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: 6914435
• 负责人: SANGLY P SRINIVAS
• 金额: $ 15.05万
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6914435
• 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 的靶向无效突变既不显示眼内压升高，也不显示 TM 形态异常，这表明 MYOC 的致病突变产生的蛋白质会对 TM 细胞产生负面影响（称为功能获得性毒性）。 这种毒性的本质是什么？毒性的分子机制是什么？在这个项目中，我们假设 MYOC 的某些突变导致肌纤蛋白展开，导致其在 TM 细胞的内质网 (ER) 中保留和聚集。 已知分泌蛋白或膜蛋白在内质网管腔中的持续积累会导致“内质网应激”。 这种应激会引发一种独特但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)，由恢复内质网稳态的机制组成。 众所周知，UPR 的不足或过度会对 ER 的部分（如果不是全部）功能产生不利影响。 因此，我们进一步假设异常肌纤蛋白的保留会诱导 UPR，进而导致 TM 细胞中 Ca 2+ 失调。 大量关于 TM 药理学的研究已清楚地表明，细胞内 Ca 2+ 升高会增加 TM 细胞的收缩性，并减少跨 TM 的流出能力。 因此，该项目的具体目标是表征 MYOC 中过度表达/突变引起的 UPR 及其机制，并确定 myocilin 诱导的 UPR 对 Ca2+ 稳态的不利影响。 由肌纤蛋白过度表达和 MYOC 突变形式诱导的 UPR 将分别在牛 TM 和 HEK-293T 细胞系的原代培养物中进行检查。 作为 UPR 的阳性对照，我们将使用已知可引起 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 信号通路成分的激活和各种 ER 特异性伴侣的激活来表征 UPR。将通过检查 SERCA Ca2+ ATP 酶的转录激活和电容性钙流入途径的结构成分来研究 Ca 2+ 失调。 我们的技术和方案包括使用定量实时 PCR、Northern 印迹、Western 印迹、共聚焦显微镜和免疫共沉淀。这些研究将导致重要知识库的发展，并影响青光眼病理生理学、诊断和治疗领域的研究。