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J. Biol. Chem., Vol. 277, Issue 9, 7076-7085, March 1, 2002
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From the Department of Cancer Endocrinology, British Columbia
Cancer Agency, Vancouver, British Columbia V5Z 4E6, Canada
Received for publication, August 27, 2001, and in revised form, December 17, 2001
The androgen receptor (AR) is a ligand-activated
transcription factor that mediates the biological responses of
androgens. However, non-androgenic pathways have also been shown to
activate the AR. The mechanism of cross-talk between the interleukin-6 (IL-6) and AR signal transduction pathways was investigated in LNCaP
human prostate cancer cells. IL-6 induced several androgen-response element-driven reporters that are dependent upon the AR, increased the
phosphorylation of mitogen-activated protein kinase (MAPK), and
activated the AR N-terminal domain (NTD). Inhibitors to MAPK and JAK
decreased the IL-6-induced phosphorylation of MAPK and activation of
the AR NTD. Immunoprecipitation and transactivation studies showed a
direct interaction between amino acids 234-558 of the AR NTD and STAT3
following IL-6 treatment of LNCaP cells. These results demonstrate that
activation of the human AR NTD by IL-6 was mediated through MAPK and
STAT3 signal transduction pathways in LNCaP prostate cancer cells.
Interleukin-6 (IL-6)1
was originally identified as a T cell-derived cytokine that induces
terminal differentiation of B cells into antibody-producing cells (1,
2). IL-6 is a multifunctional cytokine that plays an important role in
the regulation of hematopoiesis, immune response, inflammation, bone
metabolism, and neural development (3). IL-6 is produced by lymphoid,
non-lymphoid cells, and cancer cells (4). IL-6 can increase the
proliferation of some cancer cells, including prostate (5, 6), and is
considered to be an autocrine or a paracrine growth factor (7-9).
The receptor for IL-6 is composed of an IL-6 The androgen receptor (AR) is a ligand-mediated transcription factor
that belongs to the superfamily of nuclear receptors (21). These
receptors have similar structures that are composed of an N-terminal
domain (NTD) that is involved in transcriptional activation, a
DNA-binding domain (DBD), a hinge region, and a ligand-binding domain.
After the ligand binds to AR, the ligand-receptor complex translocates
to the nucleus and binds specific androgen-response elements (AREs) on
the chromosome (22). The AR can also be activated in the absence of its
cognate ligand by signaling pathways initiated by various growth
factors (23-26) and stimulation of protein kinase pathways (27, 28).
The AR has been suggested to regulate the expression of at least 60 genes in the rat prostate (29). An example of expression of a human
gene that is up-regulated by androgens in the prostate is
prostate-specific antigen (PSA).
PSA is a member of the serine protease family and is expressed in the
epithelium of "normal" prostate tissue and benign prostatic hyperplasia, prostate cancer specimens, and the LNCaP prostate cancer
cell line (30). The expression of the PSA gene is regulated at the
transcriptional level by androgen through several well characterized
AREs (31). However, in the absence of androgens, PSA has been shown to
become elevated in prostate cancer cells maintained in monolayer and
in vivo (for a review see Ref. 32). The mechanisms that
regulate the gene expression of PSA in the absence of androgen are
still unclear. Based upon these findings, we examined previously
suspected growth factors for their ability to induce PSA gene
expression prior to delineating a possible underlying mechanism. Here
we identify for the first time that the human AR NTD is activated by
IL-6 by a mechanism that is dependent upon mitogen-activated protein
kinase (MAPK) and STAT3 signal transduction pathways in LNCaP prostate
cancer cells.
Cell Culture and Materials--
Human prostate cancer LNCaP
cells were maintained in RPMI 1640 supplemented with 5% (v/v) fetal
bovine serum (FBS) (Invitrogen), penicillin (100 units/ml), and
streptomycin (100 µg/µl) at 37 °C in an atmosphere of 5%
CO2 in the air. All chemicals were purchased from Sigma,
unless stated otherwise. Insulin-like growth factor (IGF)-I,
keratinocyte growth factor (KGF), bovine serum albumin, and protease
inhibitor mixture tablets (CompleteTM) were obtained from
Roche Molecular Biochemicals. Epidermal growth factor (EGF) was
purchased from Invitrogen. IL-6 was obtained from R & D Systems
(Minneapolis, MN). The nonsteroidal antiandrogen bicalutamide was
kindly supplied by Dr. Mark Zarenda (Zeneca). Rp-(8-Br-cAMPs) and AG490 were obtained from
Calbiochem. U0126 was from Promega (Madison, WI).
Plasmids--
The human AR cDNA was a kind gift from A. O. Brinkman (Erasmus University, Rotterdam, The Netherlands). The
following plasmids have been described previously: PSA
( Transfection and Luciferase Assay--
LNCaP cells (3 × 105/well) were plated on 6-well plates and incubated with
RPMI 1640 containing 5% FBS for 24 h. Transfection was performed
by using LIPOFECTIN® Reagent (5 µl/well) (Invitrogen)
according to the methods published previously (28, 33, 34). The total
amount of plasmid DNA was prepared to 3 µg/well by addition of
control plasmid that encoded the luciferase gene but lacked the
promoter insert. After 24 h, the medium was replaced with
serum-free RPMI 1640 containing 1 mg/ml bovine serum albumin with R1881
or growth factors. Cells were collected after 24 or 48 h of
incubation using the lysis buffer provided in the luciferase kit
(Promega). Luciferase activities were measured by using Dual Luciferase
Assay System (Promega) with the aid of a multiplate luminometer (EG & G
Berthold, Germany). Luciferase activities were normalized by the
protein concentration of the samples as measured by the method of
Bradford (37). The results are presented as the fold induction that is
the relative luciferase activity of the treated cells divided by that
of the control. All transfection experiments were carried out in
triplicate wells and repeated no less than 4 times using at least 2 sets of plasmids that were prepared separately.
Northern Blot Analysis--
LNCaP cells (5 × 105) were plated in 60-mm plates in RPMI containing 5%
FBS. Immediately prior to experiments, the media were changed to RPMI
containing 5% dextran-charcoal-stripped serum for 48 h.
