Regulation of matrix turnover in mesangial cells

描述（由申请人提供）：肾小球系膜细胞在进展性肾病的肾小球硬化中起重要作用。虽然tgf - β参与了这一过程，但介导纤维形成的机制尚不清楚。在之前的资助期内，我们确定Smad信号转导通路是tgf - β 1刺激人系膜细胞I型胶原表达的关键介质。一些额外的信号机制有助于最佳的胶原生成反应。特别值得注意的是，我们定义了ERK MAP激酶途径的作用。我们发现tgf - β 1激活ERK需要Smad3，相反，ERK途径在Smad3连接区域的磷酸化中发挥作用。虽然这个磷酸化位点位于与R-Smad活化相关的c -末端Smad3结构域之外，但它显然有助于I型胶原的表达。这些结果表明，在系膜细胞中，Smad信号和ERK MAP激酶级联在多个水平上相互作用，放大tgf - β 1对I型胶原基因的激活。为了验证这一假设，我们提出了三个具体目标。在目的1中，我们将通过识别tgf - β 1刺激ERK活性的途径，并通过阻断Smad3活性来确定Smad3参与ERK激活的机制。在目标2中，我们将通过测试连接子区域突变对Smad易位、反激活活性和COL1A1或COL1A2启动子激活的影响，以及通过识别以erk依赖方式结合Smad的蛋白质，来确定Smad3连接子区域磷酸化的意义和机制。在目标3中，我们将通过检查Smad和ERK在细胞中如何以及在何处相互作用，以及抑制Smad3-ERK途径相互作用如何影响tgf - β 1对胶原I基因转录的激活，来确定Smad3-ERK途径相互作用如何调节胶原I基因表达。这些实验将为Smad作用和胶原表达的基本机制提供新的信息，并可能确定肾小球硬化治疗干预的潜在靶点。

DESCRIPTION (provided by applicant): The mesangial cell plays an important role as an effector of glomerulosclerosis in progressive kidney disease. Although TGF-beta has been implicated in this process, the mechanisms mediating fibrogenesis are not well understood. During the previous funding period, we determined that the Smad signal transduction pathway is a critical mediator of TGF-beta1-stimulated human mesangial cell collagen I expression. A number of additional signaling mechanisms contribute to optimal collagen-producing responses. Of particular note, we defined a role for the ERK MAP kinase pathway. We found that TGF-beta1 activation of ERK requires Smad3 and, conversely, that the ERK pathway plays a role in phosphorylation of the linker region of Smad3. Although this phosphorylation site is outside of the C-terminal Smad3 domain that is classically associated with R-Smad activation, it clearly contributes to collagen I expression. These results suggest the hypothesis that in mesangial cells, Smad signaling and the ERK MAP kinase cascade interact at multiple levels to amplify collagen I gene activation by TGF-beta1. To test this hypothesis, we propose three specific aims. In aim 1, we will determine the mechanism by which Smad3 participates in ERK activation by identifying the pathway(s) through which TGF-beta1 stimulates ERK activity and determining which are inactivated by blocking Smad3 activity. In aim 2, we will determine the significance and mechanism of Smad3 linker reqion phosphorylation by testing the effect of mutating the linker region on Smad translocation, transactivation activity and COL1A1 or COL1A2 promoter activation, and by identifying proteins that bind to Smad in an ERK-dependent manner. In aim 3, we will determine how Smad3-ERK pathway interactions regulate collagen I gene expression by examining how and where Smad and ERK interact in the cell, and how inhibiting Smad3-ERK pathway interactions affects the activation of collagen I gene transcription by TGF-beta1. These experiments will provide new information regarding basic mechanisms of Smad action and collagen expression, and could identify potential targets for therapeutic intervention in glomerulosclerosis.