Differential Effects Of Estradiol Research Articles

Differential effects of estradiol on neural and emotional stress response in postmenopausal women with remitted Major Depressive Disorder

BackgroundEstrogen fluctuations throughout the lifespan may contribute to major depressive disorder (MDD) risk in women through effects on brain networks important in stress responding, and mood regulation. Although there is evidence to support ovarian hormone treatment for peri-menopausal depression, postmenopausal use has not been well examined. The objective of this study was to investigate whether estrogen modulation of the neural and emotional cognitive responses to stress differs between postmenopausal women with and without MDD history. Methods60 postmenopausal women completed an fMRI psychosocial stress task, after receiving no drug or 3 months of daily estradiol (E2). fMRI activity and subjective mood response were examined. ResultsIn women without a history of MDD, E2 was associated with a more negative mood response to stress and less activity in emotional regulation regions. In women with a history of MDD, E2 was associated with a less negative mood response to stress and less activity in emotion perception regions. LimitationsThis study was limited by open-label estradiol administration and inclusion of participants using antidepressants. ConclusionsThese results support a differential effect of estrogen on emotional and neural responses to psychosocial stress in postmenopausal women with MDD history and may reflect a shift in brain activity patterns related to emotion processing following menopause.

Differential effects of estradiol on neural and emotional stress response in postmenopausal women with remitted Major Depressive Disorder

<h3>Introduction</h3> Ovarian hormones can influence neural systems involved in mood regulation, suggesting a pathway explaining why women have a greater incidence of Major Depressive Disorder (MDD) than men primarily beginning at puberty. Estrogen fluctuations may contribute to MDD risk through effects on brain networks important in emotion processing, stress responding, and mood regulation. Although there is evidence to support the use of ovarian hormone treatment for peri-menopausal depression, postmenopausal use has not been as well examined. As the neural systems involved in emotional processing and stress response are altered in MDD, it is possible that the effect of estrogen treatment on these systems may differ between postmenopausal women with and without a history of MDD. The objective of this study was to investigate whether estrogen modulation of the neural and emotional cognitive responses to stress differs between postmenopausal women with and without MDD vulnerability. <h3>Methods</h3> 60 postmenopausal women completed this fMRI study including a psychosocial stress task, the Montreal Imaging Stress Task (MIST), after receiving no drug or 3 months of daily 2mg 17β estradiol (E2). Participants included women with no personal history of MDD by SCID interview and women with remitted MDD with at least one episode in the last ten years and no depressive episodes within the last year. There were 4 participant groups: 1) MDD+/E2+ (n=10), 2) MDD+/E2- (n=12), 3) MDD-/E2= (n=20), 4) MDD-/E2- (n=18). All groups were euthymic at the time of study with non-clinical scores on the Beck Depression and Anxiety Inventories. Subjective mood response to the MIST was assessed as change score on the Stress-Arousal Checklist (SACL stress) before and after the MIST. fMRI analyses examined activity differences between the "stress and "control" conditions of the MIST using SPM 12. Second level analyses included 2 × 2 interactions (MDD history X E2 administration), whole-brain (gray matter masked), and cluster threshold corrected for multiple comparisons (p<0.005, K >102, FDR<0.05) for differences in activity during the MIST between the 4 groups and the relationship between activity during the MIST and SACL stress score in each group. <h3>Results</h3> E2 administration was associated with different effects in post-menopausal women with or without a history of MDD. In accordance with our previous work, the MDD-/E2+ group had a more negative mood response (higher SACL stress score) to the stress task than the MDD-/E2- group. In contrast, this effect was reversed in women with a history of MDD, where the MDD+/E2+ group had a less negative mood response than the MDD+/E2- group. Additionally, both groups who exhibited more negative mood responses also showed greater activity in emotional perception areas (left inferior frontal and left angular gyri) during the stress condition of the task than the groups which showed less negative mood responses. Group differences were also observed on fMRI. Women who did not receive E2 showed a positive correlation between greater right inferior frontal activity and higher SACL stress score, while the MDD+/E2- group additionally showed a positive correlation between right insula activity and SACL score. The MDD+/E2+ group showed negative correlations between activity in each area and SACL stress score in both the "stress" and "control" conditions of the MIST, while the MDD-/E2+ did not show significant correlations between activity in any area and SACL stress Score. <h3>Conclusions</h3> These results support a differential effect of estrogen administration on emotional and neural responses to psychosocial stress in postmenopausal women with and without a history of MDD. We had previously show that in women without a history of MDD, E2 is associated with a more negative mood response to stress than placebo. The results of this study replicated those findings in women without a history of MDD and show that E2 is also associated with increased activity in brain areas involved in emotion perception and response. Additionally, the result of this study support that the effect of E2 is reversed in women with a history of MDD, such that E2 is associated with a less negative mood response to stress and a differential relationship between activity in brain areas involved in emotion response. This suggests that women with a history of MDD, even while currently euthymic, have both a different mood and neural response to psychosocial stress and may show a beneficial mood effect E2 that is not seen in women without a history of MDD. These results may provide a neurological basis for recurrence risk and the beneficial mood effects of post-menopausal hormone treatment in women with a history of MDD. <h3>Funding</h3> This work was supported by the National Institutes of Aging R01AG021476, the National Institutes of Mental Health H110598, and Vanderbilt CTSA grant UL1 TR002243 from National Center for Advancing Translational Sciences.

