Stimulatory Effect Of Estradiol Research Articles

Fatty Acid Synthase Confers Tamoxifen Resistance to ER+/HER2+ Breast Cancer.

Simple SummaryOveractivation of the human epidermal growth factor receptor 2 (HER2) is one of the main drivers of tamoxifen resistance in estrogen receptor (ER)-positive breast cancer patients. Combined targeting of HER2 and ER, however, has yielded disappointing results in the clinical setting. Therefore, other potential mechanisms for tamoxifen resistance would not be overcome by solely blocking the cross-talk between ER and HER2 at the receptor(s) level. Using cell lines, animal models, and clinical data, we provide evidence to support a critical role of fatty acid synthase (FASN)—the major site for endogenous fat synthesis—in HER2-driven tamoxifen resistance. Importantly, treatment with a FASN inhibitor impeded the estrogen-like tumor-promoting effects of tamoxifen and fully restored the anti-estrogenic activity of tamoxifen in ER+/HER2-overexpressing breast cancer xenografts. We postulate FASN as a biological determinant of HER2-driven tamoxifen resistance and FASN inhibition as a novel therapeutic approach to restore tamoxifen sensitivity in endocrine-resistant breast cancer.The identification of clinically important molecular mechanisms driving endocrine resistance is a priority in estrogen receptor-positive (ER+) breast cancer. Although both genomic and non-genomic cross-talk between the ER and growth factor receptors such as human epidermal growth factor receptor 2 (HER2) has frequently been associated with both experimental and clinical endocrine therapy resistance, combined targeting of ER and HER2 has failed to improve overall survival in endocrine non-responsive disease. Herein, we questioned the role of fatty acid synthase (FASN), a lipogenic enzyme linked to HER2-driven breast cancer aggressiveness, in the development and maintenance of hormone-independent growth and resistance to anti-estrogens in ER/HER2-positive (ER+/HER2+) breast cancer. The stimulatory effects of estradiol on FASN gene promoter activity and protein expression were blunted by anti-estrogens in endocrine-responsive breast cancer cells. Conversely, an AKT/MAPK-related constitutive hyperactivation of FASN gene promoter activity was unaltered in response to estradiol in non-endocrine responsive ER+/HER2+ breast cancer cells, and could be further enhanced by tamoxifen. Pharmacological blockade with structurally and mechanistically unrelated FASN inhibitors fully impeded the strong stimulatory activity of tamoxifen on the soft-agar colony forming capacity—an in vitro metric of tumorigenicity—of ER+/HER2+ breast cancer cells. In vivo treatment with a FASN inhibitor completely prevented the agonistic tumor-promoting activity of tamoxifen and fully restored its estrogen antagonist properties against ER/HER2-positive xenograft tumors in mice. Functional cancer proteomic data from The Cancer Proteome Atlas (TCPA) revealed that the ER+/HER2+ subtype was the highest FASN protein expressor compared to basal-like, HER2-enriched, and ER+/HER2-negative breast cancer groups. FASN is a biological determinant of HER2-driven endocrine resistance in ER+ breast cancer. Next-generation, clinical-grade FASN inhibitors may be therapeutically relevant to countering resistance to tamoxifen in FASN-overexpressing ER+/HER2+ breast carcinomas.

Estradiol Upregulates c-FLIPlong Expression in Anterior Pituitary Cells

Anterior pituitary cell turnover depends on a tight balance between proliferation and apoptosis. We have previously shown that estrogens sensitize anterior pituitary cells to pro-apoptotic stimuli. c-FLIP (cellular-FLICE-inhibitory-protein) isoforms are regulatory proteins of apoptosis triggered by death receptors. c-FLIPshort isoform competes with procaspase-8 inhibiting its activation. However, c-FLIPlong isoform may have a pro- or anti-apoptotic function depending on its expression level. In the present study, we explored whether estrogens modulate c-FLIP expression in anterior pituitary cells from ovariectomized (OVX) rats and in GH3 cells, a somatolactotrope cell line. Acute administration of 17β-estradiol to OVX rats increased c-FLIPlong expression in the anterior pituitary gland without changing c-FLIPshort expression as assessed by Western blot. Estradiol in vitro also increased c-FLIPlong expression in anterior pituitary cells but not in GH3 cells. As determined by flow cytometry, the percentage of anterior pituitary cells expressing c-FLIP was higher than in GH3 cells. However, c-FLIP fluorescence intensity in GH3 cells was higher than in anterior pituitary cells. FasL increased the percentage of TUNEL-positive GH3 cells incubated either with or without estradiol suggesting that the pro-apoptotic action of Fas activation is estrogen-independent. Our results show that unlike what happens in nontumoral pituitary cells, estrogens do not modulate either c-FLIPlong expression or FasL-induced apoptosis in GH3 cells. The stimulatory effect of estradiol on c-FLIPlong expression could be involved in the sensitizing effect of this steroid to apoptosis in anterior pituitary cells. The absence of this estrogenic action in tumor pituitary cells could be involved in their tumor-like behavior.

