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Inhibition of adipogenic differentiation of bone marrow mesenchymal stem cells by all-trans retinoic acid through direct regulation of PPARγ2 by RARγ

LIU Zu-yin, LI Qing, CHEN Li-jun, CHEN Jie, LIU You-xue   

  1. Children's Nutrition Research Center, Key Laboratory of Ministry of Education in Childhood Development Diseases, Children's Hospital of Chongqing Medical University, Chongqing Stem Cell Therapy Engineering Technical Center, Chongqing 400014, China
  • Online:2015-05-28 Published:2015-06-04

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Abstract:

Objective To investigate the mechanism of regulating peroxisome proliferator-activated receptor γ2 (PPARγ2) and CCAAT enhancement binding protein α (C/EBPα) by retinoic acid receptor γ (RARγ) during the course of inhibiting adipogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) by high concentration of all-trans retinoic acid (atRA). Methods The rBMSCs were isolated, cultured, and induced in vitro. The atRA solution of 0.5 and 1.0 μmol/L was added to the medium of adipogenic induction. After being cultured for 15 d, mRNA and protein expressions of RARγ, PPARγ2, and C/EBPα were detected by the real-time quantitative polymerase chain reaction (RTPCR) and Western blotting. After being infected with the over-express RARγ (over-RARγ) adenovirus and silence RARγ (si-RARγ) adenovirus, mRNA and protein expressions of RARγ, PPARγ2, and C/EBPα were detected by the RTPCR and Western blotting. After being inducted by atRA of 1.0 μmol/L for 15 d, the coimmunoprecipitation (Co-IP) technique was employed to investigate the interaction between RARγ and PPARγ2. Results Compared with the control group, the expression of RARγ after being induced by atRA of 0.5 and 1.0 μmol/L for 15 d significantly increased (P<0.01, P<0.001), while expressions of PPARγ2 and C/EBPα significantly decreased (P<0.001). The mRNA and protein expressions of RARγ after being infected by over-RARγ adenovirus significantly increased (P<0.01), while mRNA and protein expressions of PPARγ2 and C/EBPα significantly decreased (P<0.05, P<0.01). The protein expression of RARγ after being infected by si-RARγ adenovirus significantly decreased, while expressions of PPARγ2 and C/EBPα did not change significantly. The results of Co-IP showed that RARγ directly interacted with PPARγ2 and complex was formed. The combination of RARγ and retinoic acid response element (RARE) was not found. Conclusion High concentration of atRA can inhibit the adipogenic differentiation of rBMSCs by activating the signaling pathway of retinoic acid. RARγ downregulates the expressions of PPARγ2 and C/EBPα of signaling pathway of adipogenic differentiation by directly interacting with downstream PPARγ2.

Key words: all-trans retinoic acid, RARγ, PPARγ2, bone marrow mesenchymal stem cells, Co-IP