Ignacio Camacho-Arroyo

Changes in Progesterone Receptor Isoforms Content in the Rat Brain During the Oestrous Cycle and After Oestradiol and Progesterone Treatments

We studied the effects of oestradiol and progesterone on progesterone receptor (PR) isoform content in the brain of ovariectomized rats and in intact rats during the oestrous cycle by Western blot analysis. In the hypothalamus and the preoptic area of ovariectomized rats, PR‐A and PR‐B content was increased by oestradiol, whereas progesterone significantly diminished the content of both PR isoforms after 3 h of treatment in the hypothalamus, but not in the preoptic area. In the hippocampus, only PR‐A content was significantly increased by oestradiol while progesterone significantly diminished it after 12 h of treatment. In the frontal cortex, no treatment significantly modified PR isoform content. During the oestrous cycle, the lowest content of PR isoforms in the hypothalamus was observed on diestrus day and, by contrast, in the preoptic area, the highest content of both PR isoforms was observed on diestrus day. We observed no changes in PR isoform content in the hippocampus during the oestrous cycle. These results indicate that the expression of PR isoforms is differentially regulated by sex steroid hormones in a regionally specific manner.

Ether à go-go Potassium Channels as Human Cervical Cancer Markers

Ether à go-go (EAG) potassium channels display oncogenic properties. In normal tissues, EAG mRNA is almost exclusively expressed in brain, but it is expressed in several somatic cancer cell lines, including HeLa, from cervix. Antisense experiments against eag reduce cell proliferation in some cancer cell lines, and inhibition of EAG-mediated currents has been suggested to decrease cell proliferation in a melanoma cell line. Because of the potential clinical relevance of EAG, we investigated EAG mRNA expression in the following fresh samples from human uterine cervix: 5 primary cultures obtained from cancerous biopsies, 1 cancerous fresh tissue, and 12 biopsies of control normal tissue. All of the control cervical samples came from patients with negative pap smears. Reverse transcription-PCR and Southern-blot experiments revealed eag expression in 100% of the cancerous samples and in 33% of the normal biopsies. Immunochemistry experiments showed the presence of EAG channel protein in cells from the primary cultures and in cervical cancer biopsies sections from the same patients. In addition, we looked for EAG-mediated currents in the cultures from cervical cancer cells. Here we show for the first time EAG channel activity in human tumors. Patch-clamp recordings showed typical EAG-mediated currents modulated by magnesium and displaying a pronounced Cole-Moore shift. Because EAG expression and channel activity have been suggested to be important in cell proliferation, our findings strongly support the idea of considering EAG as a tumor marker as well as a potential membrane therapeutic target for cervical cancer.

Sex differences in the regulation of progesterone receptor isoforms expression in the rat brain

We studied the effects of estradiol (E2) and progesterone (P) on progesterone receptor (PR) isoforms gene expression in the brain of ovariectomised female and gonadectomised male rats by RT-PCR analysis. In female rats, the expression of both PR isoforms was induced by E2 and down-regulated by P in the hypothalamus, whereas in the preoptic area these changes were only observed in PR-B isoform. On the contrary, in the hippocampus and the olfactory bulb, PR-A was the isoform induced by E2. In these regions, P did not modify the expression of any PR isoform. In the cerebellum and the frontal cortex of female rats, no treatment modified PR isoforms expression. In contrast with female rats, in the male rat brain, PR isoforms expression was only modified in the cerebellum, where PR-A was induced by E2. These results demonstrate a clear sexual dimorphism in the regulation of PR isoforms expression by sex steroid hormones in the rat brain, suggesting that this sex difference contributes to the sexually dimorphic effects of P in the rat brain.

Progesterone receptor isoforms are differentially regulated by sex steroids in the rat forebrain.

WE studied the effects of estradiol (E2) and progesterone (P4) on expression of genes coding for PR isoforms in the forebrain of ovariectomized rats by RT-PCR analysis. In the hypothalamus the expression of both PR isoforms was induced by E2 and down-regulated by P4. In the preoptic area these changes were only observed in the PR-B isoform. In contrast, in the hippocampus PR induction by E2 was only observed for PR-A. In this region P4 did not modify the expression of any PR isoform. These results indicate that PR isoforms expression is differentially regulated by sex steroid hormones in distinct forebrain regions and suggest that the tissue-specific regulation of either PR-A or PR-B may be involved in the physiological actions of P4 upon the rat brain.

Progesterone receptor isoforms expression pattern in the rat brain during the estrotts cycle

Progesterone receptor (PR) isoforms expression was determined in the hypothalamus, the preoptic area, the hippocampus and the frontal cerebral cortex of the rat at 12:00 h on each day of the estrous cycle by using reverse transcription coupled to polymerase chain reaction. Rats under a 14:10 h light-dark cycle, with lights on at 06:00 h were used. We found that PR-B isoform was predominant in the hypothalamus, the preoptic area and the frontal cerebral cortex. Both PR isoforms were similarly expressed in the hippocampus. The highest PR-B expression was found on proestrus day in the hypothalamus; on metestrus in the preoptic area; and on diestrus in the frontal cortex. We observed no changes in PR isoforms expression in the hippocampus during the estrous cycle. These results indicate that PR isoforms expression is differentially regulated during the estrous cycle in distinct brain regions and that PR-B may be involved in progesterone actions upon the hypothalamus, the preoptic area and the frontal cortex of the rat.

