摘要
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1μmol/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,a P2)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a P2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。
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 Smadl/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 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 （P 〈0.01 ）. BMtO 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〈0.05）. 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.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2016年第15期1722-1728,共7页
Journal of Third Military Medical University
基金
国家自然科学基金青年基金(81301573)
重庆市科委面上项目(CSTC2013jcyj A10090)~~
