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BOR - Papers in Press, published online ahead of print October 27, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.035196
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BIOLOGY OF REPRODUCTION 72, 523–529 (2005)
DOI: 10.1095/biolreprod.104.035196
© 2005 by the Society for the Study of Reproduction, Inc.

Testosterone Stimulates Follicle-Stimulating Hormone ß Transcription via Activation of Extracellular Signal-Regulated Kinase: Evidence in Rat Pituitary Cells1

D.J. Haisenleder2, L.L. Burger, K.W. Aylor, A.C. Dalkin, H.E. Walsh, M.A. Shupnik, and J.C. Marshall

Division of Endocrinology and Metabolism, Department of Medicine, and the Center for Research in Reproduction, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study investigated whether estradiol (E2) or testosterone (T) activate extracellular signal-regulated kinase (ERK) and calcium/calmodulin-dependent kinase II (Ca/CaMK II), as indicated by enzyme phosphorylation in rat pituitaries. In vivo studies used adult female rats given E2, T, or empty silastic capsules (vehicle controls). Twenty-four hours later, the rats were given a single pulse of GnRH (300 ng) or BSA-saline (to controls) and killed 5 min later. GnRH stimulated a two- to three-fold rise in activated Ca/CaMK II, and E2 and T had no effect on Ca/CaMK II activation. In contrast, both GnRH and T stimulated threefold increases in ERK activity, with additive effects seen following the combination of GnRH+T. E2 had no effect on ERK activity. In {alpha}T3 clonal gonadotrope cells, dihydrotestosterone did not activate ERK alone but enhanced and prolonged the ERK responses to GnRH, demonstrating direct effects on the gonadotrope. Thus, the ERK response to GnRH plus androgen was enhanced in both rat pituitary and {alpha}T3 cells. In vitro studies with cultured rat pituitary cells examined the effect of GnRH±T in the presence of the mitogen-activated protein (MAP) kinase kinase inhibitor, PD-098059 (PD). Results showed that PD suppressed ERK activational and FSHß transcriptional responses to T. These findings suggest that one site of T regulation of FSHß transcription is through the selective stimulation of the ERK pathway.

follicle-stimulating hormone, gonadotropin-releasing hormone, mechanisms of hormone action, signal transduction, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis and secretion of the pituitary gonadotropins (LH and FSH) are primarily regulated by GnRH from the hypothalamus and by steroids and peptides from the gonads [1, 2]. Within the pituitary, gonadal steroids (estradiol [E2] and testosterone [T]) regulate gonadotropin subunit genes directly (via steroid response elements) and indirectly, by effects on specific peptides (i.e., activin B and follistatin) that are released from gonadotrope and other pituitary cell types [2–5]. Gonadal steroids also play crucial roles in gonadotropin secretion/gene expression through stimulatory and inhibitory actions on GnRH pulsatile secretory activity [1, 6]. Together, these factors coordinate the critical LH and FSH secretory patterns necessary to maintain reproductive viability in each species.

Various intracellular signal transduction pathways have been shown to play a role in the regulation of gonadotropin subunit genes. Binding to the GnRH receptor (GnRH-R) activates specific members of the GTP-binding protein family (Gq/11, Gs). This results in activation of phospholipase C, increased phosphoinositide turnover, and a rise in intracellular diacylglycerol levels leading to the activation of protein kinase C (PKC) [7]. GnRH-R binding stimulates a rise in intracellular calcium concentration, which is derived from IP3-activated intracellular storage pools as well as influx from extracellular fluid through voltage-sensitive calcium channels [8, 9]. GnRH also activates other intracellular pathways, including cAMP/protein kinase A (PKA), extracellular signal-regulated kinase (ERK 1 and ERK 2), p38, c-Jun N-terminal kinase (JNK), and, more recently, calcium/calmodulin-dependent kinase II (Ca/CaMK II) [10–15].

