Elucidation of Regulatory Mechanisms of Osteoblastic and Chondrocytic Differentiation by Statins

辛伐他汀抑制3-羟基-3-甲基戊二酰辅酶A （HMG-CoA）还原酶，该酶催化HMG-CoA转化为甲羟戊酸盐，这是胆固醇合成的限速步骤。我们证明辛伐他汀显著抑制脂肪细胞前脂肪细胞（3T3-L1）的分化，而瘦素（脂肪细胞分化的标志）的表达被辛伐他汀抑制长达12天的培养。在小鼠基质细胞（ST2）中，辛伐他汀在成骨培养基中刺激成骨细胞分化，而在含曲格列酮的成脂培养基中，通过油红O染色和瘦素mRNA表达检测，辛伐他汀抑制脂肪细胞分化。接下来，我们阐明了辛伐他汀诱导瘦素表达减少的机制，在辛伐他汀存在或不存在的情况下，用甲羟戊酸或其代谢物的各种抑制剂治疗分化的3T3-L1脂肪细胞。辛伐他汀时间和剂量依赖性抑制…更多ed瘦素mRNA表达。在转录阻滞的3T3-L1细胞中，辛伐他汀抑制了与瘦素mRNA相关的异质核RNA，而辛伐他汀治疗未改变mRNA的稳定性。辛伐他汀对瘦素基因转录的抑制作用并没有被环己亚胺（一种蛋白质合成抑制剂）预处理所消除。添加甲羟戊酸或甲羟戊酸代谢物焦磷酸香叶基香叶基，可消除辛伐他汀诱导的3T3-L1细胞中瘦素表达的抑制。在加入香叶基转移酶I抑制剂GGTI-298时观察到表达抑制，而在加入香叶基转移酶抑制剂FTI-277时观察到表达抑制。羟基法舒地尔和蛋白烯酰化抑制剂抑制了表达。磷脂酰肌醇3-激酶（PI3K）抑制剂LY294002和wortmannin可降低3T3-L1细胞中瘦素的表达，而ERK1/2 MAP激酶途径抑制剂PD98059和p38MAP激酶途径抑制剂SB203580在最佳浓度下不影响表达。辛伐他汀剂量依赖性增加3T3-L1细胞内环AMP （cAMP）浓度。蛋白激酶A抑制剂H89完全消除辛伐他汀诱导的瘦素表达抑制。这些结果表明，辛伐他汀降低了香叶基香叶基蛋白前置化，随后PI3K失活，导致分化的3T3-L1脂肪细胞中cAMP的积累和随后PKA的激活。PKA抑制瘦素基因转录而不合成新蛋白。此外，辛伐他汀促进ATDC5细胞的软骨细胞分化。辛伐他汀的这些作用导致脂肪组织质量减少并维持骨量，强烈提示这些药物在预防和治疗肥胖相关疾病和代谢性骨病方面的有益作用。少

Simvastatin inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. We demonstrated that simvastatin at markedly inhibited adipocyte differentiation measured by Oil Red O staining in preadipocyte cells (3T3-L1), while expression of leptin, a marker of adipocyte differentiation, was suppressed by simvastatin for up to 12 days of culture. In mouse stromal cells (ST2), simvastatin at stimulated osteoblastic differentiation in osteogenic medium, while inhibiting adipocyte differentiation determined by Oil Red O staining and leptin mRNA expression in adipogenic medium containing troglitazone. We next elucidate mechanisms underlying the reduction of leptin expression induced by simvastatin, differentiated 3T3-L1 adipocytes were treated with various inhibitors with mevalonate or its metabolite in the presence or absence of simvastatin. Simvastatin time- and dose-dependently suppress … More ed leptin mRNA expression. Heterogeneous nuclear RNA related to leptin mRNA was inhibited by simvastatin, while stability of the mRNA was not changed by treatment with simvastatin in transcription-arrested 3T3-L1 cells. Simvastatin inhibition of leptin gene transcription was not abrogated by pretreatment with cycloheximide, an inhibitor of protein synthesis. Addition of mevalonate or geranylgeranyl pyrophosphate, a mevalonate metabolite, abolished simvastatin-induced inhibition of leptin expression in 3T3-L1 cells. Suppression of expression was observed upon addition of GGTI-298, a geranylgeranyl transferase I inhibitor, but not FTI-277, a farnesyl transferase inhibitor. Expression was suppressed by treatment with hydroxyfasudil, protein prenylation inhibitors. Treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, reduced leptin expression in 3T3-L1 cells, while PD98059, an inhibitor of the ERK1/2 MAP kinase pathway and SB203580, an inhibitor of the p38MAP kinase pathway, did not affect expression at optimal concentrations. Simvastatin dose-dependently increased intracellular cyclic AMP (cAMP) concentrations in 3T3-L1 cells. H89, an inhibitor of protein kinase A, completely abolished simvastatin-induced suppression of leptin expression. These results suggested that simvastatin reduced geranylgeranylprotein prenylation followed by deactivation of PI3K, leading to cAMP accumulation and subsequent activation of PKA in differentiated 3T3-L1 adipocytes. PKA inhibited leptin gene transcription without new protein synthesis. Furthermore, simvastatin promoted chondrocytic differentiation in ATDC5 cells. These effects of simvastatin cause reduction of adipocyte tissue mass and maintain bone mass, strongly suggesting salutary effects of these agents in prevention and treatment of obesity-related diseases and metabolic bone diseases. Less