Protein Inhibitor of Activated STAT-1 (Signal Transducer and Activator of Transcription-1) Is a Nuclear Receptor Coregulator Expressed in Human Testis
Jiann-an Tan,
Susan H. Hall,
Katherine G. Hamil,
Gail Grossman,
Peter Petrusz,
Jiayu Liao,
Ke Shuai and
Frank S. French
The Laboratories for Reproductive Biology Departments of
Pediatrics (J.-A.T., S.S.H., K.G.H., F.S.F.) and Cell Biology (G.G.,
P.P.) University of North Carolina School of Medicine Chapel
Hill, North Carolina 27599-7500
Department of Biochemistry
(J.L.) and Departments of Medicine and Biochemistry (K.S.)
University of California Los Angeles, California 90095-1678
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ABSTRACT
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An androgen receptor (AR) interacting protein was
isolated from a HeLa cell cDNA library by two-hybrid screening in yeast
using the AR DNA+ligand binding domains as bait. The protein has
sequence identity with human protein inhibitor of activated signal
transducer and activator of transcription (PIAS1) and human Gu RNA
helicase II binding protein (GBP). Binding of PIAS1 to human AR
DNA+ligand binding domains was androgen dependent in the yeast liquid
ß-galactosidase assay. Activation of binding by
dihydrotestosterone was greater than testosterone >
estradiol > progesterone. PIAS1 binding to full-length human AR
in a reversed yeast two hybrid system was also androgen dependent.
[35S] PIAS1 bound a glutathione
S-transferase-AR-DNA binding domain (amino acids 544634)
fusion protein in affinity matrix assays. In transient cotransfection
assays using CV1 cells with full-length human AR and a mouse mammary
tumor virus luciferase reporter vector, there was an androgen-dependent
3- to 5-fold greater increase in luciferase activity with PIAS1 over
that obtained with an equal amount of control antisense cDNA or mutant
PIAS1. Constitutive transcriptional activity of the AR N-terminal+DNA
binding domain was increased 6-fold by PIAS1. PIAS1 also enhanced
glucocorticoid receptor transactivation in response to dexamethasone
but inhibited progesterone-induced progesterone receptor
transactivation in the same assay system. mRNA for PIAS1 was highly
expressed in testis of human, monkey, rat, and mouse. In rat testis the
onset of PIAS1 mRNA expression coincided with the initiation of
spermatogenesis between 2530 days of age. Immunostaining of human and
mouse testis with PIAS1-specific antiserum demonstrated coexpression of
PIAS1 with AR in Sertoli cells and Leydig cells. In addition, PIAS1 was
expressed in spermatogenic cells. The results suggest that PIAS1
functions in testis as a nuclear receptor transcriptional coregulator
and may have a role in AR initiation and maintenance of
spermatogenesis.
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INTRODUCTION
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The androgen receptor (AR) is expressed in Sertoli cells of the
seminiferous epithelium, in peritubular myoid cells and in interstitial
cells of Leydig (1 2 3 ), indicating that androgen stimulation of
spermatogenesis (1 4 5 6 ) is mediated by regulation of gene expression
in these cell types (1 ). AR is a member of the steroid receptor
subgroup of the greater family of ligand-dependent nuclear
receptors that form dimers in complex with specific nucleotide
sequences (7 8 ) to function as transcription factors (9 10 11 12 ). Nuclear
receptors have conserved DNA- and ligand-binding domains with similar
three-dimensional structures (13 14 15 16 17 18 ). N-terminal and ligand-binding
domains contain transcriptional activation regions designated AF1 and
AF2, respectively (19 20 21 22 23 24 25 ); however, the AF2 in AR is relatively weaker
than in other steroid receptors. Nuclear receptors increase the
transcription rate of RNA by interactions with coactivators,
coactivator complexes, and general transcription factors (26 27 28 29 30 31 ). AR
is reported to interact directly with transcription factor IIF and the
TATA-box-binding protein (32 ). Transcriptional repression of specific
genes can be relieved by receptor-bound chromatin remodeling factors
and histone acetyltransferases (33 34 35 36 37 38 39 40 41 42 43 ). Acetylation of histones by
nuclear receptor-bound coactivators is believed to increase the
accessibility of nucleosomal DNA to transcription factors. Coactivators
may also control transcription by acetylation of other components of
the general transcription complex (44 ). These acetyltransferases are
expressed in most organs including testis. In addition, the AR
coactivators androgen receptor interacting nuclear protein kinase ANPK,
a Ser/Thr protein kinase (45 ), and androgen receptor assiciated
protein ARA70 (46 47 ) are expressed in testis and other organs;
however, their localization to specific cell types in testis has not
been reported.