Differential Effects of Estradiol on Reproductive Function in Camelids.

Differential Effects of Estradiol on Reproductive Function in Camelids.

Differential Effects of Estradiol and Progesterone on Cardiovascular Risk Factors in Postmenopausal Women.

ContextControlled, blinded studies of sex-hormone replacement in postmenopausal women using natural estradiol (E2) and native progesterone (P) are few.ObjectiveTo delineate the effect of E2 alone or with P on lipids and inflammatory markers.DesignA placebo-controlled, double-masked, prospectively randomized study of 40 healthy, postmenopausal volunteers assigned to four treatment groups: placebo, intramuscular E2, and/or micronized oral P for 23 (±2) days.ResultsTreatment with E2 alone compared with placebo lowered total cholesterol (TC; P = 0.006), non–high-density lipoprotein cholesterol (nonHDL-C; P = 0.004), low-density lipoprotein cholesterol (LDL-C; P = 0.012), and apolipoprotein B (Apo B; P = 0.02) levels, and raised HDL-C levels (P = 0.03 vs the 3 other groups). Conversely, addition of P to E2 reduced HDL-C levels (P = 0.015). Triglyceride concentrations manifested no effect on E2 or P. High-sensitivity C-reactive protein (hsCRP) level was highest in women with E2 and P replacement (P = 0.018 vs placebo). Leptin and IL-6 concentrations did not vary. P treatment decreased adiponectin levels (P = 0.019). Serum E2 levels correlated linearly with TC, LDL-C, nonHDL-C, Apo B (all negatively), and SHBG (positively) concentrations. P level correlated negatively with TC (P = 0.029), HDL-C (P = 0.002), and adiponectin (P = 0.002) levels.ConclusionIn this study, there were individual and interactive effects of E2 and P on key lipids in postmenopausal individuals.

Subtype-Specific Tumor-Associated Fibroblasts Contribute to the Pathogenesis of Uterine Leiomyoma.

Recent genomic studies have identified subtypes of uterine leiomyoma (LM) with distinctive genetic alterations. Here, we report the elucidation of the biological characteristics of the two most prevalent uterine leiomyoma subtypes, MED12-mutant (MED12-LM) and HMGA2-overexpressing (HMGA2-LM) uterine leiomyomas. Because each tumor carries only one genetic alteration, both subtypes are considered to be monoclonal. Approximately 90% of cells in HMGA2-uterine leiomyoma were smooth muscle cells (SMC) with HMGA2 overexpression. In contrast, MED12-LM consisted of similar numbers of SMC and non-SMC, which were mostly tumor-associated fibroblasts (TAF). Paradoxically, TAF carried no mutations in MED12, suggesting an interaction between SMC and TAF to coordinate their growth. The higher amount of extracellular matrix in MED12-LM than HMGA2-LM was partially due to the high concentration of collagen-producing TAF. SMC growth in a xenograft assay was driven by progesterone in both uterine leiomyoma subtypes. In contrast, TAF in MED12-LM proliferated in response to estradiol, whereas progesterone had no effect. The high concentration of estrogen-responsive TAF in MED12-LM explains the inconsistent discoveries between in vivo and in vitro studies on the mitogenic effect of estrogen and raises questions regarding the accuracy of previous studies utilizing MED12-LM cell culture. In addition, the differential effects of estradiol and progesterone on these uterine leiomyoma subtypes emphasize the importance of subtypes and genotypes in designing nonsurgical therapeutic strategies for uterine leiomyoma. Cancer Res; 77(24); 6891-901. ©2017 AACR.

