Isabel Méndez

Calcitriol affects hCG gene transcription in cultured human syncytiotrophoblasts

BackgroundIn pregnancy, maternal serum concentrations of calcitriol significantly rise as a result of increased renal and placental contribution in order to assure calcium supply for the developing fetus. Considering that placenta is a site for vitamin D activation, and the versatility and potency of calcitriol, it is feasible that this hormone participates in fetal/placental development and physiology. In the present work we studied calcitriol actions upon human chorionic gonadotropin (hCG) secretion and expression in cultured trophoblasts, as well as vitamin D receptor (VDR) and CYP27B1 immunolocalization in placental villi.MethodsQuantification of hCG in culture media was performed by immunoassay. Expression studies were carried out by real time PCR. Analysis of CYP27B1 and VDR localization in placental slides were performed by immunohistochemistry. Statistical significance was established by one way ANOVA using Tukey test for comparisons.ResultsCalcitriol regulated hCG in a time-dependent manner: at 6 h the secosteroid stimulated hCG, whereas longer incubations (24 h) showed opposite effects. Interestingly, calcitriol stimulatory effects on hCG were accompanied by an increase in intracellular cAMP content and were abolished by pre-incubation of the cells with a selective protein kinase A inhibitor. Immunohistochemical techniques showed differential VDR localization in the syncytiotrophoblast layer or in the vascular smooth muscle cells depending on the epitope to which the antibodies were raised (specific for the carboxy- or amino-terminal regions, respectively). CYP27B1 was immunolocalized in the syncytiotrophoblast layer of placental villi.ConclusionThe presence and location of the vitamin D activating enzyme CYP27B1 as well as the specific receptor for vitamin D were shown in placental sections. The latter, together with findings demonstrating specific effects of calcitriol acting through the VDR and the cAMP/PKA signaling pathway upon hCG expression and secretion, indicate that there is a functional vitamin D endocrine system in the placenta, and recognize calcitriol as an autocrine regulator of hCG.

Regulation of Vitamin D hydroxylases gene expression by 1,25-dihydroxyvitamin D3 and cyclic AMP in cultured human syncytiotrophoblasts.

Human placenta synthesizes and metabolizes 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)/calcitriol] through the activity of 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1) and 1,25(OH)(2)D(3)-24-hydroxylase (CYP24A1), the two key enzymes for Vitamin D metabolism. In this study, calcitriol rapidly generated intracellular cAMP accumulation in cultured human syncytiotrophoblast cells, which in turn enhanced hCG secretion, a marker of trophoblast endocrine activity. The effects of 1,25(OH)(2)D(3) upon the expression of CYP27B1 and CYP24A1 were also investigated. 1,25(OH)(2)D(3) and activators of the PKA signaling system decreased the expression of CYP27B1, whereas increased CYP24A1 gene transcription. The use of a selective inhibitor of PKA (H-89) prevented the effects of calcitriol on CYP27B1 gene and hCG secretion, but not on CYP24A1 transcription. Addition of ZK 159222, a Vitamin D receptor (VDR) antagonist, blocked the calcitriol-mediated upregulation of 24-hydroxylase gene expression but did not affect calcitriol-induced downregulation of CYP27B1 gene or hCG stimulation. In addition, our study also demonstrated a role of calcitonin on Vitamin D hydroxylases gene regulation in placenta. The overall data suggest that calcitriol downregulates CYP27B1 expression via a cAMP-dependent signaling pathway, whereas upregulates 24-hydroxylase gene expression through a VDR-dependent mechanism.

Prolactin promotes oxytocin and vasopressin release by activating neuronal nitric oxide synthase in the supraoptic and paraventricular nuclei

Prolactin (PRL) stimulates the secretion of oxytocin (OXT) and arginine AVP as part of the maternal adaptations facilitating parturition and lactation. Both neurohormones are under the regulation of nitric oxide. Here, we investigate whether the activation of neuronal nitric oxide synthase (nNOS) in the hypothalamo-neurohypophyseal system mediates the effect of PRL on OXT and AVP release and whether these effects operate in males. Plasma levels of OXT and AVP were measured in male rats after the intracerebroventricular injection of PRL or after inducing hyperprolactinemia by placing two anterior pituitary glands under the kidney capsule. NOS activity was evaluated in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei by NADPH-diaphorase histochemistry and in hypothalamic extracts by the phosphorylation/inactivation of nNOS at Ser(847). Elevated central and systemic PRL correlated with increased NOS activity in the PVN and SON and with higher OXT and AVP circulating levels. Notably, treatment with 7-nitroindazole, a selective inhibitor of nNOS, prevented PRL-induced stimulation of the release of both neurohormones. Also, phosphorylation of nNOS was reduced in hyperprolactinemic rats, and treatment with bromocriptine, an inhibitor of anterior pituitary PRL secretion, suppressed this effect. These findings suggest that PRL enhances nNOS activity in the PVN and SON, thereby contributing to the regulation of OXT and AVP release. This mechanism likely contributes to the regulation of processes beyond those of female reproduction.

