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，HMG-CoA催化HMG-CoA转化为甲羟戊酸，这是胆固醇合成的限速步骤。油红O染色显示辛伐他汀显著抑制前脂肪细胞(3T3-L1)的分化，而脂肪细胞分化的标志物瘦素的表达被辛伐他汀抑制长达12天。在小鼠基质细胞(ST2)中，辛伐他汀促进成骨细胞在成骨介质中的分化，而通过油红O染色和含曲格列酮的成脂介质中瘦素mRNA的表达来抑制脂肪细胞的分化。接下来，我们阐明了辛伐他汀降低瘦素表达的机制，在辛伐他汀存在或不存在的情况下，分化的3T3-L1脂肪细胞被不同的抑制剂与甲伐他汀或其代谢产物一起处理。辛伐他汀抑制…的时间和剂量依赖性瘦素基因表达增加。在转录受阻的3T3-L1细胞中，与瘦素mRNA相关的异质性核RNA被辛伐他汀抑制，而其mRNA的稳定性不受辛伐他汀影响。蛋白质合成抑制剂放线菌酮不能阻断辛伐他汀对瘦素基因转录的抑制作用。甲氧戊酸或甲氧戊酸代谢产物香叶基焦磷酸可阻断辛伐他汀对3T3-L1细胞瘦素表达的抑制作用。加入香叶基香叶基转移酶I抑制剂GGTI-298，但不能抑制法尼基转移酶抑制剂FTI-277的表达。羟基法舒地尔可抑制其表达，这是一种蛋白质预烯基化抑制剂。用磷脂酰肌醇3-激酶(PI3K)抑制剂LY294002和Wortmannin处理3T3-L1细胞后，Leptin的表达减少，而ERK1/2 MAP激酶途径的抑制剂PD98059和p38MAP途径的抑制剂SB203580在最佳浓度下不影响Leptin的表达。辛伐他汀呈剂量依赖性地增加3T3-L1细胞内环磷酸腺苷(CAMP)浓度。蛋白激酶A的抑制剂H89可完全阻断辛伐他汀对瘦素表达的抑制。这些结果表明，辛伐他汀抑制分化的3T3-L1脂肪细胞中香叶基香叶素蛋白的前烯基化，进而使PI3K失活，导致cAMP积聚，继而激活PKA。PKA可抑制瘦素基因转录，但无新的蛋白质合成。此外，辛伐他汀可促进ATDC5细胞向软骨细胞分化。辛伐他汀的这些作用导致脂肪细胞组织质量减少并维持骨量，强烈表明这些药物在预防和治疗肥胖相关疾病和代谢性骨病方面具有有益的作用。较少

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