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

Ilja Vietor,Igor C Oliveira,Jan Vilcek

doi:10.1074/jbc.271.10.5595

Abstract

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.

Highlights

NF-␬B proteins regulate the expression of many different genes, including genes encoding cytokines and acute phase proteins and some viral genes [1,2,3]
We have examined the ability of several members of the NF-␬B and CCAAT box enhancer binding protein (C/EBP) families to induce the transcriptional activation of a reporter construct comprising a trimerized NF-␬B binding site from the human IL-8 gene linked to the minimal promoter of the IL-8 gene and the chloramphenicol acetyltransferase (CAT) gene (3xNF-␬BCAT) [32]
Nuclear Protein Binding to NF-␬B Site from the IL-8 Promoter—To determine if increased CAT activity in FS-4 cells transfected with 3xNF-␬BCAT and C/EBP-␣ is due to the direct binding of C/EBP-␣ to the ␬B DNA element, we examined binding of recombinant C/EBP-␣ protein by electrophoretic mobility shift assays (EMSAs) with a synthetic oligonucleotide probe containing the same ␬B DNA sequence from the IL-8 gene as the 3xNF-␬BCAT construct

Summary

The abbreviations used are

C/EBP, CCAAT box enhancer binding protein; IL-8, interleukin-8; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; TNF, tumor necrosis factor; IL, interleukin; TPA, 12-0-tetradecanoylphorbol-13-acetate; USF, upstream stimulatory factor. An interaction of C/EBP-␣ with NF-␬B and/or other transcription factor(s) is likely to be involved in the transcriptional activation of the ␬B-driven construct in FS-4 cells. Our studies of transcriptional activation were confirmed by electrophoretic mobility shift assays (EMSAs), showing that in the presence of the ␬B probe nuclear extracts from unstimulated FS-4 cells formed specific complexes with recombinant C/EBP-␣ protein

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION