E2-mediated Increase Research Articles

Abstract 582: Superoxide dismutase 3 is induced by antioxidants, inhibits oxidative DNA damage, and is associated with inhibition of estrogen-induced breast cancer

Abstract Epidemiological data and studies in rodent models strongly support the role of estrogens in the development of breast cancers. Oxidant stress has been implicated in this carcinogenic process. We have recently demonstrated that antioxidants vitamin C or butylated hydroxyanisole (BHA) severely inhibit 17β-estradiol (E2)-induced breast tumor development in female ACI rats. The objective of the present study was to characterize the mechanism of antioxidant-mediated prevention of breast cancer. Female ACI rats were treated with E2; vitamin C; vitamin C + E2; BHA; and BHA + E2 for up to 8 months. mRNA and protein levels of superoxide dismutase 3 (SOD3) were suppressed in E2-exposed mammary tissues and in mammary tumors of rats treated with E2. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. 8-Hydroxydeoxyguanosine (8-OHdG) levels determined as a marker of oxidative DNA damage were higher in E2-exposed mammary tissues and in mammary tumors compared to age-matched controls. Vitamin C or BHA treatment significantly decreased E2-mediated increase in 8-OHdG levels in the mammary tissues and in MCF-10A cells. Increased nuclear translocation of SOD3 was demonstrated in vitamin C-treated mammary tissues and in MCF-10A cells. Increased DNA damage was detected after silencing SOD3. Our studies suggest that SOD3 provides protection against nuclear DNA damage. Our studies further demonstrate that SOD3 but not SOD2 and SOD1 is induced by antioxidants and is regulated through NRF2. SOD3 may thus be an important gene in defense against oxidant stress and in the prevention of estrogen-mediated breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 582. doi:1538-7445.AM2012-582

Liver kinase B1 expression (LKB1) is repressed by estrogen receptor alpha (ERα) in MCF-7 human breast cancer cells

BackgroundLiver kinase 1 (LKB1) is emerging as a multifunctional protein, acting as a key metabolic enzyme, regulator of cell polarity, and transcription factor. Altered LKB1 expression has been linked with various cancers and may be a potential prognostic marker. While the functional role of LKB1 continues to undergo intensive investigation, the molecular mechanisms that regulate its expression remain to be defined more clearly. Recent reports have established a possible link between estrogen receptor alpha (ERα) signaling and LKB1 in MCF-7 human breast cancer cells. The current study aimed to investigate whether LKB1 is transcriptionally regulated by ERα in MCF-7 cells. MethodssiRNA transfections were used to transiently knock down LKB1 and ERα. LKB1 and ERα mRNA and protein levels were evaluated by real-time PCR and Western blotting, respectively. An approximately 3 kilobase pair human LKB1 promoter construct and various truncations were generated, transfected into MCF-7 cells, and luciferase reporter assays were performed. Cells were also treated with various doses of 17-β-estradiol (E2) to evaluate the effect on LKB1 and ERα mRNA levels. ResultsLKB1 mRNA and protein levels were significantly lower in ERα-positive MCF-7 compared to ERα-negative MDA-MB-231 breast cancer cells, suggesting that ERα may act as a repressor. siRNA-mediated knock-down of ERα in MCF-7 cells significantly increased LKB1 promoter activity and expression at the mRNA and protein levels, and computational analysis revealed the presence of several putative estrogen response element (ERE) DNA binding sites in the LKB1 promoter region. In addition, treatment with E2 led to an increase in LKB1 expression, concomitant with decreased expression of ERα in MCF-7 cells. The E2-mediated increase was abrogated by pretreatment with actinomycin D, supporting that the observed changes in LKB1 levels were transcriptionally regulated. ConclusionsERα repressively modulates the expression of LKB1 at the transcriptional level. Targeting the expression of LKB1 by modulating ERα signaling may provide a potential approach to further evaluate its function in ERα-positive breast cancers.

