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Division of Reproductive Endocrinology and Infertility (J.C.H., B.R.C.), Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9032; Division of Reproductive Endocrinology and Infertility (J.W.K., G.R.A.), Department of Obstetrics and Gynecology, University of Miami, Miami, Florida 33136
Address all correspondence and requests for reprints to: George R. Attia, M.D., Division of Reproductive Endocrinology and Infertility, Cedars Medical Center, 1400 NW 12th Avenue, East Building 4th floor, Miami, Florida 33136. E-mail: gattia{at}med.miami.edu.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Luteal steroidogenesis is required for endometrial receptivity and maintenance of the early pregnancy. Although LH is essential for corpus luteum development, regression of the corpus luteum is not due to changes in LH pulsatility (12). This suggests that the actions of LH, and the maintenance of the corpus luteum, are due to intraluteal factors. Whereas luteal cell steroidogenesis is modified by growth factors, hormones, and cytokines (13), alterations in gene expression in the ovary are thought to be a major mechanism controlling the shift in ovarian steroidogenesis from a preovulatory follicle, predominantly producing estrogen, to a predominantly progesterone-producing corpus luteum (14, 15). CYP11A1 is one of three enzymes essential for progesterone synthesis, and increased expression is demonstrated in the corpus luteum (2, 3, 16).
Orphan nuclear receptors are a group of transcription factors for which a ligand has not been identified (17). Of these factors, steroidogenic factor-1 (SF-1/NR5A1) has been found to be a global regulator of endocrine development and function (14, 18, 19) and is a transcriptional regulator of several steroidogenic enzymes critical for reproductive function (20, 21, 22, 23, 24). Another orphan nuclear receptor, dosage-sensitive sex-reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1/NR0B1), colocalizes with SF-1 in steroidogenic tissues and represses the transcription of several genes encoding steroidogenic enzymes, including CYP11A1 (25).
Although SF-1 mRNA has been detected throughout the ovary, the highest levels have been detected in the theca and interstitial regions (21, 26). Furthermore, SF-1 is not expressed in the corpus luteum during pregnancy in mouse ovaries (27) and is transiently repressed in the periovulatory period (28). In contrast, CYP11A1 levels are greatly increased in the corpus luteum (2, 16, 28). This suggests that CYP11A1 expression during the periovulatory period and luteal phase may be under the regulation of transcription factors other than SF-1. Another member of the orphan nuclear receptor family is liver receptor homolog-1 (LRH-1/NR5A2). LRH-1 has been found to be expressed in multiple tissues (29, 30, 31). Recently LRH-1 expression has been demonstrated in the murine (27), equine (32), and adult human ovaries (33), suggesting that LRH-1 may play a role in ovarian steroidogenesis. LRH-1 and SF-1 share a high degree of structural similarity within the regions referred to as the hybrid P box, the A box, and the T box and are 60% homologous in their amino acid sequences. These regions are directly implicated in recognition and interaction with the canonical recognition motif for Drosophila fushi tarazu factor-1 receptors (34, 35). LRH-1 transcripts are abundant in granulosa cells and corpora lutea and absent from theca and interstitial cells (27, 36). This expression demonstrates a unique spatial and temporal expression of LRH-1 in the ovary that is distinct from SF-1 expression. To determine the role of LRH-1 in CYP11A1 regulation in the ovary, we examined the effect of LRH-1 and DAX-1 in the regulation of the CYP11A1 promoter in human granulosa cells. We also examined the effect of protein kinase A (PKA) signaling pathways on the expression of CYP11A1 in the presence of LRH-1 and DAX-1.
