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Endocrinology Vol. 143, No. 9 3351-3360
Copyright © 2002 by The Endocrine Society

ARTICLE

Müllerian Inhibiting Substance Blocks the Protein Kinase A-Induced Expression of Cytochrome P450 17{alpha}-Hydroxylase/C17–20 Lyase mRNA in a Mouse Leydig Cell Line Independent of cAMP Responsive Element Binding Protein Phosphorylation

V. Matt Laurich, Alexander M. Trbovich, Francis H. O’Neill, Christopher P. Houk, Patrick M. Sluss, Anita H. Payne, Patricia K. Donahoe and Jose Teixeira

Pediatric Surgical Research Laboratories (V.M.L., A.M.T., F.H.O., C.P.H., P.K.D., J.T.), Reproductive Endocrine Unit (P.M.S.) Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; and the Division of Reproductive Biology, Department of Gynecology/Obstetrics, Stanford University (A.H.P.), Palo Alto, California 94305

Address all correspondence and requests for reprints to: Jose Teixeira, Ph.D., Pediatric Surgical Research Laboratories/WRN1024, Massachusetts General Hospital, 32 Fruit Street, Boston, Massachusetts 02114. E-mail: teixeira{at}helix.mgh.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Müllerian inhibiting substance (MIS) is produced by fetal Sertoli cells and causes regression of the Müllerian duct in male fetuses shortly after commitment of the bipotential embryonic gonad to testes differentiation. MIS is also produced by the Sertoli cells and granulosa cells of the adult gonads where it plays an important role in regulating steroidogenesis. We have previously shown that MIS can dramatically reduce testosterone synthesis in Leydig cells by inhibiting the expression of cytochrome P450 17{alpha}-hydroxylase/C17–20 lyase (Cyp17) mRNA in vitro and in vivo. To characterize the signal transduction pathway used by MIS to control expression of endogenous Cyp17 in a mouse Leydig cell line, we demonstrate that MIS inhibits both LH- and cAMP-induced expression of Cyp17 at concentrations as low as 3.5 nM and for as long as 18 h. The induction of steroidogenic acute regulatory protein (StAR) mRNA by cAMP, however, was slightly increased by addition of MIS. Protein kinase A (PKA) inhibition with H-89 blocked Cyp17 mRNA induction, suggesting that MIS interferes with the PKA signal transduction pathway. Inhibition of Cyp17 induction was not seen with added U0126, and wortmannin inhibited the induction incompletely. In addition, phosphorylation of the cAMP responsive element binding protein (CREB) was not detected following 50 µM cAMP exposure, a concentration sufficient for Cyp17 mRNA induction. Moreover, CREB phosphorylation, which was observed with addition of 500 µM cAMP, was not inhibited by coincubation with MIS. Taken together, these results suggest that cAMP induces expression of Cyp17 by a PKA-mediated mechanism and that this induction, which is inhibited by MIS signal transduction, does not require CREB activity, and is distinct from that used to induce steroidogenic acute regulatory protein expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NORMAL MALE phenotype is dependent on the temporal exposure of target tissues to testosterone, a steroid hormone produced by the interstitial Leydig cells of the testes. Testosterone synthesis begins shortly after expression of SRY, the sex-determining gene on the Y chromosome, and the subsequent commitment of the embryonic bipotential gonads to testes differentiation. During fetal development, testosterone is essential for the differentiation of the Wolffian ducts into the male internal reproductive structures, the vasa deferens, seminal vesicles, and epididymides. Dihydrotestosterone, which is formed from testosterone by steroid 5{alpha}-reductase, induces the differentiation of the prostate and urethra from the urogenital sinus and the development of the external genitalia from the genital tubercle (1). Testosterone levels in the human male are low until puberty, except for a short period during the first postnatal months, when there is a rise in testosterone levels that may play a role in genital development (2). During puberty, serum testosterone levels rise to adult levels and result in progressive virilization. The adult testes produce approximately 6 mg of testosterone daily, which maintains the male habitus and libido and controls spermatogenesis (1).

In Leydig cells, testicular androgen production is regulated by LH, a pituitary glycoprotein that binds to its high affinity, seven transmembrane G protein-coupled receptor, which activates adenylyl cyclase and the subsequent cAMP-stimulated signal transduction pathway (3). Testosterone homeostasis is maintained through a negative feedback loop by which androgens inhibit LH secretion and also by desensitization of the Leydig cells to gonadotropin by a decrease in LH receptor (LHR) expression (4).

In the postnatal male, there is a reciprocal relationship between the serum concentrations of testosterone and another hormone critical to early mammalian sexual development, Müllerian inhibiting substance (MIS, also known as anti-Müllerian hormone or AMH) (5, 6, 7, 8). When the testosterone concentration is low, the MIS level is high; MIS levels gradually decreases until puberty as the testosterone levels increase. MIS is a glycoprotein hormone in the TGFß family of proteins that is produced by Sertoli cells of both fetal and adult testes and also by the granulosa cells of the postnatal ovary (9, 10). Signal transduction by MIS occurs when ligand binds to a cell surface complex of type I and II single-transmembrane serine/threonine kinase receptors (11, 12, 13, 14, 15, 16, 17, 18). MIS signaling causes regression of the Müllerian ducts in the bipotential urogenital ridge (reviewed in Ref. 10). In the absence of MIS signal transduction, as is the case in normal females, Persistent Müllerian Duct Syndrome (19), and mice with homozygous deletions of MIS (20) or its type II receptor (MISRII) (21), the Müllerian ducts differentiate into the female internal reproductive tract structures, the Fallopian tubes, uterus, and upper vagina.

