BioMed Central

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Reproductive Biology and Endocrinology

Open AccessResearchCalcitriol affects hCG gene transcription in cultured human syncytiotrophoblastsDavid Barrera, Euclides Avila, Guillermo Hernndez, Isabel Mndez, Leticia Gonzlez, Ali Halhali, Fernando Larrea, Anglica Morales and Lorenza Daz*

Address: Departamento de Biologa de la Reproduccin, Instituto Nacional de Ciencias Mdicas y Nutricin Salvador Zubirn, Vasco de Quiroga No. 15, Tlalpan 14000, Mxico, D.F; Mxico

Email: David Barrera - barrera1912@gmail.com; Euclides Avila - euclides_@excite.com; Guillermo Hernndez - inviernomudo@gmail.com; Isabel Mndez - isabelmendez1968@hotmail.com; Leticia Gonzlez - leti1903@yahoo.com.mx; Ali Halhali - alih@quetzal.innsz.mx; Fernando Larrea - larrea@innsz.mx; Anglica Morales - angelica170969@aol.com; Lorenza Daz* - lorenzadiaz@gmail.com

* Corresponding author

AbstractBackground: In pregnancy, maternal serum concentrations of calcitriol significantly rise as a resultof increased renal and placental contribution in order to assure calcium supply for the developingfetus. Considering that placenta is a site for vitamin D activation, and the versatility and potency ofcalcitriol, 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) andCYP27B1 immunolocalization in placental villi.

Methods: Quantification of hCG in culture media was performed by immunoassay. Expressionstudies were carried out by real time PCR. Analysis of CYP27B1 and VDR localization in placentalslides were performed by immunohistochemistry. Statistical significance was established by oneway ANOVA using Tukey test for comparisons.

Results: Calcitriol regulated hCG in a time-dependent manner: at 6 h the secosteroid stimulatedhCG, whereas longer incubations (24 h) showed opposite effects. Interestingly, calcitriolstimulatory effects on hCG were accompanied by an increase in intracellular cAMP content andwere abolished by pre-incubation of the cells with a selective protein kinase A inhibitor.Immunohistochemical techniques showed differential VDR localization in the syncytiotrophoblastlayer or in the vascular smooth muscle cells depending on the epitope to which the antibodies wereraised (specific for the carboxy- or amino-terminal regions, respectively). CYP27B1 wasimmunolocalized in the syncytiotrophoblast layer of placental villi.

Conclusion: The presence and location of the vitamin D activating enzyme CYP27B1 as well asthe specific receptor for vitamin D were shown in placental sections. The latter, together withfindings demonstrating specific effects of calcitriol acting through the VDR and the cAMP/PKAsignaling pathway upon hCG expression and secretion, indicate that there is a functional vitamin Dendocrine system in the placenta, and recognize calcitriol as an autocrine regulator of hCG.

Published: 22 January 2008

Reproductive Biology and Endocrinology 2008, 6:3 doi:10.1186/1477-7827-6-3

Received: 16 November 2007Accepted: 22 January 2008

This article is available from: http://www.rbej.com/content/6/1/3

2008 Barrera et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reproductive Biology and Endocrinology 2008, 6:3 http://www.rbej.com/content/6/1/3

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BackgroundVitamin D is metabolized to the steroid hormone 1,25-dihydroxyvitamin D3 or calcitriol, which regulates cal-cium homeostasis, modulates the immune response, andpromotes cellular differentiation, among other actions.Calcitriol, the most active vitamin D metabolite, exerts itsbiological effects by binding to the vitamin D receptor(VDR), which is a ligand-activated transcription factorthat recognizes cognate vitamin D response elements(VDREs) in target genes, and can also elicit rapidresponses mediated by membrane receptors [1]. Placentais a source and target of calcitriol [2]. In a similar mannerto the renal process, placental production of calcitriol iscatalyzed by the mitochondrial CYP27B1 [3]. In earlyreproductive events, calcitriol has shown to evoke specificbiological effects such as regulation of the decidualizationand implantation processes [4,5]. In addition, calcitriolregulates placental lactogen expression as well as proges-terone and estradiol secretion in cultured human syncyti-otrophoblasts [6,7]. Regarding other molecules that areregulated by calcitriol in the placenta, Evans et al showedthat calcitriol acts in an autocrine/paracrine fashion toregulate both acquired and innate immune responses,decreasing synthesis of cytokines such as granulocyte-macrophage colony stimulating factor 2, tumor necrosisfactor, and interleukin 6, but increasing expression ofmRNA for the cathelicidin antimicrobial peptide [8].Since human chorionic gonadotropin (hCG) is a pivotalhormone for pregnancy maintenance, the aim of thepresent work was to broaden the knowledge of calcitriolactions in the placenta, focusing in the study of its effectsupon hCG expression and secretion in cultured humansyncytiotrophoblasts. The data presented herein display afunctional vitamin D endocrine system present in humanplacenta and suggest its involvement in regulating placen-tal physiology.

