Roles of estrogen receptor alpha and beta in modulating urothelial cell proliferation

We reported previously that both subtypes of estrogen receptors, ERalpha and ERbeta, are expressed by human urothelial cells and mediate estrogen-induced cell proliferation in these cells. The aim of this study was to determine the extent to which each ER subtype contributes to urothelial cell proliferation and their possible involvement in the regulation of the cell cycle. We compared the expression of ERalpha and ERbeta mRNAs and protein quantitatively in primarily cultured human bladder urothelial cells obtained from six individuals with three immortalized urothelial (E6, E7, and UROtsa) and two bladder cancer cell lines (HTB-9 and T24). We found that all these cells express similar levels of ERbeta, but immortalized and cancer cells express much higher amounts of ERalpha than primary cells. Higher levels of ERalpha mRNA were also observed in the biopsies of bladder transitional cell carcinoma compared with sample from the same bladder unaffected by tumor. Using the ERalpha-selective agonist PPT, the ERbeta-selective agonist DPN, and specific small interfering RNA against ERalpha or ERbeta, we found that ERbeta predominantly mediates estrogen-induced G1/S transition and cell proliferation in the primary urothelial cells. By contrast, ERalpha predominantly mediates estrogen-induced G1/S transition and cell proliferation in bladder cancer cell lines. Furthermore, we found that 17beta-estradiol (E(2)) rapidly induces phosphorylation of extracellular signal-regulated kinases, but U0126, a mitogen-activated protein kinase kinase (MEK) inhibitor, does not affect E(2)-induced urothelial cell proliferation. E(2) up-regulated cyclin D1 and cyclin E expression in both the primary and bladder cancer cells, and the cancer cells have higher cyclin D1 and cyclin E expression during G0/G1 phases. Our data suggest that estrogen exerts its effects through different ER subtypes in urothelial cells. Increased expression of ERalpha may contribute to early induction of cyclin D1 and cyclin E during the cell cycle in bladder cancer cells.     

Synergistic up-regulation of prostaglandin E synthase expression in breast cancer cells by 17beta-estradiol and proinflammatory cytokines

Inflammatory mediators, such as cytokines and prostaglandins, play a fundamental role in estrogen-dependent breast cancer through their ability to up-regulate aromatase expression and subsequent local production of estrogens in the breast. To study the link between estrogens and inflammation further, we examined the regulation of prostaglandin E synthase (PTGES), a key enzyme in the production of prostaglandin E2. We found that 17beta-estradiol (E2) rapidly and robustly up-regulates PTGES mRNA and protein levels in estrogen receptor (ER)-positive breast cancer cells through ER recruitment to an essential estrogen response element located in the 5' flanking region of the PTGES gene. PTGES is also up-regulated by the proinflammatory cytokines TNFalpha or IL-1beta. Surprisingly, the combination of E2 and cytokines leads to a synergistic up-regulation of PTGES in an ER and nuclear factor-kappaB (NFkappaB)-dependent manner. This is in contrast to the mutual transrepression between ER and NFkappaB that has been well characterized in other cell types. Furthermore, we found enhanced recruitment of ERalpha as well as the NFkappaB family member, p65, to the PTGES estrogen response element by the combination of E2 and TNFalpha compared with either E2 or TNFalpha alone. The synergistic up-regulation of PTGES may result in enhanced prostaglandin E2 production, which in turn may further enhance aromatase expression and production of local estrogens. Our findings suggest that a finely tuned positive feedback mechanism between estrogens and inflammatory factors may exist in the breast and contribute to hormone-dependent breast cancer growth and progression.     

