Role of Chat-H in chemokine-induced T lymphocyte migration and trafficking

描述（由申请人提供）：在本申请中，我们建议研究新型蛋白Chat-H在调节T淋巴细胞迁移的信号通路中的作用。在初步研究中，我们利用慢病毒介导的RNA干扰（RNAi）沉默小鼠原代T细胞中Chat-H的表达，研究Chat-H缺失对T细胞功能的影响。我们发现，虽然Chat-H下调对T细胞受体（TCR）介导的信号传导没有影响，但在体外和体内趋化因子诱导的Chat-H缺陷T细胞的迁移受到显著损害。迁移和粘附缺陷与Rap-1 GTPase的激活受损相关，而Rap-1 GTPase是整合素介导的细胞粘附和迁移的关键调节因子。Chat-H与信号蛋白CasL组成相关，该复合物的形成是迁移所必需的。这些观察结果表明，Chat-H是趋化因子受体信号传导和T淋巴细胞迁移的重要调节因子，并与CasL一起调节这些过程。为了研究体内对Chat-H的需求，我们最近产生了Chat-H敲除小鼠，与先前的结果一致，初步研究表明，这些小鼠的T细胞在体外迁移中表现出受损。在这里描述的实验中，我们建议研究Chat-H调节趋化因子诱导的T淋巴细胞迁移的机制以及Chat-H缺乏在这一过程中的影响。我们将验证以下假设：Chat-H通过激活Rap1和Rap1介导的整合素激活、细胞粘附和迁移来调节趋化因子诱导的T淋巴细胞迁移。Chat-H表达不足导致整合素介导的粘附缺陷，并导致T细胞向次级淋巴器官的稳态归巢受损，以及T细胞介导的免疫反应受损。为了验证这一假设，我们将首先使用Chat-H被RNAi下调的T细胞来定义Chat-H如何调节Rap1激活和整合素介导的粘附的生化机制。为了研究体内对Chat-H的需求，我们将使用Chat-H敲除小鼠来确定Chat-H缺乏是否会影响成熟T细胞向外周淋巴器官的体内迁移和稳态运输，以及T细胞介导的免疫反应。我们预计这些实验将导致对调节淋巴细胞趋化性机制的新见解，并揭示Chat-H和CasL在趋化因子受体下游的这一过程中的重要作用。通过这些研究，我们还旨在深入了解Chat-H在慢性炎症和与白细胞粘附缺陷相关的疾病中的作用，并确定治疗人类疾病的治疗靶点。

DESCRIPTION (provided by applicant): In this application we propose to examine the role of the novel protein Chat-H in signaling pathways that regulate T lymphocyte migration. In preliminary studies, we used lentivirus-mediated RNA interference (RNAi) to silence expression of Chat-H in mouse primary T cells and study the effect of Chat-H deletion on T cell function. We found that whereas Chat-H downregulation had no effect on T cell receptor (TCR)-mediated signaling, in vitro and in vivo chemokine-induced migration of Chat-H deficient T cells was dramatically impaired. Defects in migration and adhesion correlated with impaired activation of the Rap-1 GTPase, which is a key regulator of integrin-mediated cellular adhesion and migration. Chat-H constitutively associated with the signaling protein CasL and formation of this complex was necessary for migration. These observations suggest that Chat-H is an important regulator of chemokine receptor signaling and T lymphocyte migration and it that it acts together with CasL to regulate these processes. To study the in vivo requirement for Chat-H we recently generated Chat-H knockout mice and consistent with previous results, preliminary studies show that T cells from these mice exhibit impaired in vitro migration. In the experiments described here, we propose to examine the mechanisms through which Chat-H regulates chemokine-induced T lymphocyte migration and the effect of Chat-H deficiency in this process. We will test the following hypothesis: Chat-H regulates chemokine-induced T lymphocyte migration by activating Rap1 and Rap1-mediated integrin activation, cell adhesion and migration. Deficiency in Chat-H expression results in defective integrin-mediated adhesion and as a result impaired homeostatic homing of T cells to secondary lymphoid organs, and compromised T-cell-mediated immune responses. To test this hypothesis we will use first use T cells in which Chat-H has been dowregulated by RNAi to define the biochemical mechanisms of how Chat-H regulates Rap1 activation and integrin-mediated adhesion. To study the in vivo requirement for Chat-H we will use Chat-H knockout mice to determine whether Chat-H deficiency affects in vivo migration and homeostatic trafficking of mature T cells to peripheral lymphoid organs, and T-cell-mediated immune responses. We anticipate that these experiments will lead to novel insight into the mechanisms that regulate lymphocyte chemotaxis and reveal important roles for both Chat-H and CasL in this process downstream of chemokine receptors. Through these studies we also aim towards gaining insight into the role of Chat-H in chronic inflammation and conditions associated with leukocyte adhesion deficiencies and identify therapeutic targets for treating human diseases.