Increasingly compelling evidence indicates that MIS can inhibit the synthesis of testosterone, suggesting that it might be important for testosterone homeostasis and postnatal testicular development. Overexpression of MIS in transgenic mice can lead to drastically lowered serum testosterone, undervirilization, and Leydig cell hypoplasia (22, 23). It was found that expression of steroidogenic enzyme RNAs was lower in Leydig cells isolated from the MIS-overexpressing mice (24), that testosterone secretion from cultured normal rat Leydig cells was significantly reduced after addition of MIS (25), and that MIS binds to purified prepubertal Leydig cells with an affinity of 15 nM, as measured by flow cytometry (26).

Cyp17 is the gene that encodes cytochrome P450c17, the 17{alpha}-hydroxylase/C17–20 lyase enzyme (Cyp17), the bifunctional enzyme whose lyase activity commits the 17-hydroxypregnenolone/progesterone precursor to androgen synthesis. Using the Cyp17 promoter to drive the expression of a luciferase reporter in MA-10 cells, a mouse Leydig cell tumor line, MIS was shown to inhibit transcription of luciferase (27). Additionally, injection of MIS into LH-stimulated rodents lowered serum testosterone 9-fold within 24 h indicating that MIS could exert its effects in vivo and that the observations in the MIS-overexpressing mice were not due to an MIS-mediated perturbation in Leydig cell development (28).

Relatively little is known about the molecular mechanism controlling expression of endogenous Cyp17 in rodent Leydig cells and transfection of Cyp17 promoter/luciferase reporter constructs of other species has produced conflicting results (29, 30, 31, 32, 33, 34, 35). MA-10 cells are an excellent model system for studying the molecular mechanisms of MIS-mediated inhibition of the cAMP-induced expression of Cyp17 because all the molecular components of the signal transduction pathways are present, obviating the need for transfection and/or overexpression. We elected to clone MA-10 cells in an effort to select a cell line with a more robust response to cAMP to dissect both the cAMP downstream pathway and the point(s) at which MIS exerts its inhibition. Using this system, we show that the induction of endogenous Cyp17 transcription by cAMP through the protein kinase A (PKA) pathway can be blocked by MIS and does not involve CREB phosphorylation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and reagents
Radionucleotides were purchased from Perkin-Elmer (Boston, MA). Restriction and modifying enzymes, Waymouth’s MB 752/1 medium, gentamicin, and horse serum were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA). Kinase inhibitors were from Calbiochem (San Diego, CA), Sigma (St. Louis, MO), and Promega Corp. (Madison, WI). All other chemicals were obtained from Sigma or Fisher Scientific (Pittsburgh, PA) unless otherwise noted. The steroidogenic acute regulatory protein (StAR) cDNA was provided by Dr. Douglas Stucco (Texas Tech University Health Sciences Center, Lubbock, TX). LH was provided by Dr. A. F. Parlow at the National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Peptide Program Harbor at UCLA Medical Center. Recombinant human MIS was expressed in Chinese hamster ovary cells and secreted into chemically defined serum-free media. The secreted products were then concentrated by either precipitation with ammonium sulfate or lectin-affinity chromatography, each of which was followed by anion exchange chromatography (36). Protein concentrations were determined by Bradford assays (37). The bioactivity of the MIS was then verified using an established organ culture assay, which grades Müllerian duct regression of the female 14.5-d gestation rat urogenital ridge (38).

Cell culture
The monoclonal cell line, MA-10.2 cells, was cloned from the mouse MA-10 Leydig tumor cell line (39), a gift from Dr. Mario Ascoli (University of Iowa, Ames, IA). Following trypsinization, the cell suspension was counted and serially diluted down to a final concentration of 1.5 cells/ml and then plated in 96-well plates. When a visible colony of cells had grown, the cells were subcloned again. MA-10.2 was chosen because Cyp17 mRNA expression was induced severalfold greater by cAMP than the induction observed with the parental cells and was repressed with MIS. The MA-10.2 cells were maintained in Waymouth’s MB 752/1 medium modified to contain 20 mM HEPES (pH 7.4), 50 µg/ml gentamicin, and 15% horse serum. For these experiments, MA-10.2 cells were incubated in a humidified atmosphere with 5% CO2 at 37 C and fed every other day until 80–90% confluence was reached.

Northern blot analyses
In experiments with kinase inhibitors, cells were treated for 30 min with 20 µM H-89 to inhibit PKA, 20 µM U0126 to inhibit MAPK kinse (MEK), or 10 µM wortmannin to inhibit phosphatidylinositol-3 kinase (PI3K) before any additional treatments with MIS. Total RNA was isolated using Trizol (Invitrogen Life Technologies) and quantitated by absorbance at A260. mRNA was isolated from total RNA by Poly-A+ purification (Promega Corp.). Either total RNA (10 µg) or mRNA (2 µg) samples was denatured with dimethylsulfoxide and glyoxal at 65 C, separated in a 1.5% agarose gel, blotted overnight onto nylon membranes, and UV-cross-linked. Blots were prehybridized with 100 µg/ml sonicated salmon sperm DNA in 50% formamide hybridization solution and hybridized overnight at 65 C with 2 x 106 cpm/ml with antisense riboprobes against Cyp17, MISRII (14), LHR (28), Alk2 (17), and Alk6 (18) and at 42 C with 3 x 106 cpm/ml of a random primed cDNA probe of StAR (40). Blots were washed at 65 C with 0.1x SCC (1 x SCC = 150 mM sodium chloride and 15 mM sodium citrate)-0.1% sodium dodecyl sulfate (SDS) and exposed to radiographic film with intensifying screens at -70 C. Blots were reprobed with a human ß-actin riboprobe (27) at 65 C and washed at 65 C with 0.1x SCC/0.1% SDS. Signal intensities of Northern blots were quantitated using a PhosphorImager (Amersham Pharmacia Biotech, Piscataway, NJ) or by NIH Image version 1.62 software and used to calculate fold inductions and inhibitions after normalization to actin.

RIAs and data analysis
MA-10.2 cells were plated on d 0 in duplicate at 105 cells/well in 6-well plates in 2 ml of media. On d 2, the media were changed and 35 nM MIS in the presence of absence of 50 µM 8Br-cAMP was added to the MA-10.2 cells and incubated for 24 or 48 h. For the kinase inhibitor experiments, 2 x 105 cells were plated in triplicate on d 0 and treated with inhibitors 30 min before addition of 50 µM 8Br-cAMP on d 2 and incubated for another 2 d. Culture medium was collected from treated and untreated cells and assayed by RIA in duplicate for total accumulated progesterone, 17-hydroxyprogesterone, and testosterone by the Reproductive and Endocrinology Sciences RIA Laboratory at Massachusetts General Hospital. RIA data represent the mean ± SEM of combined data from six replicate experiments. Statistical differences between mean values were analyzed by the Bonferroni/Dunn post hoc test and significance was assigned at 95% confidence.

Western blot analyses
MA-10.2 cells were plated at 2 x 106 cells in a 100-mm dish and treated 2 d later. Whole cell extracts were prepared by addition of 0.3 ml boiling lysis buffer (62.5 mM Tris, pH 6.8; 1% SDS; 10% glycerol; 5% ß-mercaptoethanol), scraped, dounce homogenized, and centrifuged at 104 x g for 10 min. Total protein (50 µg per lane) was resolved by 10% PAGE followed by electroblotting to nitrocellulose. CREB, phospho CREB (pCREB), ERK, diphospho ERK (dpERK), AKT, and phospho AKT (pAKT) were detected with antibodies and reagents from Cell Signaling Technology (Beverly, MA) according to the manufacturer’s instructions. CREB and pCREB signal intensities were measured by NIH Image version 1.62 software for normalization and calculation of fold inductions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MIS signal transduction in MA-10.2 cells
We have previously used rat R2C and mouse MA-10 Leydig cells to show that MIS regulates the expression of endogenous Cyp17 mRNA and, by employing mouse Cyp17 promoter-driven/luciferase reporter assays, showed that it does so at the transcriptional level (27). To understand the molecular mechanisms that govern the MIS-mediated regulation of Cyp17 expression, we optimized the MA-10 cell line by selecting and recloning a new line, MA-10.2 that exhibited more robust cAMP induction of Cyp17 mRNA by Northern analysis (Fig. 1AGo). Characterization of the cells by Northern analysis shows that, like the parent cells (27), MA-10.2 cells express two distinct mRNA species that hybridize to the MISRII cDNA (Fig. 1BGo). The level of expression of the MISRII mRNA was slightly lower with MIS (approximately 50%) as measured by a PhosphorImager and normalization to actin. Incubation of MA-10.2 cells with both MIS and cAMP, however, resulted in a level of expression of MISRII that was similar to that of untreated cells or cells treated with cAMP. Expression of the MIS type I receptors (MISRI) Alk2 (16, 17), and Alk6 (18) was also observed although at much lower levels than MISRII mRNA (Fig. 1Go, C and D). Expression of Alk3, another putative MISRI, as well as Alk1, 4, and 5 type I receptor mRNAs was also low (data not shown). Additionally, neither cAMP nor MIS regulated the expression of the MISRI mRNAs.



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  		Figure 1. MIS and cAMP signaling in MA-10.2 cells. Northern blot analyses of MA-10.2 cells were conducted to determine the level of expression of genes involved in MIS and cAMP signal transduction. A, Total RNA was isolated from the parental MA-10 cells and the MA-10.2 cells that were either untreated (NT) or treated with 50 µM 8Br-cAMP (cAMP), 35 nM MIS (MIS) or both (cAMP + MIS) and analyzed by Northern blot for Cyp17 mRNA expression. B, Total RNA was isolated from mouse testes and MA-10.2 cells that were not treated (NT) or treated overnight as described in A and probed with a rat MISRII riboprobe. The putative MIS type I receptors, ALK2 (C), and ALK6 (D) mRNAs were also analyzed following poly A+ selection from MA-10.2 cells treated as in A. All blots were reprobed with actin to normalize for possible loading variability.

