The restricted expression pattern, the inhibitory effect on AR function and the cofactor-dependent activity suggest an important bio-logical role of AR45 in modulating androgen action..
Trang 1tissue-specific AR45 variant
Isabelle Ahrens-Fath*, Oliver Politz, Christoph Geserick† and Bernard Haendler
Research Laboratories of Schering AG, Berlin, Germany
The surprising finding that the human genome only
codes for 20 000–25 000 genes suggests that several
processes contribute to additional complexity levels
One such mechanism is the generation of multiple
RNA forms from a single gene through use of different
promoters or alternative splicing [1–3] The
correspond-ing protein variants may exert very different and
some-times opposite biological functions [1–3] Abnormal
splicing is responsible for about 50% of genetic
disor-ders [4], including some forms of Parkinson’s Disease,
of cystic fibrosis, of polycystic kidney disease and
pro-geria [5–8] Changes in splicing patterns have also been
linked to cancer [9]; for example a role of the insulin
receptor, cyclin D1 and Mdm2 isotypes in tumour
pro-gression has been documented [10–12] Consequently,
therapeutic modalities aiming at correcting abnormal
splicing events have already been envisaged [13]
Steroid receptors belong to a unique superfamily
of ligand-activated transcription factors that have very pleiotropic effects [14] They are organized in a modular fashion and act by binding to DNA response elements found in the regulatory regions of their target genes [14] Due to the involvement of these receptors in several major diseases, extensive research has been carried out to identify agonistic and antagonistic ligands with beneficial effects An unresolved issue concerns the tissue- and cell-specific activities observed with selective receptor modulators [15–17] Different panels of cofactors may account for some of these effects [18] Another possibility arises from the existence of variant forms of steroid receptors The estrogen receptor (ER) probably belongs to the most diversified family Two isoforms, ERa and ERb, exist, and have different tissue
distri-Keywords
androgen receptor; cofactor; heart; prostate
Correspondence
B Haendler, CRBA Oncology, Schering AG,
D-13342 Berlin, Germany
Fax: +49 30 468 18069
Tel: +49 30 468 12669
E-mail: bernard.haendler@schering.de
Present address
*Paion Research Center, Berlin, Germany
†Spanish National Cancer Center, Madrid,
Spain
(Received 10 August 2004, revised 6
September 2004, accepted 9 September
2004)
doi:10.1111/j.1432-1033.2004.04395.x
A naturally occurring variant of the human androgen receptor (AR) named AR45 has been identified It lacks the entire region encoded by exon 1 of the
AR gene and is composed of the AR DNA-binding domain, hinge region and ligand-binding domain, preceded by a novel seven amino-acid long N-terminal extension A survey of human tissues revealed that AR45 was expressed mainly in heart and skeletal muscle In cotransfection experiments, AR45 inhibited AR function, an effect necessitating intact DNA- and ligand-binding properties Overexpression of AR45 reduced the proliferation rate of the androgen-dependent LNCaP cells, in line with the repressive effects of AR45 on AR growth-promoting function AR45 interacted with the AR N-terminal domain in two-hybrid assays, suggesting that AR inhibi-tion was due to the formainhibi-tion of AR–AR45 heterodimers Under condiinhibi-tions where the transcriptional coactivator TIF2 or the oncogene b-catenin were overexpressed, AR45 stimulated androgen-dependent promoters in presence
of dihydrotestosterone AR45 activity was triggered additionally by the adre-nal androgen androstenedione in presence of exogenous TIF2 Altogether, the data suggest an important role of AR45 in modulating AR function and add a novel level of complexity to the mode of action of androgens
Abbreviations
AR, androgen receptor; DAPI, 4,6-diamidino-2-phenylindole; DBD, DNA-binding domain; DHT, dihydrotestosterone; ECL,
electrochemiluminescence; ER, estrogen receptor; FBS, fetal bovine serum; GFP, green fluorescent protein; LBD, ligand-binding domain; MEM, minimal essential medium; NTD, N-terminal domain; PSA, prostate-specific antigen.
Trang 2bution [15] In addition, many ER splice variants
have been described, mainly in tumours [19] The
biolo-gical function of ER variants has only been studied in a
few cases Examples include ERa46, an ERa form
lack-ing the region encoded by the first exon, which
modu-lates the activity of ERa in MCF7 cells [20], and ERbcx,
a C-terminally truncated form containing 26 specific
amino acids, which forms heterodimers with ERa to
inhibit its function [21] The progesterone receptor exists
as two isoforms, PRA and PRB, differing by the length
of the N-terminal end and originating from translation
reinitiation at an internal methionine codon Studies
with transgenic mice show that the ratio of both forms
is essential for proper development of the mammary
gland [22] Analysis of human endometrial tumours
indicates that loss of the B-form is linked to poor
prog-nosis [23] Concerning the glucocorticoid receptor, a
splice variant named GRb that lacks the region encoded
by the most 3¢ exon and is unable to bind to
glucocortic-oids has been described Overexpression of GRb leads
to glucocorticoid resistance in a number of pathological
conditions [24] In the case of the mineralocorticoid
receptor a variant lacking the hinge and ligand-binding
regions but able to increase the activity of the full-length
receptor has been described [25]
Several variant forms of the androgen receptor (AR)
have been found A short AR-A form arising, as for
PRA from internal translational reinitiation, has been
described [26] Recent data, however, question its
exist-ence in human tissues [27] A few cases of exon
skip-ping leading to androgen insensitivity have been
reported [28] Finally, polymorphisms affecting the
length of glutamine, proline and glycine repeats exist
[28] They may lead to severe pathologies, as observed
in Kennedy’s disease, a progressive motor neuron
degeneration caused by an extended polyglutamine
repeat in the N-terminal domain of the AR
Here we describe the identification and
characteriza-tion of AR45, a human AR variant composed of a
unique N-terminal extension linked to the
DNA-bind-ing domain (DBD), hDNA-bind-inge region and ligand-bindDNA-bind-ing
domain (LBD) of the AR The restricted expression
pattern, the inhibitory effect on AR function and the
cofactor-dependent activity suggest an important
bio-logical role of AR45 in modulating androgen action
Results
Identification of AR45, a novel variant of the
human AR
5¢ Rapid amplification of cDNA ends (RACE) was
performed on human placental RNA using a reverse
primer directed against the hinge domain of the AR and a forward primer recognizing the common 5¢ end
of the reverse-transcribed RNA This allowed the amplification of a 500 bp-long DNA fragment, much shorter than the expected AR product DNA sequen-cing revealed that this fragment coded for the AR DBD and hinge region preceded by a novel, short N-terminal extension, rather than the 538 amino acid-long N-ter-minal moiety encoded by exon 1 of the AR gene (Fig 1) A primer specific for the extreme 5¢ region was then used together with a primer recognizing the 3¢ end
of the AR coding part to amplify the complete coding region of this novel AR form The deduced amino acid sequence (Fig 1A) revealed that it contained an intact DBD, hinge region and LBD However, it lacked the entire region encoded by exon 1 of the AR which was replaced by a short, unique seven amino acid-long N-terminal extension The deduced molecular mass was about 45 kDa, hence the name AR45
Homology search revealed that the AR45-specific coding region was located on chromosome Xq11, between exons 1 and 2 of the AR gene The complete DNA sequence of this novel exon, which we named 1B, together with flanking intron regions is depicted in Fig 1B Exon 1B localized 22.1 kb downstream of
AR exon 1 (Fig 1C) and was not predicted by
annota-A
B
C
Fig 1 Sequence of AR45 and position of exon 1B in the AR gene (A) Deduced amino acid sequence of AR45 The unique N-terminal extension is in bold The exon limits are indicated with converging arrowheads and the domain boundaries with colons The residues mutated for functional studies are underlined (B) DNA and deduced amino acid sequences of exon 1B The open reading frame is shown in bold capital letters The flanking intron regions are in italics (C) Localization of exon 1B in the AR gene Exon num-bers are shown above the representation, intron sizes are given below in kb These sequence data have been submitted to the DDBJ ⁄ EMBL ⁄ GenBank databases under accession number AX453758.
Trang 3tion programs of the human genome Its extremities
nevertheless conformed to the splicing rules
AR45 is mainly expressed in heart
RT-PCR was carried out on a panel of human tissues
to determine the expression pattern of AR45 mRNA
A primer corresponding to the AR45-specific upstream
region was used together with a primer recognizing the
AR common part The strongest signal was observed
in heart, followed by skeletal muscle, uterus, prostate,
breast and lung Weaker signals were seen in other
tis-sues, including testis (Fig 2A) In comparison, very
low levels of the AR transcript were detected in heart,
as compared to liver or testis (Fig 2B) The transcript
levels of the ribosomal S9 protein were determined as
a control (Fig 2C) Initial studies with an antibody
directed against the AR LBD indicated that a band of
about 45 kDa was present in human heart extracts
(not shown) However, in the absence of an antibody
recognizing the specific N-terminal extension of AR45,
it cannot be excluded that this band corresponds to a
degradation product of the AR
AR45 is bound by androgen and localizes
to the cell nucleus
The hormone-binding properties of AR45 were
assessed by competitive binding experiments in
presence of 3H-labeled R1881 and increasing amounts
of cold R1881 (Fig 3A) CV-1 cells were transfected with a pSG5-based expression vector for AR45 or AR The results showed specific R1881 binding to AR45 with a calculated IC50of 22 nm This was very close to the IC50 found for AR, which was 16 nm When cor-recting for the amount of AR45 or AR protein expressed in the cells, as determined by Western blot analysis and quantification of electrochemilumines-cence (ECL) signals, comparable levels of total [3H]R1881 bound to both forms were found (not shown) The R243H mutant form of AR45 was used
as negative control It corresponds to AR R774H, a mutant unable to bind to androgens [29,30] As expec-ted, no specific binding was measured (not shown) The subcellular localization of AR45 was deter-mined by transfecting an expression vector coding for AR45 with an N-terminally fused green fluorescent
brain
brain
testis
testis
liver
liver
pr ostate
pr ostate
lung
lung
trac hea
trac hea
m usc le
mu
sc le breast
breast
hear t
hear t
kidne y
kidne y uterus
brain testis liver pr ostate lung trac
hea mu
sc le breast hear
t kidne
y uterus
uterus
AR45
S9
A
B
C
AR
Fig 2 Analysis of tissue distribution of AR45 by RT-PCR analysis
of human RNA First-strand cDNA from the indicated organs
was used as template for PCR amplification Transcript levels of
(A) AR45, (B) AR and (C) ribosomal protein S9 (S9).
B
R1881 [M]
1e-10 1e-9 1e-8 1e-7 1e-6 1e-5 1e-4
0 5000 10000 15000 20000 25000 30000
35000
ARwt AR45
A
Fig 3 AR45 binds to androgen and localizes to the cell nucleus (A) CV-1 cells were seeded in six-well plates and transfected with
6 lg of expression plasmids for AR (d) or AR45 (m) For the bind-ing test, the indicated concentrations of unlabeled R1881 were mixed with the tracer The radioactivity of whole cell extracts was determined The results are a representative of two separate experiments (B) Subcellular localization of AR45 Left panel: PC-3 cells were seeded in 24-well plates and transfected with 2.5 lg of
an AR45–GFP expression construct and treated with R1881 Fluor-escence microscopy was used to visualize the chimeric AR45 protein Right panel: nuclear staining with DAPI.
Trang 4protein (GFP) moiety into PC-3 cells Following
R1881 treatment, we observed an exclusively nuclear
localization of AR45 (Fig 3B, left panel)
4,6-Diami-dino-2-phenylindole (DAPI) staining was performed
to visualize the cell nuclei (Fig 3B, right panel)
Transfection of a control GFP plasmid gave
fluores-cent signals in both the cytoplasm and nucleus
(not shown)
AR45 inhibits AR transcriptional activity
Initial cell-based transactivation studies with different
cell lines and with constructs containing various
andro-gen-responsive promoters showed that AR45 did not
stimulate reporter gene activity in the presence or
absence of ligand (not shown) We therefore sought to
determine whether AR45 might be an inhibitor of AR
function Transient transfection studies were performed
in CV-1 cells using AR45- and AR-expressing
mids, in the presence of the MMTV-Luc reporter
plas-mid (Fig 4A) Using increasing amounts of AR45
plasmid for transfection, a concentration-dependent
inhibition of androgen-stimulated AR activity was
observed To find out whether androgen- and
DNA-binding were important for this effect, we devised
several mutant forms of AR45 AR45 R243H corres-ponds to AR R774H, a mutant form not bound by androgens [29,30] Cotransfection experiments revealed that the AR45 R243H mutant did not inhibit AR function AR45 C31G and DR84 correspond to AR C562G and DR615, two mutants that no longer bind
to DNA [29,30] These AR45 mutants also did not repress AR activity in transactivation assays Supris-ingly, a stimulatory effect was elicited by all three AR45 mutants at the highest plasmid concentration used To show that repression was not limited to a sin-gle cell line, we performed similar cotransfection experiments in the prostate cancer cell line PC-3 and obtained comparable results (not shown)
The results were further confirmed with the prostate-specific antigen (PSA) promoter (Fig 4B) Here we also observed an inhibitory effect of AR45 on AR activity following overexpression in PC-3 cells As before, this was not seen with the mutant AR45 C31G, which does not bind to DNA
The results show that AR45 acts as an inhibitor of
AR function This may result from competition of AR45 homodimers or of AR45–AR heterodimers with
AR homodimers for binding to DNA response ele-ments
0 500 1000
1 500
2 000 2500 3000 3500
0 2000 4000 6000 8000 10000 12000 14000
0 2000 4000 6000 8000 10000
1 2000 14000
0 2000
4 000 6000 8000
1 0000 12000 14000
AR45
R243(774)H
ng 0 25 50 100
ng 0 25 50 100
0 500 1000 1500 2000 2500 3000
0 500 1000 1500 2000 2500
A
B
Fig 4 AR45 inhibits AR function (A) CV-1
cells were transfected with a reporter vector
harbouring the MMTV promoter (40 ng), an
expression vector for AR (10 ng) and with
the indicated amounts (ng) of expression
plasmids for AR45 or a mutated form
Treat-ment was with 1 n M R1881 (grey bars) or
with vehicle (white bars) The results are a
representative of three separate
experi-ments and the bars are the mean ± SEM of
sextuplicate values (B) PC-3 cells were
transfected with a reporter vector
harbour-ing the PSA promoter (40 ng), an expression
vector for AR (10 ng) and with the indicated
amounts (ng) of expression plasmids for
AR45 or a mutated form Treatment was
with 1 n M R1881 (grey bars) or with vehicle
(white bars) The results are a
representa-tive of three separate experiments and the
bars are the mean ± SEM of sextuplicate
values Position of the mutations was
indica-ted relative to the AR45 amino sequence.
For clarity, the corresponding location in the
AR was also given in parentheses.
Trang 5AR45 inhibits proliferation of LNCaP cells
We next determined the biological effects of AR
inhibi-tion by AR45 in LNCaP cells First, the endogenous
expression levels of AR45 mRNA were assessed in
LNCaP cells by RT-PCR, using similar conditions as
above Specific AR45 transcripts were detected in
LNCaP cells grown in the presence or absence of
R1881 (Fig 5A) When analyzing LNCaP nuclear
extracts with an antibody directed against the AR LBD, several protein bands migrating ahead of full-length AR were seen, including one of a size compat-ible with that of AR45 (Fig 5B, lane 4) Indeed, a pro-tein band of similar size was also seen in PC-3 cells transfected with an AR45-expressing plasmid (lane 2), but not with an AR-expressing plasmid (lane 3) Also, AR45 produced by an in vitro translation procedure migrated at the same level (lane 1) Even though the identity of the 45 kDa protein detected in LNCaP cells was not demonstrated due to the unavailability of a specific antibody, the results showed that AR45 expres-sion was at best low
Proliferation tests were carried out with LNCaP cells grown in the presence of 0.1 or 1 nm R1881 Follow-ing transfection of an AR45-expressFollow-ing plasmid, a sig-nificant reduction in the number of viable cells was measured after three days at both hormone concentra-tions, indicating that inhibition of cell proliferation had taken place (Fig 5C)
AR45 interacts with the N-terminal region
of the AR Having determined that AR45 inhibited AR activity,
we sought to find out whether this was due to a direct
0 5000 10000 15000 20000 25000 30000 35000
0 500 1000 1500 2000 2500
AR45
AR NTD
+
+
A
B
AR45
AR NTD
Fig 6 AR45 interacts with the AR NTD CV-1 cells were seeded in 96-well plates and transfected with expression vectors for AR45 and for a fusion between the AR NTD and the activation domain of NF-jB (40 ng each) Treatment was with 1 n M R1881 (A) Reporter plasmid (80 ng) containing the MMTV promoter was cotransfected (B) Reporter plasmid containing the Pem promoter (80 ng) was cotransfected The results are a representative of three separate experiments and the bars are the mean ± SEM of sextuplicate values.