ERK is a serine/threonine kinase that can be activated via signaling from tyrosine kinases, as well as seven transmembrane G protein-coupled receptors, such as GnRH-R [12, 16]. The ERK activation pathway differs between receptor subtypes. For example, growth factor receptor tyrosine kinases use the adapter molecule GRB2 and the guanine nucleotide exchange factor mSOS to activate the GTP-binding protein Ras, resulting in the cascade activation of Raf-1, mitogen-activated protein (MAP) kinase kinase (MEK), and ERK [16, 17]. In contrast, ERK activation via G-protein-coupled receptors appears to be complex, with both PKC-dependent and PKC-independent pathways used in different cell types, including the gonadotrope [18, 19]. The mechanisms used by ERK in the transmission of signals to target genes include direct activation of nuclear transcription factors and phosphorylation of downstream cytoplasmic kinases [16]. Over the past few years, ERK has been shown to regulate the expression of gonadotrope genes (i.e., {alpha}, FSHß, GnRH-R) [12, 20, 21] and phosphorylate various transcription factors (i.e., c-Fos, c-Jun, Elk-1) that may mediate actions on gonadotrope genes [16, 19, 21].

Ca/CAMK II is an important intracellular mediator of calcium signaling, secretion, and gene expression in several cell and tissue types, including the pituitary [22–24]. Recently, we showed that GnRH rapidly activates (as determined by enzyme phosphorylation) Ca/CaMK II within the rat pituitary and in LßT-2 cells, and Ca/CaMK II plays a role in GnRH-mediated increases in a {alpha}, LHß, FSHß transcription [15, 25].

Recent studies reveal that gonadal steroids can regulate ERK and members of the Ca/CaMK family in various tissues. In prostate, breast, and skeletal muscle cells, androgens activate ERK [26–28]. Similarly, E2 activates the ERK pathway in breast tumor cells, cardiac myocytes, and GnRH neuron-derived GT-1 cells [29–31]. E2 also activates Ca/ CaMK II in the hippocampus and Ca/CaMK IV in MCF-7 breast tumor cells [32, 33]. In muscle, T plays a role in the activation of the Ca/CaMK pathway by stimulating calcium influx (via actions on G-protein receptors) and calmodulin/ calcium binding [27, 34].

The present study was designed to investigate 1) whether E2 or T activate ERK and Ca/CaMK II within the rat pituitary and/or enhance activational responses to GnRH and 2) whether steroid-mediated effects on ERK or Ca/CaMK II regulate gonadotropin subunit gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Design

In vivo studies All animal procedures were conducted in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals and were approved by the University of Virginia Animal Care and Use Committee. Adult female rats were ovariectomized, jugular cannulae inserted and given phenoxybenzamine (PBZ; Day 1 = 10 mg/kg, Day 2 = 5 mg/kg i.p.) to suppress endogenous GnRH secretion [35]. Twenty-four hours later, silastic implants containing testosterone (T; serum T = 615 ± 78 pg/ml [mean ± SEM], 24 h after implant insertion) or estradiol (E2; serum E2 = 93 ± 17 pg/ml, 24 h after implant insertion) were given s.c., as previously described [35, 36]. Vehicle controls received empty implants. At selected time points after T or E2 administration (1, 6, 16, or 24 h), animals received a single i.v. pulse of GnRH (300 ng) or BSA-saline and were killed 5 min later (n = 4–8 animals per group). The GnRH treatment duration is based on our previously published reports showing that maximal ERK and Ca/CaMK II phosphorylation responses are seen 4–5 min after a single pulse of GnRH in the rat [12, 15]. Upon completing each experiment, pituitaries were collected and homogenized in tissue lysis buffer containing 50 mM Hepes, 100 mM NaCl, 2 mM EDTA, 2 µM aprotinin, 1 mM sodium vanadate, and 1% NP-40. ERK and CAMK II activity were determined by immunoblot. For some experiments, pituitary RNA was extracted to measure cellular responses to T, as indicated by alterations in FSHß and LHß primary transcripts (PT) levels.

In vitro studies To control for alterations in GnRH receptor number during the estrous cycle, pituitaries from random cyclic female rats were pooled and dissociated, then plated on coverslips in wells containing charcoal-stripped fetal bovine serum (10%) and horse serum (5%) in the culture medium (3–4 million cells per well). The following day, coverslips were transferred to wells containing T (500 pg/ml), PD-098059 (PD, 50 µM), T+PD, or vehicle (to controls), and incubated for 24 h. The selected dose for PD was based on previous studies showing that 50 µM PD completely blocks the ERK activational response to GnRH in rat pituitary cells in vitro [12] but does not suppress activation of several other protein kinase pathways in Swiss 3T3 cells [37] or JNK in LßT2 cells [13]. For studies that examined ERK responses to treatment (Fig. 5), GnRH (1 nM) was given to half of the wells from each group for 5 min before cell recovery. At completion of each experiment, cells were recovered and lysates prepared for measurement of either ERK or gonadotropin subunit mRNAs (n = 4 per group).



