Testosterone inhibits estrogen-induced mammary epithelial proliferation and suppresses estrogen receptor expression.

This study investigated the effect of sex steroids and tamoxifen on primate mammary epithelial proliferation and steroid receptor gene expression. Ovariectomized rhesus monkeys were treated with placebo, 17beta estradiol (E2) alone or in combination with progesterone (E2/P) or testosterone (E2/T), or tamoxifen for 3 days. E2 alone increased mammary epithelial proliferation by approximately sixfold (P:<0.0001) and increased mammary epithelial estrogen receptor (ERalpha) mRNA expression by approximately 50% (P:<0.0001; ERbeta mRNA was not detected in the primate mammary gland). Progesterone did not alter E2's proliferative effects, but testosterone reduced E2-induced proliferation by approximately 40% (P:<0.002) and entirely abolished E2-induced augmentation of ERalpha expression. Tamoxifen had a significant agonist effect in the ovariectomized monkey, producing a approximately threefold increase in mammary epithelial proliferation (P:<0.01), but tamoxifen also reduced ERalpha expression below placebo level. Androgen receptor (AR) mRNA was detected in mammary epithelium by in situ hybridization. AR mRNA levels were not altered by E2 alone but were significantly reduced by E2/T and tamoxifen treatment. Because combined E2/T and tamoxifen had similar effects on mammary epithelium, we investigated the regulation of known sex steroid-responsive mRNAs in the primate mammary epithelium. E2 alone had no effect on apolipoprotein D (ApoD) or IGF binding protein 5 (IGFBP5) expression, but E2/T and tamoxifen treatment groups both demonstrated identical alterations in these mRNAs (ApoD was decreased and IGFBP5 was increased). These observations showing androgen-induced down-regulation of mammary epithelial proliferation and ER expression suggest that combined estrogen/androgen hormone replacement therapy might reduce the risk of breast cancer associated with estrogen replacement. In addition, these novel findings on tamoxifen's androgen-like effects on primate mammary epithelial sex steroid receptor expression suggest that tamoxifen's protective action on mammary gland may involve androgenic effects.     

Selective estrogen receptor modulators (SERMS) and their roles in breast cancer prevention

Tamoxifen has not only proved to be a valuable treatment for estrogen receptor (ER)-positive breast cancer, but is also a pioneering medicine for chemoprevention in high-risk pre- and postmenopausal women. Insights into the pharmacology and toxicology of tamoxifen have led to the recognition of selective ER modulators (SERMs) with estrogen-like actions in maintaining bone density and in lowering circulating cholesterol, but antiestrogenic actions in the breast. Raloxifene, a related SERM, is now available to treat osteoporosis and is also being tested as a preventive for breast cancer and coronary heart disease. Emerging knowledge about the action of SERMs will provide clues for the design of mechanism-based medicines.     

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.     

Identification of novel proteins induced by estradiol, 4-hydroxytamoxifen and acolbifene in T47D breast cancer cells

Tamoxifen is currently used as adjuvant therapy for estrogen receptor (ER) positive breast cancer patients and as a chemopreventative agent. Although ER is a predictive marker for tamoxifen response, ER status fails to predict tamoxifen response in a significant number of patients highlighting the need to identify new pathways for tamoxifen sensitivity/resistance. To identify novel proteins induced by tamoxifen in breast cancer cells sensitive to tamoxifen growth inhibition, two-dimensional (2D) gel electrophoresis was used to profile proteins in T47D breast cancer cells. Six proteins were identified that were differentially regulated by 17beta-estradiol, 4-hydroxytamoxifen and the pure antagonist acolbifene (EM-652); calreticulin, synapse associated protein 1 (SYAP1), CD2 antigen binding protein 2 (CD2BP2), nucleosome assembly protein 1 like 1 (NAP1L1), d-3-phosphoglycerate dehydrogenase (3-PHGDH) and pyridoxine 5' phosphate oxidase (PNPO). At the mRNA level, these ligands differentially regulated expression of mRNAs encoding the identified proteins in T47D and MCF7 cells but had no effect on mRNA in ERalpha-negative MDA-MB-231 breast cancer cells. These novel SERM-regulated proteins may participate in new or existing pathways for sensitivity or resistance to 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.     

