Effects of the Mevalonate Pathway on Bone Formation

描述（由申请人提供）：最近，我们发现他汀类药物（抑制甲羟戊酸途径中的HMG-CoA还原酶的药物）在体外和体内刺激啮齿动物的成骨细胞分化和骨形成。其他人已经发现，含N2的双膦酸盐也靶向该途径，尽管在更远的步骤。在本申请中，我们计划研究他汀类药物影响成骨细胞分化的分子机制。心血管领域的最新观察表明，他汀类药物降低缺血性卒中的风险不是通过降低胆固醇作用，而是通过其增强内皮细胞中内皮一氧化氮合酶（eNOS）mRNA表达和一氧化氮（NO）生成的作用，这是类异戊二烯代谢物和Rho GT3介导的作用。我们认为，他汀类药物对成骨细胞分化的作用可能有类似的机制，因为1）最近发表的观察结果表明，eNOS无效突变小鼠的骨形成减少，2）我们的初步研究表明，eNOS抑制剂而不是iNOS抑制剂阻断他汀类药物刺激骨器官培养中骨形成的作用。此外，我们认为这些对成骨细胞的影响最终是通过增强BMP-2的转录介导的，从而导致成骨细胞分化和骨形成。为了确定他汀类药物刺激骨形成的分子机制，我们提出了以下目标：（1）确定抑制HMG Co-A还原酶是否是他汀类药物对骨形成的最终作用的初始作用;（2）通过确定他汀类药物是否增加骨细胞中NO的产生，检验他汀类药物通过影响eNOS表达和活性来刺激骨形成的假设，确定他汀类药物对骨的作用是否可被eNOS和iNOS的特异性抑制剂削弱，并确定他汀类药物对eNOS无效突变小鼠中的骨是否具有作用;（3）确定他汀类药物和eNOS对骨形成的作用是否最终通过使用对BMP-2无应答的体外骨细胞或体内转基因小鼠增强BMP-2转录来介导。这些实验应该提供信息的分子机制，他汀类药物和抑制HMG-CoA还原酶影响BMP信号和成骨细胞分化，并有望澄清负责正常骨形成的调控机制。

DESCRIPTION (provided by applicant): Recently, we have found that the statins, drugs that inhibit the HMG-CoA reductase enzyme in the mevalonate pathway, stimulate osteoblast differentiation and bone formation in rodents both in vitro and in vivo. Others have found that N2-containing bisphosphonates also target this pathway, albeit at more distal steps. In this application, we plan to examine the molecular mechanisms by which the statins influence osteoblast differentiation. Recent observations from the cardiovascular field show that statins reduce risk of ischemic stroke not by cholesterol-lowering actions, but rather by their effects to enhance endothelial nitric oxide synthase (eNOS) mRNA expression and nitric oxide (NO) generation in endothelial cells, an effect mediated by isoprenoid metabolites and Rho GTPase. We propose that a similar mechanism may be responsible for statin effects on osteoblast differentiation since 1) recent published observations show that eNOS null mutant mice have decreased bone formation, 2) our preliminary studies show that inhibitors of eNOS but not iNOS block effects of statins to stimulate bone formation in bone organ cultures. Further, we propose that these effects on osteoblasts are mediated ultimately by enhancing BMP-2 transcription, which leads to osteoblast differentiation and bone formation. To determine the molecular mechanisms by which statins stimulate bone formation, we propose the following aims: (1) determine if inhibition of HMG Co-A reductase is the initial effect of the statins that is responsible for their ultimate effects on bone formation; (2) examine the hypothesis that statins stimulate bone formation by effects on eNOS expression and activity, by determining if statins increase NO production in bone cells, determining if statin effects on bones can be impaired by specific inhibitors to eNOS and iNOS, and determining if statins have effects on bone in eNOS null mutant mice; (3) determine if statin and eNOS effects on bone formation are ultimately mediated through enhanced BMP-2 transcription by the use of bone cells in vitro or transgenic mice in vivo that are unresponsive to BMP-2. These experiments should provide information on the molecular mechanisms by which statins and inhibition of HMG-CoA reductase affect BMP signaling and osteoblast differentiation, and will hopefully clarify regulatory mechanisms responsible for normal bone formation.