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Am J Physiol Endocrinol Metab 293: E277-E285, 2007. First published April 3, 2007; doi:10.1152/ajpendo.00447.2006
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The regulation of FSHbeta transcription by gonadal steroids: testosterone and estradiol modulation of the activin intracellular signaling pathway

Laura L. Burger,1,2 Daniel J. Haisenleder,1,2 Gordon M. Wotton,1,2 Kevin W. Aylor,1 Alan C. Dalkin,1 and John C. Marshall1,2

1Division of Endocrinology, Department of Internal Medicine; and 2Center for Research in Reproduction, University of Virginia, Charlottesville, Virginia

Submitted 24 August 2006 ; accepted in final form 30 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent reports suggest that androgens increase FSHbeta transcription directly via the androgen receptor and by modulating activin signaling. Estrogens may also regulate FSHbeta transcription in part through the activin system. Activin signaling can be regulated extracellularly via activin, inhibin, or follistatin (FS) or intracellularly via the Smad proteins. We determined the effects of androgen and estrogen on FSHbeta primary transcript (PT) concentrations in male and female rats, and we correlated those changes with pituitary: activin betaB mRNA, FS mRNA, the mRNAs for Smads2, -3, -4, and -7, and the phosphorylation (p) status of Smad2 and -3 proteins. In males, testosterone (T) increased FSHbeta PT two- to threefold between 3 and 24 h and was correlated with reduced FS mRNA, transient increases in Smad2, -4, and -7 mRNAs, and a six- to 10-fold increase in pSmad2, and activin betaB mRNA was unchanged. In females, T also increased FSHbeta PT twofold and pSmad2 threefold but had no effect on activin betaB, FS, or the Smad mRNAs. Androgen also increased Smad2 phosphorylation in gonadotrope-derived {alpha}T3 cells. In contrast, estradiol had no effect on FSHbeta PT but transiently increased activin betaB mRNA and suppressed FS mRNA before increasing FS mRNA at 24 h and increased Smads2, -3, and -7 mRNAs and pSmad2 threefold. In conclusion, T acts on the pituitary to increase FSHbeta PT in both sexes and modulates FS mRNA, Smad mRNAs, and/or Smad2 phosphorylation. These findings suggest that T regulates FSHbeta transcription, in part, through modulation of various components of the activin-signaling system.

follicle-stimulating hormone-beta; Smad proteins

THE PRODUCTION AND SECRETION of the pituitary gonadotropins LH and FSH involve complex interactions between the hypothalamus, gonadal steroids, and peptides. Although gonadal steroids act at the level of the hypothalamus to suppress gonadotropin-releasing hormone (GnRH) pulsatility, they also have direct effects on the pituitary to regulate LH and FSH subunit synthesis. Androgens directly stimulate FSHbeta mRNA both in primary pituitary cell cultures and GnRH-deficient rats (16, 21, 36, 49, 50), which reflects a rapid and sustained increase in FSHbeta primary transcript (PT) levels (10).

The effects of estrogen on FSHbeta gene expression are less clear. Previous experiments did not show a direct effect of estrogen on FSHbeta transcription in female rat pituitary cells in vitro (40) or in female mice carrying an ovine FSHbeta transgene (24). In contrast, estrogen has been reported to suppress FSHbeta gene transcription in ovine pituitary cells (3, 32, 38) and FSHbeta mRNA in cultured female rat pituitary cells (7), and we (10) recently reported that estradiol (E2) suppressed FSHbeta PT in castrate GnRH-deficient male rats.

The mechanism(s) whereby androgens and estrogens regulate FSHbeta transcription is not well understood and may involve both classical steroid signaling and modulation of the signaling pathway for activin. Activin is a member of the TGFbeta superfamily and is produced in a variety of tissues, including the gonadotrope, where it acts in a paracrine/autocrine manner to stimulate FSHbeta transcription (4, 5, 48). Three to six androgen response elements (ARE) have been reported in the ovine and murine FSHbeta promoters, and at least one is required for androgen action (41, 45). However, there are a number of reports that androgen may also regulate FSHbeta transcription indirectly via activin. We reported that testosterone (T) rapidly and specifically increases FSHbeta PT in GnRH-deficient male rats and was associated with a rapid decline in pituitary follistatin (FS) mRNA (10). FS is a glycoprotein, produced by both the gonadotropes and the pituitary folliculostellate cells, that binds to and neutralizes the bioactivity of activin (5). Additionally, others have reported that the effects of T on FSHbeta mRNA either require activin or are blunted by FS (7, 30, 41).

