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.
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/EBPbeta and C/EBPdelta by mobility shift analysis after IL-1 treatment. A transient cotransfection approach was used to examine the effects of C/EBPbeta and C/EBPdelta overexpression. IL-1 caused a maximal 3- to 7-fold stimulation of PGHS-2 promoter activity after 2.5 h. Overexpression of murine C/EBPbeta and C/EBPdelta caused a dose-dependent increase in basal and IL-1-stimulated luciferase activity. C/EBPdelta caused a greater enhancement of basal and IL-1-stimulated promoter activity than C/EBPbeta, suggesting that C/EBPdelta is a stronger transactivator. Overexpression of p20C/EBPbeta, 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/EBPbeta and C/EBPdelta 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/EBPdelta on basal and IL-1-stimulated promoter activity. These results suggest an essential role for C/EBPbeta and C/EBPdelta in the induction of PGHS-2 gene expression by IL-1 in osteoblastic cells.
Harrison
J R
JR
Department of Orthodontics, The University of Connecticut Health Center, Farmington 06030-1850, USA.
Kelly
P L
PL
Pilbeam
C C
CC
D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D051546:Cyclooxygenase 2; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism* / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics*; D008565:Membrane Proteins; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D012333:RNA, Messenger; D020218:Response Elements; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D007375:Interleukin-1; D007527:Isoenzymes; D008565:Membrane Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D015675:Osteocalcin; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases