Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

Abstract

Objective. Scleroderma is characterized by excessive synthesis and accumulation of matrix proteins in lesional tissues. Transforming growth factor ␤ (TGF␤) plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of fibroblasts; however, the underlying mechanisms are poorly understood. We undertook this study to examine the expression and function of SMADs, recently characterized intracellular effectors of TGF␤ signaling, in scleroderma fibroblasts.Methods. Primary dermal fibroblasts obtained from 14 patients with scleroderma and from 4 healthy adult volunteers were studied. Northern analysis was used to determine the expression of endogenous SMAD messenger RNA (mRNA), and Western analysis was used to determine SMAD protein expression. Intracellular compartmentalization of cellular SMAD proteins in the presence and absence of TGF␤ was studied by antibody-mediated immunofluorescence confocal microscopy. The effect of TGF␤ blockade on SMAD subcellular distribution was determined using anti-TGF␤ antibodies as well as a dominant-negative TGF␤ receptor type II (TGF␤RII) vector to disrupt TGF␤ responses. SMAD-regulated luciferase reporter expression was examined to investigate the potential functional significance of activation and nuclear accumulation of endogenous SMADs in scleroderma fibroblasts.Results. Protein and mRNA levels of SMAD3, but not of SMAD4 or SMAD7, were variably elevated in scleroderma fibroblasts compared with those from healthy controls. In sharp contrast to control fibroblasts, which displayed predominantly cytoplasmic localization of SMAD3/4 in the absence of exogenous TGF␤, in scleroderma fibroblasts SMAD3 and SMAD4 consistently showed elevated nuclear localization. Furthermore, phosphorylated SMAD2/3 levels were elevated and nuclear localization of phosphorylated SMAD2/3 was increased, suggesting activation of the SMAD pathway in scleroderma fibroblasts. Blockade of autocrine TGF␤ signaling with antibodies or by expression of dominant-negative TGF␤RII failed to normalize SMAD subcellular distribution, suggesting that elevated nuclear SMAD import was due to alterations downstream of the TGF␤ receptors. The activity of a SMADresponsive minimal promoter-reporter construct was enhanced in transiently transfected scleroderma fibroblasts.Conclusion. This study is the first to demonstrate apparently ligand-independent constitutive activation of the intracellular TGF␤/SMAD signaling axis in scleroderma fibroblasts. SMAD signaling may be a mechanism contributing to the characteristic phenotype of scleroderma fibroblasts and playing a role in the pathogenesis of fibrosis.Scleroderma is associated with fibrosis of affected tissues due to excessive synthesis and progressive accumulation of connective tissue macromolecules (1). Scleroderma fibroblasts display features of sustained activation in vitro and in vivo. In culture, these cells show elevated synthesis of collagens, fibronectin, proteoglycans, and tissue inhibitors of metalloproteinases, constitutive secretion of i...

Mechanism of the transforming growth factor-β induction of fibronectin expression in hepatic stem-like cells

“…On the other hand, defective Smad7 induction is implicated in the exaggerated TGF-β responsiveness characteristic of hepatic cells and myofibroblasts from chronically injured livers (21,22). In the present study, we observed that the expression of Smad7 began to increase after TGF-β treatment for 4 h. These data agree well with previous results obtained for skin fibroblasts (23). In addition, our result showed an obvious Smad7 translocation from nucleus to cytoplasm after treatment with TGF-β for 4 h. In fact, Smad7 has been shown to inhibit TGF-β signaling by associating with the type I receptor and thereby interfering with Smad2/3 phosphorylation by the activated receptor complex in the cytoplasm (6,7).…”

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

“…Fibroblasts cultured from SSc biopsy samples demonstrated elevated levels of Smad1 protein, and in 6 of 7 strains tested also showed the presence of phosphorylated Smad1, indicating that activation of the Smad1 pathway persists in cultured cells. Since several previous studies have shown elevated levels of phosphoSmad3 in SSc fibroblasts (13,24), these data suggest that distinct Smad pathways may play complementary roles in the activation of cultured SSc fibroblasts. Surprisingly, however, comparison of SSc and healthy skin tissues showed similar levels of phosphorylated Smad3 (24).…”

“…The documented changes include up-regulation of ␣v␤5 and ␣v␤3 integrins, which were shown to contribute to activation of latent TGF␤ and establishment of the autocrine TGF␤ loop (10,11). Additional changes include alterations of the TGF␤ receptor ratio (12) and the presence of persistently phosphorylated Smad3 (13). Recent studies from our laboratory using an in vitro model of SSc based on the altered ratio of TGF␤ receptors have also established that activation of the Smad1 pathway may represent a novel aspect of profibrotic TGF␤ signaling that functions independently of activation of the canonical Smad2/3 pathway (14).…”

Contribution of activin receptor–like kinase 5 (transforming growth factor β receptor type I) signaling to the fibrotic phenotype of scleroderma fibroblasts

“…In addition, although it has not been shown that dcSSc fibroblasts produce higher levels of TGF␤ protein (30,31) or enhanced activation of TGF␤, it has been reported that dcSSc fibroblasts express increased levels of type I and type II TGF␤ receptors (31) and overexpress the TGF␤ ancillary receptor endoglin in a manner that increases with disease severity (13). Adding complexity to this issue, the TGF␤ signaling mediator Smad3 has been shown to be activated in leading-edge fibroblasts in a TGF␤ ligand-independent manner (30), and although 1 study has indicated that dcSSc fibroblasts possess lower levels of the Smad inhibitor Smad7 (32), other studies have reported either no such elevation or indeed increased Smad7 levels (12,17,33). Our data confirm and extend the recently reported data concerning another ALK-5 inhibitor in which blockade of acute response to recombinant TGF␤1 was examined in normal dermal fibroblasts (34).…”