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  		FIG. 5. ERK activational responses to T (500 ng/ml) or Veh for 24 h ± PD-098059 (PD, 50 µM) in female rat pituitary cells in vitro. Five minutes before cell recovery, half of the wells from each group received GnRH (1 nM). Phosphorylated (P-ERK) and total ERK (T-ERK) were measured in cell lysates. Groups marked with different letters (a–e) are significantly different (P < 0.05); n = 4 per group

Alpha T3 cells were maintained in phenol red-free Dulbecco modified Eagle medium with newborn calf serum (5% NBS). For experiments, the cells were plated in 25-mm culture dishes (400 000 cells per well) in media containing charcoal-stripped NCS (5%). For dihydrotestosterone (DHT) time course studies, cells were treated with 1 nM DHT or vehicle for durations of 2, 5, or 15 min or 1, 2, 6, or 24 h. In other studies, cells were given 1 nM DHT or vehicle and 24 h later received 10 nM GnRH (no treatment to controls). At selected posttreatment time points, cells were washed, lysed, and P-ERK and T-ERK measured. Experiments were repeated at least three times to confirm results.

Assays

Immunoblot assays Cell lysate protein from rat pituitaries (25 µg for ERK, 100 µg for CAMK II) was resolved by electrophoresis (4%–20% SDS-PAGE). For {alpha}T3 cells, 20 µg of lysate was used. Protein bands were transferred to nitrocellulose filters and immunoblotted using antibodies specific to phosphorylated ERK 1 and ERK 2 (P-ERK) and total (phosphorylated and unphosphorylated) ERK (T-ERK; Cell Signaling Tech, Beverly, MA), phosphorylated {alpha} and ß Ca/CaMK II (P-CAMK II, Promega Corp., Madison, WI) and total {alpha}, ß, {gamma}, and {delta} Ca/CAMK II (T-CAMK II; Santa Cruz Biotech, Santa Cruz, CA), as previously described [12, 15]. The secondary antibody for rat pituitary studies was horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Upstate Biotech, Lake Placid, NY). For {alpha}T3 cell studies, the secondary antibodies were HRP-conjugated donkey anti-rabbit (Amersham, Arlington Hts., IL) for P-ERK and sheep anti-mouse (Jackson Immunoresearch, West Grove, PA) for T-ERK. Bands were detected using the Super Signal Pico West chemiluminescent system (Pierce, Rockville, IL), followed by autoradiography. Protein bands were quantitated by densitometry and P-ERK and P-CAMK II bands corrected to the amount of T-ERK or T-CAMK II per sample. Treatment-induced responses were similar for both {alpha} and ß Ca/CaMK II subunits. However, as the intensity of the signal for the ß subunit was greater than that seen for the {alpha} Ca/CaMK II subunit, results were quantitated using ß Ca/CaMK II.

Quantitative reverse transcription-polymerase chain reaction LHß and FSHß primary transcript (PT) concentrations were determined by quantitative reverse transcription-polymerase chain reaction assay (RT-PCR), as previously described [38]. In brief, for each subunit, regions of intron/exon were amplified using specific oligonucleotide primers and a size-altered competitive template RNA (CT) for each gene. A four-point standard curve was generated by adding a fixed amount of pituitary RNA to an increasing amount of CT. The pituitary and CT RNAs were reverse transcribed, followed by 35 cycles of PCR in the presence of 32P-dCTP. The PCR products were separated by electrophoresis, DNA bands were excised, and 32P-dCTP incorporation determined by scintillation counting. Primary transcript concentrations were expressed as femtomoles/100 µg pituitary RNA. Intraassay coefficients of variation (%CVs) are 6.7% (LHß) and 5.0% (FSHß). To reduce the effect of interassay variation, all samples from each experiment were run within a single PCR assay.

