tTG) in TSP-2-null dermal fibroblasts and in tissues of TSP-2-null mice. tTG functions as a co-receptor for β1 and β3 integrins and stabilizes extracellular matrix proteins by introduction of isopeptide cross-links. Cell-surface tTG activity was reduced in TSP-2-null cells (0.50 ± 0.05 arbitrary units versus 0.84 ± 0.07 for wild type; P ≤ 0.05), and addition of MMP-2 to the culture medium of wild-type cells caused a 35% reduction in cell-surface tTG activity. tTG was susceptible to proteolysis by MMP-2 in vitro, and addition of the MMP inhibitor TIMP-2 to TSP-2-null cells restored tTG activity (0.3 ± 0.08 for untreated cells; 0.71 ± 0.09 with TIMP-2). TSP-2-null mice had reduced tTG activity in skin, as measured by incorporation of fluorescein isothiocyanate-labeled cadaverine, and a threefold increase in acetic acid-extracted dermal collagen. Furthermore, isopeptide cross-links were reduced in both uninjured skin and in excisional wounds of TSP-2-null mice, as determined by morphometric immunohistochemical analysis, indicating that isopeptide cross-links are important for the stabilization of the collagenous matrix in dermis. These findings provide a mechanism for the reduced adhesion of TSP-2-null fibroblasts and an explanation for the increased collagen solubility and fragility of TSP-2-null skin. Thrombospondin (TSP)-2-null dermal fibroblasts display an attachment defect that results from increased matrix metalloproteinase (MMP)-2 levels in their conditioned media. To investigate the molecular mechanisms responsible for this defect, we analyzed the activity of tissue transglutaminase (tTG) in TSP-2-null dermal fibroblasts and in tissues of TSP-2-null mice. tTG functions as a co-receptor for β1 and β3 integrins and stabilizes extracellular matrix proteins by introduction of isopeptide cross-links. Cell-surface tTG activity was reduced in TSP-2-null cells (0.50 ± 0.05 arbitrary units versus 0.84 ± 0.07 for wild type; P ≤ 0.05), and addition of MMP-2 to the culture medium of wild-type cells caused a 35% reduction in cell-surface tTG activity. tTG was susceptible to proteolysis by MMP-2 in vitro, and addition of the MMP inhibitor TIMP-2 to TSP-2-null cells restored tTG activity (0.3 ± 0.08 for untreated cells; 0.71 ± 0.09 with TIMP-2). TSP-2-null mice had reduced tTG activity in skin, as measured by incorporation of fluorescein isothiocyanate-labeled cadaverine, and a threefold increase in acetic acid-extracted dermal collagen. Furthermore, isopeptide cross-links were reduced in both uninjured skin and in excisional wounds of TSP-2-null mice, as determined by morphometric immunohistochemical analysis, indicating that isopeptide cross-links are important for the stabilization of the collagenous matrix in dermis. These findings provide a mechanism for the reduced adhesion of TSP-2-null fibroblasts and an explanation for the increased collagen solubility and fragility of TSP-2-null skin. The phenotype of the thrombospondin (TSP)-2-null mouse has provided investigators with a number of important clues to the regulation of the assembly and stabilization of the extracellular matrix (ECM). TSP-2-null fibroblasts show a marked adhesive defect, and mice that lack TSP-2 have fragile skin of low tensile strength,1Kyriakides TR Zhu YH Smith LT Bain SD Yang Z Lin MT Danielson KG Iozzo RV LaMarca M McKinney CE Ginns EI Bornstein P Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis.J Cell Biol. 1998; 140: 419-430Crossref PubMed Scopus (393) Google Scholar but heal excisional skin wounds more rapidly and with less scarring than controls.2Kyriakides TR Tam JW Bornstein P Accelerated wound healing in mice with a disruption of the thrombospondin 2 gene.J Invest Dermatol. 1999; 113: 782-787Crossref PubMed Scopus (139) Google Scholar Reduced tensile strength and tissue integrity have also been documented in the pregnant cervix3Kokenyesi R Armstrong LC Agah A Artal R Bornstein P Thrombospondin 2 deficiency in pregnant mice results in premature softening of the uterine cervix.Biol Reprod. 