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Molecular Endocrinology 16(11):2592–2602
Copyright © 2002 by The Endocrine Society
doi: 10.1210/me.2002-0011
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
Steroid hormones induce rapid membrane receptormediated effects that appear to be separate from
long-term genomic events. The membrane receptormediated effects of androgens on GT1-7 GnRHsecreting neurons were examined. We observed
androgen binding activity with a cell-impermeable
BSA-conjugated testosterone [testosterone 3-(Ocarboxymethyl)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␣-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-3BSA, all caused significant elevations in intracellular
calcium concentrations ([Ca2ⴙ]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
17␤-hemisuccinate BSA. These studies indicate that
androgen can differentially regulate GnRH secretion
and gene expression through specific membranemediated or nuclear mechanisms. (Molecular Endocrinology 16: 2592–2602, 2002)
T
tion is presumed to be mediated through membranebound 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␣-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,
HE 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 acAbbreviations: AR, Androgen receptor; [Ca2⫹]i, intracellular
calcium concentration; ConA-Rhod, concanavalin A labeled
with rhodamine; DHT, 5␣-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␣, retinoid-related orphan receptor;
SDS, sodium dodecyl sulfate; T, testosterone; T-3-BSA, testosterone 3-(O- carboxymethyl)oxime BSA conjugate; T-17BSA, testosterone 17␤-hemisuccinate BSA conjugate.
2592
Shakil et al. • Membrane vs. Nuclear AR Regulation of GnRH
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. 11–14). 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 (15–17). 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
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. 1A). Importantly,
the 110-kDa AR was also recognized by the MA1-150
Mol Endocrinol, November 2002, 16(11):2592–2602
2593
antibody in Western blot analysis of the isolated
plasma membrane fraction of GT1-7 neurons (Fig. 1A).
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. 1A), although exact quantification was difficult
from the Western blot analysis. The blots were
stripped and reprobed with the G␤-antibody, which
recognizes the heterotrimeric G protein ␤-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. 1A).
To verify the purity of the plasma membrane fraction,
we also used a nuclear receptor marker for the transcription factor retinoid-related orphan receptor-␣
(ROR␣). Copious amounts of ROR␣ 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. 1A). 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.
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 ␮M) 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. 1B), 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. 1B). 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. 1B) 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
2594 Mol Endocrinol, November 2002, 16(11):2592–2602
Shakil et al. • Membrane vs. Nuclear AR Regulation of GnRH
Fig. 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␣, a 58-kDa nuclear protein marker, and G␤, 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 ␮m each, stacked for a cross-section of ⬃5 ␮m) to enhance the T-BSA-FITC signal. Magnification, ⫻400.
Bar, 10 ␮m. C, Confocal laser scanning microscopy of GT1-7 cells transfected with an AR-GFP protein. GT1-7 cells were
transfected with 10 ␮g 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 ␮m. Magnification, ⫻630. Bar indicates 10 ␮m.
Shakil et al. • Membrane vs. Nuclear AR Regulation of GnRH
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. 1C), 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
Mol Endocrinol, November 2002, 16(11):2592–2602
2595
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. 2A). In contrast,
forskolin (1 ␮M), a adenylyl cyclase activator, increased cAMP levels approximately 10-fold in the
GT1-7 cell within 15 min (Fig. 2B). When the GT1-7
neuron was exposed to both forskolin and DHT in
combination, the levels of forskolin-stimulated
cAMP accumulation decreased significantly (Fig.
2B). 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. 2B), 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.
Membrane AR Signaling Is Coupled to a Gi
Protein and Increases [Ca2ⴙ]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
Fig. 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 ␮M) with hydroxyflutamide treatment (10 ␮M). 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 forskolininduced accumulation of cAMP by DHT. Results were plotted as the mean ⫾ SEM (n ⫽ 3).
