This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Saxena, D. Articles by Zeleznik, A. J. Search for Related Content PubMed PubMed Citation Articles by Saxena, D. Articles by Zeleznik, A. J.

Liver Receptor Homolog-1 Stimulates the Progesterone Biosynthetic Pathway during Follicle-Stimulating Hormone-Induced Granulosa Cell Differentiation

Department of Cell Biology and Physiology (D.S., L.L.-I., A.J.Z.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; and Department of Pharmacology and Cancer Biology (R.S.), Duke University Medical Center, Durham, North Carolina 27710

Address all correspondence and requests for reprints to: Anthony J. Zeleznik, Ph.D., Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 830 Scaife Hall, 3500 Terrace Street, Pittsburgh, Pennsylvania 15261. E-mail zeleznik{at}pitt.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
FSH-stimulated granulosa cell differentiation is associated with the induction of the LH receptor (LHr) as well as induction of the estrogen and progesterone biosynthetic pathways. Although activation of the cAMP-protein kinase A pathway is sufficient to stimulate progesterone production, additional pathways are required for the induction of the LHr and P450 aromatase. The orphan nuclear receptor, liver receptor homolog-1 (LRH-1), is expressed in granulosa cells and has been shown to synergize with the cAMP signaling system to regulate the gonadal type II aromatase promoter in transient transfection assays. To determine whether LRH-1 can interact with the cAMP pathway in the induction of aromatase and the LHr, we examined the effects of an adenoviral vector that directs the expression of human LRH-1 (Ad-LRH-1) on FSH-stimulated granulosa cell differentiation. Infection of undifferentiated granulosa cells with LRH-1 alone had no effect on estrogen production, progesterone production, or the expression of the LHr. However, combination of FSH stimulation and Ad-LRH-1 infection led to significantly greater progesterone production and increases in mRNA for P450 side-chain cleavage and 3ß-hydroxysteroid dehydrogenase than granulosa cells stimulated by FSH alone. However, infection with Ad-LRH-1 did not stimulate estradiol production or increases in mRNA for P450 aromatase or the LHr above that seen with FSH treatment alone. Moreover, infection with Ad-LRH-1 was able to overcome H-89 inhibition of FSH-stimulated progesterone but not estrogen production. Collectively, these observations support a direct role for LRH-1 in the induction of the progesterone but not the estrogen biosynthetic pathway during granulosa cell differentiation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
IT IS WELL ESTABLISHED that the binding of FSH and LH to their cognate receptors on ovarian granulosa cells stimulates adenylyl cyclase and the production of cAMP (1). However, although cAMP appears to be a common mediator, the responses of granulosa cells to FSH and LH differ such that activation of the FSH receptor in undifferentiated granulosa cells appears to be more effective in inducing the expression of the LH receptor (LHr) and aromatase than does activation of the LHr (2).

To identify additional signaling pathways used by the FSH receptor for the induction of P450 aromatase (P450arom) and the LHr, we used adenovirus vectors to directly regulate intracellular signaling pathways in primary cultures of undifferentiated rat granulosa cells. Using this approach, we demonstrated that protein kinase B (PKB) is an obligatory component of FSH-stimulated granulosa cell differentiation as expression of a dominant-negative mutant of PKB in undifferentiated granulosa cells completely blocked FSH-induced expression of P450arom and the LHr (3). Moreover, although activation of cAMP signaling pathway with an adenoviral vector that directs the expression of a constitutively active Gs protein was effective in stimulating progesterone production and elevating mRNA levels of cholesterol side-chain cleavage (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), expression of the constitutively active Gs did not stimulate estrogen production or increase mRNAs for P450arom or the LHr. However, expression of the constitutively active mutant of PKB synergized with Gs to promote the induction of P450arom and the LHr in undifferentiated granulosa cells (3). Collectively, these results indicate that although the expression of proteins and their mRNAs involved in progesterone production (P450scc and 3ß-HSD) can be accounted for by the Gs signaling pathway, the induction of aromatase and the LHr during granulosa cell differentiation requires activation of both the Gs and the PKB signaling pathways.

The downstream targets of PKB that may responsible for its role in the induction of aromatase and the LHr are not known. Recent studies by Clyne et al. (4) demonstrated that liver receptor homolog-1 (LRH-1) synergized with the cAMP intracellular signaling pathway in the stimulation of the gonadal type aromatase promoter II in preadipocytes. The finding that LRH-1 is expressed in granulosa cells led to the suggestion that this protein may be involved in the induction of aromatase during FSH-stimulated granulosa cell differentiation (5, 6, 7, 8). To determine the role of LRH-1 on granulosa cell differentiation and its possible interaction with PKB, we constructed an adenoviral vector that directs the expression of human LRH-1 and investigated its actions on FSH-stimulated granulosa cell differentiation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Unless otherwise noted, all reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Human FSH (AFP-4161-B; 3205 IU second IRP FSH per milligram, 225 IU second IRP LH per milligram) and antiserum to cAMP (lot CV-27) were generously provided by Dr. A. F. Parlow (National Hormone and Pituitary Program. National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Torrance, CA).