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Materials and Methods |
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Human granulosa cell tumor (HGCT) cells were isolated from a patient undergoing surgical removal of ovarian granulosa cell tumor as previously described (37). The use of ovarian tissue was approved by the Institutional Review Board of the University of Texas Southwestern Medical Center at Dallas. To obtain granulosa cell tumor cells, a portion of the tumor was dispersed into single cells using constant gentle agitation in 0.025% trypsin in DMEM and Ham’s F-12 (F-12) (Gibco BRL, Gaithersburg, MD) and antibiotics (37 C, 30 min x 8). After each time point, the cell suspension was collected, pooled, and 5% Nuserum (Becton Dickinson, Mansfield, MA) added to inactivate the trypsin. HGCT cells were pelleted and resuspended in DMEM/F-12 medium. This cell model is able to reproduce many of the differentiated functions of granulosa cells. Specifically they have maintained the production of progesterone and are able to convert androstenedione to estradiol. This cell model also responds to forskolin and dibutyryl cAMP (dbcAMP) by increasing production of progesterone and estradiol (data not shown). Cells were routinely subcultured using 0.05% trypsin and replated at a 1:3 split. All the experiments described in this study were conducted using cells in culture for 2–8 wk.
Human luteinized granulosa cells (HLGCs) were obtained by follicular aspiration from women undergoing oocyte retrieval for in vitro fertilization. Briefly, women were treated with GnRH agonist before and during follicular stimulation using recombinant human gonadotropin. After follicular aspiration, HLGCs were isolated as previously described (38). HLGCs were washed twice with (DMEM/F12) medium (Gibco) and then incubated for 30 min at 37 C in DMEM/F-12 containing 0.1% hyaluronidase to disperse HLGCs. The dispersed cells were resuspended in 20 ml medium and transferred to 50-ml tubes containing 3.5 ml Histopaque 1077 (Sigma Chemical Co., St. Louis, MO). HLGCs were separated from red blood cells by centrifugation at 600 x g for 15 min. HLGCs formed a thin layer between the Histopaque and the medium. Cells were removed, washed three times using DMEM/F-12 containing 5% fetal bovine serum; 1% ITS Plus (Collaborative Research, Waltham, MA); 2% Ultroser G (IBF Biotechnics, Sepracor, Inc., Marlborough, MA); and antibiotics. The isolated cells were used to prepare nuclear extracts.
CYP11A1-luciferase and expression vector constructs
A transient expression system using the luciferase reporter gene was used to characterize the human CYP11A1 promoter. A 4.4-kb fragment extending from position –4400 to + 63 was kindly provided by Bon-chu Chung (Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China). A 2.3-kb fragment extending from position –2298 to + 63 was cloned into a pGL-3 basic luciferase reporter plasmid at the HindIII site (Promega Corp., Madison, WI). Six deletion vectors were produced by appropriate enzyme digestion of pGL3-CYP11A1 vector (–2298 to +63): pGL3-CYP11A1 (–1850 to +63) was produced by XhoI and StuI digestion of –2.3 kb promoter fragment; pGL3-CYP11A1 (–1680 to +63) was produced by XhoI and SpeI digestion of –2.3 kb promoter fragment; pGL3-CYP11A1 (–1050 to +63) was produced by XhoI and DraI digestion of –2.3 kb promoter fragment; pGL3-CYP11A1 (–580 to +63) was produced by XhoI and PvuII digestion of –2.3 kb promoter fragment; pGL3-CYP11A1 (–340 to +63) was produced by XhoI and ApaI digestion of –2.3 kb promoter fragment; and pGL3-CYP11A1 (+50 to +63) was produced by XhoI and PvuII digestion of –2.3 kb promoter fragment.
The mutation vector (at the position of –1581/–1587) was produced by PCR amplification of pGL3-CYP11A1 (–2298/+63) vector using the following primers (for the –1581/–1587 mutants): 5'-ATTCCAGGCTCGAATTCATCATGGA-3' and 5'-TCCATGATGAATTCGAGCCTGGA-AT-3'. For the –1581/–1587 mutants, the sequence 5'-CAAGGTC-3' (–1581/–1587) was changed to 5'-CgAatTC-3', which included an EcoRI site. The pGL3-CYP11A1 construct (2.3 kb) was used as the template for PCR. The coding region of LRH-1 (provided by Dr. David Mangelsdorf, University of Texas Southwestern) was inserted into pcDNA3 (Invitrogen, Carlsbad, CA) eukaryotic expression vector.
