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）的幼年发病有关。MYOC的基因产物肌素是一种功能未知的分泌蛋白。MYOC的靶向零突变在TM中既没有显示眼压升高，也没有表现出形态学异常，这表明MYOC的致病突变产生了对TM细胞产生负面影响的蛋白质（称为功能获得毒性）。这种毒性的性质是什么？毒性背后的分子机制是什么？在这个项目中，我们假设MYOC的某些突变导致心肌蛋白展开，导致其在TM细胞的内质网（ER）中保留和聚集。分泌蛋白或膜蛋白在内质网腔内的持续积累会引起内质网应激。这种应激引发了一种独特而复杂的内质网到细胞核的信号传导，称为“未折叠蛋白反应”（UPR），由内质网恢复稳态的机制组成。普遍定期审议的不足或过调会对急诊室的某些功能（如果不是全部的话）产生不利影响。因此，我们进一步假设异常心肌的保留诱导了UPR，而UPR反过来又导致了TM细胞中ca2 +的失调。许多关于TM药理学的研究已经清楚地表明，细胞内ca2 +的升高增加了TM细胞的收缩性，并减少了TM的流出设施。因此，本项目的具体目的是表征UPR及其在MYOC中过度表达/突变导致的机制，并确定心肌素诱导的UPR对Ca2+稳态的不利影响。将分别在牛TM和HEK-293T细胞系的原代培养中检测心肌素过表达和MYOC突变形式诱导的UPR。作为UPR的阳性对照，我们将使用已知引起内质网蛋白展开的外源性药物（例如，tunicamycin）。我们将根据UPR信号通路组分的激活和各种er特异性伴侣的激活来表征UPR。Ca2+解除管制将通过检查SERCA Ca2+ atp酶的转录激活和容性钙内流途径的结构成分来研究。我们的技术和方案包括使用定量实时PCR， Northern blotting, Western blotting，共聚焦显微镜和共免疫沉淀。这些研究将为青光眼病理生理、诊断和治疗领域的研究提供必要的知识基础和影响。