全反式维甲酸抑制大鼠骨髓间充质干细胞成脂分化信号通路中Fosl1直接调控PPARγ2

目的:研究激活蛋白1(AP-1)在全反式维甲酸(ATRA)抑制大鼠骨髓间充质干细胞(BMSCs)成脂分化信号通路中的调控机制。方法:采用SD大鼠原代BMSCs,体外分离、培养和成脂诱导。油红O染色鉴定细胞脂滴形成情况。应用实时荧光定量PCR(RT-qPCR)检测脂肪细胞形成相关蛋白脂肪酸结合蛋白(FABP)、脂蛋白脂肪酶(LPL)、脂肪酸转运蛋白(CD36)、脂滴包被蛋白(perilipin)、过氧化物酶体增殖物激活受体γ2(PPARγ2)以及AP-1家族各成员(Fosl1、Fosl2、c-Fos、c-Jun、Jun B、Jun D和Fos B)的mRNA表达水平。Western blot检测相关蛋白的表达水平。染色质免疫共沉淀(Ch IP)检测相关蛋白(RARγ和Fosl1)与PPARγ2基因是否存在相互作用。结果:油红O染色显示ATRA处理组中细胞脂滴数量明显减少。BMSCs成脂诱导12 d后,与对照组相比,1μmol/L ATRA处理组FABP、LPL、CD36、perilipin和PPARγ2的mRNA表达均显著降低。RT-qPCR检测AP-1家族各转录因子表达结果显示Fosl1在ATRA处理组成脂诱导第2天、第6天和第10天表达均显著升高。Westren blot结果表明ATRA处理组Fosl1蛋白表达水平显著升高,而PPARγ2蛋白表达降低。Ch IP-qPCR实验发现Fosl1蛋白可结合在PPARγ2基因启动子区域,而RARγ蛋白未直接结合在PPARγ2基因启动子区域。结论:ATRA可抑制BMSCs成脂分化及脂质代谢相关蛋白的表达,可能与其通过上调Fosl1直接结合PPARγ2基因启动子区域、下调PPARγ2表达有关。

Fosl1 regulates PPARγ2 in ATRA-induced inhibition of adipogenic differentiation of rat bone marrow mesenchymal stem cells

AIM: To investigate the molecular mechanism of activator protein-1 (AP-1) regulating all-trans retinoic acid (ATRA)-induced inhibition of adipogenic differentiation of rat primary bone marrow mesenchymal stem cells (BMSCs).METHODS: Primary cultured BMSCs were used for induction of adipogenesis in vitro. The mRNA expression of fatty acid-binding protein (FABP), lipoprotein lipase (LPL), CD36, perilipin, peroxisome proliferator-activated receptor gamma-2 (PPARγ2) and AP-1 family members (Fosl1, Fosl2, c-Fos, c-Jun, JunB, JunD and FosB) was detected by RT-qPCR. The protein expression was determined by Western blot. Chromatin immunoprecipitation (ChIP) assay was performed to detect interactions between relevant proteins (RARγ and Fosl1) and PPARγ2 gene.RESULTS: Oil red O staining showed decreases in fat droplet formation and absorbance of the dye-triglyceride complex in ATRA treatment group. After 12 d adipogenic differentiation, compared with control group, the mRNA expression of FABP, LPL, CD36, perilipin and PPARγ2 was reduced by ATRA. In ATRA treatment group, the mRNA expression of Fosl1 was significantly increased on day 2, 6 and 10. The protein expression of Fosl1 also significantly increased in ATRA treatment group. ChIP-qPCR showed that the Fosl1 protein directly bound to the PPARγ2 promoter, while RARγ did not bind to the PPARγ2 promoter.CONCLUSION: ATRA inhibits BMSC adipogenic differentiation and reduces the expression of fat cell forming-related proteins. The mechanism may be associated with up-regulation of Fosl1 which directly bind to PPARγ2 promoter.