Cloning of human LRH-1 cDNA
Human liver poly (A)⁺ mRNA from CLONTECH (Palo Alto, CA) (250 ng) was reverse transcribed using random hexamers primers (Promega Corp., Madison, WI) and the Superscipt II reverse transcriptase in 20 µl of reaction mixture according to the instructions given by the manufacturer (Invitrogen Life Technologies, Carlsbad, CA). Resulting cDNA templates were amplified by PCR in 50 µl volume with 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO₄, 0.1% Triton X-100 (New England Biolabs, Beverley, MA) 0.25 mM of each deoxynucleotide triphosphate, 0.5 U Vent DNA polymerase (New England Biolabs), and 150 ng of each forward and reverse primers (listed below).

Specific primers were designed (LRH-1 sense start at position –16 bp and LRH-1 antisense end at position 1515 bp) according to the published cDNA sequence of the human LRH-1 (9) (GenBank database gi: 2007016). PCR was performed on an iCycler (Bio-Rad Laboratories, Inc., Hercules, CA) using the following cycling conditions: an initial cycle of 3 min at 95 C; 35 cycles of 40 sec at 95 C; 40 sec at 55 C and 2 min at 72 C; followed by an additional 10-min cycle at 72 C. The PCR product was cloned into the PCR Blunt vector (Life Technologies, Inc.-Invitrogen, Gaithersburg, MD), and the plasmid was sequenced with a 377A ABI automated DNA sequencer using the Prism Ready Reaction Dyedeoxy terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA). The amplified sequence was analyzed and found to be identical to the previous LRH-1 sequence (GenBank database gi: 2007016).

Primers were: LRH-1 sense, 5'-TAG AAT TCC ACT AAG AAT GTC TTC TAA TTC-3'; LRH-1 antisense, 5'-GAA AGC TTA GCT CCT AGG GGT TGT AAC TTA-3'.

Adenovirus vectors
An adenovirus vector that directs the expression of human LRH-1 (Ad-LRH-1) was constructed using the AdMax system (Microbix Biosystems, Inc., Toronto, Canada). The aforementioned cDNA for LRH-1 was subcloned into the adenovirus shuttle vector pDC316. Five micrograms of the pDC316 LRH-1 shuttle vector was cotransfected with 5 µg of the shuttle vector pBHGloxE, 3Cre into the human embryonic kidney cell line 293 in 60-mm culture dishes using the SuperFect reagent according to the manufacturer’s directions (Qiagen Inc., Valencia, CA). Transfected cells were maintained in DMEM containing 4.5 g/liter glucose (Life Technologies, Inc.-Invitrogen) and 10% fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA) at 37 C in 5% CO₂. Approximately 14 d after transfection when viral cytopathic effect was observed, the cells were collected and frozen on dry ice and thawed three times and then further propagated in 293 cells. When the cells exhibited complete viral cytopathic effect, the cells were collected, resuspended in PBS, frozen and thawed on dry ice three times, and then centrifuged (1000 x g, 4 C, 10 min) to remove cellular debris. Aliquots of virus stocks were diluted 50- and 100-fold in lysis solution [0.1% sodium dodecyl sulfate, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA] and incubated for 10 min at 56 C in a shaking water bath. The OD of the samples was measured at 260 nm and the value obtained was used to calculate virus content using the relationship 10¹² virus particles/ml·OD 260 U (10). An adenovirus that directs the expression of ß-galactosidase (Ad-ßgal) was generously provided by Dr. Joseph Alcorn (University of Texas Medical School, Houston, TX). An adenovirus vectors that direct the expression of a constitutively active PKB under the control of a cytomegalovirus promoter was provided by Dr. Kenneth Walsh (Boston University School of Medicine, Boston, MA) (11).

Granulosa cell culture
All procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Immature female rats (23–25 d old) were purchased from Hilltop Lab Animals (Scottdale, PA). Granulosa cells were collected from the ovaries by puncturing follicles with a 25-gauge hypodermic needle, and cells were expressed into Medium 199 (M199; Life Technologies, Inc.-Invitrogen) containing 10% FBS. Granulosa cells were seeded into 6-well (10⁶ cells/well) or 24-well (2 x 10⁵ cells/well) tissue culture plates and allowed to attach overnight. The next morning, medium and unattached cells were removed, and the granulosa cell monolayers were exposed to adenoviruses and stimulatory agents as described in the figure legends. At the end of the experiment, tissue culture medium was collected, boiled for 10 min to inactivate phosphodiesterases, and stored at –20 C for subsequent RIAs. Where indicated, total RNA was prepared from the cell monolayers using RNA-Bee (Tel-Test, Inc., Friendswood, TX) according to the manufacturer’s directions.

mRNA analysis
Samples of total RNA (1–5 µg) were analyzed for mRNAs for cytochrome P450arom, 3ß-HSD, the -subunit of inhibin, and the LHr by RNase protection assay according to the instructions provided by the supplier (Ambion, Inc. Austin, TX). Antisense RNA probes were prepared using [³²P]-CTP (PerkinElmer Life Sciences, Boston, MA) from the following cDNA inserts: P450arom (bp 1034–1295) (12); rat LHr (bp 1–622) (13); P450scc (bp 18–816) (14); 3ß-HSD (bp 453–932) (15); and cyclophylin (bp 34–142) (16). After electrophoresis (5% acrylamide containing 8 M urea), gels were dried and exposed to x-ray film for 16–96 h. Densitometric analysis of protected RNA fragments was performed using NIH Image (version 1.61).

