CCAAT Box Enhancer Binding Protein α (C/EBP-α) Stimulates B Element-mediated Transcription in Transfected Cells

A construct comprising three tandemly repeated copies of the κB element from the interleukin-8 gene linked to chloramphenicol acetyltransferase (CAT) (3xNF-κBCAT) was transcriptionally activated in normal human FS-4 fibroblasts by co-transfection with expression vectors for NF-κB p50, p65, or p52. Unexpectedly, a significant activation of 3xNF-κBCAT was also seen upon its co-transfection with the expression vector for CCAAT box enhancer binding protein α (C/EBP-α) (but not C/EBP-β or C/EBP-ẟ). Stimulation by C/EBP-α required some other factor(s) present in FS-4 cells because no transcriptional activation of 3xNF-κBCAT was seen after co-transfection with C/EBP-α 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-κB proteins, we co-transfected C/EBP-α with NF-κB p50, p65, p50 + p65, or p52 into F9 or FS-4 cells. No cooperative interaction was seen; in fact, C/EBP-α reduced p65-stimulated transcription, especially in F9 cells. Electrophoretic mobility shift assay with a κB probe revealed that the addition of recombinant C/EBP-α 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-κB proteins had no effect. Our findings show that C/EBP-α may cause activation of some κB element-containing genes lacking C/EBP binding sites. A construct comprising three tandemly repeated copies of the κB element from the interleukin-8 gene linked to chloramphenicol acetyltransferase (CAT) (3xNF-κBCAT) was transcriptionally activated in normal human FS-4 fibroblasts by co-transfection with expression vectors for NF-κB p50, p65, or p52. Unexpectedly, a significant activation of 3xNF-κBCAT was also seen upon its co-transfection with the expression vector for CCAAT box enhancer binding protein α (C/EBP-α) (but not C/EBP-β or C/EBP-ẟ). Stimulation by C/EBP-α required some other factor(s) present in FS-4 cells because no transcriptional activation of 3xNF-κBCAT was seen after co-transfection with C/EBP-α 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-κB proteins, we co-transfected C/EBP-α with NF-κB p50, p65, p50 + p65, or p52 into F9 or FS-4 cells. No cooperative interaction was seen; in fact, C/EBP-α reduced p65-stimulated transcription, especially in F9 cells. Electrophoretic mobility shift assay with a κB probe revealed that the addition of recombinant C/EBP-α 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-κB proteins had no effect. Our findings show that C/EBP-α may cause activation of some κB element-containing genes lacking C/EBP binding sites.