Antagonist-induced, activation function-2-independent estrogen receptor alpha phosphorylation

Estrogen receptor alpha (ERalpha) serine 118 (Ser118) phosphorylation modulates activation function-1 (AF1) function. Correct positioning of helix 12 promotes agonist-dependent recruitment of cyclin-dependent kinase-7 to catalyze this event. In this study we show robust cyclin-dependent kinase-7-independent, AF2 antagonist-induced Ser118 phosphorylation. Estradiol (E2) and ICI-182,780 (ICI-780) induce Ser118 phosphorylation of wild-type ERalpha and either of two helix 12 mutants, suggesting AF2-independent action, probably via shedding of 90-kDa heat shock protein. With E2 treatment, the predominantly nuclear, phosphorylated ERalpha in COS-1 cells is detergent soluble. Although levels of ICI-780-induced phosphorylation are profound, Ser118-phosphorylated ERalpha is aggregated over the nucleus or in the cytoplasm, fractionating with the cell debris and making detection in cleared lysates improbable. Selective ER modulators (SERMs) elicit a mixed response with phosphorylated ERalpha in both detergent-soluble and -insoluble compartments. Apparent ligand-induced loss of ERalpha protein from cleared lysates is thus due to ligand-induced redistribution into the pellet, not degradation. The COS-1 response to ICI-780 can be mimicked in MCF-7 cells treated with a proteasome inhibitor to block authentic ligand-induced degradation. With SERMs and antagonists, the magnitude of Ser118-phosphorylated receptor redistribution into the insoluble fraction of COS-1 cells correlates with the magnitude of authentic ERalpha degradation in MCF-7 cells. A strong inverse correlation with ligand-induced uterotropism in vivo (P < 0.0001) and direct correlation with AF2-independent transrepression of the matrix metalloprotease-1 promoter in endometrial cells in vitro are seen. These data suggest that ligand-induced Ser118 phosphorylation of ERalpha can be AF2 independent. Furthermore, they identify translocation of Ser118-phosphorylated ERalpha out of the nucleus, leading to cytoplasmic aggregation, as an antagonist pathway that may precede receptor degradation.     

Distinct mechanisms of action of selective estrogen receptor modulators in breast and osteoblastic cells

Raloxifene and idoxifene areselective estrogen receptor modulators (SERMs) that exhibittissue-specific agonist or antagonist properties via interactions withthe estrogen receptor (ER). Both compounds are similarlyosteoprotective in the ovariectomized rat in vivo as assessed bymeasurement of bone mineral density, urinary pyridinium cross-links,and serum osteocalcin, suggesting a similar mechanism of action.However, we have identified a fundamental difference in this mechanismvia the estrogen response element (ERE) in osteoblast-like cells. Withthe use of ERE-luciferase reporter constructs, raloxifene, like thecomplete ER-antagonist ICI-182780, acts as an antagonist viathe ERE in osteoblastic cells. In contrast, idoxifene, like17-estrogen itself and 4-OH-tamoxifen, acts as an agonist inosteoblastic cells via an ER/ERE-mediated mechanism. Both ICI-182780and raloxifene inhibited the ERE-dependent agonist activity of17-estradiol and idoxifene in osteoblastic cells. In contrast, inbreast cells, raloxifene, idoxifene, 4-OH-tamoxifen, and ICI-182780 hadno agonist activity and, indeed, raloxifene and idoxifene were potentantagonists of ERE-mediated 17-estradiol action, indicating anERE-dependent mode of action in these cells. Although these SERMsexhibit a similar antagonist activity profile in breast cells, they canbe distinguished mechanistically in osteoblastic cells.

     

Differential SERM effects on corepressor binding dictate ERalpha activity in vivo

Selective estrogen receptor modulators (SERMs) show differential effects upon ERalpha activation function 1 (AF-1). Tamoxifen allows strong ERalpha AF-1 activity, whereas raloxifene allows less and ICI 182,780 (ICI) allows none. Here, we show that blockade of corepressor histone de-acetylase (HDAC) activity reverses the differential inhibitory effect of SERMs upon AF-1 activity in MCF-7 cells. This suggests that differential SERM repression of AF-1 involves HDAC-dependent corepressors. Consistent with this, ICI and raloxifene are more potent than tamoxifen in promoting ERalpha-dependent sequestration of progesterone receptor-associated corepressors. Moreover, ICI and raloxifene are more efficient than tamoxifen in promoting ERalpha binding to the corepressor N-CoR in vivo and in vitro. An ERalpha mutation (537X) that increases N-CoR binding in the presence of all SERMs blocks AF-1 activity. An ERalpha mutation (L379R) that decreases N-CoR binding increases AF-1 activity in the presence of ICI and raloxifene and reverses the effect of the 537X mutation. The 537X and L379R mutations also alter the ligand preference of ERalpha action at AP-1 sites and C3 complement, an action that also involves AF-1. Together, our results suggest that differential SERM effects on corepressor binding can explain differences in SERM effects on ERalpha activity. We propose a model for differential effects of SERMs on N-CoR binding.     