 
Steroidogenesis in MA-10.2 cells in response to cAMP and MIS
We assessed the steroidogenic capacity of MA-10.2 cells following addition of LH, cAMP and MIS. When LH (5 IU/ml) was added to MA-10.2 cells, we observed an 11-fold increase in Cyp17 mRNA expression (Fig. 2AGo). Addition of MIS to LH-stimulated MA-10.2 cells resulted in an 80% reduction of Cyp17 mRNA expression (Fig. 2AGo). LH and cAMP stimulation produced similar results when MIS was added; therefore, subsequent experiments were performed with cAMP. Following 24- and 48-h incubation, conditioned media from MA-10.2 cells was collected from untreated cells, from cells treated with 50 µM 8Br-cAMP alone, and from cells treated with cAMP and 35 nM MIS for measurement of total progesterone, 17-hydroxyprogesterone, and testosterone by RIA. Media from untreated cells indicated that the cells were producing progesterone at approximately 0.15 pg/cell·d at the 24-h time point, equivalent to 7.5 ng/ml (Fig. 2BGo). By 48 h, accumulated progesterone was 4-fold less than that observed at 24 h, approximately 1.8 ng/ml. Addition of cAMP to cells resulted in variable stimulation of progesterone production that ranged from 3-fold to 20-fold and averaged approximately 10-fold. As with the untreated cells, after 48 h, total progesterone in the media of cAMP-treated cells was approximately 4-fold lower. When MIS was added with the cAMP, the progesterone concentration increased after 24 h by 3.5-fold to approximately 5 pg/cell·d. With 48 h of treatment with cAMP and MIS, progesterone was approximately half that observed at 24 h.



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  		Figure 2. Steroidogenic capacity of MA-10.2 cells. Northern blot analyses and RIAs were done to assess the steroidogenic response of MA-10.2 cells to cAMP and MIS. Expression of Cyp17 mRNA (A), analyzed by Northern analysis of total RNA from untreated cells and from cells treated overnight with LH (5 IU/ml) or with LH and 35 nM MIS. Total progesterone (B), 17OH-progesterone (C), and testosterone (D) were measured in the conditioned media of MA-10.2 cells by RIA after 24 h and 48 h. Cells were either untreated (NT, black bars), treated with 50 µM 8Br-cAMP alone (C, white bars), or with 35 nM MIS (C+M, striped bars) as indicated. Data represent the average of six experiments. 17-Hydroxyprogesterone and testosterone data were normalized to their progesterone values. Error bars represent SEM and significant differences between the MIS-treated cells and the others by the Bonferroni/Dunn post hoc test at 95% confidence are shown with an asterisk. Northern blot analyses of MA-10.2 cells were done to determine the level of expression of the LHR and StAR and their regulation by cAMP and/or MIS. Poly A+ mRNA was analyzed for the LHR (E) and total RNA for StAR (F) from cells treated as in Fig. 1Go. All blots were reprobed with actin to normalize for loading variation as shown below each blot.

 
We also assessed the effect of cAMP and MIS on the stimulation or reduction of 17-hydroxyprogesterone (Fig. 2CGo) and testosterone (Fig. 2DGo) production by MA-10.2 cells. To remove the confounding effect of the observed variable stimulation by cAMP of progesterone accumulation in the media, RIA data with both testosterone and 17-hydroxyprogesterone were normalized to their respective progesterone concentrations in a given experiment. Untreated cells produced an average 1.1 ng/ml of 17-hydroxyprogesterone or 22 fg/cell·d. Addition of cAMP increased 17-hydroxyprogesterone between 2- and 15-fold and averaged 8-fold at 24 h but when normalized to progesterone, there was little difference in stimulation to that seen with progesterone. In contrast, MIS when added with cAMP resulted in 22% of the 17-hydroxyprogesterone observed with cAMP alone. The results at 48 h were similar; induction by cAMP similar to progesterone and a decrease to 30% with addition of MIS. The effect of cAMP and MIS on testosterone production by MA-10.2 cells was more dramatic (Fig. 2DGo). Untreated cells produce approximately 4.8 ng/dl testosterone, equivalent to 1 fg/cell·d. Addition of cAMP for 24 h resulted in a range of 5- to 65-fold induction or an average 26 fg/cell·d, which was a 2.5-fold greater induction over progesterone. Incubation with of the cells with cAMP and MIS reduced the testosterone induction to 5- to 40-fold and when normalized to progesterone, only 22% of that seen with cAMP alone. By 48 h, the effects of both cAMP and MIS were amplified. The concentrations of testosterone in untreated cells were at or below the level of detection for the RIA, which is 4 ng/dl and therefore do not allow for calculating inductions by cAMP. Cells incubated with cAMP showed a range of accumulated testosterone concentrations that varied from 18–1400 ng/dl. Coincubation with MIS resulted in testosterone in a range of from 45–1020 ng/dl. When both values were normalized to progesterone, addition of MIS reduced the testosterone concentration to 35% of that observed with cAMP alone.

Binding of the pituitary hormone LH to the LHR initiates the signal transduction cascade that leads to increased testosterone production by Leydig cells. Although receptor internalization appears to account for the desensitization of Leydig cells to LH, transcription of the receptor is also down regulated in MA-10 cells after the addition of 200 µM cAMP (41, 42), but we show that overnight incubation of MA-10.2 cells with 50 µM 8Br-cAMP appears to increase only marginally the amount of LHR mRNA, which appeared to be reversed by addition of MIS (Fig. 2EGo). Phosphorimaging of the blots and normalization to actin did not indicate any significant changes, however.

