GLUCOCORTICOID-INDUCED DIFFERENTIATION OF FETAL-RAT CALVARIAL OSTEOBLASTS IS MEDIATED BY BONE MORPHOGENETIC PROTEIN-6

Glucocorticoids (GCs) at physiological concentrations promote osteobla st differentiation from fetal calvarial cells. calvarial organ culture s, and bone marrow stromal cells: however, the cellular pathways invol ved are not known. Bone morphogenetic proteins (BMPs) are recognized a s important mediators of osteoblast differentiation. Specific roles fo r individual BMPs during postembryonic membranous bone formation have yet to be determined. We recently reported that GC potentiated the ost eoblast differentiation effects of BMP-2 and BMP-4, but not of BMP-6. which, by itself, was the most potent of the three. In the present stu dy, we used fetal rat secondary calvarial cultures to study the role o f BMP-6 during early osteoblast differentiation. Treatment with the GC triamcinolone (10(-9) M) resulted in a 5- to 8-fold increase in BMP-6 steady-state messenger RNA levels, peaking at 12 h. In contrast, BMPs -2, -4, -5, -7, and transforming growth factor (TGF)-beta 1 messenger RNA levels increased by less than 2-fold, after GC treatment. compare d with untreated control cultures at 24 h. BMP-6 protein secretion inc reased 6- to 7-fold by 12 h and 12-fold (from 7.5 to 90 ng/ml) by 24 h , as measured by quantitative Western analysis. Treatment of cells wit h oligodeoxynucleotides antisense to BMP-6 diminished secretion of BMP -6 protein and significantly inhibited the GC-induced differentiation, as determined by a 10-fold decrease in the number of mineralized bone nodules, compared with controls that were treated with sense oligonuc leotides or no oligonucleotides (ANOVA, P < 0.05). The antisense oligo nucleotide inhibition of differentiation was rescued by treatment with exogenous recombinant human BMP-6. We conclude that GC-induced differ entiation of osteoblasts from the pluripotent precursors is mediated, in part, by BMP-6. These results suggest that BMP-B has an important a nd unique role during early osteoblast differentiation.