RIA
Estradiol and progesterone concentrations in culture medium were determined by RIAs as described previously (17). cAMP concentrations in culture medium were analyzed by RIA using ¹²⁵I-cAMP-TME (2-O' monosuccinyl cAMP tyrosine methyl ester) (18) and anti-cAMP in accordance to the instructions provided by the National Hormone and Pituitary Program.

Statistics
Where indicated, results were assessed for statistical significance by ANOVA, followed by comparison of group means with Fisher’s least significant difference analysis (StatView version 4.5, Abacus Concepts, Berkeley, CA). Dose-response studies were analyzed for statistical differences using ANOVA with repeated measures analysis (StatView version 4.5, Abacus Concepts).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of FSH and Ad-LRH-1 on progesterone, estradiol, and cAMP production by undifferentiated granulosa cells
Primary cultures of rat granulosa cells collected from immature female rats were stimulated by FSH or infected with Ad-LRH-1 as described in the legend. Figure 1A illustrates that FSH alone stimulated progesterone production by undifferentiated granulosa cells, compared with unstimulated controls (P < 0.005). Granulosa cells infected with Ad-LRH-1 alone did not produce progesterone above that of controls, whereas progesterone production was induced dramatically by cells stimulated by the combination of FSH and Ad-LRH-1 (P < 0.005 FSH vs. FSH+Ad-LRH-1). Figure 1B shows estradiol production by the same groups of cells. As expected, FSH stimulated the production of estradiol. Granulosa cells infected with Ad-LRH-1 alone did not produce estradiol above that of controls, and Ad-LRH-1 did not amplify the stimulatory effect of FSH on estrogen production (P = 0.5436 FSH vs. FSH+Ad-LRH-1). Figure 1C depicts cAMP production by granulosa cells under the influence of FSH and/or Ad-LRH-1. FSH alone stimulated cAMP production, compared with control cells (P < 0.001). The augmentation of FSH stimulated progesterone production by LRH-1 was not due to enhancement of cAMP because there was no significant difference between cAMP produced in response to FSH and FSH + Ad-LRH-1 (P = 0.2249).

View larger version (9K): [in this window] [in a new window]   FIG. 1. Effect of expression of Ad-LRH-1 on progesterone, estrogen, and cAMP production by undifferentiated granulosa cells. Undifferentiated rat granulosa cells were plated overnight in M199 containing 10% FBS. The next morning media and unattached cells were removed and monolayers were exposed to either Ad-ßgal or Ad-LRH-1 at a concentration of 5.0 x 1010 particles/ml for 3 h, after which virus containing medium was removed and replaced with fresh M199. After 24 h, medium was replaced with fresh medium containing 30 ng/ml testosterone with or without human FSH (100 ng/ml). Forty-eight hours later, medium was collected and analyzed for estradiol, progesterone, and cAMP content by RIA. Result shows the mean ± 1 SEM of six groups of granulosa cells.

View larger version (10K): [in this window] [in a new window]   FIG. 2. Ad-LRH-1 and constitutively active PKB increase progesterone production by undifferentiated rat granulosa cells. Undifferentiated rat granulosa cells were plated overnight in M199 containing 10% FBS. The next morning media and unattached cells were removed and monolayers were exposed to Ad-ßgal, Ad-LRH-1, or Ad-myrPKB at concentrations of 5.0 x 1010 particles/ml for 3 h, after which virus containing medium was removed and replaced with fresh M199. After 24 h, medium was replaced with fresh medium containing 30 ng/ml testosterone with or without human FSH (100 ng/ml). Forty-eight hours later, medium was collected and analyzed for estradiol, progesterone, and cAMP content by RIA. Result shows the mean ± 1 SEM of six groups of granulosa cells.

To explore further the effect of LRH-1 and PKB on granulosa cell differentiation, RNase protection assays were performed on total RNA harvested from the above cell cultures used for measurements of estrogen and progesterone. Figure 3 depicts the expression of selected mRNA associated with granulosa cell differentiation. As expected, FSH alone stimulated expression of mRNAs for P450scc, 3ß-HSD, LHr, and P450arom above those of controls (P < 0.0001). Neither Ad-myrPKB nor Ad-LRH-1 alone stimulated the expression for P450scc, 3ß-HSD, LHr, or P450arom (P < 0.0001 vs. FSH). As would be predicted from the analysis of progesterone and estrogen production, infection of granulosa cells with Ad-myrPKB + Ad-LRH-1 led to significant increases in mRNAs that encode for P450scc and 3ß-HSD (P < 0.0001 vs. control) but not P450arom or the LHr (P = 0.59 and P = 0.38, respectively, vs. control). The increase in P450scc mRNA level by Ad-LRH-1+Ad-myrPKB was greater than P450scc mRNA stimulation by FSH alone (P < 0.0001 vs. FSH), whereas 3ß-HSD mRNA stimulation by Ad-LRH-1+Ad-myrPKB and that of FSH alone were comparable (P = 0.4235, Ad-LRH-1+Ad-myrPKB vs. FSH).