Signal transducers and activators of transcription (STAT) are so named
because they serve as signal transducers in the cytoplasm and as
activators of gene transcription in the nucleus. Protein inhibitor of
activated STAT-1 (PIAS1) could provide a link between STAT-1 and AR
signaling in testis since STAT-1 is activated by cytokines and growth
factors in the testicular cells that express AR. PIAS1 was isolated
earlier by Liu et al. (48 ) from a human JY112 B cell library
by yeast two-hybrid screening for STAT-1-interacting proteins using as
bait an alternatively spliced form of STAT-1 lacking the
carboxyl-terminal transcriptional activation domain. PIAS1 was shown to
bind STAT-1 and inhibit STAT1 binding to its consensus response
element. PIAS1 inhibition of activated STAT-1 signaling was
demonstrated in cotransfection assays with interferon
-stimulated
293 cells using a STAT-1 reporter gene (48 ). STAT-1 is activated by way
of receptors that contain a gp130 transmembrane protein with associated
Janus kinases (JAK). Ligand binding to the receptor activates JAKs and
STAT monomers through specific tyrosine phosphorylation (49 50 ).
PIAS1 has close sequence identity with Gu RNA helicase II binding
protein (GBP), and the two could be the same protein. GBP, which was
isolated earlier by yeast two-hybrid screening of a human B lymphocyte
cDNA library, lacked the coding region for N-terminal amino acids 16
(51 ). It remains to be determined whether this was an incomplete cDNA
or natural gene product coding for an incomplete form of PIAS1. GBP was
shown to be highly expressed in testis. Gu RNA helicase II is a
nucleolar protein cloned from a HeLa cell expression library using
antiserum from a human with the autoimmune disease referred to as
watermelon stomach (52 53 ). The helicase has both RNA unwinding and
folding activity; however, the effect of GBP binding on Gu RNA helicase
II activity remains to be established.
Herein we present evidence that PIAS1 is a transcriptional coactivator
for AR and GR, but a corepressor of PR. PIAS1 is expressed
predominantly in testis including cell types that express AR and
mediate the actions of androgen on spermatogenesis. PIAS1/GBP may
function not only as a coregulator for nuclear receptors in the testis
but also as a modulator of activated STAT-1 and RNA helicase-mediated
processes regulating germ cell development.
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RESULTS
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Isolation of PIAS1/GBP
To identify proteins that interact with human AR, yeast
two-hybrid screening was performed using the AR DNA+ligand-binding
domain peptide (amino acids 481919) expressed in frame with the yeast
Gal4 DNA-binding domain peptide. Approximately 5 x
106 yeast colonies containing a HeLa cell cDNA
library cloned into the Gal4 activation domain were screened. Five
colonies demonstrated dihydrotestosterone (DHT)-dependent growth
and turned blue in the yeast ß-galactosidase assay. One positive
clone contained 2.2 kb of open reading frame. This cDNA sequence is
identical to the recently reported human PIAS1 except codon 119 is Glu
instead of Lys, and beginning at codon 266 the sequence is Ile Val Val
instead of Met Cys (Fig. 1
). This cDNA
sequence is also identical to the Gu RNA II helicase binding protein
(GBP) (51 ) except that its codon 613 is Ser instead of Thr. Since our
HeLa cell library clone lacked the four
NH2-terminal codons, we isolated the full-length
cDNA from a human testis cDNA library. PIAS1 coding sequence contains a
number of interesting features (48 51 ), including potential nuclear
translocation signals, potential serine/threonine phosphorylation
sites, cystine and histidine residues predicted to form a zinc finger,
an acidic region, and amino acid sequences (NTSL) that are
possible sites of Asn-glycosylation (Fig. 1
).

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Figure 1. Sequence of PIAS1 and GBP
Symbols represent the following: *, Start of GBP sequence cloned by
Valdez et al. (51 ). #, End of GBP sequence (51 ). ,
Nucleotide difference between PIAS1 and GBP. X, Serine
(PIAS1) to threonine (GBP). LXXLL motifs are underlined.
Open box, Deleted amino acid sequence of mutant PIAS1.
