Sex hormones and proteins involved in brain plasticity.

It is well known that peripheral sex steroid hormones cross the blood-brain barrier and control a broad spectrum of reproductive behaviors. However, their role in other essential brain functions was investigated since the 1980s, when the accumulation of pregnenolone and dehydroepiandrosterone in the brain of mammalian species was determined. Since then, numerous studies have demonstrated the participation of sex hormones in brain plasticity processes. Sex hormones through both genomic and non-genomic mechanisms of action are capable of inducing gene transcription or activating signaling cascades that result in the promotion of different physiological and pathological events of brain plasticity, such as remodeling or formation of dendritic spines, neurogenesis, synaptogenesis or myelination. In this chapter, we will present the effects of sex hormones and proteins involved in brain plasticity.

The Role of mPRδ and mPRε in Human Glioblastoma Cells: Expression, Hormonal Regulation, and Possible Clinical Outcome.

Glioblastomas (GBM) are the most frequent and aggressive primary tumor of the central nervous system. In recent years, it has been proposed that sex hormones such as progesterone play an essential role in GBM biology. Membrane progesterone receptors (mPRs) are a group of G protein-coupled receptors with a wide distribution and multiple functions in the organism. There are five mPRs subtypes described in humans: mPRα, mPRß, mPRγ, mPRδ, and mPRε. It has been reported that human-derived GBM cells express the mPRα, mPRß, and mPRγ subtypes, and that progesterone promotes GBM progression in part by mPRα specific activation; however, it is still unknown if mPRδ and mPRε are also expressed in this type of tumor cells. In this study, we characterized the expression and hormonal regulation of mPRδ and mPRε in human GBM cells. We also analyzed a set of biopsies from TCGA. We found that the expression of these receptors is dependent on the tumor's grade and that mPRδ expression is directly correlated to patients' survival while the opposite is observed for mPRε. By RT-qPCR, Western blot, and immunofluorescence, the expression of mPRδ and mPRε was detected for the first time in human GBM cells. An in silico analysis showed possible progesterone response elements in the promoter regions of mPRδ and mPRε, and progesterone treatments downregulated the expression of these receptors. Our results suggest that mPRδ and mPRε are expressed in human GBM cells and that they are relevant to GBM biology.

Tibolone Effects on Human Glioblastoma Cell Lines.

BACKGROUND: Ovarian steroid hormones are involved in modulating the growth of glioblastomas (the most common, aggressive, and lethal brain tumor) through the interaction with their intracellular receptors. Activation of sex hormone receptors is involved in glioblastomas progression. Tibolone (TIB) is a selective tissue estrogenic activity regulator widely prescribed to treat menopausal symptoms and to prevent bone lost. The effects of TIB on the growth of glioblastoma are unknown. AIM OF THE STUDY: To evaluate the effects of TIB on cell number, migration, and invasion of two derived human glioblastoma cell lines (U251 MG and U87), as well as the role of this steroid in estrogen and progesterone receptors activity and content. METHODS: U251-MG and U87 human glioblastoma cell lines were grown with different doses of TIB. The number of cells was determined and migration and invasion tests were carried out. Protein expression was performed by Western blot. RESULTS: We observed that TIB (10 nM) increased the number of cells by inducing proliferation with no effects on cell migration or invasion. The increase in cell proliferation induced by TIB was blocked by estrogen (ERs) or progesterone receptor (PRs) antagonists, ICI 182, 780 and RU 486, suggesting that these receptors mediate proliferating actions of TIB; TIB also modified the content of ERs and PRs by increasing ER-α, ER-ß, and PR-B, while decreased PR-A. CONCLUSION: Our results suggest that TIB increases cell number and proliferation of human glioblastoma cells through the regulation of ERs and PRs actions and content.

Hypothyroidism induces uterine hyperplasia and inflammation related to sex hormone receptors expression in virgin rabbits.

