Differential Regulation of Gonadotropin-Releasing Hormone Secretion and Gene Expression by Androgen: Membrane Versus Nuclear Receptor Activation

Tarranum Shakil, A. N. Ehsanul Hoque, Mansoor Husain and Denise D. Belsham

Departments of Physiology (T.S., D.D.B.) and Medicine (A.N.E.H., M.H.), University of Toronto and Division of Cellular and Molecular Biology (M.H., D.D.B.), Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8

Address all correspondence and requests for reprints to: Denise D. Belsham, Ph.D., Department of Physiology, University of Toronto, Medical Sciences Building 3247A, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: d.belsham{at}utoronto.ca.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid hormones induce rapid membrane receptor-mediated effects that appear to be separate from long-term genomic events. The membrane receptor-mediated effects of androgens on GT1-7 GnRH-secreting neurons were examined. We observed androgen binding activity with a cell-impermeable BSA-conjugated testosterone [testosterone 3-(O-carboxymethyl)oxime (T-3-BSA)] and were able to detect a 110-kDa protein recognized by the androgen receptor (AR) monoclonal MA1–150 antibody in the plasma membrane fraction of the GT1-7 cells by Western analysis. Further, a transfected green fluorescent protein-tagged AR translocates and colocalizes to the plasma membrane of the GT1-7 neuron. Treatment with 10 nM 5{alpha}-dihydrotestosterone (DHT) inhibits forskolin-stimulated accumulation of cAMP, through a pertussis toxin-sensitive G protein, but has no effect on basal cAMP levels. The inhibition of forskolin-stimulated cAMP accumulation by DHT was blocked by hydroxyflutamide, a specific inhibitor of the nuclear AR. DHT, testosterone (T), and T-3-BSA, all caused significant elevations in intracellular calcium concentrations ([Ca2+]i). T-3-BSA stimulates GnRH secretion 2-fold in the GT1-7 neuron, as did DHT or T. Interestingly GnRH mRNA levels were down-regulated by DHT and T as has been reported, but not by treatment with T-3-BSA or testosterone 17ß-hemisuccinate BSA. These studies indicate that androgen can differentially regulate GnRH secretion and gene expression through specific membrane-mediated or nuclear mechanisms.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE CLASSICAL MODEL of steroid hormone action involves the binding of steroids to intracellular receptors, followed by modulation of transcriptional processes after translocation of steroid-receptor complexes into the nucleus (1). In addition, there is increasing evidence for nongenomic action of steroids on cellular signaling and function. These effects, appearing within seconds to minutes after addition of the stimulus, have been described for all classes of steroids (see Refs. 2 and 3 for reviews). Examples of nongenomic steroid action include rapid aldosterone effects in lymphocytes and vascular smooth muscle cells, vitamin D3 effects in epithelial cells, progesterone action in human sperm, neurosteroid effects on neuronal function, and vascular effects of estrogens (2). Interestingly, estradiol effects via a membrane receptor have been linked to GnRH release in the rat median eminence (4). Rapid, nongenomic steroid action is presumed to be mediated through membrane-bound receptors exhibiting pharmacological properties distinct from those of the classical intracellular steroid receptors. Mechanisms of action are currently being studied with regard to signal recognition and transduction, and some receptor-second messenger cascades have been described. Whether the membrane receptors are distinct molecules from the intracellular steroid receptors is currently under intensive investigation.

Androgens have been shown to have rapid membrane receptor-mediated effects distinct from the known genomic effects. Although studies of membrane estrogen receptor effects are well documented, little is currently known about the nongenomic or cell surface receptor-mediated actions of androgen. Testosterone (T) rapidly stimulates intracellular calcium concentration ([Ca2+]i), an effect that appears to be coupled to phospholipase C via a PTX-sensitive G protein in male rat osteoblasts (5). In Sertoli cells, the increase in [Ca2+]i has been linked to T, with the potential involvement of classic intracellular androgen receptors (ARs) (6). Similarly, 5{alpha}-dihydrotestosterone (DHT) has been shown to increase [Ca2+]i in human prostate cancer cells (LNCaP), which can also be blocked by the specific AR antagonist, hydroxyflutamide (7). Nonetheless, in mouse macrophages, which lack expression of the classic AR, T-induced [Ca2+]i increases were assumed to occur via a unique androgen-binding membrane receptor, but also through a phospholipase C pathway involving an inhibitory G protein (8).

Because there are a limited number of GnRH neurosecretory cells that are dispersed throughout the preoptic area of the anterior hypothalamus (9), in vivo studies on the direct action of androgen on GnRH transcription or secretion are difficult. A model of the GnRH neuron was developed through targeted tumorigenesis of the GnRH neuron by SV-40 large T-antigen. Subsequently, a murine immortal cell line of GnRH-secreting hypothalamic neurons, the GT1 cells, was established (10). The GT1 cell line has been used extensively to study the cell biology of the GnRH neuron and has been shown to manifest many of the known traits of GnRH neurons, including the pulsatile secretion of the peptide (reviewed in Refs. 11, 12, 13, 14). The presence of a classic AR in the GnRH neuron in situ has been debated in the past. Similarly, the effects of androgen on GnRH synthesis are often contradictory in whole animal experiments (discussed at length in Ref. 15). We, and others, have previously used the GnRH-secreting GT1 hypothalamic cell line to demonstrate the expression and function of the classic nuclear AR (15, 16, 17). We also found that GnRH mRNA levels are down-regulated upon treatment with DHT (15).

In the present study, we used the GT1-7 cell line to address the question of whether androgen could have any direct effects at the level of the plasma membrane in GT1-7 hypothalamic cells. We find that DHT is capable of affecting signal transduction events initiated at the cell membrane. Furthermore, we have detected what appears to be the classic form of the AR in the plasma membrane fraction of the GT1-7 cell. Interestingly, using a BSA-conjugated androgen, we are able to link the membrane AR activation to a change in GnRH secretion, but not GnRH gene expression. These results provide the first demonstration of a direct action of androgen mediated through a membrane AR on downstream cellular functions, which include GnRH secretion in the hypothalamic GT1-7 GnRH-secreting neurons.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Localization of the AR Expressed in GT1-7 Neurons
We have previously shown that the AR, as detected by RT-PCR and the polyclonal antibody, PAR-1 (18), directed toward the classic nuclear AR, is expressed in GT1-7 cells (15). As expected, AR immunoreactivity, recognized by the AR monoclonal antibody MA1-150, is found in the total cellular protein and nuclear extract preparations from GT1-7 cells (Fig. 1AGo). Importantly, the 110-kDa AR was also recognized by the MA1-150 antibody in Western blot analysis of the isolated plasma membrane fraction of GT1-7 neurons (Fig. 1AGo). Interestingly, upon treatment with DHT (10 and 100 nM), the relative amounts of AR in the membrane fraction appear to be moderately increased after 15 min (Fig. 1AGo), although exact quantification was difficult from the Western blot analysis. The blots were stripped and reprobed with the Gß-antibody, which recognizes the heterotrimeric G protein ß-subunit, as a plasma membrane marker and loading control. This antibody detected similar amounts of a ubiquitous 40-kDa protein in the total protein and membrane fractions (<10% variability between samples), but was not found in the nuclear extract as expected (Fig. 1AGo). To verify the purity of the plasma membrane fraction, we also used a nuclear receptor marker for the transcription factor retinoid-related orphan receptor-{alpha} (ROR{alpha}). Copious amounts of ROR{alpha} at 58 kDa were detected in the nuclear extract and total protein of the GT1-7 cells, but only a faint, almost undetectable band was present in the membrane fractions (Fig. 1AGo). This demonstrates that the classic intracellular AR, or a structurally similar molecule with the same mass, can be recognized by a monoclonal AR antibody and is localized to the membrane fraction of the GT1-7 neuron.



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Figure 1. The AR Is Localized to the GT1-7 Cell Plasma Membrane

A, GT1-7 cells were treated with 10 nM or 100 nM DHT for 15 or 30 min. Plasma membrane fractions (MF) from GT1-7 cells, obtained by differential centrifugation, nuclear extract (NE), and total protein (TP) from GT1-7 were analyzed by Western blot analysis. Analysis was performed using the AR monoclonal antibody (MA1–150), that recognizes the 110-kDa AR protein. Western blots were stripped and reprobed with ROR{alpha}, a 58-kDa nuclear protein marker, and Gß, a 40-kDa membrane marker and loading control. Immunoreactive complexes were visualized by enhanced chemiluminescence. B, Confocal laser scanning microscopy of GT1-7 cells labeled with T-BSA-FITC, ConA-Rhod, a membrane marker, and BSA-FITC as a control. The GT1-7 cells were pretreated with 10 nM DHT for 15 min before fixation in 0.5% paraformaldehyde. Colocalization of the T-BSA-FITC (green) and ConA-Rhod (red) fluorescence is observed in the merge of the two images as a yellow-orange color, whereas BSA-FITC alone did not appear to have any specific fluorescent labeling. Representative photographs are composed of a series of approximately 12 optical sections (0.4 µm each, stacked for a cross-section of ~5 µm) to enhance the T-BSA-FITC signal. Magnification, x400. Bar, 10 µm. C, Confocal laser scanning microscopy of GT1-7 cells transfected with an AR-GFP protein. GT1-7 cells were transfected with 10 µg of plasmid DNA for 36 h, followed by a 15 min treatment with 10 nM DHT. The cells were then labeled with ConA-Rhod for 20 min at 15 C. The live cells were visualized with a Carl Zeiss confocal microscope. The optical section visualized is 1.0 µm. Magnification, x630. Bar indicates 10 µm.

 
To confirm these findings, we used the cell-impermeable testosterone 3-(O-carboxymethyl)oxime BSA conjugate (T-3-BSA, conjugated at the C3) tagged with fluorescein isothyocyanate (FITC) to determine whether there was any androgen binding at the cell surface. The T-BSA conjugates have been previously shown to have androgen receptor binding activity (19). Because we had some indication from the Western blot analysis that there was increased AR expression at the membrane after 15 min treatment with 10 nM DHT, we pretreated the GT1-7 cells to allow for any translocation of cellular AR to the plasma membrane. The translocation of AR upon androgen exposure has also been reported in mouse IC-20 macrophage cells (8). The GT1-7 cells were exposed to T-3-BSA-FITC (15 µM) for 30 min at room temperature, followed by incubation with concanavalin A labeled with rhodamine (ConA-Rhod), a membrane marker. The cells were then fixed with 0.5% paraformaldehyde. Using confocal scanning laser microscopy, we could detect a small amount of T-3-BSA-FITC binding to the cell surface (Fig. 1BGo), likely corresponding to the low expression levels of AR in the GT1-7 cell. When visualized in conjugation with the ConA-Rhod, distinct regions of colocalization were detected (Fig. 1BGo). Some evidence of punctate fluorescence inside the cells can also be noticed after 15 min treatment with DHT, likely due to internalization of androgen binding sites as has previously been reported (8). BSA-FITC (Fig. 1BGo) or BSA alone (data not shown) did not exhibit any binding activity.

Although both of the above experiments provide strong evidence for the presence of the classic AR at the GT1-7 cell membrane, it would be most favorable to directly label the AR in whole cells. Although we have used three different AR antibodies (polyclonal PAR-1, and monoclonal AR441 and MA1–150), we could not detect specific immunoreactivity at the plasma membrane of the GT1-7 neuron. All of the commercially available antibodies against the AR have been raised using peptides from the N-terminal domain of the protein. We suggest that this region may be masked upon insertion or docking of the AR to the cell membrane, which would make the protein inaccessible to the specific epitope of the antibody. To overcome this obstacle, we have used a green fluorescent protein-tagged AR (AR-GFP) transfected into GT1-7 cells to observe the localization of AR protein expression (20). After a 36-h transfection period, the live GT1-7 cells were treated with 10 nM DHT for 15 min, followed by incubation with ConA-Rhod for 20 min at 15 C, to avoid any internalization and thus potential toxicity caused by rhodamine. The living cells were observed using a confocal laser scanning microscope. The AR-GFP signal could be detected throughout the cell, but was observed to specifically colocalize with the ConA-Rhod signal (Fig. 1CGo), indicating that the ARs in the GT1-7 neurons are also present at the plasma membrane.

Effect of DHT on cAMP Accumulation in GT1-7 Neurons
To study the functional activity of the AR in the GT1-7 cells, we treated the cells with DHT and assessed the levels of cAMP resulting from cellular activation at the level of the membrane. DHT treatment alone, over a wide range of concentrations ranging from 100 pM to 100 nM, did not appear to affect cAMP accumulation in the GT1-7 cell, as compared with basal levels (Fig. 2AGo). In contrast, forskolin (1 µM), a adenylyl cyclase activator, increased cAMP levels approximately 10-fold in the GT1-7 cell within 15 min (Fig. 2BGo). When the GT1-7 neuron was exposed to both forskolin and DHT in combination, the levels of forskolin-stimulated cAMP accumulation decreased significantly (Fig. 2BGo). This suggests that androgen can act at the level of the membrane to inhibit forskolin-stimulated cAMP production in the GT1-7 neuron. To investigate whether this effect was occurring through the classic nuclear AR located at the membrane, we treated the cells with the specific AR antagonist, hydroxyflutamide. Remarkably, the inhibition of forskolin-stimulated cAMP accumulation by DHT was blocked by this classic AR antagonist (Fig. 2BGo), providing further evidence that the AR localized to the membrane is similar to the known nuclear AR and is capable of signal transduction upon stimulation with DHT.



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Figure 2. DHT Has No Effect on Basal cAMP Accumulation in GT1-7 Neurons, but Activation of AR Inhibits Forskolin-Induced cAMP Accumulation in GT1-7 Cells Coupled to a PTX-Sensitive G Protein

A, GT1-7 cells were plated in 24-well plates and preincubated in serum-free medium for 2 h. Cells were then incubated in 100 mM isobutylmethylxanthine (IBMX) and treated with varying doses of DHT (100 pM to 100 nM) for 15 min. The amount of cAMP in each sample was determined by RIA. DHT treatment alone did not alter basal cAMP levels. B, Cells were either treated with DHT (10 nM) ± forskolin (1 µM) with hydroxyflutamide treatment (10 µM). DHT (10 nM) inhibited forskolin-induced accumulation of cAMP in GT1-7 cells, but pretreatment with hydroxyflutamide (1 h) blocked this effect. Cells were also starved in the medium containing PTX (200 ng/ml) for 4 h. Cells were then treated ± PTX. Pretreatment with PTX blocked the inhibition of forskolin-induced accumulation of cAMP by DHT. Results were plotted as the mean ± SEM (n = 3).

 
Membrane AR Signaling Is Coupled to a Gi Protein and Increases [Ca2+]i Mobilization
The mechanism by which the classic AR can signal from the membrane is not yet known. To determine the involvement of G proteins in androgen action, GT1-7 cells were pretreated with PTX (200 ng/ml) for 4 h before DHT treatment. Treatment with PTX was able to block the DHT inhibition of forskolin-stimulated cAMP accumulation in the GT1-7 cells (Fig. 2BGo). This suggests that the membrane AR is coupled to an inhibitory G protein, Gi.

The downstream effectors of G protein signaling are numerous. An obvious question is whether androgen can alter [Ca2+]i in the GT1-7 neuron, as has been seen in numerous other cell models. We have used the calcium indicator Fura-2/AM to determine whether exposure to androgen can change [Ca2+]i mobilization. We treated the cells with DHT, T, and a BSA-conjugated testosterone (T-3-BSA), which is unable to enter the cell and therefore must act at the membrane receptor sites, over the same time course. Treatment with DHT, T (10 nM each), or T-3-BSA (400 nM) caused a significant increase in [Ca2+]i (Fig. 3Go). Cells were first exposed to vehicle as a control, followed by the specific androgen. Androgen treatment increased [Ca2+]i typically within 200 sec of treatment, although at times different cell populations showed a slower response time (Fig. 3AGo). Overall, peak [Ca2+]i achieved were similar for each androgen, whether a fast or slow response was detected (Fig. 3BGo). There were no effects seen with either BSA or vehicle alone.



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Figure 3. DHT Causes a Significant Increase in [Ca2+]i in GT1-7 Neurons

A, Fura-2/AM loaded GT1-7 neurons were continuously monitored by 340/380 ratiometric digital imaging. Representative graph showing the effect of DHT (100 nM) on [Ca2+]i as a function of raw 340:380 nM ratios for n = 12 cells. Similar results were obtained from each steroid tested although some cell populations responded slower. B, The graph indicates the mean intracellular calcium increase for each steroid tested [mean ± SEM; (n) = total number of cells analyzed in three separate experiments. *, P < 0.005].

 
Membrane Receptor Activation Results in Stimulation of GnRH Secretion, but Does Not Affect GnRH Gene Expression
To determine whether DHT was able to change GnRH secretion directly at the level of the GT1-7 GnRH neuron, we treated the cells with DHT, T (10 nM each), or T-3-BSA (100 nM) for short time periods (up to 2 h). DHT and T alone were able to stimulate GnRH secretion approximately 2-fold at 2 h. We found that T-3-BSA, acting at the cell membrane, was also able to stimulate GnRH secretion to the same levels as both DHT and T (Fig. 4Go). Forskolin, a positive control, increased GnRH secretion 2-fold as seen previously (21). Vehicle alone or BSA did not have any effect on GnRH secretion.



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Figure 4. DHT, T, or T-3-BSA All Increase GnRH Secretion from GT1-7 Neurons

GT1-7 neurons were treated for 30, 60, or 120 min with 10 nM DHT, 10 nM T, 100 nM T-3-BSA, BSA, or vehicle alone (control, C). Cells were also treated with forskolin (F, 10 µM, 2 h) (positive control, inset). Cell culture medium was then collected and assayed for GnRH-like immunoreactivity (GnRH-like IR) by RIA. Results shown are mean ± SEM [n = 3–5 independent experiments each in triplicate; P < 0.05 (T, T-3-BSA, 30 min; DHT, 1 h; T, 2 h); P < 0.005; DHT, 2 h; T-3-BSA, 1 h and 2 h].

 
We have previously shown that DHT down-regulates GnRH mRNA expression at 24 and 36 h in the GT1-7 neuron (15). To determine whether the classic nuclear AR or the membrane AR was involved in this effect, we treated the cells with both DHT, T (both at 10 nM), and T-3-BSA (100 nM). After Northern blot analysis, we found that DHT and T down-regulate GnRH mRNA at 24 and 36 h, as previously shown (15), and interestingly activation of the membrane AR by T-3-BSA had no significant effect on GnRH mRNA levels (Fig. 5Go).



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Figure 5. DHT and T, but Not T-3-BSA, Cause a Significant Decrease in GnRH mRNA Levels in GT1-7 Cells

A, Relative GnRH mRNA levels after treatment of GT1-7 cells with 10 nM DHT, 10 nM T, or 100 nM T-3-BSA at 24 and 36 h. GnRH mRNA levels were normalized to those of GAPDH used as a loading control. Data are expressed as mean ± SEM from three independent experiments. **, P < 0.005; ***, P < 0.001, vs. time-matched controls. B, Representative Northern blot of 15 µg of total GT1-7 mRNA. GnRH and GAPDH mRNAs are indicated. Film exposure time was 6 h.

