Yoshihiro Mimura

Increased Expression of Integrin αvβ3 Contributes to the Establishment of Autocrine TGF-β Signaling in Scleroderma Fibroblasts

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.

Phosphatidylinositol 3-Kinase Is Involved in α2(I) Collagen Gene Expression in Normal and Scleroderma Fibroblasts

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.

Regulation of fibrogenic/fibrolytic genes by platelet-derived growth factor C, a novel growth factor, in human dermal 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.

Constitutive Thrombospondin-1 Overexpression Contributes to Autocrine Transforming Growth Factor-β Signaling in Cultured Scleroderma Fibroblasts

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.

Matrix metalloproteinase-1 up-regulation by hepatocyte growth factor in human dermal fibroblasts via ERK signaling pathway involves Ets1 and Fli1.

In this study, we clarified the molecular mechanism(s) underlying the regulation of matrix metalloproteinase (MMP)-1 gene by hepatocyte growth factor (HGF) in cultured human dermal fibroblasts. HGF induced MMP-1 protein as well as mRNA at a transcriptional level via extracellular signal-regulated kinase (ERK) signaling pathway. The region in the MMP-1 promoter mediating the inducible responsiveness to HGF, defined by the transient transfection analysis of the serial 5′ deletion constructs, contained an Ets binding site. Mutation of this Ets binding site abrogated the HGF-inducible promoter activity. Ets1 up-regulated the expression of MMP-1 promoter activity, whereas Fli1 had antagonistic effects on them. After HGF treatment, the protein level and the binding activity of Ets1 was increased and those of Fli1 was decreased, which were canceled by PD98059. These results suggest that HGF up-regulates MMP-1 expression via ERK signaling pathway through the balance of Ets1 and Fli1, which may be a novel mechanism of regulating MMP-1 gene expression.

Clinical and laboratory features of scleroderma patients developing skeletal myopathy

Skeletal muscle involvement, or myopathy, has been a recognized feature of systemic sclerosis (SSc). We studied retrospectively 302 Japanese patients with SSc to elucidate the clinical and laboratory features in scleroderma patients developing skeletal myopathy during their clinical course. Forty-three patients (14%) developed skeletal myopathy during their course of the disease. The mean age of the patients who developed skeletal myopathy was significantly lower than that of those who did not. The ratio of male to female was significantly higher in the myopathic patients. The patients with diffuse cutaneous SSc were more likely to develop myopathy than those with limited cutaneous SSc. The prevalences of heart involvement, pulmonary fibrosis, diffuse pigmentation of the skin, and contracture of phalanges were significantly greater in those with skeletal myopathy than in those without. None of the patients with skeletal myopathy had anticentromere antibody. These findings suggested that the SSc patients with severe internal organ involvement, such as pulmonary fibrosis and heart disease, and some other complications were prone to develop skeletal myopathy during their clinical course of the disease.

High-dose intravenous immunoglobulin infusion as treatment for diffuse scleroderma

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Rheumatoid factor isotypes and anti-agalactosyl IgG antibodies in systemic sclerosis

Background  Rheumatoid factor isotypes and anti‐agalactosyl IgG antibodies (anti‐AG IgG) have been reported to be detected and correlated with the disease activity in some collagen diseases.

Rheumatoid factor isotypes in localized scleroderma

Localized sclerodema is a connective tissue disorder that is sometimes accompanied by various immunological abnormalities. In this study, we analysed serum levels of rheumatoid factor (RF) isotypes in patients with localized scleroderma and in normal controls to determine if any of these isotypes reflect the severity of the disease. IgM RF, IgG RF and IgA RF were positive in 30%, 21%, and 7% of the patients, respectively. The levels of IgM RF were significantly higher in the patients with generalized morphea (GM), the most severe form of localized scleroderma, than those with linear scleroderma (LS) (P < 0.005) or normal controls (P < 0.0005). The levels of IgG RF were significantly higher in patients with GM than normal controls (P < 0.05). The levels of IgA RF were significantly higher in patients with GM or LS than normal controls (P < 0.001 and P < 0.01, respectively). The count of sclerotic lesions was significantly higher in patients with IgM RF than those without (P < 0.05). These results suggest that the presence of RF isotypes is one of the immunological abnormalities of localized scleroderma. IgM RF seemed to be most useful of these three factors to determine the severity of disease.

Clinical and laboratory features of Japanese patients with scleroderma and telangiectasia

Backgroud.  Systemic sclerosis (SSc) is a connective tissue disease characterized by sclerotic changes of the skin and internal organs. Telangiectasia is a frequent complication of patients with SSc.