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INTRODUCTION |
The androgen receptor
(AR)1 belongs to the
superfamily of nuclear receptors that mediates the actions of
lipophilic ligands, including steroids, retinoids, vitamin D3, and
thyroid hormones (1). These receptors have distinct functional domains
that include a carboxyl-terminal ligand binding domain, a highly
conserved DNA binding domain (DBD) comprising two zinc finger motifs,
and a poorly conserved amino-terminal domain that may contain one or
more transcriptional activation domains. Binding of ligand to the
receptor results in activation or transformation such that the receptor
can effectively bind to its specific DNA element. The mechanism of
ligand-induced transformation of the AR is not clear, although it is
known that the conformation of the AR becomes more compact upon ligand
binding, heat shock proteins are dissociated, and dimerization and
phosphorylation occur before DNA binding (2). Thus, the
ligand-activated AR may stimulate or repress androgen-regulated genes.
However, it has been suggested that the AR can also be transformed in
the absence of androgen by elevation of cAMP levels and by growth
factors (3-5). The mechanism of such ligand-independent activation of
AR has not been clarified but may involve the bypassing of one of the
above-mentioned processes associated with ligand-dependent
transformation. Of these, phosphorylation has been implicated in the
ligand-independent activation of the progesterone, estrogen, and
retinoic acid receptors. On the other hand, although there are three
identified phosphorylation sites on the AR, its phosphorylation does
not appear to be essential for the induction of androgen-regulated
genes (6).
Prostate-specific antigen (PSA) is a clinically important
androgen-stimulated gene that is used to monitor treatment responses, prognosis, and progression in patients with prostate cancer. The transcriptional regulation of PSA is initially androgen-regulated and
undergoes a sharp decline after medical or surgical castration (7).
When the tumor becomes androgen-independent, PSA mRNA is
constitutively up-regulated through an unknown mechanism that presumably involves the promoter and enhancer regions of the PSA gene.
These regions have been sequenced as far as
5824 from the start site
of transcription (8, 9), and the following DNA response elements have
been characterized: 1) TATA box,
28 to
23 (10); 2) androgen
response elements (AREs),
170 to
156 (10) and
4148 to
4134 (9);
and 3) androgen response region (ARR),
395 to
376 (11). The fact
that PSA production ultimately increases in an androgen-deprived
environment suggests that other factors not directly related to
androgens but possibly acting via the AR become paramount, leading to
androgen-independent induction of PSA gene expression.
In the present study, the possibility that androgen-independent
induction of PSA gene expression by cross-talk between the AR and PKA
signal transduction pathways was investigated in prostate cancer cell
lines. The experiments confirmed that PSA gene expression was induced
by activation of PKA and demonstrated, for the first time, activation
of the amino terminus of the AR by stimulation of PKA activity.
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MATERIALS AND METHODS |
Cell Culture--
All chemicals were purchased from Sigma,
unless stated otherwise. PC3 cells between the 30th and 45th generation
were maintained in Dulbecco's modified Eagle's medium supplemented
with 5% fetal bovine serum (Life Technologies, Inc.). LNCaP cells
between the 44th and 55th generation were maintained in RPMI 1640 supplemented with 5% fetal bovine serum. When the plates or wells were
60-70% confluent with cells, the culture medium was changed to
serum-free medium containing vehicle (Me2SO), R1881, or forskolin.
Northern Blot Analysis--
Total RNA was extracted from LNCaP
cells with Trizol® (Life Technologies, Inc.) and
fractionated by electrophoresis before blotting onto
Hybond-N+ filters (Amersham Pharmacia Biotech). The
1.4-kilobase pair EcoRI fragments of the PSA cDNA and 18 S RNA were labeled with [
-32P]dCTP by Random Primers
DNA labeling kit (Life Technologies, Inc.). Hybridization was performed
according to the method described by Sato et al. (7). The
mRNA bands were quantified with the STORM 860 PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
PSA Promoter Plasmid Constructs--
PSA 5'-flanking DNA
(
630/+12) was obtained by polymerase chain reaction-mediated
amplification of human genomic DNA using oligonucleotide primers
corresponding to the PSA gene and ligated with
EcoRV-digested pBluescript (pBS) sk(
) (Stratagene, La
Jolla, CA) as described previously (12).
