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Endocrinology, doi:10.1210/en.2005-1395
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Endocrinology Vol. 147, No. 4 2028-2034
Copyright © 2006 by The Endocrine Society

Dihydrotestosterone Differentially Modulates the Mitogen-Activated Protein Kinase and the Phosphoinositide 3-Kinase/Akt Pathways through the Nuclear and Novel Membrane Androgen Receptor in C6 Cells

Joshua W. Gatson, Paramjit Kaur and Meharvan Singh

Department of Pharmacology and Neuroscience and the Institute for Aging and Alzheimer’s Disease Research, University of North Texas Health Science Center, Fort Worth, Texas 76107-2699

Address all correspondence and requests for reprints to: Dr. Meharvan Singh, Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3400 Camp Bowie Boulevard, Fort Worth, Texas 76107-2699. E-mail: msingh{at}hsc.unt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgens such as dihydrotestosterone (DHT) are known to exert their effects through the activation of intracellular receptors that regulate the transcription of target genes. Alternatively, nongenomic mechanisms, including the activation of such signaling pathways as the MAPK pathways, have been described. It is unclear, however, whether this latter mechanism of action is mediated by the classical androgen receptor (AR) or some alternative mechanism. In this study, using a glial cell model (C6 cells) that we found to express the AR, we identified that DHT increased the phosphorylation of both ERK and Akt, key effectors of the neuroprotection-associated MAPK and phosphoinositide 3-kinase signaling pathways, respectively, and ERK phosphorylation was blocked by the AR antagonist, flutamide. In contrast, the membrane-impermeable, BSA-conjugated androgen (DHT-BSA) caused a dose-dependent suppression of ERK and Akt phosphorylation, suggesting the existence of a novel membrane-associated AR that mediates this opposite effect on neuroprotective signaling. This is also supported by the observation of DHT-displaceable binding sites on the cell surface of live C6 cells. Collectively, these data support the existence of a novel membrane-associated AR in glial cells and argue for the existence of two, potentially competing, pathways in a given cell or tissue. This mutual antagonism was supported by the ability of DHT-BSA to attenuate DHT-induced ERK phosphorylation. Thus, depending on the predominance of one receptor mechanism over another, the outcome of androgen treatment may be very different and, as such, could help explain existing discrepancies as to whether androgens are protective or damage inducing.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGENS ARE classically associated with the regulation of muscle growth, spermatogenesis, growth of bone, and development of secondary sexual characteristics in males and females (1, 2). Within the brain, testosterone can be aromatized to estradiol or, alternatively, reduced at the 5{alpha} position to dihydrotestosterone (DHT) (1). DHT, however, is nonaromatizable and can elicit its effects via either genomic (classical) (1, 2) or nongenomic mechanisms (1, 3, 4). With regard to the latter, androgens have been shown to rapidly activate the phosphoinositide 3-kinase (PI-3K)/Akt and/or MAPK pathways in a variety of peripheral tissues (3, 4, 5). However, whether these nongenomic effects are mediated by the classical androgen receptor (AR) or by some alternative mechanism remains controversial and unclear.

Two isoforms of the classical AR have been described (AR-B and its N-terminally truncated form, AR-A) and are expressed in many different cell types (6, 7, 8). Although the precise role of AR-A remains unclear, it has been described that AR-A can antagonize the action of AR-B, a modulatory mechanism that may be relevant to the activation/inhibition of signaling pathways, regulation of gene transcription, as well as regulation of cell survival (9). Alternatively, the existence of a plasma membrane receptor for androgens has also been proposed (10), such as that described (or postulated) for estrogen and progesterone (11, 12, 13, 14, 15, 16, 17). This membrane AR has been described primarily in noncentral nervous system tissue, including vascular tissue, macrophages, ovary, and T cells (18, 19, 20, 21, 22, 23, 24). Of interest is that this membrane-associated AR is linked to the activation of signaling pathways that may be important in regulating cell death, survival, or growth (10).