A Multi-Oscillatory Circadian System Times Female Reproduction

Rhythms in female reproduction are critical to insure that timing of ovulation coincides with oocyte maturation and optimal sexual arousal. This fine tuning of female reproduction involves both the estradiol feedback as an indicator of oocyte maturation, and the master circadian clock of the suprachiasmatic nuclei (SCN) as an indicator of the time of the day. Herein, we are providing an overview of the state of knowledge regarding the differential inhibitory and stimulatory effects of estradiol at different stages of the reproductive axis, and the mechanisms through which the two main neurotransmitters of the SCN, arginine vasopressin, and vasoactive intestinal peptide, convey daily time cues to the reproductive axis. In addition, we will report the most recent findings on the putative functions of peripheral clocks located throughout the reproductive axis [kisspeptin (Kp) neurons, gonadotropin-releasing hormone neurons, gonadotropic cells, the ovary, and the uterus]. This review will point to the critical position of the Kp neurons of the anteroventral periventricular nucleus, which integrate both the stimulatory estradiol signal, and the daily arginine vasopressinergic signal, while displaying a circadian clock. Finally, given the critical role of the light/dark cycle in the synchronization of female reproduction, we will discuss the impact of circadian disruptions observed during shift-work conditions on female reproductive performance and fertility in both animal model and humans.

Abstract 5578: Breast cancer cell growth regulatory mechanisms of tamoxifen metabolites

Abstract The proliferation of estrogen receptor alpha (ERalpha)-positive breast cancer cells is stimulated by estradiol. Tamoxifen [(Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine] is the most commonly used endocrine treatment for ERalpha-positive breast cancer in pre- and post-menopausal women. Tamoxifen is metabolized to 4-hydroxytamoxifen and endoxifen. ICI 182780 (Faslodex or Fulvestrant) is a pure antiestrogen targeted to the ERalpha. We investigated the effects of endoxifen and 4-hydroxytamoxifen on ERalpha-positive MCF-7 breast cancer cells using cell growth assay, oncogene expression and polyamine regulatory pathways. Polyamines are ubiquitous cellular components and are essential for cell growth and differentiation. Treatment of cells for 1-5 days with 4 nM estradiol increased cells number, whereas the addition of endoxifen, 4-hydroxytamoxifen or ICI 182780 in combination with estradiol suppressed cell growth in a concentration- and time-dependent manner. Real time quantitative polymerase chain reaction (RT-qPCR) experiments showed that the addition of 4 nM estradiol to MCF-7 cells increased the expression of c-myc, c-fos and Tiff1 genes significantly, while the addition endoxifen, 4-hydroxytamoxifen, or ICI 182780 suppressed estradiol-mediated increase in gene expression. Polyamine biosynthetic enzymes were also affected by tamoxifen metabolites. Estradiol increased the activities of the polyamine biosynthetic enzyme, ornithine decarboxlase (ODC) and S-adenosylmethionine decarboxlase (AdoMetDC), whereas the antiestrogens suppressed ODC and AdoMetDC activities in a concentration- and time-dependent manner. However, these agents had little effect on the activity of the polyamine catabolic enzyme, spermidine/spermine N1-acetyl transferase (SSAT). The change in biosynthetic enzyme activities was reflected in polyamine pools, as decreased putrescine and spermidine levels. There was no significant change in spermine level. These results suggest that the mechanism of action of tamoxifen metabolites involves multiple cell growth regulatory pathways. In addition, endoxifen can act as an antiestrogen and suppress cell growth stimulatory effects of estradiol on ERalpha-positive breast cancer cells in a manner similar to that of ICI 182780. Note: This abstract was not presented at the meeting. Citation Format: T. Thomas, Huichen Hsu, PingAr Yang, Mervi T. Hyvonen, Tuomo A. Keinanen. Breast cancer cell growth regulatory mechanisms of tamoxifen metabolites. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5578. doi:10.1158/1538-7445.AM2014-5578

Estradiol and Testosterone Regulate Arginine-Vasopressin Expression in SH-SY5Y Human Female Neuroblastoma Cells Through Estrogen Receptors-α and -β

