CONTROL OF CONJUNCTIVAL GOBLET CELL MUCIN PRODUCTION

主要由结膜杯状细胞分泌的粘液层是眼表面的关键保护层，保护其免受病原体和环境挑战。 为了保护眼表，杯状细胞必须能够应对外部挑战。 这些外部挑战激活角膜中的感觉神经，以刺激杯状细胞周围的副交感神经和交感神经。 这些神经释放与杯状细胞膜中的受体相互作用的神经递质，并激活细胞内信号通路以诱导杯状细胞粘蛋白分泌。 本研究的长期目标是识别、表征由副交感神经和交感神经递质激活的信号通路中的特定细胞生化过程的功能并确定其活性，以及刺激杯状细胞粘蛋白分泌和杯状细胞增殖。 本提案的重点将是胆碱能激动剂（副交感神经通路），结膜中的主要刺激通路之一。 将确定胆碱能激动剂是否：（1）激活Ca 2 +/钙调蛋白依赖性途径以刺激杯状细胞粘蛋白分泌;（2）激活PKC依赖性途径以刺激杯状细胞粘蛋白分泌;（3）激活MAP激酶途径和应激应答性转录因子NF-κ B和AP-1以诱导杯状细胞增殖，以及（4）介导神经诱导的杯状细胞增殖。 将在体外孵育大鼠结膜，并使用酶联凝集素试验测量杯状细胞粘蛋白的分泌。 该方法将与免疫沉淀、蛋白质印迹、共聚焦免疫荧光显微镜、ELISA和电泳迁移率变动分析相结合，以确定信号通路的各个组分的识别、定位和活性，并与溴-2脱氧尿苷标记相结合，以测量细胞增殖。由于粘蛋白分泌的增加或减少与眼表疾病相关，因此杯状细胞的粘蛋白产生受到严格调节。 杯状细胞分泌和增殖的神经刺激尤其重要。 在粘蛋白过度产生的疾病中，例如巨乳头性结膜炎，持续的刺激性刺激会激活角膜中的感觉神经，以刺激结膜中的传出神经，从而增加杯状细胞分泌和增殖。 在粘蛋白缺乏的疾病中，例如麻醉角膜和疱疹性角膜炎，角膜中的感觉神经功能失调，阻止传出通路的激活并阻断杯状细胞分泌和增殖。 对刺激杯状细胞分泌和增殖的信号通路的细胞组分的研究将导致设计用于粘液过度产生和缺乏的疾病的杯状细胞特异性疗法。

The mucus layer, secreted primarily by the conjunctival goblet cells, is a critical protective layer for the ocular surface, shielding it from pathogenic and environmental challenges. To defend the ocular surface, goblet cells must be able to respond to the external challenges. These external challenges activate sensory nerves in the cornea to stimulate parasympathetic and sympathetic nerves that surround the goblet cells. These nerves release neurotransmitters that interact with receptors in the goblet cell membranes and activate intracellular signaling pathways to induce goblet cell mucin secretion. The long term objective of this research is to identify, characterize the function of, and determine the activity of the specific cellular biochemical processes in the signaling pathways activated by parasympathetic and sympathetic neurotransmitters and to stimulate goblet cell mucin secretion and goblet cell proliferation. The focus of the present proposal will be cholinergic agonists (parasympathetic pathway), one of the major stimulatory pathways in the conjunctiva. It will be determined if cholinergic agonists: (1) activate a Ca2+/calmodulin-dependent pathway to stimulate goblet cell mucin secretion; (2) activate a PKC-dependent pathway to stimulate goblet cell mucin secretion; (3) activate the MAP kinase pathway and the stress responsive transcription factors NF-kappaB and AP-1 to induce goblet cell proliferation, and (4) mediate neurally-induced goblet cell proliferation. Rat conjunctiva will be incubated in vitro and an enzyme-linked lectin assay used to measure secretion of goblet cell mucin. This method will be combined with immunoprecipitation, Western blotting, confocal immunofluorescence microscopy, ELISA, and electrophoretic mobility shift assay to determine identify, location, and activity of the individual components of the signaling pathways and bromo-2 deoxyuridine labeling to measure cell proliferation. Mucin production by the goblet cells is tightly regulated as either an increase or decrease in mucin secretion is associated with ocular surface disease. Neural stimulation of goblet cell secretion and proliferation is especially important. In diseases of mucin overproduction, such as giant papillary conjunctivitis, a constant irritative stimulus would activate sensory nerves in the cornea to stimulate efferent nerves in the conjunctiva to increase goblet cell secretion and proliferation. In diseases of mucin deficiency, such as anesthetic cornea and herpetic keratitis, sensory nerves in the cornea are rendered dysfunctional preventing activation of the efferent pathway and blocking goblet cell secretion and proliferation. Study of the cellular components of the signaling pathways that stimulate goblet cell secretion and proliferation will lead to the design of goblet cell-specific therapies for diseases of both mucus overproduction and deficiency.