To clarify the role of the classical AR in the nongenomic effects of androgens on glia, we evaluated the effects of DHT and the membrane-impermeable, BSA-conjugated androgen (DHT-BSA) on the phosphorylation of ERK and Akt, two key effectors within the MAPK and PI-3K signaling pathways, respectively. We found that although DHT induced the phosphorylation of ERK, DHT-BSA resulted in a dose-dependent suppression of both ERK and Akt phosphorylation and even blocked the effects of DHT. The suggestion that this effect of DHT-BSA was mediated by a novel membrane-associated AR was also supported by the identification of DHT-displaceable binding sites on the cell surface of live C6 cells. Collectively, these data support the existence of a novel plasma membrane-associated AR and suggest the existence of two, potentially competing, pathways within a specific cell or tissue type.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Rat glioma cells (C6; American Type Culture Collection, Manassas, VA) were propagated in DMEM (Invitrogen Life Technologies, Inc., Carlsbad, CA) supplemented with 10% charcoal-stripped fetal bovine serum (HyClone, Logan, UT) and maintained at 37 C in a humidified environment containing 5% CO2.

After treatment of the cells with the appropriate dose of hormone for the appropriate duration, the cells were harvested, homogenized, and centrifuged, and the supernatant was subsequently collected and analyzed for total protein concentration using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). Preprepared cell lysates from the prostate cancer cell line LNCaP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were obtained and used as positive controls for detection of the AR.

Treatment of cultures
C6 cells were treated with vehicle control (dimethylsulfoxide, 0.1%), dihydrotestosterone (DHT; Steraloids, Inc., Newport, RI), or 5{alpha}-androstan-17ß-ol-3-one-3-O-carboxymethyloxime (CMO):BSA (DHT-BSA; Steraloids, Inc.) at the concentrations indicated for 30 min, to assess the effects of these hormones on ERK and Akt phosphorylation. Controls for the DHT-BSA treatment included treatment with equimolar concentrations of CMO (DHT:CMO; Steraloids, Inc.) or BSA alone (Fisher Scientific, Fairlawn, NJ) for a similar duration (30 min). Inhibition of the classical AR was achieved using the AR antagonist, flutamide (3 µM; Sigma-Aldrich Corp., St. Louis, MO), which was applied 30 min before treatment with the hormone. Inhibition of MAPK kinase (MEK), the signaling protein upstream of ERK, was achieved using UO126 (10 µM; Cell Signaling Technology, Inc., Beverly, MA), which was also preincubated for a period of 30 min before hormone administration.

Western blot analysis
After treatment with hormone and/or inhibitor, C6 cells were harvested into lysis buffer containing protease and phosphatase inhibitors as described previously (25). After homogenization, samples were centrifuged at 99,000 x g for 15 min at 4 C, and the resulting supernatants were evaluated for total protein concentrations using the Bio-Rad DC (Bio-Rad Laboratories, Inc.) protein assay kit [based on the method of Lowry et al. (26)]. Sample lysates were loaded onto a sodium dodecyl sulfate/10% polyacrylamide gel, subjected to electrophoresis, and subsequently transferred onto a polyvinylidene difluoride membrane (0.22 µm pore size; Bio-Rad Laboratories, Inc.). The membrane was blocked for 6 h with a 3% BSA in 0.2% Tween-containing Tris-buffered saline solution before application of the primary antibody. The following primary antibodies were used: for the detection of the AR, anti-AR (C-19; 1:200; Santa Cruz Biotechnology, Inc.); for the detection of the phosphorylated form of Akt, rabbit antiphospho-Akt (Ser473; 1:1000; Cell Signaling Technology, Inc.), for the detection of total Akt, anti-Akt (1:1000; Cell Signaling Technology, Inc.); for the detection of the phosphorylated form of ERK1/2, rabbit antiphospho-p44/42 MAP (Thr202/Tyr204, 1:1000; Cell Signaling Technology, Inc.); and for the detection of total ERK1 and ERK2, goat anti-ERK1 (C-16; 1:500)/goat anti-ERK2 (C-14; 1:500; Santa Cruz Biotechnology, Inc.). Antibody binding to the membrane was detected using a secondary antibody (either goat antirabbit or rabbit antigoat) conjugated to horseradish peroxidase (1:20,000; Pierce Chemical Co., Rockford, IL) and visualized using enzyme-linked chemiluminescence (ECL, Amersham Biosciences, Arlington Heights, IL) with the aid of the UVP imaging system. Phospho-Akt and phospho-ERK blots were reprobed with anti-Akt or anti-ERK1/2 antibodies to ensure equal loading across lanes.

