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INTRODUCTION |
The androgen receptor
(AR)1 is a member of the
steroid receptor (SR) superfamily and plays an important role in male
sexual differentiation and prostate cell proliferation (1). The well conserved DNA binding domain (DBD) within AR has two zinc finger structures that are involved in DNA binding. The C-terminal region of
the AR, including the hinge region and the ligand-binding domain, is
responsible for the functions of dimerization and androgen binding. The
N-terminal region is involved in the transcriptional activation of
AR.
The discovery of transcriptional interference/squelching of SRs
provided the concept of the existence of transcriptional cofactors that
mediate SR function (2, 3). Recently, several putative cofactors
(either coactivators or corepressers) for SRs have been identified and
characterized (4, 5). Further studies of the interaction of SRs with
these cofactors suggested that these SR-cofactor complexes play
essential roles for the regulation of SRs target gene transcription by
interaction with general transcription factors and the remodeling of
chromatin (4, 5).
The in vivo significance of these cofactors and their
relationship to diseases, however, remains unclear. Recently, an
estrogen receptor coactivator, AIB1, was identified with higher
expression in ovarian cancer cell lines and breast cancer cells than in
other cell lines tested (6), implying that increased expression of cofactors might be involved in some hormone-responsive tumors. The
question whether cofactors of AR, the major promoter of prostate tumor
growth, can also play vital roles for the maintenance of androgen-dependent status is thus of vital interest.
Here we report for the isolation and characterization of a novel AR
coactivator, ARA55, which can bind to wild type AR (wtAR) and mutant AR
(mAR) in a ligand-dependent manner and enhance their transcriptional activities. The potential roles of ARA55 in prostate cancer is also discussed.
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EXPERIMENTAL PROCEDURES |
Expression Plasmids--
A human prostate cDNA library in
pACT2 yeast expression vector was a gift from Dr. S. Elledge. For
construction of pAS2-wtAR or mAR, C-terminal fragments (aa 595-918)
from wtAR or mAR (mART877S, point mutation threonine to serine at codon
877, from Dr. S. P. Balk (7)), respectively, were inserted in pAS2
yeast expression vector (CLONTECH). pG5CAT reporter
plasmid (CLONTECH) contains five GAL4 binding sites
upstream of the E1b TATA box, linked to the CAT gene.
Screening of Prostate cDNA Library by a Yeast Two-hybrid
System--
A pACT2-prostate cDNA library that consists of the
GAL4 activation domain (aa 768-881) fused with human prostate cDNA
library was transformed into Y190 yeast cells with a plasmid of
pAS2-mAR (mART877S) that contains GAL4DBD fused with the C-terminal
domain of this mAR. Transformants were selected for growth on synthetic dropout (SD) plates with 25 mM 3-aminotriazole and 100 nM dihydrotestosterone (DHT) lacking histidine, leucine,
and tryptophan. Colonies were also filter-assayed for
-galactosidase
activity. DNAs from positive clones were recovered from yeast,
amplified in E. coli, and confirmed by sequencing.
RACE-PCR--
The missing 5'-coding region was isolated by
5'-RACE-PCR according to the manufacturer's protocol of Marathon
cDNA Amplification Kit (CLONTECH). The
gene-specific antisense primer used for 5'-RACE-PCR was
5'-TCAGCCGAAGAGCTTCAGGAAGCAGGG-3'. The specific PCR reaction condition
was 94 °C for 1 min, 5 cycles of 94 °C for 5 s
72 °C for 3 min, 5 cycles of 94 °C for 5 s
70 °C for
3 min, then 25 cycles of 94 °C for 5 s
68 °C for 3 min. The PCR product was subcloned into pT7-Blue vector (Novagen) and sequenced.