The expression of arginine-vasopressin (AVP) is regulated by estradiol and testosterone (T) in different neuronal populations by mechanisms that are not yet fully understood. Estrogen receptors (ERs) have been shown to participate in the regulation of AVP neurons by estradiol. In addition, there is evidence of the participation of ERβ in the regulation of AVP expression exerted by T via its metabolite 5α-dihydrotestosterone (5α-DHT) and its further conversion in the androgen metabolite and ERβ ligand 3β-diol. In this study we have explored the role of ERs in the regulation exerted by estradiol and T on AVP expression, using the human neuroblastoma cell line SH-SY5Y. Estradiol treatment increased AVP mRNA levels in SH-SY5Y cells in comparison with cells treated with vehicle. The stimulatory effect of estradiol on AVP expression was imitated by the ERα agonist 4,4',4',-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol and blocked by the ER antagonist, ICI 182,780, and the ERα antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1hpyrazoledihydrochloride. In contrast, the ERβ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile reduced AVP expression, whereas the ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol enhanced the action of estradiol on AVP expression. T increased AVP expression in SH-SY5Y cells by a mechanism that was dependent on aromatase but not on 5α-reductase activity. The T effect was not affected by blocking the androgen receptor, was not imitated by the T metabolite 5α-DHT, and was blocked by the ERα antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1hpyrazoledihydrochloride. In contrast, 5α-DHT had a similar effect as the ERβ agonists 2,3-bis(4-hydroxyphenyl)-propionitrile and 3β-diol, reducing AVP expression. These findings suggest that estradiol and T regulate AVP expression in SH-SY5Y cells through ERs, exerting a stimulatory action via ERα and an inhibitory action via ERβ.

Abstract 534: GPR30 Stimulates Capillary Formation by Human Endothelial Progenitor Cells

Estradiol (E2) induces vascular repair by promoting endothelial progenitor cell (EPC) growth and capillary formation. Based on our previous findings that the capillary stimulating effects of E2 are mimicked by its non-permeable analog, BSA-tagged E2, we hypothesize that the stimulatory effects are potentially mediated via the newly discovered membrane bound estrogen receptor (ER) GPR30. To investigate this, we assessed capillary formation by EPC derived endothelial cells in response to E2, and GPR30 agonists (G1) and antagonist (G15) in a matrigel based assay. Capillary formation was assessed microscopically by quantifying junction/sprout formation and capillary length. E2 (10nM) increased EPC-induced capillary formation by 126 ± 5% and these effects were mimicked by G1 (stimulated by 133 ± 8.9% and 210 ± 7.2% by 1 and 10 nM, respectively) and propyl-pyrazole-triol [PPT; stimulated by 143 ± 9%; 100 nM; ER-α agonist], but not by diarylpropionitrile (ER-β agonist). The stimulatory effects of estradiol, G1 and PPT were significantly (p<.05) abrogated by G15 (20nM), a GPR30 antagonist, suggesting a role for GPR30 in mediating the capillary stimulatory effects of E2. Interestingly, the stimulatory of estradiol, PPT and G1 were also abrogated by methyl-piperidino-pyrazole (ER-α antagonist), implying that the stimulatory effects of E2 are mediated via cross-talk between GPR30 and membrane bound ER-α. Because, receptor tyrosine kinase (RTK), hemeoxygenase (HO), and G-protein coupled mechanisms are involved in capillary formation we investigated their role in GPR30 induced capillary formation. The stimulatory effects of G1 were blocked by SU5416 (RTK inhibitor), ZnPP (HO inhibitor), pertusis toxin (G protein pathway inhibitor), and LY294002 (Akt pathway inhibitor). Moreover, treatment of EPCs with G1 induced expression of phosphorylated-Akt and HO-1, but not MAPK. We conclude that E2 via GPR30 or GPR30-coupled ER-α promotes EPC-mediated capillary formation by a mechanism that involves non-genomic activation of RTKs and HO-1 activation. E2 in particular and GPR30 agonists in general may promote healing of injured vascular beds by promoting EPC activity leading to more rapid endothelial recovery and capillary formation following injury.

Phytoestrogen Effects on Vaginal Microbiocenosis of Sprague–Dawley Rats

Phytoestrogens present in the plants endemic for Chile were studied. The effects of phytoextract [specimen of preparation No. 181 (fraction b)] on target tissues were similar to those of estradiol. The preparation inhibited the stimulatory effect of estradiol on vaginal lactobacilli population.