Role of Sex Steroid Hormones in Bacterial-Host Interactions

Sex steroid hormones play important physiological roles in reproductive and nonreproductive tissues, including immune cells. These hormones exert their functions by binding to either specific intracellular receptors that act as ligand-dependent transcription factors or membrane receptors that stimulate several signal transduction pathways. The elevated susceptibility of males to bacterial infections can be related to the usually lower immune responses presented in males as compared to females. This dimorphic sex difference is mainly due to the differential modulation of the immune system by sex steroid hormones through the control of proinflammatory and anti-inflammatory cytokines expression, as well as Toll-like receptors (TLRs) expression and antibody production. Besides, sex hormones can also affect the metabolism, growth, or virulence of pathogenic bacteria. In turn, pathogenic, microbiota, and environmental bacteria are able to metabolize and degrade steroid hormones and their related compounds. All these data suggest that sex steroid hormones play a key role in the modulation of bacterial-host interactions.

Progesterone receptor isoforms expression in the prepuberal and adult male rat brain

Progesterone receptor (PR) isoforms expression was determined in several regions of the prepuberal and adult male rat brain by using reverse transcription coupled to polymerase chain reaction. Rats under a 14:10-h light-dark cycle, with lights on at 0600 h were used. We found that in the hypothalamus of prepuberal animals the expression of both PR isoforms was similar, whereas PR-A expression was higher than that of PR-B in adults. In the cerebellum PR-B expression was predominant in both prepuberal and adult rats. In both ages PR-A and PR-B exhibited a non-significant tendency to be predominant in the hippocampus and the preoptic area respectively. In the frontal cortex and the olfactory bulb PR isoforms were expressed at a similar level. These results indicate a differential expression pattern of PR isoforms in the male rat brain and suggest that the tissue-specific expression of PR-A and PR-B is important for the appropriate response of each cerebral region to progesterone.

Estrogens and Human Papilloma Virus Oncogenes Regulate Human Ether-à-go-go-1 Potassium Channel Expression

Ether-à-go-go-1 (Eag1) potassium channels are potential tools for detection and therapy of numerous cancers. Here, we show human Eag1 (hEag1) regulation by cancer-associated factors. We studied hEag1 gene expression and its regulation by estradiol, antiestrogens, and human papillomavirus (HPV) oncogenes (E6/E7). Primary cultures from normal placentas and cervical cancer tissues; tumor cell lines from cervix, choriocarcinoma, keratinocytes, and lung; and normal cell lines from vascular endothelium, keratinocytes, and lung were used. Reverse transcription-PCR (RT-PCR) experiments and Southern blot analysis showed Eag1 expression in all of the cancer cell types, normal trophoblasts, and vascular endothelium, in contrast to normal keratinocytes and lung cells. Estradiol and antiestrogens regulated Eag1 in a cell type-dependent manner. Real-time RT-PCR experiments in HeLa cells showed that Eag1 estrogenic regulation was strongly associated with the expression of estrogen receptor-alpha. Eag1 protein was detected by monoclonal antibodies in normal placenta and placental blood vessels. Patch-clamp recordings in normal trophoblasts treated with estradiol exhibited potassium currents resembling Eag1 channel activity. Eag1 gene expression in keratinocytes depended either on cellular immortalization or the presence of HPV oncogenes. Eag1 protein was found in keratinocytes transfected with E6/E7 HPV oncogenes. Cell proliferation of E6/E7 keratinocytes was decreased by Eag1 antibodies inhibiting channel activity and by the nonspecific Eag1 inhibitors imipramine and astemizole; the latter also increased apoptosis. Our results propose novel oncogenic mechanisms of estrogen/antiestrogen use and HPV infection. We also suggest Eag1 as an early indicator of cell proliferation leading to malignancies and a therapeutic target at early stages of cellular hyperproliferation.

Estradiol and progesterone modify microtubule associated protein 2 content in the rat hippocampus.

The molecular mechanisms involved in the regulation of synaptic plasticity and neuroprotection by estradiol (E(2)) and progesterone (P(4)) are unknown. Because these processes involve changes in cytoskeleton organization, we studied the effects of E(2) and P(4) in the expression of two cytoskeletal proteins: microtubule associated protein 2 (MAP2) and tau in the hippocampus and the frontal cortex of ovariectomized adult rats. While tau expression was unaffected by E(2) and P(4), an increase in MAP2 protein content in the hippocampus but not in the cortex was observed after E(2) and P(4) treatments. Interestingly, these steroids did not modify MAP2 mRNA content in the hippocampus. These data suggest that MAP2 is involved in the structural changes induced by E(2) and P(4) in the rat hippocampus, and that MAP2 expression is regulated by these steroid hormones at a postranscriptional level.

Progesterone receptor isoforms expression pattern in human astrocytomas

Progesterone receptors (PR) have been detected in human astrocytomas; however, the expression pattern of PR isoforms in these brain tumors is unknown. Progesterone receptor isoforms expression was studied in 13 biopsies of astrocytomas (6 grade III, and 7 grade IV) from adult Mexican patients by using reverse transcription-polymerase chain reaction and immunohistochemistry. Progesterone receptor expression was observed at mRNA and at protein levels in 66% and 83% of astrocytomas grade III, respectively, whereas 100% of astrocytomas grade IV expressed PR. Almost all PR mRNA content in astrocytomas grades III and IV corresponded to PR-B. The number of immunoreactive cells expressing PR-B was higher than that expressing PR-A in 73% of the cases. Estrogen receptor-alpha protein was only observed in 33% of astrocytomas grade III, whereas no astrocytomas grade IV expressed it. These data suggest that PR-B is the predominant isoform expressed in human astrocytomas grades III and IV, and that estrogen receptor-alpha is not expressed in astrocytomas grade IV.