Transient transfections and reporter assays
Twenty-four hours before transfection, HGCT cells were subcultured onto 12-well plates at a density of 80,000 cells/well. Fugene 6 (Roche, Indianapolis, IN) was used to transfect 0.5 µg of reporter plasmid and the indicated amounts of expression vectors. pcDNA3 empty vector was used to assure constant amounts of DNA per well for each transfection. After transfection, cells were incubated for 18 h before being treated with the agonists for 6 h in low-serum medium (DMEM/F-12 containing 0.1% Ultroser G), when indicated. Cells were assayed for reporter activity using the luciferase assay system (Promega). The ß-galactosidase enzyme assay system (Promega) was used as a positive control vector in determining transfection efficiency in earlier experiments. Because transfection efficiency was determined to be similar between the two reporter assays, additional experiments were conducted using the luciferase assay system.
Nuclear extraction and EMSA
Nuclear extract (NE) was prepared from confluent HGCT cells and HLGCs as previously described (39). Human LRH-1 was in vitro transcribed/translated using the TNT-coupled reticulocyte lysate systems following the manufacturer’s instructions (Promega). Human CYP11A1 oligonucleotide probes were designed at position –1597/–1573 and position –50/–25 (Table 1
). These oligonucleotides were annealed at 85 C for 5 min in annealing buffer [50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2 (pH 8.0)] and then slowly cooled to room temperature. The annealed oligonucleotide was end labeled with [
32P]dATP 3000 Ci/mM (Amersham Pharmacia Biotech, Piscataway, NJ) using T4 polynucleotide kinase (Invitrogen) at 37 C for 30 min. Five micrograms of NE or 5 µl of the in vitro LRH-1 protein were incubated with 30,000 cpm-labeled probe at 37 C for 30 min in 20 µl binding buffer [20 mM HEPES (pH 8.0), 1 mM EDTA, 10% glycerol, 50 mM KCl, 1 µg poly-dI.dC/dI.dC, 1 mg/ml BSA, 10 mM dithiothreitol]. The DNA-protein complexes were separated from free probe by electrophoresis using 4.5% polyacrylamide gel for 2 h at 150 V. The gel was dried and visualized after autoradiography at –70 C for 24 h.
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Data were analyzed by ANOVA using STATPAC software (Minneapolis, MN).
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Results |
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To examine the role of LRH-1 in the regulation of CYP11A1 gene expression, HGCT cells were cotransfected with LRH-1 expression vectors and CYP11A1 promoter construct. LRH-1 increased the activity of CYP11A1 promoter construct by 25-fold (Fig. 1). Previous studies demonstrated two putative SF-1 binding cis elements, (–1580 and –40) (20). To define the cis elements in the CYP11A1 promoter needed for LRH-1 transactivation, deletion analysis of the CYP11A1 promoter was performed. HGCT cells were cotransfected with LRH-1 expression vector and multiple deletion constructs of CYP11A1 promoter. LRH-1-induced CYP11A1 promoter activity was markedly decreased using the –1050 construct. In addition, the luciferase activity was comparable with basal level in +50 construct (Fig. 2). Sequence analysis of the –1680 construct and –340 construct revealed a putative SF-1 binding site between –1587 to –1581 and –42 to –35. Mutation of the upstream site from 5'-CAAGGTC-3' to 5'-CgAatTC-3' reduced the LRH-1-induced CYP11A1 promoter activity by 50% (Fig. 3).
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To examine the correlation of LRH-1 and DAX-1 on the regulation of steroidogenic enzyme expression, we examined the effect of DAX-1 on LRH-1-induced CYP11A1 promoter activity. HGCT cells were cotransfected with CYP11A1 promoter construct, LRH-1 expression vector, and increasing doses of DAX-1 expression vector. LRH-1-induced CYP11A1 activity was inhibited by DAX-1 in a concentration-dependent manner (Fig. 4).