Flow cytometry
C6 cells (106 cells) were pipetted into a 1.5-ml microcentrifuge tube, centrifuged at 250 x g for 5 min, and washed twice with 1 ml PBS. After the last wash, the cells were repelleted, suspended in 100 µl PBS, and treated with DHT-BSA-fluorescein isothiocyanate (DHT-BSA-FITC; 50 µM; Sigma-Aldrich Corp.) in the presence or absence of DHT (1 mM), for 30 min at 4 C. In parallel, cells were resuspended in PBS treated with BSA-FITC (50 µM; Sigma-Aldrich Corp.) and served as the control for the detection of nonspecific binding. After this incubation period, the cells were washed twice in PBS and resuspended in 500 µl PBS. The labeled C6 cells were injected into an EPICS XL-MCL flow cytometer (Beckman Coulter, Fullerton, CA) and analyzed with System 2 software (Beckman Coulter). Graphical representation of the data was generated using FloJo software (TreeStar, Inc., San Carlos, CA).

Statistical analysis
Densitometric analysis of the Western blots was conducted using LabWorks Image Acquisition and Analysis software (UVP, Inc., Upland, CA). Densitometric data from at least three independent experiments was subjected to ANOVA, followed by Tukey’s post hoc analysis for the assessment of group differences, and are presented as a bar graph depicting the average ± SEM, using GraphPad software (San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
C6 cells express the AR
Genomic and nongenomic pathways activated by androgens may involve one or both receptor isoforms of the AR (AR-B or AR-A). Using Western blot analysis, we evaluated whether the C6 glial cells express either of these receptor isoforms. Figure 1Go confirms the presence of immunoreactive AR and identifies two distinct bands corresponding to molecular masses of 110 and 87 kDa, respectively. These two bands are of the same size described in the literature for the full-length AR-B and the truncated AR-A. Interestingly, a doublet was observed in lysates from C6 cells. Given that protein bands may appear as doublets (such as for ERK1 and ERK2), reflecting the phosphorylated and unphosphorylated forms of the protein, we suggest that this doublet may also reflect two dominant posttranslational states of the AR. Nevertheless, the bands identified in Fig. 1Go comigrated with the AR-B and AR-A bands seen in the positive control, a cell lysate derived from the prostate cancer cell line, LNCaP cells.


Figure 1
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  		FIG. 1. The classical AR is expressed in C6 cells. Total protein from C6 cells was isolated and evaluated for expression of the AR using Western blot analysis. This analysis revealed two bands corresponding to the predicted molecular masses of AR-B (110 kDa) and AR-A (87 kDa). These bands comigrated with the AR bands observed in the prostate cancer cell line, LNCaP, that served as the positive control.

 
Nuclear AR mediates DHT-induced phosphorylation of ERK
Given that androgens have been shown to elicit activation of cell signaling pathways in a variety of tissues, we evaluated whether DHT elicits the phosphorylation of ERK, a key effector of the MAPK pathway, in glia. Treatment of C6 cells with 10 nM DHT resulted in a robust (2.5-fold) increase in the phosphorylation of ERK. This effect was inhibited by the classical AR antagonist, flutamide (Fig. 2Go). The effect of DHT on ERK phosphorylation was also blocked by the MEK1/2 inhibitor, UO126 (data not shown), suggesting that the effect of DHT on ERK required activation of the upstream signaling kinase, MEK. Interestingly, higher concentrations of DHT (0.1 and 1 µM) did not result in an increase in ERK phosphorylation (Fig. 2Go). DHT also induced an increase in Akt phosphorylation, but required a slightly higher concentration than that needed to elicit ERK phosphorylation. Another distinction was that the antagonist to the classical AR, flutamide, failed to inhibit the effect of DHT on Akt phosphorylation (Fig. 3Go).


Figure 2
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  		FIG. 2. DHT-induced ERK phosphorylation is mediated by the classical AR. C6 cells were treated with DHT (0.01, 0.1, and 1 µM) for 30 min in the presence/absence of the AR antagonist flutamide (Flut.; 3 µM). Resulting lysates (containing 60 µg total protein) were then subjected to SDS-PAGE and Western blot analysis for evaluation of ERK phosphorylation. The results demonstrate that DHT-elicits ERK phosphorylation, an effect that was blocked by the classical AR antagonist, flutamide. The upper blot and lower blot depict ERK phosphorylation and total ERK protein, respectively, from a single independent experiment. The bar graph (upper panel), however, represents densitometric analysis and statistical evaluation of data from three independent experiments and is presented as signal intensity compared with that in the sham (vehicle treated) control. Statistical significance was determined using a one-way ANOVA, followed by Tukey’s post hoc analysis for group differences (*, P < 0.01).