R1881 (M) 0 10 -10 10 -9
128
42
85
AR45
B
AR
0
200
400
600
800
1000
1200
1400
C
A
AR45 AR
actin
Fig 5 AR45 inhibits LNCaP cell proliferation (A) Total RNA was
purified from LNCaP cells grown in the presence (lane 1) or
absence (lane 2) of 0.1 n M R1881 and reverse-transcribed Primers
specific for AR45 or AR were used for PCR amplification The
lev-els of actin were determined as control (B) Nuclear extracts were
prepared from LNCaP cells grown in presence of 1 n M R1881 and
analyzed by Western blot using an antibody directed against the
LBD (lane 4) Nuclear extracts from PC-3 cells transfected with an
expression vector for AR45 or for AR were analysed in parallel
(lanes 2 and 3) In vitro translated AR45 was loaded on lane 1.
Molecular mass markers are indicated in kDa (C) LNCaP cells
grown in presence of the indicated R1881 concentrations were
seeded in 10-cm Petri dishes and transfected with 20 lg of an
AR45-expressing vector The number of viable cells was measured
after 3 days by quantifying the ATP levels (grey bars) In the control
experiment empty pSG5 plasmid was used (white bars) The
results are a representative of two separate experiments and the
bars are the mean ± SEM of quadruplicate values.
Trang 6interaction by performing a mammalian two-hybrid assay Expression vectors coding for full-length AR45 and for a fusion protein between various domains of the AR N-terminal domain (NTD) and the activation domain of NF-jB were cotransfected into CV-1 cells Using an MMTV reporter construct we observed a strong, androgen-dependent interaction between AR45 and the AR NTD (Fig 6A) No interaction was observed between AR45 and the DBD, hinge region or LBD of the AR (not shown) Additional experiments were performed with a reporter construct harbouring the androgen-dependent Pem promoter [31] The Pemgene codes for a homeobox protein of the paired-like family involved in male reproductive functions [32] and its promoter has recenly been shown to contain highly selective androgen-responsive elements [31,33]
By performing similar two-hybrid assays, we also observed an androgen–dependent interaction between AR45 and the AR NTD (Fig 6B) but not with other regions of the AR (not shown)
These results indicate that AR45 binds to androgen-responsive promoters and directly interacts with the
AR NTD
AR45 stimulates androgen-dependent promoters
in presence of cofactors and adrenal androgen The absence of stimulatory activity of AR45 might be linked to poor coactivator recruitment, due to the absence of the NTD This might be overcome by the overexpression of cofactors To test this hypothesis, we analyzed the effects of two AR cofactors known to interact with the AR LBD TIF2 and AR45 were over-expressed in CV-1 cells (Fig 7A) Subsequent dihy-drotestosterone (DHT) treatment led to a sevenfold increase of MMTV-driven reporter activity When coexpressing the oncoprotein, b-catenin, a fourfold stimulation was seen (Fig 7A) A similar induction was also seen when using the S33F b-catenin mutant,
a form with enhanced stability previously identified
in several tumours, including prostate carcinoma (Fig 7A) There was no significant effect of AR45 and cofactor overexpression on reporter gene activity in the absence of DHT (not shown) To rule out that the observed AR45 activity was cell- or promoter-specific,
we coexpressed AR45 and TIF2 in PC-3 cells, and used the PSA promoter as well as the MMTV promo-ter in the reporpromo-ter plasmids In these cells, TIF2 over-expression enhanced DHT-dependent AR45 activity sixfold on the MMTV promoter and twofold on the PSApromoter (Fig 7B,C) In the absence of DHT, no significant difference was noted for MMTV or PSA promoter activity following AR45 and TIF2
over-A
B
C
D
Fig 7 Activation of AR45 by cofactors and androstenedione
Trans-fections were carried out in 96-well plates in presence of 30 ng of
AR45 plasmid, 120 ng of cofactor plasmid and 40 ng of reporter
plasmid (A) CV-1 cells were transfected with expression vectors
for AR45 and for the indicated cofactors A reporter construct
con-taining the MMTV promoter was cotransfected Treatment was
with 1 n M DHT (grey bars) or with vehicle (white bar) (B) PC-3 cells
were transfected with expression vectors for AR45 and TIF2 A
reporter construct containing the MMTV promoter was
cotransfect-ed Treatment was with 1 n M DHT (grey bars) or with vehicle
(white bar) (C) PC-3 cells were transfected with expression vectors
for AR45 and TIF2 A reporter construct containing the PSA
promo-ter was cotransfected Treatment was with 1 n M DHT (grey bars)
or with vehicle (white bar) (D) PC-3 cells were transfected with
expression vectors for AR45 and TIF2 A reporter construct
contain-ing the MMTV promoter was cotransfected Treatment was with
1 n M androstenedione (grey bars) or with vehicle (white bar) The
results are a representative of two separate experiments and the
bars are the mean ± SEM of sextuplicate values.