The mechanism of estrogen action on FSHbeta gene expression is less well understood. Miller and Miller (32) and Strahl et al. (42) have reported an estrogen-responsive region in the ovine FSHbeta promoter, but the area does not contain an estrogen response element or bind estrogen receptor, suggesting that estrogen regulates FSHbeta expression indirectly. There is some evidence that estrogens may also regulate FSHbeta via an activin component. E2 increased pituitary FS PT and mRNA in female rats (26, 39), blunted the actions of activin on FS mRNA in female rat pituitary cells (7), and suppressed activin betaB subunit mRNA in ovine pituitary cells (3).

Activin signals intracellularly via phosphorylation of Smad proteins. Briefly, activin binds to its type II receptor subunit, which then pairs with a type I receptor subunit, forming a heteromeric complex at the cell surface. Then serine/threonine kinase activity of the type II receptor phosphorylates the type I receptor, initiating a postreceptor signaling/phosphorylation cascade. The type I receptor phosphorylates Smad2 and -3. Phosphorylated Smad2 and/or -3 then partners with Smad4, and the complex translocates to the nucleus and binds DNA to regulate gene activity (for review, see Ref. 18). Additionally, activin intracellular signaling can be regulated by "inhibitory" Smad7, which binds to the type I receptor and prevents Smad2 or -3 phosphorylation or hastens their degradation via recruitment of phosphatases.

The objective of this study was to examine androgen and estrogen regulation of FSHbeta gene transcription and whether it involves the availability of bioactive activin or the Smad intracellular signaling pathway. To this end, we characterized changes in activin betaB mRNA, FS mRNA, Smad2, -3, -4, and -7 mRNA expression, and Smad2/3 phosphorylation in pituitaries from gonadectomized GnRH-deficient male and female rats treated with physiological levels of T and E2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal models. Adult (225–250 g) male and female Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were used for all experiments. Rats were housed in a light- (lights on 0500–1700) and temperature-controlled (25°C) room and allowed access to food and water ad libitum. All surgeries were performed under isoflurane (2.5% isoflurane, balance O2; ISO-THESIA, Vetus Animal Heath, Burns Veterinary Supply, Westbury, NY) anesthesia. At the completion of experiments, rats were euthanized by decapitation under anesthesia. Trunk blood was collected for serum hormone measurements. Pituitaries were collected and bisected, and then one-half was processed immediately for protein and the other half snap-frozen in liquid nitrogen and stored at –70°C until RNA was extracted. The University of Virginia Animal Care and Use Committee approved the animal experimentation described within this report.

To isolate the effects of steroids on the pituitary, we employed a gonadectomized (GDX) GnRH antagonist-treated animal model as previously reported (9, 10, 39). Groups of male and female rats were GDX (n = 5–7/group) and given the water-soluble GnRH antagonist LRF-147 (100 µg/0.5 ml, 0.1% BSA-0.9% NaCl, sc) every 12 h. Four days after gonadectomy, male rats received two T implants each containing a 20-mm column of crystalline T that results in male T levels (~6 ng/ml after 24 h) (10). Female rats received one T implant containing a 5-mm column of crystalline T that results in female T levels (~300 pg/ml after 24 h) (51). Groups of rats were then killed 0, 3, 8, and 24 h after the onset of T treatment. To determine whether the effects of T are androgen specific, one group was also treated for 24 h with dihydrotestosterone (DHT), as previously described (25). Pituitary FSHbeta PT and activin betaB, FS, Smad2, -3, -4, and -7 mRNAs, Smad phosphorylation, serum gonadotropins, and T were measured.

To determine whether T alters Smad phosphorylation in gonadotropes, we utilized the gonadotrope derived {alpha}T3 cells. {alpha}T3 cells were plated onto 32-mm culture dishes and grown until confluent in DMEM [containing phenol red, L-glutamine, 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 µg/ml)]. Twenty-four hours before androgen treatment, the medium was replaced with phenol red-free DMEM with charcoal-stripped FBS (5% FBS). The next day, the medium was replaced with serum-free, phenol red-free DMEM for 4 h, and then cells were treated for 8 h with 100 ng/ml T or vehicle (0.2% ethanol; n = 6 wells/treatment). As a positive control for Smad phosphorylation, one to two separate wells were also treated with 30 ng/ml recombinant human activin A (R&D Systems, Minneapolis, MN) for 1 h. Upon completion, cells were washed with phosphate-buffered saline, lysed, and protein collected to determine Smad2/3 phosphorylation. Experiments were repeated at least two times to confirm results.

