Fibroblast TGF-beta/Smad Signaling in Scleroderma

描述(申请人提供)：在系统性硬化症(SSC)中，胶原蛋白的过度积累会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)在肝纤维化的发生和发展中起着重要作用。由于其在SSC中的关键作用，了解转化生长因子-β如何调节成纤维细胞的功能将有助于阐明SSC的发病机制，并为治疗干预寻找新的靶点。Smad家族细胞内转化生长因子-β信号转导通路的发现及其在多种疾病中的重要作用的认识，为研究转化生长因子-β参与自发性硬化症的发病机制提供了新的途径。建议的研究采用体外和体内相结合的方法，以回答四个重要的问题，以了解转化生长因子-β/Smad轴在SSC中的作用：哪些纤维化相关基因在人成纤维细胞中的表达受到Smad3的调节？在炎症和修复过程中，Smad通路是如何整合到正常和SSC成纤维细胞的细胞信号网络中的？Smad信号的内在改变是否与SSC成纤维细胞表型有关？Smad3在小鼠硬皮病发生发展中的作用是什么？我们的假设是，SSC的纤维化是由于成纤维细胞基因表达的持续激活，Smad3在这一过程中发挥着关键作用。这一假设将在四个相互关联的目标中进行评估。在特定目标1中，我们将使用DNA微阵列来描述在永生化的人成纤维细胞中受Smad3诱导表达调控的基因；在特定目标2中，我们将检测炎症细胞通过CD4O对成纤维细胞转化生长因子-β/Smad信号的调控。在特定的目标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中转化生长因子-β/Smad轴的调节和/或功能的内在变化，这些变化可能有助于成纤维细胞的激活。来自体外实验的观察并不总是准确地预测蛋白质在体内的功能。我们假设Smad3是转化生长因子-β信号转导的重要介体，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，在特定的目标4中，我们将在Smad3基因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。这些结果将阐明Smad信号通路在介导SSC纤维化发展中的作用，并揭示靶向治疗的可行性。

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.