Resveratrol, a Red Wine Constituent, Blocks the Antimitogenic Effects of Estradiol on Human Female Coronary Artery Smooth Muscle Cells

Antimitogenic effects of estradiol on vascular smooth muscle cells (VSMCs) may be cardioprotective, and these effects are mediated by estrogen receptor-alpha-dependent and -independent mechanisms, with the latter involving the conversion of estradiol to 2-hydroxyestradiol/2-methoxyestradiol by CYP450. Because resveratrol inhibits CYP450 and is an estrogen-receptor-alpha antagonist, resveratrol may abrogate the antimitogenic effects of estradiol. The objective of the study was to examine the interaction of pharmacologically relevant concentrations of resveratrol with estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol in human female coronary artery VSMCs. In human female coronary VSMCs, resveratrol (0.1-10 microm) alone did not influence serum-induced DNA or collagen synthesis or cell proliferation or migration; however, resveratrol abrogated the inhibitory effects of estradiol, but not 2-hydroxyestradiol or 2-methoxyestradiol, on these responses. Resveratrol also abrogated the inhibitory effects of estradiol on positive growth regulators (cyclin A, cyclin D, MAPK phosphorylation) and the stimulatory effects of estradiol on negative growth regulators (p21, p27). In microsomes and cells, dietarily relevant levels of resveratrol (0.001-1 microm) inhibited the metabolism of estradiol to 2-hydroxestradiol/2-methoxyestradiol. Propylpyrazoletriol (estrogen receptor-alpha agonist, 100 nmol/liter), but not diarylpropionitrile (estrogen receptor-beta agonist, 10 nmol/liter), inhibited VSMC mitogenesis, and this effect was blocked by resveratrol (5 micromol/liter). Higher concentrations (>25-50 microm) of resveratrol, never attainable in vivo, inhibited VSMC growth, an effect blocked by GW9662 (peroxisomal proliferator-activated receptor-gamma antagonist). In conclusion, dietarily relevant levels of resveratrol abrogate the antimitogenic effects of estradiol by inhibiting CYP450-mediated estradiol metabolism and blocking estrogen receptor-alpha.

Abstract 4603: Role of endoxifen as an antiestrogen: Effects on oncogene expression and polyamine pathway

Abstract The nonsteroid antiestrogen tamoxifen [trans-1-(4-b-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene] is the most commonly used endocrine treatment for estrogen receptor α (ERα)-positive breast cancer in pre- and post-menopausal women, and it has helped to reduce breast cancer death rate by one third. Tamoxifen is extensively metabolized, and several metabolites have been detected in human serum. It is metabolized to 4-hydroxytamoxifen and N-desmethyltamoxifen by the action of CYP2D6 and CYP3A4/5 enzymes, respectively. N-desmethyltamoxifen and 4-hydroxy-tamoxifen are further converted to endoxifen by the action of these enzymes. Steady state levels of serum tamoxifen/metabolite are achieved within 4 weeks of continuous administration of the drug. The therapeutic efficacy of tamoxifen is determined by the distribution of the drug into tissues and the availability of the parent drug and its active metabolites in target tissues. Recent reports indicate that endoxifen might act as an antiestrogen. In order to examine the mechanism of action of endoxifen on an ERα-positive breast cancer cell line, MCF-7, we determined the effects of 100, 250, 500 and 1000 nM concentrations of this agent on cell growth, c-myc oncogene expression and activity of the polyamine biosynthetic enzymes. MCF-7 breast cancer cells were treated for 1, 3 or 5 days with endoxifen. Endoxifen had a growth inhibitory effect on MCF-7 cells by day 3 and 5 of treatment, as measured by cell number. Estradiol increased cell number by >2-fold compared to untreated control by day 5 of treatment, whereas endoxifen inhibited the estrogenic effect. c-myc gene expression was increased by a significant 2- to 3-fold by 4 nM estradiol in 2 and 4 hours of treatment, while 250 and 500 nM endoxifen suppressed the increase in c-myc gene expression, as measured by real-time qPCR experiment. In the absence of estradiol, endoxifen slightly (1.5 fold) increased c-myc gene expression. Endoxifen decreased ornithine decarboxylase (ODC) and S-adenosine methyl decarboxylase (AdoMetDC) activities in a concentration- and time-dependent manner; however, it had little effect on spermidine/spermine N1-acetyl transferase (SSAT) activity. The change in biosynthetic enzyme activities were reflected in polyamine pools, as decreased putrescine and spermidine levels. There were no changes in spermine level. As expected, treatment of the cells with 4 nM estradiol induced ODC and AdoMetDC activities, and slightly decreased SSAT activity. Addition of endoxifen in combination with 4 nM estradiol led to even more dramatic decrease in ODC and AdoMetDC activities and reduction of putrescine and spermidine levels. These results support our hypothesis that endoxifen can act as an antiestrogen and suppress cell growth stimulatory effects of estradiol in ERα-positive breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4603.