The cells were treated with IL-6 in RPMI containing 5%
dextran-charcoal-stripped serum for 16 h. Total RNA was
extracted with Trizol (Invitrogen) and fractionated by electrophoresis
before blotting onto Hybond N+ filters (Amersham
Biosciences). The 1.4-kb EcoRI fragments and 1-kb
BamHI GAPDH fragments were labeled with
[ Semiquantitative RT-PCR--
Semiquantitative RT-PCR was
performed as described previously (38) with minor modifications. PSA
and GAPDH primers were as follows: PSA 418/21 sense
5'-GGCAGGTGCTTGTAGCCTCTC-3'; PSA 939/21 antisense,
5'-CACCCGAGCAGGTGCTTTTGC-3'; GAPDH, sense,
5'-CCGAGCCACATCGCTCAGAENDASH-3' and GAPDH antisense,
5'-CCCAGCCTTCTCCTGGTG-3'. For quantitation 5 µM PSA
primers (1 µl) were mixed with 2.5 µM GAPDH primers (1 µl) and then resolved on 1.3% agarose gel, and the bands were analyzed with ImageQuant 5.0 software (Molecular Dynamics, Sunnyvale, CA). The PSA fragments were normalized to GAPDH.
MTT Assays--
LNCaP cells (1 × 104) were
plated in 96-well plates in RPMI containing FBS (0.5%) in a final
volume of 0.1 ml. The next day the cells were treated with R1881,
forskolin, IL-6, or mixtures of the compounds. After 3 days in culture,
cell proliferation was assessed by adding 50 µl of MTT dye (1 mg/ml)
in serum-free media to the cells. After 4 h of incubation, the
cells were solubilized in Me2SO (150 µl per well) prior
to reading the absorbance at 570 nm using a microplate reader (Dynex Technologies).
Immunoblots--
LNCaP cells (2 × 106) were
plated on dishes (10 cm diameter) in RPMI 1640 containing 5% FBS.
Twenty four hours later, the medium was removed and replaced with RPMI
1640 (e.g. serum-free media) for 24 h prior to the
addition of IL-6, forskolin, or inhibitors. After incubation with these
compounds, whole-cell lysates were prepared as described previously
(39). Equal amounts of protein (40 µg) from each sample were
electrophoresed on SDS-PAGE (8 or 10%) followed by transfer to a
nitrocellulose membrane for Western blot analysis. Immunoblots were
blocked for 1 h in 5% nonfat dry milk (w/v) in TBST containing 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1%
Tween 20. Blots were incubated overnight with anti-phospho-p44/42 MAPK
(Thr-202/Tyr-204) antibody (1:500), anti-phospho-Stat3 (Tyr-705) antibody (1:1000) (Cell Signaling Technology, Inc., Beverly, MA), or
anti-phospho-STAT3 (Ser-727) antibody (1:1000) (Upstate Biotechnology, Inc., Lake Placid, NY), washed with TBST three times, and incubated for
1 h with the second antibody (1:2000; Cell Signaling Technology, or Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antibodies were
diluted with 5% nonfat dry milk in TBST. The protein bands were
detected by the enhanced chemiluminescence kit (Cell Signaling Technology or Amersham Biosciences). Densitometric analyses of protein
bands from scanned x-ray films were performed using the Personal
Densitometer (Molecular Dynamics).
Co-immunoprecipitations--
LNCaP cells (2 × 106) were plated on 10-cm dishes in RPMI containing 5% FBS
for 24 h before transfecting with expression vectors encoding
STAT3 (2 µg/dish) and either His-tagged AR-(1-558), AR-(1-233), AR-(234-391), or AR-(392-558) using LIPOFECTIN® reagent
(Invitrogen). After 24 h the cells were treated with IL-6 (50 ng/ml) or vehicle for 6 h before harvesting. Harvested cells were
lysed in Soft RIPA buffer (PBS, 1% sodium deoxycholate, 20 mM sodium molybdate, 50 mM NaF, 25 mM Androgen-independent Induction of the PSA Promoter Gene by
IL-6--
EGF, KGF, IGF-I, IGF-II, and IL-6 have been suspected to
play a role in the progression of prostate cancer to androgen
independence, which is clinically determined by elevating levels of
PSA. Therefore, these compounds were screened using expression of PSA
as an end point in the well differentiated LNCaP human prostate cancer
cell line to identify whether any of these growth factors may be used as a model to delineate a possible mechanism of action. LNCaP cells
were transiently transfected with the PSA ( IL-6 Increases PSA mRNA Levels in LNCaP Cells--
Because
IL-6 caused an increased in PSA reporter activity, we next examined
whether PSA mRNA levels were increased in LNCaP cells exposed to
IL-6. Northern blot analysis showed that IL-6 caused a 2-fold increase
in PSA mRNA levels compared with control values (Fig.
2A, compare 2nd and
3rd lanes). R1881 was used as a positive control and as
expected caused a robust increase in PSA mRNA levels (1st
lane). Increased levels of PSA mRNA were also detected by
semiquantitative RT-PCR using RNA isolated from LNCaP cells exposed to
two different concentrations of IL-6 (1 and 10 ng/ml) (Fig.
2B). IL-6 (10 ng/ml) caused a greater than 2-fold increase
in PSA mRNA levels in LNCaP cells. These results are consistent
with two previous reports (24, 38) using semiquantitative PCR to detect
a 2-fold increase in levels of PSA mRNA in LNCaP cells exposed to
IL-6.
IL-6 Increases Proliferation of LNCaP Cells--
The effect of
IL-6 on the proliferation of LNCaP cells is controversial with reports
of increases (5, 42, 43) and decreases (6, 24, 44, 45). To place our
results in context with other reports, we examined the effects of IL-6
on the proliferation of LNCaP cells in comparison to androgen (R1881)
and forskolin. IL-6, forskolin, and R1881 increased proliferation of
LNCaP cells as compared with control levels after 3 days (Fig.
3). IL-6 and forskolin were comparable in
promoting proliferation at the concentrations and conditions used in
this experiment. A mixture of IL-6 with R1881 did not promote further
increases in proliferation over that observed with R1881 alone. A
mixture of IL-6 and forskolin also did not increase the proliferation
of LNCaP cells over that obtained with each of the individual
compounds.
Synergistic Increases in the Induction of PSA Promoter Gene by IL-6
with Androgen or Activation of the PKA Pathway--
To determine
whether the induction of PSA by IL-6 is through a similar mechanism as
R1881, LNCaP cells were treated with a saturating concentration of
R1881 to examine whether any further increases in PSA could be induced
in the presence of the two compounds. The induction of PSA-luciferase
activity by IL-6 was dose-dependent both in the absence or
presence of R1881 (Fig. 4A).
Maximum induction of PSA reporter activity by IL-6 was obtained at the
concentration of 100 ng/ml. Combined treatments with IL-6 and R1881
resulted in synergistic increases in the activation of the PSA reporter construct over that achieved solely with a saturating concentration of
R1881.
