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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
Author(s) -
Harrison John R.,
Kelly Penny L.,
Pilbeam Carol C.
Publication year - 2000
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1359/jbmr.2000.15.6.1138
Subject(s) - ccaat enhancer binding proteins , microbiology and biotechnology , luciferase , transactivation , biology , transcription (linguistics) , promoter , transcription factor , gene expression , transfection , gene , dna binding protein , biochemistry , linguistics , philosophy
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.