Involvement of CCAAT/enhancer-binding protein and nuclear factor-kappa B binding sites in interleukin-6 promoter inhibition by estrogens

Mol Endocrinol. 1996 Jun;10(6):713-22. doi: 10.1210/mend.10.6.8776731.


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.

MeSH terms

  • Adenocarcinoma / drug therapy
  • Adenocarcinoma / pathology
  • Base Sequence
  • Binding Sites
  • Breast Neoplasms / drug therapy
  • Breast Neoplasms / pathology
  • CCAAT-Enhancer-Binding Proteins
  • Chloramphenicol O-Acetyltransferase / drug effects
  • Chloramphenicol O-Acetyltransferase / genetics
  • DNA-Binding Proteins / metabolism
  • Estradiol / metabolism*
  • Estradiol / pharmacology*
  • Genes, Reporter
  • HeLa Cells
  • Humans
  • Interleukin-6 / antagonists & inhibitors*
  • Interleukin-6 / genetics*
  • Interleukin-6 / metabolism
  • Molecular Sequence Data
  • NF-kappa B / metabolism*
  • Nuclear Proteins / metabolism
  • Promoter Regions, Genetic
  • Receptors, Estrogen / antagonists & inhibitors
  • Receptors, Estrogen / metabolism
  • Recombinant Fusion Proteins / drug effects
  • Recombinant Fusion Proteins / genetics
  • STAT3 Transcription Factor
  • Sequence Deletion
  • Trans-Activators*
  • Transfection
  • Tumor Cells, Cultured
  • Tumor Necrosis Factor-alpha / metabolism
  • Tumor Necrosis Factor-alpha / pharmacology


  • CCAAT-Enhancer-Binding Proteins
  • DNA-Binding Proteins
  • Interleukin-6
  • NF-kappa B
  • Nuclear Proteins
  • Receptors, Estrogen
  • Recombinant Fusion Proteins
  • STAT3 Transcription Factor
  • STAT3 protein, human
  • Trans-Activators
  • Tumor Necrosis Factor-alpha
  • Estradiol
  • Chloramphenicol O-Acetyltransferase