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To confirm the involvement of αvβ5 in the self‐activation system in systemic sclerosis (SSc) fibroblasts. Levels of αvβ5 expression were analyzed by immunoprecipitation. The promoter activity of the human α2(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 β (TGFβ) 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.Levels of αvβ5 expression were significantly elevated in SSc fibroblasts compared with normal fibroblasts. Treatment with anti‐αvβ5 antibody or β5 antisense oligonucleotide significantly reduced human α2(I) collagen gene promoter activity in SSc fibroblasts. In SSc fibroblasts pretreated with TGFβ1 antisense oligonucleotide, the exogenous latent TGFβ1 stimulation significantly increased human α2(I) collagen gene promoter activity; this effect was significantly reduced in the presence of anti‐αvβ5 antibody. Phosphorylation levels and DNA binding ability of Smad3 in SSc fibroblasts were significantly reduced by treatment with β5 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‐αvβ5 antibody. Anti‐αvβ5 antibody reversed the myofibroblastic features of SSc fibroblasts.Up‐regulated expression of αvβ5 contributes to the establishment of autocrine TGFβ signaling in SSc fibroblasts through activation of endogenous latent TGFβ1. [ABSTRACT FROM AUTHOR]

To investigate the effects of FK‐506 on the expression of the human α2(I) collagen gene and transforming growth factor β (TGFβ) signaling in normal and scleroderma fibroblasts.The expression levels of type I procollagen protein and α2(I) collagen messenger RNA (mRNA) were analyzed by immunoblotting and Northern blotting, respectively. The promoter activities of α2(I) collagen gene and 3TP‐Lux were determined by transient transfection assay. Interaction between TGFβ receptor type I and FK‐506 binding protein 12 (FKBP12) was evaluated by immunoprecipitation.FK‐506 did not affect the basal expression of type I procollagen protein or α2(I) collagen mRNA, but it significantly reduced the TGFβ1‐induced expression of type I procollagen protein and α2(I) collagen mRNA in normal fibroblasts. The effect of FK‐506 was regulated posttranscriptionally, but not transcriptionally. In scleroderma fibroblasts, FK‐506 significantly reduced the expression of type I procollagen protein and α2(I) collagen mRNA through posttranscriptional regulation, but not transcriptional regulation. FK‐506 increased the basal activity of the 3TP‐Lux promoter, but it did not affect the TGFβ1‐induced promoter activity in normal fibroblasts. In contrast, FK‐506 did not affect the basal or the TGFβ1‐induced 3TP‐Lux promoter activity in scleroderma fibroblasts. Furthermore, FKBP12, which protects TGFβ receptor type I from ligand‐independent activation by TGFβ receptor type II, constitutively dissociated from TGFβ receptor type I in scleroderma fibroblasts.FK‐506 inhibits α2(I) collagen gene expression by reducing the stability of mRNA without exhibiting its activation effect on TGFβ signaling in scleroderma fibroblasts. [ABSTRACT FROM AUTHOR]

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. © 2004 Wiley‐Liss, Inc. [ABSTRACT FROM AUTHOR]