Co-immunoprecipitation of AR and ARA55--
Lysates from
in vitro translated full-length AR and ARA55 were incubated
with or without 10
8 M DHT in the modified
RIPA buffer (50 mM Tris-HCL, pH 7.4, 150 mM
NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, aprotinin, leupeptin, pepstatin,
0.25% Na-deoxycholate, 0.25% gelatin) and rocked at 4 °C for
2 h. The conjugated beads were washed four times with RIPA buffer,
boiled in SDS sample buffer, and analyzed by 8% SDS-polyacrylamide gel
electrophoresis and visualized by STORM 840 (Molecular Dynamics).
Northern Blotting--
The total RNA (25 µg) was fractionated
on a 1% formaldehyde-MOPS-agarose gel, transferred onto a Hybond-N
nylon membrane (Amersham Pharmacia Biotech) and prehybridized. A probe
corresponding to the 900 bp C terminus of ARA55 was
32P-labeled in vitro using Random Primed DNA
Labeling Kit (Boehringer Mannheim) according to the manufacturer's
protocol and hybridized overnight. After washing, the blot was exposed
and quantified by PhosphorImager (Molecular Dynamics). GAPDH was used
to monitor the amount of total RNA in each lane.
Transfection Studies--
DU145 cells and PC3 cells were grown
in Dulbecco's minimal essential medium (DMEM) containing 5% fetal
calf serum (FCS). One hour before transfection, the medium was changed
to DMEM containing 5% charcoal-stripped fetal calf serum. Phenol
red-free medium was used with the E2 experiments. Cells were
transfected using the modified calcium phosphate technique for 24 h, the medium was changed, and cells were treated with either steroid
hormones or hydroxyflutamide (HF) for another 24 h. The cells were
harvested, and cell lysates were normalized by
-galactosidase
internal control and assayed for chloramphenicol
acetyltransferase (CAT) activity. CAT activity was quantified by PhosphorImager.
RT-PCR--
For RT-PCR, the total RNA (2 µg) from each sample
was reverse-transcribed using SuperScript Preamplification System (Life Technologies, Inc.) in a total reaction volume of 20 µl. cDNA (1 µl) was amplified by PCR with AmpliTaq Gold (Perkin-Elmer). The
following sense and antisense primers were used: sense primer, 5'-GCACTTCGTTTGCGGAGGC-3'; antisense primer, 5'-CCGAAGAGCTTCAGGAAGC-3'. This combination of primers amplifies 633 bp of the C-terminal region
of ARA55. After an initial denaturation at 95 °C for 9 min, 33 cycles of amplification (denaturation at 94 °C for 9 min, annealing
at 63 °C for 1 min, and extension at 72 °C for 1 min) were
followed by a terminal extension at 72 °C for 1 min. PCR products
were visualized on a 1% agarose gel containing ethidium bromide. The
600-bp fragment of GAPDH, which was amplified with 30 amplification
cycles using the sense and antisense primers for human GAPDH
(Stratagene), was used to demonstrate comparable RNA amounts and
quality among samples.
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RESULTS |
Cloning and Sequencing of ARA55--
Loss of androgen specificity
in mAR may contribute to the development of prostate cancer from an
androgen-dependent to androgen-independent state (7, 8). We
were interested to know if wtAR and mAR might exert their functions by
recruiting additional cofactors. A yeast two-hybrid system with
mART877S as a bait was used to screen the human prostate cDNA
library. As a result, six clones that interacted with mART877S were
isolated, and one of them, named ARA55, was further characterized
because its DNA sequence was highly homologous to the C terminus of
mouse hic5 (a hydrogen peroxide-inducible clone) (9).
Northern blot analysis indicated that ARA55 mRNA, with a size near
2 kilobases, could be detected in HeLa and prostate PC-3 cells but not
in other cell lines such as HepG2, H1299, MCF7, CHO, PC12, P19, and
DU145 (data not shown). The 5'-RACE-PCR technique was then used to
clone the full-length ARA55 from HeLa cells. Sequence analysis
determined that the open reading frame between the first ATG and
terminal TGA encoded 444 aa for human ARA55 with the predicted
molecular mass of 55 kDa (Fig.