 

Figure 3
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  		FIG. 3. DHT elicits an increase in phospho-Akt levels in C6 cells. Rat glioma cells were treated with DHT (0.01, 0.1, and 1 µM) for 30 min in the presence/absence of the AR antagonist flutamide (Flut.; 3 µM). Resulting lysates (containing 60 µg total protein) were then subjected to SDS-PAGE and Western blot analysis for evaluation of Akt phosphorylation. The results show that DHT elicited an increase in Akt phosphorylation, but was not inhibited by the AR antagonist, flutamide. The upper blot and lower blot depict Akt phosphorylation and total Akt protein, respectively, from a single independent experiment. The bar graph (upper panel), however, represents densitometric analysis from two independent experiments and is presented as signal intensity compared with that in the sham (vehicle treated) control.

 
Activation of a membrane AR decreased phosphorylation of ERK
In an attempt to assess whether the effect of DHT was mediated by the classical intracellular/intranuclear AR, or alternatively, if a membrane AR may be involved, we determined whether the membrane-impermeable androgen, DHT-BSA, would also elicit ERK phosphorylation. In contrast to the effect of DHT, the membrane-impermeable androgen not only failed to elicit an increase in the phosphorylation of ERK, but resulted in a substantial suppression of ERK phosphorylation, particularly at the higher concentrations of 100 nM and 1 µM (Fig. 4Go). This suppression of ERK phosphorylation by DHT-BSA was also insensitive to flutamide (Fig. 5Go). To ensure that the inhibition of ERK was not due to chemical modification of the parent DHT molecule, we evaluated the effect of DHT:CMO. As expected, the CMO group alone did not alter the ability of the androgen to elicit ERK phosphorylation (Fig. 6Go), and recapitulated the data found with DHT alone (Fig. 2Go). Furthermore, the effect of DHT:CMO on ERK phosphorylation was also blocked by the AR antagonist, flutamide (Fig. 6Go). As an added control, we determined whether the inhibitory effect of DHT-BSA could have been attributed to the bulky globulin (BSA) that was attached. Administration of BSA by itself failed to alter the basal phosphorylation state of ERK, suggesting that the inhibition was not due to BSA (Fig. 7Go).


Figure 4
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  		FIG. 4. DHT/BSA treatment suppresses phospho-ERK levels in C6 cells. C6 cells were treated with increasing concentrations of DHT-BSA (0.01, 0.1, and 1.0 µM) for 30 min. Resulting lysates (containing 60 µg total protein) were then subjected to SDS-PAGE and Western blot analysis for evaluation of ERK phosphorylation. DHT-BSA resulted in a dose-dependent decrease in phospho-ERK levels. The data revealed that DHT-BSA inhibited basal ERK phosphorylation levels in a dose-dependent manner. The upper blot and lower blot depict ERK phosphorylation and total ERK protein, respectively, from a single independent experiment. The bar graph (upper panel), however, represents densitometric analysis and statistical evaluation of data from three independent experiments and is presented as signal intensity compared with that in the sham (vehicle treated) control. Statistical significance was determined using a one-way ANOVA, followed by Tukey’s post hoc analysis for group differences (*, P < 0.001; #, P < 0.01).

 

Figure 5
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  		FIG. 5. DHT/BSA suppression of phospho-ERK levels in C6 cells is flutamide insensitive. C6 cells were treated with increasing concentrations of DHT-BSA (0.01, 0.1, and 1.0 µM) for 30 min in the presence or absence of the AR antagonist flutamide (Flut.; 3 µM). Resulting lysates (containing 60 µg total protein) were then subjected to SDS-PAGE and Western blot analysis for evaluation of ERK phosphorylation. The results demonstrated that the dose-dependent inhibition of ERK phosphorylation by DHT-BSA was not prevented by the AR antagonist flutamide. The upper blot and lower blot depict ERK phosphorylation and total ERK protein, respectively, from a single independent experiment. The bar graph (upper panel), however, represents densitometric analysis from two independent experiments and is presented as signal intensity compared with that in the sham (vehicle treated) control.

