Human fibroblasts with mutations in COL5A1 and COL3A1 genes do not organize collagens and fibronectin in the extracellular matrix, down-regulate alpha2beta1 integrin, and recruit alphavbeta3 Instead of alpha5beta1 integrin.
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Dermal fibroblasts derived from types I and IV Ehlers-Danlos syndrome (EDS) patients, carrying mutations in COL5A1 and COL3A1 genes, respectively, synthesize aberrant types V and III collagen (COLL) and show defective organization of these proteins into the extracellular matrix (ECM) and high reduction of their functional receptor, the alpha(2)beta(1) integrin, compared with control fibroblasts. EDS cells also show reduced levels of fibronectin (FN) in the culture medium and lack an FN fibrillar network. Finally, EDS cells prevalently organize alpha(v)beta(3) integrin instead of alpha(5)beta(1) integrin. The alpha(v)beta(3) integrin, distributed on the whole EDS cell surface, shows FN binding and assembly properties when the cells are treated with purified FN. Treatment of EDS cells with purified COLLV or COLLIII, but not with FN, restores the control phenotype (COLL(+), FN(+), alpha(v)beta(3)(-), alpha(5)beta(1)(+), alpha(2)beta(1)(+)). Function-blocking antibodies to COLLV, COLLIII, or alpha(2)beta(1) integrin induce in control fibroblasts an EDS-like phenotype (COLL(-), FN(-), alpha(v)beta(3)(+), alpha(5)beta(1)(-), alpha(2)beta(1)(-)). These results show that in human fibroblasts alpha(2)beta(1) integrin organization and function are controlled by its ligand, and that the alpha(2)beta(1)-COLL interaction, in turn, regulates FN integrin receptor recruitment: high alpha(2)beta(1) integrin levels induce alpha(5)beta(1) integrin organization, while low alpha(2)beta(1) integrin levels lead to alpha(v)beta(3) integrin organization. (+info)
Structurally different RGTAs modulate collagen-type expression by cultured aortic smooth muscle cells via different pathways involving fibroblast growth factor-2 or transforming growth factor-beta1.
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We have engineered polymers called ReGeneraTing Agents (RGTAs), which mimic the protecting and potentiating properties of heparan sulfates toward heparin-binding growth factors (HBGF). RGTAs have been shown to optimize cell growth and regulate collagen production in vitro. Here, we studied relationships between RGTA structure and collagen-type expression in aortic smooth muscle cells by using two RGTAs, the carboxylmethylsulfate dextran RG-1503 and the carboxylmethylsulfate dextran with added benzylamide RG-1192. RG-1192 specifically induced a fivefold decrease in collagen III synthesis. This effect was abolished by FGF-2 neutralizing antibody. RG-1192 and FGF-2 acted synergistically to decrease collagen III. RG-1192 was more effective than heparin in this process. RG-1192 increased the pericellular localization of FGF-2 and protected FGF-2 from proteolysis. Surface plasmon resonance analysis indicated a Kd of 15.7 nM for the RG-1192/FGF-2 interaction (10.6 nM for the heparin/FGF-2 interaction). The structurally different RG-1503 (without benzylamide) did not interact with FGF-2 and worked synergistically with TGF-beta1 to specifically induce a twofold increase in collagen V. RGTAs with different structures exert different modulating effects on the collagen phenotype. Selection of appropriate RGTAs, which had been shown to enhance in vivo tissue repair, may provide a mean of correcting collagen abnormalities in vascular disorders and more generally in fibrotic diseases. (+info)
Role of autoimmunity in organ allograft rejection: a focus on immunity to type V collagen in the pathogenesis of lung transplant rejection.
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Lung transplantation is the only definitive treatment modality for many forms of end-stage lung disease. However, the lung is rejected more often than any other type of solid organ allograft due to chronic rejection known as bronchiolitis obliterans (BO). Indeed, BO is the primary reason why the 5- and 7-yr survival rates are worse for the lung than for any other transplanted organ. Alloimmunity to donor antigens is established as the primary mechanism that mediates rejection responses. However, newer immunosuppressive regimens designed to abrogate alloimmune activation have not improved survival. Therefore, these data suggest that other antigens, unrelated to donor transplantation antigens, are involved in rejection. Utilizing human and rodent studies of lung transplantation, our laboratory has documented that a native collagen, type V collagen [col(V)], is a target of the rejection response. Col(V) is highly conserved; therefore, these data indicate that transplant rejection involves both alloimmune and autoimmune responses. The role of col(V) in lung transplant rejection is described in this review article. In addition, the potential role of regulatory T cells that are crucial to modulating autoimmunity and alloimmunity is also discussed. (+info)
Effects of angiotensin II receptor antagonist on expression of collagen III, collagen V, and transforming growth factor beta1 in the airway walls of sensitized rats.