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To determine whether LRH-1-enhanced CYP11A1 promoter activity was influenced by PKA, transfected cells were treated with the cAMP analog, dbcAMP. In the absence of LRH-1, dbcAMP had no effect on CYP11A1 reporter activity, compared with control. However, this activity was enhanced in the presence of LRH-1 (Fig. 5). This inhibition of CYP11A1 by DAX-1 was not overcome by the presence of the PKA pathway agonist dbcAMP (Fig. 5).
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To confirm that LRH-1 interacts directly with the two putative response element sites, wild- or mutant-type synthetic oligonucleotides encompassing the cis elements [–1597 to –1573 (Fig. 6A) and –50 to –25 (Fig. 6B)] were generated and used for EMSA. In vitro-transcribed LRH-1 proteins bound to the radiolabeled oligonucleotides and formed a specific protein/DNA complex that was completely displaced by addition of 100-fold molar excess of nonradiolabeled oligonucleotides. In addition, when the radiolabeled oligonucleotide probe was incubated with HLGCs and HGCT NEs, a similar protein/DNA complex was formed. This complex was displaced by excess nonradiolabeled oligonucleotides but not by mutant-type oligonucleotides.
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Discussion |
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The corpus luteum is a heterogeneous population of cells, with small and large luteal cells, fibroblasts, endothelial, and immune cells. Only 30% of these cells are steroidogenic (42). Within the steroidogenic cell population are the theca-derived small luteal cells and granulosa-derived large luteal cells (43). The granulosa-derived large luteal cells are responsible for the predominant production of progesterone in the corpus luteum (44). Whereas the theca-derived small luteal cells serve an important role in corpus luteum steroidogenesis by providing androgen precursors for estrogen biosynthesis, it is apparent that the cells derived from the granulosa cell population are essential for progesterone production.
Development of the corpus luteum is under the control of the preovulatory surge of gonadotropins. The LH surge induces ovulation and differentiation of the residual follicular cells that form the corpus luteum and begin to produce significant quantities of progesterone. Whereas the corpus luteum undergoes profound tissue growth and cellular proliferation, the profound increase in progesterone synthetic capability or specificity is not simply due to expansion of cell mass or volume alone.
SF-1 is a transcriptional regulator of steroidogenic enzymes essential for reproductive function, including CYP11A1 (20, 22). In contrast, whereas CYP11A1 expression is increased in the corpus luteum, SF-1 expression is significantly decreased (27, 28, 45). This suggests that other factors may regulate ovarian steroidogenesis in the corpus luteum. Because LRH-1 is abundant in human granulosa cells and LRH-1 shares the same putative binding sites as SF-1, we, therefore, hypothesize that the expression of CYP11A1 in corpus luteum could be regulated by LRH-1.
In this study, CYP11A1 gene expression in human ovarian granulosa cells is up-regulated through induction of promoter activity by LRH-1. For this study, we used HGCT cells for our experiments. These cells share significant similarity to granulosa cells as evidenced by the ability of this cell to reproduce many of the differentiated functions of granulosa cells. Specifically, they have maintained the production of progesterone and are able to convert androstenedione to estradiol. This cell model also responds to forskolin and dbcAMP by increasing production of progesterone and estradiol. In addition, HGCT cells have continued to express steroid-metabolizing enzymes that are under the control of cAMP. We previously performed a Western blot analysis for LRH-1 in the HGCT model, demonstrating low LRH-1 expression in nontransfected HGCT cells (data not shown). To validate the use of this cell model as a representative to granulosa cells, we used nuclear extract from human luteinized granulosa cells and compared its binding activity with that of the HGCT model. As shown in Fig. 6
, there is a similar binding pattern between these two cell models. Furthermore, the density of the band obtained from these two NEs was of the same density, which would argue for comparable expression of LRH-1 in both the HGCT model and the human luteinized granulosa cells. In these EMSAs using in vitro-transcribed/translated LRH-1, there was evidence of two specific complexes with both the upstream and proximal probes. This has also been observed with the mouse LRH-1 and may be due to splice variants or the result of multiple initiation start sites (27). These complexes do appear to be specific because they competed with an excess of nonradiolabeled probe.