Is the Stimulatory Effect of Estradiol on Prolactin Secretion Mediated by a Change in Dopaminergic Input to the Pituitary?

Is the Stimulatory Effect of Estradiol on Prolactin Secretion Mediated by a Change in Dopaminergic Input to the Pituitary?

Regulation of Msx2 Gene Expression by Steroid Hormones in Human Nonmalignant and Malignant Breast Cancer Explants Cultured in Vitro

Muscle segment homeobox genes, which regulate developmental programs and are expressed in embryonic and adult tissue, play a role in development of some malignancies. There are no reports on the expression of these families of genes in breast cancer tissue. The aim of this study was to compare expression of Msx2 gene in breast cancer of different genotypes as well as in surrounding nonmalignant tissues. Explants obtained during surgery were divided according to their sex steroid receptor status determined by immunocytochemistry. Four explants obtained from malignant and nonmalignant tissue of each individual patient were incubated in a control medium or with the addition of progesterone (10− 7 M) alone, estradiol 17 β (10− 5 M) or both. The relative level of Msx2 transcripts was evaluated by a semiquantitative RT-PCR and cell proliferation by Alamar Blue test. Results of RT-PCR analysis showed that the relative expression of Msx2 gene depended on the presence of ER/PR receptors both in nonmalignant and malignant tissues Relative amount of Msx2 mRNA was the highest in surrounding nonmalignant ER + /PR − and ER − /PR + tissue, whereas in ER − /PR − and ER + /PR + tissue it was 1.4–1.6-fold lower. Tumorigenesis led to about a twofold decrease in the relative amount of Msx2 mRNA except for ER + /PR + immunophenotype, where no changes were observed. Addition of estradiol or progesterone to the culture of ER − /PR − type tissue explants did not change significantly the relative amount of Msx2 gene mRNA. An opposite effect was observed in ER + /PR − type of tissue. Addition of estradiol alone, or estradiol and progesterone together to tissue culture explants decreased two to three fold the relative amount of Msx2 gene mRNA in both, malignant and surrounding tissues. Progesterone alone had no effect on Msx2 gene expression in this type of tissue. The most complicated regulation was observed in ER + /PR + type of tissue. Culture of tissue explants supplemented with estradiol significantly increased the relative amount of Msx2 gene mRNA in the surrounding tissue. Progesterone enhanced the stimulatory effect of estradiol in surrounding tissues but not in the malignant tissue. Increased expression of Msx2 correlated with an increased proliferation in ER − /PR − and ER + /PR + types, but not in ER + /PR − type of tissues. In conclusion, obtained results provide evidence that estrogen affects Msx2 gene expression. Significant changes in the relative amount of Msx2 gene mRNA and lack of canonical ERE element in 5′-upstream sequence of this gene suggest that regulation takes place indirectly probably by protein-protein interaction. The decrease in the relative amount of Msx2 gene mRNA in ER + /PR − type tumor suggests that progesterone also affects Msx2 gene expression by an indirect mechanism(s).

The combination of a novel selective estrogen receptor modulator with an estrogen protects the mammary gland and uterus in a rodent model: the future of postmenopausal women's health?

The Women's Health Initiative Study and other reports have created major uncertainty among postmenopausal women and physicians concerning hormone replacement therapy. We have thus investigated the possibility of replacing the progestin in hormone replacement therapy by a novel selective estrogen receptor (ER) modulator having potent and pure antiestrogenic activity in the mammary gland and uterus. As measured by changes in histology and Cdc47 labeling in the rat model, the present study shows that the stimulatory effect of estradiol in the mammary gland and uterus is efficiently blocked by simultaneous administration of the novel selective ER modulator EM-652, but bone mineral density is preserved and serum cholesterol is decreased. After the administration of 14C-labeled EM-652, we observed that there is no detectable radioactivity in the brain. Moreover, ER alpha immunoreactivity remained constant in the hypothalamus after EM-652 treatment, whereas ER alpha became almost undetectable in the mammary gland and uterus. The present data show the poor or absent access of EM-652 to the brain, whereas the effects of estrogens are efficiently neutralized in the mammary gland and uterus. Such data support the exciting possibility of a novel approach that could meet most of the needs of women's health at menopause, namely control of hot flushes and prevention of breast, uterine, and ovarian cancer as well as osteoporosis and potentially helping brain function and preventing Alzheimer's disease with no identifiable risk or negative effect.