 

Figure 6
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  		FIG. 6. CMO conjugation to DHT does not alter DHT’s ability to elicit ERK phosphorylation. C6 cells were treated with DHT:CMO (0.01, 0.1, and 1 µM) for 30 min in the presence or absence of the AR antagonist flutamide (3 µM). DHT:CMO was included in these studies as a control for the BSA-conjugated DHT (DHT-BSA). Resulting lysates (containing 60 µg total protein) were then subjected to SDS-PAGE and Western blot analysis for evaluation of ERK phosphorylation. The results show that the CMO moiety does not alter the ability of DHT to elicit ERK phosphorylation. The upper blot and lower blot depict ERK phosphorylation and total ERK protein, respectively, from a single independent experiment. The bar graph (upper panel), however, represents densitometric analysis and statistical evaluation of data from three independent experiments and is presented as signal intensity compared with that in the sham (vehicle treated) control. Statistical significance was determined using a one-way ANOVA, followed by Tukey’s post hoc analysis for group differences (*, P < 0.01).

 

Figure 7
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  		FIG. 7. BSA does not elicit ERK phosphorylation. C6 cells were treated with increasing concentrations of BSA alone for 30 min. Resulting lysates (containing 60 µg total protein) were then subjected to SDS-PAGE and Western blot analysis for evaluation of ERK phosphorylation. The data reveal that BSA treatment by itself failed to alter ERK phosphorylation levels. The upper blot and lower blot depict ERK phosphorylation and total ERK protein, respectively, from a single independent experiment. The bar graph (upper panel), however, represents densitometric analysis from two independent experiments and is presented as signal intensity compared with that in the sham (vehicle treated) control.

 
Activation of membrane AR results in a dose-dependent decrease in phosphorylation of Akt
To assess whether activation of the putative membrane AR influenced another growth- and/or survival-promoting signal transduction pathway in a similar manner to that observed with the MAPK pathway, we evaluated the effect of DHT-BSA on the downstream effector of the PI-3K pathway, Akt. DHT-BSA, at concentrations of 0.01, 0.1, and 1 µM resulted in a dose-dependent suppression of Akt phosphorylation (Fig. 8Go).


Figure 8
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  		FIG. 8. DHT-BSA treatment suppresses phospho-Akt levels in C6 cells. C6 cells were treated with DHT/BSA (0.01, 0.10, and 1 µM) for 30 min. Resulting lysates (containing 60 µg total protein) from the various treatment groups were subjected to SDS-PAGE and Western blot analysis for evaluation of Akt phosphorylation. Treatment of C6 cells with DHT-BSA resulted in a dose-dependent decrease in phospho-Akt levels. The data demonstrate that DHT-BSA inhibits basal Akt phosphorylation in a dose-dependent manner. The upper blot and lower blot depict Akt phosphorylation and total Akt protein, respectively, from a single independent experiment. The bar graph (upper panel), however, represents densitometric analysis and statistical evaluation of data from three independent experiments and is presented as signal intensity compared with that in the sham (vehicle treated) control. Statistical significance was determined using one-way ANOVA, followed by Tukey’s post hoc analysis for group differences (*, P < 0.001; #, P < 0.01).

 
DHT-BSA blocks DHT-induced ERK phosphorylation in a dose-dependent manner
In view of the inhibitory effects of DHT-BSA on ERK and Akt phosphorylation and the stimulatory consequence of activating the intracellular AR (using DHT), we evaluated whether activating the putative membrane AR would inhibit the classical AR-mediated induction of ERK phosphorylation. As such, we coapplied DHT and DHT-BSA to C6 cells for 30 min and found that DHT-BSA blocked the effect of DHT (10 nM) on ERK phosphorylation at all concentrations tested (0.01, 0.1, and 1 µM) and in a dose-dependent fashion (Fig. 9Go). These findings support the idea that DHT may modulate the MAPK pathway through at least two, competing pathways.


Figure 9
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  		FIG. 9. DHT-BSA blocks DHT-induced ERK phosphorylation. To determine whether DHT/BSA competitively blocks DHT’s effect on the MAPK pathway, C6 cells were treated with DHT-BSA at various concentrations (0.01, 0.1, and 1.0 µM) for 30 min in the presence or absence of DHT (10 nM, a concentration that effectively and reproducibly elicits ERK phosphorylation). As a control, BSA (1 µM) was included. Resulting lysates (containing 60 µg total protein) were then subjected to SDS-PAGE and Western blot analysis for evaluation of ERK phosphorylation. The data revealed that DHT-BSA effectively prevented the effect of DHT on ERK phosphorylation. The upper blot and lower blot depict ERK phosphorylation and total ERK protein, respectively, from a single independent experiment. The bar graph (upper panel), however, represents densitometric analysis from two independent experiments and is presented as signal intensity compared with that in the sham (vehicle treated) control.

