TGF-beta receptor signaling in scleroderma

描述（由申请人提供）：器官纤维化，一种主要的病理性 硬皮病（SSc）的表现，是过度沉积的结果， 胶原I和其它细胞外基质（ECM）蛋白。尽管 已经取得了重大进展， 和病理机制参与调节胶原基因，一个完整的 对这些过程的理解仍然缺乏。特别是， 是目前已知的分子机制，负责组成 SSc成纤维细胞对ECM蛋白的上调。这些知识对于 开发用于治疗干预的合适靶点。在过去 在资助期间，我们提出了检验自分泌TGF-B的假设， 通过TGFB受体过表达的信号传导至少部分地 负责SSc表型。为了验证这一假设，我们阻断了TGF-β 通过过表达激酶缺陷型TGF-β受体II（TBRIIdeltaK）进行信号传导。 与我们的预期相反，SSc成纤维细胞主要对此无反应。 关于胶原蛋白生产的治疗。另一方面，这种治疗 导致健康皮肤中ECM产生的显著下调 成纤维细胞这些结果导致了我们的假设的修正，并促使我们 研究可能导致SSc的替代机制 表型。为了研究TGF-B非依赖性通路，我们将重点放在CTGF上， （结缔组织生长因子）和IGFBP 5（IGF结合蛋白5）。目前的研究建议是基于我们的新观察， 人成纤维细胞中CTGF诱导ECM依赖于胰岛素信号传导 IGFBP 5还刺激成纤维细胞产生胶原蛋白。我们提出 以下具体目的是为了检验假设， TGF-B、CTGF和胰岛素/IGF通路参与SSc的调节。 表型在具体目标1中，我们将继续研究 在SSc的表现中TGF-β信号通路的组分 表型在具体目标2中，我们将确定CTGF介导的 在SSc成纤维细胞的ECM生产途径中。我们将描述 的CTGF刺激的COL 1A 2启动子，并表征的成分， 胰岛素/IGF信号通路有助于CTGF诱导 胶原在具体目标3中，我们将确定IGFBP 5的机制。 刺激SSc和健康成纤维细胞产生胶原蛋白。我们将 分析SSc和健康成纤维细胞中IGFBPs的表达模式， 利用纯化的IGFBP蛋白和相应的cDNA来探测它们在 SSc和健康成纤维细胞的胶原调节。在第四个目标中，我们将 检测TGF-β受体亚单位CTGF和 SSc皮肤中的IGF/IGFBPs。

DESCRIPTION (provided by applicant): Organ fibrosis, a major pathological manifestation of scleroderma (SSc), is the result of excessive deposition of collagen I and other extracellular matrix (ECM) proteins. Despite the significant progress that has been made towards unraveling the physiological and pathological mechanisms involved in regulation of collagen genes, a full understanding of these processes is still lacking. In particular, very little is currently known about the molecular mechanism responsible for constitutive upregulation of ECM proteins by SSc fibroblasts. Such knowledge is critical for development of suitable targets for therapeutic intervention. During the last funding period we proposed to test the hypothesis that autocrine TGF-B signaling through overexpression of TGFB receptors is at least partially responsible for the SSc phenotype. To test this hypothesis we blocked TGF-B signaling by overexpressing a kinase-deficient TGF-B receptor II (TBRIIdeltaK). Contrary to our expectations, SSc fibroblasts were mainly unresponsive to this treatment with regard to collagen production. On the other hand, this treatment resulted in a significant downregulation of the ECM production in healthy skin fibroblasts. These results led to a revision of our hypothesis and prompted us to investigate alternative mechanisms that may be responsible for the SSc phenotype. To investigate TGF-B independent pathways, we have focused on CTGF (connective tissue growth factor) and IGFBP5 (IGF binding protein 5). The current research proposal is based on our novel observations indicating that CTGF induction of ECM in human fibroblasts is dependent on insulin signaling and that IGFBP5 also stimulates collagen production by fibroblasts. We propose the following Specific Aims to test the hypothesis that interactions between TGF-B, CTGF, and insulin/IGF pathways are involved in the regulation of the SSc phenotype. In Specific Aim 1 we will continue to examine the role of the components of the TGF-B signaling pathway in the manifestation of the SSc phenotype. In Specific Aim 2 we will determine the role of the CTGF-mediated pathway in ECM production by SSc fibroblasts. We will delineate the mechanism of CTGF stimulation of the COL1A2 promoter and characterize the components of the insulin/IGF signaling pathway that contribute to CTGF induction of collagen. In Specific Aim 3 we will determine the mechanism of IGFBP5 stimulation of collagen production by SSc and healthy fibroblasts. We will analyze expression patterns of IGFBPs in SSc and healthy fibroblasts and utilize purified IGFBP proteins and corresponding cDNAs to probe their role in collagen regulation by SSc and healthy fibroblasts. In Specific Aim 4 we will examine the in vivo expression of the TGF-B receptor subunits, CTGF, and IGF/IGFBPs in SSc skin.