Oestradiol regulation of antigen presentation by uterine stromal cells: role of transforming growth factor-beta production by epithelial cells in mediating antigen-presenting cell function.

We have previously shown that oestradiol treatment of ovariectomized rats for 3 days inhibits antigen presentation by uterine stromal cells at a time when oestradiol increases the numbers of antigen-presenting cells (APC) in the uterine stroma. In the present study, we found that oestradiol treatment for 1 day is sufficient to inhibit antigen presentation by stromal cells. To define the mechanism(s) of this inhibition, we examined the effect of cytokines and found that exogenous transforming growth factor-beta (TGF-beta) inhibits antigen presentation when stromal cells from saline- but not oestradiol-treated animals are incubated with ovalbumin (OVA)-specific T cells and OVA. In contrast, antigen presentation by uterine epithelial cells was not affected by TGF-beta. In other studies, the acute inhibitory effect of oestradiol (1 day) on stromal antigen presentation is fully reversed when anti-TGF-beta antibody is added to the culture media. When given for 3 days, oestradiol inhibition of antigen presentation is partially reversed by anti-TGF-beta antibody at a time when antibodies to tumour necrosis factor-alpha and interleukin-10 have no effect. To determine whether uterine epithelial cells produce TGF-beta, epithelial cells were grown to confluence on transwell inserts. Our findings indicate that uterine epithelial cells produce biologically active TGF-beta which is preferentially released basolaterally in the direction of underlying stromal cells. When oestradiol is given to ovariectomized rats 1 day before sacrifice, TGF-beta production by epithelial cells increases within 24 hr in culture, relative to saline controls. Taken together, these results suggest that oestradiol inhibition of stromal cell antigen presentation is mediated through the stimulatory effect of oestradiol on TGF-beta production by epithelial cells.

Estrogen receptor, cyclic adenosine monophosphate, and protein kinase A are involved in the nongenomic pathway by which estradiol accelerates oviductal oocyte transport in cyclic rats.

This investigation examined the role of estrogen receptor (ER) on the stimulatory effect of estradiol (E2) on protein phosphorylation in the oviduct as well as on E2-induced acceleration of oviductal oocyte transport in cyclic rats. Estrous rats were injected with E2 s.c. and with the ER antagonist ICI 182 780 intrabursally (i.b.), and 6 h later, oviducts were excised and protein phosphorylation was determined by Western blot analysis. ICI 182 780 inhibited the E2-induced phosphorylation of some oviductal proteins. Other estrous rats were treated with E2 s.c. and ICI 182 780 i.b. The number of eggs in the oviduct, assessed 24 h later, showed that ICI 182 780 blocked the E2-induced egg transport acceleration. The possible involvement of adenylyl cyclase, protein kinase A (PK-A), protein kinase C (PK-C), or tyrosine kinases on egg transport acceleration induced by E2 was then examined. Selective inhibitors of adenylyl cyclase or PK-A inhibited the E2-induced egg transport acceleration, whereas PK-C or tyrosine kinase inhibitors had no effect. Furthermore, forskolin, an adenylyl cyclase activator, mimicked the effect of E2 on ovum transport and E2 increased the level of cAMP in the oviduct of cycling rats. Finally, we measured PK-A activity in vitro in the presence of E2 or E2-ER complex. Activity of PK-A in the presence of E2 or E2-ER was similar to PK-A alone, showing that E2 or E2-ER did not directly activate PK-A. We conclude that the nongenomic pathway by which E2 accelerates oviductal egg transport in the rat requires absolute participation of ER and cAMP and partial participation of PK-A signaling pathways in the oviduct.