Recently, we reported that activation of the protein kinase A (PKA)
pathway using forskolin resulted in androgen-independent increases in
the expression of the PSA gene (28). The optimal concentration of
forskolin required to achieve maximum induction of the PSA-luciferase
reporter was 50 µM (28). To provide insight into the
mechanism of androgen-independent induction of PSA by IL-6, the effects
of both IL-6 and forskolin treatments on the activity of the
PSA-luciferase reporter were examined. At the optimal concentration of
forskolin (50 µM), PSA-luciferase activity was increased
53-fold (Fig. 4B). Combined treatment of LNCaP cells with an
increasing concentration of IL-6 (1-100 ng/ml) and a constant concentration of forskolin (50 µM) resulted in
synergic increases in PSA-luciferase activities that were
dependent upon the concentration of IL-6.
Induction of Other Androgen-responsive Reporters by IL-6 in LNCaP
Cells--
The PSA (6.1 kb)-reporter gene construct that contains both
the enhancer and promoter regions has been reported to be highly inducible by androgens when compared with the reporter containing only
the promoter region (46). We used this longer PSA (6.1 kb) reporter to
determine whether IL-6 would also have a greater effect on this
reporter. As shown in Fig. 5A,
the PSA (6.1 kb)-luciferase reporter was induced 70-fold by R1881,
8-fold by IL-6, and 113-fold by a mixture of R1881 and IL-6. These
results suggest that the induction of PSA by IL-6 is mediated primarily
through the
To determine whether other androgen-responsive reporter constructs that
contain AREs could be induced by IL-6, two additional reporters were
evaluated in LNCaP cells. The first of these constructs was the
probasin (PB)-promoter (
The second of these reporters was the ARR3-thymidine kinase
(tk)-luciferase, which is an artificial reporter construct that contains three tandem repeats of the rat PB ARE1 and ARE2 regions upstream of a luciferase reporter (36). The effect of IL-6 on the
ARR3-tk-reporter construct was unique compared with
those obtained with PSA and PB. Although R1881 induced
ARR3-tk-luciferase activity by 627-fold, IL-6 proved to be
a very poor inducer of this construct (less than 2-fold) (Fig.
5C). However, a synergistic induction of the
ARR3-tk-luciferase reporter was observed in the presence of
both IL-6 and R1881, consistent with the results obtained with PSA and
PB reporters. The differences in the induction of these three reporter
constructs between R1881 and IL-6 demonstrate promoter-specific responses.
Bicalutamide Inhibits the Induction of PSA-Luciferase Activity by
IL-6--
AREs are present in all four of the above reporters that
respond to IL-6 either in the presence or absence of androgen,
suggesting a role for the AR in the underlying mechanism. To test this
hypothesis, we employed the non-steroidal antiandrogen, bicalutamide,
that specifically inhibits the AR while having no effect on other
steroid hormone receptors. LNCaP cells were transiently transfected
with the PSA ( IL-6 Activates the Human AR NTD--
Ligand-independent activation
of the estrogen receptor has been shown to involve the NTD for its
activation by EGF (47) and IGF-I (48). Therefore, we sought to
determine whether IL-6 targeted the AR NTD to activate this receptor.
To do this, the yeast Gal4 system was employed using a construct of the
AR NTD fused to the Gal4DBD (AR-(1-558)-Gal4DBD). LNCaP cells were
co-transfected with the expression vector encoding the
AR-(1-558)-Gal4DBD and a reporter gene containing the Gal4-binding
site (p5×Gal4UAS-TATA-luciferase). Because R1881 binds to the
ligand-binding domain of the AR, which is absent in our construct, it
was included as a negative control. Forskolin, which increases the
intracellular levels of cAMP, was previously shown to activate a
similar system (28) and was included here as a positive control.
As expected, R1881 had no effect, whereas forskolin resulted in
approximately a 6-fold increase in the activation of the AR NTD
measured as increased Gal4-luciferase activity in co-transfected LNCaP
cells (Fig. 7). Exposure of cells to IL-6
resulted in a 9-fold increase in Gal4-luciferase activity. A mixture of
IL-6 with R1881 did not significantly alter the activity of this
reporter compared with the activity achieved with IL-6 alone. However, a mixture of IL-6 and forskolin resulted in a synergistic increase (greater than 60-fold) in activity of the Gal4-luciferase reporter. These data suggest that the AR NTD is a target of the IL-6 signaling pathway and that simultaneous stimulation of the PKA pathway results in
synergistic activation of the AR NTD.
Activation of the AR NTD by IL-6 Is Mediated through the MAPK
Pathway--
Signaling to the nucleus by IL-6 has been reported to
involve various pathways in LNCaP cells including PKA, protein kinase C, phosphatidylinositol (PI) 3'-kinase, MAPK, and JAK-STAT (24, 49-51). The AR NTD contains a number of putative phosphorylation sites
for serine-proline-directed kinase, DNA-dependent kinase, protein kinase C, casein kinase I and II, PKA, MAPK, and calmodulin kinase II. This raises the possibility that IL-6 may activate the AR
NTD via a phosphorylation event involving one or more of these
pathways. To identify the signal transduction pathway involved in
activation of the AR NTD by IL-6, we employed a series of inhibitors of
various protein kinases and included forskolin as a control in these
studies. The concentration of each of the inhibitors was evaluated by
MTT assay, and no cytotoxic effects were observed (data not shown). To
determine the role of PKA in IL-6 activation of the AR NTD, LNCaP cells
were pre-treated with the PKA inhibitor, Rp-(8-bromo-cAMPs). This inhibitor blocked the
activation of the AR NTD by forskolin but had no effect on the
activation of the AR NTD by IL-6 (Fig. 8;
compare 6th with 7th and 11th with
12th lanes) or a mixture of IL-6 and forskolin
(13th and 14th lanes). These results suggest that
the PKA pathway is not involved in the activation of the AR NTD by
IL-6.
The PI 3'-kinase pathway has been reported to be a major contributor in
the signaling of IL-6 (52, 53). The role of PI 3'-kinase in the
activation of the AR NTD by IL-6 was examined using the PI 3'-kinase
inhibitor, wortmannin. A 30% decrease in the activation of the AR NTD
by IL-6 was observed in cells exposed to wortmannin (Fig. 8, compare
6th with 8th lane). This inhibitor had no effect
on the activation of the AR NTD by forskolin (Fig. 8, compare
11th with 13th lane). Thus, the PI 3'-kinase
pathway may play a role in the activation of the AR NTD by IL-6.
To evaluate the role of the MAPK pathway in the activation of the AR
NTD by IL-6, LNCaP cells were pre-treated with the MAPK kinase (MEK)
inhibitor, U0126. Interestingly, both IL-6 and forskolin activation of
the AR NTD was blocked by this inhibitor (Fig. 8, compare
6th with 9th and 11th with 14th
lanes). These results suggest that both IL-6 and forskolin
activate the AR NTD by a pathway requiring the MAPK pathway.
