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-Dihydrotestosterone in GnRH-Secreting GT1–7 Hypothalamic Neurons1
Departments of Physiology (D.D.B.) and Zoology (A.E., T.J.B.), Institute of Medical Sciences (D.R.) and Division of Reproductive Science, University of Toronto and Toronto Hospital Research Institute, Toronto, Ontario, Canada M5G 2C4
Address all correspondence and requests for reprints to: Denise D. Belsham, Ph.D., Department of Physiology/Division of Reproductive Science, University of Toronto/Toronto Hospital Research Institute, 200 Elizabeth Street, CCRW 3–831, Toronto, Ontario, Canada M5G 2C4. E-mail: d.belsham{at}utoronto.ca
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Abstract |
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-dihydrotestosterone (DHT), further demonstrating the presence of a
functional AR. Treatment of GT1–7 cells with 1 or 10 nM
DHT resulted in approximately 55% reduction in GnRH messenger RNA
measured at 24 and 36 h after treatment. This repression was
completely blocked by hydroxyflutamide, an AR antagonist. These results
provide the first demonstration that androgen acts directly through an
AR-mediated pathway to repress GnRH gene expression in hypothalamic
GnRH-secreting neurons. |
Introduction |
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Several neurotransmitter and neuropeptide systems make synaptic contacts with GnRH immunoreactive cells and are potential mediators of the gonadal steroid regulation of GnRH synthesis and secretion (10, 11, 12, 13, 14). For example, noradrenergic, GABAergic, and opiodergic neurons have been demonstrated to contain estrogen and/or progestin receptors and have also been shown to establish synaptic contacts with GnRH immunoreactive cells (15, 16, 17). Alternatively, GnRH neurosecretory cells may possess low levels of steroid hormone receptors that are near the level of detection of the techniques used.
GnRH neurosecretory cells appear to be scattered within the preoptic area of the anterior hypothalamus (18), making an in vivo approach to firmly establish the direct action of steroids on GnRH transcription or secretion technically unfeasible at present. In an attempt to produce a suitable model to study GnRH gene regulation, a directed tumorigenesis technique was used to develop a murine immortal cell line of GnRH-secreting hypothalamic neurons (GT1 cells). The cells were developed by targeting expression of the potent oncogene, SV40 T-antigen, with the regulatory region of GnRH gene (19). The GT1 cell line has been shown to faithfully exhibit many of the known characteristics of GnRH neurons (reviewed in Refs. 20–23). A number of compounds known to regulate GnRH secretion in vivo have been shown to regulate secretion of GnRH in the GT1 cells. Most substances, while stimulating GnRH release, for the most part cause an inhibition of GnRH gene expression in the GT1 cells (22, 23). It is apparent that the amount of GnRH peptide available for secretion depends upon a complex interplay of processes at both transcriptional and posttranscriptional levels (22, 23).
In the present study, we used the GT1–7 cell line to address the question of whether androgen could act directly to regulate GnRH transcription in hypothalamic cells. Poletti et al. (24) have reported that the related clonal cell line GT1–1 have a small amount of high-affinity specific androgen binding activity. We report here that GT1–7 cells also express AR messenger RNA (mRNA) and, more importantly, a functional receptor. GT1–7 cells were also found to express the recently discovered ligand-dependent AR-specific coactivator, ARA70, which functions to enhance transactivation by the AR (25). Furthermore, we show that androgen at physiologically-relevant doses down-regulates GnRH mRNA levels through an AR-dependent mechanism. These results provide the first demonstration of a direct action of androgen on GnRH gene expression in hypothalamic GnRH-secreting neurons.
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Materials and Methods |
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-dihydrotestosterone (DHT), leupeptin, antipain, and
soybean trypsin inhibitor was obtained from Sigma Chemical Co. (St.
Louis, MO). Hydroxyflutamide was a gift from Shering Plough Research
Institute (Kenilworth, NJ). [3H]R1881 (methyltrienolone),
and R1881 were from Dupont-New England Nuclear (Boston, MA).
4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF) was purchased from
Calbiochem (La Jolla, CA). DHT stock solution was prepared in absolute
ethanol. The final ethanol concentration in the treatments was 0.0001%
or 20 nM.