Interactions of ovarian steroids with pituitary adenylate cyclase-activating polypeptide and GnRH in anterior pituitary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) releases LH and FSH from anterior pituitary cells. Although this effect is relatively weak, it has a strong sensitizing action on GnRH-induced gonadotropin secretion. Here we investigated the possibility that ovarian steroids, which are well-known modulators of LH secretion, interact with PACAP and GnRH in pituitary gonadotrophs. Rat pituitary cells were treated for 48 h with vehicle, 1 nmol/l estradiol, 1 nmol/l estradiol + 100 nmol/l progesterone or 48 h with 1 nmol/l estradiol and 4 h with 100 nmol/l progesterone. The cells were stimulated for 3 h with 1 nmol/l GnRH or 100 nmol/l PACAP. Estradiol treatment alone enhanced basal as well as GnRH- or PACAP-stimulated LH secretion. LH release was facilitated by additional short-term progesterone treatment. Long-term treatment with estradiol and progesterone led to reduced LH responses to GnRH and PACAP. Neither treatment paradigms affected cAMP production. However, estradiol treatment led to enhanced cAMP accumulation in quiescent or GnRH-stimulated cells. PACAP-induced increases of cAMP production were inhibited by estradiol treatment. After 7-h preincubation with 10 nmol/l PACAP, cells responded with enhanced LH secretion to GnRH stimulation. When steroid pretreatment was performed the responsiveness of gonadotrophs to low concentrations of GnRH was still increased. In contrast, at high concentrations of GnRH the sensitizing action of PACAP on agonist-induced LH secretion was lost in steroid-treated cells. There were no significant differences between the steroid treatment paradigms. It is concluded that estradiol but not progesterone acts as a modulator of adenylyl cyclase in gonadotrophs. The stimulatory effect of estradiol is thought to be involved in its sensitizing action on agonist-induced LH secretion. The inhibitory effect of estradiol on PACAP-stimulated adenylyl cyclase activities seems to be responsible for the loss of its action to sensitize LH secretory responses to GnRH.

Almost exclusive androgenic action of dehydroepiandrosterone in the rat mammary gland.

To determine the relative role of the androgenic and/or estrogenic components of the action of dehydroepiandrosterone (DHEA) on the histomorphology and structure of the rat mammary gland, ovariectomized (OVX) female animals received DHEA administered alone or in combination with the pure antiandrogen flutamide or the pure antiestrogen EM-800 for 12 months. We have also evaluated the effect of estradiol (E2) and dihydrotestosterone constantly released from SILASTIC brand silicon implants as well as medroxyprogesterone acetate released from poly(lactide-co-glycolide) microspheres. While 1-yr OVX resulted in a severe atrophy of the mammary gland, treatment of OVX animals with DHEA stimulated lobuloalveolar and ductal growth, as well as the secretory activity of the acinar cells, thus resulting in a lobuloalveolar type of development of the mammary gland. The addition of FLU to DHEA almost completely prevented the stimulatory effect observed with DHEA alone, whereas addition of the antiestrogen EM-800 had no significant effect on the action of DHEA on the mammary gland. At the doses used, medroxyprogesterone acetate and dihydrotestosterone also stimulated ductal and alveolar development, although to a lesser degree than that achieved with DHEA. The stimulatory effect of estradiol was mainly expressed on ductal growth with a smaller stimulatory effect on lobuloalveolar development. The above-indicated stimulatory effects on lobuloalveolar development were also reflected in significant increases of the total and parenchymal gland surface areas of the mammary gland. The present study shows that androgens induce a marked lobuloalveolar type of development of the mammary gland in the rat. Moreover, these data indicate the highly predominant or almost exclusive androgenic component in the potent stimulatory action of DHEA on the histomorphology and structure of the rat mammary gland. In fact, blockade of the potential estrogenic component of DHEA action by EM-800 did not affect the stimulatory action of DHEA on mammary gland histomorphology, whereas the antiandrogen FLU almost completely blocked the effect of DHEA.

Almost Exclusive Androgenic Action of Dehydroepiandrosterone in the Rat Mammary Gland

To determine the relative role of the androgenic and/or estrogenic components of the action of dehydroepiandrosterone (DHEA) on the histomorphology and structure of the rat mammary gland, ovariectomized (OVX) female animals received DHEA administered alone or in combination with the pure antiandrogen flutamide or the pure antiestrogen EM-800 for 12 months. We have also evaluated the effect of estradiol (E2) and dihydrotestosterone constantly released from SILASTIC brand silicon implants as well as medroxyprogesterone acetate released from poly(lactide-co-glycolide) microspheres. While 1-yr OVX resulted in a severe atrophy of the mammary gland, treatment of OVX animals with DHEA stimulated lobuloalveolar and ductal growth, as well as the secretory activity of the acinar cells, thus resulting in a lobuloalveolar type of development of the mammary gland. The addition of FLU to DHEA almost completely prevented the stimulatory effect observed with DHEA alone, whereas addition of the antiestrogen EM-800 had no significant effect on the action of DHEA on the mammary gland. At the doses used, medroxyprogesterone acetate and dihydrotestosterone also stimulated ductal and alveolar development, although to a lesser degree than that achieved with DHEA. The stimulatory effect of estradiol was mainly expressed on ductal growth with a smaller stimulatory effect on lobuloalveolar development. The above-indicated stimulatory effects on lobuloalveolar development were also reflected in significant increases of the total and parenchymal gland surface areas of the mammary gland. The present study shows that androgens induce a marked lobuloalveolar type of development of the mammary gland in the rat. Moreover, these data indicate the highly predominant or almost exclusive androgenic component in the potent stimulatory action of DHEA on the histomorphology and structure of the rat mammary gland. In fact, blockade of the potential estrogenic component of DHEA action by EM-800 did not affect the stimulatory action of DHEA on mammary gland histomorphology, whereas the antiandrogen FLU almost completely blocked the effect of DHEA.

