| Objectives: To investigated 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 bone marrow mesenchymal stem cells(BMSCs)were used for induction of adipogenesis in vitro.The ATRA was added to the adipogenic induction medium at the 1st,5th,and 9th days at final concentration of 1 ?mol/L.After treatment of ATRA for 24h(i.e.,on days 2,6,and 10),the cells were harvested.mRNA expressions of fatty acid-binding protein(FABP),lipoprotein lipase(LPL),CD36,Perilipin,peroxisome proliferator-activated receptor gamma-2(PPAR?2)and AP-1 families(Fosl1,Fosl2,c-Fos,Fos B,c-Jun,JunB,JunD)were detected by the real-time quantitative polymerase chain reaction(RT-PCR).Western blot was used to measure the protein expression.Chromatin immunoprecipitation(ChIP)assay was performed to detect interactions between relevant proteins(RAR? and Fosl1)and gene(PPAR?2).Results:(1)Oil Red O staining showed decrease in fat droplet formation and absorbance of the dye-triglyceride complex in ATRA treatment group.(2)After 12 days of adipogenic differentiation,compared with the control group,the mRNA expressions of FABP,LPL,CD36,Perilipin,PPAR?2 were reduced by ATRA(PFABP<0.05,PLPL<0.001,PCD36<0.001,PPerilipin<0.05),P<0.05 considered significant difference.(3)In ATRA treatment group,Fosl1 mRNA expression significantly increased on days 2,6 and 10(Pday 2<0.01,Pday 6<0.01,Pday 10<0.001).And protein expression of Fosl1 also significantly increased.(4)ChIP-qPCR showed that the Fosl1 protein directly bound to the PPAR?2 promoter.While,RAR? did not bind to the PPAR?2 promoter.Conclusions: ATRA inhibited BMSCs adipogenic differentiation and reduced expression of fat cell forming related genes.It may be associated with upregulated Fosl1 which directly bind to PPAR?2 promoter by ATRA. |
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