Trang 7expression (not shown) Finally we tested the effects of
androstenedione on AR45 function This hormone is
synthesized primarily by the adrenal glands and is not
suppressed by chemical castration used to treat
pros-tate cancer patients, which led to speculations about a
role in refractory tumours [27] TIF2 and AR45 were
overexpressed in PC-3 cells Following
androstenedi-one treatment, we measured a twofold stimulation of
the MMTV promoter (Fig 7D)
Altogether these data document that under
condi-tions where coactivators are overexpressed, a
stimula-tion of AR45 by androgens such as DHT or
androstenedione may be observed
Discussion
Here we report the cloning and characterization of
human AR45 cDNA, which codes for a variant form
of the AR AR45 lacks the entire region encoded by
exon 1 of the AR gene and possesses instead a unique
seven amino acid-long stretch This AR45-specific
region is entirely encoded by a hitherto undescribed
exon 1B lying between the first and the second exon of
the AR gene AR45 may therefore originate from
tran-scription controlled by a novel promoter region lying
upstream of exon 1B Interestingly, the consensus
binding sequence CAAGTG for the heart-specific
tran-scription factor Nkx2.5 [34] and motifs with 90%
iden-tity to the binding site GGGRNNYCCC for p65, a
subunit of the NF-jB transcription factor that
regu-lates gene expression in heart [35], are found in the
region immediately upstream of exon 1B (not shown)
Detailed studies are now needed to determine whether
this region directs heart-selective expression of AR45
An alternative splicing event is less likely, because
no sequence corresponding to an additional, more
upstream exon was found in our 5¢ RACE-PCR
experiments Nonetheless this cannot entirely be ruled
out and tissue-specific splicing mechanisms have
already been described for other steroid receptors
[36,37]
The role of androgens in the heart has been
docu-mented by many studies Hypertension and myocardial
ischemia are associated with elevated androgen levels
[38] Direct modulatory effects of androgens and
estro-gens on the left-ventricular mass have been postulated
[39] Due to the comparatively low levels of AR in
heart, AR45 may play an important regulatory role, as
an inhibitor of AR function or possibly also as an
acti-vator in its own right, depending on the promoter
con-text and availability of cofactors AR45 may act as a
dominant-negative inhibitor of AR function As intact
ligand- and DNA-binding regions are mandatory for
this, the formation of AR45 homodimers and of AR45–AR heterodimers on DNA response elements may both account for the effect The formation of AR45–AR heterodimers is likely as previous experi-ments with a truncated rat AR form lacking most of the NTD have been shown to interact with full-length
AR in electrophoretic mobility shift assays [40,41] If such heterodimers cannot recruit the full coactivator set needed for activity, dominant-negative effects may
be observed The important role of the NTD, which
is absent in AR45, has been documented by several studies [40–42] The hormone-dependent interaction between this region and the LBD of the AR is essen-tial for activity Its disruption by introducing appropri-ate mutations in the NTD or by overexpressing an N-terminal AR peptide leads to the impairment of AR function [43,44] The amino- to carboxy-terminus inter-action probably forms a platform for recruitment of several important cofactors [45–47] Chaperones (e.g Bag1L), cofactors (e.g ARA160 and ART27) and signaling effectors (e.g Akt) have been reported to bind to the AR NTD [48] It is therefore likely that AR45 functions mainly as a repressor of AR function Our cellular assays show, however, that in an envi-ronment where a cofactor such as TIF2 or an onco-protein such as b-catenin is overexpressed, AR45 may stimulate the expression of androgen-dependent pro-moters following DHT and also adrenal androgen binding This might have implications in the progres-sion of prostate carcinoma, a disease in which andro-gens and the AR play the main role [49–51] Enhanced TIF2 protein levels have been found in the majority of recurrent prostate cancers [52] Elevated transcript levels of b-catenin, including mutant forms coding for proteins with enhanced stability, have also been found