Comparative effects of raloxifene, tamoxifen and estradiol on human osteoblasts in vitro: Estrogen receptor dependent or independent pathways of raloxifene

SERMs bind to both estrogen receptor (ER)α and β, resulting in tissue dependent estrogen agonist or antagonist responses. Both raloxifene and tamoxifen are most frequently used SERMs and exert estrogen agonistic effects on human bone tissues, but the details of their possible direct effects on human bone cells have remained largely unknown. In our present study, we examined the comparative effects of raloxifene, tamoxifen, and native estrogen, estradiol on human osteoblast cell line, hFOB in vitro. Both the cell numbers and the ratio of the cells in S phase fraction were significantly increased by the treatment of raloxifene or tamoxifen as well as estradiol treatments in hFOB. Gene profile patterns following treatment with raloxifene, tamoxifen, and estradiol demonstrated similar patterns in a microarray/hierarchal clustering analysis. We also examined the expression levels of these genes detected by this analysis using quantitative RT-PCR. MAF gene was induced by raloxifene treatment alone. GAS6 gene was induced by raloxifene and tamoxifen as well as estradiol. An estrogen receptor blocker, ICI 18, 286, inhibited an increase of GAS6 gene expression but not the levels of MAF gene mRNA expression. Results of our present study demonstrated that raloxifene exerted direct protective effects on human osteoblasts in both estrogen receptor dependent and independent manners.     

Hemodynamic effects of acute and repeated exposure to raloxifene in ovariectomized sheep

We hypothesize that administration of acute and daily doses of raloxifene will have significant effects on ovine coronary and uterine hemodynamics and that these changes are estrogen receptor dependent. Eleven ovariectomized sheep were instrumented to measure mean arterial pressure, heart rate (HR), cardiac output (CO), and coronary (CBF) and uterine artery blood flows (UBF). A dose-response curve was generated for raloxifene (1, 3, and 10 microg/kg) and compared with a standard dose of estradiol-17beta (1 microg/kg) given intravenously. In a second group of animals, raloxifene (10 microg.kg-1.day-1) was administered intravenously for 14 consecutive days, and cardiovascular responses were compared with a group of animals administered estradiol-17beta (10 microg/kg) daily for the same period. To determine whether raloxifene-related vascular responses were estrogen receptor (ER) mediated, the animals were pretreated with estrogen antagonist ICI-182,780 given intravenously. Finally, RT-PCR was preformed to determine the presence of ERalpha and ERbeta mRNA in ovine coronary and uterine vessels. Raloxifene increased CBF and UBF dose dependently with a parallel decrease in the associated vascular resistances. Acute cardiovascular responses to daily doses of raloxifene and estradiol-17beta were sustainable. In contrast to estradiol-17beta, which significantly increases CO by increasing HR but not stroke volume, raloxifene significantly increased stroke volume without a significant parallel increase in HR. ICI-182,780 abolished raloxifene-induced hemodynamic responses, and ERalpha and ERbeta mRNA are present in both ovine coronary and uterine vessels. Hence, the hemodynamic effects of raloxifene are dose dependent, sustainable, and estrogen receptor mediated.     

Target specificity of selective estrogen receptor modulators within human endometrial cancer cells

