Bone morphogenetic protein-2 restores mineralization in glucocorticoid-inhibited MC3T3-E1 osteoblast cultures.
The anti-glucocorticoid potential of BMP-2 in osteoblasts was tested in MC3T3-E1 cells using dexamethasone (1 microM) and rhBMP-2 (10 or 100 ng/ml). rhBMP-2 restored mineralization but not condensation or collagen accumulation. These results demonstrate the potential and limitations of BMPs in counteracting glucocorticoids.


BACKGROUND
Pharmacologic glucocorticoids (GCs) inhibit osteoblast function and induce osteoporosis. Bone morphogenetic proteins (BMPs) stimulate osteoblast differentiation and bone formation. Here we tested the anti-glucocorticoid potential of BMP-2 in cultured osteoblasts.


METHODS
MC3T3-E1 cells were treated with dexamethasone (DEX; 1 microM) and/or recombinant human BMP-2 (rhBMP-2; 10 or 100 ng/ml). Culture progression was characterized by cell cycle profiling, biochemical assays for DNA, alkaline phosphatase (ALP), collagen, and calcium, and by reverse transcriptase-polymerase chain reaction (RT-PCR) of osteoblast phenotypic mRNAs. Mineralization was characterized by Alizarin red and von Kossa staining and by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD).


RESULTS
DEX inhibited differentiation-related cell cycle, nodule formation, collagen accumulation, osteocalcin, and BMP-2 gene expression as well as mineralization. Replenishment of GC-inhibited cultures with 10 or 100 ng/ml rhBMP-2 dramatically rescued mineral deposition. The rhBMP-2-rescued mineral was bone-like apatite nearly identical to the mineral of control cultures. The rhBMP-2 rescue was associated with increased mRNA levels for alpha1(I) collagen, osteocalcin, and Cbfa1 types I and II, as well as ALP activity. In contrast, rhBMP-2 did not rescue the GC-inhibited differentiation-related cell cycle, nodule formation, or collagen accumulation. When administered alone, rhBMP-2 also increased the mRNA levels for alpha1(I) collagen, osteocalcin, and Cbfa1 types I and II, as well as ALP activity. However, treatment with rhBMP-2 alone inhibited cell cycle progression, nodule formation, and collagen accumulation. Surprisingly, in contrast to its rescue of mineralization in DEX-treated cultures, rhBMP-2 inhibited mineralization in the absence of DEX. In parallel to its bimodal effect on mineralization, rhBMP-2 stimulated endogenous BMP-2 mRNA in the presence of DEX, but inhibited endogenous BMP-2 mRNA in the absence of DEX.


CONCLUSIONS
Suppression of BMP-2 gene expression plays a pivotal role in GC inhibition of osteoblast differentiation. However, the inability of rhBMP-2 to rescue the entire osteoblast phenotype suggests BMP-2-independent inhibitory effects of CCs. BMP-2 exerts both positive and negative effects on osteoblasts, possibly depending on the differentiation stage and/or the existing BMP signaling.
Luppen
Cynthia A
CA
Department of Biochemistry, Institute for Genetic Medicine, University of Southern California Keck School of Medicine, Los Angeles, California 90033, USA.
Smith
Elisheva
E
Spevak
Lyudmila
L
Boskey
Adele L
AL
Frenkel
Baruch
B
D000818:Animals; D015152:Blotting, Northern; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D002113:Calcification, Physiologic / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003094:Collagen / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta
C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D005938:Glucocorticoids; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D015675:Osteocalcin; D003094:Collagen; D002118:Calcium