1A). Amino acid sequence
analysis indicated that human ARA55 shares 90.5% homology with mouse
Hic5 (9). Another interesting finding from this deduced aa
sequence was the existence of four LIM motifs in the C-terminal
regions (Fig. 1B). The LIM motif is a cysteine-rich zinc-binding motif with the consensus sequence:
CX2CX16-23HX2CX2CX2CX16-21CX2(C, H, D) (10). Although the function of the LIM motif has not been fully
defined, some data suggest that it might be involved in the
protein-protein interaction (11). Among all identified SR-associated proteins, only the thyroid hormone interacting protein 6 (Trip 6) has
these similar LIM motifs (12).

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Fig. 1.
cDNA sequence and deduced amino acid
sequence of ARA55. A, the nucleotide sequence and the
deduced amino acid sequence are presented and numbered on the
left. B, sequence alignment of the four LIM
motifs in C-terminal regions. The starting amino acids are shown on the
left, and the putative zinc fingers amino acids are
underlined.
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Interaction between ARA55 and AR Is Ligand-dependent--
We first tested whether the interaction between ARA55 and AR is
ligand-dependent in the yeast and mammalian two-hybrid
systems. Y190 yeast cells were transformed with GAL4AD fused with
ARA55251-444 and GAL4DBD fused with the C-terminal region
(aa 595-918) of mART877S or wtAR. Transformants were selected by their
growth on plates with DHT, testosterone, E2, progesterone,
dexamethasone, or vehicle (ethanol) only. Colonies were also assayed
for
-galactosidase activity. As shown in Fig.
2A, DHT and testosterone
promoted the interaction between ARA55 and wtAR or mAR at
concentrations of greater than 1 nM. E2 and progesterone
promoted the interaction only at much higher concentrations (1 µM for E2 and 0.1 µM for progesterone).
Dexamethasone and vehicle did not promote this interaction. There were
no differences between wtAR and mAR in their interactions with ARA55
(data not shown). Together, these data suggested that ARA55 can
interact with not only mAR, but also wtAR in the presence of DHT,
testosterone, and higher concentrations of E2 or progesterone and that
the presence of only the C-terminal region of ARA55 is the minimal
requirement for interaction.

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Fig. 2.
Interaction of ARA55 and AR in yeast and
mammalian systems. A, interaction in yeast system. GAL4
AR, which contains the GAL4 DBD fused to the C terminus (aa 595-918)
of wtAR or mART877S (pAS2-wtAR or -mAR), was transformed into Y190
yeast cells with a plasmid of GAL4 activation domain fused to
ARA55251-444 (pACT2-ARA55). Transformants were selected
for growth on the SD plates with serial concentrations of steroid
hormones lacking histidine, leucine, and tryptophan. Colonies were also
filter-assayed for -galactosidase activity. T,
testosterone; P, progesterone; Dex,
dexamethasone. B, the ability of a fusion protein,
comprising the full-length ARA55 and the activation domain of VP16
(VP16-ARA55), to interact with the hormone binding domain of AR fused
to the GAL4 DBD (GAL0AR) was examined in DU145 cells by determining the
CAT activity from the reporter plasmid pSG5CAT. Ten nM DHT
was used as a ligand. C, immunoprecipitation of AR and
ARA55. The in vitro translated pET-ARA55 and AR incubated
with or without 10 nM DHT were shown from lanes
1-4. The polyclonal anti-His·Tag antibodies were used for
co-immunoprecipitation, and 10 µl of protein A/G-Sepharose beads were
applied to precipitate the protein-antibody complex. The in
vitro translated AR was used in lane 1 as an input
control. Molecular mass markers are in kDa.
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Next, we tested this interaction in a mammalian two-hybrid system.