Differential Effects of Estradiol and Bisphenol A on SET8 and SIRT1 Expression in Ovarian Cancer Cells.

Exposure to estrogenic compounds has been shown to epigenetically reprogram the female reproductive tract and may contribute to ovarian cancer. The goal of this study was to compare the effect of estradiol or bisphenol A (BPA) on the expression of histone-modifying enzymes (HMEs) in ovarian cancer cells. Using 2 human ovarian cancer cell lines, we examined the expression of SET8, a histone methyltransferase, and SIRT1, a histone deacetylase, after exposure to estrogen or BPA. These experiments were carried out in complete media (fetal bovine serum) that contain natural hormones to understand the impact of additional exposure to estrogen or BPA on HME expression. We found differential expression of the HMEs in the different models examined and between the different compounds. Further, we determined that the changes in gene expression occurred via estrogen receptor signaling using the estrogen receptor antagonist, ICI 182,780 (fulvestrant).

Differential effect of estradiol and bisphenol A on Set8 and Sirt1 expression in prostate cancer

Exposure to estrogenic compounds has been shown to epigenetically reprogram the prostate and may contribute to prostate cancer. The goal of this study was to determine the effect of physiological doses of estradiol and bisphenol A (BPA) on the expression of histone modifying enzymes (HMEs) in prostate cancer. Using two human prostate cancer cell lines we examined the expression of Set8, a histone methyltransferase, and Sirt1, a histone deacetylase, after exposure to estrogen or BPA. These experiments were carried out in the presence of natural hormones to understand the impact of additional exposure to estrogen or BPA on HME expression. We found differential expression of the HMEs in the different models and between the different compounds. Further, we determined that the changes in gene expression occurred via estrogen receptor signaling using the ER antagonist, ICI 182,780 (fulvestrant). Interestingly we found that the combination of ICI with estrogen or BPA greatly affected the expression of Set8, even when the hormone alone had no effect. This study demonstrates that the effects of estrogen and BPA on HME expression vary and that the presence of both the estrogen receptor and androgen receptor may be important for therapeutic intervention.

Differential effects of estradiol on carotid artery inflammation when administered early versus late after surgical menopause

The aim of this study was to determine the effects of estrogen therapy (ET) on carotid artery inflammation when initiated early and late relative to surgical menopause. Female cynomolgus macaques consuming atherogenic diets were ovariectomized and randomized to control or oral estradiol (E2; human equivalent dose of 1 mg/d micronized E2) initiated at 1 month (early menopause, n = 24) or 54 months (late menopause, n = 40) after ovariectomy. The treatment period was 8 months. Carotid artery expression of the markers of monocyte/macrophages (CD68 and CD163), dendritic cells (CD83), natural killer cells (neural cell adhesion molecule-1), and interferon-γ was significantly lower in E2-treated animals in the early menopause group but not in the late menopause group (P < 0.05). In contrast, carotid artery transcripts for T-cell markers (CD3, CD4, CD8, and CD25), interleukin-10, type I collagen, monocyte chemoattractant protein-1, matrix metalloproteinase-9, and tumor necrosis factor-α were lower in E2-treated monkeys regardless of menopausal stage (P < 0.05). ET initiated soon after menopause inhibits macrophage accumulation in the carotid artery, an effect that is not observed when E2 is administered after several years of estrogen deficiency. No evidence for pro-inflammatory effects of late ET is observed. The results provide support for the timing hypothesis of postmenopausal ET with implications for the interpretation of outcomes in the Women's Health Initiative.

