Signaling Pathways in Renal Fibrogenesis

描述（由申请人提供）：肾纤维化的信号传导机制仍然是重要的关注和兴趣。该建议提出了一种策略，以扩展我们以前的研究，研究TGF-2如何刺激肾纤维化细胞模型中I型胶原的积累。在上一个资助期，我们证明了许多信号通路与TGF-2/Smad信号相互作用，特别关注细胞外信号调节激酶（ERK）MAP激酶的作用。我们确定Smad 3和ERK通路共同促进TGF-2刺激的肾系膜和肾小管上皮细胞胶原蛋白表达。ERK在支持Smad 3介导的胶原基因转录中起关键作用，但在Smad活化不存在的情况下不活化COL 1A 2启动子;因此，ERK对于胶原表达是必要的，但不是充分的。ERK磷酸化Smad 3的连接区（LR）结构域中的丝氨酸和苏氨酸。额外的初步研究表明，阻断1v 23整联蛋白或PI 3-激酶活性可抑制ERK活化和胶原反应。对于这一更新，我们提出的假设，从1v 23-整联蛋白ERK的粘附依赖性信号通路促进肾细胞I型胶原蛋白的表达，通过磷酸化的连接区丝氨酸和/或苏氨酸残基的Smad 3和PI 3 K，它的作用在几个点沿着途径导致ERK，是一个有前途的目标钝化胶原蛋白对TGF-2在体外和体内。为了验证这一假设，我们将追求以下目标：首先，我们将确定1v 23整合素促进TGF-2刺激的肾细胞纤维化的分子机制，建立Rac 1和PI 3 K在这一过程中的作用。第二，我们将确定的机制（S）通过ERK和Smad 3协同促进TGF-2刺激的胶原蛋白的表达，通过检查如何特定的磷酸化事件影响Smad 3信号传导的胶原蛋白I的表达，这些磷酸化依赖于PI 3 K和/或ERK。第三，我们将确定1v 23，PI 3 K和ERK如何相互作用，以调节阿霉素肾病和选择性足细胞消融的肾纤维化模型中的Smad 3 LR。这些研究将为整合素调控Smad信号提供新的见解，解决有关Smad 3 LR作用的尚未解决的问题，并提供潜在的方法来中断肾脏纤维化过程。意义：尽管进行了广泛的研究，但对TGF-2信号传导如何调节导致肾纤维化知之甚少。我们对非经典信号通路和Smad 3-LR的作用的研究将解决有关纤维化信号特异性的关键问题。通过阐明肾纤维化的信号事件，我们希望更好地了解纤维化机制，如何中断它们，并最终如何治疗它们。 公共卫生相关性：慢性进行性肾病每年花费国家数十亿美元的医疗保健费用，涉及疤痕组织成分的积累，如I型胶原蛋白。转化生长因子（TGF）-2是一种在肾脏瘢痕形成中起核心作用的蛋白质。虽然我们在了解TGF-2如何工作方面取得了很大进展，但我们还没有学会如何调节其作用以预防或减少肾瘢痕。该提案要求对目前已进入第14年的项目重新给予支持，以了解TGF-2如何刺激I型胶原蛋白的合成。通过确定细胞内导致瘢痕形成的分子事件，我们希望确定预防瘢痕形成和降低慢性肾衰竭发生率的靶点。

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. PUBLIC HEALTH RELEVANCE: Chronic, progressive kidney disease, which costs the nation many billions of dollars in health care each year, involves the accumulation of scar tissue components such as type I collagen. Transforming growth factor (TGF)-2 is a protein that plays a central role in scarring of the kidney. Although we have made much progress in understanding how TGF-2 works, we have not learned how to regulate its actions in order to prevent or minimize kidney scarring. This proposal requests renewal of support for a project presently in its 14th year, seeking to understand how TGF-2 stimulates synthesis of type I collagen. By defining the molecular events inside the cell that lead to scarring, we hope to identify targets to prevent such scarring and decrease the incidence of chronic kidney failure.