Other Plasmids--
The expression plasmid for full-length
wild-type human AR was a gift from Dr. A. O. Brinkman (Erasmus
University, Rotterdam, The Netherlands), and the expression plasmid for
full-length wild-type rat AR has been described (12).
ARR3-tk-luciferase reporter construct consists of three
congruent rat probasin AREs (
244 to
96) ligated in tandem into the
HindIII site of the pT81 luciferase vector (ATCC, Manassas,
VA) (13). The PB-luciferase reporter (
286/+28) was developed by Snoek
et al. (14). The AR1-559Gal4 plasmid was
constructed by polymerase chain reaction amplification of the
nucleotides 363 to 2039 of the human AR cDNA using primers 5'-AAA
GGA TCC GGA TGG AAG TGC AGT TAG GGC T and 5'-AAA AGG ATC CTT CAG GTC
TTC TGG GGT GGA AAG TAA TAG. The amplified DNA fragment was purified,
blunt-end ligated into the EcoRV site of Bluescript SK(
),
excised with BamHI, and cloned into the BamHI
site of pFA-CMV plasmid (Stratagene Cloning System, La Jolla,
CA). The orientation and sequences were confirmed by DNA sequence
analysis, and the expressed protein was detected by Western blotting.
The expression vector for the catalytic subunit of protein kinase A
(PKAc) was purchased from Stratagene (PathDetect CREB trans-Reporting System).
Transient Transfections and Luciferase Activity Assay--
LNCaP
cells (3 × 105) were plated on 6-well plates and
incubated in RPMI 1640 with 5% fetal bovine serum before transfection as described previously (12). The total amount of plasmid DNA used was
normalized to 3 µg/well by the addition of empty plasmid. Medium was
replaced after 24 h by serum-free RPMI 1640 containing Me2SO, R1881, or forskolin. Cells were collected after
48 h of incubation. Luciferase activities in cell lysates were
measured using the Dual Luciferase assay system (Promega, Madison, WI). The protein concentration of the cell lysates was determined by the
method of Bradford (15). Luciferase activities were normalized by the
Renilla activities and protein concentrations of the samples. The
results are presented as the fold induction, which is the relative
luciferase activity of the treated cells over that of the control
cells. All transfection experiments were carried out in triplicate
wells and repeated 2-8 times using at least 2 sets of plasmids
prepared separately.
Immunoblots--
LNCaP cells were incubated in RPMI 1640 (serum-free) for 24 h before the addition of vehicle (0.01%
Me2SO), 10 nM R1881, or 1 µM
forskolin. After incubation with compounds, whole cell lysates and
nuclear extracts were prepared (16). Western blots were performed with
40 µg of total protein/lane. Immunoblots were blocked overnight in 5% milk (w/v) in 20 mM Tris-HCl, pH 7.4, containing 500 mM NaCl (TBS). Blots were incubated for
4 h with antibodies to the AR (2 µg/ml) (PA1-111A, Affinity
Bioreagents Inc, Golden, CO). The blots were washed and incubated for
1 h with the secondary antibody (1:5000). Antibodies were diluted
in 5% milk, TBS solution. AR protein was detected using the ECL
luminescence kit (Amersham). Densitometric analyses of protein bands
from scanned x-ray films were performed using the Personal Densitometer
(Molecular Dynamics).
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts from LNCaP cells were used for EMSA studies. Nuclear extracts
were prepared from cells (17) that had been treated with 10 nM R1881 or 1 µM forskolin for 3 h. DNA
binding reactions were carried out with 10 µg of total protein from
nuclear extracts in a total volume of 60 µl containing DNA binding
buffer (10 mM HEPES, pH 7.9, 10% (v/v) glycerol, 100 mM KCl, 1 mM EDTA, 5 mM
MgCl2, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, and 2 µg of poly(dI-dC) (Amersham
Pharmacia Biotech) with approximately 1.5 fmol of double-stranded
32P-labeled PSA-ARE oligonucleotide
(5'-TTGCAGAACAGCAAGTGCTAGCTC-3'), or PSA mutant ARE
(5'-TTGCAAAAAAGCAAGTGCTAGCTC-3'). Protein-DNA complexes were separated under nondenaturing conditions in a 4% polyacrylamide gel (29:1) containing 2.5% glycerol and run in 0.5 × TBE (1× = 89 mM Tris-borate, 89 mM boric
acid, and 2 mM EDTA, pH 8.3) at 200 V. Protein-DNA
complexes were quantified with the STORM 860 PhosphorImager (Molecular Dynamics).