 
Because the T-3-BSA conjugate is used at higher concentrations than DHT or T alone, it was possible that hydrolysis of the T-3-BSA molecule could produce levels of free T capable of regulating GnRH gene expression. To confirm that there was no effect of the T-BSA conjugate on GnRH gene expression, we used another T-BSA molecule, testosterone 17ß-hemisuccinate (T-17-BSA). The two T-BSA molecules (T-3-BSA and T-17-BSA) were incubated with the GT1-7 neurons at increasing concentrations (0.01–1 µM) for 36 h. There were no significant changes in GnRH mRNA levels with either T-BSA conjugates at any of the concentrations used (Fig. 6Go). These results indicate that androgen can have distinct effects on GnRH gene expression and secretion depending upon the location of the AR when activated.



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Figure 6. Neither T-3-BSA nor T-17-BSA Cause a Significant Decrease in GnRH mRNA Levels in GT1-7 Cells

A, Relative GnRH mRNA levels after treatment of GT1-7 cells with 0.01, 0.1, or 1 µM T-3-BSA or T-17-BSA at 36 h. GnRH mRNA levels were normalized to those of GAPDH used as a loading control. Data are expressed as mean ± SEM from three independent experiments. None of the values analyzed reached statistical significance (P < 0.5). B, Representative Northern blot of 15 µg of total GT1-7 mRNA. GnRH and GAPDH mRNAs are indicated. Film exposure time was 6 h.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Androgens act on the hypothalamic-pituitary-gonadal axis through a complex feedback system, including positive and negative components. Exactly how this feedback is accomplished is not yet fully understood. Nonetheless the evidence for androgen action realized at the level of the hypothalamus is extensive from both animal and in vitro studies (for reviews see Refs. 22, 23, 24). Both stimulatory and inhibitory effects of androgens on GnRH secretion patterns have been reported depending upon the experimental regimen (23, 25). The direct vs. indirect effects of androgens on the GnRH neuron are not yet resolved due to the complexity and diversity of experimental protocols during whole-animal experiments. We have previously reported the presence of functional ARs in the GT1-7 GnRH neuron and detected a repression of GnRH mRNA expression after DHT exposure (15). No one has studied the GT1-7 cell for GnRH secretory responses to androgen. Interestingly, although we see a down-regulation of GnRH gene expression over the long term, we report here that androgens stimulate immediate GnRH secretion in the GT1-7 neuron. This apparent paradoxical observation is not unique to androgens, as activation of protein kinase C (21) and nitric oxide (26, 27) have also been shown to increase GnRH secretion and down-regulate GnRH gene expression in the GT1 GnRH neuron. Regulatory mechanisms responsible for the differential control of GnRH synthesis and secretion by the same compounds have not yet been described.

There is increasing evidence that steroid hormones may transduce their effects through steroid binding sites localized to the cell surface of many cell types (for review see Ref. 3). Although the data demonstrating membrane-mediated androgen action are limited, the most consistent effect of androgen binding at the membrane is a change in [Ca2+]i (5, 6, 7, 8, 28, 29). Because [Ca2+]i modulation is a fairly rapid response, occurring within minutes, it has been presumed that the androgen must bind to some sort of receptor at the surface of the cell to achieve this result. Interestingly, not all cell types that demonstrate a rapid androgen response express the classic nuclear AR (8). Therefore, it is not yet known whether the receptor located at the cell surface is the classic intracellular AR coupled to other signal transduction machinery located in the membrane or a unique receptor, capable of binding androgen and initiating signal transduction. There is evidence for both possibilities in the literature (6, 7, 8, 29).

In this study we have demonstrated that GT1-7 neurons have AR localized to the cell membrane. Upon isolation of the plasma membrane, we have detected what appears to be the classic intracellular AR through Western analysis utilizing an antibody toward the known AR. Perhaps most convincing is the translocation and colocalization of a GFP-tagged AR protein to the plasma membrane of the GT1-7 neuron, although, in our hands, three antibodies produced with peptides from the N terminus of the AR did not exhibit any specific immunoreactivity at the cell surface. We speculate that the region of the AR recognized by the antibodies is masked by its insertion or docking to the plasma membrane. We found that DHT inhibits forskolin-stimulated increases in cAMP. We have also shown that the down-regulation in cAMP accumulation can be blocked by the specific AR antagonist hydroxyflutamide, indicating that the classic intracellular AR is likely responsible for both the membrane and genomic effects of androgen in the GT1-7 neuron. This presumption is not unlikely as it is known that the AR is found in the cytoplasm of the cell until ligand binding and then translocates to the nucleus to regulate gene expression. We have detected an apparent increase in plasma membrane-localized AR upon treatment with DHT, indicating that androgen binding may also support translocation to the membrane. It is therefore possible that the AR could be coupled to other molecules within the cell membrane capable of signal transduction upon androgen binding. Most recently the classic AR has been found to associate with caveolin-1, a primary component of the caveolae membrane, which can be functionally described as a specialized signal-transducing domain of the plasma membrane (30).