 
C6 glial cells express binding sites for DHT-BSA on the cell surface.
To determine whether DHT-BSA binds to a specific site on the cell surface (indicative of a membrane-associated AR), we evaluated binding of the fluorescently labeled DHT-BSA (DHT-BSA-FITC) in C6 cells. Nonfixed and nonpermeabilized C6 cells were treated with DHT-BSA-FITC (50 µM) in the presence or absence of a 20-fold molar excess of DHT (1 mM). Incubation of the cells with BSA-FITC alone for 30 min at 4 C provided a measure of nonspecific binding. The fluorescence intensity histograms (Fig. 10Go) obtained through flow cytometric analysis revealed the presence of specific, DHT-displaceable binding sites on the surface of C6 cells.


Figure 10
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  		FIG. 10. DHT-BSA binds to specific sites on the cell surface of C6 cells. To determine whether DHT-BSA binds to surface (plasma membrane) receptors, C6 cells were treated with DHT-BSA-FITC (50 µM) for 30 min in the presence or absence of a 20-fold molar excess of DHT (1 mM). Samples were washed and analyzed using flow cytometric analysis. The fluorescence histogram, depicting increasing fluorescence intensity on the x-axis and cell number on the y-axis, shows significant labeling of cells with DHT-BSA-FITC. This labeling appeared to be displaced by DHT. The peak on the extreme left reflects the amount of fluorescence signal obtained when cells were incubated with BSA-FITC alone, representing nonspecific binding. The data are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we show that androgens regulate the activity of two signal transduction pathways, the MAPK and the PI-3K/Akt signaling pathways, in C6 glial cells. The existence of immunoreactive ARs (AR-B and AR-A) in C6 cells coupled with the fact that DHT-induced ERK phosphorylation was inhibited by the pharmacological antagonist of the classical AR supported the involvement of this classical receptor in regulating cell signaling. However, the opposite effect of DHT-BSA on ERK and Akt phosphorylation suggested that androgens may also regulate cell signaling via a distinct, and potentially competing, receptor mechanism. Supporting this was the finding that the inhibitory effect of DHT-BSA on ERK phosphorylation was not blocked by the classical AR antagonist, flutamide, unlike the effects of DHT. In addition, analysis of the binding of fluorescently labeled DHT-BSA to live C6 cells revealed specific, DHT-displaceable binding sites on the cell surface.

To exclude the possibility that chemical modification of the parent compound, DHT, was responsible for the observed effects of DHT-BSA, several important controls were performed. First, we ensured that the CMO linker that enables the attachment of BSA to DHT did not alter the ability of DHT to regulate cell signaling. Figure 6Go shows that DHT:CMO elicited the same dose-dependent regulation of ERK phosphorylation as the parent compound. It is also unlikely that the effect of DHT-BSA was due to the presence of free DHT present in the DHT-BSA preparation, because the effect of DHT-BSA was opposite that observed with DHT. Nevertheless, we took steps to ensure that no free DHT was present in the DHT-BSA solution. This was achieved by prefiltering DHT-BSA using a 30-kDa nominal cutoff column. The retentate was then eluted and used in the experiments. All data shown were derived from experiments in which the filtered DHT-BSA was used. Furthermore, no differences were observed between the effects of filtered DHT-BSA and unfiltered DHT-BSA (data not shown). Finally, we ensured that BSA by itself was not responsible for the effect of DHT-BSA. By testing increasing concentrations of BSA, we found no differences in ERK phosphorylation compared with controls.

Interestingly, although low concentrations of DHT elicited ERK and Akt phosphorylation, higher concentrations failed to do so. We suggest that at higher concentrations, DHT binds to the membrane AR, resulting in activation of an antagonistic mechanism that reduces and/or prevents the induction of ERK phosphorylation via the classical receptor. This assertion is consistent with the dose-response data depicted in Figs. 2Go and 4Go. That is, the inhibitory effect of DHT-BSA on either ERK or Akt phosphorylation becomes evident at the 0.1-µM concentration, and only at this same concentration (or higher) does DHT not activate ERK phosphorylation. Figure 9Go also demonstrates that DHT-BSA-induced activation of the putative membrane AR inhibits the ERK-inducing effects of DHT. Together, these data support the existence of two, competing mechanisms through which DHT regulates cell signaling. In addition, there appeared to be a slight difference in the potency by which DHT elicited ERK and Akt phosphorylation. The precise mechanism underlying this difference is still unclear, but possible explanations may include the existence of different AR subtypes or isoforms that mediate the effects of androgens on ERK and Akt separately. Ongoing research is aimed at addressing this difference.