The acute response of steroidogenic cells to cAMP is the increased delivery of cholesterol to the inner mitochondrial membrane by the activity of StAR, a protein whose expression is up-regulated by cAMP (43). Once cholesterol is transported to the inner mitochondrial membrane, it is converted to pregnenolone by the activity of the cytochrome P450 cholesterol side-chain cleavage enzyme. In rodents, pregnenolone is predominantly converted to progesterone by the activity of 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4-isomerase and progesterone is the substrate of Cyp17 (44). We previously reported that MIS also down-regulated the expression of cAMP-induced P450 side-chain cleavage and 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4-isomerase, but to a much lesser extent than that observed for Cyp17 (27). We also examined whether MIS affected the expression of StAR mRNA (Fig. 2FGo). The expected increase in StAR mRNA expression in MA-10.2 cells was observed with overnight treatment of 50 µM 8Br-cAMP and the simultaneous addition of 35 nM MIS resulted in increased StAR mRNA expression over cAMP alone in contrast with the down regulation of Cyp17 mRNA observed with MIS. MIS alone did not appear to affect StAR expression after overnight treatment.

Time course of cAMP- and MIS-regulated Cyp17 mRNA expression
The time required for MIS to inhibit the cAMP-mediated induction of endogenous Cyp17 mRNA expression in MA-10.2 cells was studied by Northern blot analyses and quantitated by phosphorimaging and normalized to actin (Fig. 3Go). Within 2 h of treatment with 50 µM 8Br-cAMP, the steady-state level of Cyp17 mRNA was increased approximately 20-fold (Fig. 3AGo). A gradual increase in Cyp17 expression was observed, with 64-fold maximal induction achieved 7 h after treatment. By the next day, expression of Cyp17 mRNA was reduced to approximately 90% of maximal. Simultaneous addition of 35 nM MIS and 50 µM 8Br-cAMP effectively blocked the cAMP-mediated stimulation of Cyp17 mRNA expression (Fig 3BGo). cAMP-stimulated levels were much lower at 2 and 4 h, approximately 20% and 13%, respectively, with MIS coincubation. As was seen in the absence of MIS, expression of Cyp17 mRNA again peaked at 7 h but was greatly reduced from the maximal expression seen with cAMP alone in Fig. 3AGo. In contrast to cells treated with cAMP alone, overnight coincubation with MIS resulted in Cyp17 mRNA expression similar to that observed in untreated cells. The addition of MIS to cells had no effect on the expression of Cyp17 mRNA at 4 h if the cells had been pretreated with cAMP 4 h earlier (Fig. 3CGo). However, overnight incubation with MIS of cells pretreated with cAMP for 4 h markedly decreased the expression of Cyp17.



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  		Figure 3. Regulation of Cyp17 mRNA expression. Northern analyses of total RNA harvested from MA-10.2 cells followed by phosphorimaging were done to determine the time course of Cyp17 mRNA induction by cAMP and its repression by MIS at various concentrations. The blots were reprobed with actin for equal loading and to normalize the PhosphorImager data. NT indicates no treatment and ON indicates overnight treatment. A, Cells were incubated with 50 µM 8Br-cAMP for the indicated times. B, Cells were treated with 50 µM 8Br-cAMP and 35 nM MIS for the indicated times. C, Cells were incubated with 50 µM 8Br-cAMP for 4 h after which time 35 nM MIS was added. Total RNA was harvested at the indicated times following MIS addition. The cAMP-labeled lane indicates RNA from cells that were not treated with MIS. D, Cells were treated with 50 µM 8Br-cAMP and increasing concentrations of MIS as indicated for 4 h after which time total RNA was harvested for Northern blot analysis.

 
Concentration of MIS required to show inhibition of Cyp17 mRNA expression
The effective concentration of MIS required to observe a difference in the cAMP-mediated induction of Cyp17 mRNA was also assessed and the results are shown in Fig. 3DGo. MA-10.2 cells were coincubated for 4 h with 50 µM 8Br-cAMP and the indicated concentrations of MIS. Compared with the induction observed with cAMP without added MIS, 33% average inhibition of Cyp17 mRNA expression was detected with as little as 3.5 nM MIS, one tenth the concentration required for complete regression of the Müllerian duct in vitro (38). There was a linear correlation between the increasing concentration of MIS and reduced induction of Cyp17 mRNA expression.

The PKA activity is required for the cAMP-mediated induction of endogenous Cyp17 expression
Because relatively little is known about the molecular mechanisms involved in the cAMP-induced expression of endogenous Cyp17 in murine cells, we next evaluated the possible cAMP signaling pathways through which MIS might interfere to inhibit Cyp17 mRNA induction (45). Figure 4Go shows the results of experiments performed with kinase inhibitors to determine which of three pathways shown to mediate the effects of cAMP in steroidogenic cells might be involved in Cyp17 mRNA induction in murine Leydig cells: PKA (30, 32, 46), cAMP-guanine nucleotide exchange factor/ERK (47, 48), and PI3K/Akt (49, 50, 51). MA-10.2 cells were treated with 20 µM H-89 to inhibit PKA activity, 20 µM U0126 to inhibit the MEK, and 10 µM wortmannin to inhibit PI3K. Following 30 min of inhibitor treatment, the cells were incubated for 2 h with or without 500 µM cAMP and the level of Cyp17 mRNA induction was assessed by Northern analysis (Fig. 4AGo). H-89 effectively blocked the cAMP-induced mRNA expression but U0126 did not and wortmannin lowered the Cyp17 mRNA concentration by approximately 50% as assessed by phosphorimaging after normalization to actin.