DHT alone does not appear to modulate cAMP accumulation, yet once cAMP levels are induced by forskolin, it precisely down-regulates this change in cAMP. This appears to be quite relevant physiologically. Many neurotransmitters are capable of increasing cAMP levels in GT1 GnRH neurons, including norepinephrine and dopamine (31, 32, 33). It is known that increases in cAMP levels result in a secretory response in the GnRH neuron (21, 34). Androgen may therefore be responsible for modulating the positive regulatory effects of other neurotransmitters on the GnRH neuron by decreasing cAMP levels in the cell. The precise mechanism responsible for the negative regulation of cAMP by androgen is not known, but certainly the control of cAMP accumulation within the cell may contribute to the overall modulation of GnRH secretion. It has recently been suggested that oscillations in cAMP levels, through activation of PKA, may result in the precise regulation of the pulsatile secretion of GnRH (35).

Similarly, a number of downstream signaling pathways regulating GnRH secretion have been proposed. Calcium has been postulated as a key factor controlling GnRH pulsatility and secretion (36, 37, 38). Recent studies indicate that there may be a direct relationship between changes in calcium levels and GnRH secretion (39). We have found that androgen can also cause an increase in [Ca2+]i, which is likely connected to the increase in GnRH secretion that we have described after treatment with the androgens DHT, T, and T-3-BSA. At this point it is difficult to determine whether the two signal transduction events, repression of forskolin-induced cAMP accumulation through a Gi protein and an increase in [Ca2+]i caused by membrane AR activation, are interrelated. We speculate that androgen signaling through inhibitory G proteins results in differential downstream effector molecule activation. Nevertheless, in rat osteoblasts, T has been found to rapidly stimulate [Ca2+]i, an effect thought to be coupled to phospholipase C via a PTX-sensitive G protein (5). T has also been linked to the protein kinase C-coupled secretion of GnRH in vivo (40). Detailed studies are currently underway to dissect the downstream mechanisms involved in the androgen-mediated regulation of the GnRH secretion in the GT1-7 neuron by membrane AR signaling. Because of the complexity of the steroid feedback system in the GnRH neuron, it is quite possible that androgens could be affecting individual signaling pathways differentially, depending upon the physiological environment at the time.

The possibility of a single hormone responsible for differential physiological effects in a single cell type is intriguing and opens new frontiers into the mechanisms involved in the control of cellular physiology. Using a homogeneous population of GnRH-synthesizing neurons, we have found that androgen can achieve diverse cellular responses by utilizing either membrane or nuclear mechanisms. We have found that membrane binding of androgen does not appear to affect the down-regulation of GnRH mRNA levels by androgen. This is not unexpected as the AR is known to act as a transcription factor and certainly is capable of altering gene expression at the genomic level. What is most interesting is that membrane-mediated signal transduction by androgen results in a change in GnRH secretion. If this is a common mechanism of cellular regulation by steroids, the complexity involved in the steroidal control of basic physiological functions is yet to be fully realized.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture and Reagents
GT1-7 cells were grown in DMEM (Life Technologies, Inc., Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum (FBS), 4.5 mg/ml glucose, and penicillin/streptomycin and maintained in an atmosphere with 5% CO2 as described (10). Cells were grown in charcoal-stripped FBS, prepared as described previously (26), during steroid treatments where indicated. DHT, T, aprotinin, leupeptin, pepstatin, BSA, and protease and phosphatase inhibitor cocktails were obtained from Sigma (St. Louis, MO). The T-BSA conjugates T-3-BSA (conjugated at the C3 and containing 20–30 mol of steroid per mole BSA), also tagged with FITC (T-3-BSA-FITC, containing ~10 mol steroid and 3 mol FITC per mole BSA) or T-17-BSA (containing 40 mol steroid per mole BSA), were obtained from Sigma. The manufacturer indicates that these conjugates are 98% pure and contain less than 0.1% free T, but may undergo hydrolysis to free T if in solution for extended periods. Hydroxyflutamide was a gift from Schering-Plough Corp. (Kenilworth, NJ). DHT and T stock solutions were prepared in absolute ethanol. The final ethanol concentration in the treatments was 20 nM. T-BSA conjugates were prepared in PBS, pH 7.4.

Western Blot Analysis
Membrane fractions of GT1-7 cells were prepared according to Song et al. (41). Briefly, cells were homogenized in 0.5 M Tris-HCl (pH 7.4) supplemented with protease inhibitors [1 µg/ml aprotinin, pepstatin and leupeptin, 1 mM phenylmethylsulfonylfluoride, 1 mM EDTA] and centrifuged at 770 x g for 10 min at 4 C. Supernatant was carefully recovered and spun again at 770 x g. The resulting supernatant was centrifuged at 20,800 x g for 15 min. The pellet was washed once, resuspended in the above buffer (0.1–0.5 ml), and stored at -80 C until assay. Nuclear extracts were prepared as previously described (42). Aliquots were taken for protein measurements using the Pierce Chemical Co. BCA Protein Assay Reagent (Pierce Chemical Co., Rockford, IL). Membrane, nuclear extract, or total proteins (40 µg) were resolved on a 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel (43) and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA). Membranes were washed briefly in PBS, blocked for 30 min, and then incubated in PBST (PBS, pH 7.4; 0.2% Tween-20; 5% powdered skim milk) for 16 h at 4 C with a rat monoclonal AR antibody, MA1-150 (1 µg/ml dilution; Affinity BioReagents, Inc., Golden, CO). Immunoreactive bands were visualized with horseradish peroxidase-labeled secondary goat antimouse antisera at 1:5000 dilution and enhanced chemiluminescence (Amersham Pharmacia Biotech, Oakville, Ontario, Canada), as described by the manufacturer. The blots were stripped (0.1 M glycine, pH 2.7) and reprobed with the mouse Gß (T-20) polyclonal antibody, which recognizes the four ß-subunit subtypes of the heterotrimeric G protein family (1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to monitor loading and transfer, and as a membrane marker. The blots were also reprobed with a mouse ROR{alpha}1 antibody (1:1000, Santa Cruz Biotechnology, Inc.), the orphan nuclear receptor, to verify the purity of the GT1-7 plasma membrane fractions.

Immunocytochemistry and Transient Transfections
GT1-7 cells were plated on four-well chamber slides (Nalge Nunc International, Naperville, IL) in DMEM overnight, washed twice with PBS+ (140 mM NaCl; 2.7 mM KCl; 6.4 mM Na2HPO4; 1.4 mM KH2PO4; 0.5 mM MgCl2; 0.9 mM CaCl2, pH 7.2) and then incubated at room temperature for 30 min with 1.5 x 10-5 M T-BSA-FITC (Sigma). BSA-FITC and BSA alone (both at 1.5 x 10-5 M) were used as controls. After two washing with PBS+, cells were then incubated with ConA-Rhod (1:100) (Vector Laboratories, Inc., Burlingame, CA), for 20 min and washed twice with PBS+ before fixation with 0.5% paraformaldehyde. The gaskets were removed from the chamber slides after the final rinse and mounted with Immuno Fluore mounting media (ICN Biochemicals, Inc., Montréal, Québec, Canada). Transient transfections were performed using the calcium phosphate precipitate method as previously described (44). Each transfection contained 10 µg of AR-GFP plasmid DNA (20). The cells were incubated for 12–14 h with plasmid DNA, followed by three PBS rinses; after another 24-h incubation in stripped medium, the cells were treated with 10 nM DHT for 15 min, washed once with PBS, and then incubated with ConA-Rhod for 20 min at 15 C.

Fixed or live cells were then analyzed with a confocal laser scanning microscope equipped with a dipping objective lens at a magnification of either x400 or x630 (LSM 510, Carl Ziess, New York, NY). FITC or GFP fluorescence was excited by the 488-nm argon laser line, whereas rhodamine was excited by the 568-nm krypton laser line. Z-series optical sections were taken at 0.4 µm (10–18 sections stacked for a total of approximately 4–7 µm to enhance the T-BSA-FITC labeling) or 1.0 µm (for the AR-GFP experiments) intervals and evaluated using Adobe Photoshop 5.0 for Windows (Adobe Systems, Mountain View, CA).

cAMP Studies
GT1-7 cells were split into 24-well plates. In the case of the dose-response curve, cells were starved for 1 h in DMEM without FBS, and then medium was replaced with 0.5 ml fresh DMEM (without FBS) with the compounds as indicated. In the experiment with the AR antagonist, cells were pretreated with 10 µM hydroxyflutamide for 1 h before treatment. In the experiment with PTX, medium was replaced with 0.5 ml fresh DMEM (without FBS), with 200 ng/ml PTX, or vehicle alone. After a 4-h PTX pretreatment, cells were washed twice with serum-free DMEM. All drugs were diluted in DMEM containing isobutylmethylxanthine (100 µM). After a 15-min incubation at 37 C, 1 ml of ice-cold ethanol was added to each well. Cells were scraped from the plate and kept at -20 C until the amounts of intracellular cAMP were determined in triplicate by RIA (Biotechnologies Inc., Stoughton, MA) according to the manufacturer’s instructions.