Because estrogen is the dominant gonadal hormone in females, we also investigated the effects of physiological levels of E2 on the regulation of FSHbeta in female rats. Groups of female rats (n = 5–6/group) were ovariectomized (OVX) and then treated for 4 days with GnRH antagonist (100 µg LRF-147, 12 h). Rats then received one silicone implant containing E2 [25-mm column of 1 mg/ml E2 in sesame oil (Sigma); silastic tubing 1.6 mm id, 3.2 mm od (Dow Corning, Midland, MI)] that resulted in proestrus E2 levels (~50 pg/ml). Rats were then killed 0, 3, 8, and 24 h after the onset of E2 treatment. Pituitary FSHbeta PT and activin betaB, FS, Smad2, -3, -4, and -7 mRNAs, Smad phosphorylation, serum gonadotropins, and E2 were measured.

Measurement of serum hormones, RNA preparation, the mRNAs for activin betaB, FS, and Smad mRNAs, and FSHbeta PT. Serum LH and FSH were measured by RIA using the standards NIDDK RP-3 for LH and NIDDK RP-2 for FSH (National Hormone and Pituitary Program). The sensitivities for the LH and FSH assays are 0.09 and 0.8 ng/ml, respectively. The coefficients of variation are 4.7 and 13.5% (intra- and interassay) for LH and 4.6 and 14.4% for the FSH assay. T and E2 were measured by RIA using kits provided by Diagnostic Products (Los Angeles, CA) and Diagnostic Systems Laboratories (Webster, TX), respectively. The sensitivities and coefficients of variation for the assays are 0.1 ng/ml, 5.0%, and 8.2% (intra- and interassay) for T and 10 pg/ml, 5.2%, and 12.1% for E2.

Total pituitary RNA was extracted using the acid guanidinium method (13). Residual genomic DNA was removed by treatment with 1 U RNase Free DNase I/µg RNA (Roche Molecular Biochemicals, Indianapolis, IN) at 37°C for 1 h. RNA preparations were confirmed to be DNA free by PCR in the absence of a preceding RT reaction. FSHbeta PT and FS mRNA were measured by quantitative RT-PCR assays, as previously described (14, 28). The mRNA for activin betaB and the Smads were determined by real time RT-PCR using an iCycler IQ (Bio-Rad, Hercules, CA) and the QuantiTech SYBR Green RT-PCR Kit (Qiagen, Valencia, CA). The real-time assay for activin betaB mRNA is based on the amplicon from our previously described quantitative RT-PCR assay (8). The areas amplified for Smad2, -3, -4, and 7 mRNAs are based on previous real-time PCR assay reported by Drummond et al. (19). Assay conditions were optimized to generate a single PCR product as determined by melt curves and agarose gel electrophoresis. The primers used were activin betaB forward (FWD) 3'-GCCAGCGGATCAGTTTTAAT-5', reverse (REV) 3'-ACTCTACCTTCTGGGTGTATAAGG-5'; Smad2 FWD 5'-TTACATCCCAGAAACACCA-3', REV 5'-CAAGCGCACTCCCCTTCCTA-3; Smad3 FWD 5'-GGCGGTCAAGAGCTTGGTGA-3, REV 5'-TGTAGTCATCCAGAGGGGGGAA-3; Smad4 FWD 5'-GCAGATAGCTTCAGGGCCTCA-3, REV 5'-CGATCTCCTCCAGAAGGATCCA-3; and Smad7 FWD 5'-CAACCCCCATCACCTTAGTCGA-3, REV 5'-CTTGCTCCTCACTTTCTGTACCA-3. PCR product identity was confirmed by DNA sequencing. Unknown samples were measured using 10–100 ng RNA against an external standard curve. All samples, including standards, were measured in triplicate. All samples from a study were measured in the same assay. Mean intra-assay coefficients of variation are 12.1, 14.8, 12.8, 12.9, and 13.8% for activin betaB and Smad2, -3, -4, and -7, respectively.