Statistical Analysis

The data were analyzed by one-way analysis of variance, with differences between treatment groups determined by Duncan multiple range test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Figure 1 shows the ERK and Ca/CaMK II activational responses, as determined by enzyme phosphorylation, to a single pulse of GnRH in the presence of E2 or T in vivo. The upper panels show representative autoradiographs of ERK (P-ERK) and Ca/CaMK II (P-CAMK) phosphorylation responses to treatments given. As shown in the lower panel, T stimulated a threefold increase in P-ERK (P < 0.05 versus Veh controls), whereas E2 had no effect. Additional increases were seen following the combined treatment of GnRH+T (P < 0.05 versus Veh+GnRH or T alone). GnRH also stimulated a two- to threefold increase in Ca/CaMK II activity (P < 0.05 versus Veh controls), but unlike results seen for ERK, neither T nor E2 had any effect on basal or GnRH-induced responses.



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  		FIG. 1. Ovariectomized (OVX) rats were given s.c. implants of estradiol (E2) or testosterone (T) for 24 h (empty implants to vehicle controls [Veh]). Five minutes before killing the animals, they received a single pulse of GnRH (G; 300 ng) or BSA-saline (C; Control). Activated (phosphorylated) ERK (P-ERK) and Ca/CaMK II (P-CAMK II), and total ERK (T-ERK) and Ca/CaMK II (T-CAMK II) were measured. P-ERK and P-CAMK for each sample were corrected to T-ERK or T-CAMK for the same sample. The data are presented as percentage increase versus Veh controls (C); n = 4–8 per group; mean ± SEM is shown. Note the different scale for each panel. Bars with different letters (a, b, or c) are significantly different (P < 0.05) from each other. A representative blot is presented in the upper panel

To characterize the time course of the ERK activational response to T, adult female rats were given T implants for 1, 6, 16, or 24 h, followed by a 5-min GnRH stimulus. As previous studies have shown that T stimulates rat FSHß transcription [38], FSHß PTs were measured in parallel groups given T implants for 1–24 h, to correlate with ERK activational responses. As shown in Figure 2, T had no effect on basal or GnRH-induced increases in ERK activation after 1 or 6 h of treatment. However, after 16 and 24 h, T stimulated a three- to fourfold increase in basal ERK activity (P < 0.05 versus Veh controls), with additional increases seen in response to GnRH (P < 0.05 versus Veh+GnRH or T alone). FSHß PT showed similar responses, increasing three- to fourfold after 16 or 24 h of T treatment. In contrast, basal or GnRH-induced Ca/CaMK II activity were not affected by 1–24 h of T treatment.



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  		FIG. 2. Time course of Ca/CaMK II and ERK activation and FSHß PT responses to T. Groups of OVX rats received T implants s.c. for 1, 6, 16, or 24 h (empty implants to controls, Veh; open bars). Some animals received a single pulse of GnRH (G; 300 ng; black bars) or BSA-saline (C; hatched bars) and were killed 5 min later. Phosphorylated and total ERK and Ca/CaMK II were measured in cell lysates (see Fig. 1 for details). Data are presented as percentage change versus Veh controls (C); n = 4–6 per group. Groups marked with different letters (a–c) are statistically different (P < 0.05) from each other. Representative blots for ERK and CAMK II are presented (Veh and 1-, 16-, and 24-h T groups shown)

Studies were conducted in {alpha}T3 cells to determine if ERK activational responses to androgen treatment could occur directly in gonadotropes. Because there are few data in the literature regarding aromatase activity within mouse gonadotrope cell lines, DHT was used as the best method to directly activate the androgen receptor. In contrast with results seen in the rat pituitary, 1 nM DHT did not stimulate an increase in ERK activity in {alpha}T3 cells, either acutely (over 15 min) or after 24 h of treatment (Fig. 3). To determine whether androgens enhance ERK responses to GnRH, cells were pretreated with DHT or vehicle for 24 h, then given GnRH for selected durations over an additional 2.5 h. This treatment paradigm was based on preliminary studies showing that 24 h of DHT pretreatment to {alpha}T3 cells is required to affect ERK activational responses to GnRH (data not shown). In vehicle-treated cells, GnRH increased ERK activation/phosphorylation, which remained elevated for 2.5 h (Fig. 4), similar to previous findings in LßT2 cells [13]. DHT enhanced and prolonged the ERK activational responses to GnRH, with significant increases in ERK activation after 1 and 1.5 h of GnRH treatment (P < 0.05 versus vehicle). Thus, while androgens play a role in the response of the ERK pathway to GnRH in {alpha}T3 cells, there is no increase in basal ERK activation, as in rat pituitary cells.