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  		Figure 4. Induction of Cyp17 mRNA by cAMP is blocked by the PKA inhibitor H-89. A combination of Northern and Western analyses and RIAs were done to determine which cAMP pathway affected by MIS might be perturbing. A, MA-10.2 cells were treated with 20 µM of the PKA-specific inhibitor H-89, 20 µM of the MEK-specific inhibitor U0126, and 10 µM of the PI3K-specific inhibitor wortmannin as indicated 30 min before addition of 500 µM 8Br-cAMP. Total RNA was prepared after 2 h cAMP treatment for Northern analysis with Cyp17 and actin riboprobes. B, Western analyses of MA-10.2 cells treated as in A show that U0126 blocks the ability of cAMP to induce MEK to phosphorylate ERK1/2, known MEK substrates. The same extracts were used to detect total ERK1/2 as a control. C, Western analysis of MA-10.2 cells treated with wortmannin inhibits PI3K, effectively blocking Akt phosphorylation and activation. Total Akt from the same extracts is shown as a control. MA-10.2 cells (2 x 105) were treated with inhibitors and 50 µM 8Br-cAMP as in A. After 2 d incubation, media were collected and assayed by RIA for total testosterone (D) and progesterone (E). The mean values of three experiments performed in duplicate with unstimulated cells (black bars) and cAMP-stimulated cells (white bars) were plotted with error bars that represent SEM. The limit of detection for the testosterone assay is 4 ng/dl, which is shown with a dashed line. Statistical significance was assessed by the Bonferroni/Dunn post hoc test and those values of cAMP-treated cells significantly from untreated cells are shown with an asterisk. Testosterone and progesterone from all 8Br-cAMP-stimulated cells treated with inhibitors was significantly different from cells treated with cAMP alone.

 
U0126 has been shown to suppress steroidogenesis by inhibiting steroidogenic factor-1 (SF-1) phosphorylation by ERKs and by inhibiting StAR expression (48). Because inhibition of MEK by U0126 did not appear to block the expression of Cyp17 mRNA, we assessed the ability of both U0126 and wortmannin to inhibit the activation of their respective downstream signaling targets in MA-10.2 cells by Western analysis to ensure that those pathways were normally active (Fig. 4Go, B and C). The cAMP-induced phosphorylation of ERK1/2 to their active forms dpERK1/2 was inhibited by addition U0126, which inhibits MEK activity directly upstream of ERK1/2 activation. Wortmannin effectively inhibited the ability of PI3K to phosphorylate the phosphoinositide-dependent kinase-1, the upstream kinase that phosphorylates and activates protein kinase B (Akt/PKB) (45) suggesting that PI3K activity is not required in cAMP-mediated induction of Cyp17 expression in MA-10.2 cells.

The effect of the inhibitors on testosterone production was also assessed (Fig. 4DGo). Culture media of MA-10 cells, incubated with or without 50 µM 8Br-cAMP and the kinase inhibitors as indicated above, was collected after 2 d and assayed by RIA. Both 20 µM H-89 and U0126 effectively blocked the ability of chronic cAMP exposure to induce testosterone production by MA-10.2 cells to less than the sensitivity of the assay at 4 ng/dl. Wortmannin at 10 µM also significantly inhibited testosterone production but not as effectively as either H-89 or U0126. Because U0126 did not inhibit the expression of Cyp17 in MA-10 cells, we expected that its inhibition of testosterone synthesis was a result of inhibiting steroidogenesis upstream (48). In Fig. 4EGo, the results of assaying the concentration of progesterone in the conditioned media from cells stimulated by cAMP shows that U0126 was blocking progesterone synthesis, which then resulted in the lower testosterone levels observed in Fig. 4DGo. H-89 appears to increase the progesterone concentration in the media by approximately 4-fold and wortmannin showed a significant 50% reduction in progesterone accumulation.

CREB phosphorylation is not required for cAMP and MIS-regulated expression of Cyp17 mRNA
Several reports indicate that SF-1 phosphorylation is the key mechanism by which cAMP induces Cyp17 expression (32, 35, 52) but mutation of the SF-1 binding site in the murine Cyp17 promoter/luciferase reporter did not diminish that induction (data not shown). Because we had previously reported that the DNA region critical for cAMP-responsive Cyp17 induction was within -346 to -245 of the murine Cyp17 proximal promoter (53), we investigated whether MIS might be inhibiting the phosphorylation of Ser133 and activation of CREB, the canonical downstream effector of LHR activation. MA-10.2 cells were incubated with either 50 µM or 500 µM cAMP for 2 h with or without 35 nM MIS and analyzed for CREB phosphorylation (Fig. 5AGo) and Cyp17 mRNA expression (Fig. 5BGo). The amount of pCREB detected in cells with addition of 50 µM 8Br-cAMP was not greater than that of untreated cells when normalized to total CREB despite the 30-fold induction of Cyp17 mRNA expression observed with 50 µM 8Br-cAMP. A 3- to 5-fold induction pCREB was observed with addition of 500 µM 8Br-cAMP that correlated well with the 55-fold induction of Cyp17 mRNA but was not diminished by addition of MIS when normalized to the respective amounts of unphosphorylated CREB. In contrast, MIS effectively blocked Cyp17 mRNA expression in cells incubated with either 50 µM or 500 µM cAMP (Fig. 5BGo).



