title: Brief bone morphogenetic protein 2 treatment of glucocorticoid-inhibited MC3T3-E1 osteoblasts rescues commitment-associated cell cycle and mineralization without alteration of Runx2.
abstract: Glucocorticoids (GCs) inhibit bone formation in vivo. In MC3T3-E1 osteoblasts, chronic administration of 1 microm dexamethasone (DEX) starting at confluency results in >98% inhibition of bone morphogenetic protein 2 (BMP-2) expression and apatite mineral deposition. Here, it is shown that brief exposure to recombinant human BMP-2 (rhBMP-2), as short as 6 h, is sufficient to induce irreversible commitment to mineralization in DEX-treated cultures. RhBMP-2 dose dependently rescued mineralization but not collagen accumulation in DEX-inhibited cultures. The selective restoration of mineralization was evident in the higher mineral to matrix ratios of DEX/rhBMP-2 co-treated cultures compared with control. We tested the involvement of the runt-related transcription factor 2 (Runx2) in the DEX inhibition and rhBMP-2 rescue of mineralization. Surprisingly, DEX did not decrease Runx2 DNA binding activity, transactivation, or association with the endogenous osteocalcin gene promoter. Furthermore, the rhBMP-2 rescue did not involve Runx2 stimulation, suggesting an important role for factors other than Runx2 in BMP-2 action. Finally, we studied the differentiation-related cell cycle, which persists during commitment to mineralization in untreated cultures, but is inhibited along with mineralization in DEX-treated cultures. Although both rhBMP-2 alone and DEX alone were antimitogenic, rhBMP-2 stimulated this cell cycle in DEX-inhibited cultures. In conclusion, brief rhBMP-2 treatment restores mineralization in DEX-inhibited MC3T3-E1 osteoblasts via a mechanism different from Runx2 stimulation. This restoration may be functionally related to the accompanying rescue of the differentiation-related cell cycle. The efficacy of short term BMP-2 treatment supports the potential of short-lived BMP vectors for skeletal therapy in both traditional and gene therapeutic approaches.
authors.affiliation: Department of Biochemistry, Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Luppen
authors.forename: Nathalie
authors.initials: N
authors.lastname: Leclerc
authors.forename: Tommy
authors.initials: T
authors.lastname: Noh
authors.forename: Artem
authors.initials: A
authors.lastname: Barski
authors.forename: Arvinder
authors.initials: A
authors.lastname: Khokhar
authors.forename: Adele L
authors.initials: AL
authors.lastname: Boskey
authors.forename: Elisheva
authors.initials: E
authors.lastname: Smith
authors.forename: Baruch
authors.initials: B
authors.lastname: Frenkel
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000494:pharmacology*; D002113:Calcification, Physiologic / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects*; D002454:Cell Differentiation; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit; D004247:DNA / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D009363:Neoplasm Proteins; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D017550:Spectroscopy, Fourier Transform Infrared; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D005938:Glucocorticoids; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D015675:Osteocalcin; D003907:Dexamethasone; D004247:DNA; D000469:Alkaline Phosphatase