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

描述（由申请方提供）：急性肾损伤（阿基）是心脏手术的并发症，具有短期发病率、治疗费用增加和长期结局不良。肾小管再生恢复肾损伤后正常的肾小管结构和肾功能。本研究的目的是探讨近端小管损伤和修复过程中Foxc 2核质穿梭的机制。Foxc 2是已知参与心血管、骨骼和肾脏发育的叉头盒（Fox）转录因子的成员，在指定间充质细胞命运中起作用。上皮肿瘤中细胞核Foxc 2的上调导致上皮标志物（E-钙粘蛋白和连环蛋白）的抑制和间充质标志物（波形蛋白和<$-Sma）的增加。然而，我们实验室最近的证据表明，细胞质Foxc 2有助于维持受损近曲小管细胞的上皮状态。因此，输出Foxc 2从细胞核可能有助于缓和去分化反应急性损伤，并促进上皮细胞在修复过程中的近端小管细胞的再分化。我的初步数据表明，在丝氨酸125磷酸化穿梭Foxc 2的细胞核在近端小管细胞。我还发现Foxc 2与小鼠近曲小管细胞质中的14- 3-3和<$-辅肌动蛋白相关。我的假设是，缺血性肾小管损伤后细胞质Foxc 2的增加促进了辅肌动蛋白的稳定和/或定位于局灶性粘连，这对FA周转和细胞形态发生/迁移至关重要。特异性aim 1决定Foxc 2核输出如何通过14-3-3和<$-辅肌动蛋白联合促进肾小管细胞修复。通过确定GFP标记的Foxc 2构建体的亚细胞定位来评价调控位点/结合配偶体，其中推定的结合/调控位点发生突变。将使用磷酸蛋白质组学方法筛选这些位点的候选调节激酶，然后进行激酶抑制剂和/或敲低研究，以确定Foxc 2对伴侣蛋白的调节以及对亚细胞（细胞质vs.细胞核）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Foxc 2和辅肌动蛋白的相互作用。通过siRNA转染对内源性Foxc 2表达的抑制将用于检查内源性Foxc 2敲低对辅肌动蛋白定位的影响。定位于细胞质的Foxc 2的过表达将决定Foxc 2对辅肌动蛋白和细胞骨架重排的上调。我们将使用辅肌动蛋白和桩蛋白染色（局灶性粘连），通过共聚焦显微镜观察和定量。将通过创伤/片层测定监测细胞迁移。特异性aim 2检查Foxc 2在体内近端小管损伤和修复过程中的作用。通过RNAi注射并通过近端小管特异性摄取的体内Foxc 2敲低将与缺血/再灌注（I/R）一起沿着进行，以模拟去分化和再分化过程。将进行蛋白质印迹以确定Foxc 2的siRNA后肾脏中的敲低水平。还将通过共聚焦显微镜和免疫印迹分析Foxc 2、14-3-3和沿着肌动蛋白以及桩蛋白染色检查局灶性粘连。TUNEL染色将用于测量细胞凋亡。累积起来，这些目标旨在更好地了解Foxc 2穿梭的机制以及Foxc 2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（阿基）的主要原因，并导致心脏手术并发症，短期发病率，治疗成本增加，长期结局不佳。肾小管再生恢复正常的肾小管结构和肾功能后，肾损伤和肾小管损伤和修复的机制知之甚少。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阿基的理解和潜在治疗。

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.