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  		Figure 5. CREB phosphorylation is not required for either cAMP or MIS to regulate Cyp17 mRNA expression. A, MA-10.2 cells were treated overnight with 50 µM 8Br-cAMP, which is sufficient for induction of Cyp17 mRNA expression, or for 2 h with 500 µM 8Br-cAMP with or without 35 nM MIS. Cells were also untreated or treated with MIS alone overnight. Protein lysates were made from the cells and analyzed by Western analysis for detection of pCREB and CREB in companion blots. The phosphorylated form of the CREB-related protein, ATF-1, is also detected by pCREB antibodies and serves as an internal control for protein loading between cAMP-treated and cAMP+MIS-treated lines. B, Northern blot analysis of RNA to detect Cyp17 mRNA expression from cells treated as indicated with or without 35 nM MIS for 4 h. Actin is shown to control for loading.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our recent characterization of the murine Cyp17 proximal promoter in vitro by deoxyribonuclease I footprinting, by EMSA, and by mutation of the SF-1 binding site in promoter/luciferase transfection experiments did not indicate any differences when either cAMP or MIS is added to MA-10 cells (data not shown). Because we show here that MIS can block the cAMP-mediated induction of Cyp17 expression, we suspected that MIS must act by interfering with the cAMP pathway upstream of transcription factor activation or binding to the Cyp17 promoter. We previously reported that induction of Cyp17 mRNA by cAMP in mouse Leydig cells requires new protein synthesis (54). Thus, it is also possible that MIS interferes with the synthesis of this as yet unidentified protein(s). We could not, however, rule out that the effect of cAMP induction of such an intermediate protein is not via the phosphorylation of CREB.

Recent reports have provided evidence for cAMP, PKA-independent pathways. For example, cAMP can also regulate the MAPK pathway by virtue of its ability to activate cAMP-guanine nucleotide exchange factors (55, 56) and signaling by other trophic pituitary hormones, TSH and FSH, can activate the PKB/Akt and PI3K pathways (51, 57). Our results with H-89 suggest that cAMP-induced expression of Cyp17 mRNA is mediated via the PKA pathway. To examine whether the phosphorylation of CREB was involved in the cAMP-mediated pathway that leads to increased expression of Cyp17 mRNA, we investigated whether 50 µM cAMP, the dose demonstrated to stimulate Cyp17 mRNA expression, activated phosphorylation of CREB. As illustrated in Fig. 5AGo, this dose of cAMP was not sufficient to allow detection of phosphorylated CREB by Western analysis and H-89, which we showed could inhibit Cyp17 expression, had no noticeable affect on CREB phosphorylation by cAMP (data not shown). This finding with endogenous cellular components is in agreement with previous reports using in vitro assays that demonstrate cAMP-mediated transcriptional activation of mouse Cyp17 (44), rat Cyp17 (35), and bovine Cyp17 (33) is not mediated by CREB. In addition, this result suggests that phosphorylated CREB may not be involved in any of the pathways leading to Cyp17 mRNA induction.

In MA-10 cells, StAR gene expression also appears to be also regulated by cAMP in a PKA-independent mechanism that requires arachidonic acid release (58) or activation of ERKs and subsequent phosphorylation of SF-1 (48), an orphan nuclear receptor critical for expression of many genes involved in steroidogenesis and development. StAR mRNA expression appears slightly induced rather than inhibited by addition of MIS, suggesting that MIS might be regulating StAR expression. Also, we have shown that in MA-10.2, addition of MIS results in a severalfold increase in progesterone in the conditioned media above that observed with cAMP alone (Fig. 2BGo). However, others have shown that in MA-10 cells progesterone concentration in the media might not be a reliable indicator of steroidogenic activity since many steroid derivatives are made (59). Also, in mice that overexpress MIS, StAR mRNA levels in testes were not significantly different (24) and cells treated with MIS alone did not show an induction of StAR mRNA (Fig. 2FGo). These data strongly suggest that the signal transduction pathway used by cAMP to induce expression of StAR mRNA is divergent from that used to induce Cyp17 mRNA and that MIS only interferes with the latter but may indirectly induce the former.

The 4-fold greater induction of progesterone that was measured in the media of cells treated with H-89 and cAMP over that of cells treated with cAMP alone as shown in Fig. 4EGo is counterintuitive. H-89 has been shown to decrease the cAMP-mediated induction of StAR in MA-10 cells (58), which would suggest a negative affect on progesterone production but that interpretation might be too simplistic. H-89 has shown a biphasic effect on progesterone production in cultured granulosa cell stimulated with FSH with lower concentrations of H-89 having a positive effect on progesterone production (60). A trivial explanation might be that H-89 inhibits the conversion of progesterone to its metabolites more strongly than it does the synthesis of progesterone and thus leads to progesterone accumulation in response to cAMP. As shown in Fig. 4Go, A and D, we have observed that the cAMP-mediated induction in Cyp17 mRNA and testosterone production is greatly inhibited by H-89. A more detailed study of the effect of H-89 on expression of the enzymes in the steroidogenic pathway is clearly needed before this conundrum of H-89-induced progesterone production can be satisfactorily explained.