DU145 cells were co-transfected with a plasmid encoding the hormone
binding domain of wtAR fused to the GAL4 DBD (GAL0AR) and with a
plasmid encoding full-length ARA55 fused to the activation domain of
VP16 (VP16-ARA55). Interaction was estimated by determining the level
of CAT activity from the reporter plasmid (Fig. 2B). A
combination of GAL0 empty vector and VP16-ARA55 did not show any CAT
activity (Fig. 2B, lane 1). A combination of
GAL0AR and VP16 vector showed a negligible amount of CAT activity (Fig.
2B, lane 2), and a significant level of CAT
activity was induced by the co-transfection of VP16-ARA55 and GAL0AR
only in the presence of 10 nM DHT (Fig. 2B,
lane 3 versus lane 4). These results indicate that ARA55 can
interact with AR and that this interaction is
ligand-dependent.
To further prove that the interaction between AR and ARA55 is the
direct interaction, a co-immunoprecipitation assay was performed using
an in vitro transcription/translation system, which
expressed high levels of AR and His·Tag fusion ARA55. A polyclonal
anti-His·Tag antibody was employed for co-immunoprecipitations, and
the resulting immune complexes were subjected to SDS-polyacrylamide gel
electrophoresis. As shown in Fig. 2C, the AR can only be
co-immunoprecipitated by incubation with ARA55 that is fused with
His·Tag, and the addition of 10
8 M DHT
enhances significantly the specific interaction between AR and ARA55
(Fig. 2C, lane 3 versus lane 4)
ARA55 Enhances AR Transcriptional Activity--
Because yeast and
mammalian two-hybrid systems and co-immunoprecipitations all indicated
that ARA55 can interact with AR in a DHT-dependent manner,
we were then interested in knowing if such an interaction can affect
further AR transcriptional activity. As shown in Fig.
3, the ligand-free AR had only
minimal MMTV-CAT reporter activity with or without the ARA55, and ARA55
alone also showed only minimal reporter activity (Fig. 3, lane
4). However, addition of 10 nM DHT resulted in a
4.3-fold increase of AR transcriptional activity and ARA55 further
increased this induction by 5.3-fold (from 4.3-fold up to 22.8-fold) in
a dose-dependent manner (Fig. 3, lanes 5-9).
The induced activity reached a plateau at the ratio of AR:ARA55 to
1:4.5. A similar induction result could also be obtained when we
replaced DU145 cells with PC-3 cells, or MMTV-CAT with a PSA-CAT
reporter plasmid that had a 2.8-kilobase promoter region of PSA gene
containing androgen responsive regions (data not shown).

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Fig. 3.
Full-length ARA55 enhances AR transcriptional
activity. DU145 cells were transiently co-transfected with 3 µg
of MMTV-CAT reporter plasmid, with or without 1 µg of AR expression
vector (pSG5AR), increasing amounts of full-length ARA55 expression
vectors (pSG5ARA55) as indicated, or 5 µg of C-terminal (aa 251-444)
region of ARA55 vectors (pSG5ARA55251-444), either in the
presence or absence of 10 nM DHT. Each relative CAT
activity is presented relative to the transactivation observed in the
absence of ARA55 and in the presence of 10 nM DHT
(lane 1). Bars represent the mean ± S.D. of
three independent experiments.
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The Effects of ARA55 on the Transcriptional Activities of wtAR and
mARs--
We then examined the effect of ARA55 on two different mARs
in the presence of serial concentrations of DHT, E2, and HF. One mAR,
mART877A, was identified in LNCaP cells and in advanced prostate cancers with a point mutation at codon 877 (threonine to alanine) (8,
13). The other mAR, mARE708K, has a point mutation at codon 708 (glutamine to lysine). This mARE708K was isolated by a yeast genetic
screening with unique characteristics of having less sensitivity to HF
and E2 as compared with wtAR (14). As shown in Fig.
