Effects of joint application of all-trans retinoic acid and bone morphogenetic protein on the expression of osteocalcin and osteogenic genes in different cells / 解放军医学杂志

ABSTRACT

[Abstract] Objective To investigate the effects of exclusive or joint application of all-trans retinoic acid (ATRA) and bone morphogenetic proteins (BMPs) on the osteocalcin (OCN) in mouse osteogenic precursor cells (MC3T3-E1) and bone marrow mesenchymal stem cells (BMSCs), and on the expressions of osteogenesis-related genes ALP, OCN and Runx2. Methods MC3T3-E1 cells and BMSCs were subcultured and stimulated with different concentrations of cytokines, and then divided into ten groups control group, ATRA group, 5ng/ml BMP2/7 group, 50ng/ml BMP2/7 group, 5ng/ml BMP2 group, 50ng/ml BMP2 group, ATRA+5ng/ml BMP2/7 group, ATRA+50ng/ml BMP2/7 group, ATRA+5ng/ml BMP2 group, and ATRA+50ng/ml BMP2 group. The morphology of the cells was observed by immunofluorescence staining. The OCN expressions in the two kinds of cells were detected by ELISA. The expressions of ALP, OCN and Runx2 genes were detected by RT-qPCR. Results Immunofluorescence staining showed that the cells grew well and were structurally intact. Exclusive use of ATRA obviously inhibited the expressions of OCN in both MC3T3-E1 and BMSCs. On the 7th day, for MC3T3-E1 cells ATRA alone group [(20.97±0.31)ng/ml] significantly decreased than control group [(33.45±0.55)ng/ml]; for BMSCs ATRA alone group [(9.90±0.16)ng/ml] significantly decreased than control group [(14.30±0.53)ng/ml] (P<0.05). Exclusive application of BMPs promoted OCN expression in a concentration-dependent manner, and BMP2/7 showed a more strong promoting role. The OCN content in MC3T3-E1 cells treated with 50ng/ml BMP2/7 was (47.13±0.59)ng/ml, and in BMSCs was (49.92±0.53)ng/ml. 50ng/ml BMP2/7 completely antagonized the inhibitory effect of ATRA on cell OCN expression (P<0.05). RT-qPCR test showed that, for MC3T3-E1 ATRA alone significantly inhibited the expressions of ALP and OCN gene (P<0.05), but promoted the expression of Runx2 gene (P<0.05). BMPs alone promoted the expression of osteogenesis-related genes in a concentration-dependent manner (P<0.05). ATRA significantly inhibited the promoting role of BMPs on the OCN gene expression, but ATRA and 50ng/ml BMP2/7 synergistically promoted the expression of ALP gene (P<0.05); for BMSCs ATRA alone significantly promoted the expression of Runx2 gene on the 7th day, but inhibited the expressions of ALP and OCN gene (P<0.05). 50ng/ml BMPs alone significantly promoted the expressions of ALP and Runx2 genes (P<0.05). 50ng/ml BMP2/7 antagonized the inhibitory effect of ATRA on ALP gene expression and promoted its expression (P<0.05). Conclusions ATRA alone may inhibit the expression of OCN in cells, and inhibits the expressions of ALP and OCN genes in cells; and BMP2/7 may significantly promote the expression of OCN in cells, and may promote the expression of osteogenesis-related genes; and 50ng/ml BMP2/7 may completely antagonize the osteogenic inhibition of ATRA on cells.