The JAK-STAT pathway is an important signal transduction pathway for
IL-6. STAT3 is a transcription factor involved in IL-6 signal
transduction. The transcriptional activity of STAT3 is regulated by
phosphorylation at two sites, tyrosine 705 and serine 727 by JAK and
MAPK, respectively (54, 55). Therefore, we investigated whether
inhibition of JAK would affect activation of the AR NTD by IL-6. The
JAK inhibitor, AG490, has been shown to block the activation of STAT3
in mycosis fungus-derived T cell lymphoma cells (56). AG490 reduced
IL-6 activation of the AR NTD by 85% (Fig. 8, compare 6th
with 10th lane). These results demonstrate that JAK is an
important pathway for activation of the AR NTD in response to IL-6.
Stimulation of the PKA Pathway Causes a Synergistic Increase in the
Activation of MAPK by IL-6--
Results presented in Fig. 8 suggest
that the MAPK and the JAK-STAT3 pathways are involved in the IL-6
activation of the AR NTD. To confirm that IL-6 activates the MAPK and
STAT3 pathways, time course studies were performed. Western blot
analyses showed that phosphorylation of MAPK (isoforms p44 and p42) and
STAT3 at tyrosine 705 were maximum after 15 min of exposure of LNCaP cells to IL-6 (Fig. 9, A and
B). At this time point, IL-6 caused an increase in
phosphorylation of MAPK that was comparable with that achieved with
forskolin (Fig. 9C, compare 3rd with 5th
lane). Simultaneous treatment of cells with IL-6 and forskolin
resulted in a synergistic increase in the phosphorylation of MAPK (Fig. 9C, compare 3rd and 5th
lanes with 7th lane). The MEK inhibitor U0126
completely blocked the phosphorylation of MAPK in cells exposed to
IL-6, forskolin, or the combined treatment of IL-6 and forskolin
(compare 3rd with 4th, 5th with 6th,
and 7th with 8th lanes). U0126 did not block the
phosphorylation of STAT3 at tyrosine 705 (Fig. 9D, compare
lane 3 with 4). Forskolin had no effect on the
phosphorylation of STAT3 at tyrosine 705 (Fig. 9D, lane 5).
Thus, exposure of LNCaP cells to IL-6 results in increased phosphorylation of MAPK and STAT3-tyrosine 705, whereas forskolin only
appears to increase the phosphorylation of MAPK.
JAK Inhibitor Blocks Phosphorylation of STAT3 by IL-6--
Because
transactivation of STAT3 is regulated by phosphorylation at tyrosine
705 and serine 727 by JAK and MAPK (54, 55), we examined
phosphorylation levels of STAT3 by Western blot analysis. Application
of a specific inhibitor of JAK, AG490, inhibited the activation of the
AR NTD by IL-6 (Fig. 8) and blocked phosphorylation of tyrosine 705 of
STAT3 (Fig. 10A, compare
lane 3 with 4). This inhibitor also blocked the
phosphorylation of MAPK in LNCaP cells treated with IL-6 as shown in
Fig. 10B (compare lane 3 with 4). By
using an antibody to phosphorylated STAT3 at serine 727, IL-6 was shown
to increase the phosphorylation of STAT3 at this serine, whereas
forskolin had no effect (Fig. 10C, compare lane 2 with 3). These results indicate that activation of the JAK
pathway by IL-6 is involved in the phosphorylation of both STAT3 and
MAPK in LNCaP cells.
Dominant Negatives of STAT3 Inhibit the Activation of the AR NTD by
IL-6--
The results above suggest that the activation of the AR NTD
by IL-6 can be blocked by inhibition of JAK. This implies a possible role for STAT3 in this mechanism. To explore this possibility, we
employed expression vectors encoding various dominant negatives of
STAT3 such that we could specifically block STAT3 (17, 26, 57, 58). The
STAT3F mutant carries a tyrosine to phenylalanine substitution at codon
705 that reduces tyrosine phosphorylation of wild-type STAT3, thereby
inhibiting both dimerization and DNA binding of STAT3. The STAT3D
mutant can be phosphorylated by IL-6 but has no STAT3 DNA binding
activity. STAT1F has a tyrosine to phenylalanine substitution at
residue 701 and inhibits the activation of transcription of the
reporter genes by interferon- Interaction between STAT3 and Amino Acids 234-558 of the AR
NTD--
The above results suggest that STAT3 may interact with the AR
NTD. To investigate this possibility further, we first determined the
region on the AR NTD to which IL-6-induced activity could be mapped. To
do expression vectors, amino acids 1-558, 1-233, 234-390, and
391-558 of the AR NTD fused to Gal4DBD (AR-(1-558)-Gal4DBD, AR-(1-233)-Gal4DBD, AR-(234-390)-Gal4DBD, and AR-(391-558)-Gal4DBD, respectively) were used. As shown in Fig.
12A, IL-6 had no effect on
the activation of Gal4-luciferase activity in cells co-transfected with
AR-(1-233)-Gal4DBD compared with the AR NTD encompassing amino acids
1-558, 234-390, and 391-558 (compare 4th with
2nd, 3rd, and 5th lanes). Thus amino
acids 234-558 of the AR NTD are required for IL-6-induced activity.
However, the most active construct was AR-(234-390)-Gal4DBD. Next we
tested whether STAT3 could be immunoprecipitated with this region of
the AR NTD. As shown in Fig. 12B, IL-6 induced
protein-protein interactions between STAT3 and the AR NTD in LNCaP
cells that were transfected with AR-(1-558)-His tag, AR-(234-390)-His
tag, and AR-(391-558)-His tag. Consistent with transactivation
studies, IL-6 did not induce protein-protein interactions in LNCaP
cells that were transfected with AR-(1-233)-His tag. As a further
control, IL-6 did not induce protein-protein interactions in LNCaP
cells that were transfected with the expression vector for His tag
(data not shown). No interaction was detected in the absence of IL-6.
These data suggest that STAT3 interacts with amino acids 234-558 of
the AR NTD in LNCaP cells exposed to IL-6. The fact interaction with
STAT3 is observed with all three fragments that contain all or part of
the AF-1 site (AR-(1-558), AR-(234-390), and AR-(391-558)) of the AR
suggests that STAT3 may interact with the AF-1 site as part of a large
multiprotein complex or alternatively STAT3 may have multiple binding
sites on both the AR-(234-390) and AR-(391-558).
