Estrogen receptors and sex hormone binding globulin in neuronal cells and tissue.

Estrogens exert a critical influence on neuronal tissues and cells. As demonstrated in many clinical studies, estrogens are neuroprotective to the extent that they improve prognosis for women with neurodegenerative diseases. Unfortunately, we still do not know exactly how these effects are mediated. Fifty years ago the first estrogen receptor was found, but since then many other new pathways of estrogen action have been identified. This review describes several of these pathways of estrogen effects and provides some conclusions and correlations about these as determined by recent studies with nerve growth factor differentiated rat pheochromocytoma cell line.

Oxytocin and Steroid Actions.

Biosynthesis and secretion of the hypothalamic nonapeptide oxytocin largely depends on steroid hormones. Estradiol, corticosterone, and vitamin D seem to be the most prominent actors. Due to their lipophilic nature, systemic steroids are thought to be capable of crossing the blood-brain barrier, thus mediating central functions including neuroendocrine and behavioral control. The actual mode of action of steroids in hypothalamic circuitry is still unknown: Most of the oxytocinergic perikarya lack nuclear steroid receptors but express proteins suspected to be membrane receptors for steroids. Oxytocin expressing neurons contain enzymes important for intrinsic steroid metabolism. Furthermore, they produce and probably liberate specific steroid-binding globulins. Rapid responses to steroid hormones may involve these binding proteins and membrane-associated receptors, rather than classic nuclear receptors and genomic pathways. Neuroendocrine regulation, reproductive behaviors, and stress response seem to depend on these mechanisms.

Estrogen dependent expression of sex hormone binding globulin in PC 12 cells.

Rat pheochromocytoma PC 12 cells are known to develop features of dopaminergic neurons upon treatment with nerve growth factor. They express in part estrogen receptors α and ß, and G-protein coupled receptor 30. Estrogens promote development of these cells and exert neuroprotective effects. Here we treated differentiated PC 12 cells with physiological concentrations of 17-ß-estradiol. We observed with immunocytochemistry cytoplasmic staining for SHBG in a portion of these cells Double immunostaining for estrogen receptor-ß revealed that some PC 12 cells contained both antigens. Numbers of estrogen receptor-ß positive cells were significantly higher after estradiol treatment; an effect that was not altered by pretreatment of cultures with tamoxifen. With reverse transcriptase polymerase chain reaction we observed sex hormone binding globulin encoding transcripts indicating intrinsic expression of the steroid binding globulin. We conclude that estrogen treatment induces SHBG expression in differentiated PC12.

Sex hormone binding globulin and corticosteroid binding globulin as major effectors of steroid action.

Contrary to the long-held postulate of steroid-hormone binding globulin action, these protein carriers of steroids are major players in steroid actions in the body. This manuscript will focus on our work with sex hormone binding globulin (SHBG) and corticosteroid binding globulin (CBG) and demonstrate how they are actively involved in the uptake, intracellular transport, and possibly release of steroids from cells. This manuscript will also discuss our own findings that the steroid estradiol is taken up into the cell, as demonstrated by uptake of fluorescence labeled estradiol into Chinese hamster ovary (CHO) cells, and into the cytoplasm where it may have multiple actions that do not seem to involve the cell nucleus. This manuscript will focus mainly on events in two compartments of the cell, the plasma membrane and the cytoplasm.

Expression of corticosteroid-binding globulin in human astrocytoma cell line.

Glial tumor cells are known to be sensitive to glucocorticoids (GC) in vivo and in vitro. Here we studied the expression of corticosteroid-binding globulin (CBG) in the low-grade malignant human astrocytoma cell line 1321N1. CBG was observed in cytoplasm of most of these cells with immunocytochemistry. RT-PCR revealed the presence of the respective mRNA. Only scattered cells contained nuclear immunoreactivity for glucocorticoid receptor as visualized by double immunostaining. Immunoreactive CBG could be recovered from the supernatant of cultures that had been exposed to 10(-5) M cortisol. Our observations indicate the endogenous expression of CBG in 1321N1 cells which may occur independently from classical glucocorticoid receptor pathways. Cortisol seems to facilitate liberation of CBG in a paracrine manner, perhaps through membrane action of the steroid. Effects of adrenal steroids on proliferation and apoptosis of certain glial tumors may in part depend on these mechanisms.

Beacon-like/ubiquitin-5-like immunoreactivity is highly expressed in human hypothalamus and increased in haloperidol-treated schizophrenics and a rat model of schizophrenia.

The beacon gene is involved in the regulation of energy metabolism, food intake, and obesity. We localized its gene product, beacon-/ubiquitin 5-like immunoreactivity in brains of normal-weight, non-psychotic individuals, adipose (BMI over 32), non-psychotic individuals, and haloperidol-treated schizophrenics. The protein was found to be highly expressed in many neurons of the paraventricular and supraoptic hypothalamic nuclei. Besides, it was detected in neurons of other hypothalamic areas (suprachiasmatic, arcuate, and ventromedial nuclei) as well as outside the hypothalamus (Nuc. basalis Meynert, thalamus, hippocampus, and some neocortical areas). A morphometric analysis of beacon-immunoreactive hypothalamic and neocortical neurons revealed that compared to normal-weight controls in haloperidol-treated schizophrenics, there was a significant increase of protein-expressing supraoptic, paraventricular, and orbitofrontal neurons. However, a significant increase in beacon-expressing supraoptic neurons was also seen in adipose, non-psychotic individuals in comparison with normal-weight controls. Haloperidol at different doses has no effect on beacon expression in SHSY5Y neuroblastoma cells, which makes the assumption unlikely that haloperidol per se is responsible for the increased neuronal expression of the peptide in schizophrenics. In rats with a neonatal lesion of the ventral hippocampus (a widely used animal model of schizophrenia), we found an increased neuronal expression of beacon in the paraventricular and supraoptic nuclei. We suppose that elevated hypothalamic expression of beacon-like protein in non-obese schizophrenics is not primarily related to metabolic alterations, but to a certain role in schizophrenia, which is possibly unrelated to aspects of weight gain and obesity. The latter assumption finds some support by data obtained in rats with ventral hippocampus lesion.