View larger version (60K): [in this window] [in a new window]   FIG. 3. Effect of expression of LRH-1 and constitutively active PKB on differentiation-associated mRNA level in undifferentiated granulosa cells. Undifferentiated rat granulosa cells were plated overnight in M199 containing 10% FBS. The next morning media and unattached cells were removed and monolayers were exposed to Ad-cytomegalovirus-ßgal, Ad-LRH-1, or Ad-myrPKB at concentrations of 5.0 x 1010 particles/ml for 3 h, after which virus containing medium was removed and replaced with fresh M199. After 24 h, medium was replaced with fresh medium containing 30 ng/ml testosterone with or without human FSH (100 ng/ml). Forty-eight hours later, total RNA was extracted from monolayers and analyzed for mRNAs by ribonuclease protection assay. Results shown are representative of three separate groups of granulosa cells.

View larger version (13K): [in this window] [in a new window]   FIG. 4. Ad-LRH-1 and Ad-myrPKB overcome the inhibition of progesterone production by H-89. Undifferentiated rat granulosa cells were plated overnight in M199 containing 10% FBS. The next morning media and unattached cells were removed and monolayers were exposed to Ad-ßgal, Ad-LRH-1, or Ad-myrPKB at concentrations of 5.0 x 1010 particles/ml for 3 h, after which virus containing medium was removed and replaced with fresh M199. After 24 h, medium was replaced with fresh medium containing 30 ng/ml testosterone with or without H-89 (10 µM). One hour later, human FSH (100 ng/ml) was added. Forty-eight hours later, medium was collected and analyzed for estradiol, progesterone, and cAMP content by RIA. Result shows the mean ± 1 SEM of six groups of granulosa cells.

View larger version (61K): [in this window] [in a new window]   FIG. 5. Ribonuclease protection analysis of mRNA associated with differentiation of granulosa cells infected with Ad-LRH-1 with or without H-89. Undifferentiated rat granulosa cells were plated overnight in M199 containing 10% FBS. The next morning media and unattached cells were removed, and monolayers were exposed to Ad-ßgal or Ad-LRH-1 at a concentration of 5 x 1010 particles/ml for 3 h, after which virus-containing medium was removed and replaced with fresh M199. After 24 h, medium was replaced with fresh medium containing 30 ng/ml testosterone with or without H-89 (10 µM). One hour later, human FSH (100 ng/ml) was added. Forty-eight hours later RNA was extracted from monolayer and analyzed for mRNAs by ribonuclease protection assay. Results shown are representative of six separate groups of granulosa cells.

View larger version (17K): [in this window] [in a new window]   FIG. 6. FSH dose-response study on estradiol and progesterone production by undifferentiated granulosa cells in absence or presence of Ad-LRH-1 and/or Ad-myrPKB. Undifferentiated rat granulosa cells were plated overnight in M199 containing 10% FBS. The next morning media and unattached cells were removed, and monolayers were exposed to each adenovirus at a concentration of 5.0 x 1010 particles/ml for 3 h, after which virus-containing medium was removed and replaced with fresh M199. After 24 h medium was replaced with fresh medium containing 30 ng/ml testosterone with human FSH at concentration of 10, 25, 50, and 100 ng/ml. Forty-eight hours later medium was collected and analyzed for estradiol and progesterone content by RIA. Result shows the mean ±1 SEM of six groups of granulosa cells. Where no error bars are shown, the SEM was less than 10% of mean.

Dose-response study of 8-bromoadenosine-cAMP (8Br-cAMP) on estrogen and progesterone production by undifferentiated granulosa cells in the absence or presence of Ad-LRH-1 and/or Ad-myrPKB
Increasing doses of 8Br-cAMP-induced progesterone production in undifferentiated granulosa cells (Fig. 7A) (P < 0.0001; 0.75 mM 8Br-cAMP vs. control). Progesterone production in response to 8Br-cAMP was slightly stimulated by Ad-myrPKB (P < 0.0001, Ad-myrPKB+8Br-cAMP vs. 8Br-cAMP+ßgal) and dramatically stimulated by 8Br-cAMP+Ad-myrPKB+Ad-LRH-1 (P < 0.0001, 8Br-cAMP+Ad-LRH-1+Ad-myrPKB vs. 8Br-cAMP+Ad-myrPKB, 8Br-cAMP+Ad-LRH-1+Ad-myrPKB vs. 8Br cAMP+ßgal). In contrast, Ad-myrPKB alone significantly enhanced 8Br-cAMP stimulated estrogen production, and this effect was only slightly enhanced by coinfection with Ad-LRH-1 (Fig. 7B) (P = 0.0154; 8Br-cAMP+Ad-LRH-1+Ad-myrPKB vs. 8Br-cAMP+Ad-myrPKB).