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BACKGROUND: Repeated attacks of bronchial asthma lead to different degrees of airway remodeling, the mechanism of which is not yet clear. Some evidences indicate that it is related to the excessive expression of some growth promotion factors. Angiotensin II is a polypeptide that may be involved in airway remodeling. To evaluate its role in airway remodeling in asthma, we observed the effects of an angiotensin II type 1 receptor antagonist (valsartan) on the expression of collagen III, collagen V, and transforming growth factor beta1 (TGF-beta1) mRNA and protein in the airway walls of sensitized rats. METHODS: Forty Wistar rats were randomly divided into 5 groups: control group, sensitized group, and valsartan groups 1, 2, and 3. The rats in the sensitized group and in valsartan groups 1, 2, and 3 were sensitized and challenged with ovalbumin. Rats in control group were sensitized and challenged with 0.9% NaCl. Rats from valsartan groups 1, 2, and 3 were drenched with valsartan (10 microg, 20 microg, or 30 microg, respectively) at the time of the ovalbumin challenges. The expression of collagen III, collagen V, and TGF-beta1 protein were detected using immunohistochemical method in combination with image analysis methods. The expression of TGF-beta1 mRNA was detected by in situ hybridization. RESULTS: The expression in the airways of collagen III and collagen V was significantly higher in rats from the sensitized group (7.73 +/- 0.81, 1.34 +/- 0.28) and from valsartan groups 1, 2, and 3 (5.73 +/- 0.64, 1.13 +/- 0.15; 4.96 +/- 0.51, 0.98 +/- 0.08; 4.43 +/- 0.35, 0.93 +/- 0.06, respectively) than those in the control group (2.65 +/- 0.38, 0.67 +/- 0.08, P < 0.05). In addition, collagen levels were significantly lower in valsartan groups 1, 2, and 3 than those from the sensitized group (P < 0.05). The expression of TGF-beta1 mRNA and protein in the airways was significantly higher in rats from the sensitized group (20.49% +/- 3.46%, 29.73% +/- 3.25%) and from valsartan groups 1, 2, and 3 (16.47% +/- 1.94%, 19.41% +/- 1.87%; 14.38% +/- 1.58%, 18.29% +/- 1.43%; 12.96% +/- 1.73%, 18.63% +/- 1.11%, respectively) than that from the control group (7.84% +/- 1.61%, 5.63% +/- 1.07%, P < 0.05). TGF-beta1 mRNA and protein levels were significantly lower in valsartan groups 1, 2, and 3 than that in the sensitized group (P < 0.05). CONCLUSIONS: Angiotensin II receptor antagonist valsartan can suppress synthesis of collagen III and collagen V by downregulating TGF-beta1 mRNA and protein expression. Valsartan can decrease airway remodeling and could play a role in asthma therapy. (+info)
Development of a functional skin matrix requires deposition of collagen V heterotrimers.
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Collagen V is a minor component of the heterotypic I/III/V collagen fibrils and the defective product in most cases of classical Ehlers Danlos syndrome (EDS). The present study was undertaken to elucidate the impact of collagen V mutations on skin development, the most severely affected EDS tissues, using mice harboring a targeted deletion of the alpha2(V) collagen gene (Col5a2). Contrary to the original report, our studies indicate that the Col5a2 deletion (a.k.a. the pN allele) represents a functionally null mutation that affects matrix assembly through a complex sequence of events. First the mutation impairs assembly and/or secretion of the alpha1(V)(2)alpha2(V) heterotrimer with the result that the alpha1(V) homotrimer is the predominant species deposited into the matrix. Second, the alpha1(V) homotrimer is excluded from incorporation into the heterotypic collagen fibrils and this in turn severely impairs matrix organization. Third, the mutant matrix stimulates a compensatory loop by the alpha1(V) collagen gene that leads to additional deposition of alpha1(V) homotrimers. These data therefore underscore the importance of the collagen V heterotrimer in dermal fibrillogenesis. Furthermore, reduced thickness of the basement membranes underlying the epidermis and increased apoptosis of the stromal fibroblasts in pN/pN skin strongly indicate additional roles of collagen V in the development of a functional skin matrix. (+info)
The transcription factor CCAAT-binding factor CBF/NF-Y and two repressors regulate the core promoter of the human pro-alpha3(V) collagen gene (COL5A3).
