Cell cycle regulation for treatment of rheumatoid arthritis

目标。在滑膜组织中强制表达细胞周期蛋白依赖性激酶抑制因子(CDKI)基因p16INK4a或p21Cip1可有效治疗类风湿关节炎(RA)动物模型。虽然CDKI的主要功能是阻止细胞周期进展，但它们的抗增殖作用伴随着对治疗关节炎症的抑制。随后，我们和其他人发现，p21Cip1不仅抑制CDKs的活性，而且下调c-jun氨基末端激酶(JNK)和其他细胞内信号通路。尽管缺乏p16INK4a与其他分子相互作用的确凿证据，p16INK4a抑制了类风湿滑膜成纤维细胞(RSF)产生的特定炎症分子。由于这些分子中有一部分是由p16INK4a和p21Cip1共同作用的，因此我们研究了CDK4/6活性对其产生的直接影响。方法：研究方法。CDKI、p16INK4a和p18INK4c基因，视网膜母细胞瘤基因…的一种结构性活性的低磷酸化形式通过腺病毒将更多的产物(Rb)、细胞周期蛋白D1和CDK4导入RSF，调节CDK4/6的活性。用合成的CDK4/6抑制剂抑制CDK4/6。用~3H-胸腺嘧啶核苷掺入法和流式细胞仪检测细胞周期。用Northern印迹分析、实时定量聚合酶链式反应和/或ELISA检测基质金属蛋白酶-3、单核细胞趋化蛋白-1和I型白介素1受体的表达水平。CDKI被免疫沉淀以揭示它们与JNK的关联。结果。P16INK4a和p18INK4c基因转移抑制细胞周期进程，抑制基质金属蛋白酶-3和单核细胞趋化蛋白-1的产生。与p21Cip1不同，这两种分子都不能抑制IL-1R1的表达，也不能与JNK结合。用合成的CDK4/6抑制剂抑制CDK4/6也下调了这些炎症介质的表达。当CDK4活性增强时，它们的表达上调。这种调节作用于基质金属蛋白酶-3的mRNA水平，而不是单核细胞趋化蛋白-1。转移活性Rb，模拟抑制依赖于CDK的Rb的磷酸化，抑制两者的产生，而不改变它们的mRNA水平。结论。抑制RSF中CDK4/6的表达可下调MCP-1和MMP3的产生，而CDK4活性增强则可上调MCP-1和MMP3的表达。MMP3的产生主要受Rb非依赖的mRNA水平上的激酶活性调节，而MCP-1的产生受Rb转录后调控。较少

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