Lipoprotein Enhancement of Ovarian Theca-Interstitial Cell Steroidogenesis: Relative Contribution of Scavenger Receptor Class B (Type I) and Adenosine 5′-Triphosphate- Binding Cassette (Type A1) Transporter in High-Density Lipoprotein-Cholesterol Transport and Androgen Synthesis

0013-7227/03/$15.00/0 Printed in U.S.A. Endocrinology 144(6):2437–2445 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2002-221110 Lipoprotein Enhancement of Ovarian Theca-Interstitial Cell Steroidogenesis: Relative Contribution of Scavenger Receptor Class B (Type I) and Adenosine 5ⴕ-TriphosphateBinding Cassette (Type A1) Transporter in High-Density Lipoprotein-Cholesterol Transport and Androgen Synthesis QIAN WU, SUSAN SUCHETA, SALMAN AZHAR, AND K. M. J. MENON Departments of Obstetrics/Gynecology and Biological Chemistry (Q.W., K.M.J.M.), University of Michigan Medical School, Ann Arbor, Michigan 48109; and Geriatric Research, Education and Clinical Center (S.S., S.A.), VA Palo Alto Health Care System, Palo Alto, California 94304 The theca-interstitial cells take up plasma high-density lipoprotein (HDL)- and low-density-lipoprotein-derived cholesterol to convert into steroid hormones. The uptake of HDLderived cholesterol is mediated by the scavenger receptor, class B, type I (SR-BI). In nonsteroidogenic cells, HDL-stimulated efflux of cholesterol has been shown to be mediated by the ATP-binding cassette A1 (ABCA1) transporter. Its expression has not been documented in steroidogenic cells. The goal of the present study was to determine: 1) the role of SR-BI in theca-interstitial cell androgen production; 2) whether thecainterstitial cells express ABCA1 transporter mRNA; and 3) the relative roles of SR-BI and ABCA1 transporter in androgen production. The ABCA1 transporter mRNA expression in rat theca-interstitial cells was shown using RT-PCR and Northern blot analyses. The role of SR-BI and ABCA1 in andro- T HECA-INTERSTITIAL CELLS PLAY a crucial role in controlling follicular growth and atresia, regulating ovarian steroidogenesis and providing a supporting structural framework for ovarian follicles (1–5). These cells are also the site of androgen production, and the thecal androgens are transferred to the granulosa cells, where they serve as substrate for estrogen biosynthesis (6 –10). Moreover, steroids and locally produced growth factors play an important role in regulating the proliferation and steroidogenic function of granulosa cells (1–5). In humans, function of the theca-interstitial cell compartment and the accompanying hyperandrogenism have been linked to pathophysiological conditions such as polycystic ovary syndrome, hyperthecosis, and anovulation (11–13). In experimental animals, a hyperandrogenic state, created by androgen administration, has also been shown to cause anovulation (14). LH is the principal hormone responsible for regulating androgen synthesis in theca-interstitial cells (15–17). HowAbbreviations: ABCA1 transporter, ATP-binding cassette A1 transporter; CE, cholesteryl ester; COE, cholesteryl oleolyl ether; DLT, dilactitol tyramine; HCD, 2-hydroxypropyl-␤-cyclodextrin; hCG, human chorionic gonadotropin; HDL, high-density lipoprotein; hHDL3, human HDL3; LDL, low-density lipoprotein; PIS, preimmune serum; SR-BI, scavenger receptor, class B, type I; TCA, trichloroacetic acid. stenedione production was also examined by treating cells with anti-SR-BI and 2-hydroxypropyl-␤-cyclodextrin in the presence and absence of human chorionic gonadotropin and/or human HDL3. The treatment of theca-interstitial cells with anti-SR-BI antibody blocked more than 90% of HDL plus human chorionic gonadotropin-stimulated androstenedione production, and selective HDL-CE uptake. On the other hand, the use of inhibitors of ABCA1 transporter function had no discernible effect on HDL-supported androgen production. These data demonstrate that, although theca-interstitial cells express both SR-BI and ABCA1 transporter mRNA, the SR-BI pathway supplies the majority of the cholesterol required for androgen production. Furthermore, the present study presents evidence for a crucial role for SR-BI in HDL-mediated androgen production. (Endocrinology 144: 2437–2445, 2003) ever, there is growing evidence that locally produced growth factors and cytokines, as well as insulin/IGF-I, play an important role in steroid production in theca-interstitial cells, either alone or in combination with trophic hormone (15–22). Theca-interstitial cells, like other steroid-producing cells, require cholesterol for the initial step in the steroidogenic process, and it seems that these cells receive cholesterol exogenously from circulating lipoproteins (23, 24). Indeed, both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) have been shown to effectively support androgen biosynthesis in these cells (23, 24). The primary pathway for cellular uptake of LDL-derived CE involves the LDL receptor and other members of the LDL receptor family (25). These receptors function via the endocytic uptake and lysosomal degradation of intact lipoprotein particles, to release cholesterol and other lipids within the cell. An alternate pathway, which occurs primarily with HDL, is the so-called selective pathway, in which HDL-CE is preferentially taken up into the cell without the parallel uptake and degradation of the intact HDL particle (26, 27). Previous studies have shown that adrenal and ovarian granulosa cells and luteal cells acquire cholesterol from HDL through the selective pathway (28, 29). A recently identified HDL receptor (the scavenger receptor, class B, type I) (SR-BI), functions as an authentic 2437 2438 Endocrinology, June 2003, 144(6):2437–2445 HDL receptor and initiates the extracellular phase of the selective CE-uptake process (30, 31). The direct involvement of SR-BI in steroidogenesis has not been established in the ovarian tissue. In addition to CE uptake, SR-BI can promote cholesterol efflux by reorganizing membrane cholesterol domains and initiating the aqueous diffusion of cholesterol to exogenous acceptors (32, 33). Recently, the ATP-binding cassette A1 (ABCA1) transporter has also been shown to play a role in cholesterol efflux in nonsteroidogenic cells, where it serves as a cholesterol efflux regulatory protein, possibly acting as an antagonist of SR-BI (34 –38). The identity and expression of ABCA1 have not yet been demonstrated in any of the steroid synthesizing tissues or cell systems. This issue is of special interest because steroid-producing cells internalize large quantities of cholesterol to satisfy their steroidogenic needs, and also because of the fact that cholesterol efflux is not an issue in these systems. Previous studies from our laboratory have shown the presence of SR-BI and gonadotropin and insulin-mediated regulation of SR-BI expression in the theca-interstitial cells, under in vivo and in vitro conditions (39, 40). The present study was undertaken to examine the role of SR-BI in androgen synthesis in theca-interstitial cells. A second goal was to determine whether ABCA1 is expressed in theca-interstitial cells and to evaluate the relative contribution of SR-BI and ABCA1 in HDL-supported androgen production. Materials and Methods Reagents (...truncated)