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RESULTS |
Effect of Forskolin on the Levels of PSA mRNA in LNCaP
Cells--
Forskolin activates adenylyl cyclase to synthesize cAMP,
which in turn stimulates PKA activity. Taking advantage of this effect, experiments were undertaken to determine whether PSA gene expression could be elevated in the absence of androgen by forskolin-induced activation of the PKA signal transduction pathway. To this end, LNCaP
cells were employed because these cells express endogenous AR and PSA.
As shown in Fig. 1A, when
LNCaP cells were exposed for 16 h to various concentrations of
forskolin (0.1 to 50 µM), the maximum induction of PSA
mRNA was achieved using a concentration of 1 µM.
Concentrations of forskolin in excess of 10 µM had little to no effect on PSA mRNA levels when compared with those in
controls, which is in agreement with other reports (18, 19). As shown in Fig. 1B, the optimal induction of PSA mRNA by
forskolin (1 µM) was comparable with that achieved with
the synthetic androgen, R1881. A mixture of R1881 and 1 µM forskolin resulted in an additive increase in the mean
levels of PSA mRNA; however, this change was not statistically
significant (Student's t test, p > 0.05). A mixture of R1881 with higher concentrations of forskolin (>10 µM) reduced PSA mRNA to levels below those of R1881
alone (data not shown), which is consistent with a previous report
(19).

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Fig. 1.
Northern blot analysis of PSA mRNA
isolated from LNCaP cells. A,
dose-dependent increases of PSA mRNA in cells treated
with various concentrations of forskolin (0.1 to 50 µM)
for 16 h. B, additive induction of PSA mRNA in
LNCaP cells treated with forskolin (FSK, 1 µM), R1881 (10 nM), or a mixture of the two
compounds for 16 h. C, time course of PSA mRNA
induction by forskolin (FSK, 1 µM) and R1881
(10 nM). RNA bands corresponding to PSA at 1.5 kilobase
pairs were quantified by scanning with a PhosphorImager, and values
represent the means ±S.E. of three to six separate experiments.
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The results of a time-course study of PSA mRNA in LNCaP cells (Fig.
1C) demonstrated that the optimal time for maximum induction was between 8 and 16 h after the addition of forskolin; after that, the levels of PSA mRNA decreased. In contrast, the induction of PSA mRNA by R1881 continued to increase for the duration of the
experiment (48 h). The transient increase in PSA mRNA induced by
forskolin, as compared with the increase obtained with R1881, may
reflect the half-lives of these compounds. R1881 is considered to be a
poorly metabolized compound, whereas forskolin is relatively labile.
Induction of Reporter Constructs Containing AREs--
To check
whether the induction of PSA mRNA may involve changes in the
activity of the PSA promoter as opposed to changes in post-transcriptional regulation, LNCaP cells were transfected with the
PSA (
630/+12) promoter-luciferase reporter plasmid. This region of
the PSA promoter contains both the ARE and ARR regions required for
androgen induction (10, 11). After transfection, LNCaP cells were
incubated in the presence of R1881 or forskolin. At the optimal
concentration of R1881 (10 nM) (12), PSA luciferase activity was increased 5-fold (Fig.
2A). In comparison, the
optimal concentration of forskolin (50 µM) increased PSA
luciferase activity by 70-fold. Elevation of PKA activity by
transfection of cells with an expression vector encoding the catalytic
subunit of PKA (pPKAc) resulted in a greater than 200-fold induction of
PSA luciferase activity. These results show that the induction of PSA
by activation of PKA is mediated, at least in part, through the
630
to +12 region of the PSA promoter.

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Fig. 2.