Pituitary protein preparation Western immunoblot assays. For protein isolation, hemi pituitaries were homogenized in tissue lysis buffer [50 mM HEPES, 100 mM NaCl, 2 mM EDTA, 1% NP-40, and protease and phosphatase inhibitor cocktails (P8340 and P5726, respectively, with stocks considered x100; Sigma, St Louis, MO)]. Pituitary protein lysates (50 µg/rat sample, 30 µg/{alpha}T3 cell lysate) were resolved by electrophoresis (12.5% SDS PAGE) and then transferred to nitrocellulose filters. Receptor-mediated Smad2 and -3 (phosphorylated and total) proteins were measured by Western immunoblot assay. For phosphorylated Smad2 and -3, primary antibodies (rabbit) were obtained from Cell Signaling Technology (Beverly, MA); each antibody recognizes a protein of 58 kDa apparent molecular weight. The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit (Upstate Biotechnology, Lake Placid, NY). As a positive control for Smad2 and -3 phosphorylation, each filter included two lanes containing 10–30 µg protein from {alpha}T3 cells that were either untreated or treated with activin A (30 ng/ml) for 1 h. Immunoactivity was detected using the Super Signal Pico West chemiluminescent system (Pierce, Rockford, IL), followed by autoradiography. Protein bands were quantitated by densitometry using TotalLab Software (Amersham Biosciences, Piscataway, NJ). Following phosphorylated Smad (pSmad) determination, filters were stripped (0.4 M glycine, 0.2% SDS, 2% Tween-20, pH 2.2) twice for 30 min and reprobed for total (t)Smad2/3. The rabbit anti-tSmad2/3 antibody (Cell Signaling) detects the conserved amino terminus of both Smad2 and Smad3, resulting in a single band for both proteins at 58 KDa. Last, as a protein loading control, blots were reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 37 KDa; Santa Cruz Biotechnology, Santa Cruz, CA).

Analysis. All data were examined by analysis of variance. Significant differences (P < 0.05) were determined post hoc by Duncan's multiple-range test. Prior to analyses, all measurements were transformed to the logarithmic scale to attain equal variation among treatments.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Regulation of the activin-signaling cascade by androgens. GnRH antagonist suppressed the post-GDX rise in serum LH to 0.18 ± 0.04 and 0.16 ± 0.01 ng/ml in males and females, respectively; LH levels for intact rats are typically 0.3–0.7 ng/ml (6, 25). In males, T implants increased T to 17.5 ± 2.0 ng/ml at 3 h before falling to 6.8 ± 0.1 ng/ml at 24 h. Similarly in females, T levels were maximal at 3 h (1.5 ± 0.3 ng/ml) and then declined to 0.29 ± 0.02 ng/ml at 24 h. Consistent with earlier reports, in male rats T significantly increased serum FSH by 3 h and remained elevated through 24 h (3.9 ± 0.2, 4.9 ± 0.1, and 5.9 ± 0.4 ng/ml at 0 h, 3-h T, and 24-h T respectively). In females, the lower dose of T tended to increase FSH, although not significantly (4.9 ± 0.5, 5.6 ± 0.7, and 5.7 ± 0.4 at 0 h, 3-h T, and 24-h T, respectively). The lack of increase in serum FSH in OVX GnRH antagonist-treated females may reflect T dose or the elevated FSH levels in these animals (intact ~2 ng/ml) due to the loss of circulating inhibin after OVX (9).

The effects of T in male and female rats on pituitary FSHbeta PT, activin betaB mRNA, and FS mRNA are shown in Fig. 1. T administration induced a rapid (<3 h) and sustained (through 24 h) increase in FSHbeta PT in both male and female rats, with the greater magnitude of increase (3-fold) in males vs. females (2-fold). Activin betaB mRNA levels tended to decline after T in both sexes but did not reach significance. As previously reported (10), the increase in FSHbeta PT in male rats was accompanied by a 50–60% reduction in FS mRNA. However, FS mRNA did not change after T in females.


Figure 1
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  		Fig. 1. The time course of testosterone (T) action on pituitary FSHbeta-subunit primary transcript (PT), activin betaB mRNA, and follistatin (FS) mRNA in gonadotropin-releasing hormone (GnRH)-deficient gonadectomized male and female rats. Rats were gonadectomized (n = 4–7/group) and given GnRH antagonist every 12 h. Four days later, rats received silastic capsules containing male or female doses of T (see MATERIALS AND METHODS) and were killed 0, 3, 8, and 24 h later. All data are presented as %0–h (±SE) controls. Points with different letters are significantly different (P < 0.05).

 
The effects of T on Smad mRNAs are shown in Fig. 2. T increased the mRNAs for Smad2, -4, and -7 at 8 h (vs. 0-h controls) in male rats but had no effect on Smad mRNA expression in female rats. The effects of T on FSHbeta PT in males have previously been shown to be androgen specific (10), and the effects of the nonaromatizable androgen DHT on female rats are shown in Table 1. Twenty-four hours of DHT increased serum FSH, FSHbeta PT, and the mRNAs for Smad3, -4, and -7 and suppressed FS mRNA at 24 h. The differences in results between T and DHT in females are likely due to androgen dosage.