Prolactin promotes cartilage survival and attenuates inflammation in inflammatory arthritis

Chondrocytes are the only cells in cartilage, and their death by apoptosis contributes to cartilage loss in inflammatory joint diseases, such as rheumatoid arthritis (RA). A putative therapeutic intervention for RA is the inhibition of apoptosis-mediated cartilage degradation. The hormone prolactin (PRL) frequently increases in the circulation of patients with RA, but the role of hyperprolactinemia in disease activity is unclear. Here, we demonstrate that PRL inhibits the apoptosis of cultured chondrocytes in response to a mixture of proinflammatory cytokines (TNF-α, IL-1β, and IFN-γ) by preventing the induction of p53 and decreasing the BAX/BCL-2 ratio through a NO-independent, JAK2/STAT3-dependent pathway. Local treatment with PRL or increasing PRL circulating levels also prevented chondrocyte apoptosis evoked by injecting cytokines into the knee joints of rats, whereas the proapoptotic effect of cytokines was enhanced in PRL receptor-null (Prlr(-/-)) mice. Moreover, eliciting hyperprolactinemia in rats before or after inducing the adjuvant model of inflammatory arthritis reduced chondrocyte apoptosis, proinflammatory cytokine expression, pannus formation, bone erosion, joint swelling, and pain. These results reveal the protective effect of PRL against inflammation-induced chondrocyte apoptosis and the therapeutic potential of hyperprolactinemia to reduce permanent joint damage and inflammation in RA.

Mechanistic Effects of Calcitriol in Cancer Biology

Besides its classical biological effects on calcium and phosphorus homeostasis, calcitriol, the active vitamin D metabolite, has a broad variety of actions including anticancer effects that are mediated either transcriptionally and/or via non-genomic pathways. In the context of cancer, calcitriol regulates the cell cycle, induces apoptosis, promotes cell differentiation and acts as anti-inflammatory factor within the tumor microenvironment. In this review, we address the different mechanisms of action involved in the antineoplastic effects of calcitriol.

Hypothalamic Dopaminergic Tone and Prolactin Bioactivity in Women with Polycystic Ovary Syndrome

BACKGROUND The present study was carried out to investigate the functional significance of the reduced dopaminergic tone in subjects affected with polycystic ovary syndrome (PCOS). METHODS Our group evaluated the response of pituitary PRL, LH, FSH, and TSH to the administration of a single 10-mg oral dose of the dopamine (DA) receptor antagonist metoclopramide in lean (n = 7) and obese (n = 8) PCOS women and in 11 regularly cycling age- and weight-matched controls (six lean and five obese). In addition, circulating PRL bioactivity was evaluated by its mitogenic activity on a lymphoma cell bioassay. RESULTS Oral administration of metoclopramide resulted in a significant increase in serum PRL in all subjects; however, the highest increments, regardless of body mass index (BMI), were observed in control women (p <0.005). Measurements of PRL mitogenic activity on the Nb2 lymphoma cell bioassay revealed a significant increase in the bioactive/immunoreactive (B/I) ratio of PRL under basal and stimulated conditions in obese PCOS subjects (p <0.05). Mean fasting glucose/insulin and glucose/insulin-AUC ratios were significantly lower (p <0.001) in obese PCOS when compared with all other groups. CONCLUSIONS These data support the existence of low DA hypothalamic tone in PCOS women that is likely involved in the inappropriate LH and PRL secretion frequently seen in this syndrome. In addition, our results suggest changes in PRL bioactivity in obese PCOS that may play a role in the development of hyperinsulinemia; however, whether PRL has a functional significance in the development of the metabolic disturbances frequently seen in PCOS remains to be elucidated.

Evidence for an altered luteinizing hormone sensitivity to naloxone in pathological hyperprolactinaemia

OBJECTIVE The underlying mechanisms involved in the pathogenesis of amenorrhoea in hyperprolactlnaemic states still remain unclear. Conflicting information exists on the role of endogenous opiates on gonadotrophin disturbances in this pathological condition. In this study we have undertaken a detailed investigation of LH and PRL secretion before and during administration of naloxone, an opioid receptor blocker, in hyperprolactinaemic women with or without ovarian function In order to assess the role of ovarian steroids upon naloxone induced LH and PRL release.