[Ca2+]i Measurements Using Fura-2 Fluorescence
With few modifications, [Ca2+]i was measured as previously described (45, 46). Briefly, cells were plated at a low density (<5 x 104 cells) on 25-mm diameter circular glass coverslips and allowed to attach overnight in regular culture media under standard culture conditions. The next day, cells were loaded with 2 µM acetoxymethylester of the fluorescent Ca2+ indicator dye Fura-2 (Fura 2-AM, Molecular Probes, Inc., Eugene, OR) for 30 min at room temperature in physiological saline solution (PSS: 140 mM NaCl; 5 mM KCl; 1.5 mM CaCl2; 1 mM MgCl2; 10 mM D-glucose; 5 mM HEPES, pH 7.4). Coverslips were then washed 10 times with PSS and mounted on a modified Leiden chamber in which the coverslip constituted the bottom and to which 0.5 ml of PSS was added. Free [Ca2+]i was measured by fluorescence ratio imaging using an Image-Master DeltaRAM digital ratio imaging system (Photon Technology International, London, Ontario, Canada) with an IX70 inverted microscope (Olympus Corp., Lake Success, NY) and an IC-200 intensified charge-coupled device camera. With alternating 340- and 380-nm excitation, Fura-2 emission (510 nm) images were acquired at 5- to 10-sec intervals at baseline and after addition to the chamber of control and experimental agents. The ratio of the emission intensities at 340 and 380 nm (340:380 ratio) constitutes a relative measure of the free [Ca2+]i (47). Single cell images and the corresponding cytoplasmic 340:380 ratios, calculated on a pixel-by-pixel basis, were collected for data processing. With the same experimental settings, Fura-2-dependent 340:380 ratios were calibrated in situ with known Ca2+ concentrations from 0 to 1 mM (48). Actual [Ca2+]i (nM) were then calculated from experimental ratios using established formulas (47).

GnRH Secretion Studies
GT1-7 cells were split into 24-well plates. Cells were preincubated in 0.5 ml DMEM with 0.1% BSA 2 h before treatment, and then with fresh DMEM/0.1% BSA and the indicated compounds for the indicated times. Medium was collected at 120 min after forskolin treatment and after 30, 60, and 120 min for all other treatments. Total cellular protein content was consistently equivalent between wells (<=10% variation). GnRH content in 300 µl aliquots of the cell culture medium samples was assayed in triplicate with a GnRH RIA kit (Peninsula Laboratories, Inc., Belmont, CA) according to the manufacturer.

Northern Analysis of GnRH mRNA Levels
GT1-7 cells were grown in DMEM containing charcoal-stripped serum for 12 h and then treated at the times and concentrations described. Total cellular RNA was isolated by the guanidinium thiocyanate phenol chloroform extraction method (49). Ten micrograms of total RNA were electrophoresed in 1% formaldehyde agarose gels and transferred to Genescreen membranes (DuPont-NEN Life Science Products, Boston, MA) by capillary blotting (50). Membranes were prehybridized for 2–6 h and hybridized for 16 h in hybridization buffer (1% wt/vol BSA, 1 mM EDTA, 0.5 M Na2HPO4, 5% wt/vol SDS, 25% formamide) at 55 C with a GnRH cDNA (51), and with {gamma}-actin cDNA (52) or a mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA generated by RT-PCR, as loading controls. The cDNA probes were labeled using random hexamers and [32P] dATP (6000 Ci/mmol, DuPont-NEN Life Science Products) incorporated with the Klenow fragment of DNA polymerase I (53). Blots were washed at high stringency (0.5x standard sodium citrate, 0.1% SDS; 50 C), and exposed to Fuji film (Fuji Photo Film Co., Ltd., Stamford, CT) at -70 C with intensifying screens for 4–24 h. Autoradiographs were scanned with a UMAX 1220 Scanner (Astra USA, Inc., Pleasanton, CA), and GnRH mRNA signals were quantified by densitometry using the NIH Image program.

Statistical Analysis
Comparisons of results between treatments over different times were made using one-way or two-way ANOVA as appropriate for the experiment. Where a significant ANOVA was identified (P < 0.05), the statistical significance of the results between individual pairs was determined using the Student’s t test. Data were analyzed using the statistical program package SPSS, Inc. 6.1.4 for Power Macintosh (University of Toronto site license).


    ACKNOWLEDGMENTS
 
We thank Dr. Pamela L. Mellon, University of California, San Diego, for generously providing the GT1-7 cells and Dr. Mark A. Trifiro for the gift of the AR-GFP construct. Thanks to Dr. Bernardo Yusta for critical reading of the manuscript.


    FOOTNOTES
 
This work was supported by the Canadian Institutes for Health Research (CIHR). D.D.B. is a CIHR Scholar and a Canada Foundation for Innovation (CFI) Researcher; A.N.E.H. is a recipient of a Department of Medicine Postdoctoral Fellowship; and M.H. is a CIHR Clinician Scientist and a recipient of a CFI New Opportunities Award.

Abbreviations: AR, Androgen receptor; [Ca2+]i, intracellular calcium concentration; ConA-Rhod, concanavalin A labeled with rhodamine; DHT, 5{alpha}-dihydrotestosterone; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; PSS, physiological saline solution; PTX, pertussis toxin; ROR{alpha}, retinoid-related orphan receptor; SDS, sodium dodecyl sulfate; T, testosterone; T-3-BSA, testosterone 3-(O- carboxymethyl)oxime BSA conjugate; T-17-BSA, testosterone 17ß-hemisuccinate BSA conjugate.

Received for publication January 9, 2002. Accepted for publication July 16, 2002.


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