TGF-beta receptor signaling in scleroderma

描述(申请人提供)：器官纤维化，一种主要的病理改变 硬皮病(SSC)的表现，是过度沉积的结果 I型胶原和其他细胞外基质(ECM)蛋白。尽管 在解开生理学方面取得的重大进展 以及涉及胶原蛋白基因调控的病理机制，完整的 对这些过程仍然缺乏了解。特别是，几乎没有 目前已知的是导致本构关系的分子机制 SSC成纤维细胞上调ECM蛋白表达。这样的知识对于 开发合适的治疗干预靶点。在过去的几年里 我们提出的资助期是为了检验自分泌转化生长因子-β的假设 通过过度表达TGFb受体的信号至少部分是 负责SSC表型。为了验证这一假设，我们阻止了转化生长因子-β 通过过表达缺乏激酶的转化生长因子-B受体II(TBRIIdeltaK)来传递信号。 与我们的预期相反，SSC成纤维细胞对此主要没有反应 关于胶原蛋白产生的治疗。另一方面，这种治疗方法 导致健康皮肤细胞外基质的产生显著下调 成纤维细胞。这些结果导致了对我们假设的修正，并促使我们 调查可能导致SSC的替代机制 表型。为了研究转化生长因子-β的非依赖性途径，我们重点研究了结缔组织生长因子 (结缔组织生长因子)和Igfbp5(IGF结合蛋白5)。目前的研究建议是基于我们新的观察结果，表明 CTGF对人成纤维细胞ECM的诱导依赖于胰岛素信号转导 而且，Igfbp5还能刺激成纤维细胞产生胶原。我们建议 以下具体目的是测试假设之间的交互作用 转化生长因子-β、结缔组织生长因子和胰岛素/胰岛素样生长因子信号转导通路参与SSC的调控 表型。在具体目标1中，我们将继续研究 转化生长因子-β信号通路在硬化性硬化症表现中的作用 表型。在具体目标2中，我们将确定CTGF介导的作用 SSC成纤维细胞产生ECM的途径。我们将描述这一机制 CTGF对COL1A2启动子的刺激作用 胰岛素/胰岛素样生长因子信号通路在CTGF诱导中的作用 胶原蛋白。在具体目标3中，我们将确定Igfbp5的作用机制。 干细胞和健康成纤维细胞对胶原生成的刺激作用。我们会 分析IGFBPs在SSC和健康成纤维细胞中的表达模式 利用纯化的IGFBP蛋白和相应的cDNA探讨其在 SSC和健康成纤维细胞对胶原蛋白的调节。在具体目标4中，我们将 检测转化生长因子-B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.