2004; 70: 385-390Crossref PubMed Scopus (29) Google Scholar and in the injured myocardium of the TSP-2-null mouse.4Schroen B Heymans S Sharma U Blankesteijn WM Pokharel S Cleutjens JP Porter JG Evelo CT Duisters R van Leeuwen RE Janssen BJ Debets JJ Smits JF Daemen MJ Crijns HJ Bornstein P Pinto YM Thrombospondin-2 is essential for myocardial matrix integrity: increased expression identifies failure-prone cardiac hypertrophy.Circ Res. 2004; 95: 515-522Crossref PubMed Scopus (154) Google Scholar At the level of the light microscope, collagen fibers are disorganized in dermis1Kyriakides TR Zhu YH Smith LT Bain SD Yang Z Lin MT Danielson KG Iozzo RV LaMarca M McKinney CE Ginns EI Bornstein P Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis.J Cell Biol. 1998; 140: 419-430Crossref PubMed Scopus (393) Google Scholar and in granulation tissue of healing skin wounds,2Kyriakides TR Tam JW Bornstein P Accelerated wound healing in mice with a disruption of the thrombospondin 2 gene.J Invest Dermatol. 1999; 113: 782-787Crossref PubMed Scopus (139) Google Scholar, 5Agah A Kyriakides TR Letrondo N Bjorkblom B Bornstein P Thrombospondin 2 levels are increased in aged mice: consequences for cutaneous wound healing and angiogenesis.Matrix Biol. 2004; 22: 539-547Crossref PubMed Scopus (56) Google Scholar and collagen fibrils show an abnormal size distribution with irregular contours,1Kyriakides TR Zhu YH Smith LT Bain SD Yang Z Lin MT Danielson KG Iozzo RV LaMarca M McKinney CE Ginns EI Bornstein P Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis.J Cell Biol. 1998; 140: 419-430Crossref PubMed Scopus (393) Google Scholar and abnormalities in fibroblast-matrix interactions in the electron microscope.6Bornstein P Kyriakides TR Yang Z Armstrong LC Birk DE Thrombospondin 2 modulates collagen fibrillogenesis and angiogenesis.J Invest Dermatol Symp Proc. 2000; 5: 61-66Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar A pathogenesis for these matrix abnormalities was suggested by the findings that matrix metalloproteinase (MMP)-2 levels are increased in cultured fibroblasts of TSP-2-null mice, and that the adhesive defect in these cells could be reversed by inhibitors of MMP-2.7Yang Z Kyriakides TR Bornstein P Matricellular proteins as modulators of cell-matrix interactions: adhesive defect in thrombospondin 2-null fibroblasts is a consequence of increased levels of matrix metalloproteinase-2.Mol Biol Cell. 2000; 11: 3353-3364Crossref PubMed Scopus (175) Google Scholar Elevated levels of MMP-2 were also documented in tissues of TSP-2-null mice after a variety of injuries, eg, in the foreign body reaction to subcutaneously implanted biomaterials,8Kyriakides TR Zhu YH Yang Z Huynh G Bornstein P Altered extracellular matrix remodeling and angiogenesis in sponge granulomas of thrombospondin 2-null mice.Am J Pathol. 2001; 159: 1255-1262Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar in healing skin wounds,5Agah A Kyriakides TR Letrondo N Bjorkblom B Bornstein P Thrombospondin 2 levels are increased in aged mice: consequences for cutaneous wound healing and angiogenesis.Matrix Biol. 2004; 22: 539-547Crossref PubMed Scopus (56) Google Scholar in the circumferentially stressed pregnant cervix,3Kokenyesi R Armstrong LC Agah A Artal R Bornstein P Thrombospondin 2 deficiency in pregnant mice results in premature softening of the uterine cervix.Biol Reprod. 2004; 70: 385-390Crossref PubMed Scopus (29) Google Scholar and in injured myocardium.4Schroen B Heymans S Sharma U Blankesteijn WM Pokharel S Cleutjens JP Porter JG Evelo CT Duisters R van Leeuwen RE Janssen BJ Debets JJ Smits JF Daemen MJ Crijns HJ Bornstein P Pinto YM Thrombospondin-2 is essential for myocardial matrix integrity: increased expression identifies failure-prone cardiac hypertrophy.Circ Res. 2004; 95: 515-522Crossref PubMed Scopus (154) Google Scholar In some of these tissues, MMP-9 was also increased. An explanation for accumulation of MMP-2 in tissues of TSP-2-null mice was provided by the demonstration that MMP-2 is normally cleared from the pericellular environment by the formation of a TSP-2-MMP-2 complex that is endocytosed by the LRP1 scavenger receptor.9Yang Z