Induction of NAD(P)H-quinone oxidoreductase 1 by antioxidants in female ACI rats is associated with decrease in oxidative DNA damage and inhibition of estrogen-induced breast cancer

Exact mechanisms underlying the initiation and progression of estrogen-related cancers are not clear. Literature, evidence and our studies strongly support the role of estrogen metabolism-mediated oxidative stress in estrogen-induced breast carcinogenesis. We have recently demonstrated that antioxidants vitamin C and butylated hydroxyanisole (BHA) or estrogen metabolism inhibitor α-naphthoflavone (ANF) inhibit 17β-estradiol (E2)-induced mammary tumorigenesis in female ACI rats. The objective of the current study was to identify the mechanism of antioxidant-mediated protection against E2-induced DNA damage and mammary tumorigenesis. Female ACI rats were treated with E2 in the presence or absence of vitamin C or BHA or ANF for up to 240 days. Nuclear factor erythroid 2-related factor 2 (NRF2) and NAD(P)H-quinone oxidoreductase 1 (NQO1) were suppressed in E2-exposed mammary tissue and in mammary tumors after treatment of rats with E2 for 240 days. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. Time course studies indicate that NQO1 levels tend to increase after 4 months of E2 treatment but decrease on chronic exposure to E2 for 8 months. Vitamin C and BHA significantly increased NQO1 levels after 120 days. 8-Hydroxydeoxyguanosine (8-OHdG) levels were higher in E2-exposed mammary tissue and in mammary tumors compared with age-matched controls. Vitamin C or BHA treatment significantly decreased E2-mediated increase in 8-OHdG levels in the mammary tissue. In vitro studies using silencer RNA confirmed the role of NQO1 in prevention of oxidative DNA damage. Our studies further demonstrate that NQO1 upregulation by antioxidants is mediated through NRF2.

Abstract 1863: Curcumin implants, not curcumin diet inhibits estrogen induced-mammary carcinogenesis

Abstract Curcumin, a naturally occurring compound, is widely known for its anti-oxidant, anti-inflammatory, anti-proliferative and various other activities in cell culture studies. However, it has elicited only limited efficacy in animal and in clinical studies despite high doses, due to its poor oral bioavailability. Recently, we showed that continuous systemic delivery of curcumin by subcutaneous polymeric implants can circumvent issues associated with bioavailability. In this study we investigated potential of curcumin delivered via the implants and diet against 17β-estradiol (E2)-mediated mammary carcinogenesis. Implants were prepared using polycaprolactone and polyethylene glycol (65:35) alone or with curcumin (20%, w/w). The implants exhibited biphasic release kinetics in vitro with a burst release (7%) in the first week, followed by a more controlled release. In vivo drug release followed the same kinetics as the in vitro release, except that the total amount released was 1.8-fold higher presumably due to sink conditions. To determine the anticarcinogenicity potential, female ACI rats were either administered curcumin via the diet or were subcutaneously grafted with two 2-cm implants (20% drug load) 4 days prior to grafting a subcutaneous E2 silastic implant (1.2 cm, 9 mg E2). Implants were changed after nearly 4 ½ months to provide higher curcumin dose at the appearance of palpable tumors. The animals were euthanized after 3 weeks, 3 months and after the tumor incidence reached >80% (6 months) in control animals. Curcumin administered via implants resulted in significant reduction in both the tumor volume (184±198 mm3 vs 280±141 mm3; p=0.0283) and tumor multiplicity (5±3 versus 2±1; p=0.001); the dietary curcumin, however, was ineffective. Further, serum E2 concentration and E2-mediated increase in plasma prolactin were also found to be significantly reduced by curcumin implants but not with dietary curcumin. Analysis of plasma by HPLC showed higher curcumin levels (2.4 nM) via implant route as compared to diet at ∼10-20 fold lower doses both after 3 weeks and 3 months of treatment. Similarly, higher levels of curcumin were detected in liver tissue also by the implant route than dietary administration after both 3 weeks (60±28 versus 40±20 ng/g) and 6 months (32±15 versus 21±9 ng/g). Dietary curcumin increased hepatic CYP1A and CYP1B1 activity without any effect on CYP3A4 activity whereas curcumin implants increased both CYP1A and CYP3A4 activity but decreased CYP1B1 activity in presence of E2. Since, CYP1A and 3A4 metabolize most of the E2 to its non-carcinogenic 2-OH metabolite and CYP1B1 produces potentially carcinogenic 4-OH metabolite, it appears that favorable modulation of these CYPs via systemic curcumin delivery could be one of the mechanisms that resulted in the reduced E2 mediated mammary carcinogenesis (Supported from CA-118114 and Agnes Brown Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1863. doi:10.1158/1538-7445.AM2011-1863

