Basal-Lateral/Endomembrane Traffic in Lacrimal Acini

描述(申请人提供)：干燥综合征(SjS)和其他免疫相关疾病在200万至400万美国人中导致泪道功能障碍、视力受损和眼表炎症。原发性泪液缺乏症(PLD)也可能与免疫相关，影响至少600万人。这些概念是由以下假设统一的：泪腺内的环境反映了涉及调节性淋巴细胞、自身免疫效应淋巴细胞和分泌上皮细胞的免疫稳态；淋巴细胞和炎症细胞介质调节上皮细胞功能；免疫稳态随着上皮细胞功能的改变而演变。然而，人们对改变上皮功能引发疾病的触发因素、女性比男性更常见的原因以及导致泪腺功能障碍的分子机制知之甚少。研究人员发现，用M受体(MAChR)激动剂卡巴胆碱慢性刺激泪腺泡细胞，会导致上皮细胞分泌停顿，还会激活异常的膜内运输程序，阻止向溶酶体移动，潜在地增加构成自身抗原的暴露，并启动先前神秘的表位的暴露。经典上与MAChR、GQ和G11偶联的G蛋白被多种介质的受体所共享，它们的信号转导受性激素的影响。因此，研究人员提出：(A)生理扰动可以启动不适当的GQ/G11信号，从而激活异常的交通程序；(B)SjS和PLD的局部环境刺激不适当的GQ/G11信号，从而导致泪腺功能障碍的功能停顿。具体目的1.激活异常膜运输程序的信号是什么？这个 中心假设是MAChR保持激活，并继续激活GQ/G11。特定目的2.慢性刺激其他受体是否也会导致功能静止并激活异常的交通程序？中心假设是，这些变化可以由利用GQ/G11的受体激动剂引起，包括组胺、5-羟色胺、前列腺素E_2和雌二醇。具体目标3.哪些交通影响因素应对这一异常计划负责？中心假设是，慢性刺激降低了动力蛋白的运动功能，但增加了P150(粘合)与动力蛋白II的联系，从而增加了动力蛋白介导的流量。具体目标4.异常程序在多大程度上改变溶酶体蛋白的运输？研究人员将使用绿色荧光蛋白-组织蛋白酶S融合蛋白 和共聚焦显微镜来检验溶酶体蛋白在内小体中积累的假说。

DESCRIPTION (provided by applicant): Sjogren's syndrome (SjS) and other immune-related diseases cause lacrimal dysfunction, impaired vision, and ocular surface inflammation in 2 to 4 million Americans. Primary lacrimal deficiency (PLD), which also may have an immune-related component, affects at least 6 million more. These concepts are unified by the hypothesis that: the milieu in the lacrimal glands reflects an immunohomeostasis involving regulatory lymphocytes, autoimmune effector lymphocytes, and secretory epithelial cells; lymphocyte and inflammatory cell mediators modulate epithelial cell functions; and the immunohomeostasis evolves in response to altered epithelial cell functions. However, little is known about the triggers that alter epithelial function to initiate the diseases, the reasons they occur more frequently in women than in men, and the molecular mechanisms that cause lacrimal dysfunction. The investigators have found that chronic stimulation of lacrimal acinar cells with the muscarinic receptor (MAChR) agonist, carbachol, causes epithelial secretory quiescence and also activates an aberrant endomembrane traffic program that blocks movement to lysosomes, potentially increasing exposure of constitutive autoantigens and initiating exposure of previously cryptic epitopes. The G proteins that classically couple to MAChR, Gq and G11, are shared by receptors for a wide variety of mediators, and their signaling is influenced by the sex hormones. Therefore, the investigators propose that: (a) physiological perturbations can initiate inappropriate Gq/G11 signaling that activates the aberrant traffic program, and (b) the local environments in SjS and PLD stimulate inappropriate Gq/G11 signaling that causes functional quiescence underlying lacrimal dysfunction. Specific Aim 1. What are the signals that activate the aberrant membrane traffic program? The central hypothesis is that MAChR remain activated and continue to activate Gq/G11. Specific Aim 2. Can chronic stimulation of other receptors also cause functional quiescence and activate the aberrant traffic program? The central hypothesis is that these changes can be elicited by agonists for receptors that utilize Gq/G11, including histamine, 5-hydroxytryptamine, PGE2, and estradiol. Specific Aim 3. What traffic effectors are responsible for the aberrant program? The central hypothesis is that chronic stimulation decreases dynein motor function but increases p150(Glued) association with kinesin II, thereby increasing kinesin-mediated traffic. Specific Aim 4. How extensively does the aberrant program alter traffic of lysosomal proteins? The investigators will use a GFP-cathepsin S fusion protein and confocal microscopy to test the hypothesis that lysosomal proteins accumulate in the endosomes.