Ligand-mediated activation of the AR results in its conformation
becoming more compact upon ligand binding, dissociation of heat-shock
proteins, homodimerization, and phosphorylation prior to DNA binding to
AREs on androgen-regulated genes such as PSA (22). The mechanisms
underlying ligand-independent activation of the AR via cross-talk with
other signaling pathways (23, 25, 27, 28) have not been elucidated. The
present studies investigating the ability of IL-6 to induce PSA gene
expression via cross-talk with the AR revealed the following. 1) IL-6
induced PSA gene expression by an AR-dependent pathway in
LNCaP cells, whereas EGF, KGF, IGF-I, and IGF-II had no effect. 2) IL-6
induction of PSA reporter activity was synergistic in the presence of
either androgen or forskolin, which stimulates the PKA pathway. 3) IL-6 induction of androgen-responsive reporter genes was gene-specific. 4)
IL-6 activated the AR NTD, and a synergistic activation was achieved
with a mixture of IL-6 plus forskolin. 5) Activation of the AR NTD by
IL-6 was blocked by inhibitors to MAPK, JAK, and dominant negatives of
STAT3. 6) Both IL-6 and forskolin increased phosphorylation of MAPK and
when used together synergistic increases were observed. 7)
Phosphorylation of MAPK by IL-6 was blocked by inhibitors to MAPK and
JAK. 8) STAT3 co-immunoprecipitated with the AR NTD.
PSA is an androgen-regulated gene in the prostate, and its promoter and
enhancer regions contains several well characterized AREs to which the
ligand-activated AR binds to initiate transcription (31, 40, 59) and
does not contain a STAT-binding site as determined by sequence
analysis. We show that IL-6 induced the activity of the PSA
( Synergistic or additive increases in the transcription of ARE-driven
reporters have been reported in the presence of androgen and compounds
that activate protein kinase pathways (34, 61-64). We observed a
synergistic induction of the ARE-driven reporters in response to a
saturating concentration of R1881 and IL-6, which may suggest that an
additional mechanism to that used solely by R1881 is involved in the
IL-6 pathway. Whole-cell levels of AR were not increased by IL-6 (data
not shown), therefore negating increased AR levels as a possible
mechanism for the synergistic increases observed. In addition, a
synergistic activation of the AR by IL-6 and forskolin was shown for
the first time. Our data are consistent in the fact that synergistic
increases were measured for the following: 1) the induction of PSA
reporter activity; 2) the induction of Gal4-luciferase reporter driven
by the AR NTD fused to Gal4DBD; and 3) the levels of phosphorylated
MAPK in cells exposed to a mixture of IL-6 and forskolin. A possible mechanism for these enhanced increases observed for PSA-luciferase activity is that IL-6 and forskolin signaling converges and amplifies the MAPK pathway leading to synergistic activation of the AR NTD.
The AR NTD interacts with co-activators (65-67), contains the AF-1
site required for transactivation (68-73), and has numerous putative
phosphorylation sites that have been proposed to be targeted by
compounds that stimulate protein kinase pathways (28, 74). Indeed,
cross-talk between the AR NTD and PKA signal transduction pathways in
LNCaP cells was recently shown by Sadar (28). Here we demonstrated that
IL-6 activates the AR NTD, and this activation was independent of the
PKA pathway (Fig. 8). Activation of the AR NTD by IL-6 and forskolin
was effectively blocked by an inhibitor of MAPK kinase. This suggests
that the MAPK pathway plays an important role not only in cross-talk
between IL-6 but also between PKA and the AR signal transduction
pathways. These data are the first to show the importance of the MAPK
pathway in the activation of the AR NTD. Whether the underlying
mechanism of MAPK involves a change in the phosphorylation state of the
AR NTD or a change in the phosphorylation state of a co-activator such
as steroid receptor co-activator-1, which is phosphorylated by MAPK
(75) and interacts with this region of the AR (65), is currently under
investigation in our laboratory.
There has been disparity in the literature concerning forskolin
activation of the MAPK pathway in LNCaP cells for reasons that remain
unknown (60, 76). Our results are consistent with those of Chen
et al. (76) showing that forskolin increases MAPK activity.
The notion that these compounds may increase PSA gene expression via
cross-talk between the MAPK and AR signal transduction pathway is also
consistent with the following reports. 1) MAPK activity is increased in
androgen-independent prostate cancer, a condition that is clinically
defined by increased expression of serum PSA levels in patients treated
with androgen withdrawal therapy (77, 78). 2) The MAPK cascade has been
shown to activate the AR in prostate cancer cells (50, 79).
The tyrosine kinases JAK1, JAK2, and Tyk2 are constitutively associated
with the gp130 (14, 15) and are activated in response to IL-6. The
JAK-STAT pathway appears to play an important role for
ligand-independent activation of the AR by IL-6 as indicated in the
studies presented here and illustrated in our present working model
(Fig. 13). Our results demonstrated
that an inhibition of JAK blocked activation of the AR NTD, tyrosine
phosphorylation of STAT3, and phosphorylation of MAPK in cells exposed
to IL-6. These results suggest that activation of JAK by IL-6 is
involved in both STAT3 and MAPK pathways in LNCaP cells. Cross-talk
between the IL-6-JAK-STAT3 and ligand-bound glucocorticoid receptor
pathways have revealed that STAT3 can act as a transcriptional
co-activator without it binding to its respective DNA-binding motif
(80). Here we show for the first time that STAT3 directly interacts with the amino acids 234-558 of the AR as suggested in
co-immunoprecipitation studies and shown in transactivation
experiments. These results are consistent with a recent report (26)
that STAT3 is required for AR-mediated gene activation in response to
IL-6 in LNCaP cells.
In summary, our results suggest that in the absence of androgens the AR
NTD can be activated by IL-6 by a pathway dependent upon MAPK and STAT3
in prostate cancer cells. Because activation of STAT3 signaling has
been shown to be important for the progression of prostate cancer cells
(51) and to induce transformation of cells (81), we propose that STAT3
plays an important role in androgen-independent growth and progression
of prostate cancer that is clinically determined by
androgen-independent increases in PSA gene expression (77). Further
support for this hypothesis can be drawn from the detection of elevated
levels of IL-6 in the serum of patients with androgen-independent
prostate cancer (82) and the fact that IL-6 increases the proliferation
of prostate cancer cells as shown here (Fig. 3) and by others (5, 6, 42, 43).
We thank N. R. Mawji for
excellent technical assistance, Dr. R. Snoek for helpful discussions,
and Dr. T. S.-Y. Kim for critical review of the manuscript.
*
This work was supported by the National Institutes of
Health, George M. O'Brien Research Center Grant P50 DK47656, and the United States Army Medical Research and Materiel Command DOD Prostate Research Program Grant DAM17-98-1-8577.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, December 19, 2001, DOI 10.1074/jbc.M108255200
The abbreviations used are:
IL-6, interleukin-6;
NTD, N-terminal domain;
MAPK, mitogen-activated protein kinase;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
AR, androgen receptor;
ARE, androgen-response elements;
JAK, Janus kinase;
STAT, signal transducers
and activators of transcription;
DBD, DNA-binding domain;
PSA, prostate-specific antigen;
FBS, fetal bovine serum;
EGF, epidermal
growth factor;
IGF, insulin-like growth factor;
KGF, keratinocyte
growth factor;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
RT, reverse transcriptase;
PKA, protein kinase A;
PI, phosphatidylinositol;
gp, glycoprotein;
tk, thymidine kinase;
PB, probasin;
MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase.
