Adaptor Molecules in TGF-beta Signaling

描述(申请人提供)：转化生长因子-β是导致慢性进行性肾脏疾病事件的关键调节因子，如细胞外基质产生、细胞凋亡和上皮-间充质转化(EMT)。虽然在理解转化生长因子受体(T？R)通过Smad信号分子传递的信号方面取得了相当大的进展，但Smad Anchor for Receptor Activation(SARA)等适配蛋白在这些信号中的作用还不清楚。我们的初步数据表明，在人肾小球系膜细胞(MC)和肾小管上皮细胞系(HKC和HK-2)中，SarA以一种依赖于转化生长因子β的方式与Smad2结合。相反，SARA在结构上与T？R结合，并与早期内小体有关。在转化生长因子-β1处理后，Smad2被磷酸化，而笼蛋白介导的内吞作用(CME)有助于将转化生长因子-β信号传递到细胞核。因此，SARA可能在与转化生长因子相关的蛋白运输中发挥核心作用，特别是通过其在T？R运输和促进Smad2活性方面的作用。除了SARA，我们的初步数据还在MC和HKC中检测到了其他适配器蛋白，这些蛋白被认为在Smad信号中发挥作用，但比SARA更不被理解。长期的转化生长因子-β1治疗导致SARA表达的最终下降，这在时间上对应于MC、HKC和HK-2的肌成纤维样表型。我们提出的假设是，通过作为支架蛋白来组装和定位T？R信号复合体，适配蛋白SARA、DAB2和Axin可能分别影响转化生长因子-β反应，并且SARA表达的丧失可能改变T？R相互作用的平衡，以调节Smad信号并增强转化生长因子-β刺激的纤维化。为了解决这一假设，我们将追求四个具体目标：(1)表征SARA、DAB2和Axin与T？R在转化生长因子-β信号转导中的相互作用，它们在T？R-复合体运输中的作用，以及它们对Smad2和Smad3介导的对转化生长因子-1的反应的影响。(2)检测SARA的丢失是否足以诱导与纤维化相关的信号或运输的改变，并确定这些变化是如何发生的。(3)明确转化生长因子β1下调SARA表达的机制及PI-3-激酶在此下调中的作用。(4)确定SARA表达的降低是否在所建立的体外和体内肾纤维化模型中起作用。这些研究将确定与转化生长因子-β信号转导和肾细胞纤维化活性相关的新事件。意义：尽管转化生长因子β是肾纤维化的重要介质，但其作用机制尚不清楚。我们的数据表明，几个T？R相互作用的接头分子之间的平衡在调节转化生长因子-β的反应中起着重要作用。具体地说，这些研究可能确定肾细胞向纤维化表型转变的新机制。了解这些机制可能预示着干预肾疤痕形成的潜在靶点。与公共健康相关的转化生长因子(TGF)是一种在全身发现的蛋白质，在肾脏和其他器官的疤痕形成中发挥核心作用。虽然我们在了解转化生长因子-β的作用机制方面取得了很大进展，但我们还没有学会如何调节它的作用，以防止或减少肾脏的疤痕形成。这个项目试图了解细胞中的一组蛋白质，即转化生长因子受体适配器蛋白，是如何帮助确定我们的细胞是否容易产生疤痕的。特别是，我们感兴趣的是了解一种名为SARA的蛋白质的表达是如何调节的，以及这种调节在瘢痕形成中扮演了什么角色。因此，实现我们的特定目标将提供关于转化生长因子-β如何发挥作用以及进展性肾脏疾病中肾脏如何受损的新的基本信息，这些信息可能对预防和治疗有用。

DESCRIPTION (provided by applicant): Transforming growth factor (TGF)-¿ is a critical regulator of events leading to chronic progressive renal disease such as extracellular matrix production, apoptosis and epithelial-mesenchymal transition (EMT). While considerable progress has been made in understanding signaling from the TGF-¿ receptor (T¿R) through the Smad signaling molecules, the role of adaptor proteins such as Smad Anchor for Receptor Activation (SARA) in these signals is not well understood. Our preliminary data indicate that, in human mesangial cells (MC) and renal tubular epithelial cell lines (HKC and HK-2), SARA binds to Smad2 in a TGF-¿-dependent manner. In contrast, SARA is constitutively bound to T¿R and is associated with early endosomes. After TGF-¿1 treatment, Smad2 is phosphorylated and clathrin-mediated endocytosis (CME) facilitates propagation of the TGF-¿ signal to the nucleus. Thus, SARA may play a central role in protein trafficking related to TGF-¿ signaling, particularly through its actions in T¿R trafficking and promoting Smad2 activity. In addition to SARA, our preliminary data have detected other adaptor proteins in MC and HKC that are proposed to function in Smad signaling but are even less well understood than SARA. Prolonged TGF-¿1 treatment leads to an eventual decline in SARA expression, which corresponds temporally to the assumption of a myofibroblastoid phenotype in MC, HKC and HK-2. We propose the hypothesis that by acting as scaffolding proteins to assemble and localize the T¿R signaling complex, the adaptor proteins SARA, Dab2 and Axin may each impact the TGF-¿ response, and the loss of SARA expression may alter the balance in T¿R interactions to modulate Smad signaling and enhance TGF-¿-stimulated fibrosis. To address this hypothesis we will pursue four specific aims: (1) Characterize the interactions of SARA, Dab2 and Axin with T¿R in TGF-¿ signaling, their role in T¿R-complex trafficking, and their effect on Smad2- vs. Smad3-mediated responses to TGF-¿1. (2) Examine whether the loss of SARA is sufficient to induce alterations in signaling or trafficking related to fibrosis, and determine how these changes occur. (3) Define the mechanism by which TGF-¿1 decreases SARA expression and the role of PI- 3-kinase in this down-regulation. (4) Determine whether a decrease in SARA expression could play a role in established in vitro and in vivo models of renal fibrogenesis. These studies will identify novel events related to TGF-¿ signaling and renal cell fibrogenic activity. SIGNIFICANCE: Although TGF-¿ is an important mediator of renal fibrogenesis, its mechanisms of action are poorly understood. Our data suggest that the balance among several T¿R-interacting adaptor molecules plays an important role in regulating TGF-¿ responses. Specifically, these studies may identify novel mechanisms whereby kidney cells transition to a fibrogenic phenotype. Understanding these mechanisms may indicate potential targets for interrupting renal scarring. PUBLIC HEALTH RELEVANCE Transforming growth factor (TGF)-¿ is a protein that is found throughout the body and plays a central role in scarring of the kidney and other organs. Although we have made much progress in understanding how TGF-¿ works, we have not learned how to regulate its actions in order to prevent or minimize scarring of the kidney. This project seeks to understand how one group of proteins in the cell, the TGF-¿ receptor adaptor proteins, helps determine whether our cells are disposed to produce scar. In particular, we are interested in understanding how the expression of one protein, called SARA, is regulated and what role this regulation plays in scarring. Carrying out our specific aims will thus provide new basic information regarding how TGF-¿ works and how the kidney is damaged in progressive kidney disease that could be useful in prevention and treatment.