Figure 2
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  		Fig. 2. The time course of T action on pituitary Smad mRNAs in GnRH-deficient gonadectomized male and female rats. Experimental details are described in Fig. 1. All data are presented as %0–h (±SE) controls. Points with different letters are significantly different (P < 0.05).

 

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  		Table 1. Mean levels of serum FSH, FSHbeta PT, and the mRNAs for FS, Smad2, Smad3, Smad4, and Smad7 in OVX female rats treated with GnRH antagonist only (0–h controls), T, or DHT for 24 h

 
To determine whether T modulates Smad activity, we used Western immunoblot assays to measure pSmad2 and -3 as well as tSmad2/3, and results are shown in Fig. 3. In light of the rapid induction of FSHbeta PT by T (2- to 3-fold increase <3h), we measured Smad phosphorylation between 0 and 8 h. At baseline, pSmad3 was barely detectable in male or female pituitary protein samples, and there did not appear to be any change with T treatment (Fig. 3A). In contrast, pSmad2 signal was easily detected and showed a significant induction by T. Between 3 and 8 h, pSmad2 increased 10- and sixfold, respectively, in male pituitaries, whereas in females the increase was not as large (1.5- to 2.5-fold) and peaked later at 8 h. T induced a small and transient increase in tSmad2/3 in female rat pituitaries at 3 h and had no effect on males. To control for protein loading and transfer, GAPDH levels were also measured for all blots, and no differences were observed between samples.


Figure 3
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  		Fig. 3. The effects of T on phosphorylated Smad 2, phosphorylated Smad3, and total Smad2 and -3 (pSmad2, pSmad3, and tSmad2/3, respectively) in pituitary protein collected from GnRH-deficient gonadectomized male and female rats (details in Fig. 1). A: representative Western blots of pituitary protein in male rats treated for 0, 3, or 8 h with T and immunostained for pSmad2, pSmad3, tSmad2/3, and GAPDH. Protein amounts are 50 µg/lane for pituitary lysates and 10 µg/lane for {alpha}T3 control lysates. B: changes in Smad2 phosphorylation and tSmad2/3 in both male and female rats were quantified by densitometry and expressed as %0–h (±SE) controls. Points with different letters are significantly different (P < 0.05).

 
To determine whether androgen-induced increases in Smad phosphorylation occur in gonadotropes, we treated {alpha}T3 cells with 100 ng/ml T for 8 h. Similarly to results in rats, T increased Smad2 phosphorylation 3.9-fold vs. vehicle-treated controls after 8 h (P < 0.05 vs. controls; Fig. 4). As in whole pituitary lysates, pSmad3 protein was very low basally and did not change with androgen treatment (results not shown). tSmad2/3 levels were also constant after T treatment.


Figure 4
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  		Fig. 4. The effects of T on pSmad2 and tSmad2/3 in protein collected from the gonadotrope-derived {alpha}T3 cell line. Representative Western blots of {alpha}T3 cells treated with either T or vehicle (6 wells/treatment are shown). Cells were plated in steroid-free medium containing 5% charcoal-stripped BSA for 24 h; the following day cells were moved into serum-free medium for 4 h and then treated with either 100 ng/ml T or 0.2% ethanol. As a positive control for Smad phosphorylation, 1–2 separate wells were also treated with 30 ng/ml recombinant human activin A for 1 h. Each lane contains 30 µg of cell lysate.

 
Regulation of the activin-signaling cascade by estrogen. GnRH antagonist suppressed the post-OVX increase in serum LH, and neither serum LH nor FSH was affected by E2 treatment (LH = 0.15 ± 0.01 and 0.17 ± 0.02 ng/ml at 0 and 24 h, respectively; FSH = 4.9 ± 0.5 and 5.4 ± 0.4 ng/ml at 0 and 24 h, respectively). Silicone implants containing E2 resulted in proestrus serum E2 levels (50.0 ± 5.0 pg/ml at 24 h). The effects of E2 on FSHbeta PT, activin betaB, FS, and Smad mRNAs are shown in Fig. 5. In contrast to prior data in male rats (10), E2 had no effect on pituitary FSHbeta PT levels in females. The effects of E2 on activin betaB and FS mRNA were biphasic. Activin betaB mRNA increased 150% at 3 and 8 h before returning to control levels, whereas FS mRNA was suppressed 70% at 3 h before increasing to 150% of controls after 24 h. Smad2, -3, and -7 mRNAs were increased between 3 and 8 h and were transient for Smad2 and -7, but the rise in Smad3 mRNA was sustained through 24 h (Fig. 5). As with the previous experiments, pSmad3 was barely detectable and did not change after E2 (Fig. 6). pSmad2 increased two- to threefold 3–8 h after E2, and tSmad2/3 increased slightly at 8 h (Fig. 6). To control for protein loading and transfer, GAPDH levels were also measured for all blots, and no differences were observed between samples (data not shown).


