Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

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Abstract
Objective Scleroderma is characterized by excessive synthesis and accumulation of matrix proteins in lesional tissues. Transforming growth factor β (TGFβ) 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 TGFβ 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 TGFβ was studied by antibody‐mediated immunofluorescence confocal microscopy. The effect of TGFβ blockade on SMAD subcellular distribution was determined using anti‐TGFβ antibodies as well as a dominant‐negative TGFβ receptor type II (TGFβRII) vector to disrupt TGFβ 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 TGFβ, 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 TGFβ signaling with antibodies or by expression of dominant‐negative TGFβRII failed to normalize SMAD subcellular distribution, suggesting that elevated nuclear SMAD import was due to alterations downstream of the TGFβ receptors. The activity of a SMAD‐responsive 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 TGFβ/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.

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