CCAAT box enhancer binding protein alpha (C/EBP-alpha) stimulates kappaB element-mediated transcription in transfected cells.
A construct comprising three tandemly repeated copies of the kappaB element from the interleukin-8 gene linked to chloramphenicol acetyltransferase (CAT) (3xNF-kappaBCAT) was transcriptionally activated in normal human FS-4 fibroblasts by co-transfection with expression vectors for NF-kappaB p50, p65, or p52. Unexpectedly, a significant activation of 3xNF-kappaBCAT was also seen upon its co-transfection with the expression vector for CCAAT box enhancer binding protein alpha (C/EBP-alpha) (but not C/EBP-beta or C/EBP-delta). Stimulation by C/EBP-alpha required some other factor(s) present in FS-4 cells because no transcriptional activation of 3xNF-kappaBCAT was seen after co-transfection with C/EBP-alpha in F9 mouse embryonic carcinoma cells, known to be deficient in several transcription factors. To determine whether transcriptional activation was the result of interaction with one of the major NF-kappaB proteins, we co-transfected C/EBP-alpha with NF-kappaB p50, p65, p50 + p65, or p52 into F9 or FS-4 cells. No cooperative interaction was seen; in fact, C/EBP- alpha reduced p65-stimulated transcription, especially in F9 cells. Electrophoretic mobility shift assay with a kappaB probe revealed that the addition of recombinant C/EBP-alpha protein to nuclear extracts from untreated FS-4 cells resulted in the appearance of four bands. Only one of these bands was supershifted by antibody to p50, whereas antibodies to p65 or other NF-kappaB proteins had no effect. Our findings show that C/EBP-alpha may cause activation of some kappaB element-containing genes lacking C/EBP binding sites.
Vietor
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Department of Microbiology and Kaplan Cancer Center, New York University Medical Center, New York, New York 10016, USA.
Oliveira
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IC
Vilcek
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J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D002467:Cell Nucleus; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D016209:Interleukin-8 / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000096:biosynthesis / Q000378:metabolism*; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D007375:Interleukin-1; D016209:Interleukin-8; D016328:NF-kappa B; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase