The constitutive secretion of latent TGF-β by many cell types in culture suggests that extracellular mechanisms to control the activity of this potent cytokine are important in the pathogenesis of the diseases in which this cytokine may be involved, including fibrotic disorders. In this study, we focused on the αvβ3 integrin, which is recently demonstrated to function as an active receptor for latent TGF-β1 through its interaction with latency-associated peptide-β1, and investigated the involvement of this integrin in the pathogenesis of scleroderma. Scleroderma fibroblasts exhibited increased αvβ3 expression compared with normal fibroblasts in vivo and in vitro. In scleroderma fibroblasts, ERK pathway was constitutively activated and such abnormality induced the up-regulation of αvβ3. Transient overexpression of αvβ3 in normal fibroblasts induced the increase in the promoter activity of human α2(I) collagen gene and the decrease in that of human MMP-1 gene. These effects of αvβ3 were almost completely abolished by the treatment with anti-TGF-β Ab or TGF-β1 antisense oligonucleotide. Furthermore, the addition of anti-αvβ3 Ab reversed the expression of type I procollagen protein and MMP-1 protein, the promoter activity of human α2(I) collagen gene, and the myofibroblastic phenotype in scleroderma fibroblasts. These results suggest that the up-regulated expression of αvβ3 contributes to the establishment of autocrine TGF-β loop in scleroderma fibroblasts, and this integrin is a potent target for the treatment of scleroderma.

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Objective. To confirm the involvement of ␣v␤5 in the self-activation system in systemic sclerosis (SSc) fibroblasts.Methods. Levels of ␣v␤5 expression were analyzed by immunoprecipitation. The promoter activity of the human ␣2(I) collagen gene was determined by transient transfection assay. Phosphorylation levels and DNA binding ability of Smad3 were investigated by immunoprecipitation and DNA affinity precipitation, respectively. The localization of active transforming growth factor ␤ (TGF␤) was determined by coculture assay using TMLC cells (mink lung epithelial reporter cells that stably express a portion of the plasminogen activator inhibitor 1 promoter). The morphologic features of cells were determined by immunofluorescence analysis.Results. Levels of ␣v␤5 expression were significantly elevated in SSc fibroblasts compared with normal fibroblasts. Treatment with anti-␣v␤5 antibody or ␤5 antisense oligonucleotide significantly reduced human ␣2(I) collagen gene promoter activity in SSc fibroblasts. In SSc fibroblasts pretreated with TGF␤1 antisense oligonucleotide, the exogenous latent TGF␤1 stimulation significantly increased human ␣2(I) collagen gene promoter activity; this effect was significantly reduced in the presence of anti-␣v␤5 antibody. Phosphorylation levels and DNA binding ability of Smad3 in SSc fibroblasts were significantly reduced by treatment with ␤5 antisense oligonucleotide. The luciferase activity of TMLC cells cocultured with SSc fibroblasts was significantly elevated compared with that of TMLC cells cocultured with normal fibroblasts and was significantly reduced in the presence of anti-␣v␤5 antibody. Anti␣v␤5 antibody reversed the myofibroblastic features of SSc fibroblasts.Conclusion. Up-regulated expression of ␣v␤5 contributes to the establishment of autocrine TGF␤ signaling in SSc fibroblasts through activation of endogenous latent TGF␤1.Scleroderma (systemic sclerosis [SSc]) is an acquired disorder that typically results in fibrosis of the skin and internal organs (1). Previous findings indicate that the pathogenesis of this disorder includes inflammation, autoimmune attack, and vascular damage, leading to fibroblast activation (2). However, cultured SSc fibroblasts, which are free of such environmental factors, continue to produce excessive amounts of extracellular matrix (ECM) proteins (3,4), suggesting that once activated, these cells establish a constitutive self-activation system. One of the major cytokines involved in this process is transforming growth factor ␤1 (TGF␤1) (5). The principal effect of TGF␤ on mesenchymal cells is its stimulation of ECM deposition, as evidenced by the findings that 1) SSc fibroblasts express elevated levels of TGF␤ receptors, and this correlates with elevated levels of ␣2(I) collagen messenger RNA (mRNA) (6-9) and 2)

TGF-β is implicated in the pathogenesis of fibrotic disorders. It has been shown that Smad3 promotes the human α2(I) collagen (COL1A2) gene expression by TGF-β1 in human dermal fibroblasts. Here, we investigated the role of phosphatidylinositol 3-kinase (PI3K) in the COL1A2 gene expression in normal and scleroderma fibroblasts. In normal fibroblasts, the PI3K inhibitor, LY294002, significantly decreased the basal and the TGF-β1-induced increased stability of COL1A2 mRNA. The TGF-β1-induced COL1A2 promoter activity, but not the basal activity, was significantly attenuated by LY294002 or the dominant negative mutant of p85 subunit of PI3K, while the constitutive active mutant of p110 subunit of PI3K did not affect the basal or the TGF-β1-induced COL1A2 promoter activity. LY294002 significantly decreased the phosphorylation of Smad3 induced by TGF-β1. Furthermore, the transient overexpression of 2xFYVE, which induces the mislocalization of FYVE domain proteins, decreased the TGF-β1-induced Smad3 phosphorylation to a similar extent to LY294002. In scleroderma fibroblasts, the blockade of PI3K significantly decreased the mRNA stability and the promoter activity of the COL1A2 gene. Furthermore, LY294002 and the transient overexpression of 2xFYVE completely diminished the constitutive phosphorylation of Smad3. These results indicate that 1) the basal activity of PI3K is necessary for the COL1A2 mRNA stabilization in normal and scleroderma fibroblasts, 2) there is an unidentified FYVE domain protein specifically interacting with Smad3, and 3) the basal activity of PI3K and the FYVE domain protein are indispensable for the efficient TGF-β/Smad3 signaling in normal fibroblasts and for the establishment of the constitutive activation of TGF-β/Smad3 signaling in scleroderma fibroblasts.