Hormone replacement therapy dependent changes in breast cancer-related gene expression in breast tissue of healthy postmenopausal women

Risk assessment of future breast cancer risk through exposure to sex steroids currently relies on clinical scorings such as mammographic density. Knowledge about the gene expression patterns in existing breast cancer tumors may be used to identify risk factors in the breast tissue of women still free of cancer. The differential effects of estradiol, estradiol together with gestagens, or tibolone on breast cancer-related gene expression in normal breast tissue samples taken from postmenopausal women may be used to identify gene expression profiles associated with a higher breast cancer risk. Breast tissue samples were taken from 33 healthy postmenopausal women both before and after a six month treatment with either 2 mg micronized estradiol [E2], 2 mg micronized estradiol and 1 mg norethisterone acetate [E2 + NETA], 2.5 mg tibolone [T] or [no HRT]. Except for [E2], which was only given to women after hysterectomy, the allocation to each of the three groups was randomized. The expression of 102 mRNAs and 46 microRNAs putatively involved in breast cancer was prospectively determined in the biopsies of 6 women receiving [no HRT], 5 women receiving [E2], 5 women receiving [E2 + NETA], and 6 receiving [T]. Using epithelial and endothelial markers genes, non-representative biopsies from 11 women were eliminated. Treatment of postmenopausal women with [E2 + NETA] resulted in the highest number of differentially ( p < 0.05) regulated genes (16.2%) compared to baseline, followed by [E2] (10.1%) and [T] (4.7%). Among genes that were significantly down-regulated by [E2 + NETA] ranked estrogen-receptor-1 ( ESR1, p = 0.019) and androgen receptor ( AR, p = 0.019), whereas CYP1B1, a gene encoding an estrogen-metabolizing enzyme, was significantly up-regulated ( p = 0.016). Mammary cells triggered by [E2 + NETA] and [E2] adjust for steroidogenic up-regulation through down-regulation of the estrogen-receptor pathway. In this prospective study, prolonged administration of [E2 + NETA] and to a lesser extent of [E2] but not [T] were associated in otherwise healthy breast tissue with a change in the expression of genes putatively involved in breast cancer. Our data suggest that normal mammary cells triggered by [E2 + NETA] adjust for steroidogenic up-regulation through down-regulation of the estrogen-receptor pathway. This feasibility study provides the basis for whole genome analyses to identify novel markers involved in increased breast cancer risk.

Differential effects of estradiol on drinking by ovariectomized rats in response to hypertonic NaCl or isoproterenol: Implications for hyper- vs. hypo-osmotic stimuli for water intake

We examined the effects of estradiol on behavioral responses to osmotic challenges in ovariectomized (OVX) rats to test the hypothesis that estradiol enhances sensitivity to gradual changes in plasma osmolality (pOsm) in stimulating water intake. Despite comparably elevated pOsm after a slow infusion of 2 M NaCl, the latency to begin water intake was significantly less in estradiol-treated OVX rats compared to that in oil vehicle-treated rats. Other groups of OVX rats were injected with isoproterenol, which increases circulating angiotensin II. These rats then were given 0.15 M NaCl to drink instead of water, to prevent decreased pOsm associated with water ingestion. Isoproterenol stimulated 0.15 M NaCl intake by both groups; however, estradiol-treated rats consumed less 0.15 M NaCl than did oil-treated rats, findings that are similar to those reported when estradiol-treated rats consumed water. The estradiol enhancement of sensitivity to increased, but not to decreased, pOsm suggests that estradiol has directionally-specific effects on osmoregulatory drinking. Moreover, the estradiol attenuation of 0.15 M NaCl intake after isoproterenol suggests that estradiol effects on osmoregulatory drinking are independent of those on volume regulatory drinking.

