Signaling Pathways in Renal Fibrogenesis

描述(由申请人提供)：肾脏纤维化的信号机制继续引起人们的关注和兴趣。这项建议提出了一种策略来扩展我们之前的研究，该研究考察了转化生长因子-2如何在肾脏纤维化的细胞模型中刺激I型胶原的积累。在之前的授权期，我们证明了许多信号通路与转化生长因子-2/Smad信号相互作用，特别是细胞外信号调节激酶(ERK)MAP激酶的作用。我们确定Smad3和ERK信号通路共同促进了转化生长因子-2刺激的肾小球系膜和肾小管上皮细胞的胶原表达。ERK在支持Smad3介导的胶原基因转录中起关键作用，但在没有Smad激活的情况下并不激活COL1A2启动子，因此它是胶原表达所必需的，但不是充分的。ERK使SMAD3链接区(LR)的丝氨酸和苏氨酸磷酸化。更多的初步研究表明，阻断1v23整合素或PI3-激酶活性会抑制ERK的激活和胶原的反应。为此，我们提出了一个假设，即从1v23-整合素到ERK的黏附依赖的信号通路通过Smad3的连接区丝氨酸和/或苏氨酸残基的磷酸化来促进肾细胞I型胶原的表达；而PI3K在体外和体内都是一个有希望的靶点，它作用于导致ERK的通路的多个点，以钝化胶原对转化生长因子-2的反应。为了验证这一假说，我们将追求以下目标：首先，我们将确定1v23整合素促进转化生长因子-2刺激的肾细胞纤维化的分子机制，确定rac1和PI3K在这一过程中的作用。其次，我们将通过研究特定的磷酸化事件如何影响Smad3信号转导的I型胶原表达，以及这些磷酸化中哪些依赖于PI3K和/或ERK，来确定ERK和Smad3协同促进转化生长因子-2刺激的胶原表达的机制(S)。第三，我们将确定1v23、PI3K和ERK如何在阿霉素肾病和选择性足细胞消融的肾纤维化模型中相互作用来调节SMAD3LR。这些研究应该为Smad信号的整合素调控提供新的见解，解决关于SMAD3受体角色的未解决的问题，并提供潜在的方法来阻断肾脏的纤维化过程。意义：尽管进行了广泛的研究，但人们对转化生长因子-2信号如何调控导致肾纤维化知之甚少。我们对非典型信号通路和SMAD3-LR的作用的研究将解决有关信号在纤维化形成中的特异性的关键问题。通过阐明肾纤维化背后的信号事件，我们希望更好地了解纤维化的形成机制，如何阻断它们，并最终如何治疗它们。

DESCRIPTION (provided by applicant): The signaling mechanisms underlying kidney fibrosis continue to be of significant concern and interest. This proposal presents a strategy for extending our previous studies that examined how TGF-2 stimulates accumulation of type I collagen in a cellular model of renal fibrogenesis. In the previous grant period, we demonstrated that numerous signaling pathways interact with TGF-2/Smad signaling, focusing particularly on the role of the extracellular signal-regulated kinase (ERK) MAP kinase. We determined that the Smad3 and ERK pathways together promote TGF-2-stimulated collagen expression in renal mesangial and tubular epithelial cells. ERK serves a critical role in supporting Smad3-mediated collagen gene transcription, but does not activate the COL1A2 promoter in the absence of Smad activation; it is thus necessary but not sufficient for collagen expression. ERK phosphorylates serines and threonines in the linker region (LR) domain of Smad3. Additional Preliminary Studies indicate that blocking 1v23 integrin or PI3-kinase activity inhibits ERK activation and the collagen response. For this renewal, we propose the HYPOTHESIS that an adhesion-dependent signaling pathway from 1v23-integrin to ERK promotes renal cell type I collagen expression through phosphorylation of linker-region serine and/or threonine residues of Smad3; and PI3K, which acts at several points along the pathway leading to ERK, is a promising target for blunting the collagen response to TGF-2 both in vitro and in vivo. To test this hypothesis, we will pursue the following AIMS: First, we will determine the molecular mechanisms by which 1v23 integrin promotes TGF-2-stimulated renal cell fibrogenesis, establishing the roles of Rac1 and PI3K in this process. Second, we will determine the mechanism(s) through which ERK and Smad3 synergize to promote TGF-2-stimulated collagen expression by examining how specific phosphorylation events affect Smad3 signaling of collagen I expression and which of these phosphorylations are dependent upon PI3K and/or ERK. Third, we will determine how 1v23, PI3K and ERK interact to modulate the Smad3 LR in the renal fibrosis models of adriamycin nephropathy and selective podocyte ablation. These studies should provide new insights into integrin regulation of Smad signaling, address unsettled issues regarding the role of the Smad3 LR, and offer potential approaches to interrupting the fibrogenic process in kidneys. SIGNIFICANCE: Despite extensive research, little is known about how TGF-2 signaling is regulated to cause renal fibrosis. Our studies of non-canonical signaling pathways and the role of Smad3-LR will address key issues regarding the specificity of signals in fibrogenesis. By elucidating the signaling events underlying kidney fibrosis we hope to better understand fibrogenic mechanisms, how to interrupt them and, ultimately, how to treat them.