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To elucidate the mechanisms underlining alpha3(V) collagen chain expression, we performed an initial analysis of the structure and function of the core promoter of the human COL5A3 gene. The core promoter, which lacks a typical TATA motif and has a high GC content, was defined within the -129 bp immediately upstream from the major transcription start site by transient transfection experiments. In this region, we identified four DNA-protein complexes, named A, B, C, and D, by a combination of DNase I footprinting and electrophoretic mobility shift assays. Electrophoretic mobility shift assays using mutant oligonucleotide revealed that the complexes A, B, C, and D bind to -122 to -117, the -101 to -96, the -83 to -78, and the -68 to -57 bp, respectively. The competition assays using consensus oligonucleotides and supershift assays with specific antibodies showed that complex A consists of CBF/NF-Y. In a chromatin immunoprecipitation assay, CBF/NF-Y protein directly bound to this region, in vivo. Functional analysis showed that CBF/NF-Y activated the gene, whereas the proteins of complexes B and C repressed its activity. Furthermore, overexpression of a mutant form of the CBF-B/NF-YA subunit, which forms CBF/NF-Y with CBF-A/NF-YB and CBF-C/NF-YC subunits, inhibited promoter activity. (+info)
Constitutive release of alpha4 type V collagen N-terminal domain by Schwann cells and binding to cell surface and extracellular matrix heparan sulfate proteoglycans.
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During peripheral nerve development, Schwann cells synthesize collagen type V molecules that contain alpha4(V) chains. This collagen subunit possesses an N-terminal domain (NTD) that contains a unique high affinity heparin binding site. The alpha4(V)-NTD is adhesive for Schwann cells and sensory neurons and is an excellent substrate for Schwann cell and axonal migration. Here we show that the alpha4(V)-NTD is released constitutively by Schwann cells both in culture and in vivo. In cultures of neonatal rat Schwann cells, alpha4(V)-NTD release is increased significantly by ascorbate treatment, which facilitates collagen post-translational modification and collagen trimer assembly. In peripheral nerve tissue, the alpha4(V)-NTD is localized to the region of the outer Schwann cell membrane and associated extracellular matrix. The released alpha4(V)-NTD binds to the cell surface and extracellular matrix heparan sulfate proteoglycans of Schwann cells. Pull-down assays and immunofluorescent staining showed that the major alpha4(V)-NTD-binding proteins are glypican-1 and perlecan. alpha4(V)-NTD binding occurs via a mechanism that requires the high affinity heparin binding site and that is blocked by soluble heparin, demonstrating that binding to proteoglycans is mediated by their heparan sulfate chains. (+info)
Type V collagen controls the initiation of collagen fibril assembly.
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Vertebrate collagen fibrils are heterotypically composed of a quantitatively major and minor fibril collagen. In non-cartilaginous tissues, type I collagen accounts for the majority of the collagen mass, and collagen type V, the functions of which are poorly understood, is a minor component. Type V collagen has been implicated in the regulation of fibril diameter, and we reported recently preliminary evidence that type V collagen is required for collagen fibril nucleation (Wenstrup, R. J., Florer, J. B., Cole, W. G., Willing, M. C., and Birk, D. E. (2004) J. Cell. Biochem. 92, 113-124). The purpose of this study was to define the roles of type V collagen in the regulation of collagen fibrillogenesis and matrix assembly. Mouse embryos completely deficient in pro-alpha1(V) chains were created by homologous recombination. The col5a1-/- animals die in early embryogenesis, at approximately embryonic day 10. The type V collagen-deficient mice demonstrate a virtual lack of collagen fibril formation. In contrast, the col5a1+/- animals are viable. The reduced type V collagen content is associated with a 50% reduction in fibril number and dermal collagen content. In addition, relatively normal, cylindrical fibrils are assembled with a second population of large, structurally abnormal collagen fibrils. The structural properties of the abnormal matrix are decreased relative to the wild type control animals. These data indicate a central role for the evolutionary, ancient type V collagen in the regulation of fibrillogenesis. The complete dependence of fibril formation on type V collagen is indicative of the critical role of the latter in early fibril initiation. In addition, this fibril collagen is important in the determination of fibril structure and matrix organization. (+info)