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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。我们证明，辛伐他汀在显着抑制脂肪细胞分化的油红O染色测量前脂肪细胞（3 T3-L1），而瘦素的表达，脂肪细胞分化的标志物，抑制辛伐他汀长达12天的文化。在小鼠基质细胞（ST 2）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抑制脂肪细胞分化（油红O染色和瘦素mRNA表达）。我们接下来阐明了辛伐他汀诱导瘦素表达减少的潜在机制，在存在或不存在辛伐他汀的情况下，用甲羟戊酸或其代谢物的各种抑制剂处理分化的3 T3-L1脂肪细胞。辛伐他汀呈时间和剂量依赖性抑制 ...更多信息艾德瘦素mRNA表达。辛伐他汀可抑制与瘦素mRNA相关的核内异质性RNA的表达，而在转录停滞的3 T3-L1细胞中，辛伐他汀处理并不改变瘦素mRNA的稳定性。辛伐他汀对瘦素基因转录的抑制作用并不因放线菌酮（一种蛋白质合成抑制剂）的预处理而消失。甲羟戊酸或香叶基香叶基焦磷酸（甲羟戊酸代谢产物）的加入，消除了辛伐他汀诱导的3 T3-L1细胞中瘦素表达的抑制。在加入GGTI-298（一种香叶基香叶基转移酶I抑制剂）后观察到表达抑制，但未观察到FTI-277（一种法呢基转移酶抑制剂）。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（PI 3 K）抑制剂LY 294002和渥曼青霉素处理3 T3-L1细胞可降低瘦素的表达，而ERK 1/2 MAP激酶通路抑制剂PD 98059和p38 MAP激酶通路抑制剂SB 203580在最佳浓度下不影响瘦素的表达。辛伐他汀剂量依赖性地增加3 T3-L1细胞内cAMP浓度。蛋白激酶A抑制剂H89可完全阻断辛伐他汀对瘦素表达的抑制作用。这些结果表明，辛伐他汀减少香叶基香叶基蛋白异戊二烯化，随后失活的PI 3 K，导致cAMP积累和随后激活PKA在分化的3 T3-L1脂肪细胞。PKA抑制瘦素基因的转录而不产生新的蛋白质合成。此外，辛伐他汀促进ATDC 5细胞的软骨细胞分化。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作用。少