AIMS: In women, uterine alterations have been associated with sex steroid hormones. Sex hormones regulate the expression of thyroid hormone receptors (TRs) in the uterus, but an inverse link is unknown. We analyzed the impact of hypothyroidism on histological characteristics, vascular endothelial growth factor (VEGF-A), progesterone receptors (PR), estrogen receptors (ER), thyroid hormone receptors (TRs), perilipin (PLIN-A), and lipid content in the uterus of virgin rabbits. MAIN METHODS: Twelve Chinchilla-breed adult female rabbits were grouped into control (n = 6) and hypothyroid (n = 6; 0.02% of methimazole for 30 days). The thickness of endometrium and myometrium, number of uterine glands, and infiltration of immune cells were analyzed. The expression of VEGF-A, PR, ERα, and PLIN-A was determined by RT-PCR and western blot. The uterine content of triglycerides (TAG), total cholesterol (TC), and malondialdehyde (MDA) was quantified. KEY FINDINGS: Hypothyroidism promoted uterine hyperplasia and a high infiltration of immune cells into the endometrium, including macrophages CD163+. It also increased the expression of VEGF-A, TRA, and ERα-66 but reduced that of PR and ERα-46. The uterine content of PLIN-A, TAG, and TC was reduced, but that of MDA was augmented in hypothyroid rabbits. SIGNIFICANCE: Our results suggest that uterine hyperplasia and inflammation promoted by hypothyroidism should be related to changes in the VEGF-A, PR, ER, and TRs expression, as well as to modifications in the PLIN-A expression, lipid content, and oxidative status. These results suggest that hypothyroidism should affect the fertility of females.

Testosterone Promotes Glioblastoma Cell Proliferation, Migration, and Invasion Through Androgen Receptor Activation.

Glioblastomas (GBM) are the most frequent and aggressive human brain tumors due to their high capacity to migrate and invade normal brain tissue. Epidemiological data report that GBM occur in a greater proportion in men than in women (3:2), suggesting the participation of sex hormones in the development of these tumors. It has been reported an increase in testosterone (T) levels in patients with GBM. In addition, androgen receptor (AR) is overexpressed in human GBM, and genetic silencing of AR, and its pharmacological inhibition, induce GBM cell death in vivo and in vitro. However, the role of T in proliferation, migration and invasion in human GBM cell lines has not been evaluated. We observed that T increased the number of U87, U251, and D54 cells derived from human GBM due to an increase in cell proliferation. This induction was blocked with flutamide, an antagonist of AR. T also induced migration and invasion of GBM cells that flutamide partially blocked. These data suggest that T through AR contributes to the progression of GBM by promoting proliferation, migration, and invasion.

Progesterone promotes cell migration, invasion and cofilin activation in human astrocytoma cells.

Astrocytomas are the most common and aggressive primary brain tumors in humans. Invasiveness of these tumors has been attributed in part to deregulation of cell motility-dependent cytoskeletal dynamics that involves actin-binding proteins such as cofilin. Progesterone (P4) has been found to induce migration and invasion of cells derived from breast cancer and endothelium. However, the role of P4 in migration and invasion of astrocytoma cells as well as its effects on astrocytomas cytoskeleton remodeling is not known. In this work we evaluated these aspects in D54 and U251 cells derived from human astrocytomas from the highest degree of malignancy (grade IV, glioblastoma). Our results showed that in scratch-wound assays P4 increased the number of D54 and U251 cells migrating from 3 to 48 h. Both RU486, a P4 receptor (PR) antagonist, and an oligonucleotide antisense against PR significantly blocked P4 effects. Transwell assays showed that P4 significantly increased the number of invasive cells at 24h. As in the case of migration, this effect was blocked by RU486. Finally, by Western blotting, an increase in the cofilin/p-cofilin ratio at 15 and 30 min and a decrease at 30 and 60 min in U251 and D54 cells, respectively, was observed after P4, P4+RU486 and RU486 treatments. These data suggest that P4 increases human astrocytoma cells migration and invasion through its intracellular receptor, and that cofilin activation by P4 is independent of PR action.

Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity.

In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone.

C/EBPß Isoforms Expression in the Rat Brain during the Estrous Cycle.