in prostate tumours [53] In addition, a nuclear colo-calization with the AR and a strictly ligand-dependent interaction have been reported for b-catenin [54] Enhanced TIF2 or b-catenin expression may therefore lead to aberrant AR45 activity and the stimulation of prostate tumour cell proliferation Our additional find-ing that androstenedione, a low-affinity adrenal andro-gen, may stimulate AR45 when cofactors are overexpressed, suggests a mechanism by which treat-ment-refractory prostate tumours may bypass testo-sterone deprivation In the light of a recent study demonstrating that the AR is a major player in both early and late androgen-independent stages of prostate cancer [50], a survey of AR45 levels in tumour resec-tions (surgical removal of tissue) should help clarify the role of this variant form in disease progression
In conclusion, we have identified AR45, a naturally occurring variant of the AR It may either repress or
Trang 8stimulate AR activity, depending on the respective
lev-els of each protein and of cofactors such as TIF2 and
b-catenin This study raises the possibility of a role of
AR45 in modulating androgen effects in heart, where
this form is most abundantly expressed, or in prostate
tumours, where aberrant expression of cofactors may
modify its activity
Experimental procedures
Chemicals and plasmids
Methyltrienolone (R1881), dihydrotestosterone (DHT) and
Dupont NEN (Boston, MA, USA) RPMI 1640, minimal
essential medium (MEM), OPTI-MEM, streptomycin,
peni-cillin, geneticin and l-glutamine were obtained from Gibco
BRL Life Technologies (Eggenstein, Germany) Fetal
bovine serum (FBS) was from PAA (Pasching, Austria)
(Mannheim, Germany) The oligonucleotides were
pur-chased from MWG Biotech (Ebersberg, Germany) or Roth
(Karlsruhe, Germany) Plasmids were from Stratagene
(pSG5, pCMV-BD; Amsterdam, the Netherlands),
Invitro-gen (pCR-TOPO II; Karlsruhe, Germany) or Promega
(pGL3-Basic, pGL3-Promoter; Mannheim, Germany)
Cloning procedures and site-directed
mutagenesis
Human placental RNA (1 lg; Stratagene) was
reverse-tran-scribed using the 5¢-SMART RACE kit (BD Bioscience
Clontech; Heidelberg, Germany) and used as the template
for PCR amplification This was performed with the
Advantage-2 PCR kit (BD Bioscience Clontech) using a
reverse primer directed against the AR hinge region (5¢-CA
GATTACCAAGCTTCAGCTTCCG-3¢) and the forward
5¢-Smart II primer that binds to the common end of the
SMART cDNA population Reaction conditions were: five
separation, a 500 bp-long DNA fragment was generated, as
visualized after gel electrophoresis It was cloned into the
pCR-TOPO II plasmid and sequenced The corresponding
full-length cDNA was amplified from placental DNA using
a forward primer derived from the specific 5¢ extremity and
a reverse primer recognizing the 3¢ end of the AR This
allowed the amplification of 1200 bp-long fragment which
was purified on a gel, cloned and sequenced
Cloning of TIF2 and b-catenin coding sequences was
carried out using primers flanking the coding region and
human universal cDNA (QUICK-Clone II, BD Bioscience
Clontech) as the template PCR amplification was
per-formed with the Herculase enhanced DNA polymerase (Stratagene) Following purification on an agarose gel, the amplified DNA was cloned into pCR-TOPO II (Invitrogen) and sequenced
Site-directed mutagenesis of AR45 to generate the C31G, DR84 and R243H forms, and of b-catenin to generate the S33F form was performed with the QuickChange kit (Strat-agene) using appropriate mutating oligonucleotides, follow-ing the manufacturer’s instructions
RT-PCR analysis of AR45 mRNA expression
AR45 RNA levels were determined by semiquantitative RT-PCR Total RNA from different human organs was obtained from BD Bioscience Clontech LNCaP total RNA was purified using the RNeasy kit (Qiagen, Hilden, Ger-many) Reverse-transcription was carried out using the First-Strand cDNA kit (Stratagene) and semiquantitative PCR analysis performed with the Advantage-2 kit (Strata-gene) The following primers were used for AR45: 5¢-ACA GGGAACCAGGGAAACGAATGCAGAGTGCTCCTGA CATTGCCTGT-3¢ and 5¢-TCACTGGGTGTGGAAATA GATGGGCTTGA-3¢ Reaction conditions were: five cycles
used for AR, 5¢-GGGTGAGGATGGTTCTCCCC-3¢ and 5¢-CTGGACTCAGATGCTCC-3¢ The reaction conditions were as above, except that the annealing temperatures were
