Bone morphogenetic protein 2 induces cyclo-oxygenase 2 in osteoblasts via a Cbfal binding site: role in effects of bone morphogenetic protein 2 in vitro and in vivo.
We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-E1 cells transfected with Pluc. Deletion analysis identified the -300/-213-bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of core-binding factor activity 1 (muCbfal) consensus sequence (5'-AACCACA3') at -267/-261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfal site was inhibited or supershifted by specific antibodies to Cbfal. In cultured osteoblasts from calvariae of COX-2 knockout (-/-) and wild-type (+/+) mice, the absence of COX-2 expression reduced the BMP-2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP-2 induced osteoclast formation in cells from COX-2(+/+) mice but not in cells from COX-2(-/-) mice. In vivo, BMP-2 (10 microg/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (microCT), was decreased by 78% in COX-2(-/-) mice compared with COX-2(+/+) mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfal binding site and that the BMP-2 induction of COX-2 can contribute to effects of BMP-2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP-2 stimulation of ectopic bone formation in vivo.
Chikazu
Daichi
D
Department of Medicine, University of Connecticut Health Center, Farmington 06030, USA.
Li
Xiaodong
X
Kawaguchi
Hiroshi
H
Sakuma
Yoko
Y
Voznesensky
Olga S
OS
Adams
Douglas J
DJ
Xu
Manshan
M
Hoshio
Kazuto
K
Katavic
Vedran
V
Herschman
Harvey R
HR
Raisz
Lawrence G
LG
Pilbeam
Carol C
CC
D016475:3T3 Cells; D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000502:physiology*; D002113:Calcification, Physiologic; D050660:Core Binding Factors; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000096:biosynthesis; D024202:Electrophoretic Mobility Shift Assay; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D008156:Luciferases / Q000096:biosynthesis; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D010006:Osteoblasts / Q000201:enzymology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta
C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D050660:Core Binding Factors; D007527:Isoenzymes; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D008156:Luciferases; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone