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.
Highlights
Glucocorticoids (GCs)1 are potent anti-inflammatory agents for the treatment of diseases such as rheumatoid arthritis, systemic lupus erythematosus, asthma, and some types of cancer
Inhibition of Mineralization by Chronic DEX Treatment Is Corrected by Chronic bone morphogenetic protein (BMP)-2 Treatment Commencing 48 h after DEX—We have previously shown that Recombinant human bone morphogenetic protein 2 (BMP-2) (rhBMP-2) counteracts GC-mediated inhibition of mineralization in MC3T3-E1 osteoblast cultures [16]
Local administration of rhBMP-2 has been demonstrated effective in counteracting the inhibitory effect of GCs on osteotomy healing in a rabbit model [27]
Summary
Reagents—To maintain the MC3T3-E1 cell line, ␣-minimum essential medium and penicillin/streptomycin were obtained from Invitrogen Corp. (Carlsbad, CA). Runx Electromobility Shift Assay (EMSA)—Cultures for whole cell extract preparation were washed with phosphate-buffered saline, and the cell layers were scraped and centrifuged at 3,000 rpm for 5 min at 4 °C. EMSA was performed with 15 g of whole cell extract and 80 fmol of an end-labeled 23-base pair oligonucleotide probe containing the Runx2-binding OSE2 site from the mouse osteocalcin gene 2 (OG2) promoter [34]. Resulting chromatin solution was sonicated using a Virsonic 60 sonicator (VirTis Co., Gardiner, NY; 4 pulses at 4 watts, 10 s each) and centrifuged at 16,000 ϫ g for 10 min to remove cell debris At this point, samples were diluted to adjust the absorbance to 0.25 A260 units and 100 l was further diluted 10 times with IP buffer (16.7 mM Tris-HCl, pH 8.1, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl) and protease inhibitors. Mean Ϯ S.D. were compared using the Student’s t test and the differences were considered significant when p Յ 0.05
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