Northern blot analysis
Northern blot analysis was performed as previously described
(27). Briefly, total cellular RNA was isolated by the guanidinium
thiocyanate phenol chloroform extraction method (28). Ten micrograms of
total RNA were size fractionated in 1% agarose-formaldehyde gels and
transferred to Genescreen membranes (Dupont-New England Nuclear) by
capillary blotting (29). The filters were hybridized with rat GnRH
complementary DNA (cDNA) (30), rat androgen receptor cDNA (31), human
ARA70 cDNA (25), and with human 
-actin cDNA (32) to control for
variations in gel loading and transfer efficiency. GnRH and -actin
probes were routinely hybridized concurrently to minimize error caused
by stripping and reprobing of the membrane. Prehybridization for 2–16
h and hybridization for 16 h was conducted in a 25% formamide
hybridization buffer at either 50 C (for ARA70 or AR cDNA) or 55 C (for
GnRH and actin cDNA). The cDNA probes were labeled using random
hexamers and [32P]dATP (6000 Ci/mmol, Dupont-New England
Nuclear) incorporated with the Klenow fragment of DNA polymerase I
(33); specific activities of 0.5–1 x 109 dpm/µg
were routinely obtained. Blots were washed at high stringency (55 C;
0.5 x SSC, 0.1% SDS) and exposed to Fuji film at -70 C with
intensifying screens for 4–48 h. Autoradiographs were scanned with a
Hewlett Packard ScanJet 3p scanner and levels of GnRH, AR, ARA70, or
actin mRNA were quantified using the NIH Image densitometry program.
Statistical significance of the results was determined by comparing the
t values using the Student’s t test, as
indicated.
Cytosol preparation
Cell cultures were grown to about 80–90% confluence and
maintained in sFBS-RPMI medium for 48 h before cytosol
preparation. Cultures were washed twice with ice-cold PBS, scrapped,
and collected by brief centrifugation at 400 x g for 5
min. Cell pellets were resuspended in 0.5 ml of TEGDMo buffer [10
mM Tris-HCl, pH 7.4, 1.5 mM
Na2EDTA, 10% (vol/vol) glycerol, 0.1 mM DTT,
and 40 mM sodium molybdate (Na2
MoO4); containing 0.6 mM AEBSF, 10 µg/ml
leupeptin, 10 µg/ml antipain, and 100 µg/ml soy bean trypsin
inhibitor] and homogenized at 4 C using a Teflon pestle. The
homogenate was centrifuged at 104,000 x g for 45 min
at 4 C, and the supernatant, representing the cytosolic fraction,
collected. Protein content was measured using the Coomasie blue protein
assay (34).
Androgen binding analysis
Androgen binding assays were performed basically as previously
described (35). Briefly, aliquots of cytosol extracts (40 µg
protein/incubant), prepared as above, were incubated for 16 h at 4
C with increasing concentrations (0.1–12 nM final) of the
radiolabeled synthetic androgen, [3H]R1881 (specific
activity, 86 Ci/mmol). Parallel incubations were conducted in the
presence of 1 µM unlabeled R1881 to determine nonspecific
binding at each concentration of [3H]R1881. All incubates
also contained 1 µM triamcinolone acetonide to suppress
possible binding of R1881 to the progestin receptor. Bound
[3H]R1881 was separated from unbound by gel filtration on
7 x 32 mm Sephadex LH-20 columns (Pharmacia, Baie d’Urfe,
Québec, Canada) at 4 C. Aliquots of incubate were loaded onto the
columns and washed into the column bed with 100 µl TEGDMo. After
sample application, the macromolecular fraction containing bound
[3H]R1881 was eluted into scintillation vials with 400
µl TEGDMo. After overnight extraction into 5 ml Betacount liquid
scintillation fluid (ICN, Montréal, Québec, Canada),
radioactivity was quantified at 50% efficiency. A small aliquot (20
µl) of residual incubation mixture was taken at the end of the
incubation period to determine the actual [3H]R1881
concentration. Specific binding was calculated as the difference
between total binding (measured in the absence of R1881) and
nonspecific binding (measured in the presence of R1881). Binding data
were analyzed by the method of Scatchard using a computer-assisted
nonlinear curve fitting method (LIGAND).
