Nuclear-Cytoplasmic Shuttling of Foxc2 via 14-3-3 Determines Proximal Tubule Cell

描述(由申请人提供)：急性肾损伤(AKI)是心脏手术的一种并发症，具有短期发病率、治疗成本增加和长期预后差的特点。肾损伤后，肾小管再生可恢复正常的肾小管结构和肾功能。本研究的目的是探讨FOXC2在近曲小管损伤和修复过程中核质穿梭的机制。FOXC2是Forkhead box(FOX)转录因子的成员，已知参与心血管、骨骼和肾脏的发育，在决定间充质细胞命运方面发挥作用。在上皮性肿瘤中，核FOXC2表达上调导致上皮标记物(E-钙粘附素和连接素)的抑制和间质标记物(波形蛋白和β-Sma)的增加。然而，我们实验室最近的证据表明，胞质FOXC2有助于维持受损近端小管细胞的上皮状态。因此，从细胞核输出FOXC2可能有助于缓和急性损伤的去分化反应，并在修复过程中促进近端小管细胞上皮的再分化。我的初步数据表明，丝氨酸125的磷酸化将FOXC2送出近端小管细胞的细胞核。我还发现FOXC2与小鼠近端小管细胞胞浆中的14-3-3和？-肌动蛋白结合。我的假设是，缺血性肾小管损伤后胞浆FOXC2的增加促进了肌动蛋白的稳定和/或局部粘连的定位，在局灶性粘连中，肌动蛋白对FA的周转和细胞的形态形成/迁移至关重要。特异的AIM1决定FOXC2核出口如何通过14-3-3和？-肌动蛋白结合促进肾小管细胞修复。将通过确定GFP标记的FOXC2结构的亚细胞定位来评估调控位点/结合伙伴，其中假定的结合/调控位点发生突变。将使用磷酸化蛋白质组学方法筛选这些位点的候选调节蛋白，然后进行激酶抑制和/或敲除研究，以确定FOXC2对伙伴蛋白的调节以及对亚细胞(细胞质与核)定位的影响。还将检查两个调控位点的突变体，以确定14-3-3 通过免疫共沉淀调节FOXC2和肌动蛋白相互作用。通过siRNA转染抑制内源性FOXC2的表达将被用来检测内源性FOXC2基因敲除对肌动蛋白定位的影响。FOXC2定位于细胞质的过表达将决定FOXC2对肌动蛋白和细胞骨架重排的上调。我们将使用肌动蛋白和帕西林染色(灶性粘连)，通过共聚焦显微镜观察和定量。细胞迁移将通过损伤/薄片分析进行监测。特异性AIM2检测FOXC2在体内近端小管损伤和修复中的作用。在体内，通过注射RNAi并通过近端小管特异性摄取FOXC2基因，将在缺血/再灌注(I/R)时进行，以模拟去分化和再分化过程。Western blotting将确定FOXC2的siRNA在肾脏中的敲除水平。FOXC2、14-3-3和β-肌动蛋白的免疫荧光共聚焦显微镜和免疫印迹分析以及局部粘连的巴西林染色也将被检测。TUNEL染色将被用来检测细胞凋亡。总的来说，这些目的是为了更好地了解FOXC2的穿梭机制以及FOXC2在上皮损伤和近端小管修复中的作用。 公共卫生相关性：缺血/再灌注是住院患者急性肾损伤(AKI)的主要原因，并会导致心脏手术的并发症，具有短期发病率、治疗成本增加和长期预后差的特点。肾损伤后肾小管再生可恢复正常的肾小管结构和肾功能，但肾小管损伤和修复的机制尚不清楚。对机制和潜在治疗靶点的广泛研究将促进对近端肾小管再生中AKI的理解和潜在的治疗。

DESCRIPTION (provided by applicant): Acute kidney injury (AKI) is a complication of cardiac surgery with short-term morbidity, increased costs of treatment, and poor long-term outcome. Tubular regeneration restores normal tubular architecture and renal function following kidney injury. The goal of the proposed research is to investigate the mechanism of Foxc2 nuclear-cytoplasmic shuttling via during proximal tubule injury and repair. Foxc2 is a member of the Forkhead box (Fox) transcription factors known to be involved in cardiovascular, skeletal, and kidney development, playing a role in specifying mesenchymal cell fates. Upregulation of nuclear Foxc2 in epithelial tumors leads to repression of epithelial markers (E-cadherin and catenins) and an increase in mesenchymal markers (vimentin and ¿-Sma). However, recent evidence from our laboratory has shown that cytoplasmic Foxc2 helps maintain the epithelial state in injured proximal tubule cells. Thus, exporting Foxc2 from the nucleus may serve to moderate the de-differentiation response to acute injury and to promote epithelial re-differentiation during the repair process in proximal tubule cells. My preliminary data suggests that phosphorylation at serine 125 shuttles Foxc2 out of the nucleus in proximal tubule cells. I have also discovered that Foxc2 associates with 14- 3-3 and ¿-actinins in the cytoplasm of mouse proximal tubule cells. My hypothesis that the increase in cytoplasmic Foxc2 that is seen following ischemic tubular injury promotes ¿-actinin stabilization and/or localization to focal adhesions, where it is critical for FA turnover and cell morphogenesis/migration. Specific aim1 determines how Foxc2 nuclear export promotes tubular cell repair by 14-3-3 and ¿-actinin association. Regulatory sites/binding partners will be evaluated by determining the subcellular localization of GFP-tagged Foxc2 constructs in which the putative binding/regulatory site is mutated. Candidate regulatory kinases for these sites will be screened using a phosphoproteomics approach followed by kinase inhibitor and/or knock- down studies to determine regulation of partner proteins with Foxc2 and impact on subcellular (cytoplasmic vs. nuclear) localization. Mutants at two regulatory sites will also be examined to determine if 14-3-3 modulates Foxc2 and ¿-actinin interaction by co-immunoprecipitation. Inhibition of endogenous Foxc2 expression by siRNA transfection will be utilized to examine the effect of endogenous Foxc2 knockdown on ¿-actinin localization. Overexpression of Foxc2 localized to the cytoplasm will determine upregulation of Foxc2 on ¿-actinin and cytoskeleton rearrangements. We will use ¿-actinin and paxillin staining (focal adhesions) viewed and quantified by confocal microscopy. Cell migration will be monitored by wounding/sheet assays. Specific aim2 examines the role of Foxc2 during injury and repair of the proximal tubule in vivo. Knockdown of Foxc2 in vivo by RNAi injection with uptake specifically by the proximal tubule will be performed along with Ischemia/Reperfusion (I/R) to simulate the de-differentiation and re-differentiation process. Western blots will be performed to determine the level of knockdown in the kidney after siRNA of Foxc2. Immunofluorescence by confocal microscopy and western blot analysis of Foxc2, 14-3-3, and ¿-actinin along with paxillin staining for focal adhesions will also be examined. TUNEL staining will be utilized to measure cellular apoptosis. Cumulatively, these aims are designed to better understand the mechanisms of Foxc2 shuttling and the role of Foxc2 in epithelial injury and repair of the proximal tubule. PUBLIC HEALTH RELEVANCE: Ischemia/reperfusion is a major cause of acute kidney injury (AKI) in hospitalized patients and causes complications in cardiac surgery with short-term morbidity, increased costs of treatment, and poor long-term outcome. Tubular regeneration restores normal tubular architecture and renal function following kidney injury and the mechanisms of tubular injury and repair are poorly understood. Extensive examination of the mechanisms and potential therapeutic targets will advance the understanding and potential cure for AKI in proximal tubular regeneration.