Receptor Alpha mRNA Research Articles

Androgen receptor overexpression induces tamoxifen resistance in human breast cancer cells

Although the androgen receptor (AR) is a known clinical target in prostate cancer, little is known about its possible role in breast cancer. We have investigated the role of AR expression in human breast cancer in response to treatment with the antiestrogen tamoxifen. Resistance to tamoxifen is a major problem in treating women with breast cancer. By gene expression profiling, we found elevated AR and reduced estrogen receptor (ER) alpha mRNA in tamoxifen-resistant tumors. Exogenous overexpression of AR rendered ERalpha-positive MCF-7 breast cancer cells resistant to the growth-inhibitory effects of tamoxifen in anchorage-independent growth assays and in xenograft studies in athymic nude mice. AR-overexpressing cells remained sensitive to growth stimulation with dihydrotestosterone. Treatment with the AR antagonist Casodex (bicalutamide) reversed this resistance, demonstrating the involvement of AR signaling in tamoxifen resistance. In AR-overexpressing cells, tamoxifen induced transcriptional activation by ERalpha that could be blocked by Casodex, suggesting that AR overexpression enhances tamoxifen's agonistic properties. Our data suggest a role for AR overexpression as a novel mechanism of hormone resistance, so that AR may offer a new clinical therapeutic target in human breast cancers.

FGF-1 induces expression of LXRα and production of 25-hydroxycholesterol to upregulate the apoE gene in rat astrocytes

Fibroblast growth factor 1 (FGF-1) enhances apolipoprotein E (apoE) expression and apoE-HDL biogenesis in autocrine fashion in astrocytes (Ito, J., Y. Nagayasu, R. Lu, A. Kheirollah, M. Hayashi, and S. Yokoyama. Astrocytes produce and secrete FGF-1, which promotes the production of apoE-HDL in a manner of autocrine action. J. Lipid Res. 2005. 46: 679-686) associated with healing of brain injury (Tada,T., J-i. Ito, M. Asai, and S. Yokoyama. Fibroblast growth factor 1 is produced prior to apolipoprotein E in the astrocytes after cryo-injury of mouse brain. Neurochem. Int. 2004. 45: 23-30). FGF-1 stimulates mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) to increase cholesterol biosynthesis and phosphatidylinositol 3-OH kinase (PI3K)/Akt to enhance apoE-HDL secretion (Ito, J., Y. Nagayasu, K. Okumura-Noji, R. Lu, T. Nishida, Y. Miura, K. Asai, A. Kheirollah, S. Nakaya, and S. Yokoyama. Mechanism for FGF-1 to regulate biogenesis of apoE-HDL in astrocytes. J. Lipid Res. 2007. 48: 2020-2027). We investigated the mechanism for FGF-1 to upregulate apoE transcription. FGF-1 increased apoE and liver X receptor alpha (LXRalpha) mRNAs in rat astrocytes. Increase of LXRalpha mRNA was suppressed by inhibition of the FGF-1 receptor-1 and MEK/ERK but not by inhibition of PI3K/Akt. The increases of apoE mRNA and apoE-HDL secretion were both inhibited by downregulation or inhibition of LXRalpha, while they were partially suppressed by inhibiting cholesterol biosynthesis. We identified the liver X receptor element responsible for activation of the rat apoE promoter by FGF-1 located between -450 and -320 bp, and the direct repeat 4 (DR4) element in this region (-448 to -433 bp) was responsible for the activation. Chromatin immunoprecipitation analysis supported that FGF-1 enhanced association of LXR with the rat apoE promoter. FGF-1 partially activated the apoE promoter even in the presence of an MEK inhibitor that inhibits the FGF-1-mediated enhancement of cholesterol biosynthesis. On the other hand, FGF-1 induced production of 25-hydroxycholesterol by MEK/ERK as an sterol regulatory element-dependent reaction besides cholesterol biosynthesis. We concluded that FGF-1-induced apoE expression in astrocytes depends on LXRalpha being mediated by both LXRalpha expression and an LXRalpha ligand biosynthesis.

Substrates for vocal‐acoustic plasticity: Neuroestrogen and steroid hormone receptor expression in midshipman fish.