Induction of the activities of
androgen-responsive promoters by forskolin and PKA. LNCaP cells
were transfected with 1.0 µg of either PSA ( 630 to +12)
(A)-, PB (B)-, or ARR3-tk-luciferase
reporter constructs (C) and either with or without with the expression
vector for the catalytic subunit of PKA (PKAc, 0.5 µg). Cells were
incubated with forskolin (FSK, 50 µM), R1881
(10 nM), or vehicle (Me2SO, 0.5%) for 48 h under serum-free conditions and harvested, and luciferase activities
were measured. The normalized luciferase activities were divided by the
normalized activity of cells transfected with reporter plasmid and with
empty PKAc plasmid to give the fold induction.
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To determine whether other reporter constructs that contain AREs could
be induced by forskolin, two additional reporters were tested in LNCaP
cells. The first of these was the probasin promoter (PB-luciferase), a
naturally occurring androgen-regulated promoter from the rat. As shown
in Fig. 2B, the PB-luciferase reporter construct was induced
65-fold by R1881, 35-fold by forskolin, and greater than 80-fold by
overexpression of pPKAc. The second of these reporters was the
ARR3-tk-luciferase, which is an artificial reporter
construct that contains three repeats of the rat probasin ARE1 and ARE2
region ligated in tandem with a luciferase reporter (13). Transfection
of the ARR3-tk-luciferase reporter construct yielded very
different results to those obtained with the PSA and PB reporters.
Although R1881 induced ARR3-luciferase activity by
233-fold, forskolin and pPKAc proved to be very poor inducers of this
construct, each resulting in a very small increase in activity (Fig.
2C). The differences between R1881 and forskolin/PKA in the
induction of reporter constructs containing AREs demonstrates promoter specificity.
The Role of the AR in Forskolin Induction of Reporter Constructs
Containing AREs in PC3 Cells--
PC3 cells are of human prostate
cancer origin but are poorly differentiated and do not express AR or
PSA. Therefore, by using PC3 cells, it was possible to define the role
of AR in the forskolin induction of ARE-containing promoters more
precisely. The results in Fig. 3
(upper ABC panels) show that AR-deficient PC3 cells could
not be induced by forskolin to increase the activities of PSA-, PB-,
and ARR3-luciferase reporters. However, when these cells
were transfected with an expression vector for wild-type human AR,
PSA-luciferase activity was induced 3.5-fold by forskolin (Fig. 3,
lower panel A). Induction of the PSA-reporter construct by
R1881 requires greater than 24 h and was therefore not included. As shown in the results in Fig. 3, lower panel B,
PB-luciferase activity was induced 5.2-fold by R1881 and 2.3-fold by
forskolin in PC3 cells transfected with AR. Fig. 3, lower panel
C, shows that ARR3-luciferase activity was induced
73-fold by R1881 and only 2.9-fold by forskolin. The fact that all
three of these promoters were induced by forskolin only in the presence
of transfected wild-type human AR demonstrates an
AR-dependent mechanism.

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Fig. 3.
Forskolin induction of the activities of
androgen-responsive promoters in PC3 cells requires the AR. Cells
were transfected with PSA-luciferase (1.0 µg) (A),
PB-luciferase (1.0 µg) (B), or
ARR3-tk-luciferase (1.0 µg) reporter constructs
(C) in the absence (upper panel) or presence
(lower panel) of the expression vector for the human
wild-type AR (0.5 µg) for 5 h and then incubated with forskolin
(FSK, 50 µM), R1881 (10 nM), or
vehicle (Me2SO, 0.5%) for an additional 16 h under
serum-free conditions. The normalized luciferase activities were
divided by the normalized activity of cells transfected with reporter
plasmid and exposed to Me2SO (Control) to give
the fold induction.
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Effect of Forskolin on Whole Cell and Nuclear Levels of AR
Protein--
To test the possibility that forskolin may alter the
cellular levels and/or localization of the AR, whole cell and nuclear levels of AR protein were determined by Western blots. LNCaP cells were
incubated with Me2SO, R1881, or forskolin, and then whole cell lysates and nuclear extracts were prepared from cells harvested at
different time points. When cells were exposed to forskolin, whole cell
levels of AR remained relatively constant for the 50-h duration of the
experiment (Fig. 4A). In
androgen-treated cells, there was a transient increase in whole cell
levels of AR, which returned to control levels after 24 h. The
increased levels of AR protein in extracts prepared from cells exposed
to R1881 may reflect stabilization of the protein in the presence of
ligand (20, 21).