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  		FIG. 3. ERK time course responses to 1 nM DHT in {alpha}T3 cells. Cells received DHT for 2, 5, or 15 min (m) or 1, 2, 6, or 24 h; controls received vehicle (Veh) treatment for 24 h. Phosphorylated (P-ERK) and total ERK (T-ERK; phosphorylated and unphosphorylated ERK) were measured in cell lysates by immunoblot. No significant treatment effects were seen, and fold changes in ERK activation (P-ERK corrected to T-ERK for each sample) for Veh and DHT-treated groups are included; mean ± SEM shown (n = 4 per group). The study was repeated three times to confirm data and results were similar for each experiment



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  		FIG. 4. ERK activational responses to 10 nM GnRH ± 1 nM DHT or vehicle (Veh) in {alpha}T3 cells. Control (Con) groups did not receive GnRH (media only). Cells were pretreated with DHT or Veh for 24 h before giving GnRH, for durations indicated. Percent increases in ERK activation (P-ERK corrected to T-ERK for each sample) for Veh and DHT-treated groups are shown. * P < 0.5 versus Veh-pretreated cells, same GnRH treatment time point; n = 4–5 per group. The study was repeated three times to confirm data and results were similar for each experiment

To determine whether T-stimulated phosphorylation of ERK involves the MEK activation pathway, pituitary cells from randomly cyclic female rats were treated in vitro with T±GnRH in the presence or absence of the MEK-specific inhibitor, PD-098059. The results are shown in Figure 5. Similar to findings in vivo, GnRH stimulated a fourfold increase in ERK activity (P < 0.05 versus Veh controls). T also increased ERK activity (threefold, P < 0.05), with additional increases seen in response to GnRH (P < 0.05 versus Veh+GnRH or T alone). PD administration reduced basal ERK activity and completely blocked the response to GnRH (P < 0.05 versus Veh controls). PD also suppressed the ERK responses to T. However, while values in T plus PD ± GnRH were similar to ERK activity levels seen in Veh controls, enzyme activity in androgen-treated groups remained twofold greater than Veh+PD groups (P < 0.05). These data suggest that T stimulates the ERK pathway primarily via activation of MEK and to a lesser extent via alternate mechanism(s).

To investigate the mechanistic link between T actions on the ERK pathway and physiological regulation of gonadotropin subunit genes, further in vitro studies examined whether PD blocks the stimulatory effect of T on FSHß transcription. Similar to our previous in vivo results [38], T increased FSHß PT levels threefold (Fig. 6; P < 0.05 versus control). PD administration reduced basal FSHß PT (P < 0.05 versus control) as well as T-induced increases in FSHß PT (P < 0.05 versus T alone). Although the magnitudes of the inhibitory responses to PD were similar in both control and T-treated groups, FSHß PT levels remained higher in the presence of T (P < 0.05 versus PD alone), similar to results seen for ERK activity (Fig. 5). The effects of T or PD were selective, as demonstrated by LHß PT being unchanged by either treatment.



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  		FIG. 6. LHß and FSHß transcriptional responses to T±PD-098059. Rat pituitary cells were treated with T or vehicle ± PD in vitro (see Fig. 5 for details). After 24 h of treatment, cells were recovered and LHß and FSHß primary transcripts measured. Groups marked with different letters (a–c) are different (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study are the first to show that androgens stimulate ERK activity in normal pituitary cells, as indicated by an increase in ERK phosphorylation. These effects are both kinase specific, as Ca/CaMK II was not altered by T, and steroid-specific, because E2 was ineffective. Also of note, the data reveal that blocking activation of the ERK pathway (via the MEK inhibitor, PD) partially suppressed androgen-induced FSHß transcription. These findings suggest that ERK plays a role in the previously described androgen-induced stimulation of FSHß transcription in the rat [39]. However, the results of other recent studies suggest that direct actions on the FSHß promoter are also a factor in androgen regulation of FSHß gene expression [40]. Further, in the presence of GnRH plus T, the stimulation of ERK activation was higher than by either hormone alone. This observed effect demonstrates that T and GnRH cooperate to stimulate ERK activation, primarily via MEK phosphorylation.