In a teleost fish, the midshipman Porichthys notatus, androgens and estrogen rapidly modulate vocal motor patterning and contribute to seasonal changes in the frequency encoding of the eighth nerve. To identify potential sites of action of these steroids, as well as local estrogen synthesis (via conversion from testosterone by the enzyme aromatase), in situ hybridization was employed to identify estrogen receptor alpha (ER), androgen receptor (AR), and aromatase mRNA distribution in vocal and auditory circuitry. AR expression was more widespread than ER and found at all levels of the vocal motor pathway and in vocal‐acoustic integration centers, while aromatase was particularly abundant in the vocal motor system. Both AR and ER were identified in midbrain and forebrain auditory nuclei and in the saccular epithelium of the inner ear, the main auditory end organ in midshipman and most teleosts. Aromatase was found in ganglion nerve cells of the eighth nerve, suggesting that the ear itself is a local source of estrogen driving peripheral auditory plasticity. In sum, expression of steroid receptors in vocal and auditory nuclei, and the inner ear, support neurophysiological evidence for the role of estrogen and androgens as modulators of vocal output and auditory encoding in midshipman fish. [Work supported by NIMH, NIH, and NSF.]

Effects of estradiol, sex, and season on estrogen receptor alpha mRNA expression and forebrain morphology in adult green anole lizards

Steroid hormones, especially estradiol, facilitate reproductive behaviors in male and female rodents and birds. In green anole lizards estradiol facilitates receptivity in females but, unlike in some other species, is not the activating hormone for courtship and copulatory behavior in males. Instead, testicular androgens directly facilitate male courtship and copulation. Yet, activity of the estradiol synthesizing enzyme aromatase is higher in the brain of male than female green anoles, and it is increased during the breeding compared to the non-breeding season. The functional relevance of these differences in local estradiol production is unknown. They might prime the male forebrain to facilitate production of appropriate sexual behaviors, perhaps by modifying morphology of relevant brain regions. In addition, we recently reported increased expression of estrogen receptor alpha (ERα) in selected brain regions in females compared to males [Beck LA, Wade J (2009b) Sexually dimorphic estrogen receptor α mRNA expression in the preoptic area and ventromedial hypothalamus of green anole lizards. 55:398–403]. Thus, it is possible that the hormone serves to downregulate its receptor in males to inhibit the expression of estradiol-dependent receptive behaviors. To begin to address these ideas, the present study examines the effects of estradiol treatment, sex, and season on forebrain morphology and ERα mRNA abundance in three regions important for anole reproductive behavior—the preoptic area, ventromedial amygdala, and ventromedial hypothalamus. While a number of effects of sex and season on forebrain morphology were detected, direct effects of estradiol treatment on these measures were minimal. ERα expression was greatest in the ventromedial hypothalamus, and a large female-biased sex difference was detected in this area alone; it resulted from estradiol-treated animals. These results indicate a sex- and region-specific mechanism by which estradiol can modify ERα expression in the green anole and could impact the expression of female-typical receptivity.

Sexually dimorphic estrogen receptor α mRNA expression in the preoptic area and ventromedial hypothalamus of green anole lizards

Estradiol (E2) is important in activation of male reproductive behaviors, and masculinizes morphology of associated brain regions in a number of mammalian and avian species. In contrast, it is testosterone, rather than its metabolites, that is the most potent activator of male sexual behavior in green anole lizards. As in other vertebrate groups, however, E2 is critical for receptivity in females of this species. Aromatase, the enzyme which converts testosterone to E2, is more active in the male than female green anole brain, and appears to be actively regulated on a seasonal basis, suggesting some role for E2 in males. This study was designed to enhance our understanding of potential E2 actions by localizing and quantifying relative levels of estrogen receptor-alpha (ERα) mRNA in forebrain regions involved in masculine and feminine behaviors in anoles. These areas include the preoptic area (POA), ventromedial amygdala (AMY) and ventromedial hypothalamus (VMH). In situ hybridization was conducted in adult males and females collected during both breeding and non-breeding seasons. ERα mRNA was expressed in each brain region across sexes and seasons. However, expression was up to 3 times greater in the VMH compared to the POA and AMY. In the POA and VMH, expression was higher in females compared to males, independent of season. The increased receptor expression in females is consistent with E2 playing a larger role in female than male reproductive behaviors.