Abstract 5543: Partial inhibition of estrogen-induced breast cancer by tamoxifen in female ACI rats: Implications of oxidant stress

Abstract Epidemiological and experimental evidence strongly supports the role of estrogens in the etiology of breast cancer. Both estrogen receptor [ER]-dependent and ER-independent mechanisms are implicated in estrogen-induced breast carcinogenesis. Tamoxifen, a selective ER modulator is widely used as chemoprotectant in human breast cancer. It binds to ERs and interferes with normal binding of estrogen to ER and the resultant ER-dependent processes. Tamoxifen treatment for six months has previously been reported to result in complete abrogation of 17β-estradiol (E2)-induced breast cancer in female ACI rats. We investigated the effect of tamoxifen on breast tumor incidence in female ACI rats after treatment with tamoxifen for 8 months. Female ACI rats were treated with E2 (3 mg pellet, s.c.), tamoxifen (40 mg pellet, s.c.) or with a combination of E2 and tamoxifen. At the time of surgery, breast tumors were detected in the 44% of rats co-treated with tamoxifen + E2 compared to 82% in rats treated with E2 alone. The tumors were confirmed as breast adenocarcinomas by histopathological examination of paraffin-embedded tissues. Immunohistochemical and real time-PCR analysis indicated increased expression of ER-α and progesterone receptor in E2-treated mammary tissue compared to age-matched controls and inhibition of these receptors in the mammary tissue of tamoxifen-treated groups. Cytochrome P450 3A4 (CYP 3A4), and CYP 2D6, the main enzymes involved in tamoxifen metabolism were unchanged in the mammary tissue of all the treatment groups compared to controls. Tissue levels of oxidative stress markers were quantified in the mammary tissues of rats. Superoxide dismutase 2, catalase and glutathione peroxidase expressions were differentially altered in E2, tamoxifen and tamoxifen + E2 co-treated groups compared to age-matched controls. Co-treatment with tamoxifen did not decrease E2-mediated increases in 8-iso-prostane F2α levels, a marker of lipid peroxidation/oxidative stress. Oxidative stress-mediated DNA damage marker, 8-hydroxydeoxyguanosine, was quantified in the mammary tissues of all the treatment groups. Co-treatment of tamoxifen with E2 did not inhibit E2-mediated increase in 8-hydroxydeoxyguanosine levels in mammary tissue. In summary, our studies suggest that inhibition of ER-mediated processes by tamoxifen does not completely abrogate estrogen-dependent breast tumor development in a rat model of estrogen-induced breast cancer. Our results further demonstrate that tamoxifen does not decrease E2-mediated oxidant stress and DNA damage. Thus, chronic exposure to estrogen-mediated oxidative stress may contribute to the development of breast cancer in rats co-treated with E2 and tamoxifen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5543. doi:10.1158/1538-7445.AM2011-5543

Abstract 4212: Prevention of estrogen-induced breast cancer by antioxidants: Role of N-quinone oxidoreductase 1 (NQO1)