Membrane receptors have also been proposed for estrogen and progesterone, but only the membrane progesterone receptor has been successfully cloned. Zhu and colleagues (17) characterized a novel membrane-associated progesterone receptor that appears not to exhibit the stereotypical modular structure seen with other members of the steroid hormone receptor superfamily, but, instead, contains a seven-transmembrane-spanning domain. As such, this membrane progesterone receptor was reported to be coupled to the Gi/o class of G proteins. Based upon this observation, we postulated that the inhibitory effects of DHT-BSA on ERK and Akt phosphorylation could also be regulated through Gi/o. To test this hypothesis, we evaluated whether pertussis toxin (a Gi/o inhibitor) prevented the effect of DHT-BSA. Pertussis toxin (50 µM) failed to prevent DHT-BSA-mediated suppression of either ERK or Akt phosphorylation (data not shown), leading to our conclusion that this novel membrane AR was not coupled to the Gi/o class of G proteins, at least in the C6 glial cell model.

Activation of the ERK/MAPK pathway is associated with various cellular responses, including the induction of cell differentiation, increased cell growth/proliferation, as well as the regulation of cell viability (27, 28). Similarly, activation of the PI-3K/Akt signaling pathway results in numerous effects on the cell, including the regulation of cell growth, motility, and survival (29). Various studies have shown that depending on the cell type, androgens can either cause a decrease or an increase in phospho-ERK levels, (5, 22, 23, 24, 30, 31) and, as a consequence, may result in varied cellular responses. In the brain, this variability in androgen function has also been observed, such that depending on the experimental model used or region of the brain evaluated, androgens can either exert protective influences (32, 33) or be damage promoting (34). For example, in a kainic acid model of hippocampal injury, DHT was found to reduce the amount of hippocampal neuron damage (33), whereas in a middle cerebral artery occlusion model of stroke, elevated androgen levels were associated with greater amounts of cortical cell death (34). This discrepancy may be related to the relative abundance of the classical intracellular/intranuclear AR and the membrane AR identified here. Specifically, the protective effects of androgens may be seen only under conditions where the classical AR predominates, resulting in increased activation of ERK and/or Akt, which, in turn, favors the promotion of cell survival. In contrast, if the membrane AR predominates, one might predict that elevated androgens may result in increased vulnerability to insult or injury due to the suppression of neuroprotective signaling pathways.

Another critical aspect in understanding the neurobiology of androgens is the possibility that glia may respond differently to androgens than do neurons. Of importance is that the AR is present not only in neurons, but in glial cells as well. In fact, brain astrocytes express high amounts of AR (35). The functional significance of this expression in glia may be inferred from the observation that after stroke, AR levels are up-regulated in glial cells within the hippocampus and parietal cortex (36). Thus, androgens and the AR may play an important role in regulating brain vulnerability to injury by acting at ARs on both neurons as well as glia.

In summary, our data support the existence of a novel membrane-associated AR in glial cells, in addition to the classical AR. Furthermore, the data argue for the existence of two, potentially competing, pathways in a given cell or tissue. Thus, the ratio of one receptor type over another may be predictive of whether androgens are beneficial or detrimental and, as such, could help explain existing discrepancies as to whether androgens are protective or damage inducing. Such information may also be instrumental in helping design appropriate therapeutic regimens that employ androgens for the treatment of various diseases of the brain.


   Footnotes
 
This work was supported by funds from the National Institutes on Aging (Grants AG-22550 and AG-23330), a National Alliance for Research on Schizophrenia and Depression-sponsored Young Investigator Awarded (to M.S.), and the National Science Foundation SCORE (School’s Cooperative Opportunities for Resources and Education) grant.

J.G., P.K., and M.S. have nothing to declare.

First Published Online January 12, 2005

Abbreviations: AR, Androgen receptor; CMO, 5{alpha}-androstan-17ß-ol-3-one-3-O-carboxymethyloxime; DHT, dihydrotestosterone; FITC, fluorescein isothiocyanate; MEK, MAPK kinase; PI-3K, phosphoinositide 3-kinase.

Received November 3, 2005.

Accepted for publication January 4, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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