Study of Molecular and Granular Layer of Dentate Gyrus of Female Rats Neonatally Treated with Estrogen

The proliferation of hippocampal dentate gyrus granule cells was investigated using 3H-thymidine incorporation in control and estrogen-treated rats. Newborn 3-day old female Wistar rats were treated with a single dose of 1 mg of estradiol and 30 μCi 3H-thymidine, and were sacrificed when 10 days old. The total number of neurons and the number of labeled granule cells in the granular layer and its subdivisions of both suprapyramidal and infrapyramidal limbs were analyzed using a stereological method. In both limbs, the total number of neurons as well as the total number of labeled granule cells in the granular layer were significantly increased in treated rats compared to corresponding controls. The thicknesses of the molecular and the granular layers and their subdivisions of both suprapyramidal and infrapyramidal limbs were analyzed using a stereological method. In treated female rats the molecular layer (ML) in both limbs was significantly decreased, and the granular layer (GL) was significantly increased in suprapyramidal limb. However, in the infrapyramidal limb an increased number of labeled cells in treated animals were significant in all particular zones of the granular layer. In the suprapyramidal limb's granular layer a significant increase in labeled cells was observed in subgranular zone (SGZ). Our results suggest a differential effect of estradiol on thicknesses of the ML and the GL, and dentate gyrus granule cells proliferation through the early rat life.

Differential Effects of Estradiol on Encoding Properties of TMJ Units in Laminae I and V at the Spinomedullary Junction in Female Rats

To determine whether estrogen status modulated dorsal horn neural activity relevant to temporomandibular joint (TMJ) processing single units were recorded in superficial and deep laminae at the trigeminal subnucleus caudalis/upper cervical cord (Vc/C1-2) junction of ovariectomized (OvX) female rats under barbiturate anesthesia after 17beta-estradiol (E2) treatment for 2 days. E2 dose-dependently enhanced the response to intra-TMJ stimulation by adenosine triphosphate (ATP) of neurons classified as nociceptive specific (NS), but not wide dynamic range (WDR), in superficial laminae. ATP caused similar responses among NS and WDR neurons from deep laminae in all groups. By contrast, the cutaneous receptive field areas of WDR, but not NS, units in superficial and deep laminae were enlarged in high E2-treated (HE2) compared with low E2-treated (LE2) females. Units from untreated or vehicle-treated male rats displayed responses similar to those of LE2 females. TMJ units in superficial laminae from females were more likely to receive convergent cutaneous input and respond to jaw movement than males, independent of E2 treatment. Western blot analysis revealed similar levels of P2X2 and P2X3 receptor protein in Vc/C1-2 or trigeminal ganglion samples in all groups. Immunohistochemistry revealed dense terminal labeling for P2X3 receptors in superficial laminae and moderate labeling in deep laminae at the Vc/C1-2 junction. These data indicated a significant linkage between estrogen status and the magnitude of articular input evoked by ATP from TMJ neurons in the superficial laminae at the Vc/C1-2 junction, whereas estrogenic modulation of TMJ neurons in deep laminae affected only the convergent input from overlying facial skin.

Differential effects of estradiol, raloxifene and tamoxifen on estrogen receptor expression in cultured human skin fibroblasts

The balance between ER-alpha and ER-beta in fibroblasts may be crucial in the physiological response to ligands. Up- or down-regulation of the ERs in response to different compounds could mediate the reversal of certain age-related changes in skin and connective tissue. The time-dependent effects of 17-beta estradiol, raloxifene and tamoxifen on ER-alpha and ER-beta mRNA expression in the skin fibroblast cultures were performed. Experiments were carried out in primary cultures of human skin fibroblasts obtained from postmenopausal women. The cells were cultured in medium containing: 2 micromol/l estradiol (E2), 4 micromol/l tamoxifen (Tx) or 4 micromol/l raloxifene (Rx) for 7, 24 and 32 h. ER-alpha and ER-beta mRNAs were measured by quantitative assays based on reverse transcription (RT) of the mRNA and polymerase chain reaction (PCR) amplification of the cDNA. We suggest that ER-alpha and ER-beta are co-expressed in human postmenopausal skin fibroblast and documented that the level of mRNA expression of ERs in this tissue is estradiol, raloxifene or tamoxifen regulated as a mechanism to control the action of those ligands on the cell. On the basis of ER mRNA expression levels, fibroblast response to estradiol appears to be modulated by up-regulation of ER-beta rather than ER-alpha. Two of the examined SERMs appear to have different response to modulation of ERs: response of raloxifen is modulated by up-regulation of ER-beta, and no changes in expression of ER-alpha and tamoxifen response seem to be modulated by ER down-regulation in short-term or up-regulation during longer treatment.