Progesterone Receptor B Recruits a Repressor Complex to a Half-PRE Site of the Estrogen Receptor α Gene Promoter

In the present study, we demonstrate that elevated levels of the progesterone receptor (PR)-B isoform in breast cancer cells induces down-regulation of estrogen receptor (ER) alpha mRNA and protein content, causing concomitant repression of the estrogen-regulated genes insulin receptor substrate 1, cyclin D1, and pS2, addressing a specific effect of PR/PR-B on ERalpha gene transcription. ERalpha gene promoter activity was drastically inhibited by PR-B overexpression. Promoter analysis revealed a transcriptionally responsive region containing a half-progesterone response element (PRE) site located at -1757 bp to -1752 bp. Mutation of the half-PRE down-regulated the effect induced by PR/PR-B overexpression. Moreover chromatin immunoprecipitation analyses revealed an increase of PR bound to the ERalpha-regulatory region encompassing the half-PRE site, and the recruitment of a corepressor complex containing nuclear receptor corepressor (NCoR) but not silencing mediator of retinoid and thyroid hormone receptor and DAX1, concomitantly with hypoacetylation of histone H4 and displacement of RNA polymerase II. Furthermore, NCoR ablation studies demonstrated the crucial involvement of NCoR in the down-regulatory effects due to PR-B overexpression on ERalpha protein and mRNA. We also demonstrated that the ERalpha regulation observed in MCF-7 cells depended on PR-B expression because PR-B knockdown partially abrogates the feedback inhibition of ERalpha levels after estrogenic stimulus. Our study provides evidence for a mechanism by which overexpressed PR-B is able to actively repress ERalpha gene expression.

Effects of Maternal Exposure to Diethylstilbestrol on Epididymal Development in Rat Offspring

In our previous study, prenatal diethylstilbestrol (DES) exposure (days 7-21 of gestation) suppressed plasma testosterone levels and histological development in the epididymis of rat offspring. In this study, we measured cell proliferation in epididymal ductules and the expression of steroid hormone receptors and 5alpha-reductase 1 in the epididymis to assess the effect of DES on epididymal development in the offspring. Prenatal DES exposure did not alter the cell division index, but suppressed the expression of androgen receptor mRNA at 15 weeks after birth, and stimulated estrogen receptor alpha mRNA at 6 weeks. These results suggest that prenatal DES exposure results in the retardation of epididymal tissue maturation by disruption of the postnatal expression of steroid hormone receptors.

Effects of ovariectomy on PPARα, SREBP-1c, and SCD-1 gene expression in the rat liver

To investigate whether estrogen deficiency modifies the expression of important genes involved in hepatic lipid regulation, PPAR alpha, SREBP-1c, and SCD-1, in association with fat accumulation in the liver of ovariectomized rats. Thirty female Sprague-Dawley rats were divided into three groups: sham-operated (n = 12), ovariectomized (n = 12), and ovariectomized with 17beta-estradiol replacement (n = 6). All animals were killed 8 weeks after surgery. In addition to liver triacylglycerol determination, transcripts levels and protein content of peroxisome proliferator-activated receptor alpha, liver sterol regulatory element-binding protein 1c, and stearoyl coenzyme Adesaturase 1 were quantified by quantitative real-time polymerase chain reaction and Western blot, respectively. As expected, liver triacylglycerol levels were higher (51%; 21.9 +/- 2.6 vs 14.5 +/- 1.2 mg/g; P < 0.01) in ovariectomized compared with sham-operated rats. Peroxisome proliferator-activated receptor alpha mRNA levels were 66% lower (P < 0.01), whereas sterol regulatory element-binding protein 1 and stearoyl coenzyme A desaturase 1 transcript levels were 80% and 41% higher (P < 0.05), respectively, after estrogen removal. Our data on gene expression obtained with quantitative real-time polymerase chain reaction for peroxisome proliferator-activated receptor alpha and sterol regulatory element-binding protein 1c were confirmed by Western blots. All the effects of ovariectomy were prevented by 17beta-estradiol replacement, indicating a role for estrogens in the prevention of hepatic fat accumulation. Our results suggest that a reduction in lipid oxidation and an increase in lipogenesis are defective mechanisms leading to lipid accumulation in the liver of ovariectomized rats. We conclude that estrogen deficiency induced by ovariectomy changes the expression of genes that favor the development of a steatotic phenotype.