Activation of the Androgen Receptor N-terminal Domain by
Interleukin-6 via MAPK and STAT3 Signal Transduction Pathways*
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INTRODUCTION
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subunit ( chain or
gp80) and a signal transducer, gp130 (
chain) (10, 11). The mRNA
encoding IL-6 subunit is detectable in both cell lines and specimens
of prostate cancer (12, 13). Upon binding of IL-6 to its receptor, a
homodimer of gp130 is formed that results in activation of Janus
kinases (JAKs) (14-16). Once gp130 is tyrosine-phosphorylated, it
recruits signal-transducing molecules, SHP-2 (protein tyrosine phosphatase 2), and signal transducers and activators of transcription 3 (STAT3) (for a review see Ref. 17). Monomers of STAT3 protein exist
in the cytoplasm of non-stimulated cells, and when activated by tyrosine phosphorylation by JAK in response to IL-6 they
dimerize through SH2-phosphotyrosyl interactions that lead to nuclear
translocation and DNA binding to initiate transcription
(18-20).
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630/+12)-luciferase (28, 33, 34), PB-luciferase (35),
ARR3-tk-luciferase (36), AR-(1-558)-Gal4DBD, Gal4DBD (the
control vector), and p5×Gal4UAS-TATA-luciferase (28).
AR-(1-233)-Gal4DBD, AR-(234-390)-Gal4DBD, and AR-(391-558)-Gal4DBD plasmids were constructed by PCR of the nucleotides 363-1062, 1063-1533, 1534-2037, respectively, of the human AR cDNA using primers 5'-AAA AGG ATC CGG ATG GAA GTG CAG TTA GGG CT and 5'-TTT GGA
TCC TCA GTT GTC AGA AAT GGT CGA AGTBGCC, or 5'-AAA AGG ATC CGG GCC AAG
GAG TTG TGT AAG GCA GT and 5'-AAA AGG CTT CAG GTC TTC TGG GGT GGA AAG
TAA TAG as described previously (28). PSA (6.1 kb)-luciferase was
kindly provided by Dr. J.-T. Hsieh (the University of Southwestern
Medical Center, Dallas, TX). The expression vectors for
dominant-negative STATs (pCAGGS-Neo-HA-STAT3F, pCAGGS-Neo-HA-STAT3D, and pCAGGS-Neo-HA-STAT1F), wild-type STAT3 (pCAGGS-Neo-HA-STAT3), and
the control vector (pCAGGS-Neo) were kindly provided by Dr. M. Hibi and
Dr. T. Hirano (Osaka University Graduate School of Medicine, Japan).
-32P]dCTP by Random Primers DNA labeling kit
(Invitrogen). Hybridizations were performed according to the method
described previously (33). The mRNA bands were quantified with the
STORM 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
-glycerophosphate, 1 mM EDTA, 1% Nonidet
P-40 and protease inhibitors). Cell lysates were passed several times through a 301/2-gauge needle to disrupt the nuclei.
Immunoprecipitations were performed using anti-His antibody conjugated
to agarose (Santa Cruz Biotechnology). Immune complexes were analyzed
by SDS-PAGE/immunoblot assay with anti-STAT3 (Upstate Biotechnology,
Inc., Lake Placid, NY).
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630/+12)-luciferase reporter plasmid. This region of the PSA promoter has been partially characterized and contains several AREs that are required for androgen
induction (31, 40). Maximum induction (6-fold) of PSA-luciferase
reporter activity by the synthetic androgen, R1881, was obtained at 1 nM and remained elevated at 10 nM. These
results are consistent with previous reports (28, 33, 34). All studies measuring activities of the PSA promoter were performed in parallel with saturating concentrations of R1881 (10 nM), which was
included as a positive control. Exposure of transiently transfected
LNCaP cells to IL-6 (50 ng/ml) resulted in a 12-fold increase in
PSA-reporter activity relative to the control (Fig.
1). EGF, KGF, IGF-I, and IGF-II yielded
negligible to no effect on PSA-luciferase activities. Our results with
EGF are consistent with the report showing that this growth factor has
no effect in the absence of androgen on the secretion of PSA in LNCaP
cells (41). These results show that IL-6 causes androgen-independent
increases in PSA-luciferase activity, whereas other previously
suspected growth factors did not affect the activity of this reporter
in LNCaP cells.
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Fig. 1.
Effects of R1881 and growth factors on
PSA-luciferase activity in LNCaP cells. LNCaP cells were
transiently transfected with PSA ( 630/+12)-luciferase (1 µg/well)
for 24 h and then incubated with R1881 (10 nM), growth
factors (50 ng/ml), or vehicle for an additional 48 h under
serum-free conditions. The total amount of plasmid DNA transfected was
normalized to 3 µg/well by addition of the empty vector. The
error bars represent the mean ± S.E. of three
independent experiments.
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Fig. 2.
IL-6 increases PSA mRNA levels in LNCaP
cells. A, Northern blot analysis of PSA mRNA levels
in LNCaP cells exposed to R1881 (10 nM) and IL-6 (5 ng/ml);
and B, semiquantitative RT-PCR for the measurement of PSA
mRNA levels in LNCaP cells exposed to IL-6 (1 and 10 ng/ml) in RPMI
containing 5% dextran-charcoal-stripped serum for 16 h
prior to isolation of total RNA (described under "Materials and
Methods").
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Fig. 3.
IL-6 increases the proliferation of LNCaP
cells. Cells were treated with R1881 (10 nM),
forskolin (FSK, 1 µM), IL-6 (50 ng/ml), IL-6
plus R1881 (50 ng/ml and 10 nM, respectively), or IL-6 plus
FSK (50 ng/ml and 1 µM, respectively). After 3 days in
culture, cell proliferation was assessed by the MTT assay. Fold
induction represents the mean of the treated value for absorbance
divided by the mean value for the control. Student's t
test, two-tailed: ***, p < 0.0001; **,
p < 0.05. Error bars signify the mean ± S.E., n = 5.
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Fig. 4.
Effects of IL-6 and R1881 or forskolin on
PSA-luciferase activity in LNCaP cells. LNCaP cells were
transiently transfected with PSA ( 630/+12)-luciferase (1 µg/well)
for 24 h and then pre-treated with IL-6 (ng/ml) for 2 h
before the addition of R1881 (10 nM) (A) or
vehicle or forskolin (50 µM) (B) or vehicle
and then incubated for an additional 48 h under serum-free
conditions. The error bars represent the mean ± S.E.
of three independent experiments.
