engleski

BMP-2 Induces COX-2 in Osteoblasts via a Cbfa1 Binding Site: Role in Effects of BMP-2 In Vitro and In Vivo

We tested the hypothesis that induction of cyclooxygenase (COX)-2 mediates some effects of bone morphogenetic protein-2 (BMP-2) on bone. BMP-2 induced COX-2 mRNA and prostaglandin 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 to -213 bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of a Cbfa1 consensus sequence (5&#8217 ; -AACCACA-3&#8217 ; ) at -267/-261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfa1 site was inhibited or supershifted by specific antibodies to Cbfa1. 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 alkaline phosphatase 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 microCT, was decreased by 78% in COX-2-/- mice compared to COX-2+/+ mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfa1 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.

MC3T3-E1 cells; prostaglandin G/H synthase; prostaglandin E2 (PGE2); COX-2 knockout; promoter regulation

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