CGRP CONTROL IN TRIGEMINAL NEURONS IN VITRO AND IN VIVO

描述（由申请人提供）：拟开展的研究旨在了解NO和TNFalpha在培养三叉神经细胞和TMJ炎症体内模型中调节CGRP表达的机制。CGRP被认为在TMJ疾病（TMJDs）的潜在病理中起关键作用。高水平的神经肽CGRP存在于滑液期间TMJDs与疼痛有关。除CGRP外，据报道，NO和TNF水平在TMJ炎症期间升高。在TMJDs期间控制CGRP表达的细胞机制尚不清楚。在本课题中，我将在培养的三叉神经节神经元和体内TMJ模型中验证NO和TNF直接刺激CGRP基因表达的假设。此外，还将探讨NO和TNF在TMJ炎症发病机制中的作用。第一个目的提出的研究将确定NO和TNF单独或与其他炎症介质联合对三叉神经细胞CGRP释放的影响。第二个目标将集中于确定CGRP启动子中的基础（非刺激）、NO-和tnf反应性调控位点。三叉神经原代培养将瞬时转染cgrp -荧光素酶报告基因结构，并测量报告基因活性。第三个目的是阐明三叉神经中NO和TNF信号通路。特异性环化酶和激酶抑制剂和激活剂将用于确定参与调节CGRP的主要信号通路。个体途径的进一步研究将利用磷特异性抗体和信号通路检测试剂盒。在第四个目标中，NO和TNF对CGRP表达的调节将扩展到TMJ炎症的体内模型。并研究NO和TNF在体内TMJ炎症发病机制中的作用，以及信号通路特异性抑制剂是否能减轻TMJ炎症。这些研究的结果将为控制三叉神经细胞中CGRP基因表达的细胞和分子机制提供有价值的见解，这可能导致TMJDs和其他炎症性疾病的新治疗策略的发展。

DESCRIPTION (provided by applicant): The goal of the proposed research is to understand the mechanisms by which NO and TNFalpha regulate CGRP expression in cultured trigeminal neurons and an in vivo model of TMJ inflammation. CGRP is thought to play a key role in underlying pathology of TMJ disorders (TMJDs). High levels of the neuropeptide CGRP present in the synovial fluid during TMJDs are associated with pain. In addition to CGRP, the levels of NO and TNF have been reported to be elevated during TMJ inflammation. The cellular mechanisms by which CGRP expression is controlled during TMJDs are not known. In this proposal, I will test the hypothesis that NO and TNF directly stimulate CGRP gene expression in cultured trigeminal ganglia neurons and in vivo TMJ models. In addition, the role of NO and TNF in the pathogenesis of TMJ inflammation will be investigated. Studies proposed in the first aim will determine the effect of NO and TNF alone or in combination with other inflammatory mediators on CGRP release from trigeminal neurons. The second aim will focus on identifying the basal (unstimulated), and NO- and TNF-responsive regulatory sites in the CGRP promoter. Primary trigeminal cultures will be transiently transfected with CGRP-luciferase reporter constructs and reporter activity measured. The goal of the third aim is to elucidate the pathways involved in NO and TNF signaling in trigeminal neurons. Specific cyclase and kinase inhibitors and activators will be used to identify the major signaling pathway(s) involved in regulating CGRP. Further studies of individual pathways will utilize phosphospecific antibodies and signaling pathway detection kits. In the fourth aim, NO and TNF regulation of CGRP expression will be extended to in vivo models of TMJ inflammation. Also, the role of NO and TNF in the pathogenesis of TMJ inflammation in vivo will be investigated and whether specific inhibitors of signaling pathways can reduce TMJ inflammation. Results from these studies will provide valuable insight into the cellular and molecular mechanisms that control CGRP gene expression in trigeminal neurons that may lead to the development of novel therapeutic strategies for TMJDs and other inflammatory diseases.