630 to +12 region of the PSA promoter and does not
encompass DNA elements upstream in the enhancer region.
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Fig. 5.
Effect of IL-6 on the activities of
androgen-inducible reporters in LNCaP cells. LNCaP cells were
transiently transfected with PSA (6.1 kb) (A), PB
( 286/+28) (B), or ARR3-tk-luciferase
(C) (1 µg/well) for 24 h and treated with R1881 (10 nM), IL-6 (50 ng/ml), a mixture of R1881 (10 nM) and IL-6 (50 ng/ml), or vehicle for an additional
48 h under serum-free conditions. The error bars
represent the mean ± S.E. of three independent experiments.
286/+28) which is a naturally occurring
androgen-regulated promoter from the rat that contains ARE1 and ARE2
(35). As shown in Fig. 5B, the PB-luciferase reporter construct was induced 146-fold by R1881, 8-fold by IL-6, and 218-fold by a mixture of R1881 and IL-6. These results show that IL-6 induces androgen-independent increases of PB-luciferase activity when used
solely, and synergistic increases when used in combination with R1881.
These results are consistent with those obtained using the
PSA-luciferase reporters.
630/+12)-luciferase reporter and treated with
bicalutamide and IL-6. Expectedly, bicalutamide blocked the induction
of the PSA-luciferase activity by R1881 by 84%. Bicalutamide also
blocked the induction of PSA-luciferase activities by IL-6 by 70%
(Fig. 6). These results suggest that the
induction of PSA promoter activity by IL-6 is dependent upon a
functional AR.
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Fig. 6.
Inhibitory effect of bicalutamide on the
induction of PSA-luciferase by IL-6. LNCaP cells were transiently
transfected with PSA ( 630/+12)-luciferase (1 µg/well) for 24 h
and then pre-treated with bicalutamide (10 µM) or vehicle
for 2 h before the addition of IL-6 (50 ng/ml) and R1881 (10 nM) and then incubated for an additional 48 h under
serum-free conditions. The error bars represent the
mean ± S.E. of three independent experiments.
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Fig. 7.
Effect of IL-6 on the activity of the human
AR NTD. Transactivation assays were performed in LNCaP cells
co-transfected with the 5×Gal4UAS-TATA-luciferase (1 µg/well) and
AR-(1-558)-Gal4DBD (50 ng/well) or Gal4DBD for 24 h prior to
incubation with R1881 (10 nM), forskolin (50 µM), IL-6 (50 ng/ml), a mixture of IL-6 and R1881, a
mixture of IL-6 and forskolin, or vehicle for an additional 24 h.
The total amount of plasmid DNA transfected was normalized to 3 µg/well by addition of the empty vector. The error bars
represent the mean ± S.E. of three independent experiments and
are normalized to respectively treated Gal4DBD (vector alone)
values.
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Fig. 8.
Effects of inhibitors on the activity of the
AR NTD. Transactivation assays were performed as described in Fig.
7 with LNCaP cells pre-treated for 2 h with each inhibitor,
Rp-(8-bromo-cAMPs) (RP, 200 µM), wortmannin (WM, 100 nM),
U0126 (10 µM), and AG490 (50 µM), prior to
the addition of IL-6 (50 ng/ml), forskolin (FSK, 50 µM), or vehicle and then incubated for an additional
24 h. The error bars represent the mean ± S.E. of
three independent experiments.
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Fig. 9.
Effect of IL-6 on phosphorylation of MAPK and
STAT3 in LNCaP cells. Time course studies in LNCaP cells showing
IL-6-induced phosphorylation of MAPK (A) and STAT3
(B). LNCaP cells were incubated in serum-free conditions for
24 h and then treated for the indicated times with IL-6 (50 ng/ml). Effect of an inhibitor of MEK on IL-6-induced phosphorylation
of MAPK (C) and STAT3 (D). LNCaP cells were
incubated in serum-free conditions for 24 h and then treated with
IL-6 (50 ng/ml), forskolin (50 µM), or vehicle for 15 min, with or without pretreatment with U0126 (10 µM; 30 min) or vehicle. Phosphorylation of MAPK or STAT3 was detected by
Western blot analysis using anti-phospho-MAPK or STAT3 antibodies
(P-MAPK or P-STAT3[Tyr-705]). Membranes were
stripped and re-blotted with anti-MAPK or STAT3 antibodies that
detected total MAPK (T-MAPK) or total STAT3
(T-STAT3).
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Fig. 10.
Effect of a JAK inhibitor on IL-6-induced
phosphorylation of STAT3-tyrosine 705 (A) and MAPK
(B) in LNCaP cells. LNCaP cells were incubated in
serum-free conditions for 24 h and then treated with IL-6 (50 ng/ml) or vehicle for 15 min, with or without pretreatment with AG490
(50 µM) or vehicle. C, IL-6 induced
phosphorylation of STAT3 at serine 727. LNCaP cells were incubated in
serum-free conditions for 24 h and then treated with IL-6 (50 ng/ml), forskolin (50 µM), or vehicle for 15 min. Western
blots were prepared as described in Fig. 9.
(57). To evaluate the role of STAT3 in
the activation of the AR NTD by IL-6, the activation of the
AR-(1-558)-Gal4DBD with the Gal4-luciferase reporter was examined in
LNCaP cells transiently co-transfected with these dominant-negative
STAT3 constructs. Overexpression of STAT3F and STAT3D (Fig.
11) suppressed activation of the AR NTD
by IL-6. Dominant-negative STAT1 (STAT1F) did not reduce activation of
the AR NTD by IL-6. These results indicate that STAT3 plays a role in
the activation of the AR NTD by IL-6.
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Fig. 11.
Effects of dominant-negative STATs on the
activity of AR NTD induced by IL-6. Transactivation assays were
performed in LNCaP cells co-transfected with 0.5 µg of plasmid DNA
encoding dominant negatives of STAT3 (STAT3F or STAT3D), a
dominant-negative STAT1 (STAT1F), or Neo (vector alone) as described in
Fig. 7. After 24 h, serum-starved cells were treated with IL-6 (50 ng/ml) or vehicle and then incubated for an additional 24 h. The
error bars represent the mean ± S.E. of three
independent experiments.
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Fig. 12.
Effect of IL-6 on the interaction between
amino acids 234 and 558 of the AR NTD and STAT3. A,
mapping of the AR NTD required for IL-6-induced activity.
