Ali Halhali

Estradiol and progesterone synthesis in human placenta is stimulated by calcitriol

Calcitriol exerts a diverse range of biological actions including the control of growth and cell differentiation, modulation of hormone secretion, and regulation of reproductive function. The placenta synthesizes calcitriol through the expression of CYP27B1, but little is known about local actions of this hormone in the fetoplacental unit. The objective of this study was to investigate the effects of calcitriol upon progesterone (P(4)) and estradiol (E(2)) secretion in trophoblasts cultured from term human placenta. Cells were incubated in the presence of calcitriol for 18 h and pregnenolone or androstenedione were subsequently added as substrates for the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) or P450-aromatase (CYP19), respectively. Calcitriol stimulated in a dose-dependent manner E(2) and P(4) secretion. The use of a selective inhibitor of PKA prevented the effects of calcitriol upon E(2) secretion, but not on P(4). These results show that calcitriol is a physiological regulator of placental E(2) and P(4) production and suggest a novel role for calcitriol upon placental steroidogenesis.

Calcitriol inhibits TNF-α-induced inflammatory cytokines in human trophoblasts

Elevated placental proinflammatory cytokine release is associated with miscarriage, preterm labor and preeclampsia. Specifically, tumor necrosis factor-alpha (TNF-alpha)-induced cytokines may threaten pregnancy outcome. Since trophoblasts produce calcitriol, a hormone with strong immunosuppressive properties, we assessed the effects of this secosteroid on inflammatory cytokines induced in trophoblasts by challenge with TNF-alpha. The effects of calcitriol on synthesis of mRNAs encoding interleukin-6 (IL-6), interferon-gamma (IFN-gamma), and TNF-alpha were measured by real time RT-PCR. Secreted cytokines were quantified by ELISA. The effects of TNF-alpha on CYP24A1, chorionic gonadotropin (hCG), 3beta-hydroxysteroid dehydrogenase (HSD3B1) and P(450)-aromatase (CYP19) mRNA expression were also studied. TNF-alpha stimulated IL-6, IFN-gamma and its own expression more than 3-fold over controls (P<0.05). Calcitriol inhibited the expression profile of inflammatory cytokine genes in a dose-response manner (P<0.05). This effect was prevented by addition of the vitamin D receptor antagonist TEI-9647. TNF-alpha also significantly inhibited expression of hCG, HSD3B1 and CYP19 genes, and stimulated CYP24A1 gene expression. These data show that calcitriol prevents TNF-alpha induction of inflammatory cytokines through a process likely to be mediated by the vitamin D receptor. We conclude that TNF-alpha inhibits placental hormone synthesis and stimulates calcitriol catabolism by regulating enzymes involved in these processes.

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.

Newborn Birth Weight Correlates with Placental Zinc, Umbilical Insulin-Like Growth Factor I, and Leptin Levels in Preeclampsia

BACKGROUND The aim of the present study was to determine whether newborn birth weights were associated with insulin-like growth factor I (IGF-I), zinc, and leptin in preeclamptic and normotensive pregnancies. METHODS This study was done cross-sectionally and included 15 women with moderate preeclampsia (PE group) and 11 normotensive pregnant women (NT group) of similar gestational age. Maternal and umbilical cord serum levels of zinc, IGF-I, IGF binding proteins (IGFBPs), leptin, and placental zinc were assessed in each group. RESULTS As compared with the NT group, the PE group had significantly (p <0.05) lower newborn birth weight [3.33 (3.11-3.55) vs. 2.80 (2.40-3.17) kg], maternal IGF-I [303 (276-364) vs. 198 (153-244) ng/mL], and umbilical IGF-I [87 (71-126) vs. 44 (26-98) ng/mL]. In addition, low IGFBP-3 and high IGFBP-1 were observed in the PE group. In the PE group, birth weight correlated positively with placental zinc (rho = 0.56, p = 0.04), maternal IGF-I (rho = 0.76, p = 0.004), umbilical cord IGF-I (rho = 0.75, p = 0.005), and umbilical cord leptin (rho = 0.61, p = 0.02) levels. Stepwise multiple regression analysis showed that among these variables, umbilical IGF-I was the major predictor of birth weight. In the NT group, birth weight did not correlate with any of these variables. CONCLUSIONS The present study indicates that moderate preeclampsia is associated with low newborn birth weight, IGF-I, IGFBP-3, and high IGFBP-1 without significant changes in zinc and leptin levels. In addition, umbilical IGF-I was the major predictor of newborn birth weight.