Identification of selective estrogen receptor modulators by their gene expression fingerprints

Clinical studies have shown that estrogen replacement therapy (ERT) reduces the incidence and severity of osteoporosis and cardiovascular disease in postmenopausal women. However, long term estrogen treatment also increases the risk of endometrial and breast cancer. The selective estrogen receptor (ER) modulators (SERMs) tamoxifen and raloxifene, cause antagonistic and agonistic responses when bound to the ER. Their predominantly antagonistic actions in the mammary gland form the rationale for their therapeutic utility in estrogen-responsive breast cancer, while their agonistic estrogen-like effects in bone and the cardiovascular system make them candidates for ERT regimens. Of these two SERMs, raloxifene is preferred because it has markedly less uterine-stimulatory activity than either estrogen or tamoxifen. To identify additional SERMs, a method to classify compounds based on differential gene expression modulation was developed. By analysis of 24 different combinations of genes and cells, a selected set of assays that permitted discrimination between estrogen, tamoxifen, raloxifene, and the pure ER antagonist ICI164384 was generated. This assay panel was employed to measure the activity of 38 compounds, and the gene expression fingerprints (GEFs) obtained for each compound were used to classify all compounds into eight groups. The compound's GEF predicted its uterine-stimulatory activity. One group of compounds was evaluated for activity in attenuating bone loss in ovariectomized rats. Most compounds with similar GEFs had similar in vivo activities, thereby suggesting that GEF-based screens could be useful in predicting a compound's in vivo pharmacological profile.     

Endometrial effects of selective estrogen receptor modulators (SERMs) on estradiol-responsive gene expression are gene and cell-specific

Three selective estrogen receptor modulator (SERM) drugs which included 4-OH-tamoxifen (Tam), EM-800 (EM) and GW 5638 (GW) were investigated to determine their ability to inhibit estradiol-responsive gene expression in sheep endometrium. The uteri of ovariectomized ewes (10 ewes per SERM group) were infused with 10⁻⁷ M SERMs for 24 h prior to hysterectomy. Five ewes from each group received 50 μg 17β-estradiol (E2) and the remaining five ewes received vehicle 18 h prior to hysterectomy. Northern blot analyses and in situ hybridization demonstrated that E2 treatment increased estrogen receptor (ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and cyclophilin (CYC) mRNA levels in most endometrial cells examined. Tam and GW exhibited characteristics similar to E2 by increasing ER gene expression, but they antagonized the E2-induced increases in PR and CYC mRNA levels. EM acted as an E2-agonist of GAPDH gene expression, but antagonized the E2 up-regulation of ER, PR and CYC gene expression in most endometrial cells. Immunohistochemistry determined that EM decreased ER protein levels in the glandular epithelium, and the SERMs investigated antagonized increases in PR protein levels in endometrium. In conclusion, GW and EM exhibit fewer agonist effects than Tam on endometrial gene expression. EM demonstrated the greatest antagonism of E2-enhanced levels of ER, PR and CYC, likely due to the inhibition of ER gene expression at both mRNA and protein levels.     

Myometrial effects of selective estrogen receptor modulators on estradiol-responsive gene expression are gene and cell-specific

We examined in vivo effects of selective estrogen receptor modulators (SERMs) 4-OH-tamoxifen (Tam), GW 5638 (GW) and EM-800 (EM) on myometrial gene expression. The uteri of ovariectomized ewes were infused with 10⁻⁷ M of one SERM via indwelling catheters for 24 h preceding hysterectomy. Half of the ewes in each SERM group received an intramuscular injection of 50 μg 17β-estradiol (E2) 18 h prior to hysterectomy. Northern blot analysis and in situ hybridization demonstrated that E2 increased estrogen receptor (ER), progesterone receptor (PR) and cyclophilin (CYC) gene expression in the cells of both inner layer of myometrium (IM) and outer layer of myometrium (OM) as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in OM. Tam also increased ER mRNA levels in OM. EM appeared to increase ER gene expression, but antagonized E2’s up-regulation of PR and CYC gene expression in both IM and OM. Tam and GW also antagonized E2 up-regulation of PR gene expression in OM but not IM. No SERM affected GAPDH gene expression with or without E2. Immunohistochemistry indicated that E2 increased nuclear ER and PR protein levels in both IM and OM. EM was unique in up-regulating ER protein levels, opposite to its effects in endometrial cells. All SERMs tested antagonized this increase in PR immunostaining preferentially in OM compared to the IM layer. These results illustrate gene and cell layer-specific effects of SERMs in sheep myometrium.     

