Fluid Shear Stress-induced Cyclooxygenase-2 Expression Is Mediated by C/EBP β, cAMP-response Element-binding Protein, and AP-1 in Osteoblastic MC3T3-E1 Cells

• Published in: The Journal of biological chemistry 2001-03, Vol.276 (10), p.7048-7054
• Main Authors: Ogasawara, Aichi, Arakawa, Toshiya, Kaneda, Toshio, Takuma, Taishin, Sato, Takuya, Kaneko, Hironori, Kumegawa, Masayoshi, Hakeda, Yoshiyuki
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Blotting, Western; C/EBP^b protein; C/EBPb protein; CCAAT-Enhancer-Binding Protein-beta - metabolism; Cell Line; Cell Nucleus - metabolism; CREB protein; Cyclic AMP Response Element-Binding Protein - metabolism; Cyclooxygenase 2; fluid shear stress; Genetic Vectors; Isoenzymes - biosynthesis; Isoenzymes - genetics; Luciferases - metabolism; Mice; Mutation; Osteoblasts - metabolism; Plasmids - metabolism; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases - biosynthesis; Prostaglandin-Endoperoxide Synthases - genetics; Reverse Transcriptase Polymerase Chain Reaction; Stress, Mechanical; Time Factors; Transcription Factor AP-1 - metabolism; Transcriptional Activation; Transfection; Up-Regulation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M008070200

Mechanical loading is crucial for maintenance of bone integrity and architecture, and prostaglandins are an important mediator of mechanosensing. Cyclooxygenase-2 (COX-2), an inducible isoform of prostaglandin G/H synthase, is induced by mechanical loading-derived fluid shear stress in bone-forming cells such as osteoblasts and osteocytes. In this study, we investigated transcription factor and transcriptional regulatory elements responsible for the shear stress-induced COX-2 expression in osteoblastic MC3T3-E1 cells. When the cells were transfected with luciferase-reporter plasmids including the 5′-flanking region of the murine cox-2 gene, the fluid shear stress increased the luciferase activities, consistent with the induction of COX-2 mRNA and protein expression. Deletion analysis of the promoter region revealed that the shear stress-induced luciferase responses were regulated by two regions, −172 to −100 base pair (bp) and −79 to −46 bp, of the cox-2 promoter, in which putativecis-elements of C/EBP β, AP-1, cAMP-response element-binding protein (CREB), and E box are included. Mutation of sites of C/EBP β, AP-1, and/or cAMP-response element decreased the shear stress-induced luciferase activities, whereas mutation of the E box did not affect the responses. In an electrophoretic mobility shift assay, shear stress enhanced nuclear extract binding to double-stranded oligonucleotide probes containing C/EBP β and AP-1-binding motifs, and the bands of the complexes were supershifted by the addition of antibody specific for each regulator. Although the binding activity of CREB toward its probe was unaffected by shear stress, the phosphorylation of CREB was enhanced by the stress. These data suggest that C/EBP β, AP-1, and CREB play crucial roles in the shear stress-induced cox-2 expression in osteoblasts.