Shaded box, Acidic amino acid sequence. Amino acids in
italics, C2X21C2 domain. Double
underline, NTSL repeat. The cDNA nucleotide sequence
cloned by J.-A. Tan et al. was included in GenBank with
the accession number AF167160.
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AR-PIAS1 Interaction in Yeast Is Androgen Dependent
Androgen dependency of the AR interaction with PIAS1 was
analyzed in the yeast liquid ß-galactosidase assay with increasing
concentrations of DHT or testosterone (T). Yeast strain Y190
transformed with pBDGAL-AR 481919 and pGADGH-PIAS1 was grown in
selective medium with or without androgen. At 0.01 µM
DHT, the AR-PIAS1 interaction stimulated a 5-fold increase in
ß-galactosidase activity over the control with no steroid added while
0.01 µM T stimulated a 2-fold increase (Fig. 2A
). ß-Galactosidase activities
increased to a maximum 8-fold at 1 µM DHT and T. The
results indicate that the AR-PIAS1 interaction in yeast is androgen
dependent. Ligand specificity of the AR-PIAS1 interaction in yeast was
tested with different steroid hormones (Fig. 2B
). ß-Galactosidase
activity was increased about 2-fold at 1 µM progesterone
(P), and at the same concentration estradiol
(E2), dehydroepiandrosterone (DHEA),
dexamethasone (DEX), or hydroxyflutamide (OH-FL) did not increase
ß-galactosidase activity. Activation of AR binding of PIAS1 by
DHT > T > P > E is consistent with the order of AR
binding affinity and agonist activity of these steroids in mammalian
cells (54 ). Hydroxyflutamide binds AR with an affinity lower than
estradiol (55 ) and has weak agonist activity at high concentrations in
mammalian cells; however it is reported to lack both agonist and
antagonist activity in Saccharomyces cerevisiae (56 ).

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Figure 2. Androgen Dependence of PIAS1 Binding to AR Amino
Acids 481919 (DNA and Ligand Binding Domains) in a Yeast Two-Hybrid
Liquid ß-Galactosidase Assay
A, Androgen dependence of PIAS1 binding to AR amino acids 481919. B,
Steroid specificity of AR 481919 activation for PIAS1 binding. C,
Androgen dependence of PIAS1 binding to full-length AR in a reverse
two-hybrid liquid ß-galactosidase assay. In Fig. 2C the basal
activity with AR and PIAS1 in the absence of steroid was about twice
that in Fig. 2, A or B.
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Androgen-dependent binding of full-length AR and PIAS1 (amino acids
5651) was tested in the yeast reversed two-hybrid liquid
ß-galactosidase assay by coexpression of the two fusion proteins. In
the presence of full-length AR-Gal activation domain and PIAS1-Gal DNA
binding domain, DHT (0.01 µM) stimulated a 4.6-fold
increase in ß-galactosidase activity that did not increase further at
higher concentrations of DHT up to 1.0 µM (Fig. 2
C). T
(0.01 µM) stimulated a 3.4-fold increase in
ß-galactosidase activity that increased to 6.5-fold at 1.0
µM, indicating full-length AR interacts with PIAS1 in an
androgen-dependent manner.
PIAS1 Interacts Directly with AR in Vitro
PIAS1 interacted with AR DNA-binding domain
in vitro. In affinity matrix assays, full-length
[35S] PIAS1 (amino acids 1651) bound
glutathione-S-transferase (GST)-AR (amino acids 544634)
(Fig. 3
). This region of AR includes the
entire DNA-binding domain and small portions of the N-terminal and
hinge regions (10 ). PIAS1 N-terminal amino acids 1318 had AR binding
activity similar to that of full-length PIAS1, while binding of the
C-terminal peptide 318651 was not increased over the
GST-glutathione-Sepharose control (Fig. 3
). It should be noted that
peptide fragment 1318 contains three LXXLL motifs beginning at amino
acids 19, 146, and 293 (Fig. 1
). The motifs beginning at amino acids 19
and 293 are conserved among the PIAS family (48 ). Since the PIAS1
C-terminal peptide did not appear to interact with AR in this assay,
binding of a PIAS1 fragment containing amino acids 173 contiguous
with 564651 suggested the LXXLL motif beginning at residue 19
contributes to the AR interaction. LXXLL motifs in the p160
coactivators interact with other nuclear receptors at a hydrophobic
region of the ligand- binding domain that contains the activation
function AF2 (57 ). Further experiments will be required to establish
the role of these motifs in the AR DNA-binding domain interaction.
