Myocilin-induced ER Stress in Trabecular Meshwork Cells

肌纤蛋白诱导的小梁网细胞内质网应激

• 批准号: 6774686
• 负责人: SANGLY P SRINIVAS
• 金额: $ 14.65万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6774686
• 关键词: apoptosis; calcium flux; confocal scanning microscopy; dexamethasone; endoplasmic reticulum; gene expression; gene mutation; glaucoma; glycoproteins; homeostasis; immunoprecipitation; intraocular pressure; molecular chaperones; northern blottings; polymerase chain reaction; tissue /cell culture; trabecular meshwork; tunicamycin; western blottings

DESCRIPTION (provided by applicant): Certain mutations of MYOC, a gene expressed in trabecular meshwork (TM cells), are associated with the juvenile onset of POAG (primary open angle glaucoma). The gene product of MYOC, myocilin, is a secretory protein of unknown function. Targeted null mutations of MYOC show neither an elevated intraocular pressure nor manifest morphological abnormalities in the TM, indicating that the disease-causing mutations of MYOC produce proteins that cause negative effects in TM cells (called gain-of-function toxicity). What is the nature of this toxicity? What is the molecular mechanism underlying the toxicity? In this project, we hypothesize that certain mutations of MYOC result in unfolding of myocilin leading to its retention and aggregation in the endoplasmic reticulum (ER) of the TM cells. Persistent accumulation of secretory or membrane proteins in the ER lumen is known to cause "ER stress." This stress elicits a unique but complex ER-to-nucleus signaling called "Unfolded Protein Response" (UPR) consisting of mechanisms to restore homeostasis in the ER. A deficiency or overshoot in UPR is known to adversely affect some, if not all of the functions of the ER. Therefore we further hypothesize that retention of aberrant myocilin induces UPR which, in turn, leads to Ca 2+ deregulation in TM cells. A number of studies on the pharmacology of TM have clearly demonstrated that elevated intracellular Ca 2+ increases the contractility of the TM cells and decreases the outflow facility across TM. Thus, the specific aims of this project are to characterize UPR and its mechanisms resulting from overexpression/mutations in MYOC, and to determine the adverse effects of myocilin-induced UPR on Ca2+ homeostasis. UPR induced by overexpression of myocilin and mutant forms of MYOC will be examined in primary cultures of bovine TM and HEK-293T cell line, respectively. As a positive control of UPR, we will employ exogenous drugs known to cause protein unfolding in the ER (e.g., tunicamycin). We will characterize UPR in terms of activation of components of UPR signaling pathways and on activation of various ER-specific chaperones. Ca 2+ deregulation will be investigated by examining transcriptional activation of SERCA Ca2+ ATPase and structural components of capacitative calcium influx pathways. Our techniques and protocols include use of quantitative real-time PCR, Northern blotting, Western blotting, confocal microscopy, and coimmuniprecipitation. These studies will lead to the development of an essential knowledge base and influence research in the field of glaucoma pathophysiology, diagnostics, and therapeutics.

描述（由申请人提供）：Myoc的某些突变，一种在小梁网络（TM细胞）中表达的基因（TM细胞），与POAG的少年发作（主要的敞开角度青光眼）有关。肌动蛋白的基因产物是一种分泌蛋白，具有未知功能的分泌蛋白。 MYOC的靶向零突变既不显示升高的眼内压，也没有表现出TM的形态异常，表明肌动物的致病突变产生的蛋白质会在TM细胞中引起负面影响（称为功能性毒性获得）。 这种毒性的性质是什么？毒性的基础机制是什么？在这个项目中，我们假设MYOC的某些突变导致肌动蛋白展开导致其在TM细胞内质网（ER）中的保留和聚集。 众所周知，ER管腔中分泌或膜蛋白的持续积累会引起“ ER应激”。 这种应力引起了一种独特但复杂的ER到核信号传导，称为“展开的蛋白质反应”（UPR），该信号由恢复ER中稳态的机制组成。 已知UPR中的缺陷或过时会对某些ER的功能产生不利影响。 因此，我们进一步假设保留异常的肌动蛋白会诱导UPR，从而导致TM细胞中的Ca 2+失控。 许多关于TM药理学的研究清楚地表明，升高的细胞内Ca 2+会增加TM细胞的收缩力，并减少TM跨TM的流出设施。 因此，该项目的具体目的是表征UPR及其由MYOC中过表达/突变引起的机制，并确定肌动蛋白诱导的UPR对Ca2+稳态的不利影响。 在牛TM和HEK-293T细胞系的原发性培养物中将检查由肌动蛋白和突变体形式过表达引起的UPR。 作为对UPR的阳性对照，我们将采用已知在ER中引起蛋白质展开的外源性药物（例如，衣霉素）。 我们将根据UPR信号通路的激活和各种ER特异性伴侣的激活来表征UPR。 Ca 2+放松管制将通过检查SERCA CA2+ ATPase的转录激活以及电容性钙涌入途径的结构成分。 我们的技术和协议包括使用定量实时PCR，Northern印迹，Western印迹，共聚焦显微镜和共发育沉淀。这些研究将导致基本知识基础的发展，并影响青光眼病理生理学，诊断和治疗学领域的研究。