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Fig. 4.
Cellular localization of the AR in LNCaP
cells treated with forskolin. A, AR protein levels in
whole cell lysates; B, nuclear extracts prepared from cells
exposed to vehicle (0.01% Me2SO), forskolin
(FSK, 1 µM), or R1881 (10 nM) and
analyzed by Western blotting using an antibody raised against the amino
terminus of the human AR. Protein bands detected at 110-112 kDa were
scanned by laser densitometry and plotted as fold induction (divided by
the protein band obtained for vehicle-treated cells). Samples were
harvested at various time points to a maximum of 48 h after the
addition of compounds to the cells. Values represent the means ±S.E.
of three or four separate experiments.
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Nuclear extracts prepared from cells exposed to forskolin demonstrated
a transient increase in nuclear levels of AR protein (Fig.
4B). After 3 h, forskolin-treated cells were
characterized by a 5-fold increase in nuclear AR protein compared with
control levels. R1881 increased nuclear levels of AR protein within 90 min after its addition to the cells. The nuclear level of AR protein at
3 h was 38-fold higher in R1881-treated cells than in control cells analyzed at the same time point. In the continuing presence of
R1881, nuclear levels of AR remained elevated in LNCaP cells for at
least 48 h.
Inhibitory Effect of Bicalutamide on the Induction of PSA mRNA
by Forskolin--
The induction of PSA by forskolin appears to be
dependent on the presence of the AR, as implied by the above
experiments. To further test the role of the AR in this mechanism, the
antiandrogen, bicalutamide, was employed. As shown in the Northern blot
in Fig. 5, preincubation of LNCaP cells
with bicalutamide blocked the induction of PSA mRNA by forskolin
(compare lane 5 to lane 6). Induction of PSA
mRNA by R1881 was also prevented by bicalutamide, as expected
(compare lane 2 to lane 4). These results
demonstrate that the induction of PSA mRNA by forskolin requires a
functional AR.

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Fig. 5.
Inhibitory effect of bicalutamide on the
induction of PSA mRNA by forskolin. LNCaP cells were
preincubated with bicalutamide (BIC, 100 µM)
for 2 h before the addition of forskolin (FSK, 1 µM), R1881 (10 nM), or vehicle
(Me2SO, 0.01%) and then incubated for an additional
16 h. At the end of the incubation period, cells were harvested,
RNA was isolated, and Northern blots were performed using radiolabeled
PSA probes. Each lane contains 20 µg of total RNA.
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Inhibitory Effect of Bicalutamide on the Forskolin Induction of
Reporter Constructs Containing AREs--
To clarify whether the
induction of transfected androgen-responsive reporter constructs (PSA,
PB, and ARR3) by forskolin was dependent upon the presence
of a functional AR, LNCaP cells were preincubated with bicalutamide.
Because lengthy exposure of LNCaP cells to bicalutamide causes cell
death, a relatively short 24-h incubation period was employed with no
evidence of cytotoxicity (data not shown). As shown in Fig.
6A, PSA-luciferase activity was induced approximately 70-fold by forskolin, and this induction was
blocked (approximately 80%) by preincubation of cells with bicalutamide. Results with R1881 are not shown because no induction was
observed with this construct at 24 h. The induction of PB- (Fig.
6B) and ARR3-luciferase (Fig. 6C)
reporters by R1881 was completely blocked (100%) by bicalutamide,
whereas forskolin induction of these reporters was only blocked by 80%
with bicalutamide. Thus, bicalutamide blocks the forskolin-induced
luciferase activities of ARE-containing reporters, consistent with a
role for the AR in this mechanism.

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Fig. 6.
Inhibitory effect of bicalutamide on the
induction of PSA-, PB-, and ARR3tk-luciferase
activities by forskolin. LNCaP cells were transfected with PSA
(1.0 µg) (A)-, PB (1.0 µg) (B)-, or
ARR3tk-luciferase (1.0 µg) (C)- reporter
constructs for 24 h and then preincubated with bicalutamide
(BIC, 50 µM) for 2 h before the addition
of forskolin (FSK, 50 µM), R1881 (10 nM), or vehicle (Me2SO, 0.5%) and then
incubated for an additional 24 h under serum-free conditions. The
normalized luciferase activities were divided by the normalized
activity of cells transfected with reporter plasmid and exposed to
Me2SO (control) to give the fold-induction.