Also, the binding of PIAS1 318651 to GST-glutathione-Sepharose may
have obscured a low level of binding to AR 544634 in this system.

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Figure 3. Direct Interaction of AR and PIAS1 in
Vitro
Binding of [35S]PIAS1 and PIAS1 peptide fragments to
GST-AR amino acids 544634 (DNA binding domain including 16 amino
acids each of flanking N-terminal and hinge sequence) in
glutathione-Sepharose affinity matrix assay. Binding of full-length
[35S] PIAS1, amino acids 1651 (lanes 1 and 2), amino
acids 1318 (lanes 3 and 4), amino acids 318651 (lanes 5 and 6), and
amino acids 173 contiguous with 564651 (lanes 7 and 8) GST control
(-) GST-AR 544634 (+).
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PIAS1 Expression in Testis and Epididymis
Earlier studies on GBP mRNA expression using a poly (A) RNA blot
(CLONTECH Laboratories, Inc., Palo Alto, CA) indicated
much higher mRNA levels in human testis than in other human tissues
including spleen, thymus, prostate, uterus, small intestine, colon, and
leukocytes (51 ). However, subsequent identification of the PIAS family,
PIASx
, PIASxß, PIASy, and PIAS3, with sequence similarity to PIAS1
(48 ) raised the possibility that the 500-bp GBP probe used in these
earlier studies might have cross-hybridized with other members of the
PIAS family. To distinguish PIAS1 from other members of the PIAS
family, we prepared a more specific PIAS1 probe containing nucleotides
15872101. Northern blotting of human testis poly (A) RNA revealed a
2.5-kb mRNA consistent in size and intensity with the GBP Northern blot
reported earlier (51 ) (Fig. 4A
). In
addition there were weaker bands of approximately 4 and 5 kb. mRNAs of
similar size were detected in blots of human epididymis poly (A) RNA;
however, the 2.5-kb mRNA was much less abundant than in testis. In
Macaca mulatta testis total RNA, the abundance of the 2.5-kb
message was similar to that in human testis, but the mRNA signals in
epididymis, seminal vesicle, and prostate were weak (Fig. 4B
). In rat
testis, expression of PIAS1 mRNA was relatively low at 20 and 25 days
and increased in intensity from 30 to 75 days consistent with
expression in developing spermatogenic cells (Fig. 4C
).

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Figure 4. Northern Hybridization of PIAS1 mRNA in Primate And
Rodent Male Reproductive Tract Using a Specific 32P-Labeled
PIAS1 Probe (Nucleotides 15872101)
A, Poly (A) RNA (5 µg per lane) isolated from human testis
(58-yr-old) and epididymis (62-yr-old). B, Total RNA (10 µg per lane)
isolated from prostate (P), epididymis (E), testis (T), and seminal
vesicle (SV) of a fertile 10-yr-old Macaca mulatta. C,
Age-dependent expression of PIAS1 in rat testis during sexual
development. Total RNA (10 µg per lane) from testes of Sprague Dawley
rats 2075 days of age.
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Expression of PIAS1 protein was localized in human and mouse testis by
immunostaining using anti-PIAS1-specific antibodies raised against the
C-terminal region of PIAS1 (amino acids 549650) (Fig. 5
). Staining of adjacent tissue sections
demonstrated that AR and PIAS1 are both expressed in Sertoli cells and
Leydig cells. In contrast to AR, which was expressed only in Sertoli
cells of the seminiferous epithelium, PIAS1 was also expressed in
spermatogenic cells including spermatocytes and round spermatids. PIAS1
was predominantly nuclear in spermatogenic cells and Sertoli cells but
both cytoplasmic and nuclear in Leydig cells.

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Figure 5. Immunohistochemical Staining of PIAS1 Protein in
Somatic and Germ Cells of Mouse and Human Testis (A, B, and D) Using a
Specific Polyclonal Antibody to PIAS1 Amino Acids 549650 Raised in
Rabbit
For comparison, immunolabeling of AR is shown (C). A, PIAS1 in mouse
testis. Sertoli cell and spermatogenic cell nuclei are labeled
(brown reaction product) in all stages of
spermatogenesis. Elongated spermatids (blue areas
nearest the lumen) did not stain. Magnification: x143. B, PIAS1 in
human testis (81-yr-old). Note abundant staining in spermatogenic cell
nuclei with exception of condensed spermatids and in the cytoplasm of
interstitial (Leydig) cells. Magnification: x143. C, In the human
testis, AR antibody labeled nuclei of Sertoli cells (arrowheads),
Leydig cells, peritubular myoid cells and vascular smooth muscle cells.