A previously published report showed that only 0.84% of female rat pituitary cells express aromatase, that the enzyme is not selectively found in any specific cell type, and that it does not change throughout the estrous cycle [41]. Those findings suggest that aromatase does not play a major role at the level of the pituitary in the female rat. Also of note, data presented in Figure 1 clearly demonstrate that the ERK activational response to testosterone is an androgen effect and is not due to aromatization to estradiol. Similar to results seen in rat pituitary cells in vivo, preliminary data in {alpha}T3 cells reveal that estradiol (in contrast with DHT) does not alter ERK responses to GnRH (not shown).

In recent years, considerable evidence has accumulated regarding steroid regulation of various intracellular pathways, including members of the MAP kinase family. For both T and E2, these effects can occur in either a steroid receptor-dependent or receptor-independent manner depending on the target cell [29, 34]. Putative mechanisms of action include cytoplasmic estrogen receptor- (ER) or androgen receptor- (AR) induced activation of the ERK cascade or steroid binding to a G-protein-coupled plasma membrane receptor(s) [27, 34, 42]. Among possible candidates, a functional sex hormone-binding globulin receptor has been identified within various tissues, with data suggesting it is either a G-protein receptor or linked to one [34, 43].

In MCF-7 cells, E2 rapidly (within 30 min) stimulates an increase in intracellular calcium and ERK activity [29]. Similar rapid stimulation of the ERK pathway by E2 has been observed in cortical neurons [42]. E2 activation of the ERK pathway stimulates cell proliferation in many tissues, including the breast and heart [30, 44]. Other investigations reveal that ERK can play a role in ER-mediated transcriptional actions by phosphorylating ER coactivators and by stimulating assembly of receptor-coactivator complexes [45].

In the prostate, androgens stimulate the ERK pathway via the AR, resulting in the phosphorylation of critical factors that mediate androgen-induced cell proliferation, including the AR itself, SRC-1, and Elk-1 [26, 34, 46]. In contrast, in skeletal muscle cells, androgens can stimulate a rapid rise in intracellular calcium and ERK activity via activation of a G-protein-linked receptor [27]. Similar to observations for E2, androgens stimulate the ERK pathway by activating the intracellular tyrosine kinase, c-Src [34, 43]. Most of the previously described ERK activational responses to androgen are rapid, in contrast with the slower responses seen in the present study for the pituitary. Whether the actions of T on ERK in the rat pituitary are nuclear AR dependent will require further investigation. However, recent findings by Okada et al. [47] show that androgen receptors are present in the gonadotrope and androgens stimulate AR translocation into the nucleus in both rat gonadotropes and gonadotrope-derived {alpha}T3 and LßT2 cells.

As discussed above, T can act on target cells rapidly, within a few minutes via a plasma membrane-associated receptor(s) or via a conventional AR-mediated mechanism, which is slower and occurs over hours [34]. An example of the former is the rapid T-induced increase in intracellular calcium seen in various tissues [34]. The present data (Fig. 2) reveal that a minimum of 6–16 h is required to activate ERK. This slower time course is consistent with T acting through an AR-dependent or genomic-related mechanism.

Of interest, our results showed that T stimulated an increase in basal ERK activation in the rat pituitary and responses to GnRH were maintained or reduced over time. However, in {alpha}T3 cells, the ERK activational response to androgen differed, enhancing and prolonging the effects of GnRH, but a response to androgen alone was not seen. It is likely that the ERK responses to T seen in rat pituitary cells reflect actions on gonadotropes as well as on other cell types. One possible explanation for the differential responses could be that intracellular signal transduction mechanisms differ between primary rat cells and mouse-derived cell lines, as has been shown for other intracellular signal pathways [48–50]. Another possibility is that the androgen effect on basal ERK activity in rat pituitary cells is indirect. In the breast, ER activation stimulates the secretion of growth factors, leading to a rise in ERK activity via a paracrine regulatory mechanism [44]. In the case of the pituitary, T may stimulate the expression/secretion of specific growth factors (i.e., EGF, IL-6, TGFß) in other pituitary cell types, resulting in the activation of ERK within gonadotropes and possibly other pituitary cell types. Whether T is acting directly or indirectly in the rat gonadotrope remains to be resolved. However, it is important to point out that the T/ERK response occurs directly in gonadotropes because the T-induced increase in FSHß transcription is inhibited by the MEK blocker, PD (Fig. 6). Furthermore, the overall ERK stimulation by GnRH plus androgen is greater in both rat pituitary cells (i.e., increased basal ERK activation with similar stimulation by GnRH) and clonal pituitary cells (i.e., greater stimulation by GnRH in the presence of DHT).