Selective estrogen receptor modulators (SERMs) are estrogen receptor (ER) ligands that function as antagonists in some tissues, but have either partial or full agonist activity in others. SERMs often display variable partial agonist activity in uterine tissues and this activity can be displayed in uterine cell lines such as the human Ishikawa endometrial adenocarcinoma cell line. In this study, we compared the effects of several ER ligands including some SERMs on alkaline phosphatase (AP) activity and the expression of an ER target gene, the progesterone receptor (PR), in Ishikawa cells. As expected, estradiol (E2) was a potent and efficacious activator of both AP activity and PR mRNA expression. 4-Hydroxytamoxifen (4OHT) stimulated AP activity to a level 47% of that of E2 (100nM), while CP 336156 (lasofoxifene) increased AP activity 18%. A benzothiophene, such as LY 117018, a raloxifene analog, stimulated AP even less with values approximately 11% of E2-stimulated levels. A pure antiestrogen, ICI 182,780 did not stimulate AP activity. Interestingly, when we examined the ability of these compounds to increase the expression of the ER target gene, PR, a different rank order of efficacy was detected. After E2, CP 336156 was the most efficacious in increasing PR mRNA with a maximal stimulation of 20% of E2 levels, while 4OHT stimulated only 17%. LY 117018 increased PR mRNA expression 8% while ICI 182,780 did not increase PR mRNA expression at all. These data illustrate the target specificity that a SERM is able to display within a single cell type independent of "tissue specificity" and differential levels of expression of various cofactors. While 4OHT is 160% more active than CP 336156 in terms of inducing AP activity in the Ishikawa cells, CP 336156 has equivalent activity as 4OHT when one examines the ability of these SERMs to induce PR mRNA expression. Since the stimulation of Ishikawa cells by ER ligands is often used to assess the potential in vivo uterotrophic activity, these data indicate that examination of several endpoints in these cells may be necessary in order to fully characterize the activity of SERMs.     

Comparative study of the short-term effects of a novel selective estrogen receptor modulator, ospemifene, and raloxifene and tamoxifen on rat uterus

To investigate the differential short-term effects of selective estrogen receptor (ER) modulators (SERMs) on uterus, we treated adult ovariectomized rats with a novel SERM, ospemifene (Osp), two previously established SERMs (tamoxifen and raloxifene (Ral)) and estradiol. The expression of two estrogen-regulated early response genes c-fos and vascular endothelial growth factor (VEGF), and DNA synthesis were analysed at 1-24 h after treatment of ovariectomized rats. Induction of c-fos mRNA by each of the SERMs showed a biphasic pattern with peaks at 3 and 20 h, respectively. The maximum level of VEGF mRNA was observed at 1 h after raloxifene and 6 h after tamoxifen or ospemifene treatment. Maximum levels of the c-fos and VEGF mRNA after raloxifene treatment were higher than those seen after treatments with E2 or a corresponding dose of tamoxifen or ospemifene. DNA synthesis was significantly increased by ospemifene, tamoxifen and raloxifene both in luminal and glandular epithelium. The stimulation was transient, peaking at 16 h. In comparison, the maximum level observed at 16 h after E2 treatment sustained at least until 24 h. DNA synthesis in stromal cells was increased by the SERMs but not by E2 at 24 h. When treated together with E2, the SERMs were able to antagonise E2-stimulated DNA synthesis at 16 h. Our results demonstrate that the initial response of uterus to ospemifene, raloxifene and tamoxifen includes activation of early response genes and even transient stimulation of DNA synthesis in spite of their different long-term effects. However, the early stimulatory events may be mediated by different mechanisms leading to diverging pathways in various tissue compartments and development of differential SERM-specific long-term responses of uterus.     

Estradiol reduces F2alpha-isoprostane production in cultured human endothelial cells

Free radical-generated F(2alpha)-isoprostanes are a group of compounds with vasoconstrictor properties. To investigate whether estradiol exerts antioxidant actions modifying F(2alpha)-isoprostane production, cultured human umbilical vein endothelial cells were exposed to estradiol and other compounds and F(2alpha)-isoprostanes were measured in culture medium. Exposure to 1 and 10 nM estradiol for 24 h reduced F(2alpha)-isoprostane production by 36 and 49%, respectively (P < 0.001 vs. control). Exposure to antiestrogens alone (ICI-182780 or EM-652) slightly reduced F(2alpha)-isoprostanes (P < 0.05 vs. control), but much less than exposure to estradiol (P < 0.05). ICI-182780 reversed the estradiol-induced reduction of F(2alpha)-isoprostane concentration (P < 0.05). Along with time-course analysis, these results suggest that estradiol effects were mediated through estrogen receptor-dependent and -independent mechanisms. Progestogens alone (progesterone or medroxyprogesterone acetate) did not modify F(2alpha)-isoprostane production at any of the tested concentrations (1, 10, and 100 nM). Progesterone completely reversed estradiol-induced reduction of F(2alpha)-isoprostane production (P < 0.05 vs. control and estradiol), but medroxyprogesterone acetate did not (P < 0.05 vs. control).