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Forskolin Increases AR·ARE Complex Formation--
To determine
whether forskolin increases DNA binding activity of AR protein to the
PSA ARE, EMSAs were employed using radiolabeled oligonucleotides of the
PSA-ARE promoter with nuclear extracts from LNCaP cells. Nuclear
extracts from cells treated for 3 h with forskolin (maximum
nuclear levels of AR protein) showed an increase in AR·ARE complex
formation (Fig. 7, lane 2)
compared with control (lane 4). In comparison, cells treated
with R1881 (lane 3) also had an increase in AR·ARE complex
formation compared with control (lane 4). As expected,
substitution with a mutated PSA-ARE showed an 85-90% decrease in
complex formation with the nuclear extracts prepared from
forskolin-treated (lane 6), R1881-treated (lane
7), and control cells (lane 8). These results show that forskolin increases the DNA binding activity of the AR.

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Fig. 7.
Formation of AR·ARE complexes in the
presence of nuclear extracts from LNCaP cells treated with
forskolin. EMSA were performed using radiolabeled PSA-ARE
oligonuclotides with nuclear extracts isolated from LNCaP cells
incubated with compounds for 3 h. Lanes 1 and
5, no nuclear protein, only bovine serum albumin (10 µg);
lanes 2 and 6, forskolin (1 µM);
lanes 3 and 7, R1881 (10 nM);
lanes 4 and 8, control (Me2SO).
Lanes 1-4, PSA-ARE; lanes 5-8, mutated PSA-ARE.
Protein-DNA complexes were quantified by scanning with a
PhosphorImager.
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PKA Activates the Amino-terminal Domain of the Human AR--
There
are two potential PKA phosphorylation sites on the human AR, and both
of these reside within the amino-terminal domain (amino acids 16 and
213). Therefore, the amino-terminal domain of the AR may be the target
of PKA for the induction of ligand-independent activation. To test this
hypothesis, amino-terminal fragments of the human AR were cloned into
the carboxyl terminus of Gal4 DBD. Expression vectors for these
chimeric proteins were cotransfected into LNCaP cells with a reporter
gene containing the Gal4-binding site as cis-acting elements
(p5xGal4UAS-TATA-luciferase). As shown in Fig.
8, R1881 did not significantly change the
activity of this reporter when comparing the
AR1-559Gal4DBD (lane 4) to that of Gal4DBD
lacking the amino terminus of the AR (lane 3). In contrast,
upon the addition of forskolin, the Gal4 DBD fused to residues 1-559
of the human AR, activating the Gal4-luciferase reporter 60-fold
(lane 6) over levels achieved with the Gal4 DBD lacking the
amino terminus of the AR (lane 5). These results indicate
that the amino-terminal domain of the AR is targeted by the PKA
pathway.

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Fig. 8.
Activation of the amino terminus of the AR by
forskolin. Transactivation assays with human
AR1-559-Gal4 DBD chimera and the Gal4 reporter construct.
Transactivation assays were performed in LNCaP cells transfected with
the 5xGal4UAS-TATA-luciferase reporter, Gal4DBD, and
AR1-559-Gal4 DBD and exposed to R1881 or forskolin.
Lanes 1-2, control (Me2SO, 0.5%); lanes
3-4, R1881 (10 nM); lanes 5-6, forskolin
(50 µM); lanes 1, 3, and
5, Gal4 DBD (0.5 µg); lanes 2, 4,
and 6, AR1-559-Gal4 DBD (0.5 µg); lanes
1-6, 5xGal4UAS-TATA-luciferase reporter (1.0 µg).
