HSP27 in kidney Epithelial to Mesenchymal Transformation

描述（由申请人提供）： 该K 08奖项的目的是使候选人成为慢性肾脏病（CKD）领域肾脏病理学和分子生物学的独立研究者。候选人提出了一个多学科指导下的研究计划博士贝克尔，Hullett（生物化学），Oberley（病理学），艾肯（分子生物学）和巴肯（教师发展）。实验室工作将补充分子生物学，病理学和生物化学课程，旨在加强候选人的培训，作为一个医生，科学家。CKD是一个全球性的公共卫生问题，结果不佳，成本高。新出现的证据表明，肾小管上皮细胞-肌成纤维细胞转分化（EMT）是肾小管间质纤维化的重要事件，肾小管间质纤维化是各种肾脏损伤的常见最终途径。EMT涉及肾小管上皮细胞（TEC）转化为具有间充质形态的细胞、肌动蛋白应力纤维的形成、细胞粘附的丧失和细胞迁移的增加。热休克蛋白27（HSP 27）是一种应激蛋白，通过抑制细胞凋亡、控制氧化还原状态和调节肌动蛋白丝动力学发挥其细胞保护作用。尽管它的细胞保护作用和与肌动蛋白丝的相互作用，热休克蛋白27尚未在EMT的研究。本研究的目的是（1）研究EMT过程中HSP 27的mRNA和蛋白水平及定位;（2）分析导致HSP 27活化的信号传导过程;（3）研究HSP 27过表达是否延迟/阻止EMT。我们已经成功地再现了NRK 52 E细胞（大鼠近端TEC细胞系）中的EMT用TGF-（1。从培养的细胞中提取mRNA和蛋白质后，将通过实时PCR、免疫印迹（IB）和免疫组织化学（IHC）技术以及EMT（m-平滑肌肌动蛋白和ecadherin）标志物在不同时间点研究HSP 27 mRNA和蛋白质水平。将通过荧光和免疫金电子显微镜（EM）分析HSP 27细胞定位。HSP 27-肌动蛋白相互作用和促分裂原活化蛋白激酶（MAPK）通路将通过IB、IHC和EM使用鬼笔环肽和总/磷酸特异性抗体以及MAPK通路抑制剂进行评估。通过重组病毒介导的HSP 27基因转移获得HSP 27的过表达。这一建议将使我们能够确定HSP 27过表达是否可以预防/延迟EMT。

DESCRIPTION (provided by applicant): The objective of this K08 Award proposal is to enable the candidate to become an independent investigator in renal pathology and molecular biology in the field of chronic kidney disease (CKD). The candidate proposes a research plan under the multidisciplinary mentorship of Drs. Becker, Hullett (biochemistry), Oberley (pathology), Aiken (molecular biology) and Bakken (faculty development). The laboratory efforts will be supplemented with coursework in molecular biology, pathology and biochemistry designed to enhance the candidate's training as a physician-scientist. CKD is a worldwide public health problem with poor outcomes and high costs. Emerging evidence suggests that tubular epithelial-myofibroblast transdifferentiation (EMT) is an important event in renal tubulointerstitial fibrosis, a common final pathway to various injuries to the kidney. EMT involves the transformation of tubular epithelial cells (TEC) into cells with mesenchymal morphology, formation of actin stress fibers, loss of cell adhesions and increased cell migration. Heat shock protein 27 (HSP27) is a stress protein that exerts its cytoprotective effects by apoptosis inhibition, control of the redox status and modulation of actin filament dynamics. Despite its cytoprotective roles and interactions with actin filaments, HSP27 has not been studied in EMT. The aims of this proposal are (1) to study HSP27 mRNA and protein levels and localization during EMT (2) to dissect the the signaling events that lead to HSP27 activation (3) to investigate whether overexpression of HSP27 delays/arrests EMT. We have successfully reproduced EMT in NRK52E cells (rat proximal TEC cell line) after six days of treatment with TGF-(1. Following the extraction of mRNA and protein from cultured cells, HSP27 mRNA and protein levels will be studied at various time-points by real-time PCR, immunoblot (IB) and immunohistochemistry (IHC) techniques, together with markers of EMT (m-smooth muscle actin and ecadherin). HSP27 cellular localization will be analyzed by fluorescence and immunogold electron microscopy (EM). HSP27-actin interactions and the mitogen-activated protein kinase (MAPK) pathway will be assessed by IB, IHC and and EM using phalloidin and total/phosphospecific antibodies, with inhibitors of the MAPK pathway. Overexpression of HSP27 will be obtained by recombinant virus-mediated HSP27 gene transfer. This proposal would allow us to determine whether HSP27 overexpression can prevent/delay EMT.