STAT Regulation by Polycystin-1

常染色体显性遗传病(ADPKD)是最常见的危及生命的遗传病。我们最近发现，ADPKD中受影响的蛋白多囊蛋白-1(Polycystin-1，PC1)通过纤毛机械转导在肾上皮细胞中调节STAT6的活性。PC1经历了依赖于流动的蛋白分解，从膜上释放出细胞质的尾巴，然后是核转位。PC1尾巴与STAT6和转录共激活因子P100结合，刺激STAT6依赖的基因表达。当液体停止流动时，STAT6从初级纤毛转移到细胞核。核型PC1尾巴在ADPKD的囊壁上皮细胞中高表达。PC1尾巴的表达刺激MDCK细胞的增殖，并导致斑马鱼胚胎的肾囊形成。此外，MDCK细胞对白介素4(IL4)和IL13的反应类似于免疫细胞，因为它们激活STAT6，并上调IL4/13受体链的表达。这些结果强烈地表明，PC1在正常健康的肾脏中起着沉默STAT6活性的作用。腔内液体流动停止，例如由于肾脏损伤，会触发PC1裂解、STAT6激活和增殖反应。我们假设在ADPKD中缺乏功能性PC1会导致结构性的STAT6活性和异常的增殖性“修复”反应，从而导致囊性生长。我们的初步结果显示，一种临床批准的药物，已知抑制STAT6，在多囊小鼠模型中强烈抑制肾脏生长并保护肾功能。我们现在建议详细研究PC1调节STAT6活性的机制，并检验我们关于这一新的信号通路在肾损伤修复和ADPKD中的作用的假设。在目标1中，我们将表征STAT6在表达STAT6响应GFP报告的MDCK细胞中的调节。我们将研究分化状态、PC1 Tail表达、根尖液流量和IL4/13的影响。我们将测试STAT6是否在划伤模型中被激活。我们将研究STAT6激活对IL4/13和IL4/13受体链的表达和定位/分泌的影响。对ADPKD组织和多囊小鼠模型的分析将揭示IL4/13受体在囊中是否上调，以及它们是否将IL4/13分泌到管腔。最后，我们将验证我们的假设，即IL4/13/STAT6/PC1通路在小鼠肾缺血/再灌注损伤模型中被激活。在目标2中，我们将测试STAT6基因缺失的小鼠与多囊小鼠模型的杂交是否会导致肾囊性疾病的抑制。此外，我们还将研究STAT6抑制剂对肾囊性疾病的改善作用，并阐明其作用机制。在目标3中，我们将建立一个转基因小鼠系，以肾脏特异性和多西环素诱导的方式过表达可溶性PC1尾巴。根据我们在MDCK细胞和斑马鱼上的研究结果，我们预计这些动物将发展为肾囊性疾病，并将在机制上最接近于人类ADPKD。

Autosomal-dominant (ADPKD) is the most common life-threatening genetic disease. We have recently found that polycystin-1 (PC1), the protein affected in ADPKD, functions in the regulation of STAT6 activity by ciliary mechanotransduction in renal epithelial cells. PC1 undergoes flow-dependent proteolytic cleavage which releases its cytoplasmic tail from the membrane, followed by nuclear translocation. The PC1 tail binds to STAT6 and the transcriptional co-activator P100, and stimulates STAT6-dependent gene expression. STAT6 translocates from primary cilia to nuclei upon cessation of fluid flow. The nuclear PC1 tail is highly expressed in cyst-lining epithelial cells in ADPKD. Expression of the PC1 tail stimulates proliferation in MDCK cells and results in renal cyst-formation in zebrafish embryos. Furthermore, MDCK cells respond to interleukin-4 (IL4) and IL13 similar to immune cells in that they activate STAT6, and up-regulate the expression if IL4/13 receptor chains. These results strongly suggest that PC1 functions to silence STAT6 activity in the normal healthy kidney. Cessation of lumenal fluid flow, e.g. due to renal injury, triggers PC1 cleavage, STAT6 activation and a proliferative response. We hypothesize that lack of functional PC1 in ADPKD leads to constitutive STAT6 activity and an aberrant proliferative "repair" response leading to cyst growth. Our preliminary results show that a clinically approved drug, known to inhibit STAT6, strongly inhibits renal growth and preserves renal function in a polycystic mouse model. We now propose to study in detail the mechanism of the regulation of STAT6 activity by PC1 and to test our hypotheses regarding the role of this novel signaling pathway in renal injury repair and ADPKD. In Aim 1, we will characterize the regulation of STAT6 in MDCK cells expressing a STAT6-responsive GFP reporter. The effects of the state of differentiation, PC1 tail expression, apical fluid flow and IL4/13 will be investigated. We will test whether STAT6 is activated in a scratch-wounding model. We will investigate the expression and localization/secretion of IL4/13 and IL4/13 receptor chains in response to STAT6 activation. Analysis of ADPKD tissue and polycystic mouse models will reveal whether IL4/13 receptors are up-regulated in cysts and whether they secrete IL4/13 into the lumen. Finally, we will test our hypothesis that the IL4/13/STAT6/PC1 pathway is activated in a mouse model of renal ischemia/reperfusion injury. In Aim 2, we will test whether crossing of STAT6 null mice with polycystic mouse models will result in suppression of renal cystic disease. Furthermore, we will investigate the amelioration of renal cystic disease by treatment with the STAT6 inhibitor and delineate the mechanism of action. In Aim 3, we will generate a transgenic mouse line over-expressing the soluble PC1 tail in a kidney-specific and doxycycline-inducible manner. Based on our results in MDCK cells and zebrafish, we anticipate that these animals will develop renal cystic disease and will mimic human ADPKD most closely mechanistically.