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DISCUSSION |
Steroid hormone receptors are considered ligand-activated
transcription factors. However, recent evidence shows that the human estrogen and the chicken, rat, and rabbit progesterone receptors can
mediate extracellular signals in the absence of cognate ligand by
dopamine, epidermal growth factor, heregulin, gonadotropin-releasing hormone, tumor growth factor
, insulin and insulin-like growth factor I, cAMP, okadaic acid, and vanadate (22-26). Other human steroid hormone receptors such as the glucocorticoid, progesterone, and
mineralocorticoid receptors, apparently are not activated in the
absence of ligand by these compounds (27, 28).
Recently, it has been suggested that the human AR can also be activated
in the absence of its cognate ligands by insulin-like growth factor I,
keratinocyte growth factor, epidermal growth factor, and compounds that
elevate cAMP (4, 5). However, induction of PSA gene expression by
androgen-independent activation of the AR in human prostate cancer
LNCaP cells has not been previously reported. Therefore, the present
studies investigated androgen-independent induction of PSA gene
expression via cross-talk between the AR and PKA signal transduction
pathways, and these studies revealed the following: 1) PSA gene
expression is induced by activation of PKA; 2) PKA induction of
androgen-responsive reporter genes is promoter-specific; 3) induction
of PSA gene expression by PKA requires a functional AR; and 4) PKA
appears to target the amino terminus region of the AR.
Forskolin induction of PSA gene expression in LNCaP cells was shown by
both Northern blot and the PSA reporter gene construct. The transient
induction of PSA mRNA by activation of PKA using forskolin was
shown to be dose-dependent, with the optimal concentration at 1 µM. At higher concentrations of forskolin, the
levels of PSA mRNA did not plateau, but rather these concentrations
resulted in a decrease in the induction of PSA mRNA. Forskolin
concentrations of 10 µM or higher did not induce PSA
mRNA above control levels. High concentrations of forskolin also
decreased induction of PSA mRNA by R1881. This lack of induction of
PSA mRNA at high concentrations of forskolin could not be explained
by cytotoxic effects. However, these results are consistent with the
report of Blok et al. (19), showing that a high
concentration of forskolin decreases the phosphorylation of AR, thereby
attenuating its DNA binding activity.
In agreement with the results showing the induction of PSA mRNA by
forskolin, PSA reporter activity was also induced by forskolin and
overexpression of PKAc in LNCaP cells. The PB-luciferase reporter was
also induced by forskolin in LNCaP and PC3 cells that were transfected
with wild-type human AR. Curiously, the powerful androgen-responsive ARR3-tk-luciferase reporter construct, which contains three
repeats of the probasin ARE1 and ARE2, was poorly induced by forskolin and PKAc relative to R1881. One explanation for this weak induction may
be a requirement of different factors for the thymidine kinase minimal
promoter, as compared with the PSA and PB natural promoters.
Evidence that forskolin activates the AR to induce PSA gene expression
is based on the following: 1) the induction of PSA gene expression by
forskolin in LNCaP cells was blocked by bicalutamide; 2) the induction
of PSA and other ARE reporter gene constructs by forskolin or PKAc in
PC3 cells did not occur in the absence of transfected human AR; and 3)
an increase in AR-ARE complex formation was observed using nuclear
extracts from cells treated with forskolin.
Bicalutamide is an antiandrogen that prevents dissociation of the heat
shock protein complex from the AR, thereby preventing DNA binding
activity and possibly AR nuclear translocation (29-31). It has been
employed in numerous studies to determine the role of the AR in the
activation of androgen-responsive reporter gene constructs (4, 5). In
this study, application of bicalutamide blocked forskolin induction of
PSA mRNA and androgen responsive reporter activities. These results
show that the induction of PSA gene expression by forskolin is
dependent upon a functional AR and imply cross-talk between the AR and
PKA signal transduction pathways.
Further evidence that the AR is required for forskolin induction of
reporter gene constructs containing AREs can be drawn from studies
using the PC3 cell line. These cells are devoid of AR and therefore
provide a good model for studying the requirement of AR in the
induction of these reporters by forskolin. In the absence of
transfected AR, forskolin induction of PSA-, PB-, and ARR3-luciferase activities did not occur. However, if the
wild-type human AR was expressed in these cells, forskolin was able to
induce these reporters. These data are consistent with the conclusion that forskolin activates the AR to induce androgen-responsive genes. In
addition, these data demonstrate that the induction of PSA gene
expression in LNCaP cells is not unique to the mutated AR endogenously
expressed in this cell line (32).