Differential effects of estradiol and raloxifene on collagen biosynthesis in cultured human skin fibroblasts

The dose-dependent effect of a 24 h treatment with estradiol (E(2)) (1, 2, 5, 10 nM) and raloxifene (Rx) (1, 5, 10, 20 microM) on ER alpha and ER beta mRNA expression, collagen bio-synthesis, prolidase activity, MMP-2, MMP-9, insulin-like growth factor I receptor expression (IGF-1R) and beta1-integrin expressions in cultured fibroblasts obtained from postmenopausal women were examined. Both ligands increased mRNA expression of ER compared to control. Rx at 5 and 10 microM concentrations had greater stimulative effect on collagen biosynthesis, prolidase activity and IGF-1R expression compared to E(2) at 2 and 5 nM concentration. Both studied ER ligands had no effect on beta1-integrin receptor expressions. MMP-2 expression was not detected in human skin fibroblast culture. In contrast to estradiol raloxifene inhibited the expression of MMP-9. Raloxifene had stronger positive stimulative effects on collagen biosynthesis, through different biochemical mechanisms, than estradiol in human skin fibroblasts and might reverse some of the postmenopausal changes in skin or connective tissue. Increase of collagen synthesis induced by raloxifene may be activated by both estrogen receptor dependent and independent pathways such as up-regulation of estrogen receptors, up-regulation of IGF receptor, transcriptional regulation of collagen genes by estrogen receptor-raloxifene complex, increasing of prolidase activity or finally by inhibition of MMP-9 expression.

Differential effects of estradiol on the adrenocorticotropin responses to interleukin-6 and interleukin-1 in the monkey.

Endotoxin and the inflammatory cytokines interleukin (IL)-1 and IL-6 are potent activators of the hypothalamic-pituitary-adrenal (HPA) axis. Although estradiol (E(2)) has been shown to enhance the HPA response to certain types of stress, previous studies in the rodent have shown that HPA responses to endotoxin and to IL-1 were enhanced by ovariectomy and attenuated by E(2). The mechanisms underlying these observations are unclear, but there is evidence that E(2) may have direct inhibitory effects on IL-6 synthesis and release. Because endotoxin and IL-1 both stimulate IL-6, it is possible that the E(2)-induced suppression of the HPA response to endotoxin and IL-1 results from decreased IL-6 release. We have therefore examined the ACTH response to IL-6 and IL-1beta in six ovariectomized rhesus monkeys with and without 3 weeks of E(2) replacement. In the first study, plasma ACTH levels peaked at 60 min after iv injection of 6 microg recombinant human IL-6. Both the ACTH response, over time, and the area under the ACTH response curve were significantly higher in the E(2)-treated animals (P < 0.05). The peak ACTH level was 66 +/- 16 pg/ml without E(2) vs. 161 +/- 69 pg/ml with E(2). In the second study, iv infusion of recombinant human IL-1beta (400 ng) produced plasma IL-6 levels comparable with those seen after IL-6 injection in the first study. In the IL-1 study, however, there was a significant attenuation of the ACTH response, over time, in the E(2)-treated animals (P < 0.001); the peak ACTH level was 83 +/- 34 pg/ml vs. 13 +/- 4.4 pg/ml after E(2). The IL-6 response was similarly attenuated (P < 0.001); the peak IL-6 level was 614 +/- 168 pg/ml vs. 277 +/- 53 pg/ml after E(2) treatment. Our results demonstrate that physiological levels of E(2) enhance the ACTH response to IL-6 but attenuate the ACTH response to IL-1. The attenuated ACTH response to IL-1 was accompanied by a blunted IL-6 response. Our results suggest that the blunted HPA response to IL-1 can be explained, at least in part, by E(2)-induced alterations in IL-6 release. It remains to be determined whether E(2) affects other inflammatory mediators that also participate in this process.