Expression of Glut-4 and Glut-1 transporters in rat diaphragm muscle

Glucose transporters (Gluts) are a family of membrane proteins responsible for the transport of glucose across cellular membranes. Generally, alterations of Gluts expression in limb skeletal muscle have been reported. However, the changes of Glut isoforms in respiratory muscle which contracts with a duty cycle have rarely been studied. This study was performed to evaluate at the light microscopy level the expression of Glut-4 and Glut-1 transporters in normal and denervated diaphragm by immunohistochemistry method with specific Gluts antibodies. The results showed Glut-4 immunoreactivity in both the cell periphery and the interior of myocytes. Glut-1 was also present in the cell border and in the interior of myocytes in control diaphragm. However, Glut-4 staining was stronger than Glut-1 staining in control diaphragm. In denervated hemidiaphragm, the Glut-4 immunolabelling decreased and Glut-1 increased. These data indicated that (1) Glut-4 and Glut-1 transporters were observed in diaphragm; and (2) there were alterations in the expression of both glucose transporters after denervation. These alterations in Glut isoforms after denervation may be associated with the removal of innervation itself, and/or may partly result from passive stretch imposed by inspiratory activation of the contralateral side.     

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.     

Estrogen Receptor β Signaling Induces Autophagy and Downregulates Glut9 Expression

Glut9 is highly expressed in the human kidney proximal convoluted tubular and plays a crucial role in the regulation of plasma urate levels. The gene effects were stronger among women. Our results show that 17-β-estradiol (E2) through ER (estrogen receptor) β downregulates Glut9 protein expression on human renal tubular epithelial cell line (HK2). Intriguingly, E2 does not affect the expression of Glut9 mRNA. ERβ is linked to PTEN, the PTEN gene negatively regulates the PI3K/AKT pathway, and the PI3K/AKT pathway inhibition may lead to autophagy. Further study indicates that ERβ may affect the expression of Glut9 though autophagy.     

Estrogen increases proenkephalin messenger ribonucleic acid levels in the ventromedial hypothalamus of the rat

The effects of estrogen on proenkephalin (PE) gene expression were measured in neurons of the ventromedial hypothalamus. Slot blot hybridization analysis indicates that the levels of PE mRNA in the ventromedial hypothalamus of ovariectomized rats increase 3.1-fold after 2 weeks of estrogen replacement. In situ hybridization reveals that the estrogen-inducible enkephalinergic neurons are located in the ventrolateral aspect of the ventromedial nucleus, a subnucleus known to contain many estrogen-concentrating neurons. The increase in PE mRNA levels is due to both a 63% increase in the number of detectable PE mRNA-containing neurons and a 2.0-fold increase in the levels of PE mRNA per enkephalinergic neuron (1.63 x 2.0 = 3.3-fold overall induction). This estrogen-regulated enkephalinergic cell group may represent part of the neural network mediating estrogen's effects on reproductive behavior and/or other neuroendocrine processes.     

Estrogen interaction with the anterior pituitary of female rats differential cytosol binding, nuclear translocation and stimulation of RNA synthesis by 17β-estradiol and tamoxifen