Figure 5
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  		Fig. 5. The time course of estradiol (E2) action on pituitary FSHbeta-subunit PT and the mRNAs for activin betaB, FS, and the Smads in GnRH-deficient ovariectomized (OVX) female rats. OVX rats (n = 4–6/group) were given GnRH antagonist every 12 h. Four days later rats received silastic capsules containing E2 and were killed 0, 3, 8, and 24 h later. All data are presented as %0–h (±SE) controls. Points with different letters are significantly different (P < 0.05).

 

Figure 6
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  		Fig. 6. The effects of E2 on pSmad2 and tSmad2/3 in pituitary protein collected from GnRH-deficient OVX female rats. Changes in t- and pSmad2 immunostaining after E2 treatment were quantified by densitometry and expressed as %0–h (±SE) controls. Points with different letters are significantly different (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present results extend previous data and show that physiological T concentrations act directly on the pituitary to increase FSHbeta transcription in both male and female rats. Furthermore, the effects of T are androgen specific, as nonaromatizable DHT increased FSHbeta PT and E2 did not. Potentially, androgens could increase FSHbeta transcription directly via the classical ligand-bound steroid receptor-signaling pathway, by enhancing the activity of other signaling systems (e.g., activin/Smad), or by a combination thereof. Spady et al. (41) and Thackery et al. (45) have reported that androgen increased the activity of ovine and mouse FSHbeta promoter-reporter constructs transfected into pituitary-derived LbetaT2 cells. Additionally, they identified three and six AREs in the ovine and murine FSHbeta promoters, respectively, and mutations in the most distal ARE in both, as well as most proximal ARE in the sheep, rendered the promoter constructs androgen insensitive. These data indicate that androgen acts, at least in part, through binding to its own receptor and interacting with androgen-responsive gene sequences to stimulate FSHbeta transcription.

However, data reported here as well as in a number of earlier studies (7, 10, 30, 41) indicate that androgen may also regulate FSH indirectly via modulating the activity of activin. In male rats, the T-induced increase in FSHbeta PT is temporally correlated with a suppression of FS mRNA, suggesting that T may also act indirectly by increasing the bioavailability of pituitary activin. Similarly, the administration of exogenous FS to pituitary cell cultures has been shown to block androgen-induced FSH secretion (7, 30) . Also, Spady et al. (41) found that FS treatment or mutation of an activin-responsive Smad binding element (SBE) in the ovine FSHbeta promoter abrogated the induction of the promoter by androgen. In contrast, our earlier studies in primary rat pituitary cells (10) revealed that, despite addition of exogenous FS, T treatment still increased FSHbeta transcription. The differences between these two studies are likely due to experimental methodologies. Specifically, we cotreated rat pituitary cells with 30 ng/ml recombinant human FS ± androgen, whereas Spady et al. pretreated LbetaT2 cells for 20 h with 250 ng/ml rhFS before treatment with androgen ± FS. Therefore, in our paradigm the activin intracellular signaling system was likely undisturbed when cells were treated with androgen, which may account for why we still observed an increase in FSHbeta PT, whereas they found that androgen-induced FSHbeta promoter activity was highly dependent on activin and presumably its intracellular signaling system. Accordingly, we aimed to determine whether androgen affected components of the activin-signaling system and whether those changes were correlated with FSHbeta transcription.

We found that T rapidly increased FSHbeta PT in both male and female rats but had sex-specific effects on FS and Smad mRNAs. In male rats, T suppressed FS mRNA and transiently increased the mRNAs for Smad2, -4, and -7. In contrast, in female rats a lower T dose increased FSHbeta PT but had no effect on FS or Smad mRNA expression. The differences in FS and Smad mRNA expression between the sexes may reflect androgen dosage, as female rats treated with DHT responded in a fashion similar to males, with a larger increase in serum FSH and FSHbeta PT, suppression of FS mRNA, and increased Smad3 and -4 mRNAs. However, it is important to note that in female rats T stimulation of FSHbeta transcription occurs in the absence of androgen-specific changes in the activin-signaling pathway.