Activation of adenosine deaminase in MCF-7 cells through IGF-estrogen receptor alpha crosstalk

Adenosine deaminase (ADA) regulates cellular levels of adenosine and deoxyadenosine, and 17beta-estradiol (E(2)) induces ADA mRNA in MCF-7 human breast cancer cells. IGF-I also induces ADA gene expression in these cells, and induction of this response through IGF activation of estrogen receptor alpha (ERalpha) was further investigated. IGF and other polypeptide growth factors induce reporter gene expression in MCF-7 cells cotransfected with ERalpha expression plasmid and pADA211, a construct containing the -211 to +11 region of the ADA gene promoter which is required for high basal and E(2)-inducible activity. Deletion analysis of this promoter demonstrates that IGF activates ERalpha/Sp1 interactions with multiple GC-rich sites in the promoter and this response is abrogated in cells transfected with ERalpha containing mutations at Ser(118) or Ser(163). IGF induces both MAPK (mitogen-activated protein kinase) and PI3-K (phosphatidylinositol-3-kinase) phosphorylation cascades in MCF-7 cells; however, using a series of inhibitors and dominant negative constructs, our results show that induction of ADA by IGF activation of ERalpha/Sp1 is dependent on the MAPK signaling pathway.     

Estradiol regulates corticotropin-releasing hormone gene (crh) expression in a rapid and phasic manner that parallels estrogen receptor-alpha and -beta recruitment to a 3',5'-cyclic adenosine 5'-monophosphate regulatory region of the proximal crh promoter

In the central nervous system, CRH regulates several affective states. Dysregulation of neuronal crh expression in the paraventricular nucleus of the hypothalamus correlates with some forms of depression, and amygdalar crh expression may modulate levels of anxiety. Because estrogens modulate these states, we sought to determine 17beta-estradiol (E2) effects on crh expression. CRH mRNA levels were measured in the AR-5 amygdaloid cell line by RT-PCR analysis. They increased by 1 min of E2 treatment, suggesting that crh behaves as an immediate-early gene. After peaking at 3 min, CRH mRNA returned to basal levels and then increased by 60 min. To dissect some of the molecular mechanisms underlying these events, we measured occupancy of the crh promoter by estrogen receptors (ERs) and coactivators, using chromatin immunoprecipitation. Because this promoter does not contain palindromic estrogen response elements, we targeted the region of a cAMP regulatory element (CRE), implicated in crh regulation. The temporal pattern of the mRNA response was mimicked by recruitment of ERalpha and -beta, phospho-CRE-binding protein, coactivators steroid receptor coactivator-1 and CRE-binding protein-binding protein (CBP), and an increase in histone 3 and 4 acetylation. Lastly, ERalpha and -beta loading were temporally dissociated, peaking at 1 and 3 min, respectively. The ER peaks were associated with coactivators and acetylation patterns. ERalpha associated with phospho-CRE-binding protein, CBP, steroid receptor coactivator-1, and increased acetylated histone 3. ERbeta associated with CBP and increased acetylated histone 4. The tight temporal correlation between E2-induced CRH mRNA levels and promoter occupancy by ERs strongly suggest that E2 regulates crh expression through an ERalpha- and/or ERbeta-CRE alternate pathway.     

Vascular endothelial growth factor receptor-2 expression is induced by 17beta-estradiol in ZR-75 breast cancer cells by estrogen receptor alpha/Sp proteins

Vascular endothelial growth factor receptor-2 kinase insert domain receptor (VEGFR2/KDR) is critical for angiogenesis, and VEGFR2 mRNA and protein are expressed in ZR-75 breast cancer cells and induced by 17beta-estradiol (E2). Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich region is required for both basal and hormone-induced transactivation, and mutation of one or both of the GC-rich motifs at -58 and -44 results in loss of transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that Sp1, Sp3, and Sp4 proteins bind the GC-rich region of the VEGFR2 promoter. Results of the chromatin immunoprecipitation assay also demonstrate that ERalpha is constitutively bound to the VEGFR2 promoter and that these interactions are not enhanced after treatment with E2, whereas ERalpha binding to the region of the pS2 promoter containing an estrogen-responsive element is enhanced by E2. RNA interference studies show that hormone-induced activation of the VEGFR2 promoter constructs requires Sp3 and Sp4 but not Sp1, demonstrating that hormonal activation of VEGFR2 involves a nonclassical mechanism in which ERalpha/Sp3 and ERalpha/Sp4 complexes activate GC-rich sites where Sp proteins but not ERalpha bind DNA. These results show for the first time that Sp3 and Sp4 cooperatively interact with ERalpha to activate VEGFR2 and are in contrast to previous results showing that several hormone-responsive genes are activated by ERalpha/Sp1 in breast cancer cell lines.     