Estrogen enhances growth hormone receptor expression and growth hormone action in rat osteosarcoma cells and human osteoblast-like cells.

Postmenopausal bone loss is primarily due to estrogen deficiency. Recent clinical observation demonstrate that GH increases bone mass in GH deficient patients. The present study investigates whether estrogen regulates GH action and GH receptor expression in osteoblasts. 17 beta-estradiol or GH added to the culture medium as single substances did not influence rat osteosarcoma cell proliferation nor human osteoblast-like (hOB) cell proliferation. However, together they synergistically induced osteoblast proliferation (rat osteosarcoma cells 160.1 +/- 15.5% of control cells; human osteoblast-like cells 159.6 +/- 5.1% of control cells). 17 beta-estradiol stimulated 125I-GH binding and GH receptor (GHR) mRNA levels in rat osteosarcoma cells. The stimulatory effect of estradiol was time dependent, reaching a peak after 8 h of incubation with 17 beta-estradiol (binding 216.9 +/- 27.8% and mRNA 374.6 +/- 30.8% of control). The finding that estradiol stimulated 125I-GH binding was confirmed in human osteoblast-like cells. In these cells, 17 beta-estradiol (10(-12) M) increased 125I-GH binding to 203.8 +/- 3.6% of control levels. We conclude that estrogen stimulates GH activity as well as GH binding and GHR mRNA levels in osteoblasts. These findings indicate that estrogen potentiates the effect of GH at the receptor level.

Age-related changes in axonal transport.

In rats the rate of axonal transport (AT) or radiolabeled material decreased in the ventral roots of the spinal cord and the vagal and hypoglossal nerves with aging. A maximum AT deceleration in old age was observed in the vagus. The uncoupling of oxidative phosphorylation, inhibition of glycolysis and hypoxia induced a greater AT deceleration in old rats as compared to adults. Small doses of sodium fluoride accelerated AT, and this correlated with a rise in cAMP levels in ventral roots. High doses of sodium fluoride decelerated AT more markedly in old rats. It was shown that anabolic hormones (sex steroids and thyroxine) accelerated AT in both adult and old rats, whereas insulin induced a rise in AT rate in only adults. The catabolic steroid, hydrocortisone decelerated AT. In old rats castration diminished AT, while thyroidectomy had no effect. It was also shown that hydrocortisone and testosterone were transported along axons, reached fibers of the skeletal muscles, and hyperpolarized the plasma membrane. In old age the latent period was extended. Following 73 to 74 days of irradiation, AT slowed down in all the nerves studied in both adult and old rats. Following irradiation hormonal effects on AT changed, for example, the stimulatory effect of estradiol became weak, especially in old rats. Changes in AT could be an important mechanism of disordering the growth of neurons and innervated cells in old age.

Functional heterogeneity with respect to oestrogen treatment in prolactin cell subpopulations separated by Percoll gradient centrifugation

The effects of oestradiol on prolactin gene expression were studied by quantitative in situ hybridization histochemistry in different prolactin pituitary cell (sub)populations, which had been obtained by separation on a discontinuous Percoll gradient. When cells were incubated in vitro in the presence of oestradiol (10(-8) M) for a period of 4, 24, 48 and 72 h, there was an increase in the amount of prolactin mRNA, from 24 h on, only in high-density prolactin cells and lactotrophs of the total cell suspension. In contrast, the amount of prolactin mRNA in lactotrophs of low density did not change upon treatment with oestradiol. Pharmacological treatment with 50 micrograms oestradiol/day (s.c.) of random cycling female rats in vivo for 14 days increased the total number of prolactin gene-expressing cells and more lactotrophs were recovered at high density after Percoll gradient centrifugation. These results suggest a preferential stimulatory effect of oestradiol on prolactin gene transcription on a subpopulation of lactotrophs. Changes observed in prolactin cell layers after oestradiol treatment in vivo may represent a preferential effect in situ on a particular mammotroph cell subpopulation.