cycles The amplification products were separated on a 1%
bromide
Western blot analysis
(NuPAGE Novex, Invitrogen), the proteins were trans-ferred onto poly(vinylidene difluoride) membranes (Roche Molecular Biochemicals) Incubation with the primary anti-body AR C-19 (sc-815; Santa Cruz Biotechnology, Santa
dilution For the secondary, peroxidase-labeled anti-rabbit whole IgG (A-0545, Sigma-Aldrich, Munich, Germany), incubation was for 1 h at room temperature and at a
1 : 5000 dilution Detection was performed using the electro-chemiluminescence (ECL) kit and ECL hyperfilms (Amer-sham Pharmacia Biotech, Freiburg, Germany)
In vitro translation
AR45 cDNA cloned into the pCRII-TOPO plasmid (1 lg)
Reticulocyte Lysate system and the SP6 RNA polymerase,
Trang 9following the manufacturer’s instructions (Promega) The
Cell culture and transfections
Cell lines were obtained from the German Collection of
Microorganisms and Cell Cultures (Braunschweig,
cell line derived from PC-3 cells CV-1 cells were grown at
R1881
For the transactivation assays, the CV-1 and PC-3 cells
were seeded in 96-well plates at a concentration of 10 000–
15 000 cells per 100 lL per well in medium supplemented
used Transfections were carried out 18–19 h later using
FuGene 6 in OPTI-MEM and 40 ng of reporter plasmid
based on pGL3-Basic For LNCaP cells, seeding was in
with X-treme GENE (Roche Molecular Biochemicals) using
10 lg of reporter plasmid Expression plasmids for AR45,
AR or cofactors were cotransfected as indicated The
amount of transfected plasmids was kept constant by
add-ing the appropriate concentrations of pSG5 containadd-ing a
neutral insert Induction was performed 5 h later by adding
1 nm (CV-1 and PC-3 cells) or 2 nm R1881 (LNCaP cells)
Alternatively 1 nm androstenedione was used Measurement
of luciferase activity was carried out after 18 h in a
Lumi-count luminometer, following the addition of 100 lL of
LucLite Plus reagent (both Packard, Dreieich, Germany)
The activity of a constitutively active luciferase vector was
determined in parallel to assess transfection efficiency For
all points the average value of six wells treated in parallel
was taken The experiments were repeated independently at
least three times
For the androgen binding test, CV-1 cells were seeded in
mutant corresponding to the nonandrogen-binding form
R774H was used in the control experiment The level of
AR45 or AR was determined by Western blot analysis as
above Quantification of the ECL signal was performed in
a Kodak Image Station (Rochester, NY, USA)
For determination of the nuclear localization, PC-3
cells in 0.5 mL) were seeded in 24-well
plates on glass coverslips, transfected with 2.5 lg of a GFP-AR45 expression construct and treated 5 h later with 1 nm R1881 After 24 h, the cells were fixed with
microscopy at 507 nm Alternatively, staining with DAPI (Calbiochem, Bad Soden, Germany) was performed to visualize the nuclei
LNCaP cells were seeded into 10-cm Petri dishes Transfection was with 20 lg
of pSG5-AR45 plasmid in Lipofectamine 2000 (Invitrogen) The number of viable cells was determined after 3 days in a Lumicount luminometer (Packard) using the Cell Titer-Glo assay (Promega)
The two-hybrid assay was performed in CV-1 cells in the
with FuGene reagent using 80 ng of reporter plasmid,
40 ng of pSG5-AR45 and 40 ng of a pCMV-BD-based plasmid containing different domains of the human AR Treatment was with 1 nm R1881 and luciferase activity was determined after 23 h
Androgen-binding assay
200 lL of different concentrations of cold R1881 were
transferred into scintillation tubes, supplemented with
Rodgau-Ju¨gesheim, Germany) and incubated for 18 h in the dark Scintillation counting was performed in a 1500 Liquid Scintillation Analyzer (Packard)
Acknowledgements
We are indebted to Prof G Stock and Dr U.-F Habe-nicht for continuous interest in this project We thank Prof W.-D Schleuning and Dr K Bosslet for support, and Dr D Zopf, Dr K Barbulescu and Dr D Mum-berg for fruitful discussions The expert technical assistance of F Knoth, I Schu¨ttke and M Wostrack was much appreciated
The PC-3⁄ AR cell line and the human AR cDNA were obtained from Prof A Cato (FZ Karlsruhe, Germany), the PSA promoter construct from Prof J Trapman (Erasmus Medical Center, Rotterdam, the Netherlands)
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