BIOLOGY OF REPRODUCTION 56, 272-278 (1997) Estrogen Stimulation of P450 Cholesterol Side-Chain Cleavage Activity in Cultures of Human Placental Syncytiotrophoblasts' Jeffery S. Babischkin,3 Randall W. Grimes,3 Gerald J. Pepe, 4 and Eugene D. Albrecht2,3 Departments of Obstetrics/Gynecology/Reproductive Sciences and Physiology,' Center for Studies in Reproduction, University of Maryland School of Medicine, Baltimore, Maryland 21201 Department of Physiology Eastern Virginia Medical School, Norfolk, Virginia 23501 ABSTRACT biochemical differentiation of the latter cells that is mani- fested as an increase in expression of the LDL receptor and Downloaded from https://academic.oup.com/biolreprod/article/56/1/272/2760805 by guest on 29 September 2021 The present study determined whether estrogen has a role in regulating the P450 cholesterol side-chain cleavage enzyme P450,,, enzyme system. (P450_cc) and/or de novo/deesterification cholesterol pathways Recently, we reported that LDL uptake was increased in involved in progesterone biosynthesis within human syncytiotro- human syncytiotrophoblast cells cultured with estrogen [7] phoblasts. Human placental syncytiotrophoblasts were cultured and suggested that estrogen also regulates key steps in the for 48 h with estradiol, and P450,,,cc activity was determined by placental progesterone pathway in human pregnancy. How- the formation of progesterone from 25-hydroxycholesterol. Es- ever, it remains to be determined whether estrogen also reg- tradiol at 10 7 or 10 6 M and 25-hydroxycholesterol increased ulates the P450,s,-catalyzed utilization of cholesterol sub- mean ( SE) progesterone production by syncytiotrophoblasts strate in human syncytiotrophoblasts. Moreover, although (ng/0.5 x 106 cells) to a value (19.2 - 1.1) that was 104% (p the de novo and deesterification pathways appear to have < 0.001) higher than that of the untreated controls (9.4 + 0.8) a minor role in providing cholesterol substrate for steroid- and 52% higher (p < 0.001) than with 25-hydroxycholesterol ogenesis within the human trophoblast [8, 9], it is not alone (12.6 0.9). The stimulation of progesterone secretion known whether these cholesterol synthetic pathways are apparently was not the result of a change in progesterone me- regulated by estrogen. tabolism to its principal metabolite, because 20a-hydroxypregn- In the present study, therefore, we determined the effect 4-en-3-one represented a minor secretory component (0.7-1.7 of estrogen on P450,.c activity and mRNA levels, on the ng/0.5 x 106 cells) under these conditions, and levels were not de novo pathway involving the rate-limiting 3-hydroxy-3- substantially altered by estrogen. In contrast to the stimulatory methylglutaryl coenzyme-A (HMG-CoA) reductase en- effect of estradiol on P450scc activity, estrogen did not alter ei- zyme, and on the deesterification/esterification pathway in- ther the P450c c mRNA levels or the activities of 3-hydroxy-3- volving the rate-limiting cholesterol ester hydrolase/acyl methylglutaryl coenzyme-A reductase and cholesterol ester hy- coenzyme A-cholesterol acyltransferase (ACAT) in cultures drolase-rate-limiting enzymes for the de novo and deesterifi- of human syncytiotrophoblasts. cation pathways, respectively, for cholesterol formation in syn- cytiotrophoblasts in culture. Collectively, these results indicate that estrogen regulates the P450,cc component of the progester- MATERIALS AND METHODS one biosynthetic pathway, which we suggest signals functional/ Placental Collection and Trophoblast Preparation biochemical differentiation of syncytiotrophoblasts during pri- mate pregnancy. Human placentas were obtained immediately after un- complicated elective cesarean section at term, and cytotro- INTRODUCTION phoblasts were isolated essentially as described by Kliman et al. [10]. Briefly, placental villous tissue was dispersed at We have recently shown that there was an estrogen-de- 37°C in calcium-magnesium-free Hanks' Balanced Salt So- pendent developmental increase in low-density lipoprotein lution (GIBCO, Grand Island, NY), containing 4.0 mM so- (LDL) receptor and P450 cholesterol side-chain cleavage dium bicarbonate, 10.0 mM HEPES, 50 lpg/ml gentamicin (P450,,c) mRNA levels [1, 2] and LDL uptake [3, 4] in pla- sulfate, 50 IU/ml penicillin G, 50 pLg/ml streptomycin sul- cental syncytiotrophoblasts during advancing stages of ba- fate, 0.125% trypsin (bovine pancreas type III; Sigma boon pregnancy when estrogen levels become elevated. Chemical Co., St. Louis, MO), and 1500 kunitz U/mg DNa- Therefore, we have proposed that estrogen has a central se I (bovine pancreas type IV; Sigma). The cell suspensions integrative role in regulating those components of the pro- obtained from digestion were then layered onto a 5-70% gesterone biosynthetic pathway critical to the cellular up- (v:v) Percoll (Pharmacia LKB Biotechnology, Piscataway, take of substrate cholesterol and its metabolism to proges- NJ) gradient, and a cytotrophoblast-enriched cell fraction