Involvement of CCAAT Enhancer Binding Protein Transcription Factors in the Regulation of Prostaglandin G/H Synthase 2 Expression by Interleukin‐1 in Osteoblastic MC3T3‐E1 Cells

• Published in: Journal of bone and mineral research 2000-06, Vol.15 (6), p.1138-1146
• Main Authors: Harrison, John R., Kelly, Penny L., Pilbeam, Carol C.
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; CCAAT enhancer binding protein; CCAAT-Enhancer-Binding Proteins; Cell Line; Cyclooxygenase 2; cyclo‐oxygenase; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation - drug effects; Humans; Interleukin-1 - metabolism; Interleukin-1 - pharmacology; interleukin‐1; Isoenzymes - genetics; Membrane Proteins; Mice; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; osteoblast; Osteoblasts - cytology; Osteoblasts - drug effects; Osteoblasts - metabolism; Osteocalcin - genetics; prostaglandin G/H synthase; Prostaglandin-Endoperoxide Synthases - genetics; Response Elements; RNA, Messenger; Skeleton and joints; Transcription Factors - genetics; Transcription Factors - metabolism; Vertebrates: osteoarticular system, musculoskeletal system
• ISSN: 0884-0431; 1523-4681
• DOI: 10.1359/jbmr.2000.15.6.1138

Interleukin‐1 (IL‐1) stimulates prostaglandin production in bone by a rapid and transient activation of prostaglandin G/H synthase 2 (PGHS‐2) gene expression. In osteoblastic MC3T3‐E1 cells, IL‐1 caused a transient increase in PGHS‐2 messenger RNA (mRNA), which peaked at 2 h. IL‐1 caused a 2‐ to 4‐fold activation of a 371‐base pair (bp) murine PGHS‐2 promoter/luciferase construct in stable transfectants. This response mapped to a proximal promoter element(s) located between −150 and −40 bp. This region contains a putative CCAAT enhancer binding protein (C/EBP) site (centered at −135 bp), which shows enhanced binding of C/EBPβ and C/EBPδ by mobility shift analysis after IL‐1 treatment. A transient cotransfection approach was used to examine the effects of C/EBPβ and C/EBPδ overexpression. IL‐1 caused a maximal 3‐ to 7‐fold stimulation of PGHS‐2 promoter activity after 2.5 h. Overexpression of murine C/EBPβ and C/EBPδ caused a dose‐dependent increase in basal and IL‐1‐stimulated luciferase activity. C/EBPδ caused a greater enhancement of basal and IL‐1‐stimulated promoter activity than C/EBPβ, suggesting that C/EBPδ is a stronger transactivator. Overexpression of p20C/EBPβ, a dominant negative inhibitor of C/EBP function, blocked the stimulation of PGHS‐2 promoter activity by IL‐1 and blocked the ability of overexpressed C/EBPβ and C/EBPδ to increase basal and IL‐1‐stimulated promoter activity. Mutagenesis of the C/EBP site reduced, but did not abolish, the stimulation of PGHS‐2 promoter activity by IL‐1 and blunted the effect of overexpressed C/EBPδ on basal and IL‐1‐stimulated promoter activity. These results suggest an essential role for C/EBPβ and C/EBPδ in the induction of PGHS‐2 gene expression by IL‐1 in osteoblastic cells.