Fibroblast TGF-beta/Smad Signaling in Scleroderma

描述（由申请人提供）：在系统性硬化症（SSc）中，胶原蛋白的过度积累导致纤维化和随后的皮肤和内脏功能障碍。转化生长因子- β (tgf - β)在纤维化的启动和传播中起着关键作用。由于其在SSc中的关键作用，了解tgf - β如何调节成纤维细胞功能将澄清SSc的发病机制并确定治疗干预的新靶点。最近发现的细胞内tgf - β信号转导Smad家族及其在多种疾病中的重要作用的认识，为研究tgf - β参与SSc的发病机制提供了新的途径。拟议的研究利用体外和体内方法的结合，以回答四个重要的问题，以理解tgf - β /Smad轴在SSc中的作用：在人成纤维细胞中，哪些纤维化相关基因的表达受到Smad3的调节？在炎症和修复过程中，Smad通路如何整合到正常和SSc成纤维细胞的细胞信号网络中？Smad信号的内在改变是否与SSc成纤维细胞表型有关？Smad3在小鼠硬皮病发生中的功能作用是什么？我们的假设是，SSc中的纤维化是由于成纤维细胞基因表达的持续激活，而Smad3在这一过程中起关键作用。该假设将在四个相互关联的目标中进行评估。在Specific Aim 1中，我们将使用DNA微阵列来分析通过诱导Smad3在永生化人成纤维细胞中表达而调节的基因；在特异性目标2中，我们将研究炎症细胞通过cd40对成纤维细胞tgf - β /Smad信号的调节。在Specific Aim 3中，我们将表征SSc成纤维细胞和组织中的Smad通路，以确定SSc中tgf - β /Smad轴的调节和/或功能的内在改变，这些改变可能有助于成纤维细胞的激活。来自体外实验的观察结果不能总是准确地预测蛋白质在体内的功能。我们假设Smad3是tgf - β信号的重要介质，缺乏Smad3的小鼠对病理性组织纤维化的易感性降低。为了直接验证这一假设，在Specific Aim 4中，我们将在Smad3缺失的小鼠中诱导皮肤纤维化，以确定Smad3在这一过程中的作用。该结果将阐明Smad信号通路在SSc纤维化发生中的作用，并揭示治疗靶向的可行性。

DESCRIPTION (provided by applicant): In systemic sclerosis (SSc), excessive accumulation of collagen leads to fibrosis and subsequent dysfunction of skin and internal organs. Transforming growth factor-beta (TGF-beta) plays a pivotal role in initiating and propagating fibrosis. Because of its key role in SSc, understanding how TGF-beta regulates fibroblast functions would clarify the pathogenesis of SSc and identify novel targets for therapeutic intervention. The recent discovery of the Smad family of intracellular TGF-beta signal transducers and the recognition of their major roles in multiple diseases, provides novel approaches for investigating the involvement of TGF-beta in pathogenesis of SSc. The proposed studies utilize a combination of in vitro and in vivo approaches in order to answer four important questions fundamental to understanding the role of the TGF-beta/Smad axis in SSc: What are fibrosis-related genes whose expression in human fibroblasts is modulated by Smad3? How is the Smad pathway integrated into the cellular signaling network of normal and SSc fibroblasts during inflammation and repair? Are intrinsic alterations in Smad signaling implicated in the SSc fibroblast phenotype? What is the functional role of Smad3 in the development of scleroderma in mice? Our hypothesis is that fibrosis in SSc is due to sustained activation of fibroblast gene expression, and that Smad3 plays a pivotal role in this process. The hypothesis will be evaluated in four interrelated aims. In Specific Aim 1, we will use DNA microarrays to profile genes whose expression is modulated by inducible expression of Smad3 in immortalized human fibroblasts; In Specific Aim 2, we will examine modulation of fibroblast TGF-beta/Smad signaling by inflammatory cells through CD4O. In Specific Aim 3, we will characterize the Smad pathway in SSc fibroblasts and tissues in order to identify intrinsic alterations in the regulation and/or function of the TGF-beta/Smad axis in SSc that may contribute to fibroblast activation. Observations derived from in vitro experiments cannot always accurately predict protein function in vivo. We hypothesize that Smad3 is an essential mediator of TGF-beta signaling, and mice lacking Smad3 have reduced susceptibility to pathological tissue fibrosis. To test this hypothesis directly, in Specific Aim 4 we will induce cutaneous fibrosis in Smad3-null mice in order to determine the role of Smad3 in this process. The results will clarify the role of Smad signal pathway in mediating the development of fibrosis in SSc, and reveal the feasibility of therapeutically targeting.