Development of new anti-rheumatic drugs

新型抗风湿药的开发

基本信息

批准号：
21249060
负责人：
KOHSAKA Hitoshi
金额：
$ 29.45万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2009
资助国家：
日本
起止时间：
2009 至 2011
项目状态：
已结题

项目摘要

Up-regulated expression of cyclin-dependent kinase inhibitor(CDKI) p16^<INK4a>, which inhibits CDK4/6, in the synovial tissues as well as systemic administration of small molecule(sm) CDK4/6 inhibitors suppress animal models of rheumatoid arthritis. Although both inhibit cell cycle progression, they exert differential effects especially in modulating inflammatory mediator production. This part of the present studies was conducted to investigate how p16^<INK4a> expression modulates inflammatory cytokine production from macrophages in relation to simple CDK4/6 kinase inhibition. Forced expression of p16^<INK4a> in bone marrow-derived macrophages(BMM) suppressed LPS-induced expression of IL-6 but not TNFα. This was not observed in RSF. A sm CDK4/6 inhibitor and shRNA to knock down CDK4 failed to suppress IL-6 production, demonstrating that the inhibition did not depend on decrease in CDK4/6 kinase activity. The p16INK4a expression accelerated LPS-induced IRAK1 degradation, and suppressed … More the p38 MAPK/AP-1 pathway but not the NFκB pathway. Direct down-regulation of IRAK1 with small hairpin RNA also induced specific inhibition of the AP-1 pathway. The accelerated IRAK-1 degradation should be mediated by proteasomal degradation because a proteosome inhibitor restored p38 MAPK activation in p16INK4a-expressing BMM. Also, IRAK1 gene overexpression restored IL-6 production in p16INK4a-expressing THP-1 macrophage cell line. Finally, p16INK4a knock down with si RNA enhanced IL-6 production from senescent BMM that expressed endogenous p16INK4a. These results showed that p16INK4a suppressed IL-6 production from macrophages in a CDK4/6-independent manner. This was due to proteosome-mediated IRAK1 degradation and following suppression of the AP-1 pathway. Thus, unlike sm CDK inhibitors, p16INK4a could exert anti-inflammatory effects on synovial macrophages.Triggering receptor expressed on myeloid cells(TREM)-1 is the other therapeutic target we are pursuing. It is expressed by macrophages and neutrophils. Its activation augments inflammatory cytokine production triggered by Toll-like receptor engagement. In a mouse sepsis model, blockade of TREM-1 by administration of a TREM-1 extracellular domain/Ig Fc domain fusion protein(TREM-1-Ig) prolonged survival of affected mice. This indicated TREM-1 blockade suppressed pathological inflammation with maintaining minimal inflammatory cytokine production for anti-microbial defense. We reported previously that TREM-1 is expressed on synovial macrophages in the rheumatoid joints and that TREM-1 blockade ameliorated mouse collagen(CII)-induced arthritis(CIA), which is an animal model of rheumatoid arthritis. However, since a ligand for TREM-1 was unknown, physiological roles of TREM-1-ligand and interactions between TREM-1 and TREM-1-ligand remained to be clarified. This part of the studies was conducted to identify the TREM-1-ligand molecule for discerning its involvement in arthritis. To search for cells expressing the TREM-1-ligand, various types of cells were incubated with TREM-1-Ig. It bound to mouse B cells and A20 B-cell lymphoma cells. Expression cloning using A20 cell cDNA library led us to identify a gene encoding the TREM-1-ligand. Furthermore, we raised anti-TREM-1-ligand blocking monoclonal antibody(mAb). Administration of this antibody to CIA mice ameliorated the disease. Anti-TREM-1-ligand mAb treatment exerted no apparent effects on T and B cell responses to CII. Thus, in analogous to the effect of TREM-1-Ig, this effect appeared attributable to attenuation of the inflammatory responses rather than prevention of the adaptive immune responses. Identification of the human TREM-1-ligand in the future study will warrant establishment of a new anti-rheumatic therapy that is not associated with a risk of serious infection. Less

在滑膜组织中抑制CDK4/6的细胞周期蛋白依赖性激酶抑制剂（CDKI）p16^<ink4a>的上调表达，以及全身给药小分子（SM）CDK4/6抑制剂抑制类风湿性关节炎动物模型。尽管两者都抑制了细胞周期的进展，但它们在调节炎症介质产生时会执行差异效应。进行了本研究的这一部分是为了研究p16^<ink4a>表达如何调节与简单CDK4/6激酶抑制有关的巨噬细胞的炎症细胞因子产生。在骨髓来源的巨噬细胞（BMM）中，p16^<ink4a>的强制表达抑制了LPS诱导的IL-6的表达，而不是TNFα。在RSF中未观察到这一点。 SM CDK4/6抑制剂和SHRNA击倒CDK4无法抑制IL-6的产生，这表明抑制作用不取决于CDK4/6激酶活性的下降。 P16INK4A表达加速了LPS诱导的IRAK1降解，并抑制了……更多的是p38 MAPK/APK/AP-1途径，而不是NFκB途径。与小发夹RNA对IRAK1的直接下调还引起了对AP-1途径的特异性抑制作用。加速的IRAK-1降解应通过蛋白体降解介导，因为蛋白体抑制剂恢复了表达P16INK4A的BMM中的P38 MAPK激活。同样，IRAK1基因过表达还恢复了表达P16INK4A的THP-1巨噬细胞系中的IL-6产生。最后，P16INK4A用Si RNA击倒，从表达内源性P16INK4A的Senscent BMM产生IL-6。这些结果表明，P16INK4A以CDK4/6独立的方式抑制了巨噬细胞的IL-6产生。这是由于蛋白体介导的IRAK1降解以及AP-1途径抑制后。与SM CDK抑制剂不同，P16INK4A可以对滑膜巨噬细胞产生抗炎作用。在髓样细胞上表达的受体-1是我们追求的另一个治疗靶标。它由巨噬细胞和中性粒细胞表达。 IT激活增强了由Toll样受体参与触发的炎症细胞因子产生。在小鼠败血症模型中，通过施用TREM-1细胞外域/Ig Fc结构域融合蛋白（TREM-1-Ig）延长了受影响小鼠的生存率的阻断。这表明TREM-1阻断抑制了病理感染，从而维持抗微生物防御的最小炎性细胞因子产生。我们先前报道说，TREM-1是在类风湿关节中的滑膜巨噬细胞上表达的，而TREM-1封锁改善了小鼠胶原蛋白（CII）诱导的关节炎（CIA），这是类风湿关节炎的动物模型。但是，由于用于TREM-1的配体是未知的，因此TREM-1--配体的物理作用以及TREM-1和TREM-1-配体之间的相互作用尚待澄清。进行了这一部分研究，以鉴定TREM-1-配体分子以辨别其参与关节炎。为了搜索表达TREM-1--凸的细胞，将各种类型的细胞与TREM-1-Ig孵育。它与小鼠B细胞和A20 B细胞淋巴瘤细胞结合。使用A20细胞cDNA库克隆的表达克隆导致我们识别编码TREM-1----凸的基因。此外，我们提出了抗Trem-1----------封闭单克隆抗体（MAB）。对CIA小鼠的这种抗体的给药改善了该疾病。抗Trem-1-rigand mab处理对T和B细胞对CII的反应没有外观影响。与TREM-1-Ig的效果相似，这种作用似乎归因于炎症反应的衰减，而不是预防适应性免疫调查。在未来研究中对人类TREM-1--配体的识别将保证建立一种与严重感染风险无关的新抗风湿疗法。较少的