Effect of Glucocorticoids on Transcriptional Status of HLA-G in Human Trophoblast Cells from Full Term Placenta

There are evidences which suggest that HLA-G molecule plays an important role in immune tolerance, protecting the potentially allogenic fetus from maternal immune attack. Regulation of HLA-G expression is not well characterized; however, studies suggest a possible role of glucocorticoids in modulation of HLA-G gene expression. Therefore, we tested this hypothesis by inducing the HLA-G expression levels in full term placenta using two glucocorticoids -Dexamethasone and Hydrocortisone, JEG-3 and JAR cell lines were used as a positive and negative controls. Cultured trophoblast cells were treated with Dexamethasone and hydrocortisone. HLA-G transcription was determined by semi-quantitative RT-PCR. Choriocarcinoma JEG-3 (HLA-G ) and JAR (HLA-G ) cell lines were obtained from American Type Culture Collection (ATCC). The level of HLA-G mRNA transcripts in trophoblast cells were elevated by Dexamethasone and hydrocortisone in dose and time dependent manners. Glucocorticoids have an up-regulatory effect on HLA-G transcripts in trophoblast cells. immunoglobulin-like transcripts 2, 4 (ILT2, ILT4), and killer immunoglobulin receptor 2DL4 (KIR2DL4) expressed on these immune cells (Rajagopalan et al. 1999; Yan et al. 2005). The expression of this molecule is limited to a few other adults or fetal tissues including oocytes, thymus and activated monocytes (Moreau et al. 1999). In contrast to classical HLA class-I genes, the primary transcript of the nonclassical HLA-G gene gives rise to four membrane bound isoforms ( HLA-G1, HLA-G2, HLA-G3, HLA-G4 and three soluble isoforms HLA-G5, HLA-G6 and HLA-G7 through alternative splicing mechanisms (Paul et al. 2000). In case of pregnancy all the isoforms were seen in first trimester and second trimester placenta and the expression reduced during gestation but in recurrent spontaneous abortion an altered expression of HLA-G was seen (Aldrich et al. 2001; Ober et al. 2006). In present study, glucocorticoids are used to induce HLA-G expression in trophoblast cells obtained from full term pregnancy. Glucocorticoids (Dexamethasone and Hydrocortisone) have potent anti-inflammatory and immunosuppressive properties. Glucocorticoids (GC) is a key regulator of placental gene expression and are used to improve the outcome of pregnancy in women undergoing assisted conception by in vitro fertilization-embryo transfer (IVF-ET) Address of Corresponding author Prof. Suraksha Agrawal Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow 226 014, Uttar Pradesh, India Phone: 091-522 -668004-8 Ext 4338 (O), 4346, 4347, 4339(R) Fax: 091-522 -6680973/6680017 E-mail: [email protected] 224 ARIZ AKHTER, VINITA DAS, AMITA PANDEY AND SURAKSHA AGRAWAL (Boomsma et al. 2007), and in women with a history of recurrent miscarriages (Quenby et al. 2005). These hormones are expressed by the placental tissues widely in addition to their appropriate receptors. They act as positive or negative regulators of transcription of genes such as collagen, fibronectin, integrin, prostaglandin dehydrogenase (Patel et al. 1999), human chorionic gonadotropin (HCG) (Ringler et al. 1989) plasminogen activator inhibitor-1 (Ma et al. 2002), and glucose transporters (Hahn et al. 1999). Through semiquantitative determination of mRNA levels, Moreau and co-workers (Moreau et al. 2001) had demonstrated for the first time that HLA-G mRNA expression in choriocarcinoma cells (JEG-3) as well as in cultured trophoblast cells obtained from 1 trimester terminated pregnancy explants can be upregulated by glucocorticoids such as dexamethasone and hydrocortisone. Expression of HLA-G protein has also been shown to be upregulated by the effect of progesterone (Yie et al. 2006) which is another important steroid hormone, as well as by reversal of DNA methylation (Moreau et al. 2002) emphasizing the role of epigenetic mechanisms involved in the regulation of HLA-G expression. In order to examine the effects of glucocorticoides on the expression of HLA-G mRNA during full term pregnancy. We conducted this study using dexamethasone and hydrocortisone as candidate glucocorticoids and induced HLA-G mRNA expression in cultured trophoblast cells obtained from term placenta. We have used JEG-3 (HLAG) and JAR (HLA-G) cell lines as positive and negative controls. These cell lines are established from human choriocarcinoma cells derived from first trimester trophoblast (Kitano et al. 1988). They are potentially suitable “in vitro” models for the regulation of the expression of HLA-G in placenta. MATERIALS AND METHODS Purification of Villous Cytotrophoblasts from Term Placenta Term placental tissues (N=20) obtained immediately after uncomplicated cesarean sections. These samples were collected from “Queen Mary’s Hospital, Lucknow”, Uttar Pradesh, India. Cytotrophoblast cell isolation and primary culture were performed as described (Kliman et al. 1986; and Alsat et al. 1991) with some modification. Briefly, chorionic villi were minced into small pieces and incubated for 30 minutes at 37oC in 0.25% trypsin, 0.02% EDTA. The resultant cell suspension was filtered through muslin cloth, washed, and layered over a discontinuous (5-70% in 5% steps) percoll gradient (Sigma-Aldrich). After centrifugation at 800g for 25min, the cells at the interface was removed, washed, and re-suspended in RPMI1640 media with 2mM L-glutamine, and antibiotics at a concentration of 1x10 cells/ml. The cell suspension was allowed to settle on 35mm plastic culture dishes for 30 min. at room temperature so that contaminating macrophages got adhered to the plates. The non-adherent cells were then plated on to 35mm culture dishes, which were pre-coated with laminin (20μg/ml, sigma Chemicals Co, St. Louis, MO, Cat No. L2020) for 45min at room temperature. The histopathology of all the cells was carried out to confirm that at least 90% of the cells were trophoblast cells. The human gestational choriocarcinoma cell lines ‘hcc’, JEG-3 (HTB-36) and JAR (HTB-144) were obtained from the American type culture collection (ATCC, Rockville, MD, USA). These cells were cultured in RPMI-1640 (Sigma-Aldrich), containing 10% fetal bovine serum (Life technology) and 100 IU/ml streptomycin-penicillin solution (Sigma-Aldrich) and other supplements.