Elucidation of Regulatory Mechanisms of Osteoblastic and Chondrocytic Differentiation by Statins

他汀类药物对成骨细胞和软骨细胞分化的调节机制的阐明

基本信息

批准号：
18592045
负责人：
HORIUCHI Noboru
金额：
$ 2.49万
依托单位：
Ohu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18592045/
关键词：
Dentistry Differentiation Cell and Tissue Lipid Osteoblast Chondrocyte Statin Adipocyte

项目摘要

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

辛伐他汀抑制 3-羟基-3-甲基戊二酰辅酶 A (HMG-CoA) 还原酶，该还原酶催化 HMG-CoA 转化为甲羟戊酸，这是胆固醇合成的限速步骤。我们通过油测量证明，辛伐他汀显着抑制脂肪细胞分化。前脂肪细胞 (3T3-L1) 中的红 O 染色，而瘦素（脂肪细胞分化标志物）的表达，在培养长达 12 天的小鼠基质细胞 (ST2) 中，辛伐他汀在成骨培养基中刺激成骨细胞分化，同时在含有曲格列酮的成脂培养基中抑制脂肪细胞分化（通过油红 O 染色和瘦素 mRNA 表达测定）。接下来阐明辛伐他汀诱导瘦素表达减少的机制，用各种方法处理分化的 3T3-L1 脂肪细胞在辛伐他汀存在或不存在的情况下，甲羟戊酸或其代谢物抑制剂会以时间和剂量依赖性方式抑制瘦素 mRNA 的表达。在转录抑制的 3T3-L1 细胞中用辛伐他汀治疗，辛伐他汀对瘦素基因转录的抑制作用不会被消除。用放线菌酮（一种蛋白质合成抑制剂）进行预处理，添加甲羟戊酸或香叶基香叶基焦磷酸（一种甲羟戊酸代谢物），消除了辛伐他汀诱导的 3T3-L1 细胞中瘦素表达的抑制作用。 I 抑制剂，但不是法尼基转移酶抑制剂 FTI-277。用羟基法舒地尔、蛋白质异戊二烯化抑制剂治疗可抑制表达。用磷脂酰肌醇 3-激酶 (PI3K) 抑制剂 LY294002 和渥曼青霉素治疗可降低 3T3-L1 细胞中的瘦素表达，而 ERK1/2 MAP 激酶途径抑制剂 PD98059 则可降低瘦素表达。 SB203580，p38MAP 激酶抑制剂辛伐他汀在最佳浓度下不影响表达，剂量依赖性地增加 3T3-L1 细胞中的环 AMP (cAMP) 浓度，这是一种蛋白激酶 A 抑制剂，完全消除了辛伐他汀诱导的瘦素表达抑制。辛伐他汀减少了香叶基香叶基蛋白异戊二烯化，随后使 PI3K 失活，导致 cAMP 积累并随后激活分化中的 PKA 3T3-L1 脂肪细胞强烈抑制瘦素基因转录，而没有新的蛋白质合成。此外，辛伐他汀促进 ATDC5 细胞中的软骨细胞分化。辛伐他汀的这些作用导致脂肪细胞组织量减少并维持骨量，表明这些药物在预防和维持骨量方面具有有益作用。肥胖相关疾病和代谢性骨病的治疗较少。