Rho GTPase-mediated pathways in autoimmune arthritis

描述(申请人提供)：Rho GTP酶(包括RAC和RhoA)是在生物学中发挥基础作用的分子开关，因为它们能够调节细胞骨架动力学和信号通路。Rac和RhoA针对不同的效应器，从而调节不同的信号事件。Rac和RhoA也相互交叉调节，因为激活的Rac抑制RhoA的活性，反之亦然。RhoA的主要效应器之一是Rho激酶(或岩石)。Rho GTP酶的激活在免疫应答中起着关键作用。鸟嘌呤核苷酸交换因子(GEF)是调节Rho GTP酶激活的主要蛋白质。在分离与TH-17细胞分化所需的转录因子IRF4相互作用的蛋白过程中，我们克隆了一种新的环境蛋白，我们将其命名为IBP(IRF-4结合蛋白)，也称为Def6。由于Def6是该基因的正式名称，IBP此后将被称为Def6。Def6在T细胞中高表达，并在TCR参与时激活RAC，但不激活RhoA。在与IRF4相互作用的核中也可以发现Def6。我们之前曾报道，Def6基因缺陷的小鼠会自发发展成狼疮样综合征。我们最近发现，TCR转基因(DO11.10)Def6缺陷小鼠会自发地发展成类风湿性关节炎。这些小鼠的自身免疫反应是由于CD4+T细胞对自身抗原产生IL-17和IL-21的异常能力。在分子水平上，Def6缺陷的T细胞表现出缺陷的RAC激活和IRF4与IL-17和IL-21启动子结合的能力增强。与Rac激活抑制RhoA诱导的通路的观点一致，我们最近观察到Def6的缺失也会导致ROCK2活性的增加。重要的是，我们发现ROCK2使IRF4磷酸化，并调节其控制IL-17和IL-21产生的能力。与这些发现一致的是，摇滚抑制剂法舒地尔阻止了在没有Def6的情况下检测到的IL-17和IL-21的异常产生，并防止了Def6缺陷DO11.10小鼠的关节炎的发展。我们现在提出，Def6-ROCK2-IRF4轴调节IL-17和IL-21的产生，ROCK抑制剂代表着治疗自身免疫性关节炎的一种新方法。这项建议的具体目标是：1)研究Def6和ROCK2之间的相互作用；2)描述T细胞ROCK2在自身免疫性关节炎中的作用；3)广泛评估ROCK抑制剂在自身免疫性关节炎中的有效性。

DESCRIPTION (provided by applicant): Rho GTPases (which include Rac and RhoA) are molecular switches that play a fundamental role in biology due to their ability to regulate both cytoskeletal dynamics and signaling pathways. Rac and RhoA target different effectors and thus regulate distinct signaling events. Rac and RhoA also cross-regulate each other since activated Rac inhibits RhoA activity and vice versa. One of the major effectors of RhoA is the Rho kinases (or ROCKs). Activation of Rho GTPases plays a key role in immune responses. Guanine nucleotide exchange factors (GEFs) are the major proteins regulating the activation of Rho GTPases. In the course of isolating proteins interacting with IRF4, a transcription factor required for the differentiation of TH-17 cells, we cloned a novel GEF that we termed IBP (IRF-4 Binding Protein), also known as Def6. Since Def6 is the official name of the gene, IBP will be hereafter referred to as Def6. Def6 is highly expressed in T cells, and, upon TCR engagement, activates Rac but not RhoA. Def6 can also be found in the nucleus where it interacts with IRF4. We previously reported that Def6-deficient mice spontaneously develop a lupus-like syndrome. We have recently found that TCR transgenic (DO11.10) Def6-deficient mice spontaneously develop RA-like arthritis. The autoimmune responses in these mice are due to an aberrant ability of CD4+ T cells to produce IL-17 and IL-21 in response to self-antigens. At a molecular level, Def6-deficient T cells exhibit defective Rac activation and an enhanced ability of IRF4 to bind to the IL-17 and IL-21 promoters. Consistent with the notion that Rac activation inhibits RhoA- induced pathways, we have recently observed that the absence of Def6 also leads to increased ROCK2 activity. Importantly, we have found that ROCK2 phosphorylates IRF4 and regulates its ability to control the production of IL-17 and IL-21. In line with these findings, Fasudil, a ROCK inhibitor, blocked the aberrant IL-17 and IL-21 production detected in the absence of Def6 and prevented development of arthritis in Def6-deficient DO11.10 mice. We now propose that the Def6-ROCK2-IRF4 axis regulates the production of IL-17 and IL-21 and that ROCK inhibitors represent a novel approach for the treatment of autoimmune arthritis. The specific goals of this proposal are: 1) To investigate the interplay between Def6 and ROCK2, 2) to delineate the role of T-cell ROCK2 in autoimmune arthritis and 3) to broadly assess the effectiveness of ROCK inhibitors in autoimmune arthritis.