Abstract The importance of estrogens in the etiology of breast cancer is widely recognized. However, the exact mechanisms underlying the initiation and progression of estrogen-related cancers are not clear. Literature evidence and our studies strongly support the role of estrogen metabolism-mediated oxidative stress in estrogen-induced breast carcinogenesis. The objective of the current study was to characterize the role of NQO1 in the prevention of estrogen-induced DNA damage and mammary tumorigenesis by antioxidants vitamin C or butylated hydroxyanisole (BHA). NQO1 converts 17β-estradiol (E2)-quinones back to E2-catechols, thus making E2-quinones unavailable for oxidant damage. Female ACI rats were treated with E2 (3 mg); vitamin C (1% in drinking water); E2 + vitamin C; BHA (0.7% in diet); and E2 + BHA for up to 8 months. The female ACI rat is an established animal model to study the mechanisms of E2-induced breast cancer. No tumors were detected in the sham control, vitamin C and BHA treatment groups whereas in the E2 + vitamin C, E2 + BHA, and E2 treatment groups, the breast tumor incidences were 29, 24 and 82% respectively. mRNA and protein expression of NQO1 was analyzed in breast and breast tumor tissues of rats treated with E2 and antioxidants vitamin C or BHA. NQO1 was suppressed in E2-exposed mammary tissue after treatment of rats with E2 for 8 months and in mammary tumors. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. NQO1 enzyme activity was unchanged in mammary tissue of rats from all the treatment groups. However, NQO1 enzyme activity was decreased in E2-induced mammary tumors. Catechol-O-methyltransferase (COMT) activity was unchanged in the mammary tissue of all the groups except in the E2-treated group. Vitamin C or BHA co-treatment significantly decreased E2-mediated increase in 8-hydroxydeoxyguanosine (8-OHdG, an oxidative DNA damage marker) levels in mammary tissue. To investigate the relationship between NQO1 and 8-OHdG damage, human breast epithelial cell line MCF10A was treated with E2, vitamin C, BHA, dicumarol (NQO1 inhibitor), E2 + vitamin C, and E2 + BHA. E2 and dicumarol significantly increased 8-OHdG levels in MCF-10A cells whereas co-treatment with E2 and antioxidants reverted them back to the normal levels. Our studies suggest a protective role of NQO1 in E2-induced DNA damage and breast carcinogenesis. NQO1 may thus have a unique potential in the development of therapeutic strategies for the prevention of estrogen-induced neoplasia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4212. doi:10.1158/1538-7445.AM2011-4212

17-Beta-estradiol increases neuronal excitability through MAP kinase-induced calpain activation.

17-Beta-estradiol (E2) is a steroid hormone involved in numerous brain functions. E2 regulates synaptic plasticity in part by enhancing NMDA receptor function and spine density in the hippocampus, resulting in increased long-term potentiation and facilitation of learning and memory. As the calcium-dependent neutral protease, calpain, is also involved in these processes, we tested whether E2 could activate calpain and examined the functional consequences of E2-mediated calpain activation in hippocampus. Calpain activity was analyzed by a fluorescence resonance energy transfer (FRET)-based assay that allows both quantitative determination and spatial resolution. E2 rapidly activated calpain in cultured cortical and hippocampal neurons, prominently in dendrites and dendritic spines. E2-induced calpain activation was mediated through mitogen-activated protein kinase (MAPK), as it was completely blocked by MEK inhibitors. It was also calcium-independent, as it was still evident in presence of the calcium chelator, BAPTA-AM. Activation of ERalpha and ERbeta receptors by specific agonists stimulated calpain activity. Finally, the rapid E2-mediated increase in excitability in acute hippocampal slices was prevented by a membrane-permeable calpain inhibitor. Furthermore, E2 treatment of acute hippocampal slices resulted in increased actin polymerization and membrane levels of GluR1 but not GluR2/3 subunits of AMPA receptors; both effects were also blocked by a calpain inhibitor. Our results indicate that E2 rapidly stimulates calpain activity through MAP kinase-mediated phosphorylation, resulting in increased membrane levels of AMPA receptors. These effects could be responsible for E2-mediated increase in neuronal excitability and facilitation of cognitive processes.

