Roles of Tuberin (TSC2), Hamartin (TSC1), and Rheb in Renal Cyst Pathogenesis

描述(申请人提供)：结节性硬化症(TSC)是由分别编码Hamartin(TSC1)和Tuberin(TSC2)的TSC1或TSC2基因的种系失活突变引起的。携带TSC1或TSC2基因突变的人类和小鼠都会患上囊性肾病。TSC1和TSC2蛋白作为异源二聚体抑制Rheb，Rheb是tuberin的GTPase激活域的靶标。Rheb激活雷帕霉素(MTOR)复合体1(TORC1)的哺乳动物靶点。我们和其他人观察到常染色体显性遗传性多囊肾病(ADPKD)患者囊中mTOR通路过度激活的证据，表明TSC/Rheb/mTOR通路参与了ADPKD的发病。除了在ADPKD包囊和PKD1失活小鼠的包囊中mTOR激活外，另有两条证据表明TSC蛋白和Rheb与ADPKD的发病密切相关：TSC2蛋白被发现调节多囊蛋白1(PC1)的膜定位，PC1的过度表达的羧基末端被发现与TSC2共免疫沉淀。这些数据导致了一种假设，即PC1的失活足以激活mTOR。然而，我们发现PKD-/-MEF没有mTOR激活的证据，并且mTOR只在ADPKD包囊的一部分中高活性。由于雷帕霉素和其他TORC1抑制剂目前正在对ADPKD患者进行临床试验，因此阐明在ADPKD囊肿子集中激活mTOR的未完全了解的机制具有特别重要的临床意义。在未发表的工作中，我们发现Hamartin(TSC1)定位于初级纤毛的基底层，并且TSC1缺失和TSC2缺失的小鼠胚胎成纤维细胞(MEF)的纤毛细胞比例高于对照MEF。这些数据为AIM 2提供了基础，首次将TSC途径与纤毛联系起来，提示Rheb可能通过多个机制影响囊变发生。我们的中心假设是Rheb的激活和PKD1的突变失活协同促进了ADPKD的囊变发生。为了验证这一点，我们提出了以下目标：目标1：定义ADPKD中mTOR激活的机制。目的2：确定TSC蛋白调控纤毛形成的机制。目的3：探讨Rheb激活是否增强了PKD1诱导的囊性病变。 公共卫生相关性：确定ADPKD中mTOR激活的机制以及TSC蛋白和Rheb在纤毛形成中的作用可能对全球约400万至600万ADPKD患者具有关键的治疗意义，特别是因为mTOR抑制剂已经在PKD的临床试验中。

DESCRIPTION (provided by applicant): Tuberous sclerosis complex (TSC) is caused by germline inactivating mutations in the TSC1 or TSC2 genes, which encode hamartin (TSC1) and tuberin (TSC2), respectively. Both humans and mice with mutations in TSC1 or TSC2 can develop cystic kidney disease. The TSC1 and TSC2 proteins function as a heterodimer to inhibit Rheb, which is the target of tuberin's GTPase activating domain. Rheb activates the mammalian target of rapamycin (mTOR) complex 1 (TORC1). We and others have observed evidence of mTOR pathway hyperactivation in cysts from autosomal dominant polycystic kidney disease (ADPKD) patients, suggesting that the TSC/Rheb/mTOR pathway is involved in ADPKD pathogenesis. In addition to the mTOR activation in ADPKD cysts and in cysts from mice with Pkd1 inactivation, two other lines of evidence suggest that the TSC proteins and Rheb are closely linked with the pathogenesis of ADPKD: the TSC2 protein has been found to regulate membrane localization of Polycystin 1 (PC1), and the overexpressed carboxyl-terminus of PC1 has been found to co-immunoprecipitate with TSC2. These data have led to the hypothesis that inactivation of PC1 is sufficient to activate mTOR. However, we have found that Pkd1-/- MEFs do not have evidence of mTOR activation and that mTOR is hyperactive in only a subset of ADPKD cysts. Elucidating the incompletely understood mechanisms through which mTOR is activated in a subset of ADPKD cysts, which is the focus of Aim 1, has particular clinical importance since rapamycin and other TORC1 inhibitors are now in clinical trials for patients with ADPKD. In unpublished work, we have found that hamartin (TSC1) localizes to the basal body of the primary cilium, and that Tsc1-null and Tsc2-null mouse embryonic fibroblasts (MEFs) have a higher fraction of ciliated cells than control MEFs. These data, which provide the foundation for Aim 2, link the TSC pathway with the cilium for the first time and suggest that Rheb may impact cyst pathogenesis through multiple mechanisms Our central hypothesis is that activation of Rheb cooperates with mutational inactivation of PKD1 to promote cyst pathogenesis in ADPKD. To test this, we propose the following Aims: Aim 1: To define the mechanisms through which mTOR is activated in ADPKD. Aim 2: To identify the mechanisms through which the TSC proteins regulate ciliary formation. Aim 3: To determine whether Rheb activation potentiates Pkd1-induced cyst pathogenesis. PUBLIC HEALTH RELEVANCE: Determining the mechanisms of mTOR activation in ADPKD and the role of the TSC proteins and Rheb in ciliary formation may have key therapeutic implications for the estimated 4-6 million ADPKD patients worldwide, particularly since mTOR inhibitors are already in clinical trials for PKD.