Limited remyelination in Theiler's murine encephalomyelitis due to insufficient oligodendroglial differentiation of nerve/glial antigen 2 (NG2)‐positive putative oligodendroglial progenitor cells

Limited remyelination is a key feature of demyelinating Theiler's murine encephalomyelitis (TME). It is hypothesized that a dysregulation of differentiation of oligodendroglial progenitor cells (OPCs) represents the main cause of insufficient regeneration in this model of multiple sclerosis. TME virus (TMEV)-infected SJL/J mice were evaluated by footprint analysis, light and electron microscopy, immunohistology, confocal immunofluorescence and RT-qPCR at multiple time points ranging from 1 h to 196 days post infection (dpi). Footprint analysis revealed a significantly decreased stride length at 147 and 196 dpi. Demyelination progressively increased from 14 towards 196 dpi. A mild amount of remyelination was detected at 147 and 196 dpi. Early onset axonal injury was detected from 14 dpi on. TMEV RNA was detectable throughout the observation period and markedly increased between 7 and 28 dpi. Intralesional nerve/glial antigen 2 (NG2)-positive OPCs were temporarily increased between 28 and 98 dpi. Similarly, a transient upregulation of NG2 and platelet-derived growth factor alpha-receptor mRNA was noticed. In contrast, intralesional 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)-positive oligodendrocytes were decreased between 56 and 196 dpi. Although CNPase mRNA remained unchanged, myelin basic protein mRNA and especially its exon 2 containing splice variants were decreased. Glial fibrillary acidic protein (GFAP)-positive astrocytes and GFAP mRNA were increased in the late phase of TME. A mildly increased colocalization of both NG2/CNPase and NG2/GFAP was revealed at 196 dpi. Summarized, the present results indicated a dysregulation of OPC maturation as the main cause for the delayed and limited remyelination in TME. A shift of OPC differentiation from oligodendroglial towards astrocytic differentiation is postulated.

Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction

BackgroundDevelopment of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously.Principal FindingsWe report here that Meltrin β, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor α mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin β–deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin β–deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 knockout mice, which lead us to investigate a possible link between Meltrin β and ephrin-A5-Eph signaling in the development of the NMJ. Meltrin β and EphA4 interacted with each other in developing motor neurons, and both of these proteins localized in the NMJ. Coexpression of Meltrin β and EphA4 strongly blocked vesicular internalization of ephrin-A5–EphA4 complexes without requiring the protease activity of Meltrin β, suggesting a regulatory role of Meltrin β in ephrin-A5-Eph signaling.ConclusionMeltrin β plays a regulatory role in formation of the NMJ. The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph. We propose that Meltrin β stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.

Water temperature and concomitant waterborne ethinylestradiol exposure affects the vitellogenin expression in juvenile brown trout ( Salmo trutta)

Environmental estrogens have the potential to considerably affect the reproduction and development of aquatic vertebrates by interfering with the endocrine system. In addition to the potential risk of environmental estrogens, increasing water temperatures as a result of global warming have become a serious problem in many rivers and streams. To assess the degree of estrogenic exposure, the analysis of the estrogen-dependent protein vitellogenin (Vtg) is a frequently used biomarker in field studies. Little, however, is known regarding the potential interaction between ambient water temperature and the Vtg production induced by waterborne environmental estrogens. In order to test the influence of temperature on Vtg synthesis, we exposed juvenile brown trout to an environmentally relevant concentration of ethinylestradiol (EE 2) and held them either at low or high temperatures (12 and 19 °C, respectively), but also at temperature cycles of 12–19 °C in order to simulate the field situation. The EE 2 exposure caused a 7–74-fold increase of hepatic Vtg mRNA. The synthesis of Vtg mRNA was clearly stimulated in fish held at higher water temperatures (12–19 °C and 19 °C, respectively). On the protein level, Vtg showed a similar pattern; the higher the temperature, the higher the concentration of Vtg in the plasma. The experiment further revealed a temperature-dependent increasing amount of hepatic estrogen receptor alpha mRNA (ERα) after exposure to waterborne EE 2. The gene expression of estrogen receptor beta-1 (ERβ-1) and the glucocorticoid receptor (GR) in the liver of EE 2 exposed fish, however, showed no treatment-related alterations. In line with observed constant bile cortisol concentrations, our data do not indicate corresponding stress related effects on hepatic Vtg production. The present survey, however, clearly demonstrates that increased temperature significantly elevates the estrogen-induced expression of Vtg and therefore has to be considered when interpreting environmental monitoring studies.