Expression of sex hormone-binding globulin, oxytocin receptor, caveolin-1 and p21 in leiomyoma.

BACKGROUND: Interaction of sex hormone-binding globulin (SHBG) and oxytocin (OT) is among the factors that control smooth muscle proliferation and tumor growth through the oxytocin receptor (OTR). Also, a close functional interaction of OTR and caveolin-1 has been shown to modulate cell growth and proliferation. METHODS: We studied surgical samples from 23 leiomyoma patients (aged 33-66 years) with immunocytochemistry. Specimens from five patients (34-76 years), who had hysterectomy for other reasons, served as controls. Tissue samples were cut into serial 1-microm thick sections for co-localization of SHBG, OTR, proliferation marker p21 and caveolin-1. RESULTS: SHBG was found in smooth muscle cells in all samples. OTR staining occurred in most of these cells in myomas, while controls contained only scattered cells positive for OTR. There were no apparent differences in immunostaining for p21, while immunoreactivity for caveolin-1 was observed in most cells in myomas and in only few cells in controls. Caveolin-1 was mostly co-localized with SHBG and OTR in myoma samples whereas controls showed this co-localization only occasionally. CONCLUSIONS: Our observations indicate an interaction of SHBG and OTR, associated with caveolin-1, which may account in part for known non-genomic actions of ovarian steroids. Growth of leiomyomas may be linked to these mechanisms.

Identification of sex hormone-binding globulin in the human hypothalamus.

Gonadal steroids are known to influence hypothalamic functions through both genomic and non-genomic pathways. Sex hormone-binding globulin (SHBG) may act by a non-genomic mechanism independent of classical steroid receptors. Here we describe the immunocytochemical mapping of SHBG-containing neurons and nerve fibers in the human hypothalamus and infundibulum. Mass spectrometry and Western blot analysis were also used to characterize the biochemical characteristics of SHBG in the hypothalamus and cerebrospinal fluid (CSF) of humans. SHBG-immunoreactive neurons were observed in the supraoptic nucleus, the suprachiasmatic nucleus, the bed nucleus of the stria terminalis, paraventricular nucleus, arcuate nucleus, the perifornical region and the medial preoptic area in human brains. There were SHBG-immunoreactive axons in the median eminence and the infundibulum. A partial colocalization with oxytocin could be observed in the posterior pituitary lobe in consecutive semithin sections. We also found strong immunoreactivity for SHBG in epithelial cells of the choroid plexus and in a portion of the ependymal cells lining the third ventricle. Mass spectrometry showed that affinity-purified SHBG from the hypothalamus and choroid plexus is structurally similar to the SHBG identified in the CSF. The multiple localizations of SHBG suggest neurohypophyseal and neuroendocrine functions. The biochemical data suggest that CSF SHBG is of brain rather than blood origin.

Estradiol control of expression and levels of estradiol-binding proteins in the medial preoptic area, medial hypothalamus and pituitary.

The brains of mammals have at least three estradiol-binding proteins: estradiol receptor-alpha (ERalpha), ERbeta, and sex hormone-binding globulin (SHBG). In this study we compare the effects of estradiol treatment on the expression of mRNA for these three estradiol-binding proteins in two reproductively important brain areas, the medial preoptic area-anterior hypothalamus (MPOA-AH) and medial hypothalamus (MH) as well as in the hippocampus in ovariectomized rats, using the reverse transcriptase-polymerase chain reaction (RT-PCR). We also used surface-enhanced laser desorption ionization time of flight (SELDI-TOF) mass spectrometry (MS) to analyze the effects of estradiol in ovariectomized rats on SHBG levels in the MPOA-MH as well as the neurohypophysis. In vivo estradiol treatment in ovariectomized rats eliminated or significantly reduced expression of all three estradiol-binding proteins in both the MPOA-AH and MH. This change in ERalpha, ERbeta, and SHBG expression did not occur in the hippocampus. Both Northern blot and DNA sequence analysis confirmed the results of the RT-PCR for SHBG. SELDI-TOF MS analysis demonstrated that in vivo estradiol treatments resulted in dramatically decreased levels of SHBG in the hypothalamus and that a reduction in SHBG mRNA by estradiol treatment also resulted in a reduction in SHBG protein levels. Estradiol treatment also eliminated detectable SHBG from the neurohypophysis, suggesting that estradiol controls SHBG levels in this release site. That in vivo estradiol treatments had the same inhibitory effects on mRNA levels for SHBG and both ERs suggests similar translational control mechanisms for all three steroid-binding proteins in the brain. That estradiol treatments also reduced pituitary SHBG suggests that such treatment releases SHBG from the neurohypophysis.