Adipogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells Is Directly Inhibited By ATRA With Interaction Between Rargama And Ppargama2

Objectives To investigate the roles of retinoic acid receptor y (RARy) on peroxisome proliferator-activated receptor y2 (PPARy2) and CCAAT enhancement binding protein a (C/EBPa) in order to reveal the underline inhibition mechanisms of atRA on adipogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs).Methods rBMSCs isolated from the bone marrow of 3-week-old rats were cultured and induced to differentiate into adipocytes. (1) During adipogenic induction, atRA was added to the medium at final concentrations of 0.5,1.0 μmol/L, respectively, for 15 days continuous culture. The expressions of RARy, PPARy2 and C/EBPa were analyzed by Real-time quantitative polymerase chain reaction (RT-PCR) at the end of induction. Meanwhile, Western-blot was used to detect the proteins of RARy, PPARy2 and C/EBPa. (2) After infected with the over-express RARy and silence RARy adenovirus, the expressions of RARy, PPARy2, and C/EBPa was analyzed by RT-PCR and Western-blot once more. (3) After 15 days induction at 1.0 μmol/L of atRA, co-immunoprecipitation (Co-IP) technique was employed to investigate the interaction relationship between RARy and PPARy2 using PPARy2 antibody.Results (1) Compared with the control groups, ie atRA at 0 μmol/L, RT-PCR and Western-blot identified significantly increased expression of RARy (P<0.01, P<0.001, respectivly). Nevertheless, PPARy2 and C/EBPa were significantly down-regulated (P<0.001). (2) The recombinant over-RARy adenovirus infection significantly increased the levels of RARy mRNA and protein expression in the rBMSCs compared with the RFP control group (P<0.01). The expression levels of PPARy2 and C/EBPa of rBMSCs were also obviously down-regulated (P<0.05, P<0.01). However, the levels of PPARγ2 and C/EBPa protein expression were not significantly changed in rBMSCs compared with the RFP-RARy control group when the RARy expression was blocked by the si-RARy adenovirus. (3)The results of Co-IP showed that RARy protein interacted with protein PPARy2 via forming RARy-PPARy2 complexes.Conclusions (1) Higher concentrations of atRA (0.5-5.0 μmol/L) can inhibit the adipogenic differentiation of rBMSCs. It is achieved by activating RARy expression of retinoic acid signal pathway. (2) The mechanism of the above process might be directly contributed to protein-protein interaction between RARy and PPARy2 by forming RARy-PPARy2 complexes, which down-regulates C/EBPa.