Comparative contents of mRNAs of sex steroid receptors and enzymes of their metabolism in arterial walls of men

The potential role of estrogens in regulation of metabolism in arteries of men was studied. Contents of mRNAs of sex hormone receptors, of some enzymes of their metabolism, and of some potential markers of the hormone effects were determined by real-time polymerase chain reaction in fragments of 18-54-year-old men's large arteries with and without atherosclerotic lesions. Contents of estrogen receptor alpha (ERalpha) and transferrin receptor mRNAs were significantly different in undamaged fragments of the aorta and of the carotid and coronary arteries. Contents of some mRNAs in the carotid artery and aorta were found to correlate, which suggested a similarly directed regulation of their expressions. The levels of ERalpha and aromatase mRNAs negatively correlated with the blood plasma concentration of estradiol. Levels of steroid sulfatase and aromatase mRNAs were lower and the level of estrogen sulfotransferase mRNA was higher in blood vessel fragments with atherosclerotic lesions than in undamaged fragments. It is suggested that large arteries should be different in sensitivity to estrogens and that atherosclerotic lesions could lead to local suppression of the effect of estrogen on the cells of arteries.

Effects of implants of trenbolone acetate, estradiol, or both, on muscle insulin-like growth factor-I, insulin-like growth factor-I receptor, estrogen receptor-α, and androgen receptor messenger ribonucleic acid levels in feedlot steers1

We previously showed that a combined trenbolone acetate (TBA)/estradiol-17beta (E2) implant significantly increases IGF-I mRNA levels in the LM of feedlot steers by 28 d after implantation. Here we compare the effects of E2 (25.7 mg), TBA (120 mg), and combined TBA (120 mg)/E2 (24 mg) implants on IGF-I, IGF-I receptor (IGFR-1), estrogen receptor (ER)-alpha and androgen receptor (AR) mRNA levels in the LM of steers. Twenty yearling crossbred steers with an average initial BW of 421.1 +/- 3.6 kg were stratified by BW and randomly assigned to 1 of 4 treatments: 1) nonimplanted, control; 2) implanted with TBA and E2; 3) implanted with E2; or 4) implanted with TBA. Steers were weighed weekly starting on d 0, and muscle biopsy samples were taken from each steer on d 0 (before implantation), 7, 14, and 28. Ribonucleic acid was prepared from each sample and real-time reverse transcription-PCR was used to determine the levels of IGF-I, IGFR-1, ER-alpha, and AR mRNA. Body weight of implanted steers, adjusted by using d-0 BW as a covariant, tended (P = 0.09) to be greater than that of control steers. On d 7 and 28, IGF-I mRNA levels were greater (58 and 78%, respectively; P < 0.009) in E2-implanted animals than in control steers. Similarly, on d 28 the LM IGF-I mRNA level was 65% greater (P = 0.017) in TBA/E2-implanted steers than in control animals. In contrast, the TBA implant did not increase (P = 0.99) LM IGF-I mRNA levels after 28 d of implantation. Muscle IGFR-1, AR, and ER-alpha mRNA levels were not different (P > 0.47) in any of the treated groups compared with the control group. These data suggest that E2 is responsible for the increased muscle IGF-I mRNA level observed in steers implanted with a combined TBA/E2 implant.

Intrahepatic expression of interferon alpha &amp; interferon alpha receptor m-RNA can be used as predictors to interferon response in HCV and HCC patients

