Glucocorticoid-Induced Differentiation of Fetal Rat Calvarial Osteoblasts Is Mediated by Bone Morphogenetic Protein-6

Abstract

Glucocorticoids (GCs) at physiological concentrations promote osteoblast differentiation from fetal calvarial cells, calvarial organ cultures, and bone marrow stromal cells; however, the cellular pathways involved are not known. Bone morphogenetic proteins (BMPs) are recognized as important mediators of osteoblast differentiation. Specific roles for individual BMPs during postembryonic membranous bone formation have yet to be determined. We recently reported that GC potentiated the osteoblast 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 study, we used fetal rat secondary calvarial cultures to study the role of 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)-beta1 messenger RNA levels increased by less than 2-fold, after GC treatment, compared with untreated control cultures at 24 h. BMP-6 protein secretion increased 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 with 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 oligonucleotides or no oligonucleotides (ANOVA, P < 0.05). The antisense oligonucleotide inhibition of differentiation was rescued by treatment with exogenous recombinant human BMP-6. We conclude that GC-induced differentiation of osteoblasts from the pluripotent precursors is mediated, in part, by BMP-6. These results suggest that BMP-6 has an important and unique role during early osteoblast differentiation.

Potent induction of chondrocytic differentiation of human adipose‐derived adult stem cells by bone morphogenetic protein 6

“…In addition, the findings of Awad and colleagues (13) and the present results are consistent with the results of studies of calvarial rat MSCs, which indicate that glucocorticoids are strong inducers of the bone phenotype (36)(37)(38)47). However, the latter studies indicate that BMP-6 is also up-regulated by glucocorticoids, strongly suggesting that BMP-6 is required for endochondral ossification and plays an integral role in bone formation (36)(37)(38)47). Conversely, the data from the present study suggest that BMP-6 signaling may occur via different mechanisms in ADAS cells as compared with MSCs, since major differences between these cell types in the induction of chondrogenesis in response to BMP-6 were observed.…”

“…This conclusion of a reduction in chondrogenesis can also be gleaned from results of the present study, by comparing the decrease in COL2A1 gene expression when dexamethasone is added to TGF␤1 compared with the TGF␤1 condition alone, suggesting that dexamethasone somewhat inhibits chondrogenesis. In addition, the findings of Awad and colleagues (13) and the present results are consistent with the results of studies of calvarial rat MSCs, which indicate that glucocorticoids are strong inducers of the bone phenotype (36)(37)(38)47). However, the latter studies indicate that BMP-6 is also up-regulated by glucocorticoids, strongly suggesting that BMP-6 is required for endochondral ossification and plays an integral role in bone formation (36)(37)(38)47).…”

“…The results suggest that the response of ADAS cells to known chondrogenic agents differs considerably from the response of other mesenchyme-derived progenitor cells, such as MSCs. Notably, and in contrast to the osteogenic effects of BMP-6 on many cell types (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49), the effects of BMP-6 on ADAS cells in 3-D culture are highly chondrogenic, and among the growth factors investigated in this study, BMP-6 was shown to be the most potent regulator of chondrogenesis.…”

Expression and Synthesis of Bone Morphogenetic Proteins by Osteoclasts: A Possible Path to Anabolic Bone Remodeling

“…A single immunoreactive band at 36 kDa was detected for BMP-4, probably representing a dimeric conformation of the mature peptide (Suzuki et al 1993). For BMP-6, multiple bands were detected at 21 kDa (also detected in recombinant BMP-6), 23 kDa, 36 kDa, z45 kDa, 69 kDa, and 90 kDa in cultured osteoclasts, as previously reported in other cells (Boden et al 1997;Rickard et al 1998;Akiyoshi et al 2004). For BMP-7, multiple bands were detected at 18 kDa (monomer), 23 kDa (glycosylated form), 40 kDa (prodomain), 70 kDa, and an additional high-molecularmass band (.125 kDa), as seen in other studies Ct method), normalized to an endogenous control (GAPDH) and relative to a calibrator (osteoblast RNA sample).…”

Regulation of Osteogenesis by Fetuin

“…The addition of BMPs to RBMC enhances osteogenesis (37). Similarly, glucocorticoid stimulates BMPs-2, 4, 5, 6, and 7 production in cultured fetal calvarial cell, which contribute to osteogenic commitment in vitro (38). Osteogenesis in dex-RBMC proceeded further in the presence of anti-TGF-␤1 antibodies, suggesting the cytokine was specifically required in the later phases of matrix deposition and mineralization.…”