View larger version (14K): [in this window] [in a new window]   FIG. 7. Effect of 8Br-cAMP dose response on progesterone and estradiol production by undifferentiated granulosa cells expressing in absence or presence of Ad-LRH-1 and/or Ad-myrPKB. Undifferentiated rat granulosa cells were plated overnight in M199 containing 10% FBS. The next morning media and unattached cells were removed and monolayers were exposed to each adenovirus at a concentration of 5.0 x 1010 particles/ml for 3 h, after which virus- containing medium was removed and replaced with fresh M199. After 24 h medium was replaced with fresh medium containing 30 ng/ml testosterone with 8Br-cAMP at concentrations of 0, 0.1, 0.5, and 0.75 mM. Forty-eight hours later medium was collected and analyzed for estradiol and progesterone content by RIA. Result shows the mean ±1 SEM of six groups of granulosa cells. Where no error bars are shown, the SEM was less than 10% of mean.

View larger version (17K): [in this window] [in a new window]   FIG. 8. Dose-response study of LRH-1 on estrogen and progesterone production by undifferentiated granulosa cells in the absence or presence of Ad-myrPKB and/or FSH. Undifferentiated rat granulosa cells were plated overnight in M199 containing 10% FBS. The next morning media and unattached cells were removed, and monolayers were exposed to Ad-ßgal and Ad-myrPKB at concentrations of 5.0 x 1010 particles/ml with different dilutions of Ad-LRH-1 (0, 1:1000, 1:500, 1:200, 1:100, and 1:50) for 3 h (where 1:100 dilution of Ad-LRH-1 corresponds to 5.0 x 1010 particles/ml). Thereafter, virus-containing medium was removed and replaced with fresh M199. After 24 h medium was replaced with fresh medium containing 30 ng/ml testosterone with or without 100 ng/ml FSH as indicated in the figure. Forty-eight hours later medium was collected and analyzed for estradiol and progesterone content by RIA. Result shows the mean ±1 SEM of six groups of granulosa cells. Where no error bars are shown, the SEM was less than 10% of mean.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The selective effect of LRH-1 on the progesterone biosynthetic pathway (P450scc and 3ß-HSD mRNA) with no apparent effect on mRNAs for P450arom or the LHr is surprising in view of fact that the promoters of genes involved in both estrogen and progesterone biosynthesis contain LRH-1 response elements (19, 20). Furthermore, the rat LHr promoter contains an LRH-1 motif (AAGTCA) in a region (–173 bp) that is essential for transcriptional activity of the LHr gene (21, 22). A number of possible explanations exist for the paradox that neither mRNA for aromatase nor the LHr appear to be regulated by LRH-1 in undifferentiated rat granulosa cells. First, the primary difference between our study and previous studies is that the adenoviral vector approach has allowed us to monitor the expression of endogenous, fully chromatinized genes in their natural genomic context. By contrast, transiently transfected reporter plasmids are expressed episomally and may not be decorated with chromatin-associated proteins to the same extent as fully integrated genes. This could result in the inability of LRH-1 to gain access to the endogenous aromatase and LHr promoters as well as influencing the recruitment of coactivators required for the initiation of transcription (23, 24). A second possibility is that individual promoters could differ in their absolute responsiveness to LRH-1. For example, Sirianni et al. (25) demonstrated in transiently transfected HEK 293 cells that the relative increase in the transcription of the luciferase reporter gene driven by the P450scc promoter in response to LRH-1 is substantially greater than that driven by the 3ß-HSD promoter in response to LRH-1. To address this possibility of differential responsiveness of individual gene promoters to LRH-1 would require assessing the activities of simultaneously transfected reporter constructs for P450scc and P450arom in undifferentiated granulosa cells. To the best of our knowledge, a study such as this has not been reported in the literature. The possibility for differential sensitivities of individual genes for LRH-1 could impart a physiologically relevant control mechanism as the absolute level of mRNA for LRH-1 in granulosa cells is increased by FSH treatment (7).

Our observation that infection of undifferentiated granulosa cells with Ad-myrPKB + Ad-LRH-1 stimulates progesterone production and mRNA for P450scc and 3ß-HSD without increasing cAMP production together with the findings that LRH-1 can overcome the inhibition of H-89 on FSH-induced progesterone production and mRNAs for P450scc and 3ß-HSD poses an interesting question regarding the mechanism by which cAMP/PKA signaling regulates the progesterone biosynthetic pathway in granulosa cells. It is well known that elevation of intracellular cAMP concentrations rapidly stimulates steroid production in adrenal and gonadal cells through the mobilization of intracellular cholesterol to the inner mitochondrial membrane. This trafficking of cholesterol to the inner mitochondrial membrane mediated by steroidogenic acute regulatory protein (StAR) (26). The finding that StAR is rapidly phosphorylated in cAMP-stimulated cells in a PKA-dependent manner (27) provides an explanation to account for the acute effects of the cAMP/PKA signaling pathway. However, phosphorylation of StAR by PKA is not an absolute requirement for its activity because mutation of the putative sites for PKA phosphorylation in StAR reduced pregnenolone production by only 50% in COS-1 cells cotransfected with StAR and cytochrome P450scc enzyme constructs (27). Alternately, the cAMP-PKA intracellular signaling system is involved in the transcriptional regulation of the StAR gene (28). It is possible that PKB could substitute for PKA in the phosphorylation of transcription factors such as cAMP response element binding protein (29) or other members of this protein family that are involved in the transcriptional regulation of the StAR gene (30). If this is so, it will be important to determine the relative roles of PKA and PKB in the physiological control of the StAR gene.

