Novel therapeutic approaches to bone diseases using cell cycle factors Cdk4/6

本研究旨在开发利用细胞周期因子CDK4/6治疗骨骼疾病的新方法。首先，我们研究了几种CDK抑制剂对成骨细胞分化的影响。我们发现一些CDK抑制剂可以改变成骨细胞的分化。此外，为了确定参与CDK抑制促进成骨分化的下游分子，我们进行了DNA微阵列分析。从上调的基因中，我们选择了几个调控成骨分化的基因作为CDK抑制的下游效应基因。在这些基因中，我们集中在锌指蛋白或G蛋白偶联受体等，考虑到表达的变化和从文献中收集的发现。实时定量RT-PCR分析证实，部分候选基因表达上调。接下来，为了研究这些基因与CDK抑制成骨分化促进因子的功能相关性，我们通过小RNA干扰(SiRNA)敲除了候选基因。此外，我们还建立了几只双突变小鼠来研究CDK的体内抑制作用。其中一只双突变小鼠证实CDK抑制可部分挽救部分基因敲除小鼠的表型。综上所述，本研究结果提示，CDKs的调节可能导致治疗骨分解代谢障碍的新疗法。

This study aimed to develop novel therapeutic approaches to bone diseases using cell cycle factors, Cdk4/6. First, we investigated the effects of several Cdk inhibitors on osteoblast differentiation. We discovered that some Cdk inhibitors could alter osteoblast differentiation. In addition, to identify downstream molecules involved in the promotion of osteogenic differentiation by Cdk inhibition, we performed a DNA microarray analysis. From the up-regulated genes, we selected several genes as candidates which could regulate osteogenic differentiation as downstream effectors of Cdk inhibition. Among these genes, we focused on Zinc finger proteins or G protein-coupled receptors etc., in consideration of both the expression change and findings gathered from the literature. Real-time quantitative RT-PCR analyses confirmed that that there were up-regulations of some of candidate genes. Next, to investigate the functional relevance of such genes to the osteogenic differentiation promo by Cdk inhibition, we knocked down candidate genes through small RNA interference (siRNA). Moreover, we generated several double mutant mice to investigate the effects of Cdk inhibition in vivo. One of double mutant mice proved that the phenotype in some knockout mice was partially rescued by Cdk inhibition. In conclusion, the results of this study imply that the modulation of Cdks may lead to novel therapeutics to treat bone catabolic disorders.