title: Involvement of CCAAT/enhancer-binding protein and nuclear factor-kappa B binding sites in interleukin-6 promoter inhibition by estrogens.
abstract: Bone loss observed in postmenopausal women is clearly associated with a decrease in estrogen levels. Interleukin 6 (IL-6), a multifunctional cytokine involved in osteoclast differentiation, is secreted by osteoblasts and appears to be a key molecule in the osteoporotic process. As previous reports have shown that the human IL-6 promoter is inhibited by estradiol, we investigated the mechanism of estradiol (E2)-mediated IL-6 inhibition in human cells. Analysis of the IL-6 secretion as a function of time in osteoblastoma Saos-2 cells, using an IL-6 ELISA test, showed that a maximal E2 inhibition of tumor necrosis factor-alpha (TNF alpha) induction could be monitored between 2 and 24 h of treatment. IL-6 inhibition was clearly estrogen agonist-specific in Saos-2 and MCF7 cells. Transient transfections of HeLa cells with a pIL-6/CAT plasmid and an estrogen receptor (human ER) expression vector, confirmed the role of the human ER in inhibition of the IL-6 promoter. Deletion and mutational analysis of the promoter highlighted the role of the -185/-60 region and showed that in both MCF7 and HeLa cells, the nuclear factor-IL 6 (NF-IL6) site cooperates with the nuclear factor-kappa B (NF-kappa B) motif to produce maximal induction by TNF alpha, whereas the CCAAT/enhancer-binding protein (C/EBP) site displayed different cooperative effects toward NF-kappa B depending on the cell line used. In HeLa cells, but not in MCF7 cells, we defined an essential role for the C/EBP site by showing that the E2 sensitivity was clearly dependent on its integrity. In these cell lines, the NF-kappa B site mutation abrogated both the TNF alpha-and E2- sensitivity of the construct.
authors.affiliation: Roussel Uclaf, Romainville, France.
authors.forename: R
authors.initials: R
authors.lastname: Galien
authors.forename: H F
authors.initials: HF
authors.lastname: Evans
authors.forename: T
authors.initials: T
authors.lastname: Garcia
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy / Q000473:pathology; D001483:Base Sequence; D001665:Binding Sites; D001943:Breast Neoplasms / Q000188:drug therapy / Q000473:pathology; D022762:CCAAT-Enhancer-Binding Proteins; D015500:Chloramphenicol O-Acetyltransferase / Q000187:drug effects / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism* / Q000494:pharmacology*; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011960:Receptors, Estrogen / Q000037:antagonists & inhibitors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics; D050796:STAT3 Transcription Factor; D017384:Sequence Deletion; D015534:Trans-Activators; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology; D000093242:Interleukin-6 Inhibitors
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D015500:Chloramphenicol O-Acetyltransferase; D004268:DNA-Binding Proteins; D004958:Estradiol; D015850:Interleukin-6; D016328:NF-kappa B; D009687:Nuclear Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D050796:STAT3 Transcription Factor; D015534:Trans-Activators; D014409:Tumor Necrosis Factor-alpha; D000093242:Interleukin-6 Inhibitors; C494086:STAT3 protein, human