Because the changes in FS and Smad mRNAs are not required for androgen induction of FSHbeta transcription, it is possible that androgens could modulate inhibitor Smad7 gene expression, which is known to be involved in negative feedback of activin intracellular signaling (18). Smad7 mRNA is present in both normal pituitary cells and gonadotrope cell lines and is stimulated by activin, a response that can be blocked by FS (5). Overexpression of Smad7 protein in LbetaT2 cells disrupts activin-induced increases in FSHbeta gene expression (4, 20). However, we observed only modest changes in Smad7 mRNA concentration in male rats, and no differences were seen in females. Thus, alterations in the endogenous inhibitor of activin signaling, Smad 7, likely do not serve to modulate androgen action. There are no reports of hormone-modulated changes in Smad2, -3, or -4 mRNA in gonadotropes, but in other cell types TGFbeta family members have been reported to increase these RNAs in a feed-forward mechanism to regulate the sensitivity of their signaling pathways (1). Overexpression of Smad3 alone or in combination with Smad4 increases FSHbeta promoter activity in LbetaT2 cells (4, 20, 22, 29, 43, 44). Smad2 overexpression also increases FSHbeta promoter activity, but only in combination with Smad4 (4, 20, 22, 29, 43). Additionally, FSHbeta promoter activity is attenuated when Smad2 or -3 protein is suppressed (4, 20, 29).

Alternatively to changes in Smad gene expression, androgens could regulate the FSHbeta gene via modulation of Smad (-2 and/or -3) phosphorylation. In pituitary cells, activin-induced increases in FSHbeta transcription have been correlated to increased phosphorylation of Smad2 and -3 (4, 20). We investigated the effects of T on phosphorylation of the COOH-terminal SSXS motif of Smad2 and -3, which is regulated by the type I receptor. Despite data suggesting that Smad3, in combination with Smad4, plays the major role in transmitting the activin signal for the FSHbeta gene (22, 43, 44), we were unable to detect pSmad3 signal either basally or after androgen exposure in pituitary lysates or {alpha}T3 cells. This did not appear to reflect limitations in our Western blot assay, as we had no trouble observing activin-induced Smad3 phosphorylation in {alpha}T3 cells. Therefore, we conclude that basal pSmad3 levels in the rat pituitary are low and unaffected by T. In contrast, pSmad2 levels were easily detected and increased after T administration; Smad2 phosphorylation increased more rapidly and to a greater degree in males vs. females, which again may reflect androgen dosage. One limitation of in vivo studies is that the pituitary is composed of multiple cell types, and it cannot be determined by Western blotting whether androgen induction of Smad2 phosphorylation is occurring in gonadotrope cells. To address this issue, we treated gonadotrope-derived {alpha}T3 cells with T and found that androgen also increases Smad2 phosphorylation in these cells, indicating that the changes we observed in vivo likely occur, at least in part, in the gonadotropes.

It is unusual to report that T increases just Smad2 phosphorylation and not Smad3. Ordinarily, a stimulus (activin or TGFbeta) acting though the type I receptors will increase phosphorylation of both Smad2 and Smad3. One explanation for our observation of an increase in pSmad2, but not pSmad3, may be pSmad3 Ab sensitivity. As noted earlier, it was difficult to detect basal pSmad3 protein in either pituitary or {alpha}T3 cell lysates. Because we are measuring relatively small increases in Smad phosphorylation, compared with Smad activation by activin, it is possible that our assay is not sensitive enough to detect Smad3 phosphorylation in response to T. Alternatively, T may increase Smad2 phsophorylation through some unknown mechanism. There are several reports of differential activation of Smad2 and -3 in both hepatic and renal cells, which may be dependent on cell cycle stage, intracellular, and/or extracellular matrix environment and may also be independent of TGFbeta or activin signaling (31, 33, 37, 46). There also appears to be differential actions of Smad2 and -3 on FSHbeta transcription. Both Bernard (4) and Suszko et al. (43) report that abrogation of Smad2 or -3, by RNAi, reduces both basal and activin-induced FSHbeta promoter activity, but only depletion of Smad3 reduces the magnitude increase in FSHbeta response to activin. Both authors interpret these findings to indicate that, although Smad2 may not be as important in activin-induced FSHbeta transcription, it does play a role in maintaining basal FSHbeta levels. Additionally, Lamba et al. (29) found that overexpression of Smad2 in combination with Smad4 increased FSHbeta promoter-reporter activity in LbetaT2 cells and that a combination of all three Smads (Smad2, -3, and -4) acted synergistically to increase FSHbeta promoter activity fivefold greater than Smad3 and -4 alone. They also identified Smad2 in the transcriptional complex as a heterotrimer of Smad2, -3, and -4, bound to the SBE of the mouse FSHbeta promoter, and hypothesized that the trimer containing Smad2, -3, and -4 may either recruit more diverse coactivators or enhance the affinity of these regulators to the FSHbeta promoter when Smad2 is present in the complex (29). Therefore, T-induced Smad2 phosphorylation may increase FSHbeta transcription, in part, by either augmenting basal transcription and/or acting synergistically with pSmad3 and Smad4.