Collectively, our findings indicate that activation of the cAMP/PKA signaling pathway in granulosa cells is sufficient for the expression of P450scc and 3ß-HSD and that LRH-1 is directly downstream of PKA in this effector pathway. Conversely, although the regulation of aromatase and the LHr genes during FSH-stimulated granulosa cell differentiation also requires the cAMP/PKA signaling pathway, unlike that of P450scc and 3ß-HSD, the induction of mRNAs for aromatase and the LHr is refractory to overexpression of LRH-1. Thus, it appears that signaling through LRH-1 is one potential mechanism by which the progesterone and estrogen biosynthetic pathways could be regulated differentially by FSH and cAMP. In this regard, Wang et al. (31) have shown recently that Leydig cells from dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1)-deficient male mice overexpress mRNA for P450arom but not mRNA for P450scc or 3ß-HSD. Furthermore, overexpression of DAX-1 in human embryonic kidney inhibited steroidogenic factor-1 (SF-1)-stimulated P450arom promoter activity but not P450scc or StAR promoter activity (30). Thus, absence or presence of DAX-1-mediated repression of SF-1 (and perhaps LRH-1) signaling could regulate FSH-stimulated P450arom expression independently of P450scc and 3ß-HSD expression in granulosa cells. Consistent with this notion, Yazawa et al. (32) recently demonstrated that FSH stimulation of undifferentiated rat granulosa cells rapidly decreased DAX-1 mRNA and protein levels.

In summary, we show that overexpression of LRH-1 differentially regulates FSH-mediated induction of the estrogen and progesterone biosynthetic pathways, thereby providing a possible mechanism for differential control of estrogen and progesterone synthesis in granulosa cells. The ability to use sense and antisense adenoviral vectors to selectively increase or diminish the levels of DAX-1, SF-1, and LRH-1 in granulosa cells may provide additional clues in the elucidation of the signaling pathways that underlie FSH-stimulated granulosa cell differentiation.

	Footnotes

 
This work was supported by National Institutes of Health (NIH) Grant HD 16842 (to A.J.Z.) and NIH Training Grant HD 07332 (to D.S.).

Abbreviations: Ad-ßgal, Adenovirus that directs the expression of ß-galactosidase; Ad-LRH-1, adenoviral vector that directs the expression of human LRH-1; Ad-myrPKB, adenovirus vectors that direct the expression of constitutively activated PKB; 8Br-cAMP, 8-bromoadenosine-cAMP; DAX-1, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1; FBS, fetal bovine serum; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; LHr, LH receptor; LRH-1, liver receptor homolog-1; M199, Medium 199; P450arom, P450 aromatase; P450scc, P450 side-chain cleavage; PKA, protein kinase A; PKB, protein kinase B; SF-1, steroidogenic factor-1; StAR, steroidogenic acute regulatory protein.

Received April 2, 2004.

