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.