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: 6686910
• 负责人: SANGLY P SRINIVAS
• 金额: $ 14.6万
• 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/6686910
• 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（一种在小梁网（TM细胞）中表达的基因）的某些突变与POAG（原发性开角型青光眼）的青少年发病相关。MYOC的基因产物myocilin是一种功能未知的分泌蛋白。 MYOC的靶向无效突变既没有显示出眼内压升高，也没有显示出TM中的明显形态异常，表明MYOC的致病突变产生了在TM细胞中引起负面影响的蛋白质（称为功能获得性毒性）。 这种毒性的性质是什么？毒性的分子机制是什么？在这个项目中，我们假设MYOC的某些突变导致肌球蛋白的解折叠，导致其在TM细胞的内质网（ER）中的滞留和聚集。 ER腔中分泌蛋白或膜蛋白的持续积累已知引起“ER应激”。这种压力激发了一种独特但复杂的ER到细胞核的信号传导，称为“未折叠蛋白反应”（UPR），由恢复ER内稳态的机制组成。 已知UPR中的不足或过冲会对ER的一些功能（如果不是全部功能的话）产生不利影响。 因此，我们进一步推测，异常肌钙蛋白的滞留诱导UPR，进而导致TM细胞中Ca 2+的失调。 大量关于TM药理学的研究表明，细胞内Ca 2+升高可增加TM细胞的收缩力，降低跨TM的流出功能。 因此，本项目的具体目标是表征MYOC中过表达/突变导致的UPR及其机制，并确定肌球蛋白诱导的UPR对Ca 2+稳态的不利影响。 将分别在牛TM和HEK-293 T细胞系的原代培养物中检查由肌球蛋白和MYOC突变形式的过表达诱导的UPR。 作为UPR的阳性对照，我们将使用已知引起ER中蛋白质解折叠的外源性药物（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。通过检测SERCA Ca 2 + ATP酶的转录激活和容量性钙内流途径的结构成分来研究Ca 2+的失调。 我们的技术和方案包括使用定量实时PCR、北方印迹、Western印迹、共聚焦显微镜和共沉淀。这些研究将导致一个重要的知识基础的发展，并影响青光眼病理生理学，诊断和治疗领域的研究。