Differential Effects of Estradiol on the Adrenocorticotropin Responses to Interleukin-6 and Interleukin-1 in the Monkey

Differential Effects of Estradiol on the Adrenocorticotropin Responses to Interleukin-6 and Interleukin-1 in the Monkey

Differential effects of estradiol and its analogs on cyclin D1 and CDK4 expression in estrogen receptor positive MCF-7 and estrogen receptor-transfected MCF-10AEwt5 cells.

Estradiol stimulates the growth of a majority of human breast tumors containing the estrogen receptors (ERs), proteins that mediates estrogen function. Opposing effects of estradiol have been found in cells expressing endogenous ER and those containing a transfected ER. To understand the role of estradiol structure in diverse estrogenic responses related to cell cycle regulation, we evaluated the effects of estradiol and its analogs on cell cycle progression, and the expression of cyclin D1 and the cyclin dependent kinase 4 (CDK4) in ER-positive MCF-7 and ER-transfected MCF-10AEwt5 cells. Four analogs of estradiol, with a re-positioned or a deleted hydroxyl group, were used. Our results show that estradiol and all of the analogs facilitated cell cycle progression of MCF-7 cells. In contrast, only estradiol inhibited the cell cycle progression of MCF-10AEwt5 cells significantly. Western blot analysis revealed that cyclin D1 protein increased to the maximal level by 6 h after the initiation of cell cycle from G1 phase of MCF-7 cells. The least effective analog in inducing cyclin D1 was 3-hydroxyestratriene. However, this analog was most effective at inducing CDK4, contributing to its efficacy in facilitating MCF-7 cell cycle. In contrast to MCF-7 cells, the level of cyclin D1 protein was not influenced significantly by estradiol or its analogs in MCF-10AEwt5 cells. Sucrose gradient sedimentation analysis of ER from MCF-7 cells showed that the major peak of [3H]-estradiol bound to ER could be displaced by a 10-fold excess of unlabelled estradiol or any of the analogs. In contrast, several analogs were less effective than unlabelled estradiol in competitive displacement of [3H]-estradiol bound to ER from MCF-10AEwt5 cells. These data indicate that the induction of cyclin D1 is an important part of the growth stimulatory effects of estrogens in MCF-7 cells, but it may not be involved in growth inhibition of MCF-10AEwt5 cells. Our results also show that estrogenic compounds interact with ER from MCF-10AEwt5 cells with altered ligand binding affinity, possibly due to the absence or dysfunction of certain transcription factors or ER-associated proteins that co-regulate ER function.

Gonadal steroid modulation of vasopressin secretion in response to osmotic stimulation.

As deficiencies in osmotic stimulation of vasopressin (VP) messenger RNA (mRNA) content in castrated rats have been reported, experiments were performed to determine whether castration altered osmotically stimulated VP release in vitro. Perifused explants of the hypothalamo-neurohypophyseal system were obtained from sham and gonadectomized male rats. There were no significant differences in VP release stimulated by a ramp increase in the osmolality of the culture medium between the two groups. As testosterone was undetectable in the perifusion medium, the effect of addition of testosterone on osmotically stimulated VP release was evaluated. Testosterone (3 ng/ml) and its metabolites, estradiol (50 pg/ml) and dihydrotestosterone (DHT; 3 ng/ml), inhibited osmotically stimulated VP release in hypothalamo-neurohypophyseal system explants. The osmotically induced increase in VP mRNA content was also inhibited by testosterone and estradiol, but not by DHT. Neither estradiol nor DHT affected stimulus-secretion coupling of hormone secretion, because they did not inhibit KCl (25 mM)-stimulated VP release. BSA conjugates of estradiol (200 nM) and DHT (10 mM) also inhibited osmotically stimulated VP release, and VP mRNA content was inhibited by BSA-estradiol, but not by BSA-DHT, suggesting nongenomic actions of the steroids. The differential effects of estradiol and DHT on VP mRNA imply distinct actions for these steroids, and the DHT mechanism uncouples regulation of VP release from VP mRNA content.