2596 Mol Endocrinol, November 2002, 16(11):2592–2602
Shakil et al. • Membrane vs. Nuclear AR Regulation of GnRH
treatment. Treatment with PTX was able to block the
DHT inhibition of forskolin-stimulated cAMP accumulation in the GT1-7 cells (Fig. 2B). 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. 3). 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. 3A). Overall, peak [Ca2⫹]i achieved were
similar for each androgen, whether a fast or slow response was detected (Fig. 3B). There were no effects
seen with either BSA or vehicle alone.
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. 4). 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.
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-3BSA had no significant effect on GnRH mRNA levels
(Fig. 5).
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 17␤-hemisuccinate (T-17-BSA). The two T-BSA molecules (T-3BSA and T-17-BSA) were incubated with the GT1-7
Fig. 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].
neurons at increasing concentrations (0.01–1 ␮M) for
36 h. There were no significant changes in GnRH
mRNA levels with either T-BSA conjugates at any of
the concentrations used (Fig. 6). These results indicate
that androgen can have distinct effects on GnRH gene
expression and secretion depending upon the location
of the AR when activated.
DISCUSSION
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–
24). Both stimulatory and inhibitory effects of androgens on GnRH secretion patterns have been reported
Shakil et al. • Membrane vs. Nuclear AR Regulation of GnRH
Fig. 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 ␮M,
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].
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 (5–8, 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
Mol Endocrinol, November 2002, 16(11):2592–2602
2597
Fig. 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 ␮g of total GT1-7 mRNA. GnRH and GAPDH
mRNAs are indicated. Film exposure time was 6 h.
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
(6–8, 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
2598 Mol Endocrinol, November 2002, 16(11):2592–2602
Fig. 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 ␮M 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 ␮g of total GT1-7 mRNA.
GnRH and GAPDH mRNAs are indicated. Film exposure time
was 6 h.
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
Shakil et al. • Membrane vs. Nuclear AR Regulation of GnRH
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 (31–33). 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 (36–38). 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-3BSA. 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 mem-
Shakil et al. • Membrane vs. Nuclear AR Regulation of GnRH
brane 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.
Mol Endocrinol, November 2002, 16(11):2592–2602
2599
reprobed with the mouse G␤ (T-20) polyclonal antibody,
which recognizes the four ␤-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␣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 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-3BSA-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.
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 ⫻ 10⫺5 M T-BSA-FITC (Sigma). BSA-FITC and BSA alone
(both at 1.5 ⫻ 10⫺5 M) were used as controls. After two
washing with PBS⫹, cells were then incubated with ConARhod (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 ␮g 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 ⫻400 or ⫻630 (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 ␮m (10–18 sections stacked for a
total of approximately 4–7 ␮m to enhance the T-BSA-FITC
labeling) or 1.0 ␮m (for the AR-GFP experiments) intervals
and evaluated using Adobe Photoshop 5.0 for Windows
(Adobe Systems, Mountain View, CA).
Western Blot Analysis
cAMP Studies
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
␮g/ml aprotinin, pepstatin and leupeptin, 1 mM phenylmethylsulfonylfluoride, 1 mM EDTA] and centrifuged at 770 ⫻ g for
10 min at 4 C. Supernatant was carefully recovered and spun
again at 770 ⫻ g. The resulting supernatant was centrifuged
at 20,800 ⫻ 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 ␮g) 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 ␮g/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
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 ␮M 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 ␮M). 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.
MATERIALS AND METHODS
Cell Culture and Reagents
[Ca2ⴙ]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 ⫻ 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 ␮M acetoxymethylester of the fluorescent Ca2⫹
2600 Mol Endocrinol, November 2002, 16(11):2592–2602
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 (ⱕ10%
variation). GnRH content in 300 ␮l 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 charcoalstripped 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 ␥-actin cDNA (52) or a mouse glyceraldehyde-3phosphate dehydrogenase (GAPDH) cDNA generated by RTPCR, 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 (0.5⫻ 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 ap-
Shakil et al. • Membrane vs. Nuclear AR Regulation of GnRH
propriate 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.
Received January 9, 2002. Accepted July 16, 2002.
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:
[email protected].
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.
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