Estrogens increase the expression of fibulin-1, an extracellular matrix protein secreted by human ovarian cancer cells.

Ovarian cancers have a high ability to invade the peritoneal cavity and some are stimulated by estrogens. In an attempt to understand the mode of action of estrogens on these cancer cells and to develop new markers, we have characterized estrogen-regulated proteins. This study was aimed at identifying a protein secreted by ovarian cancer cells whose level was increased by estradiol [Galtier-Dereure, F., Capony, F., Maudelonde, T. & Rochefort, H. (1992) J. Clin. Endocrinol. Metab. 75, 1497-1502]. By using microprotein sequencing, the 110-kDa protein was identified as fibulin-1, a protein of the extracellular matrix that binds to fibronectin, laminin, and nidogen. The amount of immunoprecipitated fibulin-1 secreted into the medium and present in the cell extract was increased up to 10-fold by estradiol in three estrogen-responsive ovarian cancer cell lines. By immunohistochemistry fibulin-1 was located in the stroma of several ovarian cancers and cysts. The findings highlight a potential role for fibulin-1 in the spread of ovarian cancer in the peritoneal cavity and/or in distal metastases.     

In vivo profiling of estrogen receptor/specificity protein-dependent transactivation

17beta-Estradiol (E2) activates the estrogen receptor (ER) through multiple genomic and nongenomic pathways in various tissues/organs. ERalpha/specificity protein-dependent activation of E2-responsive genes containing GC-rich promoters has been identified in breast and other cancer cell lines, and in this study, we describe transgenic animals overexpressing a transgene containing three tandem GC-rich sites linked to a minimal TATA or thymidine kinase promoter and a luciferase gene. Several mouse lines expressing the transgenes were characterized and, in line 15, E2 induced a 9-fold increase in luciferase activity in the female mouse uterus, and the synthetic estrogens bisphenol A and nonylphenol also induced uterine luciferase activity. The pure antiestrogen ICI 182,780 induced luciferase activity in the mouse uterus, and similar results were observed for ICI 182,780 in breast cancer cells transfected with this construct. Differences in the ER agonist and antagonist activities of E2, nonylphenol, bisphenol A, and ICI 182,780 were investigated in the male testis and penis and the male and female stomach in line 15 transgenic mice. All of these tissues were hormone responsive; however, the patterns of induced or repressed luciferase activity were ligand structure, tissue, and sex dependent. These results demonstrate for the first time hormonal activation or repression of a GC-rich promoter in vivo, and the results suggest that the ERalpha/specificity protein pathway may contribute to E2-dependent induction and repression of genes.     

Stimulation of the novel estrogen receptor-alpha intronic TERP-1 promoter by estrogens, androgen, pituitary adenylate cyclase-activating peptide, and forskolin, and autoregulation by TERP-1 protein

The estrogen receptor-alpha (ERalpha) pituitary-specific variant, TERP-1, is regulated dramatically by physiological status. We examined hormonal regulation of the TERP-1 promoter in transient transfection assays in GH3 somatolactotrope cells. We found that 17beta-estradiol (E2), genistein, androgen, pituitary adenylate cyclase-activating peptide, and forskolin (FSK) all stimulated TERP-1 promoter activity, whereas progesterone had no effect. ERalpha bound to a palindromic estrogen response element (ERE) and two half-site EREs; mutation of any of these sites decreased basal expression and completely obliterated E2 stimulation. In contrast, mutation of an activator protein-1 site decreased basal and FSK-stimulated promoter activity, but not E2 or androgen stimulation. The pure antiestrogen ICI 182,780 suppressed E2 and genistein, but not FSK or androgen, stimulation. Similarly, mutation of the ERE palindrome or half-site EREs suppressed promoter stimulation by E2 and genistein, but not by androgen or FSK. Because TERP-1 levels regulate ERalpha function on model promoters, we tested TERP-1 modulation of its own and other physiological promoters. TERP-1 suppressed basal and E2-stimulated expression of its own promoter. TERP-1 suppression required the ERE regions of the promoter, and the dimerization domain of TERP-1. TERP-1 overexpression also suppressed E2 stimulation of the progesterone receptor and prolactin promoters. Thus, estrogens, androgen, and FSK can stimulate TERP-1 promoter activity, and increased TERP-1 expression modulates E2 stimulation of physiological promoters. These data suggest that TERP-1 regulation may play a significant role in modifying pituitary ERalpha responses.     