Accepted for publication April 21, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Conti M 2002 Specificity of the cyclic adenosine 3',5'-monophosphate signal in granulosa cell function. Biol Reprod 67:1653–1661[Abstract/Free Full Text]
2. Bebia Z, Somers JP, Liu G, Ihrig L, Shenker A, Zeleznik AJ 2001 Adenovirus-directed expression of functional luteinizing hormone (LH) receptors in undifferentiated rat granulosa cells: evidence for differential signaling through follicle-stimulating hormone and LH receptors. Endocrinology 142:2252–2259[Abstract/Free Full Text]
3. Zeleznik AJ, Saxena D, Little-Ihrig L 2003 Protein kinase B is obligatory for follicle-stimulating hormone-induced granulosa cell differentiation. Endocrinology 144:3985–3994[CrossRef][Medline]
4. Clyne CD, Speed CJ, Zhou J, Simpson ER 2002 Liver receptor homologue-1 (LRH-1) regulates expression of aromatase in preadipocytes. J Biol Chem 277:20591–20597[Abstract/Free Full Text]
5. Schoonjans K, Annicotte JS, Huby T, Botrugno OA, Fayard E, Ueda Y, Chapman J, Auwerx J 2002 Liver receptor homolog 1 controls the expression of the scavenger receptor class B type I. EMBO Rep 3:1181–1187[CrossRef][Medline]
6. Liu DL, Liu WZ, Li QL, Wang HM, Qian D, Treuter E, Zhu C 2003 Expression and functional analysis of liver receptor homologue 1 as a potential steroidogenic factor in rat ovary. Biol Reprod 69:508–517[Abstract/Free Full Text]
7. Falender AE, Lanz R, Malenfant D, Belanger L, Richards JS 2003 Differential expression of steroidogenic factor-1 and FTF/LRH-1 in the rodent ovary. Endocrinology 144:3598–3610[Abstract/Free Full Text]
8. Hinshelwood MM, Repa JJ, Shelton JM, Richardson JA, Mangelsdorf DJ, Mendelson CR 2003 Expression of LRH-1 and SF-1 in the mouse ovary: localization in different cell types correlates with differing function. Mol Cell Endocrinol 207:39–45[CrossRef][Medline]
9. Nitta M, Ku S, Brown C, Okamoto AY, Shan B 1999 CPF: an orphan nuclear receptor that regulates liver-specific expression of the human cholesterol 7-hydroxylase gene. Proc Natl Acad Sci USA 96:6660–6665[Abstract/Free Full Text]
10. Mittereder N, March L, Trapnell BC 1996 Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J Virol 70:7498–7509[Abstract]
11. Fujio Y, Walsh K 1999 Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage-dependent manner. J Biol Chem 274:16349–16354[Abstract/Free Full Text]
12. Hickey GJ, Krasnow JS, Beattie WG, Richards JS 1990 Aromatase cytochrome P450 in rat ovarian granulosa cells before and after luteinization: adenosine 3',5'-monophosphate-dependent and independent regulation. Cloning and sequencing of rat aromatase cDNA and 5' genomic DNA. Mol Endocrinol 4:3–12[Abstract/Free Full Text]
13. McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH 1989 Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science 245:494–499[Abstract/Free Full Text]
14. John ME, John MC, Ashley P, MacDonald RJ, Rutter WJ 1984 Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450. Proc Natl Acad Sci USA 81:5628–5632[Abstract/Free Full Text]
15. Lorence MC, Naville D, Graham-Lorence SE, Mack SO, Murry BA, Trant JM, Mason JI 1991 3ß-Hydroxysteroid dehydrogenase/delta 5–4-isomerase expression in rat and characterization of the testis isoform. Mol Cell Endocrinol 80:21–31[CrossRef][Medline]
16. Danielson PE, Forss-Petter S, Brow MA, Calavetta L, Douglass J, Milner RJ, Sutcliffe JG 1998 p1B15: a cDNA clone of the rat mRNA encoding cyclophilin. DNA 7:261–267
17. Zeleznik AJ, Resko JA 1980 Progesterone does not inhibit gonadotropin-induced follicular maturation in the female rhesus monkey. Endocrinology 106:1820–1826[Abstract/Free Full Text]
18. Steiner AL, Parker CW, Kipnis DM 1972 Radioimmunoassay for cyclic nucleotides. I. Preparation of antibodies and iodinated cyclic nucleotides. J Biol Chem 247:1106–1113[Abstract/Free Full Text]
19. Parker KL, Schimmer BP 1997 Steroidogenic factor 1: a key determinant of endocrine development and function. Endocr Rev 18:361–377[Abstract/Free Full Text]
20. Michael MD, Michael LF, Simpson ER 1997 A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450 (CYP19) gene. Mol Cell Endocrinol 134:147–156[CrossRef][Medline]
21. Tsai-Morris CH, Buczko E, Wang W, Xie XZ, Dufau ML 1991 Structural organization of the rat luteinizing hormone (LH) receptor gene. J Biol Chem 266:11355–11359[Abstract/Free Full Text]
22. Tsai-Morris CH, Xie X, Wang W, Buczko E, Dufau ML 1993 Promoter and regulatory regions of the rat luteinizing hormone receptor gene. J Biol Chem 268:4447–4452[Abstract/Free Full Text]
23. Soeth E, Thurber DB, Smith CL 2002 The viral transactivator E1A regulates the mouse mammary tumor virus promoter in an isoform- and chromatin-specific manner. J Biol Chem 277:19847–19854[Abstract/Free Full Text]
24. Keeton EK, Fletcher TM, Baumann CT, Hager GL, Smith CL 2002 Glucocorticoid receptor domain requirements for chromatin remodeling and transcriptional activation of the mouse mammary tumor virus promoter in different nucleoprotein contexts. J Biol Chem 277:28247–28255[Abstract/Free Full Text]
25. Sirianni R, Seely JB, Attia G, Stocco DM, Carr BR, Pezzi V, Rainey WE 2002 Liver receptor homologue-1 is expressed in human steroidogenic tissues and activates transcription of genes encoding steroidogenic enzymes. J Endocrinol 174:R13–R17
26. Stocco DM, Clark BJ 1996 Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev 17:221–244[Abstract/Free Full Text]
27. Arakane F, King SR, Du Y, Kallen CB, Walsh LP, Watari H, Stocco DM, Strauss III JF 1997 Phosphorylation of steroidogenic acute regulatory protein (StAR) modulates its steroidogenic activity. J Biol Chem 272:32656–32662[Abstract/Free Full Text]
28. Reinhart AJ, Williams SC, Stocco DM 1999 Transcriptional regulation of the StAR gene. Mol Cell Endocrinol 151:161–169[CrossRef][Medline]
29. Du K, Montminy M 1998 CREB is a regulatory target for the protein kinase Akt/PKB. J Biol Chem 273:32377–32379[Abstract/Free Full Text]
30. Stocco DM, Clark BJ, Reinhart AJ, Williams SC, Dyson M, Dassi B, Walsh LP, Manna PR, Wang XJ, Zeleznik AJ, Orly J 2001 Elements involved in the regulation of the StAR gene. Mol Cell Endocrinol 177:55–59[CrossRef][Medline]
31. Wang ZJ, Jeffs B, Ito M, Achermann JC, Yu RN, Hales DB, Jameson JL 2001 Aromatase (Cyp19) expression is up-regulated by targeted disruption of Dax1. Proc Natl Acad Sci USA 98:7988–7993[Abstract/Free Full Text]
32. Yazawa T, Mizutani T, Yamada K, Kawata H, Sekiguchi T, Yoshino M, Kajitani T, Shou Z, Miyamoto K 2003 Involvement of cyclic adenosine 5'-monophosphate response element-binding protein, steroidogenic factor 1, and Dax-1 in the regulation of gonadotropin-inducible ovarian transcription factor 1 gene expression by follicle-stimulating hormone in ovarian granulosa cells. Endocrinology 144:1920–1930[Abstract/Free Full Text]