Strickland DK Bornstein P Extracellular matrix metalloproteinase 2 levels are regulated by the low density lipoprotein-related scavenger receptor and thrombospondin 2.J Biol Chem. 2001; 276: 8403-8408Crossref PubMed Scopus (237) Google Scholar These findings, although implicating proteolysis by MMP-2 in the generation of matrix abnormalities in TSP-2-null mice, raise the question of the target of that proteolysis. MMP-2 is capable of degrading many matrix proteins, including type I collagen and fibronectin,10Mott JD Werb Z Regulation of matrix biology by matrix metalloproteinases.Curr Opin Cell Biol. 2004; 16: 558-564Crossref PubMed Scopus (857) Google Scholar, 11Aimes RT Quigley JP Matrix metalloproteinase-2 is an interstitial collagenase: inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4- and 1/4-length fragments.J Biol Chem. 1995; 270: 5872-5876Crossref PubMed Scopus (827) Google Scholar but there is evidence that much of the proteolytic activity of MMP-2 in the extracellular milieu is neutralized by the tight-binding inhibitor, TIMP-2.12Lambert E Dassé E Haye B Petitfrère E TIMPs as multifacial proteins.Crit Rev Oncol Hematol. 2004; 49: 187-198Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar However at the cell surface, pro-MMP-2 is activated by MT1-MMP in a complex with TIMP-2 and free, active MMP-2 is released.13Werb Z Vu TH Rinkenberger JL Coussens LM Matrix-degrading proteases and angiogenesis during development and tumor formation.APMIS. 1999; 107: 11-18Crossref PubMed Scopus (143) Google Scholar There is ample evidence for a role of MMP-2 and other MMPs in regulating cell adhesion and other cellular functions by proteolysis of cell-surface proteins.14Ray JM Stetler-Stevenson WG Gelatinase A activity directly modulates melanoma cell adhesion and spreading.EMBO J. 1995; 14: 908-917Crossref PubMed Scopus (172) Google Scholar, 15Sternlicht MD Werb Z How matrix metalloproteinases regulate cell behavior.Annu Rev Cell Dev Biol. 2001; 17: 463-516Crossref PubMed Scopus (3171) Google Scholar Our attention was drawn to the possibility that tissue transglutaminase (transglutaminase 2; tTG) might serve as a target of the elevated levels of MMP-2 in TSP-2-null mice for several reasons. As shown by Akimov and Belkin,16Akimov SS Belkin AM Cell-surface transglutaminase promotes fibronectin assembly via interaction with the gelatin-binding domain of fibronectin: a role in TGFbeta-dependent matrix deposition.J Cell Sci. 2001; 114: 2989-3000Crossref PubMed Google Scholar cell-surface tTG interacts with β1 and β3 integrins and mediates their association with the gelatin-binding domain of fibronectin, which lacks intrinsic integrin-binding sites. tTG also increases the size of focal adhesions and increases adhesion-dependent phosphorylation of focal adhesion kinase. These properties are not shared by a functional homologue of tTG, factor XIIIA, and do not depend on the catalytic activity of tTG.16Akimov SS Belkin AM Cell-surface transglutaminase promotes fibronectin assembly via interaction with the gelatin-binding domain of fibronectin: a role in TGFbeta-dependent matrix deposition.J Cell Sci. 2001; 114: 2989-3000Crossref PubMed Google Scholar Reduced levels of tTG could account for both reduced cell adhesion and abnormal collagen fibrillogenesis in TSP-2-null mice, since it is now recognized that assembly of collagen monomers into fibers occurs in close association with the cell surface,17Birk DE Trelstad RL Extracellular compartments in tendon morphogenesis: collagen fibril, bundle, and macroaggregate formation.J Cell Biol. 1986; 103: 231-240Crossref PubMed Scopus (280) Google Scholar, 18Canty EG Lu Y Meadows RS Shaw MK Holmes DF Kadler KE Coalignment of plasma membrane channels and protrusions (fibripositors) specifies the parallelism of tendon.J Cell Biol. 2004; 165: 553-563Crossref PubMed Scopus (225) Google Scholar and this association could involve collagen-binding β1 integrins. Finally, tTG is known to stabilize the ECM by the introduction of γ-glutamyl-ε-lysyl isopeptide bond cross-links into many ECM proteins, including collagens and