Estradiol Increases Guanosine 5′-Triphosphate Cyclohydrolase Expression Via the Nitric Oxide-Mediated Activation of Cyclic Adenosine 5′-Monophosphate Response Element Binding Protein

A number of studies have demonstrated that estradiol can stimulate endothelial nitric oxide synthase expression and activity, resulting in enhanced nitric oxide (NO) generation. However, its effect on the NO synthase cofactor, tetrahydrobiopterin are less clear. Cellular tetrahydrobiopterin levels are regulated, at least in part, by GTP cyclohydrolase 1 (GCH1). Thus, the purpose of this study was to determine the effect of estradiol on GCH1 expression and the regulatory mechanisms in pulmonary arterial endothelial cells. Our data indicate that 17beta-estradiol (E2) increases GCH1 transcription in a dose- and time-dependent manner, whereas estrogen receptor antagonism or NO synthase inhibition attenuated E2-stimulated GCH1 expression. Analysis of the GCH1 promoter fragment responsive to E2 revealed the presence of a cAMP response element, and we found that E2 triggers a rapid but transient elevation of phospho-cAMP response element-binding protein (CREB; <1 h) followed by a second sustained rise after 6 h. EMSA analysis revealed an increase in the binding of CREB during E2 treatment and mutation of the cAMP response element in the GCH1 promoter attenuated the E2-mediated increase in transcription. Furthermore, inhibition of the cAMP-dependent kinase, protein kinase A (PKA) completely abolished the E2-stimulated GCH1 promoter activity, whereas the stimulation of cAMP levels with forskolin increased GCH1 promoter activity, indicating the key role of cAMP in regulating GCH1 promoter activity. In conclusion, our results demonstrate that estradiol can modulate GCH1 expression via NO-mediated activation of CREB in pulmonary arterial endothelial cells. These findings provide new insight into the vascular protective effect of estradiol.

Progesterone reverses 17β‐estradiol‐mediated neuroprotection and BDNF induction in cultured hippocampal slices

Due to the many similarities in mechanisms of action, targets and effects, progesterone (P4), estrogen and neurotrophins have been implicated in synaptic plasticity as well as in neuroprotection and neurodegeneration. In this study, we examined the interactions between 17beta-estradiol (E2) and P4 and brain-derived neurotrophic factor (BDNF) on both plasticity and excitotoxicity in rat cultured hippocampal slices. First, we evaluated the neuroprotective effects of E2 and P4 against N-methyl-D-aspartate (NMDA) toxicity in cultured rat hippocampal slices. As previously reported, pretreatment with 10 nm E2 (24 h) was neuroprotective against NMDA toxicity. However, P4 (10 nm) added 20 h after E2 treatment for 4 h reversed its protective effect. In addition, the same E2 treatment resulted in an increase in BDNF protein levels as well as in activation of its receptor, TrkB, while addition of P4 attenuated E2-mediated increase in BDNF and TrkB levels. Furthermore, E2-mediated neuroprotection was eliminated by a BDNF scavenger, TrkB-Fc. Our results indicate that E2 neuroprotective effects are mediated through the BDNF pathway and that, under certain conditions, P4 antagonizes the protective effect of estrogen.