This article has been cited by other articles:

				C. Labelle-Dumais, J.-F. Pare, L. Belanger, R. Farookhi, and D. Dufort Impaired Progesterone Production in Nr5a2+/ Mice Leads to a Reduction in Female Reproductive Function Biol Reprod, August 1, 2007; 77(2): 217 - 225. [Abstract] [Full Text] [PDF]

				J. Guo, S.-X. Tao, M. Chen, Y.-Q. Shi, Z.-Q. Zhang, Y.-C. Li, X.-S. Zhang, Z.-Y. Hu, and Y.-X. Liu Heat Treatment Induces Liver Receptor Homolog-1 Expression in Monkey and Rat Sertoli Cells Endocrinology, March 1, 2007; 148(3): 1255 - 1265. [Abstract] [Full Text] [PDF]

				D. Saxena, R. Escamilla-Hernandez, L. Little-Ihrig, and A. J. Zeleznik Liver Receptor Homolog-1 and Steroidogenic Factor-1 Have Similar Actions on Rat Granulosa Cell Steroidogenesis Endocrinology, February 1, 2007; 148(2): 726 - 734. [Abstract] [Full Text] [PDF]

				J. Weck and K. E. Mayo Switching of NR5A Proteins Associated with the Inhibin {alpha}-Subunit Gene Promoter after Activation of the Gene in Granulosa Cells Mol. Endocrinol., May 1, 2006; 20(5): 1090 - 1103. [Abstract] [Full Text] [PDF]

				S. A. Pangas and A. Rajkovic Transcriptional regulation of early oogenesis: in search of masters Hum. Reprod. Update, January 1, 2006; 12(1): 65 - 76. [Abstract] [Full Text] [PDF]

				M. D. Serafica, T. Goto, and A. O. Trounson Transcripts from a human primordial follicle cDNA library Hum. Reprod., August 1, 2005; 20(8): 2074 - 2091. [Abstract] [Full Text] [PDF]

				F.-Q. Yu, C.-S. Han, W. Yang, X. Jin, Z.-Y. Hu, and Y.-X. Liu Activation of the p38 MAPK pathway by follicle-stimulating hormone regulates steroidogenesis in granulosa cells differentially J. Endocrinol., July 1, 2005; 186(1): 85 - 96. [Abstract] [Full Text] [PDF]

				W. H Walker and J. Cheng FSH and testosterone signaling in Sertoli cells Reproduction, July 1, 2005; 130(1): 15 - 28. [Abstract] [Full Text] [PDF]

				W. Wang, C. Zhang, A. Marimuthu, H. I. Krupka, M. Tabrizizad, R. Shelloe, U. Mehra, K. Eng, H. Nguyen, C. Settachatgul, et al. The crystal structures of human steroidogenic factor-1 and liver receptor homologue-1 PNAS, May 24, 2005; 102(21): 7505 - 7510. [Abstract] [Full Text] [PDF]

				Y. Park, E. T. Maizels, Z. J. Feiger, H. Alam, C. A. Peters, T. K. Woodruff, T. G. Unterman, E. J. Lee, J. L. Jameson, and M. Hunzicker-Dunn Induction of Cyclin D2 in Rat Granulosa Cells Requires FSH-dependent Relief from FOXO1 Repression Coupled with Positive Signals from Smad J. Biol. Chem., March 11, 2005; 280(10): 9135 - 9148. [Abstract] [Full Text] [PDF]

				K. Schoonjans, L. Dubuquoy, J. Mebis, E. Fayard, O. Wendling, C. Haby, K. Geboes, and J. Auwerx Liver receptor homolog 1 contributes to intestinal tumor formation through effects on cell cycle and inflammation PNAS, February 8, 2005; 102(6): 2058 - 2062. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Saxena, D. Articles by Zeleznik, A. J. Search for Related Content PubMed PubMed Citation Articles by Saxena, D. Articles by Zeleznik, A. J.