Acinar Biology and Pancreatic Disease

描述(申请人提供)：胰腺外分泌腺泡细胞制造、储存和释放大量水解酶进入肠道，这些酶对营养物质的消化和吸收是必不可少的。由于食物来自细胞材料，腺泡细胞保留了保护机制，以确保消化酶在到达肠道之前不会过早激活。事实上，腺泡分泌途径的异常失调和酶的过早激活与胰腺炎和胰腺癌的发展有关，每年有超过48,000名美国人受到影响。人们普遍认为，消化酶的过早激活是由于腺泡内分泌和溶酶体途径的异常相互作用所致。因此，了解协调这些通路正常相互作用的基本分子机制对于理解胰腺疾病的病理生理学至关重要。本研究探讨了腺泡细胞调节消化酶在分泌途径和溶酶体途径中的运输，从而调节消化酶分泌的一条重要的调控途径。我们最近报道，腺泡细胞根据胞外调节蛋白VAMP2和VAMP8的表达表达两种不同的酶原颗粒(ZGS)。此外，我们的数据表明VAMP8 ZGS的成熟受肿瘤蛋白D52(又名CRHSP-28)的指导，我们已经证明D52在腺泡细胞中唯一表达，并直接调节钙依赖的分泌。出乎意料的是，D52被定位于腺泡中一个独特的内小体和溶酶体相关的小室，我们称之为内溶酶体小室。此外，在CHO-K1细胞中，D52直接调节溶酶体膜的胞吐作用，当过度表达时，会导致细胞质小泡的大量积累。这项研究旨在阐明VAMP2和VAMP8阳性ZGS在腺泡分泌反应中的确切功能，并将检验D52调节通过内切酶系统运输的核心假设，即D52通过独特的溶酶体相关途径控制VAMP8阳性ZGS的生物发生/成熟。具体目标1将验证这样的假设，即VAMP2阳性的ZGS介导消化酶的早期即时释放，而VAMP8阳性的ZGS介导腺泡刺激后酶原分泌的持续平台期。特殊目的2将验证一种假设，即在腺泡过度刺激期间，激活PIKfyve产生PtdIns(3，5)P2调节内切溶酶体向溶酶体的运输并抑制顶端分泌。具体目标3将检验这样一种假设，即D52激活“次要调节途径”，介导重要调节蛋白迅速插入颗粒胞吐所必需的顶膜。了解腺泡蛋白如何独特地协调分泌或溶酶体间隔的基本分子原理，是开发旨在治疗胰腺疾病的治疗策略的关键。

DESCRIPTION (provided by applicant): Exocrine pancreatic acinar cells manufacture, store and release large quantities of hydrolytic enzymes into the intestine that are essential for nutrient digestion and absorption. As food is derived from cellular material, acinar cells retain protective mechanisms to ensure digestive enzymes are not prematurely activated until reaching the intestine. Indeed, aberrant dysregulation of the acinar secretory pathway and premature activation of enzymes has been tied to the development of pancreatitis and pancreatic cancer, which affect over 48,000 Americans each year. It is widely believed that premature digestive enzyme activation is caused by abnormal interactions of the secretory and lysosomal pathways in acini. Thus, knowledge of the basic molecular mechanisms which orchestrate the normal interactions of these pathways is critical to understanding the pathophysiology of pancreatic disease. This proposal investigates a previously unrecognized and important regulatory pathway by which acinar cells modulate digestive enzyme trafficking within the secretory and lysosomal pathways and accordingly regulate digestive enzyme secretion. We recently reported that acinar cells express two distinct populations of zymogen granules (ZGs) based on the expression of the exocytic regulatory proteins VAMP2 and 8. Moreover, our data indicate that maturation of VAMP8 ZGs is directed by Tumor Protein D52 (aka CRHSP-28), which we have shown is uniquely expressed in acini and directly regulates Ca2+-dependent secretion. Unexpectedly, D52 was localized to a unique endosome and lysosome related compartment in acini that we term the endo-lysosomal compartment. Moreover, in CHO-K1 cells, D52 directly regulates lysosmal membrane exocytosis and when over-expressed induces a massive accumulation of cytoplasmic vesicles. This proposal aims to delineate the precise functional roles of VAMP2- and VAMP8- positive ZGs in the acinar secretory response and will test the central hypothesis that D52-regulated trafficking through the endo-lysosomal system controls the biogenesis/maturation of VAMP8-positive ZGs within a unique lysosome-related pathway. Specific Aim 1 will test the hypothesis that VAMP2-positive ZGs mediate the early immediate release of digestive enzymes, whereas VAMP8-positive ZGs mediate the sustained plateau phase of zymogen secretion following acinar stimulation. Specific Aim 2 will test the hypothesis that activation of PIKfyve to produce PtdIns(3,5)P2 in acini regulates endo-lysosomal trafficking to the lysosome and inhibits apical secretion during acinar hyperstimulation. Specific Aim 3 will test the hypothesis that D52 activation of the "minor regulated pathway" mediates the rapid insertion of important regulatory proteins into the apical membrane necessary for granule exocytosis. Understanding the basic molecular principles of how acini uniquely orchestrate the secretory or lysosomal compartments is key to the development of therapeutic strategies aimed at treating pancreatic disease.