It is known that Smad binding to the activin-responsive region of the FSHbeta promoter may not be enough to stimulate transcription of the gene. Transcription factors such the bicoid-related homeodomain factor Pitx2 or the TALE homeodomain proteins Pbx1 and Prep1 have been reported to be important partners with the Smads in stimulating FSHbeta promoter activity (2, 44). It is also possible that androgen receptor (AR) might partner with the Smads in regulating FSHbeta transcription. In prostate cell lines, AR has been reported (12, 23, 27) to form protein-protein interactions with either Smad3 or Smad4 and to modulate either AR interacting with androgen-responsive DNA elements or Smads interacting with Smad-responsive regions of DNA. Of note, the activin-responsive region of the rodent FSHbeta promoter that contains the SBE (–266/–269 bp; 29, 43, 44) is within a larger hormone response element (–274/–260 bp) that confers both androgen and progesterone sensitivity on the FSHbeta promoter (34, 35, 45, 47). It remains to be seen whether AR, or other steroid receptors, acting through protein-protein interactions, can be part of and/or regulate the transcriptional complex that binds to the activin response region of the FSHbeta promoter.

We also examined the effects of estrogen on FSHbeta transcription in female rats. In female rats, E2 had no effect on FSHbeta transcription, which contrasts with previous results, where E2 markedly suppressed FSHbeta PT in male rats (10). The differences between the two studies may be due to sex differences but may also reflect estrogen dose. The current study used a physiological proestrus amount (~50 pg/ml), whereas in the prior study in male rats circulating E2 levels were supraphysiological at ~120 pg/ml. The effects of E2 on FSHbeta transcription in vivo are likely indirect, since E2 does not stimulate FSHbeta mRNA synthesis in female rat pituitary fragments or a murine FSHbeta promoter-reporter construct in LbetaT2 cells (40, 45). E2 has been shown to decrease activin betaB mRNA and increase pituitary FS mRNA and PT in female rats (15, 26, 39). Surprisingly, we found that E2 had a biphasic effect on both activin betaB and FS mRNAs, increasing betaB mRNA at 3 and 8 h and initially suppressing FS at 3 h before rebounding to 150% of controls by 24 h. Coincident with the increase in betaB and decline in FS mRNAs was a sustained increase in Smad3 mRNA and transient increases in Smad2 and -7 mRNAs. Similar to males, Smad3 phosphorylation was extremely low and was unchanged after E2. In contrast, pSmad2 and tSmad2/3 were increased after E2. As E2 did not alter FSHbeta transcription, this suggests either that the actions on activin, FS, and Smad2 and -3 (all of which could increase FSH) may be balanced by stimulation of the inhibitory Smad7. Alternatively, the changes in Smad mRNAs and phosphorylation may be occurring in other pituitary cell types, as estrogen is known to act through the TGFbeta pathway to modulate lactotrope growth and differentiation (17).

In conclusion, androgens rapidly increase FSHbeta PT in both male and female rats. The effects of T on FSHbeta transcription are androgen specific, as they can be reproduced by DHT but not E2, and are correlated with modest, but significant, changes in the activin-signaling system. However, although changes in Smad mRNAs and protein phosphorylation may be part of and/or facilitate androgen action of FSHbeta transcription, they are not required.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Child Health and Human Development Grants HD-11489 and HD-33039 (to J. C. Marshall), by postdoctoral fellowship T32-HD-07382 (to G. M. Wotton), and by the Core Laboratories of Specialized Collaborative Centers Program for Research in Reproduction Center Grant U54-HD-28934.


   ACKNOWLEDGMENTS
 
We thank the University of Virginia, Center for Research in Reproduction Ligand Preparation and Assay Core, for conducting the RIAs; Dr. D. Bernard, Population Council's Center for Biomedical Research at Rockefeller University, for assistance with the pSmad2 and -3 immunoassays; Dr. P. Mellon for generating the {alpha}T3 cell line; Dr. M. Shupnik and H. Walsh for their assistance with the {alpha}T3 cells; and Vanessa Greenberg for assistance with the Smad mRNA PCR assays.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. L. Burger, Univ. of Virginia, Dept. of Internal Medicine, P. O. Box 801412, Charlottesville, VA 22908 (e-mail: lburger{at}virginia.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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