Role of Collagen Binding Receptors in Glomerulosclerosis

摘要 肾小球硬化是终末期肾病的特征之一，其特征是 用细胞外基质成分(主要是胶原蛋白)取代肾小球组织导致 肾小球功能丧失。这项资助的目标是研究其背后的分子机制。 调节受损肾小球胶原代谢以设计更有效的治疗方法预防 肾小球硬化。尽管许多途径都与启动和进展有关 对于肾小球纤维化，我们主要关注胶原结合受体整合素α1β1(Itgα1β1)。这种受体扮演着一种 通过重新招募和激活酪氨酸磷酸酶TCPTP从而下调 促纤维化受体的磷酸化，包括表皮生长因子受体(EGFR)。此外，Itgα1β1为阴性 在转录和翻译水平上调节胶原蛋白水平。 最近，我们开始研究itgα1β1控制细胞核内胶原合成的机制。 水平。由于许多转录因子和/或核糖核蛋白的核转位和激活 在酪氨酸磷酸化的调控下，我们分析了酪氨酸磷酸化的核蛋白水平。 野生型和ITGα1KO系膜细胞鉴定仅在ITGα1KO细胞中酪氨酸磷酸化的蛋白质。我们 确定在肉瘤中融合的核糖核蛋白(FUS)为候选。我们在ITGα1KO系膜中证明了 细胞，总水平和酪氨酸磷酸化的核FU水平的增加与 胶原蛋白的产生和FUS的下调减少了胶原蛋白的合成。有趣的是，FUS包含两个 可被EGFR磷酸化和TCPTP去磷酸化的酪氨酸和细胞核水平 FU与活化的EGFR水平相关。基于这些观察和发现，FUS是 在受损的人和小鼠肾脏中，FUS表达上调，我们认为FUS是胶原的正调节因子 并在肾小球硬化过程中发挥促纤维化作用。我们假设ITGα1β1 负向调节EGFR依赖的FUS酪氨酸磷酸化和功能 以独立的方式。因此，itgα1β1介导的FUS去磷酸化是一种重要的，但 以前未描述的机制，以选择性地减少FUS激活和随后的进展到 纤维化症。这笔赠款的目的是定义FUS对肾小球疾病的贡献，并 确定阻断其功能是否有利于肾小球硬化的治疗。 在目标1中，我们将在体外确定FUS转录控制胶原产生的机制。 并确定抑制FUS是否可以改善胶原合成。在目标2中，我们将确定 利用遗传学和药理学方法研究FUS在肾小球损伤进展中的作用。我们会调查的 野生型和特异型α1KO小鼠与全局FUSKO小鼠以及高表达小鼠的杂交反应 野生型FUS或突变型FUS不再能够移位到细胞核以致肾小球损伤。到时候我们会的 将这些发现的相关性转化为更具临床相关性的环境，通过调查对 未经治疗或新生细胞穿透治疗的野生型和ITGα1KO小鼠肾小球损伤 能够防止FUS核转位的多肽。 了解核糖核蛋白FUS如何控制肾小球硬化和 通过创新地提供核运输修饰剂来探索其抑制的后果将 为治疗和预防肾小球硬化提供了一种全新的方法。

Abstract Glomerulosclerosis is one of the hallmarks of end stage kidney disease and it is characterized by the replacement of the glomerular tissue with extracellular matrix components (mainly collagens) leading to the loss of functioning glomeruli. The goal of this grant is to investigate the molecular mechanisms that underlie the modulation of collagen turnover in injured glomeruli to devise more effective therapies to prevent glomerulosclerosis. Although many pathways have been implicated in both initiation and progression to glomerular fibrosis, we focus on the collagen binding receptor integrin α1β1 (Itgα1β1). This receptor plays an anti-fibrotic action by recruiting and activating the tyrosine phosphatase TCPTP thus downregulating the phosphorylation of pro-fibrotic receptors, including the EGF receptor (EGFR). Moreover, Itgα1β1 negatively regulates collagen levels at both transcriptional and translational levels. Recently, we started to investigate the mechanisms whereby Itgα1β1 controls collagen synthesis at the nuclear level. As nuclear translocation and activation of many transcription factors and/or ribonucleoproteins are regulated by tyrosine phosphorylation, we analyzed the levels of tyrosine phosphorylated nuclear proteins in wild type and Itgα1KO mesangial cells to identify proteins tyrosine phosphorylated only in Itgα1KO cells. We identified the ribonucleoprotein Fused in Sarcoma (FUS) as a candidate. We show that in Itgα1KO mesangial cells, increased levels of total and tyrosine phosphorylated nuclear FUS are associated with increased collagen production and downregulation of FUS decreases collagen synthesis. Interestingly, FUS contains two tyrosines that can be phosphorylated by EGFR and dephosphorylated by TCPTP and the levels of nuclear FUS are associated with levels of activated EGFR. Based on these observations and the finding that FUS is upregulated in injured human and mouse kidneys, we propose that FUS is a positive regulator of collagen synthesis and plays a pro-fibrotic action in the course of glomerulosclerosis. We hypothesize that Itgα1β1 negatively regulates FUS tyrosine phosphorylation and function in an EGFR-dependent and - independent manner. Thus, Itgα1β1-mediated dephosphorylation of FUS represents an important, but previously undescribed mechanism to selectively reduce FUS activation and consequent progression to fibrosis. The aims of this grant are designed to define the contribution of FUS to glomerular disease and to determine whether blocking its function is beneficial for the treatment of glomerulosclerosis. In Aim 1 we will determine in vitro the mechanisms whereby FUS transcriptionally controls collagen production and determine whether inhibiting FUS ameliorates collagen synthesis. In Aim 2 we will determine the role of FUS in the progression to glomerular injury using a genetic and pharmacological approach. We will investigate the response of wild type and Itgα1KO mice crossed with global FUSKO mice, as well as mice overexpressing wild type FUS or mutated FUS no longer able to translocate to the nucleus to glomerular injury. We will then translate the relevance of these findings to a more clinically relevant setting, by investigating the response to glomerular injury in wild type and Itgα1KO mice untreated or treated with newly generated cell-penetrating peptides able to prevent FUS nuclear translocation. Understanding how the ribonucleoprotein FUS controls collagen production in glomerulosclerosis and exploring the consequences of its inhibition based on innovative delivery of nuclear transport modifiers will offer an entirely novel approach for the treatment and, ideally, prevention of glomerulosclerosis.