Prolactin-derived vasoinhibins increase anxiety- and depression-related behaviors

The hormone prolactin (PRL) regulates neuroendocrine and emotional stress responses. It is found in the hypothalamus, where the protein is partially cleaved to vasoinhibins, a family of N-terminal antiangiogenic PRL fragments ranging from 14 to 18kDa molecular masses, with unknown effects on the stress response. Here, we show that the intracerebroventricular administration of a recombinant vasoinhibin, containing the first 123 amino acids of human PRL that correspond to a 14kDa PRL, exerts anxiogenic and depressive-like effects detected in the elevated plus-maze, the open field, and the forced swimming tests. To investigate whether stressor exposure affects the generation of vasoinhibins in the hypothalamus, the concentrations of PRL mRNA, PRL, and vasoinhibins were evaluated in hypothalamic extracts of virgin female rats immobilized for 30min at different time points after stress onset. The hypothalamic levels of PRL mRNA and protein were higher at 60min but declined at 360min to levels seen in non-stressed animals. The elevation of hypothalamic PRL did not correlate with the stress-induced increase in circulating PRL levels, nor was it modified by blocking adenohypophyseal PRL secretion with bromocriptine. A vasoinhibin having an electrophoretic migration rate corresponding to 17kDa was detected in the hypothalamus. Despite the elevation in hypothalamic PRL, the levels of this hypothalamic vasoinhibin were similar in stressed and non-stressed rats. Stress reduced the rate of cleavage of PRL to this vasoinhibin as shown by the incubation of recombinant PRL with hypothalamic extracts from stressed rats. These results suggest that vasoinhibins are potent anxiogenic and depressive factors and that stress increases PRL levels in the hypothalamus partly by reducing its conversion to vasoinhibins. The reciprocal interplay between PRL and vasoinhibins may represent an effective mechanism to regulate anxiety and depression.

Maternal Protein Restriction During Pregnancy and/or Lactation Negatively Affects Follicular Ovarian Development and Steroidogenesis in the Prepubertal Rat Offspring

BACKGROUND AND AIMS Maternal protein restriction during rat pregnancy and lactation is associated with alterations in reproductive function of female offspring including delayed onset of puberty, decreased fertility and premature reproductive aging. These alterations may be related to ovarian prepubertal development, distribution of follicle populations and their steroidogenic capacities. We undertook this study to evaluate the ovarian function of prepubertal female offspring exposed to maternal protein restriction during pregnancy and/or lactation. METHODS Adult female Wistar rats were fed a control (C-20% casein diet) or restricted isocaloric diet (R-10% casein) during pregnancy--first letter--and lactation--second letter, to form four groups, CC, RR, CR, RC. Ovaries were collected from 21-day-old female offspring. Preantral and antral follicles were quantified and mRNA expression of key genes involved in follicular development and steroidogenesis (gonadotropin receptors, StAR, P450scc and P450 aromatase) was evaluated. Serum gonadotropin levels were measured. RESULTS Significantly decreased numbers of preantral and antral follicles were observed in CR and RC ovaries compared with CC. LH levels were lower and FSH higher in CR pups. mRNA expression of LH receptor (LH-R) was decreased in RR in comparison with the other groups. CR and RC expressed higher StAR, RC increased and RR decreased P450scc, whereas RR and CR decreased aromatase expression in comparison with CC. CONCLUSIONS Maternal protein restriction influences prepubertal ovarian follicular number and steroidogenic function in the rat offspring, although RR and CR nutritional schemes have similar outcomes, the mechanisms affecting ovarian function are at different levels of the hypothalamic-pituitary-ovarian axis.

Calcitriol stimulates prolactin expression in non-activated human peripheral blood mononuclear cells: Breaking paradigms

Calcitriol, the hormonal form of vitamin D(3), exerts immunomodulatory effects through the vitamin D(3) receptor (VDR) and increases prolactin (PRL) expression in the pituitary and decidua. Nevertheless, the effects of calcitriol upon lymphocyte PRL have not been evaluated. Therefore, we investigated calcitriol effects upon PRL in resting and phytohemagglutinin-activated human peripheral blood mononuclear cells (PBMNC) and Jurkat T lymphoma cells. Immunoblots showed constitutive expression of the 50-kDa VDR species in activated PBMNC and Jurkat cells, while a 75-kDa species was recognized in both resting and activated-PBMNC. Only in resting PBMNC calcitriol significantly stimulated PRL expression in a dose-dependent manner. The positive control CYP24A1, a highly VDR-responsive gene, was stimulated by calcitriol, effect that was stronger in resting than in activated-PBMNC (P<0.05), and without effect in Jurkat cells. Calcitriol upregulation of PRL and CYP24A1 was significantly inhibited by the VDR antagonist TEI-9647. EMSA showed that resting PBMNC contain a protein that binds to DR3-type VDRE. Cell activation reduced basal CYP24A1 while induced CYP27B1, VDR and pregnane X receptor (PXR) expression. In summary, calcitriol stimulated PRL and CYP24A1 gene expression in quiescent lymphocytes through a VDR-mediated mechanism. Our results suggest that the 75-kDa VDR species could be participating in calcitriol-mediated effects, and that activation induces factors such as PXR that restrain VDR transcriptional processes. This study supports the presence of a functional VDR in quiescent lymphocytes, providing evidence to reevaluate the VDR paradigm that assumes that lymphocytes respond to calcitriol only after activation. Altogether, our results offer new insights into the mechanisms whereby PRL is regulated in immune cells.