Objective      To determine the effect of all-trans retinoic acid (ATRA) on bone morphogenetic protein 9 (BMP9)-induced osteogenic and adipogenic differentiation in preadipocytes and explore the underlying molecular mechanism.       Methods      Expression of retinoic acid receptors（RARs）in 3T3-L1 preadipocytes was measured by RT-PCR. The 3T3-L1 preadipocytes were infected by recombinant adenovirus BMP9 and treated with 1μmol/L ATRA. Alkaline phosphatase (ALP) activities were tested at different time points (days 5

7 and 9 after treatment). The protein level of osteopontin (OPN)

osteocalcin (OC) and adipocyte fatty acid binding protein（aP2）was detected by Western blotting after treatment of BMP9 and/or ATRA for 12 d．The mineralization and lipid accumulation in preadipocytes were tested with Alizarin red staining and Oil red O staining after the treatment for 14 d. The activation of BMPR-Smad signaling was studied with BMPR-Smad binding site luciferase reporter assay

and Western blotting was performed to determine the phosphorylation of Smad1/5/8.       Results      Except retinoic X receptor γ

the other 5 subtypes of RARs were expressed in the preadipocytes. BMP9 increased the ALP activity (P0.01)

elevated the expression of OPN and OC as well as the deposition of mineral calcium. Addition of ATRA enhanced BMP9-induced osteogenic differentiation in the preadipocytes compared to the cells treated by BMP9 or ATRA alone (P0.01). BMP9 also induced aP2 expression and fat accumulation

but this effect was suppressed by ATRA. ATRA upregulated the expression of BMP9 at mRNA and protein levels

and promoted BMP9-induced phosphorylation of Smad-1/5/8. This synergistic action of ATRA and BMP9 was consistently found in BMPR-Smad luciferase reporter assay. Luciferase activity was significantly increased in ATRA and BMP9 group compared to BMP9 alone (P0.05).       Conclusion      ATRA enhances BMP9-induced osteogenic differentiation and blocks BMP9-induced adipogenic differentiation in 3T3-L1 preadipocytes

which might be partly medicated by BMP9-induced BMPR-Smad signaling.