Regulation of matrix turnover in mesangial cells

描述（由申请人提供）：肾小球系膜细胞在进行性肾脏疾病中作为肾小球硬化的效应物发挥重要作用。虽然TGF-β参与了这一过程，但其介导纤维化的机制还不清楚。在之前的资助期间，我们确定Smad信号转导通路是TGF-β 1刺激的人肾小球系膜细胞胶原I表达的关键介质。许多额外的信号传导机制有助于最佳的胶原蛋白产生反应。特别值得注意的是，我们确定了ERK MAP激酶通路的作用。我们发现TGF-β 1激活ERK需要Smad 3，相反，ERK通路在Smad 3连接区的磷酸化中起作用。 尽管该磷酸化位点在与R-Smad活化典型相关的C-末端Smad 3结构域之外，但它明显有助于胶原I表达。这些结果表明，在肾小球系膜细胞，Smad信号和ERK MAP激酶级联反应在多个水平上相互作用，以放大TGF-β 1激活胶原蛋白I基因的假设。为了验证这一假设，我们提出了三个具体目标。在目标1中，我们将通过鉴定TGF-β 1刺激ERK活性的途径并确定哪些途径通过阻断Smad 3活性而失活，来确定Smad 3参与ERK激活的机制。目的2：通过检测Smad 3连接区的突变对Smad转位、转录激活活性和COL 1A 1或COL 1A 2启动子激活的影响，以及通过鉴定以ERK依赖方式与Smad结合的蛋白质，来确定Smad 3连接区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哪里相互作用，以及抑制Smad 3-ERK途径相互作用如何影响TGF-β 1对I型胶原基因转录的激活，确定Smad 3-ERK途径相互作用如何调节I型胶原基因表达。这些实验将提供有关Smad作用和胶原蛋白表达的基本机制的新信息，并可以确定肾小球硬化症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.