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气道疾病的常见组成部分，并且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过度分泌的药物治疗的分子靶点。使用 SPOC1 细胞作为气道粘蛋白分泌的模型，我们发现位于顶膜的磷脂酶 C 偶联 P2Y2 受体形成这些细胞中的主要调节途径。由于所有气道上皮细胞可能都具有细胞外 ATP 和 UTP 的 P2Y2 受体，因此选择性抑制粘蛋白分泌的靶标需要位于远离 PLC、IP3 和 DAG 生成激活的点。 PLC 途径的两个下游元件 Ca2+ 蛋白激酶 C (PKC) 在激活粘蛋白释放方面似乎是独立的，初步数据强烈表明 nPKCdelta（一种 Ca2+ 不敏感亚型）仅由激动剂激活。该提案将测试导致粘蛋白分泌的 Ca2+ 和 PKC 途径是否完全相加和独立，或者它们是否以接近粘蛋白颗粒/质膜融合和胞吐作用的速率限制步骤收敛。这种近端分泌屏障的一个可能候选者是由肌动蛋白微丝组成的皮质细胞骨架。具体目标 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 是否确实介导粘蛋白分泌激动剂的作用，并将检查 nPKCdelta 被激活的方式。 Specific Aim II 通过探测粘蛋白颗粒穿过皮质微丝屏障和质膜的转运来测试 Ca2+- 和 PKC 激活的粘蛋白分泌途径之间的独立性。我们分别关注 scinderin（一种凝溶胶蛋白相关的 F 作用切断酶）和 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位于由 nPKCdelta 和/或 Ca2+ 调节的途径中。最后，我们将利用 BAPTA 和 EGTRA 的不同结合动力学来测试胞吐作用最后步骤中 PKC 和 Ca2+ 之间的独立程度。