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.

Inhibition of NFκB Activity through Maintenance of IκBα Levels Contributes to Dihydrotestosterone-mediated Repression of the Interleukin-6 Promoter

“…This apparent contradiction can be resolved if we assume that the contribution from the Ϫ225/Ϫ160 fragment involves synergistic interaction with NFB and that DHTs major effect is to inhibit this synergistic interaction. In fact, it has been demonstrated that NF-IL6, whose response element is located immediately adjacent to Ϫ160, synergizes with NFB to amplify the response mediated by the NFB site on the IL-6 promoter (56,57,77). This would be consistent with the observation that deletion of the Ϫ225/Ϫ160 fragment, which alters the context of the NF-IL6 site, results in marked diminution of the PMA-induction of the promoter.…”

“…Several investigators have noted the NF-IL6 site is required for strong NFB-mediated induction of the IL-6 promoter (56,57). Therefore, when we delete the promoter to Ϫ160, which alters the region of the NF-IL6 response element, we lose the synergistic effect of NF-IL6 necessary for NFB-mediated activation of the IL-6 promoter.…”

Cyanidin-3-O-β-glucoside inhibits LPS-induced expression of inflammatory mediators through decreasing IκBα phosphorylation in THP-1 cells

“…The expression of cytokines IL-6 and TNFa is regulated directly by the upstream factor NF-jB [34][35][36][37]. LPS promotes the inflammatory response through the NF-jB pathway by activating phosphorylation of IjBa.…”

Estrogen Receptor (ER)-α, but Not ER-β, Mediates Regulation of the Insulin-like Growth Factor I Gene by Antiestrogens

“…By mutation analysis, we showed that both C/EBP sites have a major contribution to the raloxifene inducibility of the IGF-I promoter. C/EBPs are transcriptional regulators that are highly expressed in liver and have previously been shown to be involved in ER-mediated promoter activation (31)(32)(33). C/EBP-␤ has been shown to be necessary for ER-␣ mediated down-regulation of the interleukin 6 promoter and to cooperate with a nuclear factor B site (32).…”

“…C/EBPs are transcriptional regulators that are highly expressed in liver and have previously been shown to be involved in ER-mediated promoter activation (31)(32)(33). C/EBP-␤ has been shown to be necessary for ER-␣ mediated down-regulation of the interleukin 6 promoter and to cooperate with a nuclear factor B site (32). Stein and Yang (31) demonstrated a direct in vitro interaction between a glutathione S-transferase-ER-␣ fusion protein and C/EBP␤.…”