Magnification: x550. D, PIAS1-immunoreactivity in within the human
seminiferous tubule is present in nuclei of Sertoli cells
(arrowheads), spermatocytes, and round spermatids.
Magnification: x550.
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PIAS1 Enhances AR and GR Transactivation
To determine whether the androgen dependent interaction of AR with
PIAS1 influences AR transactivation, transient cotransfection assays
were performed in CV1 cells. Native PIAS1 mRNA was not detected by
Northern hybridization in CV1 cell total RNA (10 µg) (not shown). In
assays with full-length AR and mouse mammary tumor virus
(MMTV)-luciferase reporter vector, DHT (0.1 nM) stimulated
a 117-fold increase in luciferase activity over the minus DHT
background (Fig. 6A
). PIAS1 enhancement
of luciferase activity showed a dose-dependent increase from 15 µg
pSG(l)-PIAS1 vector DNA/6-cm dish, 2- to 4-fold higher than with an
equal weight of antisense PIAS1 cDNA (Fig. 6A
). However, with the
higher expressing pSG-PIAS1 vector (Fig. 6B
, inset),
luciferase activity reached a peak at 0.5 µg and was lower at 1.0
µg/dish (Fig. 6B
). Dose responses with the two vectors correlated
with expression levels of the 76-kDa protein (Fig. 6
, A and B,
insets). The pSG(1 )-PIAS1 vector (Fig. 6A
, inset)
expressed less of the 76-kDa protein and also a smaller form of PIAS1
probably resulting from translation initiation at an internal
methionine (see Materials and Methods). Larger amounts of
this vector that might result in decreased coactivation were not
tested. In subsequent assays, similar results were obtained with the
two expression vectors, although the fold stimulation over background
was somewhat higher with pSG(l)-PIAS1. These results with the two
expression vectors suggest that AR coactivator function is reduced at
higher concentrations of the 76-kDa PIAS1. Recently, Moilanen et
al. reported in transient cotransfection assays with
PIASx
, another member of the PIAS family, that AR coactivator
function decreased with transfection of larger amounts of the PIASx
expression vector (48 58 ).

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Figure 6. Concentration-Dependent PIAS1 Enhancement of
Androgen-Dependent AR Transactivation in Transient Cotransfection
Assays
Full-length PIAS1 expression vectors were cotransfected into CV1 cells
with pSG-AR, 0.1 µg, and MMTV-luciferase, 2.5 µg/6 cm dish, and the
cells were incubated in the presence and absence of 0.1 nM
DHT as described in Materials and Methods. Luciferase
activity is expressed as mean and SE light units and fold
increase over background luciferase activity in the absence of DHT. At
PIAS1 concentrations with maximum coactivator activity, PIAS1
background was about 2-fold higher than with AR alone or AR and
antisense PIAS1. A, pSG(l)-PIAS1; B, pSG-PIAS1. Broken
lines indicate light units obtained with equal weights of the
control vector containing PIAS1 in the reverse (antisense) orientation.
The pSG(l)-PIAS1 (see Materials and Methods) was the
less efficient of the two vectors in expressing the 76-kDa protein and
also expressed a smaller form of the protein as shown by Western blot
of protein extracts from transfected COS cells (insets).
Thus, larger amounts of the pSG(l)-PIAS1 vector could be transfected
without diminishing PIAS1 enhancement of DHT-dependent AR
transactivation.
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In the process of screening a human testis cDNA library for full-length
PIAS1 cDNAs, a mutant full-length PIAS1 containing an in-frame 588-bp
internal deletion (codons 341536) was isolated and cloned into pSG5
as described in Materials and Methods. This mutant did not
enhance the DHT-dependent AR increase in luciferase activity (Fig. 7A
).

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Figure 7. Transient Cotransfection Assays of PIAS1 Effect on
Human AR Transactivation
Assay data were obtained under conditions described in Fig. 6 and in
Materials and Methods. In panels A and B, pSG(1 )-PIAS1,
3 µg/6-cm dish, was used, and similar results were obtained with the
pSG-PIAS1 vector, 0.5 µg/6 cm dish. A, Deletion of PIAS1 amino acids
341536 abolished enhancement of AR transactivation. CONTROL: 0.1 µg
pSG-AR, 2.5 µg MMTV-luciferase, ±0.1 nM DHT.