To determine whether T stimulation of ERK phosphorylation involves activation of MEK 1/2, in vitro studies were conducted in the presence of PD. As shown in Figure 5, PD reduced basal and completely blocked GnRH-induced ERK activation, as previously shown [12]. Although PD also suppressed the stimulatory response to T, the compound was less effective than for GnRH alone. This could suggest that T increases ERK activity via a secondary pathway that is independent of MEK 1/2. One possible explanation is that T may influence cross-talk between intracellular pathways or activate an uncharacterized member of the MEK family that is resistant to PD inhibition, but still activates ERK. Another possibility is that T may suppress one or more MAP kinase phosphatases (MKPs), resulting in a rise in steady-state ERK activity.

Data obtained in various tissues reveal that ERK activity is the product of both input signals from the ERK activation cascade and the expression of the various MKP subtypes present in the tissues [51]. One particular family of MKPs, the dual-specificity MKPs, are potential targets for androgen suppression because they are regulated by factors that influence ERK or other MAP kinase pathways. Two members of this group (i.e., MKP 1 and MPK 2) are present in the rat pituitary and gonadotrope-derived cell lines [51, 52]. Recent data reveal that GnRH stimulates MKP 2 mRNA and protein expression in {alpha}T-3 cells [52]. Gonadal steroids can also regulate MKPs, as suggested by studies demonstrating that E2 suppresses MKP-1 in rat prostate cells [53].

Testosterone regulates the expression of several gonadotrope-related genes in either a positive or negative manner [1–3, 39, 40, 54, 55]. Spady et al. [40] identified two androgen-responsive elements within the ovine FSHß promoter, which are highly conserved in other species, including the rat. Their data also showed that androgen stimulation of the ovine FSHß promoter is dependent on an activin-responsive element. We showed that T suppressed LHß PT, and FS, and inhibin ßB mRNAs in male rats in vivo [39]. T also selectively stimulated a rise in FSHß PT, and these effects were androgen specific, as T and DHT induced similar responses. Also of note, the FSHß transcriptional response over longer durations (i.e., 24 h) was not mediated by the coincident reduction in FS production because treating cells with excess FS for 24 h did not block the androgen-stimulated increase in FSHß PT [39]. In the present study, we observed a T-induced increase in FSHß PT after 16 h of treatment, coincident with the T-stimulated rise in ERK activity (Fig. 2). These findings, along with results showing that MEK blockade suppressed T-induced increases in ERK (Fig. 5) and T stimulation of FSHß PT (Fig. 6), support a mechanistic link for ERK activation in androgen stimulation of FSHß transcription. However, because FSHß PT levels in T+PD-treated cells remained significantly elevated versus PD alone (Fig. 6), it is likely that T regulates FSHß transcription by more than one signal pathway, including direct actions on the promoter and via activin [40].

In summary, these results demonstrate that T selectively stimulates the activation of ERK within the rat pituitary. Although the site(s) of T action on the ERK pathway remain to be determined, the present observations clearly demonstrate the involvement of a site(s) upstream from MEK activation, and involvement of other regulatory sites is possible. These findings also suggest that ERK plays a major role in the regulation of the rat FSHß gene by testosterone.


   FOOTNOTES
 
1 Supported by USPHS grants HD-33039 and HD-11489 to J.C.M. and NICHD/NIH through a cooperative agreement (U54-HD28934) as part of the Specialized Cooperative Centers Program in Reproductive Research (J.C.M., D.J.H., M.A.S.). Back

2 Correspondence: D.J. Haisenleder, Aurbach Medical Research Building, P.O. Box 801412, University of Virginia Health Sciences Center, Charlottesville, VA 22908. FAX: 434 243 9143; djh2q{at}virginia.edu Back

Received: 12 August 2004.

First decision: 13 September 2004.

Accepted: 14 October 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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