Affirmative Action of Osteopontin on Endothelial Progenitors

Gender differences in susceptibility to coronary heart disease have been firmly established: the incidence of various manifestations of coronary atherosclerosis is less in premenopausal women than age-matched men. The protective role of estrogen covers a broad range of mechanisms including improvement of endothelial regeneration after arterial injury. Mediated primarily by the estrogen receptor alpha (ERα), 17β-estradiol (E2) stimulates migration and proliferation of endothelium. This effect involves among others, eNOS activity,1 prostacyclin, endothelin, bFGF,2 and VEGF-R2 signaling.3 Estrogen also stimulates the recruitment of endothelial progenitor cells (EPCs), which in experimental and emerging clinical studies appear to be important effectors of endothelium repair (for review see Besler et al4). In rodents, E2 increases the number of circulating EPCs and enhances their adherence and homing to the region of vascular damage. This process is ERα dependent and EPCs are believed to play a nonredundant role in the E2 enhanced reendothialization.1 Likewise, in premenopausal women a higher number of circulating EPCs is found when compared to age-matched men. The level of circulating EPCs synchronizes with menstrual cycle and is the highest during the fertile period. In addition, male EPCs showed a lower adherence capacity as compared to premenopausal female EPCs, an effect that was abolished by E2 supplementation.5 See accompanying article on page 2131 The presumed clinical relevance of EPC recruitment, for instance in restenosis, is underscored by …

Estradiol increases proteinuria and angiotensin II type 1 receptor in kidneys of rats receiving L-NAME and angiotensin II

Prospective, placebo-controlled clinical trials suggest that estrogen may have adverse effects on the vascular system in women. The goal of this study was to determine if 17beta-estradiol (E2) would have adverse effects on the renovasculature in a rat model of renal injury characterized by low nitric oxide (NO) and high angiotensin II (AngII). We studied female Wistar rats that were sham-operated (sham), ovariectomized (OVX), or ovariectomized and replaced with E2 (OVX/E2). All rats were maintained on a high salt diet and renovascular injury was caused by treating rats with an inhibitor of NO synthase, N(omega)-nitro-L-arginine-methyl-ester (L-NAME), for 14 days, plus AngII on days 11 through 14. L-NAME/AngII treatment, as compared to placebo, caused proteinuria, glomerular injury, and fibrinoid necrosis of renal arterioles in sham-operated rats. Ovariectomy reduced L-NAME/AngII-induced renal damage, whereas E2 treatment increased L-NAME/AngII-induced damage in OVX rats. In rats treated with L-NAME/AngII, levels of AngII type 1 receptor (AT(1)R) protein were higher in the renal cortex of sham and OVX/E2 rats than in OVX rats. AT(1)R protein correlated with renal injury. E2 treatment also increased expression of AT(1)R mRNA. Thus, under conditions of low NO and high AngII, E2 exacerbated renal injury. E2-mediated increases in renal cortical AT(1)R expression may represent a novel mechanism for the adverse renovascular effects of estrogen.

A+U-rich-element RNA-binding factor 1/heterogeneous nuclear ribonucleoprotein D gene expression is regulated by oestrogen in the rat uterus

Oestrogen-mediated gene expression is regulated at both the transcriptional and post-transcriptional levels. The molecular mechanism of transcriptional regulation has been well characterized. On the other hand, there is little understanding of the mechanism of post-transcriptional regulation. To clarify the mechanism of oestrogen-mediated post-transcriptional regulation, we focused on A+U-rich-element RNA-binding factor 1/heterogeneous nuclear ribonucleoprotein D (AUF1/hnRNP D), which is known as a regulator of cytosolic mRNA degradation and nuclear pre-mRNA maturation. However, little is known about the expression levels and the regulation of AUF1/hnRNP D mRNA in tissues. We further investigated the expression levels of AUF1/hnRNP D isoform mRNAs to determine whether AUF1/hnRNP D gene expression is regulated by oestrogen in the ovariectomized adult female rat uterus. Uterine AUF1/hnRNP D mRNA was induced by a single subcutaneous injection (1μg/kg) of 17β-oestradiol (E2), reaching a peak level within 6h. Furthermore, we observed that the E2-induced AUF1/hnRNP D isoform mRNAs are p45 and p40 transcripts, and that E2-mediated induction is suppressed by the oestrogen receptor antagonist ICI 182,780. Finally, using the transcriptional inhibitor actinomycin D, we confirmed that the E2-mediated increase in AUF1/hnRNP D mRNA is caused by E2-dependent AUF1/hnRNP D mRNA stabilization.

