Endocrine Journal 2008, 55 (3), 575–581 Inhibitory Effects of Cholesterol Sulfate on Progesterone Production in Human Granulosa-like Tumor Cell Line, KGN RYO TSUTSUMI, HISAHIKO HIROI, MIKIO MOMOEDA, YUMI HOSOKAWA, FUMIKO NAKAZAWA, MINAKO KOIZUMI, TETSU YANO, OSAMU TSUTSUMI AND YUJI TAKETANI Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan Abstract. Cholesterol sulfate (CS) is a component of cell membranes that plays a role in stabilizing the cell membrane. We previously reported that CS increased in the endometrium during implantation, suggesting that CS plays an important role in reproduction. It has been reported that CS regulates progesterone and pregnenolone production in the placenta, adrenal glands and ovary. The regulatory mechanisms of steroid hormone production by CS, however, are still unknown. In the present study, we investigated the effect of CS on the expression of progesterone production-related genes in KGN cells, derived from human granulosa-like tumor. KGN cells were cultured with CS (10 µM) or cholesterol (10 µM) in the presence of 8-bromo-cAMP (1 mM). Progesterone levels in the culture media were measured by enzyme linked fluorescent assay at 24 h after treatment of CS and cAMP. Total RNAs were extracted for quantitative real time RT-PCR with specific primer of StAR protein, P450scc, HSD3B2, ferredoxin and ferredoxin reductase. Whole cell lysates were extracted for western blot analysis with antibody for StAR protein. Progesterone concentration in the culture medium increased to 38-fold by treatment of cAMP. CS significantly reduced progesterone concentration by 30% compared with those of cAMP treatment (p<0.05), while cholesterol did not change the progesterone concentration. CS treatment down- regulated the expression of StAR mRNA and P450scc mRNA was to 54% and 60%, respectively (p<0.05). Western blot analysis revealed that the amount of StAR protein was also reduced by CS treatment. The expression of HSD3B2 mRNA was up-regulated to 3.4-fold by treatment of cAMP. The expression of ferredoxin and ferredoxin reductase mRNA was not affected by CS treatment. These data implied that CS has an inhibitory effect on progesterone production by regulating the expression of StAR and P450scc gene expression. Key words: Cholesterol sulfate, Progesterone, StAR protein, P450scc, KGN cells (Endocrine Journal 55: 575–581, 2008) CHOLESTEROL sulfate(CS), made by sulfonation nizing epithelia and display abnormal barrier in the of cholesterol, is a component of cell membranes and skin [2]. has a stabilizing role, such as protecting erythrocytes We previously reported that CS increased in rabbit from osmotic lysis and regulating sperm capacitation endometrium during implantation. Concentration of [1]. CS can regulate the activity of selective protein CS was relatively low in nonpregnant rabbit en- kinase C and modulate the specificity of phosphatidyli- dometrium, but abruptly increased at day 5 of pregnan- nositol 3-kinase and thus involved in signal transduc- cy, at the beginning of implantation [3]. During the tion [1]. Patients with recessive x-linked ichthyosis implantation phase, CS concentration in rabbit en- (fish skin disease) accumulate CS in squamous kerati- dometrium increased to 10 times that in nonpregnant rabbit endometrium [4]. These data indicate that CS plays important roles in reproduction. Received: June 4, 2007 Accepted: February 8, 2008 Steroidogenesis is one of the essential steps in repro- Correspondence to: Mikio MOMOEDA, M.D., Department of duction. CS stimulates progesterone production in pla- Obstetrics and Gynecology, Faculty of Medicine, University of cental trophoblast cells, adrenal cells and ovarian cells Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. [5–8]. There are two different theories about involve- 576 TSUTSUMI et al. ment of CS in adrenal steroid synthesis. First, CS can say (Arklay, Kyoto, Japan). Progesterone concentra- serve as a substrate for mitochondrial P450side chain tions were normalized with respect to total cellular cleavage (P450scc), resulting in the formation of preg- protein content as determined by DC protein assay nenolone sulfate [7]. Second, CS has the ability to in- (Bio-Rad, Hercules CA, USA). hibit side chain cleavage of free or unconjugated cholesterol in the formation of pregnenolone [9]. Inter- Isolation of total RNA and cDNA synthesis estingly, this inhibition occurs upon intramitochondrial cholesterol translocation and does not involve the func- Total RNA was extracted from KGN cells using tion of P450scc [9, 10]. The effect of CS on steroido- RNeasy mini kit (Qiagen, Hilden, Germany) according genesis is still controversial, and to date few studies to the manufacturer’s instructions. Complementary attempted to elucidate the mechanism of the effect of DNA was synthesized from 500 ng of total cellular CS on steroidogenesis. RNA using the First Strand cDNA synthesis kit (Toyo- KGN cells were derived from human ovarian granu- bo, Osaka, JAPAN). losa tumor [11]. KGN cells