Western blot analysis
Cytosolic proteins (20 µg) were resolved on an 8% SDS-PAGE
gel and transferred to Immobilon-PVDF membrane (Millipore Corp.,
Bedford, MA). The membranes were incubated in TBST blocking solution
[50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.2%
(vol/vol) Tween 20, and 5% powdered skim milk] for 1 h with 1
µg/ml PAR-1 (36) polyclonal rabbit antihuman AR antisera.
Immunoreactive bands were visualized with horseradish
peroxidase-labeled secondary goat antirabbit antisera at 1:10,000
dilution and enhanced chemiluminescence (Amersham Life Science, Inc.,
Oakville, ON) as described by the manufacturer.
Plasmids and transfections
An expression vector with the mouse mammary tumor virus long
terminal repeat (MMTV) promoter linked to the luciferase gene was
transfected into either GT1–7, PC-3(AR)2 or DU145 cells.
Transfections into GT1–7 cells were performed using a modified calcium
phosphate precipitate method containing 15 µg of plasmid DNA (37).
The cells were incubated 12–16 h with the DNA, washed three times with
PBS, and then treated with 1 nM DHT for 24 h.
PC-3(AR)2 or DU145 cells were transfected with 10 µg DNA
and then incubated 5 h in MEM containing 5% FBS, followed by
three PBS rinses, glycerol shocked for 3 min, washed with three PBS
rinses, then treated with 1 nM DHT for 24 h in RPMI
1640 medium with 5% FBS. After harvesting, cells were resuspended in
0.1 ml buffer containing 0.25 mM Tris-HCl, pH 7.4. Protein
extracts were prepared by freeze-thawing as described (38) and protein
concentrations were determined using the Coomasie blue procedure (34).
Extracts (0.05 ml) were added to 10 mM MgCl2,
2.5 mM luciferin and 6 mM ATP and assayed for
luciferase activity using a Berthold LB 9501/16 luminometer (Fisher
Scientific, Unionville, Ontario, Canada). Luciferase values were
normalized to the amount of protein recovered per plate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), as has been reported previously
(31). The same three transcripts were detected in LNCaP cells, whereas
no bands were detected for DU145 cells, and a smaller band, 3.8 kb, was
detected in PC-3(AR)2 cells, as expected (data not shown).
These Northern blots were also probed for ARA70 to determine if the AR
coactivator was also expressed in GT1–7 cells. A single band at 3.6 kb
was observed in GT1–7 and PC-3(AR)2 (Fig. 1B).
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). Two
additional bands corresponding to smaller proteins were also observed
in GT1–7 cells, likely representing AR degradation products. As
expected, no immunoreactive bands were evident in DU145 cells.
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). The data
were obtained from three independent saturation curves.
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). In PC-3(AR)2 cells,
induction of luciferase activity by 1 nM DHT was 22-fold.
No induction was seen upon transfection of this same reporter gene into
DU145 cells, which do not express the AR. Results represent three (for
control cell lines) to five (for GT1–7 cells) independent experiments
performed in triplicate.
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, A and B). GnRH mRNA levels
fell to approximately 45% of basal gene expression at 24–36 h, and
then returned to normal by 48 h. Repression of GnRH mRNA levels
was found to be statistically significant (P 
0.005)
at 24 and 36 h for both 1 and 10 nM DHT treatments
(Fig. 5A). Similar results were obtained when treatment of the cells
was performed using DME medium with either charcoal-stripped or regular
FBS, indicating that endogenous androgen in FBS did not interfere in
our experiments. Vehicle alone, 20 nM ethanol, did not
change GnRH mRNA levels at any time point. Further, upon reprobing the
Northern blots with the 18S cDNA, we found that the 18S and -actin
signals correlated accurately. This indicates that in the GT1–7 cell
line, DHT does not affect -actin mRNA levels.
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). These results demonstrate that the
repression of GnRH synthesis occurs directly through the action of the
AR, and further confirms that the GnRH-secreting GT1–7 neurons are
directly responsive to androgen.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we have found that the GnRH-secreting neuronal cell line, GT1–7, expresses the fully functional AR. The GT1–7 cell line possesses a low, but detectable, level of androgen binding activity (12.4 fmol/mg protein, approximately 3300 ARs per cell) consistent with previous studies in a related clonal GT1–1 cell line (24). The levels of androgen binding activity in other cell types appear to be higher; nevertheless, the binding affinity of the AR expressed in the GT1–7 cells is similar to that reported for other AR-positive cell lines (35). We have shown in transfection analysis that the levels of AR in the GT1 cells are sufficient to significantly induce an androgen responsive reporter gene construct, MMTV-luciferase upon treatment with DHT.