Signal transduction by members of the TGFß family of glycoprotein homodimers, among which MIS, activin, inhibin, bone morphogenetic proteins (BMPs), nodal, and the many growth and differentiation factors (GDFs) are examples of members, occurs by a well-conserved sequence of events that is initiated by ligand binding to a heteromeric complex of single transmembrane, serine/threonine kinases (reviewed in Ref. 15). The signal transduction pathways employed by the TGFß family fall into two distinct sets: the TGFß/activin group of type I receptors (Alk5/Alk1, and 4) and the MIS/BMP/GDF group (ALK1, 2, 3, and 6). The ligand specificity within the family is determined by the type II receptor, which can bind ligand cooperatively with the type I receptor in the case of MIS/BMP/GDF or else recruits the appropriate type I receptor into the complex as with TGFß/Activin. The ligand-bound type II receptor phosphorylates the type I receptor, activating its latent kinase for subsequent downstream signaling via intracellular Smad proteins that translocate to the nucleus to affect gene transcription.

Smads fall into three different classes, receptor-regulated R-Smads, inhibitory Smads, and the common Smad4. The R-Smads 2 and 3 are phosphorylated by the TGFß/activin type I receptors Alk4 and 5, whereas the R-Smads 1, 5, and 8 are phosphorylated by the MIS/BMP/GDF type I receptors Alk1, 2, 3, and 6. These phosphorylated Smads then dimerize with the common Smad4 to form heteromeric complexes that translocate to the nucleus and effect their respective activities by binding to the Smad-responsive DNA element, alone with a relatively loose specificity or in a supercomplex with cofactors that can modulate ligand-specific gene expression such as FAST-1 and CREB-binding protein/p300. In addition, MIS has also been shown to modulate other signaling pathways such as nuclear factor-{kappa}B (61), and ß-catenin (62) as well as cell cycle progression by up-regulation of p16, a member of the INK4a family of cyclin-dependent kinase inhibitors (63). Clues as to how the MIS pathway will perturb the cAMP-induced pathway will be important in understanding the changes and balance achieved in steroid production during various stages of development.

Recent experiments studying the bovine and rat Cyp17 promoter suggest that SF-1 mediates the cAMP-induced expression of Cyp17 mRNA through a PKA-dependent mechanism (32, 35) or that phosphorylation of SF-1 in response to cAMP occurring through the MAPK pathway is the key mechanism for induction (52). The synergistic effect of added cAMP and SF-1 to cells on the expression of inhibin {alpha} promoter-driven/luciferase reporter constructs in granulosa cells has been shown to correlate with the formation of a stabilized complex that can recruit coactivators such as CREB-binding protein and induce histone acetylation (64). Because our results thus far have indicated that SF-1 binding to the murine Cyp17 promoter is unchanged by addition of cAMP or MIS (data not shown), it will be important to investigate these observations with regard to cAMP-induced expression of the endogenous Cyp17 mRNA, which may be tissue specific, in this new subclone of MA-10 cells that expresses endogenous CREB and SF-1 (65) and do not require overexpression. The findings described here encourage and provide the tools for future studies devoted to identifying the downstream proteins activated by PKA involved in the induction of Cyp17 in Leydig cells and the mechanism by which MIS abrogates this induction.


   Acknowledgments
 
We would like to thank Dr. David T. MacLaughlin and Nima Pahlavan for MIS production and Dr. Douglas Stocco for the murine StAR cDNA, Dr. Mario Ascoli for the MA-10 cells, and Dr. A. F. Parlow for LH. We are also indebted to Sheila Mallette and Joe Moy for the RIA data. We are grateful to Drs. David T. MacLaughlin, Shyamala Maheswaran, and Trent R. Clarke for preliminary reviews of the manuscript.


   Footnotes
 
This work was supported in part by research grants from NCI (R29-CA-79459 to J.T. and R01-CA-17393 to P.K.D.) and by NICHD/NIH through cooperative agreement (U54-HD-28138 to J.T. and P.K.D. and U54-HD-31398 to A.H.P.) as part of the Specialized Cooperative Centers Program in Reproduction Research. A.M.T. was supported by a Lalor Foundation postdoctoral fellowship.

Abbreviations: Alk, Activin group of type I receptors; BMP, bone morphogenetic protein; CREB, cAMP responsive element binding protein; Cyp17, cytochrome P450 17{alpha}-hydroxylase/C17–20 lyase; dpERK, diphospho ERK; GDF, growth and differentiation factor; LHR, LH receptor; MEK, MAPK kinase; MIS, Müllerian inhibiting substance; MISRI and MISRII, MIS type I and II receptors; p, phospho; PI3K, phosphatidylinositol-3 kinase; PKA, protein kinase A; SDS, sodium dodecyl sulfate; SF-1, steroidogenic factor-1; StAR, steroidogenic acute regulatory protein.

Received December 3, 2001.

Accepted for publication May 10, 2002.


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 Introduction
 Materials and Methods
 Results
 Discussion
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