A+U-rich-element RNA-binding factor 1/heterogeneous nuclear ribonucleoprotein D gene expression is regulated by oestrogen in the rat uterus.

Oestrogen-mediated gene expression is regulated at both the transcriptional and post-transcriptional levels. The molecular mechanism of transcriptional regulation has been well characterized. On the other hand, there is little understanding of the mechanism of post-transcriptional regulation. To clarify the mechanism of oestrogen-mediated post-transcriptional regulation, we focused on A+U-rich-element RNA-binding factor 1/heterogeneous nuclear ribonucleoprotein D (AUF1/hnRNP D), which is known as a regulator of cytosolic mRNA degradation and nuclear pre-mRNA maturation. However, little is known about the expression levels and the regulation of AUF1/hnRNP D mRNA in tissues. We further investigated the expression levels of AUF1/hnRNP D isoform mRNAs to determine whether AUF1/hnRNP D gene expression is regulated by oestrogen in the ovariectomized adult female rat uterus. Uterine AUF1/hnRNP D mRNA was induced by a single subcutaneous injection (1 microg/kg) of 17beta-oestradiol (E2), reaching a peak level within 6 h. Furthermore, we observed that the E2-induced AUF1/hnRNP D isoform mRNAs are p45 and p40 transcripts, and that E2-mediated induction is suppressed by the oestrogen receptor antagonist ICI 182,780. Finally, using the transcriptional inhibitor actinomycin D, we confirmed that the E2-mediated increase in AUF1/hnRNP D mRNA is caused by E2-dependent AUF1/hnRNP D mRNA stabilization.

Sex Steroid-Binding Protein and Its Membrane Receptor in Estrogen-Dependent Breast Cancer: Biological and Pathophysiological Impact

Data obtained in our laboratory about the membrane receptor for sex steroid-binding protein (SBP-R) in human breast cancer are reported. SBP-R was detected in MCF-7 cells (estrogen receptor positive, ER+), while MDA-MB 231 cells (ER-) did not bind SBP. MCF-7 cells treated with SBP and E2 showed a marked increase of intracellular cAMP, and a significant reduction of both E2 induced cell proliferation and E2-mediated increase of progesterone receptor (PGR). The inhibition of E2 effects in MCF-7 cells was shown to be highly specific for SBP and mediated by protein kinase A, the target of cAMP. Membrane SBP-R was also evaluated in primary breast cancers. SBP-R was detectable only on ER+/PR+ samples and SBP-R+ samples presented a lower proliferation rate than negative samples. Our data, thus suggest that SBP-R and ER could be functionally related and also that SBP could modulate estrogen action at target cell site.

Estrogen regulation of epidermal growth factor receptor messenger ribonucleic acid.

Previous studies have demonstrated that 17 beta-estradiol (E2) causes a 3-fold increase in epidermal growth factor (EGF) receptors in uterine membranes. We now report that the increase in uterine EGF receptor levels is due to an increase in the steady-state levels of EGF receptor mRNA. After a single E2 injection, EGF receptor mRNA levels, as determined by RNA blots, increase 3- to 4-fold between 1 and 3 h, remain elevated at 6 h, and decline between 12 and 18 h. The effect is specific for E2 since the nonestrogenic hormones progesterone, dexamethasone, 5 alpha-dihydrotestosterone, and the inactive stereoisomer of E2, 17 alpha-estradiol, are without effect. E2-Mediated increases in EGF receptor mRNA levels are blocked by actinomycin D but not by puromycin. Taken together, these results indicate that E2 regulates the level of EGF receptor by increasing the steady-state concentration of EGF receptor mRNA in vivo.