Gonadal Steroid Modulation of Vasopressin Secretion in Response to Osmotic Stimulation

As deficiencies in osmotic stimulation of vasopressin (VP) messenger RNA (mRNA) content in castrated rats have been reported, experiments were performed to determine whether castration altered osmotically stimulated VP release in vitro. Perifused explants of the hypothalamo-neurohypophyseal system were obtained from sham and gonadectomized male rats. There were no significant differences in VP release stimulated by a ramp increase in the osmolality of the culture medium between the two groups. As testosterone was undetectable in the perifusion medium, the effect of addition of testosterone on osmotically stimulated VP release was evaluated. Testosterone (3 ng/ml) and its metabolites, estradiol (50 pg/ml) and dihydrotestosterone (DHT; 3 ng/ml), inhibited osmotically stimulated VP release in hypothalamo-neurohypophyseal system explants. The osmotically induced increase in VP mRNA content was also inhibited by testosterone and estradiol, but not by DHT. Neither estradiol nor DHT affected stimulus-secretion coupling of hormone secretion, because they did not inhibit KCl (25 mm)-stimulated VP release. BSA conjugates of estradiol (200 nm) and DHT (10 mm) also inhibited osmotically stimulated VP release, and VP mRNA content was inhibited by BSA-estradiol, but not by BSA-DHT, suggesting nongenomic actions of the steroids. The differential effects of estradiol and DHT on VP mRNA imply distinct actions for these steroids, and the DHT mechanism uncouples regulation of VP release from VP mRNA content.

Insulin-like growth factor-binding protein-3 production by MCF-7 breast cancer cells: stimulation by retinoic acid and cyclic adenosine monophosphate and differential effects of estradiol.

The insulin-like growth factors (IGFs) stimulate the proliferation of human breast cancer cells, including the estrogen-dependent cell line MCF-7. These cells secrete regulatory IGF-binding proteins (IGFBPs) which may enhance or attenuate IGF-stimulated cell proliferation. In this study, we have used RIA to quantify the production and regulation of IGFBP-3 and IGFBP-6 by MCF-7 cells in vitro. Under basal (serum- and phenol red-free) conditions, IGFBP-3 and IGFBP-6 accumulated in 72 h-conditioned MCF-7 medium to concentrations of approximately 0.18 nM and 0.02 nM, respectively. Treatment with retinoic acid (RA, 100 nM) increased medium concentrations of IGFBP-3 to 175 +/- 8% (mean +/- SE, n = 4), and IGFBP-6 to 217 +/- 20% of control values. Forskolin (0.5 microM) or dibutyryl cAMP (db-cAMP, 1 mM) increased both proteins 2- to 3-fold. In the presence of 100 nM RA, the stimulation elicited by these agents was enhanced, with IGFBP-3 levels increasing to 6-fold above that seen with RA alone. IGFBP-6 increased 12-fold with RA + forskolin and 20-fold with RA + dbcAMP. Estrogen (10 nM estradiol) reduced basal IGFBP-3 levels by 25% but increased IGFBP-6 1.5- to 2-fold. The stimulatory effect of RA + forskolin on IGFBP-3 was partially reversed by estrogen, whereas RA + forskolin-stimulated IGFBP-6 levels were further increased by estrogen. Increased IGFBP-3 and -6 production in response to RA + forskolin was accompanied by a decrease in IGF-stimulated thymidine incorporation into DNA; by contrast, the bioactivity of an IGF analog that does not bind with IGFBPs, [Gln3, Ala4, Tyr15, Leu16]IGF-I, was unchanged under these conditions. These data demonstrate that modulating the production of IGFBPs can lead to changes in the sensitivity of breast cancer cells to IGFs, and as a result change the cell proliferative effects of these growth factors. Further, IGFBP-3 and IGFBP-6 are differentially regulated by estrogen. Dissecting the roles of the individual IGFBPs is essential to understanding how such differential regulation will ultimately affect IGF-stimulated cell proliferation in breast cancer.