fibronectin.19Lorand L Graham RM Transglutaminases: crosslinking enzymes with pleiotropic functions.Nat Rev Mol Cell Biol. 2003; 4: 140-156Crossref PubMed Scopus (1192) Google Scholar Indeed, we show in this study that tTG is susceptible to proteolysis by MMP-2 in vitro, and that tTG activity is reduced in both fibroblasts in culture and in tissues from TSP-2-null mice. A reduction in tTG activity could therefore account for the inferior structural properties of connective tissues, as summarized above, and for the increased extractability of collagen from the dermis of TSP-2-null mice, as demonstrated in this study. Furthermore, the content of isopeptide cross-links is reduced in TSP-2-null skin and excisional skin wounds. We therefore propose that a reduction in intact tTG protein and activity is a major cause of the connective tissue abnormalities in TSP-2-null mice. All mice used in this study were on a mixed C57BL6/129SvEMS+Ter background. After euthanasia, mice (four of each genotype) were shaved and skinned. Subcutaneous fat was removed by scraping, and the skin was treated with 0.25% w/v trypsin in Dulbecco's modified Eagle's medium (DMEM) at 4°C overnight to detach the epidermis from the dermis. The dermis was then cut into small pieces with a scalpel and digested with 0.25% w/v collagenase in DMEM at 37°C for 3 to 5 hours. The resulting cells were collected by centrifugation, washed twice with DMEM, plated, and maintained in DMEM supplemented with 10% fetal calf serum. Cell attachment assays were performed in 96-well plates coated with fibronectin or vitronectin as previously described.7Yang Z Kyriakides TR Bornstein P Matricellular proteins as modulators of cell-matrix interactions: adhesive defect in thrombospondin 2-null fibroblasts is a consequence of increased levels of matrix metalloproteinase-2.Mol Biol Cell. 2000; 11: 3353-3364Crossref PubMed Scopus (175) Google Scholar Briefly, cultured fibroblasts were trypsinized, washed, and 100 μl of a cell suspension (104 cells) was added to each well. Cells were allowed to attach for 1 hour at 37°C, unattached cells were removed by washing the plates with DMEM, and attachment was quantified by addition of 20 μl of Cell Titer 96 solution (Promega, Madison, WI). After incubation at 37°C for 1 hour, the absorbance at 650 nm was measured in a microplate reader (Molecular Devices, Sunnyvale CA). MMP-2 (25 to 100 ng/ml) (a generous gift from Dr. C.M. Overall, University of British Columbia, Vancouver, BC, Canada) was added to the culture medium for 9 hours to determine its effects on tTG activity and cell attachment. Fibroblasts were suspended in 0.1 mmol/L biotinylated cadaverine (Molecular Probes, Eugene, OR) and plated in fibronectin-coated 96-well plates at 2 × 103 cells/well. After incubation at 37°C for 2 hours, the cells were washed and lysed with deoxycholate. Incorporation of biotinylated cadaverine into soluble protein was detected by addition of avidin-horseradish peroxidase and tetra-methyl benzidine, followed by measurement of absorbance at 450 nm. DNA concentrations of the cell lysates were measured in a fluorimeter using SYBR green dye, and were used to normalize the cell content of the wells. Antibodies to laminin 5 (DAKO, Glostrup, Denmark) were used to test for contamination of fibroblasts with epidermal keratinocytes. Recombinant mouse TIMP-2 (Chemicon, Temicula, CA) was used to inhibit MMP-2 activity. Pro-MMP-2 was activated for 30 minutes at 4°C with 1 mmol/L p-aminophenylmercuric acetate (Sigma Chemical Co., Saint Louis, MO). Purified tTG (5 μg; Diarect Ag, Freiburg, Germany) was incubated with activated MMP-2 (0.3 μg) for 1 hour at 37°C in a buffer containing 0.05 mol/L Tris-HCl, pH 7.5, 50 mmol/L NaCl, 1 mmol/L CaCl2, and 10 μmol/L ZnCl2. The digestion products were analyzed by 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Western analysis for tTG was performed with a polyclonal antibody (Neo-markers, Fremont, CA). Three mice of each genotype were euthanized, shaved, and the skins were removed. After removal of subcutaneous tissue by scraping, the skins were weighed, minced, and