Smad1 pathway is activated in systemic sclerosis fibroblasts and is targeted by imatinib mesylate

Abstract

Objective. Activation of Smad1 signaling has recently been implicated in the development of fibrosis. The goal of the present study was to gain further insights into activation of the Smad1 pathway in fibrosis in systemic sclerosis (SSc) and to determine whether this pathway is targeted by the antifibrotic drug imatinib mesylate.Methods. Levels of phosphorylated Smad1 and total Smad1 were examined in SSc and control skin biopsy samples by immunohistochemistry and in cultured fibroblasts by Western blotting. Activity of the CCN2 promoter was examined by a luciferase reporter gene assay. Interactions of Smad1 with the CCN2 promoter were examined by in vitro and in vivo DNA binding assays. Expression of the nonreceptor tyrosine kinase c-Abl and Smad1 was blocked using respective small interfering RNA.Results. Total and phosphorylated Smad1 levels were significantly elevated in SSc skin biopsy samples and in cultured SSc fibroblasts and correlated with elevated CCN2 protein and CCN2 promoter activity. DNA binding assays demonstrated that Smad1 was a direct activator of the CCN2 gene. Small interfering RNA-mediated depletion of Smad1 in SSc fibroblasts normalized the production of CCN2 and collagen. Imatinib mesylate blocked activation of the Smad1 pathway in transforming growth factor ␤-stimulated control fibroblasts and reversed activation of this pathway in SSc fibroblasts. Likewise, blockade of c-Abl abrogated activation of the Smad1 pathway in SSc fibroblasts.Conclusion. Our findings demonstrate that activation of Smad1 signaling occurs in a subset of SSc patients and contributes to persistent activation of SSc fibroblasts. Demonstration that the Smad1/CCN2 pathway is blocked by imatinib mesylate further clarifies the mechanism of the antifibrotic effects of this compound.

Phosphorylation of Fli1 at Threonine 312 by Protein Kinase C δ Promotes Its Interaction with p300/CREB-Binding Protein-Associated Factor and Subsequent Acetylation in Response to Transforming Growth Factor β

“…Thus, the TGF-␤-dependent synchronized regulation of the DNA binding activity of transcriptional activators, such as Smads, and repressors, such as Fli1, ultimately results in the increased expression of ECM genes. Clinical studies of cultured scleroderma fibroblasts and skin biopsies strongly support the primary role of TGF-␤ in the development of pathological fibrosis (3,18,22,29,30,40). The non-Smad signaling molecules, such as PKC ␦, represent attractive novel therapeutic targets for the treatment of this disease.…”

Cytokines in the immunopathology of systemic sclerosis

“…This small molecule inhibitor of intracellular kinase modulates the fibrotic process in SSc by interfering selectively with both TGF-β and PDGF signalling pathway (PDGFR/c-Abl and Smad1/ CCN2 pathway) [111,112]. In in vitro studies, the inhibition of the pathway results in a decrease in type I collagen and fibronectin-1 synthesis by dermal fibroblasts and myofibroblasts [113].…”

Imatinib and the Treatment of Fibrosis: Recent Trials and Tribulations

“…Pannu et al [19] observed that activation of TGF-β-mediated Smad1 signaling occurs in a subset of SSc patients and contributes to persistent activation of SSc fibroblasts. This Smad1/CCN2 pathway is blocked by imatinib mesylate [19].…”

“…Pannu et al [19] observed that activation of TGF-β-mediated Smad1 signaling occurs in a subset of SSc patients and contributes to persistent activation of SSc fibroblasts. This Smad1/CCN2 pathway is blocked by imatinib mesylate [19]. Bhattacharyya et al [18•] delineated a novel signaling mechanism of TGF-β and were able to position early growth response factor 1 downstream of c-Abl in a TGF-β-mediated fibrotic response of murine embryonic fibroblasts.…”