The interaction of tamoxifen (trans-1-(p-β-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene) with the cytosol estrogen receptor of the anterior pituitary of female rats was studied. No differences were recorded between incubations of cytosol samples with 17β-[³H]estradiol performed in the presence or absence of unlabeled 17β-estradiol and tamoxifen, respectively, thus suggesting that these interactions were at common receptor sites and excluding possible cooperative interactions. Competition experiments and Scatchard plot analysis of saturation experiments add further evidence for common receptor sites. A dissociation constant for tamoxifen of K_d = 2 nM was recorded. Tamoxifen was found to be bound to a moiety sedimenting in the 4–5 S region, on a 6–24% linear sucrose density gradient at low salt concentrations, whereas 17β-estradiol sedimented in the 8–9 S area. These data suggest possible conformational changes of the receptor in the presence of tamoxifen. Furthermore, nuclear estrogen receptor levels remained elevated for at least 80 h after the application of tamoxifen alone or in a combination with 17β-estradiol, and a concomitant inhibition of cytosol receptor replenishment was noted. Tamoxifen and 17β-estradiol, respectively, were found to stimulate progesterone receptor levels when applied through 5 days. Tamoxifen plus 17β-estradiol administration elevated progesterone receptor contents above those found for each of the two compounds alone. On the other hand, tamoxifen enhanced the 17β-estradiol-induced prolactin serum levels, but did not stimulate prolactin serum levels by itself. These data combine to suggest that tamoxifen interacts with common estrogen receptor sites at the rat anterior pituitary.     

Glucose catabolic gene mRNA levels in skeletal muscle exhibit non-coordinate expression in hyperglycemic mice.

To assess the correlation between hyperglycemia and glucose catabolic gene levels in diabetic and healthy mice, we determined mRNA levels of pivotal proteins such as glucose transporters, hexokinase II, glycogen synthase, glutamine:fructose-6-phosphate amidotransferase and uncoupling proteins. Both KK and KKAy mice showed marked decreases of Glut1 and Glut4 mRNA levels in soleus compared to C57BL; db/db and ob/ob mice exhibited significantly decreased Glut4 mRNA levels, but not Glut1, in soleus. KK and KKAy mice showed a decrease of soleus HKII gene level, which may indicate decreased intracellular catabolism of glucose. Likewise, GS mRNA level was decreased in soleus muscle tissue in KK and KKAy mice. GFAT mRNA levels was no different between hyperglycemic and normoglycemic mice. In contrast, UCP2 and UCP3 mRNA levels were higher in KK and KKAy mice. Conversely, db/db and ob/ob mice showed a significant decrease in UCP3 mRNA. Individual correlation analysis indicated that the decrease in Glut4 gene levels was only observed in hyperglycemic mice. The more important observation is that the glucose catabolic genes do not exhibit any clear coordinate expression. Abnormal expression of glucose catabolic genes may contribute to hyperglycemia and muscle insulin resistance in these four strains.     

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.     

Interactions of tamoxifen in the chicken

The triphenylethylene antiestrogens are very potent antagonists of estrogen action in the chicken and manifest little agonist activity compared to their action in other species. The estrogen antagonism is most probably mediated by the estrogen receptor, to which tamoxifen binds with a Ki of 2.6 nM. Tamoxifen is readily metabolized by liver to 4-hydroxytamoxifen, which binds the liver nuclear estrogen receptor with a Ki of 0.1 nM. The Kd of the receptor is 0.7 nM. Estrogen receptor concentrations in liver from immature chickens are relatively low both in nuclear and cytosol fractions. Treatment with estradiol results in 10-fold up-regulation of the nuclear levels to give a total receptor concentration of about 2 pmol/g tissue. Tamoxifen can promote this up-regulation to a limited extent, but interpretation of experimental results is compromised by difficulties with exchange assays in the face of the very high binding affinity of 4-hydroxytamoxifen. Tamoxifen also binds with high affinity (Kd 2-4 nM) and distinctive specificity to antiestrogen binding sites (AEBS) present in a wide variety of chicken tissues and in the highest concentration in the liver (800 pmol/g tissue). Liver and serum contain ether-soluble components which can compete for binding of [3H]tamoxifen to the AEBS. The serum AEBS inhibitory activity is chromatographically heterogeneous and is associated with a sterol-like fraction as well as with a fatty-acid-containing fraction. Tamoxifen treatment of cockerels results in dose- and time-dependent decreases in serum free and esterified cholesterol, and in phospholipids and triglycerides. These changes may reflect estrogen-receptor-independent interactions of tamoxifen.