4, wtAR and these mARs responded well to
DHT at 10
11 to 10
8 M, and ARA55
enhanced their transactivation by 4-8-fold. In the presence of E2 or
HF, wtAR was slightly active at high concentrations (10
7
M for E2 and 10
5 M for HF).
Co-transfection of wtAR with ARA55 at a 1:4.5 ratio, however, increased
AR transcriptional activity in the presence of 10
7
M for E2 or 10
6 to 10
5
M for HF. Compared with wtAR, mART877A responded much
better to E2 and HF, and ARA55 could further enhance significantly its transcriptional activity. Interestingly, whereas mARE708K could respond
well to DHT induction and ARA55 could also further enhance its
transcriptional activity, mARE708K showed very poor response to E2 and
HF even at the high concentrations and ARA55 did not have any effect on
its transactivation. These data suggest that although mutations at AR
may still play important roles for the AR transcriptional activity,
ARA55 may be needed for the proper or maximal DHT-, E2-, or HF-mediated
AR transcriptional activity.

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Fig. 4.
The effects of ARA55 on the
transcriptional activities of wild type and mutant ARs. DU145
cells were transiently co-transfected with 1 µg of wtAR
(pCMVAR), LNCaP mutant AR (pCMVm-ART877A), or mARE708K
(pCMVmARE708K), and 4.5 µg of ARA55, or empty
expression vector in the presence or absence of DHT,
E2, or HF, at the indicated concentrations. Three µg of MMTV-CAT
plasmid was used as a reporter. Each relative CAT activity is presented
relative to the transactivation observed in the absence of ligand.
Bars represent the mean ± S.D. of three independent
experiments.
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Does ARA55 Function as a Specific Coactivator for AR?--
As most
of the identified cofactors for SRs are general and not specific to
only one receptor (15), we were interested in knowing if ARA55 was a
specific coactivator for AR. To discover the answer, we tested the
effect of ARA55 on transcriptions mediated by glucocorticoid receptor
(GR), progesterone receptor (PR), and estrogen receptor (ER) in DU145
cells. As shown in Fig. 5, ARA55 turns
out to be relatively specific to AR, although it also enhances GR and
PR to a lesser degree, and has only a marginal effect on ER. While we
do not know if the environmental conditions we used in the prostate
DU145 cells were optimal for PR, GR, and ER, our data did indicate that
ARA55 could activate PR and GR to a greater degree when compared with
another AR coactivator, ARA70 (16). ARA70 showed much higher
specificity to AR (Fig. 5), which may suggest ARA55 has more
flexibility to interact with other SRs.

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Fig. 5.
The effects of ARA55 and ARA70 on the
transcriptional activities of AR, GR, PR, and ER. DU145 cells were
transiently co-transfected with 3 µg of reporter plasmids (MMTV-CAT
for AR, GR, and PR, and ERE-CAT for ER), 1 µg of each receptor in
pSG5, and 4.5 µg of ARA55 (or 3 µg of ARA70), or empty pSG5 vector,
in the presence of 10 nM cognate ligand. Relative CAT
activity is presented relative to the transactivation observed in the
absence of ARA55. Bars represent the mean ± S.D. of at
least four independent experiments.
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ARA55 Expression in the Prostate--
The mARs (mART877S and
mART877A) were identified in advanced prostate cancer patients and
could respond to antiandrogens or estrogen as well as androgen. Because
ARA55 could enhance the transactivation of these mARs as well as wtAR,
we were interested in knowing whether the prostate cancer cells or the
prostate tissue could express the ARA55. Northern blot analysis
demonstrated that DU145 and LNCaP cells did not, but PC-3 cells did
express ARA55 mRNA (Fig.
6A). Next, we examined ARA55
mRNA expression in the clinical prostate samples and the cell
lines, using RT-PCR (Fig. 6B), because these clinical
samples were obtained by needle biopsy and the amount of the extracted
RNA was not sufficient for Northern blotting analysis. As the Northern
blotting demonstrated, PC-3 cells expressed ARA55 mRNA, but DU145
and LNCaP cells did not. Every clinical prostate sample, including
normal, benign hypertrophy, and cancer, expressed ARA55 mRNA, but
there were some differences in its expression level among the samples.
This heterogeneity of ARA55 expression might be one of the factors that
cause the different clinical courses of prostate cancer.