terone thereafter within the placenta ([5, 6] for reviews). was isolated by centrifugation at 1200 x g for 20 min at Moreover, because the ontogenetic increase in receptor-me- room temperature. diated LDL uptake and the P450scc enzyme occurred within syncytiotrophoblasts, we have proposed that after morpho- logical differentiation of cytotrophoblasts into syncytiotro- Culture phoblasts, there is a further estrogen-regulated functional/ Purified cytotrophoblasts (0.5-2.0 x 106 cells/ml) were incubated in 1 ml Dulbecco's Modified Eagles Medium Accepted September 11, 1996. (DMEM) containing 10% (v:v) fetal bovine serum, 44 mM Received July 8, 1996. sodium bicarbonate, 25 mM HEPES, 50 Rpg/ml gentamicin 'This work was supported by NIH Research Grant R01 HD-13294. 'Correspondence: Eugene D. Albrecht, Department of Obstetrics, Gv- sulfate, 50 IU/ml penicillin G, and 50 ,ag/ml streptomycin necology, and Reproductive Sciences, University of Maryland School of sulfate in 24-well culture plates (CoStar, Cambridge, MA) Medicine, Bressler Research Laboratories 11-017, 655 West Baltimore for 72 h at 37°C in a humidified atmosphere of 10% CO2: Street, Baltimore, MD 21201. FAX: (410) 706 5747. 90% air to allow for cell attachment, aggregation, and for- 272 P450,CC IN HUMAN PLACENTAL SYNCYTIOTROPHOBLASTS 273 mation of syncytia. Syncytiotrophoblast cultures were (Eastman Kodak, Rochester, NY). The level of mRNA ex- washed, incubated in serum-free DMEM for 48 h, and then pression was analyzed by autoradiographic densitometry incubated for 48 h in the presence of serum-free DMEM using a model 620 Video Densitometer (Bio-Rad, Rich- and various concentrations of estradiol-173 or other ster- mond, CA). oids (Sigma), 100 pxg/ml aminoglutethimide (Sigma), or 62 RIM 25-hydroxycholesterol (Steraloids, Wilton, NH) to as- HMG-CoA Reductase Activity sess P450,cc activity. Because 25-hydroxycholesterol readi- HMG-CoA reductase activity was measured by the for- ly traverses the plasma and mitochondrial membranes to 4 4 gain access to the terminal oxidase of the P450,cc, its con- mation of ['1 C]mevalonolactone from [1 C]HMG-CoA es- version into progesterone provides an index of P450,cc ac- sentially as described by Kubo and Strott [16]. Trophoblast tivity [11, 12]. In those cultures in which P450.cc mRNA cells were sonicated and then incubated (60 min; 37°C) in expression, HMG-CoA reductase, cholesterol ester hydro- 100 mM potassium phosphate buffer, pH 7.4, containing 20 lase, and ACAT were determined, the first 48-h serum-free mM glucose-6-phosphate (Sigma), 2.5 mM NADP (Sigma), 3.5 U/ml glucose-6-phosphate dehydrogenase (Sigma), 50 DMEM interval was omitted. At the end of all treatment Downloaded from https://academic.oup.com/biolreprod/article/56/1/272/2760805 by guest on 29 September 2021 periods, culture media were stored at -20°C until assayed mM NaCl, 5 mM dithiothreitol (Boehringer Mannheim), 10 mM EDTA, 0.1% (v:v) Triton X-100 (Sigma), and 2.5-200 for progesterone and 20a-hydroxypregn-4-en-3-one. Cells 4 were washed with 0.9% NaCl, and either RNA was isolated ,uM [' C]HMG-CoA (New England Nuclear-Dupont Co., for Northern blot analysis of P450,cc or cells were assessed Wilmington, DE). Reactions were terminated by addition of 0.05 ml of 5 N HCI, and after addition of 40 000 d.p.m. for activity of cholesterol metabolism enzymes as described 3 below. A small fraction of cells was also solubilized in 0.1 [ H]mevalonic acid (New England Nuclear-Dupont) as re- covery indicator, media were applied to 1-ml Bio-Rex 5 N NaOH for protein determination [13] or suspended in 1 14 3 ml 0.01 M Tris buffer and stored at -20°C until assayed (Bio-Rad) columns, and [ C/ H]mevalonolactone was elut- fluorometrically for cellular DNA content [14]. ed with 2 ml water. More than 95% of mevalonolactone was eluted from the column while 99% of [14C]HMG-CoA RIA was retained. HMG-CoA reductase specific activity was de- termined by the amount of ['4 C]mevalonolactone formed The contents of progesterone and 20a-hydroxypregn-4- in the presence of cells minus that measured in the absence en-3-one in the media were determined by RIA after puri- of cells. fication of samples on Sephadex LH-20 columns (Isolab, Inc., Akron, OH), as described previously [15], using high- Cholesterol Ester Hydrolase Activity ly specific antisera to progesterone (generously provided by Gordon Niswender, Colorado State University, Boulder, Neutral pH cholesterol ester hydrolase activity was mea- CO) and 20a-hydroxypregn-4-en-3-one (Endocrine Sci- sured in trophoblast cells by the formation of ['4 C]oleic ences, Tarzana, CA). acid from ['4 C-oleoyl]cholesteryl oleate by methods adapt- ed from Wittmaack et al. [17] and Miller and Small [18]. Northern Analysis of P450,,. Cells were incubated in 0.05 M Tris-HCI buffer (pH 7.4 at 37°C) containing 4.2 mg/ml fatty acid-free BSA (Sigma) RNA was prepared and analyzed by Northern blot hy- and 2.5-150 M [14 C-oleoyl]cholesteryl oleate (Amer- bridization as described previously [1].