Transactivation assays were performed in LNCaP cells co-transfected
with the 5×Gal4UAS-TATA-luciferase (1 µg/well) and
AR-(1-558)-Gal4DBD (50 ng/well), AR-(1-233)-Gal4DBD (50 ng/well),
AR-(234-390)-Gal4DBD (50 ng/well), and AR-(391-558)-Gal4DBD (50 ng/well) or Gal4DBD (50 ng/well) for 24 h prior to incubation with
IL-6 (50 ng/ml) or vehicle for an additional 24 h. The total
amount of plasmid DNA transfected was normalized to 3 µg/well by
addition of empty vector. The error bars represent the
mean ± S.E. of three independent experiments. RLU,
relative luminescent unit(s). B, physical interaction
between the AR NTD and STAT3 in LNCaP cells exposed to IL-6. LNCaP
cells co-transfected with an expression vector for STAT3 (2 µg/dish)
and either His-tagged AR-(1-558), AR-(1-233), AR-(234-391), and
AR-(392-558) prior to exposure to IL-6 (50 ng/ml) or vehicle for
6 h. Extracts were immunoprecipitated (IP) with
anti-His antibody. Immune complexes were analyzed by
SDS-PAGE/immunoblot assay with anti-STAT3.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
630/+12)-luciferase reporter by a mechanism dependent upon the AR in
human LNCaP prostate cancer cells, whereas previously suspected growth
factors, EGF, KGF, IGF-I, and IGF-II, had no effect. These results are
in conflict to a previous report (23) that IGF-I, KGF, and EGF can
activate the AR to increase an ARE-driven reporter gene construct in
prostate cancer cell lines. A possible explanation for these
disparities may include promoter or cell specificity because the
earlier studies (23) generally used an artificial reporter containing
two AREs in Du145 cells transfected with an expression vector for the
AR. Support for this hypothesis can be drawn from our studies showing
that the ARR3-tk-luciferase reporter was poorly induced by
IL-6, whereas the PSA and PB reporters were strongly induced. Such
promoter-specific responses via a non-ligand-activated AR
versus the ligand-activated AR have been reported previously
in prostate cancer cells exposed to butyrate and compounds that
stimulate the PKA pathway (28, 34). A possible mechanism underlying
promoter-specific responses to the ligand-activated AR
versus the non-ligand-activated AR may involve recruitment
of different co-activators. Ligand-independent activation of the
AR may cause the AR to adopt a different conformation to that assumed
by the ligand-bound AR. This could result in the exposure of different
regions of the AR for alternative protein-protein interactions.
Alternatively, the discrepancy with the reported results may be due to
cell specificity, because the previous studies (51, 60) were performed
in poorly differentiated human prostate Du145 cells, rather than the
well differentiated LNCaP cells used here, and differences in MAPK and
STAT3 signaling have been noted between these two cell lines.
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Fig. 13.
A hypothetical working model of signal
transduction pathways leading to ligand-independent activation of the
AR by IL-6 in LNCaP cells. IL-6-mediated and forskolin-mediated
signals are illustrated. The IL-6 signaling cascade induces
up-regulation of AR-regulated genes such as PSA by ligand-independent
activation of the AR, which is stimulated by both phosphorylation of
STAT3 and MAPK. Stimulation of the PKA pathway using forskolin results
in phosphorylation of MAPK. Therefore, the IL-6 and forskolin signaling
pathways may converge and amplify the MAPK pathway leading to
synergistic activation of the AR NTD.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Cancer
Endocrinology, British Columbia Cancer Agency, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada. Tel.: 604-877-6036; Fax:
604-877-6011; E-mail: msadar@bccancer.bc.ca.
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M. Estrada, A. Espinosa, M. Muller, and E. Jaimovich Testosterone Stimulates Intracellular Calcium Release and Mitogen-Activated Protein Kinases Via a G Protein-Coupled Receptor in Skeletal Muscle Cells Endocrinology, August 1, 2003; 144(8): 3586 - 3597. [Abstract] [Full Text] [PDF]
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X. Yu, R. H. Kennedy, and S. J. Liu JAK2/STAT3, Not ERK1/2, Mediates Interleukin-6-induced Activation of Inducible Nitric-oxide Synthase and Decrease in Contractility of Adult Ventricular Myocytes J. Biol. Chem., April 25, 2003; 278(18): 16304 - 16309. [Abstract] [Full Text] [PDF]
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L. Jia, J. Kim, H. Shen, P. E. Clark, W. D. Tilley, and G. A. Coetzee Androgen Receptor Activity at the Prostate Specific Antigen Locus: Steroidal and Non-Steroidal Mechanisms Mol. Cancer Res., March 1, 2003; 1(5): 385 - 392. [Abstract] [Full Text] [PDF]
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S. O. Lee, W. Lou, M. Hou, F. de Miguel, L. Gerber, and A. C. Gao Interleukin-6 Promotes Androgen-independent Growth in LNCaP Human Prostate Cancer Cells Clin. Cancer Res., January 1, 2003; 9(1): 370 - 376. [Abstract] [Full Text] [PDF]
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S. L. Planey, A. Derfoul, A. Steplewski, N. M. Robertson, and G. Litwack Inhibition of Glucocorticoid-induced Apoptosis in 697 Pre-B Lymphocytes by the Mineralocorticoid Receptor N-terminal Domain J. Biol. Chem., October 25, 2002; 277(44): 42188 - 42196. [Abstract] [Full Text] [PDF]
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J. Reid, I. Murray, K. Watt, R. Betney, and I. J. McEwan The Androgen Receptor Interacts with Multiple Regions of the Large Subunit of General Transcription Factor TFIIF J. Biol. Chem., October 18, 2002; 277(43): 41247 - 41253. [Abstract] [Full Text] [PDF]
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J. D. Debes, L. J. Schmidt, H. Huang, and D. J. Tindall p300 Mediates Androgen-independent Transactivation of the Androgen Receptor by Interleukin 6 Cancer Res., October 15, 2002; 62(20): 5632 - 5636. [Abstract] [Full Text] [PDF]
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T. Ueda, N. R. Mawji, N. Bruchovsky, and M. D. Sadar Ligand-independent Activation of the Androgen Receptor by Interleukin-6 and the Role of Steroid Receptor Coactivator-1 in Prostate Cancer Cells J. Biol. Chem., October 4, 2002; 277(41): 38087 - 38094. [Abstract] [Full Text] [PDF]
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M. Sharma, W. W. Chuang, and Z. Sun Phosphatidylinositol 3-Kinase/Akt Stimulates Androgen Pathway through GSK3beta Inhibition and Nuclear beta -Catenin Accumulation J. Biol. Chem., August 16, 2002; 277(34): 30935 - 30941. [Abstract] [Full Text] [PDF]
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A. C. B. Cato, A. Nestl, and S. Mink Rapid Actions of Steroid Receptors in Cellular Signaling Pathways Sci. Signal., June 25, 2002; 2002(138): re9 - re9. [Abstract] [Full Text] [PDF]
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