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Fig. 6.
ARA55 mRNA expression in the prostate
cancer cell lines and clinical prostate samples. Northern blotting
and RT-PCR were performed using total RNA from each sample as described
under "Experimental Procedures." A, Northern blotting of
prostate cancer cell lines. Lane 1, PC-3; lane 2,
DU145; lane 3, LNCaP. GAPDH was used as a control.
B, RT-PCR of prostate cancer cell lines and human prostate
samples. The expected RT-PCR product of ARA55 was 633 bp; M,
1-kb ladder (Life Technologies, Inc.); lane 1, PC-3;
lane 2, DU145; lane 3, LNCaP; lane 4,
normal prostate; lane 5, benign prostatic hypertrophy;
lanes 6-11, prostate cancer. GAPDH was used as a
control.
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DISCUSSION |
The unique sequence of ARA55 places this AR coactivator outside
the family of common SR coactivators that include SRC-1 (17), TIF2/GRIP1 (18, 19), and ACTR/AIB1/RAC3 (4, 6, 20). For example, ARA55
lacks some common motifs (such as basic helix-loop-helix (bHLH) domain
and Per-AhR-Sim (PAS) domain) that are shared by SR coactivators.
Instead, ARA55 does have three LIM motifs in the interaction domain of
the C-terminal region. The LIM motif is a cysteine-rich motif that is
found in several proteins (including Trip 6), with diverse functions
and subcellular distributions. The biochemical properties and the
function of the LIM motifs have not been fully defined, and it has been
suggested that their main function is in developmental regulation (10,
21). However, Schmeichel and Beckerle reported that LIM motifs might be
involved in protein-protein interaction (11). Therefore, the LIM motifs in the C-terminal region of ARA55 may contribute to its interaction with AR.
There is also no homology between ARA55 and the first identified AR
coactivator, ARA70 (16, 22-26). Although both AR coactivators enhance
AR transcriptional activity in DU145 cells, they show distinct
differences: 1) ARA55 is more general to SRs whereas ARA70 is more
specific to AR; 2) ARA55 has a lesser effect than ARA70 on AR-mediated
transactivation in the presence of E2 and HF; and 3) ARA55 is a
TGF-
-inducible gene. The precise role of these two cofactors may
affect the different physiological influences on prostate cells.
One of the very interesting features of ARA55 sequence is its high
homology to mouse hic5, a well documented TGF-
1-inducible gene (9).
Whereas previous studies suggested that Hic5 may play important roles
in the growth-inhibitory pathway associated with senescence (27), the
linkage of hic5 to steroid hormones and their receptors has not been
demonstrated. Our findings here may therefore provide a potential
connection to allow TGF-
1 to increase AR transcriptional activity
via induction of ARA55 in prostate.
Prostate cancer is the most frequently diagnosed malignant tumor and
the second leading cause of cancer death in American men (28). Today,
the only effective treatment for advanced prostate cancer is hormonal
therapy that combines surgical or chemical castration with
administration of antiandrogens, such as HF or E2. Although hormonal
therapy is very effective and most of the patients initially respond to
this treatment, the vast majority of them may relapse within 18 months
(29). The mechanisms by which prostate cancer cells become resistant to
hormonal therapy remain unclear. One popular hypothesis to explain how
prostate can progress from androgen dependent- to androgen
independent-state is that mutations in AR may change this receptor's
sensitivity to other steroid hormones or antiandrogens, such as E2 and
HF. As ARA55 was able to induce transcriptional activity of both wtAR and mAR in the presence of DHT, E2, and HF, and ARA55 was expressed differently in various prostate tumor cells, ARA55 may therefore, play
some important roles for the progression of prostate cancer and its
resistance to hormonal therapy. Further studies of blocking the
function of ARA55 (e.g. a small peptide or compound that can interfere with the interaction of AR and ARA55) may provide a novel
clue for the development of treatments of the advanced prostate cancer.