Decreased fractional urinary calcium excretion and serum 1,25-dihydroxyvitamin D and IGF-I levels in preeclampsia.

During preeclampsia several alterations of calcium metabolism have been described, the most common of them is hypocalciuria, which pathophysiology is still unclear. In order to assess the contribution of calciotropic hormones to urinary calcium excretion, a cross-sectional study was done including 26 preeclamptic Mexican women (PE group) and 26 normotensive control pregnant women (NT group). Total and fractional urinary calcium excretion were significantly lower (P<0.0001) in the PE group than in the NT group (82+/-7 versus 171+/-7 mg/24h and 0.62+/-0.38 versus 1.38+/-0.71%, respectively), without significant differences in creatinine clearance, urinary sodium excretion and phosphate tubular reabsorption. In addition, serum 1,25-(OH)(2)D and IGF-I levels were significantly (P<0.05) lower in the PE than in NT group (43+/-9 versus 50+/-9 pg/mL and 195+/-67 versus 293+/-105 ng/mL, respectively), without significant differences in serum PTH levels. In the NT group, association analysis showed that total and fractional urinary calcium excretions positively correlated with serum levels of 1,25-(OH)(2)D (P<0.01) and IGF-I (P<0.001). In the PE group, total urinary calcium excretion positively correlated only with serum 1,25-(OH)(2)D (P<0.05). In conclusion, the results obtained in this study confirm that PE is associated with hypocalciuria and suggest that 1,25-(OH)(2)D and/or IGF-I may be involved in the regulation of urinary calcium excretion.

Calcitriol downregulates TNF-α and IL-6 expression in cultured placental cells from preeclamptic women.

Placenta is an important source and target of hormones that contribute to immunological tolerance and maintenance of pregnancy. In preeclampsia (PE), placental calcitriol synthesis is low; whereas pro-inflammatory cytokines levels are increased, threatening pregnancy outcome. Previously, we showed that calcitriol inhibits Th-1 cytokines under experimental inflammatory conditions in normal trophoblasts. However, a study of the regulation of inflammatory cytokines by calcitriol in trophoblasts from a natural inflammatory condition, such as PE, is still lacking. Therefore, the aim of the present study was to investigate calcitriol effects upon TNF-α, IFN-γ, IL-6 and IL-1β in cultured placental cells from preeclamptic women by using qPCR and ELISA. Placentas were collected after cesarean section from preeclamptic women and enriched trophoblastic preparations were cultured in the absence or presence of different calcitriol concentrations during 24h. In these cell cultures, pro-inflammatory cytokines TNF-α and IL-6 secretion and mRNA expression were downregulated by calcitriol (P<0.05). No significant effects of calcitriol upon IFN-γ and IL-1β were observed. In addition, basal expression of TNF-α, IL-6 and IL-1β decreased as the cells formed syncytia. Our study supports an important autocrine/paracrine role of placental calcitriol in controlling adverse immunological responses at the feto-maternal interface, particularly in gestational pathologies associated with exacerbated inflammatory responses such as preeclampsia.

