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. 2005 Jan;25(1):472–487. doi: 10.1128/MCB.25.1.472-487.2005

FIG.3.

FIG.3.

Suppression of 1,25(OH)2D3-induced 24(OH)ase transcription using C/EBP DN or osteoblastic cells from C/EBPβ−/− mice. (A) C/EBP DN inhibits C/EBPβ enhancement and suppresses 1,25(OH)2D3-induced 24(OH)ase transcription. (Left panel) Representative autoradiogram. COS-7 cells were cotransfected with the CAT reporter plasmid [4 μg of the rat 24(OH)ase promoter construct −1367/+74 and the hVDR expression plasmid in the presence or absence of pMEX C/EBPβ and/or C/EBP DN]. Empty vectors were used to keep the total DNA concentration the same. Cells were treated with vehicle or 1,25(OH)2D3 (10−8 M). (Right panel) Graphic representation of the results of three separate experiments are expressed as mean percentages of the maximal response ± SE. C/EBPβ-mediated enhancement of transcription (third bar) and 1,25(OH)2D3-induced 24(OH)ase transcription (second bar) are significantly inhibited by C/EBP DN (second bar versus fourth bar and third bar versus fifth bar; P < 0.05). (B) Reduced VDR-mediated 24(OH)ase transcription and mRNA in osteoblastic cells isolated from C/EBPβ−/− mice. (Left and right panels) Primary osteoblastic cells were prepared from C/EBPβ−/− mice and control littermates (WT). (Left panel) Osteoblastic cells were transfected with the rat 24(OH)ase promoter (−1367/+74) and treated with 1,25(OH)2D3 (10−8 or 10−7 M). A significant inhibition of 1,25(OH)2D3-induced 24(OH)ase transcription was observed using cells from C/EBPβ−/− mice (P < 0.05 compared to values for the wild type). (Right panel) RT-PCR analysis of 24(OH)ase mRNA. A significant inhibition of 1,25(OH)2D3-induced 24(OH)ase mRNA was observed using osteoblastic cells from C/EBPβ−/− mice (P < 0.05 compared to values for the wild type). (C) Northern blot analysis of 24(OH)ase mRNA in the kidneys of C/EBPβ−/− and wild-type mice. C/EBPβ−/− or wild-type mice (3 to 4 months old) received either vehicle or 1,25(OH)2D3 (45 ng) 16 h before termination. A representative Northern blot is shown. There was no significant difference in 24(OH)ase mRNA levels induced by 1,25(OH)2D3 in the kidneys of the C/EBPβ−/− mice and in wild-type mice (P > 0.5).