Cell cycle regulation for treatment of rheumatoid arthritis

细胞周期调节治疗类风湿性关节炎

基本信息

批准号：
15390311
负责人：
KOHSAKA Hitoshi
金额：
$ 7.81万
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 (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Objective. Forced expression of cyclin-dependent kinase inhibitor (CDKI) gene, p16INK4a or p21Cip1 in the synovial tissues was effective in treating animal models of rheumatoid arthritis (RA). Although the primary function of CDKIs is halting cell cycle progression, their anti-proliferative effect accompanied suppression of inflammation in the treated joints. Subsequently, we and others found that p21 Cip1 does not only inhibit activitity of CDKs but downmodulates c-Jun N-terminal kinase (JNK) and other intracellular signaling pathways. Despite lack of solid evidence for interaction of p16INK4a with other molecules, p16INK4a suppressed specific inflammatory molecules produced by rheumatoid synovial fibroblasts (RSF). Since, some of these molecules were shared by the effects of p16INK4a and p21 Cip1, direct effects of CDK4/6 activity on their production were studied. Methods. Genes for CDKIs, p16INK4a and p18INK4c, a constitutively active, hypophosphorylated form of retinoblastoma gene … More product (Rb), cyclin D1 and CDK4 were transferred into RSF with adenoviruses to modulate CDK4/6 activity. A synthetic CDK4/6 inhibitor was used to inhibit CDK4/6. Cell cycle was studied with 3H-thymidine incorporation and flow cytometry. Expression levels of matrix metalloproteinase (MMP)-3, monocyte chemoattaractant protein (MCP)-1 or type I interleukin 1 receptor (IL-1R1) was determined with Northern blot analyses, real-time polymerase chain reaction, and/or ELISAs. CDKIs were immunoprecipitated to reveal their association with JNK. Results. Gene transfer of p16INK4a as well as p18INK4c inhibited cell cycle progression and suppressed MMP-3 and MCP-1 production. Unlike p21Cip1, neither molecules inhibit IL-1 R1 expression or bind to JNK. Inhibition of CDK4/6 by a synthetic CDK4/6 inhibitor also downregulated these inflammatory mediators. They were upregulated when CDK4 activity was augmented. This regulation functioned at the mRNA level for MMP-3, but not for MCP-1. Transfer of active Rb, which mimicked inhibition of CDK-dependent Rb phosphorylation, suppressed production of both without changing their mRNA levels. Conclusions. Inhibition of CDK4/6 in RSF downregulated MCP-1 and MMP-3 production, which was upregulated by augmented CDK4 activity. MMP-3 production was primarily regulated by the kinase activity at the mRNA level in an Rb-independent manner, while MCP-1 production was controlled posttranscriptionally by Rb. Less

客观的。在滑膜组织中细胞周期蛋白依赖性激酶抑制剂（CDKI）基因，p16INK4A或p21CIP1的强迫表达有效地治疗类风湿关节炎的动物模型（RA）。尽管CDKI的主要功能是停止细胞周期的进程，但其抗增殖效果抑制了处理的关节中的炎症。随后，我们和其他人发现，p21 CIP1不仅抑制CDK的活性，而且抑制了C-Jun N末端激酶（JNK）和其他细胞内信号传导途径。尽管缺乏证据表明P16INK4A与其他分子相互作用，但P16INK4A抑制了类风湿滑膜成纤维细胞（RSF）产生的特定炎症分子。由于这些分子中的某些分子是通过p16ink4a和p21 CIP1的影响共享的，因此CDK4/6活性对其生产的直接影响是研究的。方法。 CDKIS，P16INK4A和P18INK4C的基因是一种组成型活性，低磷酸化形式的视网膜细胞瘤基因…更多的产物（RB），Cyclin D1和CDK4转移到带有腺病毒的RSF中，以调节CDK4/6活性。合成CDK4/6抑制剂用于抑制CDK4/6。细胞周期与3H-胸腺苷掺入和流式细胞仪进行了研究。基质金属蛋白酶（MMP）-3的表达水平，单核细胞化学量核酸蛋白（MCP）-1或I型白介素1受体（IL-1R1）的表达水平通过北印迹分析，实时聚合酶链链反应和/或ELISAS确定。对CDKI进行了免疫沉淀，以揭示其与JNK的关联。结果。 P16INK4A以及P18INK4C的基因转移抑制了细胞周期的进展，并抑制了MMP-3和MCP-1产生。与P21CIP1不同，分子均不抑制IL-1 R1表达或与JNK结合。合成CDK4/6抑制剂对CDK4/6的抑制也下调了这些炎症介质。当CDK4活动增加时，它们进行了更新。该调节在mMP-3的mRNA水平上起作用，但在MCP-1上不起作用。活性Rb的转移，它模仿了CDK依赖性RB磷酸化的抑制作用，抑制了两者的产生而没有改变其mRNA水平。结论。 RSF中CDK4/6的抑制下调了MCP-1和MMP-3的产生，通过增强CDK4活性进行了更新。 MMP-3的产生主要由RB独立于mRNA水平的激酶活性调节，而MCP-1产生在RB后转录后控制。较少的