Yeh and Chang (25) have reported that coexpression of ARA70 and ARs in DU145 cells increased, by 10-fold, the transactivational activity of the AR in response to DHT. ARA70 expression in GT1–7 cells may also contribute to the ability of these cells to respond to androgen despite the low level of AR expression. If the levels of AR detected in the GT1–7 cells are representative of those in GnRH neurons in situ, our results may explain why steroid receptors have not yet been detected. The apparent absence of steroid receptors is likely due to the scarcity and scattered distribution of the GnRH neuron itself and/or to limitations in the sensitivity of the detection methods used. Furthermore, it is possible that only subgroups of the GnRH neurons may contain steroid receptors, as is noticed with progestin receptors (8). Thus classical approaches such as immunocytochemistry, which will detect only the cells with the highest levels of receptors, or autoradiography, which often relied on tritium labeled ligands, requiring very thin tissue sections and long exposure times for optimal results, may not have been sensitive enough to detect very low levels of receptors within GnRH neurons. As an example of experimental limitations, estrogen receptors were previously undetectable by standard methods in bone cells. Nevertheless, a low level of receptors, sufficient to mediate a hormonal response but requiring more sensitive methods for detection, has since been demonstrated in these cells (43, 44). Further, the sex steroid environment of the GnRH neuron may also dictate the levels of the respective receptor expressed, as autoregulation of steroid receptor gene expression is common (45, 46). Castration of the animals, frequently performed in steroid sensitivity experiments, may have altered the GnRH or steroid hormone receptor levels as well.
The in vivo studies of hypothalamic GnRH mRNA levels following castration and steroid hormone replacement, as revealed mainly by in situ hybridization, have generally yielded conflicting results. While some studies indicate castration increases GnRH mRNA levels (47, 48, 49), others find no change (50, 51), or even a decrease (52) relative to sham operated controls. With regards to the steroid hormonal regulation of GnRH gene expression, Park et al. (52) reported an increase in GnRH mRNA levels with testosterone treatment, Spratt and Herbison (53) found no effect of DHT, and Toranzo et al. (47) found that DHT decreased GnRH mRNA levels in both sexes, to precastration levels. There are several possible explanations for these discrepancies. Spratt and Herbison (53) effectively argue that, during the first 3–4 weeks post castration, the hypothalamic-pituitary-gonadal axis is in a state of flux, resulting in fluctuations in GnRH synthesis. This would not be surprising given the spectrum of neural systems that are thought to act to regulate the GnRH cell in vivo and the finding that GnRH may act to autoregulate its own synthesis (54). Although a 7-week castration period may be required to equilibriate the system, it has been suggested that the lack of DHT response in these animals may be due to a decreased sensitivity to androgen (53). In comparison, the animals exhibiting a decrease in GnRH mRNA levels upon exposure to DHT were only 2 weeks post castration (47). Another factor to consider with in vivo studies is the anatomical location of the GnRH neuron. Cells within the rostral preoptic area responded to castration with an elevation of GnRH expression, whereas the ones in the medial septum and the diagonal band of Broca remained unchanged by either castration or hormonal treatment (53). This suggests a heterogeneity in the regulation of GnRH gene expression within the GnRH neuronal population in the brain.
It is quite possible that the interaction of the GnRH neuron with other neurons, known to synapse with and effect GnRH neuronal function, could mediate an indirect sensitivity to androgen. Because it was believed that GnRH-secreting neurons did not contain androgen receptors, other neurons, belonging to a wide variety of neuromodulatory systems, emerged as potential sex steroid-sensitive interneurons, able to synapse with and modify the physiology of the GnRH neuron (42). A large number of neurotransmitter and neuropeptides have been implicated in the control of reproductive function, as they have been found to regulate GnRH synthesis and secretion. The presence of steroid receptors within the GnRH neuron does not exclude the possibility that sex steroids may also be acting alone or in combination within the interneurons to modulate the production of their respective signaling molecules necessary for the precise control of GnRH and reproductive function.