If the AR is activated by forskolin, an increase in AR-ARE complex
formation should be observed. Application of EMSA confirmed that indeed
an increase in AR-ARE complex formation did occur when nuclear extracts
from LNCaP cells treated with forskolin were used together with PSA-ARE
oligonucleotides. Unexpectedly, there was more AR-ARE complex formation
when using nuclear extracts from forskolin-treated cells than from
R1881-treated cells. This was not anticipated because the data in Fig.
4B shows a 40-fold increase in nuclear levels of AR protein
in R1881-treated cells, as opposed to a 5-fold increase in nuclear AR
levels in forskolin-treated cells. Although AR DNA binding activity is
dependent on the amount of AR present in the assay, other factors are
also crucial for DNA binding activity. Thus, it is conceivable that
forskolin-transformed AR may have a greater affinity for the PSA-ARE
than the AR activated by R1881. Such enhancement of affinity may result
from differential interactions with other proteins or modulation of the
receptor that may include a change in its phosphorylation state.
Upon establishing that forskolin elevates PSA gene expression by an
AR-dependent pathway, the next step was to map what region of the AR was targeted. To do this, chimeric fragments of the AR
receptor fused to the DBD of the Gal4 protein, together with the Gal4
reporter system, were employed (Fig. 8). These data provide the first
demonstration of activation of the amino-terminal domain of the AR by
stimulation of the PKA signal transduction pathway. In vivo,
the transcriptional activation of the AR requires the AF-1 in the
amino-terminal domain (28, 33-37), which has been mapped to two
discrete overlapping regions between amino acids 110 to 379 and 369 to
494 (33, 36). Although distinct transactivation regions may be active
for specific responsive promoters, ligand-dependent versus ligand-independent activation of the AR also may
target different transactivation regions. This would result in
recruitment of different co-activators or altered interactions with the
basal transcription machinery. Studies examining ligand-independent activation of the estrogen receptor by growth factors and PKA have
determined that epidermal growth factor (38) and insulin growth factor
(39) act primarily by means of the transactivation domain AF-1 (in the
amino terminus), whereas PKA acts through the transactivation domain
AF-2 (in the carboxyl terminus) (38). Hence, the estrogen receptor can
be activated through three signaling molecules, estradiol, cAMP, and
growth factors, each acting through a different discrete domain.
Although this study shows forskolin activation of the amino terminus of
the AR in LNCaP cells, further experiments are in progress to map the
precise site.
Whether forskolin/PKA directly alters the phosphorylation state of the
amino-terminal domain of the AR is not known. There is reason to
believe that a change in the phosphorylation state may be involved
because this domain contains numerous potential serine phosphorylation
sites for not only PKA but also for mitogen-activated protein kinase,
DNA-dependent protein kinase, protein kinase C, casein
kinase II, and serine-proline-directed kinase (40). Such a mechanism
could potentially affect the ability of the receptor to dissociate from
the heat shock proteins, shuttle to and from the nucleus, bind DNA,
interact with other transcription factors, or activate particular
responsive genes. However, early work examining AR phosphorylation
strongly indicates that PKA does not directly alter the total
phosphorylation state of the AR (41). In addition, although there are
different phosphorylated isoforms of the AR (21, 42, 43) and
phosphorylation increases with ligand (44, 45), AR transactivation does
not appear to be affected by phosphorylation. Therefore, a more likely
scenario may involve the changes in the phosphorylation state of a
co-regulator or another protein factor that binds to the amino-terminal
domain of the AR, thereby increasing AR transactivation activity. Such
a hypothesis may also help to explain the promoter specificity of AR
that is activated by forskolin seen in Fig. 2.
In summary, the data presented here provides evidence that the amino
terminus of the AR can be activated by PKA or elevation of cAMP by
forskolin in LNCaP cells to initiate transcription of some genes
containing AREs, such as PSA. Identification of such a mechanism may be
of critical importance in the understanding of the molecular changes
that are involved in the progression of prostate cancer to androgen
independence. Through mapping of the site on the AR that is targeted by
PKA, new approaches to averting androgen-independent prostate cancer
might be developed.