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??)信号在膀胱发生中的机制。肿瘤坏死因子?通过其受体和受体相关蛋白形成膜结合的促生存复合体I以及细胞质促死亡复合体II的能力，帮助癌细胞逃脱凋亡。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?？可以促进ADPKD的进展，我们的初步研究进一步发现：1)TNF?？总是存在于ADPKD肾脏的囊肿液中；2)多囊蛋白功能丧失导致TNF?？受体I (TNFR-I)；3)在Pkd1突变的MEK细胞中复合物I和复合物II蛋白组分上调；4)最重要的是TNF?？单独使用即使高浓度也不能诱导正常或Pkd1突变的上皮细胞死亡，而TNF?？瞬时转染的Smac只诱导Pkd1突变型囊上皮细胞死亡，而对Pkd1野生型肾上皮细胞无影响。此外，以下证据支持TNF?？和Id2信号通路：1)TNF？通过IKK激活mTOR ？mTOR通过调节Id2的表达调控乳腺上皮细胞的功能分化；2) NF-?受体激活剂B配体（RANKL）， TNF家族分子，通过Id2触发标记的Id2核易位，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生存复合体I和细胞质促死亡复合体II调控囊性上皮细胞凋亡，并通过mTOR或rankl介导的Id2信号传导进一步调控囊性上皮细胞的增殖和分化。为了验证我们的假设，我们提出了三个目标：1)研究TNF的作用机制？肾上皮细胞存活的信号调控2)确定IAP拮抗剂Smac-mimetic在调节TNF中的潜在作用和机制。依赖性囊肾上皮细胞死亡；3)探讨TNF?？信号通路通过mTOR和RANKL介导的Id2信号通路调控囊性上皮细胞的增殖和分化。完成该项目将进一步阐明多囊蛋白下游通路在膀胱形成中的作用，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。

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.