STAT Regulation by Polycystin-1

常染色体显性（ADPKD）是最常见的危及生命的遗传性疾病。我们最近发现在ADPKD中受影响的蛋白polycytin -1 （PC1）在肾上皮细胞中通过纤毛机械转导调节STAT6活性。PC1经历流动依赖的蛋白水解裂解，使其细胞质尾部从膜上释放出来，随后发生核易位。PC1尾部结合STAT6和转录共激活子P100，刺激STAT6依赖性基因的表达。当液体停止流动时，STAT6从初级纤毛转移到细胞核。细胞核PC1尾部在ADPKD的囊衬上皮细胞中高度表达。PC1尾部的表达刺激MDCK细胞的增殖并导致斑马鱼胚胎肾囊肿的形成。此外，MDCK细胞对白细胞介素-4 （IL4）和IL13的反应类似于免疫细胞，它们激活STAT6，上调IL4/13受体链的表达。这些结果强烈提示PC1在正常健康肾脏中具有抑制STAT6活性的功能。如由于肾损伤导致的管腔液流动停止，可触发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STAT6活性和异常的增殖“修复”反应，从而导致囊肿生长。我们的初步结果表明，一种临床批准的药物，已知可以抑制STAT6，在多囊小鼠模型中强烈抑制肾脏生长并保持肾功能。我们现在建议详细研究PC1调控STAT6活性的机制，并验证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Aim 1中，我们将描述STAT6在表达STAT6应答型GFP报告基因的MDCK细胞中的调控。研究分化状态、PC1尾表达、根尖液流动和IL4/13的影响。我们将在抓伤模型中测试STAT6是否被激活。我们将研究IL4/13和IL4/13受体链在STAT6激活下的表达和定位/分泌。分析ADPKD组织和多囊小鼠模型将揭示IL4/13受体在囊肿中是否上调，以及它们是否将IL4/13分泌到管腔中。最后，我们将在小鼠肾缺血/再灌注损伤模型中验证il /13/STAT6/PC1通路被激活的假设。在Aim 2中，我们将测试STAT6缺失小鼠与多囊小鼠模型杂交是否会抑制肾囊性疾病。此外，我们将研究STAT6抑制剂治疗对肾囊性疾病的改善作用，并描述其作用机制。在Aim 3中，我们将以肾特异性和强西环素诱导的方式产生过表达可溶性PC1尾部的转基因小鼠系。基于我们在MDCK细胞和斑马鱼中的结果，我们预计这些动物将发展为肾囊性疾病，并在机制上与人类ADPKD最接近。

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.