全反式维甲酸调控BMP9诱导3T3-L1前脂肪细胞成骨和成脂分化

目的 研究全反式维甲酸(all-trans retinoic acid,ATRA)调控骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导前脂肪细胞成骨和成脂分化的作用及相关机制.方法 首先用RTPCR检测维甲酸受体在3T3-L1前脂肪细胞中的表达;用BMP9编码序列的重组腺病毒感染3T3-L1细胞,再以1 μmol/L的ATRA干预不同时间后,利用碱性磷酸酶(alkaline phosphatase,ALP)活性测定和染色、Western blot检测骨桥素(osteopontin,OPN)、骨钙素(osteocalcin,OC)和脂肪酸结合蛋白(adipocyte fatty acid binding protein,aP2)表达,茜素红染色检测钙盐沉积水平,油红O染色检测脂质积聚研究BMP9和ATRA对前脂肪细胞成骨和成脂分化的作用;利用荧光素酶报告质粒和Western blot检测ATRA对BMP9介导的BMPR-Smad信号通路活性的影响.结果 前脂肪细胞中,除视黄醇受体γ外其余5种维甲酸受体均有表达;BMP9能增加前脂肪细胞ALP活性(P＜0.01),并促进OPN及OC蛋白表达和钙盐沉积,ATRA能进一步增强该作用(P＜0.01);BMP9能促进前脂肪细胞中脂质积聚,并诱导aP2表达,但该作用被ATRA抑制;ATRA还能够上调细胞中BMP9的mRNA和蛋白表达,BMP9合并ATRA组的Samd1/5/8磷酸化水平明显强于BMP9组,BMPR-Smad报告质粒荧光素酶活性也呈相同变化趋势(P＜0.01).结论 在前脂肪细胞中,ATRA能增强BMP9的成骨诱导活性,并抑制其诱导的成脂分化;ATRA的作用可能与进一步激活BMP9介导的BMPR-Smad信号有关.

All-trans retinoic acid regulates BMP9-induced osteogenic and adipogenic differentiation in 3T3-L1 preadipocytes

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 1 μmol/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 (P ＜ 0.01),elevated theexpression 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 (P ＜0.01).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 (P＜O.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.