This report describes an investigation of the role of 1, 25-dihydroxyvitamin D (VD(3)) in the regulation of estrogen receptor-alpha (ER) in the ER-positive breast cancer cell line, MCF-7. Treatment of cells with 10 nM VD(3) resulted in a 50% decline in the concentration of ER protein at 24 h. Scatchard analysis showed a corresponding decrease in the number of estradiol binding sites and no alteration in the binding affinity of estradiol for the ER (K(d) = 0.08 nM in VD(3)-treated cells compared with K(d) = 0.07 nM in control cells). Vitamin D treatment also caused a 50% decrease in the steady state amount of ER mRNA, which was maximal by 18 h. In vitro transcription run-on experiments demonstrated a decrease of approximately 60% in transcription of the estrogen receptor gene. Transient transfections using an ER promoter-CAT construct also demonstrated a 40% decrease in CAT activity after VD(3) treatment. Sequence analysis identified a potential vitamin D response element (nVDRE) within the ER promoter. When this element was mutated, the ability of VD(3) to block transcription from the ER promoter was lost. When the nVDRE was placed upstream of a heterologous promoter, nVDRE-SV40-CAT, treatment with VD(3) resulted in a 50% decrease in CAT activity. Interestingly, co-transfection of either the ER promoter-CAT or the nVDRE-SV40-CAT construct and a vitamin D receptor expression vector into COS-1 or CV-1 cells showed an approximately 4-fold increase in CAT activity after VD(3) treatment. Taken together these data suggest that VD(3) inhibition of ER gene transcription is mediated through a nVDRE in the ER promoter. Inhibition appears to be cell specific.

The role of insulin-like growth factor-I (IGF-I) in regulating estrogen receptor-alpha (ER-alpha) gene expression and activity was investigated in the human breast cancer cell line MCF-7. Treatment of cells with 40 ng/ml IGF-I resulted in a 60% decrease in ER-alpha protein concentration by 3 h, and the amount of ER-alpha remained suppressed for 24 h. A multiple-dose ligand-binding assay demonstrated that the decrease in ER-alpha protein corresponded to a similar decrease of 50% in estradiol-binding sites with no effect on the binding affinity of ER-alpha. The dissociation constant of the estradiol-ER-alpha complex in the absence of IGF-I (K(d) = 3 x 10(-10) +/- 0.5 x 10(-10) M) was similar to the dissociation constant in the presence of IGF-I (K(d) = 6 x 10(-10) +/- 0.3 x 10(-10) M). The decrease in ER-alpha protein concentration was paralleled by an 80% decrease in the steady-state amount of ER-alpha mRNA by 3 h. The IGF-I induced decrease in ER-alpha mRNA was due to the inhibition of ER-alpha gene transcription. When an 128-base pair ER-alpha-promoter-CAT construct was transfected into MCF-7 cells, treatment with IGF-I resulted in a 40% decrease in CAT activity. In contrast to the effects on ER-alpha, treatment with IGF-I induced two endogenous estrogen-regulated genes, progesterone receptor and pS2, by 4- and twofold, respectively. The pure antiestrogen ICI-164, 384 blocked this induction, suggesting that ER-alpha mediates the effects of IGF-I. Transient co-transfections of wild-type ER-alpha and an estrogen response element-CAT reporter into COS-1 cells demonstrated that IGF-I increased reporter gene activity. This effect was also blocked by ICI 164,384. Protein kinase A and phosphatidylinositol 3-kinase inhibitors blocked the IGF-I effects on ER-alpha expression and activity, suggesting that these kinases may be involved in the cross-talk between the IGF-I and ER-alpha pathways.

The role of transforming growth factor-beta1 (TGFbeta1) in the regulation of estrogen receptor (ER) expression in MCF-7 cells was investigated. After treatment of the cells with 100 pM TGFbeta1, ER protein declined by about 30% at 6 h from a concentration of 413.5 fmol/mg protein in control cells to 289.5 fmol/mg protein in treated cells. The concentration of receptor remained suppressed for 24 h. Scatchard analysis demonstrated that the decrease in ER protein corresponded to a decrease in estradiol-binding sites, with no effect on the binding affinity of the ER. The dissociation constant of the estradiol-ER complex was 0.117 nM in TGFbeta1-treated cells compared to 0.155 nM in control cells. Treatment with TGFbeta1 did not influence the half-life of the ER. In TGFbeta1-treated cells, as well as in control cells, the half-life of the receptor was approximately 4 h. In contrast to the effect on ER concentration, TGFbeta1 treatment resulted in a greater decrease in the steady state level of ER messenger RNA (approximately 75%) at 6 h. By 24 h, a small recovery in the amount of messenger RNA was observed. Transcription run-on experiments demonstrated a decrease of approximately 70% in the level of ER gene transcription at 3 h. Transient transfections using an ER promoter-chloramphenicol acetyltransferase construct demonstrated that after TGFbeta1 treatment, chloramphenicol acetyltransferase activity decreased by 50%, suggesting that TGFbeta1 inhibition of the ER gene transcription is mediated through the ER promoter. Although treatment with TGFbeta1 decreased the ER concentration, the growth factor had no effect on the activity of ER, as measured by its effects on estradiol induction of progesterone receptor and pS2, suggesting that TGFbeta1 does not inhibit proliferation of MCF-7 cells by blocking ER activity.

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