17beta-Estradiol inhibits cytokine induction of the human E-selectin promoter.
Estradiol has been shown to decrease levels of the cell adhesion molecule E-selectin in cultured cells and in women on hormone replacement therapy. We set out to determine if the mechanism of estradiol action on E-selectin is at the level of its promoter. It was found that estradiol repressed the cytokine-stimulated induction of luciferase activity driven by the human E-selectin promoter in a reporter plasmid (hE-sel-LUC) in co-transfected human hepatoma cells (Hep G2) and human umbilical cord endothelial cells (ECV-304). Repression by estradiol was dependent on the presence of transfected estrogen receptor (ER) alpha or beta expression vectors. The ER antagonist ICI-182,780 blocked the repression by estradiol, confirming the receptor-dependence of the effect. The intact DNA-binding domain of ERalpha was required for estradiol repression of the cytokine-induced stimulation of the promoter in each cell line as demonstrated by the inability of an ER construct with two point mutations in the DNA-binding domain to inhibit reporter activity. Mutation of the NFK-B site at -94 to -85 within the E-selectin promoter led to less stimulation of hE-sel-LUC by interleukin one beta (IL-1beta). Estradiol did not inhibit this IL-1beta stimulated luciferase activity, indicating that the NFK-B site is necessary for ER-mediated inhibition of this promoter. Mutation of the AP-1 site at -500 to -494 within the E-selectin promoter had no effect on the ability of IL-1beta to stimulate its transcription, and estradiol repressed this activation in an ER-dependent manner with identical efficacy and potency in comparison with the wild-type promoter. Therefore, the E-selectin promoter is down-regulated by estradiol working through either ERalpha or ERbeta and requires the NFK-B site at -94 to -85 within the promoter.
Tyree
Curtis M
CM
Department of New Leads Discovery, Ligand Pharmaceuticals Inc., 10255 Science Center Road, San Diego, CA 92121, USA. ctyree@neurogeneticsinc.com
Zou
Aihua
A
Allegretto
Elizabeth A
EA
D001483:Base Sequence; D002460:Cell Line; D016207:Cytokines / Q000502:physiology*; D017931:DNA Primers; D019040:E-Selectin / Q000235:genetics*; D004958:Estradiol / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D011401:Promoter Regions, Genetic; D011960:Receptors, Estrogen / Q000502:physiology
D016207:Cytokines; D017931:DNA Primers; D019040:E-Selectin; D011960:Receptors, Estrogen; D004958:Estradiol; D008156:Luciferases