Chronic hepatitis C Virus (HCV) is the leading cause of liver cirrhosis worldwide and in Egypt. Patients with cirrhosis secondary to chronic HCV infection are at increased risk for developing Hepatocellular carcinoma (HCC) in which Interferon therapy is the only effective anti-viral therapy. The current study aimed to investigate the expression IFN-&#945;and IFN-&#945;Receptor genes in liver biopsies from patients with HCV and HCC. Correlation of their expression with the clinical, histopathological progress of the disease and the effectiveness of IFN therapy in HCV patients after a period of 6 months follow-up was done. Expression of IFN-&#945; and IFN&#945;-Rc m-RNA was investigated by RT-PCR using liver biopsy specimens from 30 HCV patients including 7 patients complicated with HCC. Liver biopsies were also subjected to formalin fixation for complete histopathological examination. Ninety seven percent of patients expressed Interferon Alpha m-RNA while 30% only expressed Interferon Alpha Receptor m-RNA. Responders and non-responders to Interferon therapy were divided according to their HCV RNA after six-months follow up period of interferon therapy. Responders showed significantly lower mean age, better histopathological states and higher incidence of expression of IFN Alpha Receptor mRNA. Regardless of the response to interferon, histological activity index scores and the degree of fibrosis showed a significant inverse correlation to the presence of IFN&#945;-R m-RNA. IFN&#945;-R mRNA expression decreases with the histological progress of the disease, suggesting that lower expression of the IFN&#945;-Rc may be partially responsible for the unfavorable response to interferon in these patients. African Journal of Health Sciences Vol. 14 (1-2) 2007: pp. 86-96

Ontogenic profile of estrogen receptor alpha and beta mRNA and protein expression in fetal rat brain

It has been shown that increased estrogen can down-regulate its receptor, but there is no data to determine if that mechanism acts in the fetal brain as a consequence of high maternal estrogen levels. The aim of this study was to explore the expression of estrogen receptor (ER) in the developing fetal brain at 16, 19 and 21 days gestation (dg). The results revealed that both ERalpha and ERbeta isoforms, and some of their variants, were present in rat fetal brain at the transcript level and at the protein level. PCR results showed that the amount of ERalpha and ERbeta mRNA did not change significantly between 16, 19 and 21 dg; however, changes in protein expression were apparent. Two bands were detected for ERalpha protein by immunoblotting: the expression of the 73 kDa band, relative to the expression of actin, decreased significantly between 16 and 21 dg, while expression of the 67 kDa band did not change. Multiple variants of ERbeta were detected, including wild type ERbeta, ERbeta2 and ERbetaDelta5; the amounts of all decreased significantly, relative to the amount of actin, between 16 and 21 dg. A decrease in protein expression of some of the ER variants without an equivalent decrease in the amount of mRNA suggests that high levels of estrogen might triggered posttranslational modifications of ER, including the ER ubiquitin-dependent proteolysis. Overall, these findings suggest that the fetal brain might be less responsive to estrogens during late pregnancy in the rat, thereby minimizing the known harmful effects of high levels of circulating maternal estrogens.

Steroid receptor expression in the developing copulatory system of the green anole lizard ( Anolis carolinensis)

In adulthood, the copulatory system in male green anole lizards is characterized by the presence of two hemipenes, each controlled by ipsilateral muscles. These structures are present in both sexes early in development, but prior to hatching regress completely in females. Embryonic treatment with steroid hormones alters the morphology of the copulatory system, suggesting active roles for both androgens and estrogens in sexual differentiation. To elucidate the timing and sites of steroid hormone action in the embryonic copulatory system, the distributions of androgen receptor (AR) and estrogen receptor-alpha (ERα) mRNA expression were examined. In situ hybridization was conducted on the rostral tail of anoles at three stages spanning differentiation of the copulatory structures: embryonic days (E) 13, 18, and 24 (hatching occurs at approximately E34). At E13, males expressed significantly higher levels of AR mRNA in both hemipenes and muscles than did females, while females at the same age tended to express higher levels of ERα mRNA in these structures. By E18, hemipenes and copulatory muscles were regressed in most females, and were not present in any females at E24. In males, no effect of age was detected on the expression of either AR or ERα. These data suggest that peripheral copulatory structures in the embryonic anole are direct targets for the actions of both androgens and estrogens in sexual differentiation, consistent with the idea that estradiol facilitates regression in females and androgen promotes survival in males. However, the issue of whether or not a critical period exists remains open.

