CCAAT BOX ENHANCER-BINDING PROTEIN ALPHA(C EBP-ALPHA) STIMULATES KAPPA-B ELEMENT-MEDIATED TRANSCRIPTION IN TRANSFECTED CELLS/

A construct comprising three tandemly repeated copies of the kappa B e lement from the interleukin-8 gene linked to chloramphenicol acetyltra nsferase (CAT) (3xNF-kappa BCAT) was transcriptionally activated in no rmal human FS-4 fibroblasts by co-transfection with expression vectors for NF-kappa B p50, p65, or p52, Unexpectedly, a significant activati on of 3xNF-kappa BCAT 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 transc riptional activation of 3xNF-kappa BCAT was seen after co-transfection with C/EBP-alpha in F9 mouse embryonic carcinoma cells, known to be d eficient in several transcription factors. To determine whether transc riptional activation was the result of interaction with one of the maj or NF-kappa B proteins, we co-transfected C/EBP-alpha with NF-kappa B p50, p65, p50 + p65, or p52 into F9 or FS-4 cells. No cooperative inte raction was seen; in fact, C/EBP-alpha reduced p65-stimulated transcri ption, especially in F9 cells. Electrophoretic mobility shift assay wi th a kappa B probe revealed that the addition of recombinant C/EBP-alp ha protein to nuclear extracts from untreated FS-4 cells resulted in t he appearance of four bands. Only one of these bands was supershifted by antibody to p50, whereas antibodies to p65 or other NF-kappa B prot eins had no effect. Our findings show that C/EBP-alpha may cause activ ation of some kappa B element-containing genes lacking C/EBP binding s ites.