extracted in phosphate-buffered saline at 4°C overnight with gentle stirring. The mince was then centrifuged, the supernate discarded, and the sediment extracted with 0.5 N of acetic acid (30:1, v/w) for 2 days at 4°C with gentle stirring. The mixture was centrifuged, the supernate was filtered, and NaCl was added to a final concentration of 10% to precipitate the collagen. After centrifugation, the supernatant was discarded, 0.1 N acetic acid was added to the collagen pellet, and the slurry dialyzed against 0.1 N of acetic acid to dissolve the salt-free collagen. The mince was then subjected to a second extraction and purification. The resulting soluble collagens were combined and lyophilized. Four mice of each genotype were euthanized and shaved, and skin was removed from the back. Skins were minced and homogenized with a Polytron homogenizer in 2 ml of phosphate-buffered saline containing 2 mmol/L phenylmethyl sulfonyl fluoride and 1 μg/ml each of aprotinin, leupeptin, and pepsin A (Sigma Chemical Co.). Protein content was determined by the bicinchoninic acid method according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA). tTG activity in the skin homogenate was measured as described above. Four mice of each genotype, ∼3 months of age and sex-matched, were anesthetized with avertin and 6-mm excisional wounds were made as described previously.2Kyriakides TR Tam JW Bornstein P Accelerated wound healing in mice with a disruption of the thrombospondin 2 gene.J Invest Dermatol. 1999; 113: 782-787Crossref PubMed Scopus (139) Google Scholar Wounded animals were housed individually. Uninjured skin and excised wounds were fixed in 10% zinc-formalin buffer (Z-fix; Anatech, Battle Creek, MI), paraffin-embedded, and sectioned at 5 μm thickness, as described previously.2Kyriakides TR Tam JW Bornstein P Accelerated wound healing in mice with a disruption of the thrombospondin 2 gene.J Invest Dermatol. 1999; 113: 782-787Crossref PubMed Scopus (139) Google Scholar The measurement of tTG activity in situ was performed as previously described.20Johnson TS Abo-Zenah H Skill JN Bex S Wild G Brown CB Griffin M El Nahas AM Tissue transglutaminase: a mediator and predictor of chronic allograft nephropathy?.Transplantation. 2004; 77: 1667-1675Crossref PubMed Scopus (22) Google Scholar Briefly, cryostat sections were washed with a solution containing a cocktail of protease inhibitors and then incubated with fluorescein isothiocyanate cadaverine (Molecular Probes) in 2.5 mmol/L CaCl2 for 1 hour at room temperature. For negative controls, CaCl2 was replaced with 2 mmol/L ethylenediamine tetraacetic acid. Visualization and quantification were performed using fluorescence microscopy. Analysis of cross-link formation was quantified by immunodetection of isopeptide bonds with specific antibodies to ε-lysyl γ-glutaminyl cross-links (Abcam, Cambridge, MA) in paraffin-embedded sections, as described previously.21Agah A Kyriakides TR Lawler J Bornstein P The lack of thrombospondin-1 (TSP1) dictates the course of wound healing in double-TSP1/TSP2-null mice.Am J Pathol. 2002; 161: 831-839Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 22Grenard P Bresson-Hadni S El Alaoui M Chevallier DA Vuitton DA Ricard-Blum S Transglutaminase-mediated cross-linking is involved in the stabilization of extracellular matrix in human liver fibrosis.J Hepatol. 2001; 35: 367-375Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar All examinations were performed with a Nikon Eclipse 800 microscope (Tokyo, Japan). For morphometric analyses, images were captured with a digital camera and analysis was performed with Metamorph software (Universal Corp., West Chester, PA), as described previously.8Kyriakides TR Zhu YH Yang Z Huynh G Bornstein P Altered extracellular matrix remodeling and angiogenesis in sponge granulomas of thrombospondin 2-null mice.Am J Pathol. 