Regulation of 25-hydroxyvitamin D3 1α-hydroxylase, 1,25-dihydroxyvitamin D3 24-hydroxylase and vitamin D receptor gene expression by 8-bromo cyclic AMP in cultured human syncytiotrophoblast cells ☆

In vitro differentiation of human trophoblast cells is a dynamic process accompanied by increasing intracellular levels of cyclic AMP (cAMP). Signaling through cAMP in this tissue is central to hormone expression and cytodifferentiation. In the present study, we analyzed transcriptional regulation of key enzymes involved in vitamin D endocrine system during in vitro syncytiotrophoblast formation. Total RNA was isolated from human trophoblast cells and subjected to reverse transcription, polymerase chain reaction and Southern blot analysis using specific primers and radiolabeled probes. During syncytium formation 25-hydroxyvitamin D(3) 1alpha-hydroxylase (CYP27B1) was decreased while vitamin D receptor (VDR) gene remained unaffected. No 1,25-dihydroxyvitamin D(3) 24-hydroxylase (CYP24) transcription signal was detected. Nevertheless, incubations in the presence of 8-bromo cAMP (1.5mM) resulted in CYP24 induction and CYP27B1 inhibition, respectively. The overall data showed that cultured human syncytiotrophoblasts express key enzymes involved in vitamin D metabolism, as well as VDR. The results support previous findings that human placenta is a source of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)), which synthesis is regulated by common growth and developmental factors. The data also suggest a tissue-dependant differential regulation of CYP27B1 gene expression by cAMP.

Calcitonin gene- and parathyroid hormone-related peptides in preeclampsia: effects of magnesium sulfate

Objective To determine whether circulating levels of calcitonin gene-related peptide (CGRP) and parathyroid hormone-related peptide (PTHrP) are altered in preeclampsia, and to assess the effects of magnesium sulfate therapy on circulating levels of these two peptides. Methods The study population included 25 women with preeclampsia and 25 normotensive controls of similar gestational age. The effects of magnesium sulfate therapy were evaluated in 17 of the 25 preeclamptic women. Circulating levels of immunoreactive CGRP and PTHrP, including calcium, magnesium, and phosphate in the maternal and umbilical cord serum were measured. Results The frequency of preeclampsia subjects with nondetectable PTHrP (under 3 pg/mL) was significantly higher (92% versus 48%, P < .001), whereas maternal serum CGRP levels were significantly lower (50 ± 19 versus 90 ± 23 pg/mL, P < .001). Similarly, the frequency of newborns with nondetectable PTHrP levels in umbilical serum was significantly higher (68% versus 36%, P < .05), whereas the levels of CGRP were significantly lower (67 ± 17 versus 79 ± 16 pg/mL, P < .05). Magnesium sulfate treatment resulted in a significant increase in maternal circulating CGRP levels (64 ± 17 versus 47 ± 18 pg/mL, P < .05) with no changes in PTHrP. Conclusion Maternal circulating PTHrP and CGRP concentrations were significantly lower in women with preeclampsia, which may contribute to the development and maintenance of hypertension during pregnancy. Furthermore, magnesium sulfate therapy increased the levels of CGRP in the maternal circulation.

Longitudinal changes in maternal serum 1,25-dihydroxyvitamin D and insulin like growth factor I levels in pregnant women who developed preeclampsia: comparison with normotensive pregnant women.

This study was undertaken to determine the longitudinal changes of serum 1,25-dihydroxyvitamin D (1,25-(OH)(2)D) and insulin like growth factor I (IGF-I) levels at 20.7, 27.6, and 35.5 week periods of gestation in 40 pregnant women who remained normotensive (NT) and in 10 women who developed preeclampsia (PE). As compared with the first period, significant increases (P < 0.01) in maternal serum 1,25-(OH)(2)D and IGF-I were observed in the NT group. In the PE group, a similar increase in serum 1,25-(OH)(2)D was observed. In contrast, significant (P < 0.05) lower IGF-I levels were observed in the PE group at the moment of diagnosis. In addition a high incidence of subjects with low increase in IGF-I levels (<percentile 10) was found in the PE group (30% versus 5%, P = 0.02). In conclusion, circulating levels of 1,25-(OH)(2)D were not alterated in women before they developed PE. In the opposite, the high percentage of PE women with low increase in circulating IGF-I levels between the 20th and 35th week of pregnancy suggests early alterations of IGF-I synthesis in women developing PE.