The CCAAT/enhancer-binding protein beta (C/EBPß) is a transcription factor expressed in different areas of the brain that regulates the expression of several genes involved in cell differentiation and proliferation. This protein has three isoforms (LAP1, LAP2, and LIP) with different transcription activation potential. The role of female sex hormones in the expression pattern of C/EBPß isoforms in the rat brain has not yet been described. In this study we demonstrate by western blot that the expression of the three C/EBPß isoforms changes in different brain areas during the estrous cycle. In the cerebellum, LAP2 content diminished on diestrus and proestrus and LIP content diminished on proestrus and estrus days. In the prefrontal cortex, LIP content was higher on proestrus and estrus days. In the hippocampus, LAP isoforms presented a switch on diestrus day, since LAP1 content was the highest while that of LAP2 was the lowest. The LAP2 isoform was the most abundant one in all the three brain areas. The LAP/LIP ratio changed throughout the cycle and was tissue specific. These results suggest that C/EBPß isoforms expression changes in a tissue-specific manner in the rat brain due to the changes in sex steroid hormone levels presented during the estrous cycle.

Sex hormones and expression pattern of cytoskeletal proteins in the rat brain throughout pregnancy.

Pregnancy involves diverse changes in brain function that implicate a re-organization in neuronal cytoskeleton. In this physiological state, the brain is in contact with several hormones that it has never been exposed, as well as with very high levels of hormones that the brain has been in touch throughout life. Among the latter hormones are progesterone and estradiol which regulate several brain functions, including learning, memory, neuroprotection, and the display of sexual and maternal behavior. These functions involve changes in the structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity is regulated by estradiol and progesterone. We have found that the expression pattern of Microtubule Associated Protein 2, Tau, and Glial Fibrillary Acidic Protein changes in a tissue-specific manner in the brain of the rat throughout gestation and the start of lactation, suggesting that these proteins participate in the plastic changes observed in the brain during pregnancy. This article is part of a Special Issue entitled 'Pregnancy and Steroids'.

Expression pattern of Tau in the rat brain during pregnancy and the beginning of lactation.

Pregnancy involves changes in brain function that implicate a re-organization in neuronal cytoskeleton. We analyzed the content of the microtubule associated protein Tau (65kDa isoform) and its phosphorylated form (PhosphoTau) in several rat brain regions throughout pregnancy and on day 2 of lactation by Western blot. In hypothalamus the content of Tau increased on days 2 and 18 of gestation compared with days 14, 21 and in lactation. PhosphoTau content increased throughout pregnancy. In preoptic area Tau content did not show significant changes throughout pregnancy or lactation, however, the content of PhosphoTau presented a decrease on day 21 of gestation. In hippocampus Tau content decreased on day 14 until day 21 compared with day 2 of gestation, however, in lactation day 2 the content of Tau increased meanwhile PhosphoTau content progressively increased throughout pregnancy. In frontal cortex Tau content decreased on day 21 of gestation compared with days 2, 14 and 18, with an increase in lactation, whereas PhosphoTau did not show significant changes. In cerebellum Tau protein decreased on days 14, 18 and 21 of pregnancy with an increase in lactation. PhosphoTau content increased throughout pregnancy and on day 2 of lactation. PhosphoTau/Tau ratio changes in each brain area along pregnancy and in lactation. Our data suggest that Tau expression and its phosphorylation pattern change in a tissue-dependent manner throughout pregnancy and the beginning of lactation in the rat brain.

Short- and long-term treatment with estradiol or progesterone modifies the expression of GFAP, MAP2 and Tau in prefrontal cortex and hippocampus.

AIMS: We analyzed the effects of the short- and long-term administration of estradiol (E2) or progesterone (P4) after ovariectomy on the expression of MAP2, Tau and GFAP in prefrontal cortex and hippocampus. MAIN METHODS: Sprague Dawley rats were ovariectomized and immediately treated with E2 or P4 for 2 or 18 weeks. At the end of treatments, hippocampus and prefrontal cortex were excised, proteins were extracted and MAP2, Tau and GFAP were analyzed by Western blot. KEY FINDINGS: MAP2 and Tau content was not modified by E2 in the prefrontal cortex. On the contrary, P4 decreased MAP2 content after a short-term treatment, while it increased that of MAP2 and TAU in this brain region after a long-term treatment. E2 increased MAP2 content in hippocampus. In this region, short-term administration of P4 increased that of MAP2. GFAP content was diminished after a long-term administration of P4 in hippocampus. SIGNIFICANCE: Current data emphasize the importance of short- and long-term sex steroid treatment on neuronal and glial cytoskeletal proteins expression.