2001; 159: 1255-1262Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Four sections per sample were analyzed. All results are expressed as means ± SEM. Statistical significance was evaluated by the two-tailed unpaired Student's t-test for comparisons between the means of two groups. A value of P < 0.05 was considered to be statistically significant. To evaluate the basis for the adhesive defect observed in TSP-2-null cells, dermal fibroblasts were isolated from both wild-type (WT) and TSP-2-null mice. Cell-surface-mediated tTG activity was measured by incorporation of biotinylated cadaverine (a substrate for tTG) into soluble proteins. Cell-surface tTG activity was significantly reduced in mutant in comparison with WT cells (0.50 ± 0.05 arbitrary units versus 0.84 ± 0.07 for WT, P ≤ 0.05; Figure 1). To assess possible contamination with keratinocytes, which are rich in tTG, fibroblasts were plated on chamber slides at a density of 105 cells/well. Cells were allowed to attach and were then stained with an antibody to laminin 5, a keratinocyte marker. Less than 1% of the cell population stained positively (data not shown). To determine whether the reduced adhesion of TSP-2-null fibroblasts could be attributed directly to increased MMP-2 levels, we subjected purified tTG to proteolysis by MMP-2. Pro-MMP-2 was incubated with p-aminophenylmercuric acetate and the conversion of zymogen to the active form of the enzyme was confirmed by gelatin zymography (data not shown). The proteolytic digest of tTG was subjected to SDS-PAGE and Western blot analysis. As shown in Figure 2, additional low molecular weight fragments of tTG were generated by MMP-2. To provide additional support for the possibility that increased MMP-2 activity is responsible for the reduced activity of cell-surface tTG in TSP-2-null fibroblasts, exogenous, purified active MMP-2 was added to WT fibroblasts. Addition of 100 ng/ml of MMP-2 to cultured WT dermal fibroblasts caused a 35% reduction in tTG activity (Figure 3A). The addition of MMP-2 also resulted in a dose-dependent reduction in the adhesive properties of WT cells (Figure 3B). In support of these findings, the MMP-2 inhibitor, TIMP-2, increased tTG activity in TSP-2-null fibroblasts to levels similar to those found in WT cells (0.68 ± 0.07 arbitrary units, TIMP-2; 0.70 ± 0.1; Figure 4). We conclude, on the basis of the results shown in Figure 1, Figure 2, Figure 3, Figure 4, that the adhesive defects observed in TSP-2-null fibroblasts are a consequence of partial proteolysis of tTG by the elevated levels of MMP-2 present in these cells.Figure 4Addition of the MMP-2 inhibitor, TIMP-2, to TSP-2-null fibroblasts increases cell-surface tTG activity. The bar graphs represent the averages of four determinations; mean ± SEM; *P ≤ 0.05. A.U., arbitrary units. This experiment was repeated twice with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether the reduced tTG activity documented in cultured TSP-2-null fibroblasts could also account for some of the phenotypic features of TSP-2-null mice, we first assessed the ability of 0.5 N acetic acid to extract dermal collagen. The extractability of collagen from TSP-2-null skin was found to be increased threefold in comparison with that from WT skin (Table 1). Examination of the collagens by SDS-PAGE failed to reveal lower molecular weight bands in the extracts from TSP-2-null skin, as might be expected if the increased solubility of TSP-2-null collagen resulted from partial pro-teolysis by MMP-2 (data not shown). Furthermore, measurement of tTG activity, based on incorporation of biotinylated cadaverine into proteins solubilized from homogenates of dermis, indicated that the enzymatic activity was significantly reduced in skin of mutant mice, in comparison with WT skin (Figure 5). These results support a role for tTG in stabilizing the ECM in dermis.Table 1Extractability of Collagen from Mouse SkinGenotype*n = 3 each group.Skin (g)†Wet weight.Extracted collagen (mg)Collagen yield (mg/g skin)TSP2+/+2.7 ± 1.64.2 ± 3.11.3 ± 0.7TSP2−/−3.2 ± 1.816 ± 9.84.2 ± 2.1‡P ≤ 0.05.Collagen was extracted with 0.5 N of acetic acid at 4°C. Mice averaged 25 to 30 g in weight and varied between 3 and 4 months of age. Values are means ± SD. The extractability of TSP2-null skin exceeded that of WT by threefold.* n = 3 each group.