HSP27 in kidney Epithelial to Mesenchymal Transformation

描述(由申请人提供)： 这项K08奖提案的目的是使候选人成为慢性肾脏疾病(CKD)领域的肾脏病理学和分子生物学的独立研究员。候选人提出了一个在贝克尔博士、休利特博士(生物化学)、奥伯利博士(病理学)、艾肯博士(分子生物学)和巴肯博士(教师发展)的多学科指导下的研究计划。实验室工作将辅以分子生物学、病理学和生物化学方面的课程，旨在加强候选人作为内科科学家的培训。慢性肾脏病是一个世界性的公共卫生问题，结果差，成本高。新的证据表明，肾小管上皮-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导致肾脏各种损伤的常见最终途径。EMT涉及肾小管上皮细胞(TEC)向间充质细胞的转化，肌动蛋白应激纤维的形成，细胞黏附的丧失和细胞迁移的增加。热休克蛋白27(HSP27)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细丝动力学来发挥其细胞保护作用。尽管HSP27具有细胞保护作用以及与肌动蛋白细丝的相互作用，但尚未在EMT中得到研究。本研究的目的是(1)研究HSP27在EMT中的表达水平和定位；(2)剖析导致HSP27激活的信号事件；(3)研究HSP27的过表达是否延迟/抑制EMT。我们成功地在NRK52E细胞(大鼠近端TEC细胞系)中复制了EMT。1.从培养细胞中提取mRNA和蛋白后，采用实时荧光定量PCR、免疫印迹(IB)和免疫组织化学(IHC)技术，结合EMT的标志物(m-平滑肌肌动蛋白和粘附素)，研究不同时间点HSP27的mRNA和蛋白水平。Hsp27细胞的定位将通过荧光和免疫金电子显微镜(EM)进行分析。IB、IHC和EM将使用鬼臼毒素和总/磷酸特异性抗体以及MAPK途径的抑制剂来评估HSP27-肌动蛋白的相互作用和丝裂原激活的蛋白激酶(MAPK)途径。通过重组病毒介导的HSP27基因转移可获得HSP27的高表达。这一建议将使我们能够确定HSP27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.