17␤-Estradiol (E2) induces expression of several genes via estrogen receptor (ER)-Sp1 protein interactions with GC-rich promoter elements in which The estrogen receptor (ER)1 is a member of the nuclear receptor superfamily of transcription factors that exhibit several common structural/functional domains (1, 2). Estrogens, the endogenous ER ligands, influence gene expression and physiological responses in multiple target tissues (3, 4), and mechanisms associated with selective estrogen and antiestrogen action have been extensively investigated. 17␤-Estradiol (E2)-induced transactivation in specific cell types is modulated by multiple factors, including expression of the ER, coactivator and corepressor proteins, other nuclear factors, and accessibility of responsive elements in target gene promoters (5-9). The recent discovery of a second form of the ER (i.e. ER ␤ ) extends the potential tissue-specific selectivity for induction of estrogenic and antiestrogenic responses (10, 11).ER ␣ and ER ␤ exhibit both differential and overlapping expression in various tissues and cell lines (10 -21). For example, in rats, ER ␤ mRNA transcripts were highly expressed in the prostate and ovary; moderate expression was observed in testis, uterus, lung, and bladder; and low ER ␤ expression was observed in the spinal cord, various brain sections, pituitary, epididymis, and thymus (10). In many of these same tissues, both ER ␤ and ER ␣ are co-expressed; however, it was evident that in tissues such as the epididymis, uterus, kidney, and adrenal, which express high levels of ER ␣ mRNA, low to nondetectable ER ␤ mRNA was detected. The functional tissuespecific differences in ER subtypes have not yet been delineated; however, estrogenic responses observed in murine neuronal cells, vascular tissue, and the male reproductive tract of ER ␣ knockout mice suggest that in these tissues, ER ␤ is active in addition to ER ␣ (22-24).There is considerable overlap in the ligand binding and functional properties of ER ␣ and ER ␤ (12,(25)(26)(27)(28)(29)(30). Both proteins bind a diverse spectrum of ligands with similar (but not identical) relative binding affinities; ER ␣ and ER ␤ form homo-and heterodimers that bind estrogen-responsive elements (EREs) in gel mobility shift assays. ER ␣ and ER ␤ activate transcription from ERE-and AP1-containing constructs; however, the effects are ligand-, cell type-, and promoter-dependent (11, 25, 28 -34). A recent study reported that mutation of a conserved tyrosine to asparagine in the ligand binding domains of ER ␣ and ER ␤ conferred hormone-independent activation properties to both ER subtypes (31).Recent studies in our laboratory have demonstrated physical and functional interactions between ER ␣ and the nuclear transcription factor Sp1 (35). E2 induced reporter gene activity in breast cancer cells transfected with pSp1, a construct containing a consensus GC-rich oligonucleotide insert linked to a bacterial CAT reporter gene (35). Sp1 protein plays an important role in regulation of mammalian and viral gene...

No abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands suppress 17␤-estradiol (E)-induced responses in the rodent uterus and mammary tumors and in human breast cancer cells. Treatment of ZR-75, T47D, and MCF-7 human breast cancer cells with TCDD induces proteasomedependent degradation of endogenous estrogen receptor ␣ (ER␣).The proteasome inhibitors MG132, PSI, and PSII inhibit the proteasome-dependent effects induced by TCDD, whereas the protease inhibitors EST, calpain inhibitor II, and chloroquine do not affect this response. ER␣ levels in the mouse uterus and breast cancer cells were significantly lower after cotreatment with E plus TCDD than after treatment with E or TCDD alone, and our results indicate that AhR-mediated inhibition of E-induced transactivation is mainly due to limiting levels of ER␣ in cells cotreated with E plus TCDD. TCDD alone or in combination with E increases formation of ubiquitinated forms of ER␣, and both coimmunoprecipitation and mammalian two-hybrid assays demonstrate that TCDD induces interaction of the AhR with ER␣ in the presence or absence of E. In contrast, E does not induce AhR-ER␣ interactions. Thus, inhibitory AhR-ER␣ cross talk is linked to a novel pathway for degradation of ER␣ in which TCDD initially induces formation of a nuclear AhR complex which coordinately recruits ER␣ and the proteasome complex, resulting in degradation of both receptors.Estrogenic hormones induce their tissue-specific responses through binding the estrogen receptor (ER), which is a ligandactivated transcription factor and a member of the nuclear receptor (NR) superfamily (3, 15). The two ER subtypes (ER␣ and ER␤) and other NRs exhibit modular structures containing N-terminal activation function 1 (AF1) and C-terminal AF2, which also contains the ligand-binding domain, a DNAbinding domain (DBD), and an adjacent hinge region. Most early-stage mammary tumors are ER positive and are responsive to endocrine therapies which target ER␣ and/or E biosynthesis (5, 13, 47). Selective ER modulators, such as tamoxifen, are extensively used for treating early-stage breast cancer, and the primary modes of action of selective ER modulators involve competitive binding to the ER and subsequent inhibition of one or more steps in ER-mediated transactivation. Several studies show that there are important mechanistic differences among antiestrogens, and this is consistent with their tissuespecific ER antagonist-agonist activities (20,52,53). For example, the "pure" antiestrogens ICI 164,384 and/or ICI 182,780 not only bind ER␣ with high affinity but induce a rapid proteasome-dependent degradation of the receptor, and this is observed in breast cancer cells and human tumors (41, 52). Rapid degradation of ER␣ protein in cells or tumors treated with ICI 164,384 or 182,780 may play an important role in the antiestrogenic activity of these compounds.Studies in this laboratory have investigated inhibition of ER signaling through cross talk with the ligand-activated aryl hydrocarbon rece...

Vascular endothelial growth factor (VEGF) is expressed in multiple hormone-dependent cancer cells/tumors. Treatment of ZR-75 breast cancer cells with 17b-estradiol (E2) induced a greater than fourfold increase of VEGF mRNA levels. ZR-75 breast cancer cells were transfected with pVEGF1, a construct containing a À2018 to þ 50 VEGF promoter insert, and E2 induced reporter gene (luciferase) activity. Deletion and mutation analysis of the VEGF gene promoter identified a GC-rich region (À66 to À47) which was required for E2-induced transactivation of pVEGF5, a construct containing the minimal promoter (À66 to þ 54) that exhibited E2-responsiveness. Interactions of nuclear proteins from ZR-75 cells with the proximal GC-rich region of the VEGF gene promoter were investigated by electrophoretic mobility shift and chromatin immunoprecipitation assays. The results demonstrate that both Sp1 and Sp3 proteins bound the GCrich motif (À66 to À47), and estrogen receptor a (ERa) interactions were confirmed by chromatin immunoprecipitation. Moreover, E2-dependent activation of constructs containing proximal and distal GC/GT-rich regions of the VEGF promoter was inhibited in ZR-75 cells transfected with small inhibitory RNAs for Sp1 and Sp3. These results were consistent with a mechanism of hormone activation of VEGF through ERa/Sp1 and ERa/Sp3 interactions with GC-rich motifs.

Regulation of estrogen receptor alpha (ERalpha) plays an important role in hormone responsiveness and growth of ER-positive breast cancer cells and tumors. ZR-75 breast cancer cells were grown under conditions of normoxia (21% O(2)) or hypoxia (1% O(2) or cobaltous chloride), and hypoxia significantly increased hypoxia-inducible factor 1alpha protein within 3 h after treatment, whereas ERalpha protein levels were dramatically decreased within 6-12 h, and this response was blocked by the proteasome inhibitor MG-132. In contrast, hypoxia induced only minimal decreases in cellular Sp1 protein and did not affect ERalpha mRNA; however, hypoxic conditions decreased basal and 17beta-estradiol-induced pS2 gene expression (mRNA levels) and estrogen response element-dependent reporter gene activity in ZR-75 cells. Although 17beta-estradiol and hypoxia induce proteasome-dependent degradation of ERalpha, their effects on transactivation are different, and this may have implications for clinical treatment of mammary tumors.