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

Abstract

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.