INTRACELLULAR PATHWAYS MEDIATING AIRWAY MUCIN SECRETION

介导气道粘蛋白分泌的细胞内途径

• 批准号: 6027990
• 负责人: C. William Davis
• 金额: $ 20.02万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-15 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6027990
• 关键词: athymic mouse; calcium flux; chickens; chronic obstructive pulmonary disease; exocytosis; gelsolin; microfilaments; molecular pathology; mucins; phospholipase C; protein binding; protein kinase C; second messengers; secretion; tissue /cell culture; trachea; transfection /expression vector

Mucin hyperproduction is a common component of obstructive airways disease and comprises a principle problem faced by patients and their attending physicians. A major long-term goal of this laboratory is the identification of molecular targets for pharmacologic therapies against mucin hypersecretion. Using SPOC1 cells as a model for airway mucin secretion, we have shown that phospholipase C-coupled P2Y2 receptors localized in the apical membrane form the dominate regulatory pathway in these cells. Because all airway epithelial cells likely possess P2Y2 receptors for extracellular ATP and UTP, targets for the selective inhibition of mucin secretion need to be located at points distal to activation of PLC, and IP3 and DAG generation. The two downstream elements of the PLC pathway, Ca2+-protein kinase C (PKC), appear to be independent in their activation of mucin release, and preliminary data suggest strongly that nPKCdelta, a Ca2+-insensitive isoform, is uniquely activated by agonist. This proposal will test whether the Ca2+ and PKC pathways leading to mucin secretion are fully additive and independent, or whether they converge at a rate limiting step proximal to mucin granule/plasma membrane fusion and exocytosis. A likely candidate for this proximal barrier to secretion is the cortical cytoskeleton, comprised of actin microfilaments. Specific Aim 1 will use wild-type and mutant nPKCdelta retroviral expression vectors to test whether nPKCdelta, in fact, mediates the effects of agonist of mucin secretion, and it will examine the means by which nPKCdelta is activated. Specific Aim II tests the independence between Ca2+- and PKC-activated mucin secretory pathways by probing the transit of mucin granules across the cortical microfilament barrier and the plasma membrane. We focus on the roles of scinderin, a gelsolin-related, F-acting severing enzyme, and Rab3 isoforms, respectively. In each case, we will test whether these elements participate in the regulation of agonist-stimulated exocytosis and whether they lie in the pathways modulated by nPKCdelta and/or Ca2+. Lastly, we will exploit the different binding kinetics of BAPTA and EGTRA to test the degree of independence between PKC and Ca2+ in the final steps of exocytosis.

粘蛋白过度产生是阻塞性呼吸道疾病的常见组成部分，也是患者及其主治医生面临的主要问题。该实验室的一个主要长期目标是确定针对粘蛋白高分泌的药物治疗的分子靶点。利用SPOC1细胞作为呼吸道粘蛋白分泌的模型，我们发现位于顶膜的磷脂酶C偶联的P2Y2受体在这些细胞中形成了主要的调节途径。由于所有的呼吸道上皮细胞可能都有细胞外ATP和UTP的P2Y2受体，因此选择性抑制粘蛋白分泌的靶点需要位于PLC激活、IP3和DAG产生的远端。PLC途径的两个下游元件，即钙-蛋白激酶C(PKC)，在激活粘蛋白释放方面似乎是独立的，初步数据有力地表明，nPKC Delta是一个钙不敏感的亚型，是唯一被激动剂激活的。这项建议将测试导致粘蛋白分泌的钙和PKC通路是否完全相加和独立，或者它们是否在粘蛋白颗粒/质膜融合和胞吐作用附近的限速步骤汇聚。这种近端分泌屏障的一个可能的候选者是由肌动蛋白微丝组成的皮质细胞骨架。特殊目的1将使用野生型和突变型nPKCDelta逆转录病毒表达载体来测试nPKCDelta是否确实介导了粘蛋白分泌激动剂的作用，并将检测nPKCDelta被激活的方式。特殊目的II通过探测粘蛋白颗粒穿过皮质微丝屏障和质膜来测试钙激活和PKC激活的粘蛋白分泌途径之间的独立性。我们的重点是闪光蛋白，一种明胶蛋白相关的F作用断裂酶，以及Rab3亚型的作用。在每种情况下，我们将测试这些元件是否参与激动剂刺激的胞吐作用的调节，以及它们是否存在于nPKC Delta和/或Ca~(2+)调节的通路中。最后，我们将利用BAPTA和EGTRA不同的结合动力学来测试PKC和钙在胞吐最后步骤中的独立程度。