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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

滑膜组织中细胞周期蛋白依赖性激酶抑制剂（CDKI）p16 β<INK4a>（其抑制CDK 4/6）的上调表达以及小分子（sm）CDK 4/6抑制剂的全身施用抑制类风湿性关节炎的动物模型。虽然两者都抑制细胞周期进程，但它们发挥不同的作用，特别是在调节炎症介质产生方面。本研究的这一部分是为了研究p16 β表达如何<INK4a>调节巨噬细胞产生的炎性细胞因子，与简单的CDK 4/6激酶抑制有关。骨髓<INK4a>源性巨噬细胞（BMM）中p16^的强制表达抑制LPS诱导的IL-6表达，但不抑制TNFα表达。在RSF中没有观察到这一点。SmCDK 4/6抑制剂和敲低CDK 4的shRNA不能抑制IL-6的产生，表明抑制作用不依赖于CDK 4/6激酶活性的降低。p16 INK 4a的表达加速了LPS诱导的IRAK 1降解，并抑制了IRAK 1的表达。 ...更多信息 p38 MAPK/AP-1通路而非NFκB通路。用小发夹RNA直接下调IRAK 1也诱导AP-1途径的特异性抑制。IRAK-1降解的加速应该是由蛋白酶体降解介导的，因为蛋白酶体抑制剂恢复了表达p16 INK 4a的BMM中p38 MAPK的激活。此外，IRAK 1基因过表达恢复了表达p16 INK 4a的THP-1巨噬细胞系中IL-6的产生。最后，用siRNA敲低p16 INK 4a增强表达内源性p16 INK 4a的衰老BMM的IL-6产生。这些结果表明，p16 INK 4a以CDK 4/6非依赖性方式抑制巨噬细胞产生IL-6。这是由于蛋白体介导的IRAK 1降解和AP-1途径的抑制。因此，与sm CDK抑制剂不同，p16 INK 4a可以对滑膜巨噬细胞发挥抗炎作用。髓样细胞表达的触发受体（TREM）-1是我们正在寻找的另一个治疗靶点。它由巨噬细胞和中性粒细胞表达。它的激活增强了由Toll样受体参与触发的炎性细胞因子的产生。在小鼠脓毒症模型中，通过施用TREM-1细胞外结构域/IG Fc结构域融合蛋白（TREM-1-IG）阻断TREM-1延长了受影响小鼠的存活。这表明TREM-1阻断抑制病理性炎症，同时维持最小的炎性细胞因子产生用于抗微生物防御。我们先前报道了TREM-1在类风湿性关节中的滑膜巨噬细胞上表达，并且TREM-1阻断剂改善了小鼠胶原蛋白（CII）诱导的关节炎（CIA），这是类风湿性关节炎的动物模型。然而，由于TREM-1的配体是未知的，TREM-1-配体的生理作用以及TREM-1和TREM-1-配体之间的相互作用仍有待澄清。进行这部分研究是为了鉴定TREM-1-配体分子，以辨别其参与关节炎。为了寻找表达TREM-1-配体的细胞，将各种类型的细胞与TREM-1-IG一起孵育。它与小鼠B细胞和A20 B细胞淋巴瘤细胞结合。利用A20细胞cDNA文库进行表达克隆，我们鉴定了编码TREM-1-配体的基因。此外，我们还制备了抗TREM-1配体阻断单克隆抗体（mAb）。给CIA小鼠施用这种抗体改善了疾病。抗TREM-1-配体mAb处理对T和B细胞对CII的应答没有明显影响。因此，与TREM-1-IG的作用类似，该作用似乎可归因于炎症反应的减弱而不是适应性免疫反应的预防。在未来的研究中，对人TREM-1配体的鉴定将保证建立一种与严重感染风险无关的新的抗风湿治疗。少