Calcium Dynamics in Interstitial Cells of Cajal

描述（由申请人提供）：胃肠道运动的控制需要几种细胞类型（如平滑肌和肠神经）之间复杂的相互作用。近年来，我们在这个复杂的系统中加入了Cajal间质细胞（ICC）。ICC调节平滑肌功能，ICC的丧失与几种运动障碍有关。国际商会网络不断更新换代，维护网络需要严格监管国际商会的扩散。Ano-1，一个Ca2+激活的Cl-通道，最近被发现在肠肌层的ICC上选择性表达，以及在一些肿瘤中，包括胃肠道间质瘤。该提案的总体目标是确定Ano1在ICC功能中的作用。该提案的中心假设是，Ano-1在ICC生理学中具有双重的新作用。它是调控ICC增殖的关键分子，也是慢波复极化所需的离子通道。中心假设将在两个具体目标中得到检验。特异性目标1将测试Ano1调节ICC增殖的假设，特异性目标2将测试Ano1是慢波复极化所必需的假设。第一个具体目的得到了初步数据的支持，这些数据表明，肌膜中的Ano1仅在ICC上表达，Ano1可用于检测运动障碍中ICC的缺失，Ano1的缺乏与ICC增殖的减少有关，Ano1表达的增加与ICC增殖的增加有关。通过对基因敲除小鼠与野生型小鼠的生物信息学分析，初步数据还提供了证据，表明当Ano1缺失时，与增殖控制相关的关键分子，包括CDK1/cdc2，均下调。第二个具体目标得到了初步数据的支持，这些数据表明Cl通道阻滞剂改变慢波平台期的持续时间，当Ano-1表达减少时慢波延长，而在Ano-1表达后慢波正常化。PI将通过创新技术的组合来检验中心假设，包括免疫组织化学数据的3D重建、Western blots、RT-PCR、单细胞PCR、定量PCR、慢病毒和si/shRNA敲除技术、电穿孔、敲除小鼠、器官型和单细胞培养、微阵列数据的生物信息学分析、蛋白质组学、Ca2+和Cl-成像、微电极记录和膜片钳技术。本研究的成功完成既有基础意义又有临床意义。我们对新发现的Ano1蛋白的研究将提供有关ICC网络如何维持和慢波调节的机制信息。具有直接临床意义的是，我们的工作将告知临床医生和病理学家使用Ano1抗体来评估运动障碍中的ICC，并为未来的治疗干预提供目标。我们的工作还具有胃肠道以外的广泛意义。由于Ano-1在包括胃肠道间质瘤在内的许多器官和肿瘤中表达，我们的发现可能适用于胃肠道外的其他器官。

DESCRIPTION (provided by applicant): The control of gastrointestinal (GI) motility requires a complex interaction between several cell types such as smooth muscle and enteric nerves. In recent years we have added interstitial cells of Cajal (ICC) to this complex system. ICC regulate smooth muscle function and loss of ICC is associated with several motility disorders. ICC networks are constantly turning over and maintenance of the networks requires tight regulation of ICC proliferation. Ano-1, a Ca2+ activated Cl- channel has recently been found to be selectively expressed on ICC in the muscle layers of the gut as well as in several tumors, including gastrointestinal stromal tumors. The overall objective of this proposal is to determine the role of Ano1 in ICC function. The central hypothesis of this proposal is that Ano-1 has dual, novel roles in ICC physiology. It acts as a key molecule in the regulation of ICC proliferation and as an ion channel required for the repolarization of the slow wave. The central hypothesis will be tested in two specific aims. Specific Aim 1 will test the hypothesis that Ano1 regulates proliferation of ICC and Specific aim 2 will test the hypothesis that Ano1 is required for repolarization of the slow wave. The first specific aim is supported by preliminary data that show that Ano1 in the tunica muscularis is expressed only on ICC, that Ano1 may be used to detect loss of ICC in motility disorders, that a lack of Ano1 is associated with a decrease in proliferation of ICC and that an increase in expression of Ano-1 is associated with an increase in ICC proliferation. The preliminary data also provide evidence, from bioinformatics analysis of microarray data comparing knockout to wild type mice, that key molecules associated with the control of proliferation, including CDK1/cdc2, are down-regulated when Ano1 is absent. The second specific aim is supported by preliminary data that show that Cl- channel blockers alter the duration of the plateau of the slow wave, that the slow wave is prolonged when Ano-1 expression is decreased and that it normalizes after Ano-1 is expressed. The PI will test the central hypothesis by a combination of innovative techniques including 3D reconstructions of immunohistochemical data, Western blots, RT-PCR, single cell PCR, quantitative PCR, lentivirus and si/shRNA knock down techniques, electroporation, knockout mice, organotypic and single cell cultures, bioinformatic analysis of microarray data, proteomics, Ca2+ and Cl- imaging, microelectrode recordings and patch clamp techniques. Successful completion of the proposed studies has both basic significance and clinical impact. Our work on the newly discovered protein Ano1 will provide mechanistic information on both how ICC networks are maintained and on the regulation of the slow wave. Of immediate clinical significance our work will inform clinicians and pathologists on use of antibodies to Ano1 to assess ICC in motility disorders and provide targets for future therapeutic interventions. Our work also has broad implications beyond the GI tract. As Ano-1 is expressed in many organs and tumors including gastrointestinal stromal tumors, our findings will likely apply to several other organs outside of the GI tract.