MethodsReagentsCulture media, fetal bovine serum (FBS) and Trizol werefrom Invitrogen (NY, USA). TaqMan Master reaction, Taq-Man probes and the transcriptor RT system were fromRoche (Roche Applied Science, IN, USA), calcitriol(1 ,25-dihydroxycholecalciferol) was kindly donatedfrom Hoffmann-La Roche Ltd (Basel, Switzerland). 3-Iso-butyl-1-methylxanthine (IBMX), 8-Bromo cAMP (8-Br-cAMP), H-89 and the enzymes used for cell cultures werefrom Sigma-Aldrich (MO, USA). Immunoassay for hCGwas from Immunometrics Ltd, (London, UK). CYP27B1antibody (sheep anti-murine 25-hydroxyvitamin D-1 -hydroxylase) was from The Binding Site (Birmingham,UK). The VDR antibodies (rabbit polyclonal anti-VDR N-20 sc-1009 and anti-VDR C-20 sc-1008), as well as the sec-ondary antibodies rabbit anti-sheep-horseradish peroxi-dase, and mouse anti-rabbit IgG-HRP were purchased

from Santa Cruz Biotechnology (CA, USA). DAB (3,3'-diaminobenzidine tetrahydrochloride) was from ZymedLaboratories Inc. (CA, USA).

ImmunohistochemistryThis study was approved by the Institutional Human Eth-ical Committee (Hospital de Gineco-Obstetricia "LuisCastelazo Ayala", IMSS, Mxico), and written informedconsents forms were obtained from each placental donor.Term placentae (3742 weeks of gestation) were acquiredfrom uncomplicated pregnancies.

Fresh placental tissue from 5 term placentas was embed-ded in paraffin after fixation in 4% paraformaldehyde in0.1 M phosphate buffer, pH 7.4. Serial sections (7 m)were obtained according to standard procedures. Slideswere treated with methanol-hydrogen peroxide in orderto block the endogenous peroxidase activity. Normal rab-bit serum and 1% BSA were used as blockers to decreasenon-specific signal. Slides were then incubated with pri-mary antibodies (anti-CYP27B1, anti-VDR-N and anti-VDR-C) during 45 minutes at room temperature, fol-lowed by further washing and incubation with secondaryantibodies for another 45 minutes. Staining was devel-oped using DAB substrate and the chromogen was con-trasted with Mayer's hematoxylin. Immunolabelingspecificity was tested by omitting the primary antibody.

Trophoblast cell cultureTrophoblasts were cultured as previously described[3,9,10]. Briefly: Villous cytotrophoblasts were obtainedby enzymatic dispersion and cells were separated on den-sity Percoll gradients. Trophoblasts were plated at a den-sity of 8 105 cells/mL in supplemented medium[(DMEM) 100 U/ml penicillin, 100 mg/ml streptomycin,0.25 mg/ml Fungizone], containing 20% heat-inactivatedFBS. Incubations were performed in humidified 5% CO2-95% air at 37C. The morphological aspects of cells wereexamined daily, secreted hCG was measured by immu-noassay (EIA) following manufacturer instructions andresults were normalized against total protein content. Pro-tein was determined by the method of Bradford [11].

Calcitriol effects on hCG secretionTwo days-cultured trophoblasts were incubated in thepresence of different concentrations of calcitriol or etha-nol as vehicle, in serum-free DMEM-F12 during 6 h or 24h. Additional experiments were performed incubating thecells with a selective protein kinase A inhibitor (H-89).Incubations were stopped by media collection, cell lysiswith RIPA buffer (9.1 mM dibasic sodium phosphate, 1.7mM monobasic sodium phosphate, 150 mM NaCl, 1%Nonidet P-40, 0.1% SDS, pH 7.4) was used for proteindetermination and hCG was quantified in culture media.

Reproductive Biology and Endocrinology 2008, 6:3 http://www.rbej.com/content/6/1/3

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Calcitriol effects on hCG expressionFor expression studies 3 106 cells were plated in 25 cm2

cell culture flasks and subjected to the same treatments asstated above. Total RNA was extracted using Trizol and 1

g was reverse transcribed using the transcriptor RT sys-tem. Real-time PCR was carried out using the LightCycler2.0 from Roche (Roche Diagnostics, Mannheim, Ger-many), according to the following protocol: activation ofTaq DNA polymerase and DNA denaturation at 95C for10 min, proceeded by 45 amplification cycles consistingof 10 s at 95C, 30 s at 60C, and 1 s at 72C. The primerpair was targeted to the