Bone Morphogenetic Protein 2 Induces Cyclo-oxygenase 2 in Osteoblasts via a Cbfa1 Binding Site: Role in Effects of Bone Morphogenetic Protein 2 In Vitro and In Vivo

Abstract

We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (HMP) 2 on bone. BIMP-2 induced COX-2 mKNA and prostaglandin (PG) production in cultured ostcoblasts. BMP-2 increased luciferase activity in calvarial ostcoblasts froni mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-El 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 (niuCbfal ) consensus seyuerice (5'-AACCACA-3') at -267/-261 bp decreased HMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonuclcotide spanning the Cbfal site was inhibited or supershifted by specific antibodies to Cbfal. I n 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. RMP-2 (10 pg/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. ILliIieralization of pellets, measured by microcomputed tomography (pCT), 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 viva (J Bone

Inhibition of cyclooxygenase-2 impacts chondrocyte hypertrophic differentiation during endochondral ossification

“…However, taking the previously reported involvement of canonical Wnt-signalling in the transcriptional regulation of RUNX2 expression upon BMP-2 ligand binding into account (Dong et al, 2006), this provides, at least in part, a mechanistic explanation for how inhibition of COX-2 activity reduces -catenin levels and leads to reduced chondrocyte hypertrophic maturation. Combined with data from other groups (Chikazu et al, 2002;Arikawa et al, 2004;Javed et al, 2009;Steinert et al, 2009), our fi ndings strongly support a functional relation between COX-2, PGE 2 and BMP-2 in chondrocyte hypertrophy. Chondrocyte hypertrophic differentiation is induced by BMP-2 during chondrogenesis (Steinert et al, 2009).…”

Selective and non‐selective cyclooxygenase inhibitors delay stress fracture healing in the rat ulna

“…The vital role of COX-2 in bone formation is also demonstrated in several experimental models. 34,43,44 Both non-selective and selective COX-2 inhibitors also reduce mechanically induced lamellar bone formation when given prior to mechanical loading. 3 In these experiments, selective COX-2 inhibitors demonstrate a greater effect.…”

The BMP signaling and in vivo bone formation

“…Mad, a Drosophila homologue of Smad, was shown to directly bind to the enhancer of these genes and GCCGnCGC (GCCG motif) was identified as the consensus binding site. It has been reported that Smads 1 and 5 interact with bone-specific transcription factor Runx2 (Hanai et al, 1999;Lee et al, 2000;Zhao et al, 2003) and activate the transcription of target genes such as COX-2 and type X collagen (Col-X) in osteoblasts or in chondrocytes (Chikazu et al, 2002;Leboy et al, 2001). The association of Smad1 with homeodomain-containing proteins suggests another important mechanism of BMP signaling in osteoblasts and in chondrocytes.…”