Journal of Steroid Biochemistry & Molecular Biology 121 (2010) 448–451 Contents lists available at ScienceDirect Journal of Steroid Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb Effects of calcitriol on calbindins gene expression and lipid peroxidation in human placenta  Ali Halhali ∗ , Ana Guadalupe Figueras, Lorenza Díaz, Euclides Avila, David Barrera, Guillermo Hernández, Fernando Larrea Departamento de Biología de la Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan 14000, México D.F., Mexico article info Article history: Received 27 October 2009 Received in revised form 11 February 2010 Accepted 1 March 2010 Keywords: Calcitriol Calbindins Malondialdehyde Placenta abstract Pregnancy is associated with increased maternal calcitriol levels and placenta is an extrarenal source of this hormone. Calbindin-D 9k and calbindin-D 28k are vitamin D-dependent. Since calbindin-D 28k has been considered as an antioxidant factor, the aim of the present work was to investigate the effects of calcitriol on calbindins gene expression and lipid peroxidation in cultured syncytiotrophoblast cells obtained from healthy human placentas. Gene expression of calbindins was evaluated using RT and real- time PCR techniques. Malondialdehyde (MDA) levels were used as lipid peroxidation marker. The results of the present study showed that cultured syncytiotrophoblast cells expressed the mRNA of calbindin- D 9k and calbindin-D 28k . In addition, calcitriol stimulated gene expression of both calbindins in a dose- dependent manner. Placental MDA levels were not significantly different at physiological concentrations of calcitriol (10 -11 M and 10 -9 M). However, the use of calcitriol at 10 -7 M resulted in significantly higher MDA levels (P < 0.05). In conclusion, the results showed that cultured syncytiotrophoblast cells expressed calbindin-D 9k and calbindin-D 28k genes, which were stimulated by calcitriol. In addition, the results suggest that calcitriol may be considered as pro-oxidant when used at pharmacological doses. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Calcitriol (1,25-(OH) 2 D), the hormonal form of vitamin D, is involved in calcemic and non-calcemic functions [1]. In addition to kidney, which represents the main source of circulating calcitriol, other tissues are able to synthesize this hormone [2,3]. During preg- nancy, the placenta is considered an extrarenal source of calcitriol [4–7]. The locally produced calcitriol regulates placental vitamin D-dependent functions since the receptor of this hormone (VDR) has been demonstrated in human placenta [8]. Calbindin-D 9k and calbindin-D 28k (9000 Mr and 28,000 Mr cal- cium binding proteins, respectively) are considered among the most sensitive proteins to calcitriol stimulatory effects [9]. In mam- malian tissues, calbindin-D 9k has been described in the intestine, uterus and placenta, while calbindin-D 28k has been located in the kidney, brain, osteoclasts and pancreas [10]. In addition to their participation in buffering cytosolic calcium and transcellular trans- port of this ion, calbindin-D 28k may prevent lipid peroxidation  Special issue selected article from the 14th Vitamin D Workshop held at Brugge, Belgium on October 4–8, 2009. ∗ Corresponding author at: Department of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, Tlalpan 14000, México D.F., Mexico. Tel.: +52 55 54 87 09 00x2417; fax: +52 55 56 55 98 59. E-mail address: ali.halhalib@quetzal.innsz.mx (A. Halhali). since its expression in pancreatic islet -cells abolishes the proin- flammatory cytokine-induced production of lipid hydroperoxide [11]. Regarding calbindin-D 9k , we did not find information about its relation with lipid peroxidation. Interestingly, calbindin-D 9k and calbindin-D 28k have been found in cultured trophoblast cells [12,13]. In addition, it has been reported that calcitriol stimulates calbindin-D 28k mRNA and protein in human choriocarcinoma cell line JEG-3 [14]. Concerning the relation between oxidative stress and vitamin D, it has been shown that calcitriol exhibits antioxi- dant [15] and prooxidant effects [16]. Since calcitriol effects upon calbindin-D 9k and calbindin-D 28k synthesis and oxidative stress have not been studied in human placenta, the present work was undertaken in order to evaluate the effects of this hormone on gene expression of these two calcium binding proteins and lipid peroxidation in cultured syncytiotrophoblast cells. 2. Materials and methods 2.1. Cytotrophoblastic cell isolation and culture This study protocol was approved by the Human Ethical Com- mittee of the Institute, and written informed consent form was obtained from each placental donor. Term placentas were obtained from normal pregnant women. The isolation and culture of cytotro- phoblasts were performed as previously described [17,18]. Briefly, villous cytotrophoblasts were obtained by enzymatic dispersion 0960-0760/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2010.03.008