High tumoral levels of Kiss1 and G-protein-coupled receptor 54 expression are correlated with poor prognosis of estrogen receptor-positive breast tumors

KiSS1 is a putative metastasis suppressor gene in melanoma and breast cancer-encoding kisspeptins, which are also described as neuroendocrine regulators of the gonadotropic axis. Negative as well as positive regulation of KiSS1 gene expression by estradiol (E(2)) has been reported in the hypothalamus. Estrogen receptor alpha (ERalpha level is recognized as a marker of breast cancer, raising the question of whether expression of KiSS1 and its G-protein-coupled receptor (GPR54) is down- or upregulated by estrogens in breast cancer cells. KiSS1 was found to be expressed in MDA-MB-231, MCF7, and T47D cell lines, but not in ZR75-1, L56Br, and MDA-MB-435 cells. KiSS1 mRNA levels decreased significantly in ERalpha-negative MDA-MB-231 cells expressing recombinant ERalpha. In contrast, tamoxifen (TAM) treatment of ERalpha-positive MCF7 and T47D cells increased KiSS1 and GPR54 levels. The clinical relevance of this negative regulation of KiSS1 and GPR54 by E(2) was then studied in postmenopausal breast cancers. KiSS1 mRNA increased with the grade of the breast tumors. ERalpha-positive invasive primary tumors expressed sevenfold lower KiSS1 levels than ERalpha-negative tumors. Among ERalpha-positive breast tumors from postmenopausal women treated with TAM, high KiSS1 combined with high GPR54 mRNA tumoral levels was unexpectedly associated with shorter relapse-free survival (RFS) relative to tumors expressing low tumoral mRNA levels of both genes. The contradictory observation of putative metastasis inhibitor role of kisspeptins and RFS to TAM treatment suggests that evaluation of KiSS1 and its receptor tumoral mRNA levels could be new interesting markers of the tumoral resistance to anti-estrogen treatment.     

Requirement of Ras-dependent pathways for activation of the transforming growth factor beta3 promoter by estradiol

It has been previously observed that the transforming growth factor beta3 (TGFbeta3) gene can be activated by both estradiol (E(2)) and selective estrogen receptor modulators (SERMs) in vivo but that only SERMs have a potent stimulatory effect on the TGFbeta3 promoter in cultured cells. We demonstrate in this report that E(2) can act also as a potent inducer of the TGFbeta3 promoter via a novel and specific estrogen receptor (ER)alpha-mediated mechanism. Our results show that treatment with epidermal growth factor or transfection of a constitutively active Ras mutant allows E(2) to induce the TGFbeta3 promoter via ERalpha in cotransfected HeLa and osteosarcoma MG63 cells. Both protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors can block the combined stimulatory effect of E(2) and epidermal growth factor/Ras. However, E(2) induction of the TGFbeta3 promoter was found to be unaffected by mutation of ERalpha serine 118, a well-characterized target of MAPK. Progressive deletion analysis of the ERalpha amino-terminal region delineated three separate domains modulating the E(2)/activated Ras response, revealing a complex functional organization of the ERalpha A/B domain required for regulation of the TGFbeta3 promoter. In addition, PKC and MAPK inhibitors had no effect on the induction of TGFbeta3 promoter activity by the SERM EM-652. These results indicate that induction of the TGFbeta3 promoter by the E(2)/ERalpha complex requires the concomitant activation of PKC and MAPK signaling and provide a novel framework for the design of more effective estrogen-based therapeutic strategies.