are undifferentiated and maintain physiological characteristics of ovarian cells, Quantitative reverse transcription polymerase chain including the expression of functional FSH receptor reaction and the expression of aromatase [11, 12]. We used KGN cells as a model of human ovary. To investigate The amounts of steroidogenic acute regulatory pro- the role of CS in ovarian steroidogenesis, we studied tein (StAR), P450scc, 3beta-hydroxysteroid dehydro- the effect of CS for progesterone production with KGN genase type2 (HSD3B2), ferredoxin, ferredoxin cells and analyzed the expression of steroid regulating reductase and glyceraldehyde-3-phosphate dehydroge- genes in KGN cells to clarify the underlining mecha- nase (GAPDH) mRNA in KGN cells was quantified by nism. real time polymerase chain reaction (PCR) with the Light Cycler rapid thermal cycler system (Roche Diag- nostics, Lewes, UK). It was performed in a 20 µl vol- Materials and Methods ume containing 3 µl cDNA, 500 nM primer pairs, 1xSYBR green master mix (Nippon Gene, Tokyo, Ja- Culture of KGN cells pan). Following 10 min of denaturing at 95°C, 35 cy- cles of amplification were conducted (95 denaturation KGN cells, a human ovarian granulosa-like tumor for 15 sec, 64 annealing for 10 sec, and 72 extension cell line [11] kindly provided by Prof. Hajime Nawata for 10 sec). The primers for the PCR reactions are (Kyushu University), were cultured in DMEM/F12 shown in Table 1. The PCR products were subcloned (phenol red free) supplemented with 10% FBS and an- into pCR2.1-TOPO vector with TOPO TA cloning kit tibiotics in a 5% CO2 atmosphere at 37°C. KGN cells (Invitrogen, Carlsbad CA, USA), and these vectors (4 × 105 viable cells) were cultured on 6-well plate for were used for the standard of quantitative real time 48 h with DMEM/F12 (phenol red free) supplemented PCR. We confirmed the identity of the product by with Charcoal/Dextran treated 10% FBS. After 48 h DNA sequencing. preincubation, the medium was changed to FBS free DMEM/F12 and then treated with CS (Sigma, St Lou- Western blot analysis is, USA) at a dose of 10 µM or cholesterol (Sigma) at a dose of 10 µM in the presence of 1 mM of 8-bromo- KGN cells were scraped into RIPA buffer and al- cAMP (Sigma) in DMEM/F12 for an additional 24 h lowed to stand on ice for 15 min. After centrifuging at and 48 h. Culture media were collected at the end of 15,000 rpm for 15 min, the supernatant was removed the assay and stored at –20°C until use. for western blot analysis. Total cellar protein mixed with 2× sample buffer (containing 0.25 M Tris–HCl, Progesterone quantification 4% SDS, 10% sucrose, 0.004% bromophenol blue, 10% beta-2 mercaptoethanol), were separated electro- Progesterone concentrations in the culture medium phoretically on a 12.5% SDS-polyacrylamide gel. The were determined using enzyme linked fluorescent as- proteins on the gel were transferred to a PVDF mem- CHOLESTEROL SULFATE AND PROGESTERONE 577 Table 1. Primers used for the quantitative real-time PCR Gene Accession No. Primer sequences Fragment size (bp) StAR NM_000349 F 5'-GGCATCCTTAGCAACCAAGA-3' 200 R 5'-TCTCCTTGACATTGGGGTTC-3' P450scc NM_000781 F 5'-GCAACGTGGAGTCGGTTTAT-3' 268 R 5'-TGCAGGACACTGACGAAGTC-3' HSD3B2 NM_000198 F 5'-GCGGCTAACGGGTGGAATCT-3' 270 R 5'-CATTGTTGTTCAGGGCCTC-3' Ferredoxin NM_004109 F5'-TGGCTTGTTCAAACCTGTCAC-3' 223 R5'-GAGGTCTTGCCCACATCAAT-3' Ferredoxin reductase NM_004110 F5'-CCTGAGCACCTGGAGAGAAC-3' 195 R5'-CTGGAGACCCAAGAAATCCA-3' GAPDH NM_002046 F5'-ACCACAGTCCATGCCATCAC-3' 450 R5'-TCCACCACCCTGTTGCTGTA-3' brane, which was then incubated in 5% skim milk– TBS/0.1% Tween20 (TBST) at room temperature (RT) for 1 h to block any nonspecific reaction. The mem- brane was incubated in 5 µg/ml of anti StAR antibody PA1-560(ABR USA) at RT for 1 h. After three washes in TBST, the membrane was incubated with peroxi- dase-conjugated anti-rabbit IgG goat IgG (diluted to 1 : 3000) (Santa Cruz Biotechnology, CA, USA) for 1 h at RT. After three washes in TBST the immuno- complexes were visualized using the ECL plus West- ern Blot system (Amersham Pharmacia Biotech, Buckinghamshire, England). Fig. 1. Effects of CS, cholesterol and cAMP on progesterone Statistical Analysis production. KGN cells were cultured with cholesterol sulfate (CS), Statistically significant differences between treat- cholesterol (chol), cAMP, cAMP + CS and cAMP + Cholesterol. Progesterone concentrations in culture me- ment groups were identified using one way ANOVA dia were determined at 24 h. Treatment of CS with test. All statistical tests were conducted using Statview cAMP significantly reduced progesterone levels com- (SAS Institute USA). Data were presented as pared with treatment of cAMP. CS suppressed progest- mean ± S.E., and P<0.05 were considered to be statisti- erone production by about 70%. On the other hand, cally significant. Data shown are representative of four Cholesterol with cAMP did not change progesterone levels significantly. Data are represented as means ± or more independent experiments.