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Dihydrosphingosine 1-phosphate has a potent antifibrotic effect in scleroderma fibroblasts via normalization of phosphatase and tensin homolog levels.

ABSTRACT: Previous studies have revealed a phosphatase and tensin homolog (PTEN)-dependent interaction between the sphingolipid agonist dihydrosphingosine 1-phosphate (dhS1P) and the transforming growth factor beta/Smad3 signaling pathway. This study was undertaken to examine responses of systemic sclerosis (SSc) fibroblasts to sphingosine 1-phosphate (S1P) and dhS1P and to gain further insight into the regulation of the S1P/dhS1P/PTEN pathway in SSc fibrosis.Fibroblast cultures were established from skin biopsy samples obtained from patients with SSc and matched healthy controls. Western blotting and quantitative polymerase chain reaction were used to measure protein and messenger RNA levels, respectively. PTEN protein was examined in skin biopsy samples by immunohistochemistry.PTEN protein levels were low in SSc fibroblasts and correlated with elevated levels of collagen and phospho-Smad3 and reduced levels of matrix metalloproteinase 1 (MMP-1). Treatment with dhS1P restored PTEN levels and normalized collagen and MMP-1 expression, as well as Smad3 phosphorylation status in SSc fibroblasts. S1P was strongly profibrotic in SSc and control fibroblasts. Distribution of S1P receptor isoforms was altered in SSc fibroblasts, which had reduced levels of S1P receptor 1 and S1P receptor 2 and elevated levels of S1P receptor 3. Only depletion of S1P receptor 1 abrogated the effects of dhS1P and S1P in control dermal fibroblasts. In contrast, depletion of either S1P receptor 1 or S1P receptor 2 prevented the effects of S1P and dhS1P in SSc fibroblasts.Our findings demonstrate that PTEN deficiency is a critical determinant of the profibrotic phenotype of SSc fibroblasts. The antifibrotic effect of dhS1P is mediated through normalization of PTEN expression, suggesting that dhS1P or its derivatives may be effective as therapeutic antifibrotic agents. The distribution and function of S1P receptors differ in SSc and healthy fibroblasts, suggesting that alteration in the sphingolipid signaling pathway may contribute to SSc fibrosis.

SUBMITTER: Bu S

PROVIDER: S-EPMC3034368 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Dihydrosphingosine 1-phosphate has a potent antifibrotic effect in scleroderma fibroblasts via normalization of phosphatase and tensin homolog levels.

Bu Shizhong S Asano Yoshihide Y Bujor Andreea A Highland Kristin K Hant Faye F Trojanowska Maria M

Arthritis and rheumatism 20100701 7

<h4>Objective</h4>Previous studies have revealed a phosphatase and tensin homolog (PTEN)-dependent interaction between the sphingolipid agonist dihydrosphingosine 1-phosphate (dhS1P) and the transforming growth factor beta/Smad3 signaling pathway. This study was undertaken to examine responses of systemic sclerosis (SSc) fibroblasts to sphingosine 1-phosphate (S1P) and dhS1P and to gain further insight into the regulation of the S1P/dhS1P/PTEN pathway in SSc fibrosis.<h4>Methods</h4>Fibroblast c ...[more]

PMID: 20309867

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Project description:OBJECTIVE:Emerging evidence supports a role for epigenetic regulation in the pathogenesis of scleroderma (SSc). We aimed to assess the role of methyl-CpG-binding protein 2 (MeCP2), a key epigenetic regulator, in fibroblast activation and fibrosis in SSc. METHODS:Dermal fibroblasts were isolated from patients with diffuse cutaneous SSc (dcSSc) and from healthy controls. MeCP2 expression was measured by qPCR and western blot. Myofibroblast differentiation was evaluated by gel contraction assay in vitro. Fibroblast proliferation was analysed by ki67 immunofluorescence staining. A wound healing assay in vitro was used to determine fibroblast migration rates. RNA-seq was performed with and without MeCP2 knockdown in dcSSc to identify MeCP2-regulated genes. The expression of MeCP2 and its targets were modulated by siRNA or plasmid. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) using anti-MeCP2 antibody was performed to assess MeCP2 binding sites within MeCP2-regulated genes. RESULTS:Elevated expression of MeCP2 was detected in dcSSc fibroblasts compared with normal fibroblasts. Overexpressing MeCP2 in normal fibroblasts suppressed myofibroblast differentiation, fibroblast proliferation and fibroblast migration. RNA-seq in MeCP2-deficient dcSSc fibroblasts identified MeCP2-regulated genes involved in fibrosis, including PLAU, NID2 and ADA. Plasminogen activator urokinase (PLAU) overexpression in dcSSc fibroblasts reduced myofibroblast differentiation and fibroblast migration, while nidogen-2 (NID2) knockdown promoted myofibroblast differentiation and fibroblast migration. Adenosine deaminase (ADA) depletion in dcSSc fibroblasts inhibited cell migration rates. Taken together, antifibrotic effects of MeCP2 were mediated, at least partly, through modulating PLAU, NID2 and ADA. ChIP-seq further showed that MeCP2 directly binds regulatory sequences in NID2 and PLAU gene loci. CONCLUSIONS:This study demonstrates a novel role for MeCP2 in skin fibrosis and identifies MeCP2-regulated genes associated with fibroblast migration, myofibroblast differentiation and extracellular matrix degradation, which can be potentially targeted for therapy in SSc.

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Dihydrosphingosine 1-phosphate has a potent antifibrotic effect in scleroderma fibroblasts via normalization of phosphatase and tensin homolog levels.

Publications

Dihydrosphingosine 1-phosphate has a potent antifibrotic effect in scleroderma fibroblasts via normalization of phosphatase and tensin homolog levels.

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