Localized scleroderma (LSc) is a connective tissue disorder limited to skin and subcutaneous tissue, which may share pathogenic processes with systemic sclerosis (SSc). We previously demonstrated that upregulated expression of integrin alphavbeta5 might contribute to autocrine TGF-beta signaling in SSc fibroblasts. Based on these data, we presently focused on alphavbeta5 and assessed its involvement in pathogenesis of LSc. We initially demonstrated that LSc fibroblasts might be activated by the stimulation of autocrine TGF-beta. Consistent with SSc fibroblasts, expression levels of alphavbeta5 were elevated in LSc fibroblasts in vitro and in vivo. Anti-alphavbeta5 antibody partially reversed expression levels of type I procollagen and MMP-1 and constitutive DNA-Smad3 binding in LSc fibroblasts. In LSc fibroblasts pretreated with antisense TGF-beta1, exogenous latent TGF-beta1 stimulation increased expression of type I procollagen in an alphavbeta5-dependent manner. The luciferase activities of TMLC cells, Mv1Lu cells stably expressing a portion of the plasminogen activator inhibitor 1 promoter, co-cultured with LSc fibroblasts were significantly elevated compared with those co-cultured with normal fibroblasts and were significantly reduced in the presence of anti-alphavbeta5 antibody. Anti-alphavbeta5 antibody reversed the myofibroblastic features of LSc fibroblasts. These results indicate that upregulated expression of alphavbeta5 contributes to autocrine TGF-beta signaling in LSc fibroblasts.

Platelet-derived growth factor (PDGF) is a potent mitogenic and chemotactic cytokine, and PDGF-C is a novel growth factor belonging to the PDGF family. In this study, to determine whether this growth factor can contribute to fibrosis and tissue remodeling, we examined the effect of PDGF-CC on the expression of fibrogenic/fibrolytic genes such as type I collagen, fibronectin (FN), matrix metalloproteinases (MMPs), and their inhibitors (TIMPs) in normal human dermal fibroblasts in vitro. PDGF elevated the levels of MMP-1 or TIMP-1 protein as well as mRNA, whereas this cytokine had no influence on the expression of type I collagen, FN, or TIMP-2. PDGF-CC also increased the levels of MMP-1 catalytic activity in the cultured media and mRNA expression, which was paralleled that on the levels of promoter activation. Additionally, PDGF-CC induced the mitogenic and migratory activity of human dermal fibroblasts in a dose-dependent manner. On the other hand, we also determined the specificity of the inhibitory effect of monoclonal antibodies against PDGF-CC generated by immunizing balb/c mice with recombinant human PDGF-CC. This antibody could inhibit the regulatory effects of MMP-1 or TIMP-1 synthesis as well as the mitogenic effects on human dermal fibroblasts induced by PDGF-CC, whereas this antibody did not affect those induced by other PDGF forms such as PDGF-AA, -AB, or -BB. These results suggest that this cytokine plays a role in the tissue remodeling.

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The extracellular matrix (ECM) glycoprotein thrombospondin-1 (TSP-1) has been reported to activate the latent complex of transforming growth factor-beta (TGF-beta), the major effects of which in mesenchymal cells is stimulation of the synthesis of ECM. Previous reports suggested the involvement of an autocrine TGF-beta loop in the pathogenesis of scleroderma. In this study, we examined whether TSP-1 plays a role in maintaining the autocrine TGF-beta loop in scleroderma. TSP-1 expression was increased in scleroderma patients compared with in healthy controls in vivo and in vitro. TGF-beta blocking antibody or TGF-beta1 antisense oligonucleotide markedly reduced the up-regulated TSP-1 expression in scleroderma fibroblasts but had little effect on normal fibroblasts. The expression of TSP-1 is up-regulated in scleroderma fibroblasts, possibly at the post-transcriptional level just like in normal fibroblasts stimulated with exogenous TGF-beta1. TSP-1 blocking peptide or antisense oligonucleotide had an inhibitory effect on the up-regulated alpha2I collagen and phosopho-Smad3 levels in scleroderma fibroblasts but had little effects on normal fibroblasts. The transient overexpression of TSP-1 up-regulated alpha2I collagen and phospho-Smad3 levels in normal fibroblasts but had no major effect on scleroderma fibroblasts. Furthermore, these effects of transiently overexpressed TSP-1, which possibly occurred via the activation of latent TGF-beta1, were abolished by the TGF-beta1 antisense oligonucleotide. These results indicate that the constitutive overexpression of TSP-1 may play an important role in autocrine TGF-beta signaling and accumulation of ECM in scleroderma fibroblasts.