ADPKD: Understanding mechanisms, Discovering treatments.

描述（由申请人提供）：本提案的目的是继续我们的研究，了解肿瘤坏死因子-α（TNF？？）信号传导。肿瘤坏死因子？通过其受体和通过受体相关蛋白质形成膜结合的促存活复合物I以及细胞质促死亡复合物II的能力，帮助癌细胞逃避凋亡。细胞凋亡抑制剂（IAP）拮抗剂Smac-mimetic能够靶向上述TNF？介导的复合物诱导癌细胞死亡。我们的研究表明TNF？可促进ADPKD的进展，我们的初步研究进一步发现：1）TNF？多囊蛋白功能丧失导致TNF？？受体I（TNFR-I）; 3）复合物I和复合物II中的蛋白组分在Pkd 1突变的MEK细胞中上调; 4）最重要的是，TNF？单独不能诱导正常或Pkd 1突变上皮细胞死亡，即使在高浓度，而TNF？与瞬时转染的Smac一起仅诱导Pkd 1突变型囊性上皮细胞死亡，而对Pkd 1野生型肾上皮细胞没有影响。此外，以下证据支持TNF？和Id 2信号通路：1）TNF？通过IKK激活mTOR mTOR通过调节Id 2的表达调节乳腺上皮细胞的功能分化; 2）NF-？B配体（RANKL）是一种TNF家族分子，通过触发Id 2的显著核转位，经由Id 2调节乳腺上皮细胞增殖。基于这些发现，我们假设：TNF？？及其受体能够通过膜结合的促存活复合物I和胞质促死亡复合物II调节囊性上皮细胞凋亡，并通过mTOR或RANKL介导的Id 2信号传导进一步调节囊性上皮细胞增殖和分化。为了验证我们的假设，我们提出了三个目标：1）研究TNF？？2）探讨IAP拮抗剂Smac模拟物在调节TNF？？依赖性囊性肾上皮细胞死亡; 3）研究TNF？？信号通过mTOR和RANKL介导的Id 2信号调节囊性上皮细胞增殖和分化。完成该项目将进一步阐明多囊蛋白下游途径在囊肿形成中的作用，并将确定可能作为减缓疾病进展的有效靶点的关键调控成分。 公共卫生相关性：本研究的目的是进一步了解肿瘤坏死因子-α（TNF？？）信号传导。我们的一般假设是TNF？IAP拮抗剂Smac-mimetic可诱导TNF？依赖于囊性上皮细胞死亡，以进一步防止囊肿扩张/形成。完成该项目将进一步阐明多囊蛋白下游途径在囊肿形成中的作用，并将确定可能作为减缓疾病进展的有效靶点的关键调控成分。本研究的结果将为ADPKD的研究开辟新的途径，并为开发有效的治疗方法奠定基础。

DESCRIPTION (provided by applicant): The objective of this proposal is to continue our studies on understanding the mechanisms of tumor necrosis factor-alpha (TNF??) signaling in cystogenesis. TNF?? through its receptor and through the ability of the receptor associated proteins to form the membrane bound pro-survival complex I as well as the cytoplasmic pro-death complex II help cancer cells to escape apoptosis. The inhibitor of apoptosis (IAP) antagonist, Smac-mimetic, is able to target the above TNF??-mediated complexes to induce cancer cell death. Our studies have suggested that TNF?? can promote ADPKD progression and our preliminary studies have further found: 1) TNF?? is always present in cyst fluid from ADPKD kidneys; 2) Loss of polycystin function leads to increased expression of TNF?? receptor I (TNFR-I); 3) The components of proteins in complex I and complex II are upregulated in Pkd1 mutant MEK cells; 4) Most importantly, TNF?? alone could not induce normal or Pkd1 mutant epithelial cell death even at high concentrations, while, TNF?? together with transiently transfected Smac could induce only Pkd1 mutant cystic epithelial cell death but had no effect on Pkd1 wild type kidney epithelial cells. Furthermore, the following evidence supports the connection between TNF?? and Id2 signaling pathways: 1) TNF?? activates mTOR through IKK? and mTOR regulates the functional differentiation of mammary epithelial cells through regulating the expression of Id2; 2) Receptor activator of NF-?B ligand (RANKL), a TNF family molecule, regulates mammary epithelial cell proliferation via Id2 by triggering marked nuclear translocation of Id2. Based on these findings, we hypothesize that: TNF?? and its receptor are able to regulate cystic epithelial cell apoptosis through the membrane bound pro-survival complex I and cytoplasmic pro-death complex II and further regulate cystic epithelial cell proliferation and differentiation through mTOR or RANKL-mediated Id2 signaling. To test our hypothesis, we propose three aims: 1) To Investigate the mechanism(s) of TNF?? signaling in regulation of kidney epithelial cell survival; 2) To determine the potential role and mechanism of the IAP antagonist, Smac-mimetic, in regulating TNF??-dependent cystic kidney epithelial cell death; 3) To investigate whether TNF?? signaling regulates cystic epithelial cell proliferation and differentiation through mTOR and RANKL mediated Id2 signaling. Accomplishing this project will further elucidate the role of the pathways downstream of the polycystins in cystogenesis and will identify key regulatory components that may serve as effective targets to slow disease progression. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to continue our studies on understanding the mechanisms of tumor necrosis factor-alpha (TNF??) signaling in cystogenesis. Our general hypothesis is that TNF?? through its receptor to form a membrane bound pro-survival complex protects the cystic epithelial cell apoptosis and IAP antagonist, Smac-mimetic, can induce TNF?? dependent cystic epithelial cell death to further prevent cyst expansion/formation. Accomplishing this project will further elucidate the role of the pathways downstream of the polycystins in cystogenesis and will identify key regulatory components that may serve as effective targets to slow disease progression. The information from our studies will be likely to open new avenues in the research of ADPKD and establish the foundation for developing effective therapies of the disease.