All of the above-mentioned factors, contributing to the complex nature of the action of steroid hormones in the hypothalamus, are generally overcome using a homogeneous population of GnRH-secreting neurons, such as the GT1–7 cell line. Further, the in vivo studies cannot yet distinguish if the effect of androgen is mediated directly at the level of the GnRH neuron. On the other hand, one must also consider that the GT1–7 cell line represents a single population of neurons outside of its natural environment and cell contacts, and thus may not respond exactly like a GnRH neuron in vivo. Nevertheless, androgen receptors are found within the GT1–7 cells, indicating that androgen likely contributes to the physiology of the GnRH neuron.
We have demonstrated that GnRH gene expression is repressed upon DHT treatment of GT1–7 neurons. Because there has been some resurgence of the possibility of membrane receptors for steroids within the brain, we needed to determine whether the repression of GnRH mRNA was through the canonical 110K AR protein. We used a specific antagonist of the AR, hydroxyflutamide, to demonstrate that the effect on GnRH gene expression is through the classic nuclear AR. However, these data do not exclude additional effects of androgens mediated through membrane interactions, not necessarily noticed at the level of transcription. We also find that repression of GnRH gene expression subsides between 36–48 h. Although DHT is likely to be rapidly metabolized, we have found that daily replacement of the androgen does not chronically inhibit GnRH gene expression (D.D.B., data not shown). Addition of DHT may be required more often, perhaps at 12-h intervals to maintain suppressed GnRH mRNA levels, or alternatively, there may be a desensitization of the cells to androgen over the 48-h time course.
Like other steroid hormones, androgen receptors are capable of binding specific regions of DNA, termed an androgen responsive element (ARE), within the regulatory region of the gene to modify transcriptional activity. We intend to determine whether repression of GnRH gene expression occurs through a transcriptional mechanism at the level of the GnRH 5' regulatory region. As a classic ARE cannot be found within the promoter of the rat GnRH gene, we postulate that there may be a novel region that confers negative regulation of the GnRH gene within the promoter region (nARE). Kepa et al. (55), in a preliminary study, localized a region within the GnRH promoter that conferred androgen responsiveness in the GT1–7 cells after cotransfection of an expression vector containing the complete coding region of the rat AR. Further studies will be pursued to analyze the GnRH gene promoter region to determine if the repression of GnRH gene expression by DHT occurs at the transcriptional level.
Our study demonstrates that there is indeed a possibility that the GnRH
neuron is directly influenced by androgen in vivo. Using the
immortalized GnRH-secreting GT1–7 cell line, we have determined that
GnRH mRNA levels are significantly down-regulated upon treatment with
DHT. We have found that there are ARs expressed in the GT1–7 neurons
capable of transcriptional activation of a reporter gene, although a
previous study reported that the GnRH neuron did not contain
immunoreactivity for AR (9). The apparent absence of the AR in the
GnRH-secreting neuron may have been due to the increased sensitivity
required to detect low levels of ARs in the GnRH neuron, as seen in
GT1–7 cells. Development of more sensitive techniques may be necessary
for the colocalization of the AR to the GnRH neuron in the rodent
hypothalamus. It has been postulated that the effects of androgens are
linked to their aromatization to estrogens. DHT is not normally
aromatized. Further, it was found that the GT1–1 cells did not have
aromatase activity but did have the necessary 5-reductase enzyme
activity to efficiently convert testosterone to DHT, a more potent
androgen (24). Our results suggest that there must be a specific
receptor for androgen within the GnRH-secreting GT1–7 neurons
responsible for the repression of GnRH mRNA levels. We have also
determined that repression of GnRH gene expression occurs through the
action of the AR using a specific antagonist of AR activity, indicating
that perhaps there are direct effects of androgen on reproductive
function, not requiring aromatization to estrogen for a physiological
response.
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Acknowledgments |
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Footnotes |
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Received September 26, 1997.
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References |
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-aminobutyric acid neurons in the
medial preoptic area are synaptic targets of dopamine axons originating
in anterior periventricular areas. J Neuroendocrinol 5:71–79[Medline]
-, ß, and -actin mRNAs: skeletal but not cytoplasmic
actins have an amino-terminal cysteine that is subsequently removed.
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