Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts
Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts
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DOI：
10.1002/art.11157
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发表时间：
2003-07-01
影响因子：
--
通讯作者：
Varga, J
中科院分区：
文献类型：
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作者：
Mori, Y;Chen, SJ;Varga, J
Objective.硬皮病的特征是病变组织中基质蛋白的过度合成和积累。转化生长因子β（TGF β）通过诱导和维持成纤维细胞的活化在纤维化的发病机制中起着核心作用;然而，对其潜在机制的了解甚少。我们进行了这项研究，以检查的表达和功能的SMADs，最近的特点是细胞内效应的TGF β信号，在硬皮病成纤维细胞。研究了从14名硬皮病患者和4名健康成人志愿者中获得的原代真皮成纤维细胞。北方分析用于确定内源性SMAD信使RNA（mRNA）的表达，并且Western分析用于确定SMAD蛋白的表达。通过抗体介导的免疫荧光共聚焦显微镜研究了在存在和不存在TGF β的情况下细胞SMAD蛋白的细胞内区室化。TGF β阻断对SMAD亚细胞分布的影响使用抗TGF β抗体以及显性负性TGF β受体II型（TGF β RII）载体来破坏TGF β应答。通过检测SMAD调控的荧光素酶报告基因的表达，探讨内源性SMAD在硬皮病成纤维细胞中的激活和核积累的潜在功能意义。与健康对照组相比，硬皮病成纤维细胞中SNIAD 3的蛋白和mRNA水平显著升高，但SMAD 4或SMAD 7的蛋白和mRNA水平没有显著升高。与对照成纤维细胞形成鲜明对比，对照成纤维细胞在不存在外源性TGF β的情况下主要显示SMAD 3/4的细胞质定位，在硬皮病成纤维细胞中SMAD 3和SMAD 4一致地显示升高的核定位。此外，磷酸化SMAD 2/3水平升高，磷酸化SMAD 2/3的核定位增加，表明硬皮病成纤维细胞中SMAD途径的激活。用抗体或显性阴性TGF β RII表达阻断自分泌TGF β信号传导不能使SMAD亚细胞分布正常化，表明核SMAD输入升高是由于TGF β受体下游的改变。SMAD应答性最小启动子-报告基因构建体在瞬时转染的硬皮病成纤维细胞中的活性增强。本研究首次证实了硬皮病成纤维细胞内TGF β/SMAD信号轴的明显配体非依赖性组成性激活。SMAD信号可能是促成硬皮病成纤维细胞特征性表型的机制，并在纤维化的发病机制中发挥作用。
Objective. Scleroderma is characterized by excessive synthesis and accumulation of matrix proteins in lesional tissues. Transforming growth factor beta (TGFbeta) 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 TGFbeta 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 TGFbeta was studied by antibody-mediated immunofluorescence confocal microscopy. The effect of TGFbeta blockade on SMAD subcellular distribution was determined using anti-TGFbeta antibodies as well as a dominant-negative TGFbeta receptor type II (TGFbetaRII) vector to disrupt TGFbeta 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 SNIAD3, 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 TGFbeta, 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 TGFbeta signaling with antibodies or by expression of dominant-negative TGFbetaRII failed to normalize SMAD subcellular distribution, suggesting that elevated nuclear SMAD import was due to alterations downstream of the TGFbeta 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 TGFbeta/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.