† Wet weight.‡ P ≤ 0.05. Open table in a new tab Collagen was extracted with 0.5 N of acetic acid at 4°C. Mice averaged 25 to 30 g in weight and varied between 3 and 4 months of age. Values are means ± SD. The extractability of TSP2-null skin exceeded that of WT by threefold. tTG activity in intact, uninjured skin was found to be below the level for reliable detection by fluorescence microscopy. However, the enzyme has been reported to be induced in response to injury.23Haroon ZA Hettasch JM Lai TS Dewhirst MW Greenberg CS Tissue transglutaminase is expressed, active, and directly involved in rat dermal wound healing and angiogenesis.FASEB J. 1999; 13: 1787-1795Crossref PubMed Scopus (227) Google Scholar We therefore chose a well-characterized injury model, full-thickness excisional skin wounds, to compare the activity of this enzyme in WT and TSP-2-null-mice. To detect endogenous tTG activity, cryosections of day 10 wounds, a time at which levels of MMP-2 are elevated, were incubated with fluorescein isothiocyanate-labeled cadaverine, and in situ tTG activity was tested. Reduced tTG activity was evident by fluorescence microscopy in sections of granulation tissue of TSP-2-null wounds, in comparison with sections of WT wounds (Figure 6). Levels of isopeptide bond cross-links were also evaluated in skin and excisional wounds of TSP-2-null and WT mice. Uninjured skin and day 10 wound sections were stained with antibodies to cross-links and peroxidase-conjugated secondary antibodies, visualized by light microscopy, and quantified with Metamorph software. In comparison with WT tissues, isopeptide cross-links in TSP-2-null mice were reduced in both uninjured skin (Figure 7; A, B, E) and in day 10 wounds (Figure 7; C, D, F). The TSP-2-null mouse has proved to be a useful experimental animal in which to study the role of angiogenesis in reactions to injury, tumor growth and metastasis, and the mechanisms involved in the assembly and stabilization of the ECM. In previous publications, both from our own laboratory and in collaboration with other scientists, we had identified elevated levels of MMP-2 in cultured fibroblasts and in tissues of TSP-2-null mice,3Kokenyesi R Armstrong LC Agah A Artal R Bornstein P Thrombospondin 2 deficiency in pregnant mice results in premature softening of the uterine cervix.Biol Reprod. 2004; 70: 385-390Crossref PubMed Scopus (29) Google Scholar, 4Schroen B Heymans S Sharma U Blankesteijn WM Pokharel S Cleutjens JP Porter JG Evelo CT Duisters R van Leeuwen RE Janssen BJ Debets JJ Smits JF Daemen MJ Crijns HJ Bornstein P Pinto YM Thrombospondin-2 is essential for myocardial matrix integrity: increased expression identifies failure-prone cardiac hypertrophy.Circ Res. 2004; 95: 515-522Crossref PubMed Scopus (154) Google Scholar, 5Agah A Kyriakides TR Letrondo N Bjorkblom B Bornstein P Thrombospondin 2 levels are increased in aged mice: consequences for cutaneous wound healing and angiogenesis.Matrix Biol. 2004; 22: 539-547Crossref PubMed Scopus (56) Google Scholar, 7Yang Z Kyriakides TR Bornstein P Matricellular proteins as modulators of cell-matrix interactions: adhesive defect in thrombospondin 2-null fibroblasts is a consequence of increased levels of matrix metalloproteinase-2.Mol Biol Cell. 2000; 11: 3353-3364Crossref PubMed Scopus (175) Google Scholar, 8Kyriakides TR Zhu YH Yang Z Huynh G Bornstein P Altered extracellular matrix remodeling and angiogenesis in sponge granulomas of thrombospondin 2-null mice.Am J Pathol. 2001; 159: 1255-1262Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 9Yang Z Strickland DK Bornstein P Extracellular matrix metalloproteinase 2 levels are regulated by the low density lipoprotein-related scavenger receptor and thrombospondin 2.J Biol Chem. 2001; 276: 8403-8408Crossref PubMed Scopus (237) Google Scholar and considered the relation between the increase in protease activity and the reduced cell adhesion and tissue integrity observed in these mice. In this study we have shown that a target of MMP-2 is cell-surface tTG on fibroblasts, an
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