title: 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.
authors.affiliation: Department of Orthopaedic Surgery, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, Georgia 30033, USA.
authors.forename: S D
authors.initials: SD
authors.lastname: Boden
authors.forename: G
authors.initials: G
authors.lastname: Hair
authors.forename: L
authors.initials: L
authors.lastname: Titus
authors.forename: M
authors.initials: M
authors.lastname: Racine
authors.forename: K
authors.initials: K
authors.lastname: McCuaig
authors.forename: J M
authors.initials: JM
authors.lastname: Wozney
authors.forename: M S
authors.initials: MS
authors.lastname: Nanes
mesh_terms: D000818:Animals; D015153:Blotting, Western; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002454:Cell Differentiation / Q000187:drug effects; D006224:Cricetinae; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D012886:Skull / Q000196:embryology*; D013997:Time Factors
chemical_list: C525816:BMP6 protein, human; C525818:Bmp6 protein, rat; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins; D005938:Glucocorticoids; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011994:Recombinant Proteins