Previous studies demonstrated two putative SF-1 binding sites in the CYP11A1 promoter region (46, 47) that are located at the upstream (–1580) and proximal (–40) regions. Through site-directed mutagenesis, a previous study examining the regulatory role of these two SF-1 binding sites revealed that the proximal site was important for basal CYP11A1 gene expression, whereas the upstream site was located in the cAMP-responsive region of the promoter and was important for hormonal response through adenylate cyclase activity (22). In this study, through deletion analysis of the CYP11A1 promoter construct, loss of the upstream segment revealed a significant decrease in LRH-1-induced CYP11A1 expression, whereas loss of both binding sites resulted in LRH-1-induced CYP11A1 expression equivalent to basal levels of the wild-type promoter construct. Site-directed mutagenesis of the upstream putative LRH-1 binding site revealed a similar reduction in activity, indicating that LRH-1 regulation of CYP11A1 promoter activity in human ovarian granulosa cells functions through the same mechanism as SF-1. EMSA confirmed the LRH-1 interaction with the putative binding sites.
DAX-1 has previously been demonstrated to block steroidogenesis at multiple levels, including inhibition of CYP11A1 synthesis in the presence or absence of a PKA agonist (48). DAX-1 has been shown to repress SF-1-mediated transcriptional activity (49, 50). In the present study, DAX-1 inhibited LRH-1 stimulated CYP11A1 in a dose-dependent manner. This inhibition was maintained in the presence of a PKA agonist. This inhibition may be mediated through direct interaction of LRH-1 with DAX-1 (51).
It is unlikely that SF-1 and LRH-1 are redundant transcriptional regulators within the ovary. There appears to be differential regulation of gonadogenesis and gonadal steroidogenesis by SF-1 and LRH-1. The fact that SF-1 knockout mice fail to develop gonads suggests a lack of expression or regulatory function of LRH-1 in the developing gonad and again demonstrates the importance of the temporal expression of these transcriptional regulators (14, 41). Furthermore, corpus luteum steroidogenesis in rodents is under control of estradiol and prolactin (52), and androgen biosynthesis in rodents occurs using progesterone and 17-hydroxyprogesterone as precursors instead of pregnenolone and 17-hydroxypregnenolone, suggesting that the regulation of ovarian steroidogenesis is species dependent and may not accurately reflect in vivo progesterone biosynthesis in humans. The role of these orphan receptors in gonadogenesis and gonadal steroidogenesis will be further elucidated with the development of ovary-specific SF-1, LRH-1, and double-knockout animal models.
In conjunction with our previous finding of greater LRH-1 expression in the human corpus luteum, compared with SF-1 (41), along with increased CYP11A1 expression in human granulosa cells in the presence of LRH-1, the regulation of CYP11A1 in the corpus luteum appears to be under the control of LRH-1. In addition, with our previous findings of LRH-1 regulation of 3ß-hydroxysteroid dehydrogenase (41) and steroidogenic acute regulatory protein (40), transcriptional regulation of the enzymes necessary for progesterone biosynthesis in the corpus luteum appears to be under the control of LRH-1.
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Acknowledgments |
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Footnotes |
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Abbreviations: CYP11A1, Cholesterol side-chain cleavage cytochrome P450; DAX-1, dosage-sensitive sex-reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1; dbcAMP, dibutyryl cAMP; F-12, Ham’s F-12; HGCT, human granulosa cell tumor; HLGC, human luteinized granulosa cell; LRH-1, liver receptor homolog-1; NE, nuclear extract; PKA, protein kinase A; SF-1, steroidogenic factor-1.
Received February 25, 2004.
Accepted December 9, 2004.
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References |
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1-fetoprotein locus is activated by a nuclear receptor of the DrosophilaFTZ-F1 family. Mol Cell Biol 16:3853–3865[Abstract]
-hydroxylase gene. Proc Natl Acad Sci USA 96:6660–6665This article has been cited by other articles:
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