Regulation of Estrogen Receptor Alpha Expression in Breast Cancer Cells by Sulforaphane

Sulforaphane (SUL, 1‐isothiocyanato‐4‐(methylsulfinyl) butane) is an isothiocyanate derived from glucoraphanin that is abundant in broccoli and broccoli sprouts and which has a variety of potential chemopreventive actions. SUL is capable of inhibiting cancer at both early (initiation) and late (promotion) stages of experimental tumorigenesis. In this study we analyzed the effects of SUL on proliferation of several human breast cancer cell lines and evaluated the effect of SUL on estrogen receptor alpha (ERα) protein and mRNA expression in MCF‐7 cells. SUL at doses between 10 and 30 μM inhibited proliferation of neoplastic breast cell lines to a greater extent than non‐neoplastic MCF‐10F breast cells. SUL at doses between 2.5 and 30 μM decreased ERα protein expression in MCF‐7 cells. This effect of SUL on ERα expression was apparent under complete and estrogen‐depleted growth conditions and was accompanied by a decrease in progesterone receptor protein expression. At SUL doses of 30μM and greater, SUL treatment was associated with suppression of ERα mRNA transcription. In contrast, at doses of SUL less than 30μM, there was no effect of SUL on ERα mRNA expression. However, the proteasome inhibitor MG132 reversed the suppression of ERα protein levels in MCF‐7 cells treated with SUL at the lower SUL doses. Thus, the downregulation of ERα protein expression appears to be due to multiple dose‐dependent mechanisms involving changes in mRNA expression and proteasome activity. The effect of SUL on hormone‐responsiveness of human breast cancer cells warrants further study.

MicroRNAs in facial development

Cranial neural crest cell migration is a prerequisite for normal craniofacial morphogenesis in animals. A new study shows that the movement of a specific subset of cranial neural crest cells is exquisitely sensitive to concentrations of a microRNA that targets platelet-derived growth factor receptor alpha mRNA.

Oestrogen and progesterone receptor binding capacity and oestrogen receptor alpha expression (ERα mRNA) along the cervix of cycling ewes

The aim of the present work was to study the oestrogen receptor (ER) and progesterone receptor (PR) binding capacity and the oestrogen receptor alpha (ERalpha) mRNA concentration in cranial and caudal cervix during the ovine oestrous cycle. Cervical samples of synchronised Corriedale ewes were obtained on Day 1 (n=7), 6 (n=6) or 13 (n=7) after oestrus detection (Day 0). The ER and PR binding capacity by ligand-binding assay and the ERalpha mRNA concentration by solution hybridisation in both cranial and caudal zones of the cervix were determined. The ER and PR binding capacity were higher (P<0.005) on Day 1 than on Days 6 and 13 in both cranial and caudal zones. The ERalpha mRNA concentrations were higher (P<0.0001) on Day 1 than on Days 6 and 13 only in the caudal zone. The PR binding capacity and ERalpha mRNA concentration were higher (P<0.005) in the caudal than in the cranial zone on Day 1. The ER and PR expression in the ovine cervix varied during the oestrous cycle in agreement with the known upregulation exerted by oestrogen and downregulation exerted by progesterone. Differences in ER and PR expression along the longitudinal axis of the ovine cervix were found, reflecting histological and functional differences between the cranial and caudal zones.

Up‐regulation of estrogen receptor‐β expression during osteogenic differentiation of human periodontal ligament cells

Estrogen has been shown to up-regulate the expression of osteoblastic phenotypes of human periodontal ligament cells via binding to estrogen receptors and may also help periodontal tissue regeneration. However, which subtype of estrogen receptor (alpha or beta) is predominately expressed in human periodontal ligament cells, and how estrogen receptor expression is regulated during the osteogenic differentiation of human periodontal ligament cells, is still unclear. This study aimed to explore the expression and regulation of estrogen receptor subtypes in human periodontal ligament cells and during their osteogenic differentiation. Human periodontal ligament cells derived from 10 individual age-matched donors (five male and five female donors) were cultured. Human periodontal ligament cells under osteogenic induction (group M) and the corresponding controls (group C) were harvested on days 7, 14 and 21 for estrogen receptor detection. Both estrogen receptor-alpha and estrogen receptor-beta mRNAs were expressed in human periodontal ligament cells from all of the 10 donors. Protein only of estrogen receptor-beta (not of estrogen receptor-alpha) was detected and was shown to be located in the nuclei of human periodontal ligament cells. The expression levels of estrogen receptor-beta mRNA and protein from both male and female donors in group M were significantly higher compared with group C during the 21-d study period. In comparison, the expression level of estrogen receptor-alpha mRNA of the donors was not significantly different from that of the controls during osteogenic differentiation and no estrogen receptor-alpha protein was detected. The results suggest that estrogen receptor-beta may be the predominant subtype expressed in human periodontal ligament cells and may actively participate in the osteogenic differentiation process of human periodontal ligament cells, both in male and in female subjects.