ANTISENSE: control + 3 µg pSG-antisense PIAS1. SENSE: control + 3
µg pSG(l)-PIAS1. MUTANT: control + 3 µg pSG-mutant
PIAS1. B, Steroid induction of AR coactivation by PIAS1 paralleled the
relative AR binding affinity of the steroid. Concentrations were 0.1
nM DHT and 10 nM E2, P, DEX, or
OH-FL. 1. CONTROL: as in panel A or ±E2, P, DEX, or OH-FL
2. ANTISENSE: control + 3 µg pSG-antisense PIAS1. 3. SENSE: control +
3 µg pSG(l)-PIAS1. C, PIAS1 enhanced the constitutive transcriptional
activity of AR N-terminal and DNA binding domains (amino acids 1660).
CONTROL: 10 ng pCMV-AR amino acids 1660, 2.5 µg MMTV-luciferase,
±0.1 nM DHT. ANTISENSE: control + 3 µg pSG-antisense
PIAS1. SENSE: control + 3 µg PIAS1. Numbers in
parentheses indicate the fold increase over background in the
absense of DHT.
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We tested the steroid specificity for AR transactivation enhancement
with PIAS1 in cotransfection assays. Luciferase activity was increased
62-fold with 0.1 nM DHT as compared with 15-fold with 10
nM E2, 5-fold with 10 nM
P, and none with OH-FL or DEX (Fig. 7B
). In assays with DHT,
E2 and P, pSG(l)-PIAS1 enhanced AR induced
luciferase activity 3- to 5-fold over the level with an equal amount of
antisense control. Relative potency of steroids for activation of
transcription was similar to that for activation of AR binding to PIAS1
in the yeast two-hybrid assay and corresponded to the relative AR
binding affinities for these steroids (59 ).
AR has a strong AF1 domain in the N-terminal region, as evidenced by
transactivation with the AR N-terminal and DNA-binding domain fragment
(amino acids 1660). Because it lacks the ligand-binding domain, this
AR fragment is constitutively active in transient cotransfection
assays. We tested the effect of PIAS1 on the transcriptional activity
of AR (1660) to further localize the site of PIAS1-AR interaction. In
this cotransfection assay the amount of AR expression vector
transfected was reduced to 10 ng since the activity of this fragment is
diminished at higher concentrations. Luciferase activity of AR N
terminus and DNA-binding domain was increased 6-fold in the presence of
pSG(l)-PIAS1 (3 µg) but was unchanged by an equal amount of the PIAS1
antisense vector (Fig. 7C
). Since PIAS1 interacted with the AR DNA and
ligand-binding domain fragment in the yeast two-hybrid assay, this
result is consistent with a site of interaction in the AR DNA-binding
domain but does not rule out other sites within the N- and C-terminal
regions of AR.
To establish the specificity of PIAS1 activity, we tested its influence
on human glucocorticoid receptor (GR) and progesterone receptor (PR)
transactiva-tion using the MMTV-luciferase reporter. PIAS1 +
pSGhGR in the presence of DEX (10 nM) induced a
423-fold increase in luciferase activity over the minus DEX control
(Fig. 8A
). This was twice the fold
increase observed in the presence of GR alone or GR with antisense
PIAS1 and was similar to PIAS1 enhancement of AR transcriptional
activity in the same experiment. In contrast, PIAS1 inhibited
progesterone-induced PR transactivation (Fig. 8B
). In the same
assay system using 0.1 µg PR expression vector (pRSVhPRB) -/+ 10
nM progesterone, ligand-dependent PR stimulation of
luciferase activity in the presence of pSG-PIAS1 (0.11.0 µg) was
610 times lower than the control with PR and empty pSG5 parent
vector.

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Figure 8. Transient Cotransfection Assays of PIAS1 Effects on
GR and PR Transactivation
A, PIAS1-enhanced DEX-dependent GR transactivation in transient
cotransfection assays. Each 6-cm dish contained 0.1 µg pSG-hGR
[plusm] 10 nM DEX and 3 µg pSG(l)-PIAS1 (SENSE) or an
equivalent amount of pSG(l)-antisense PIAS1 (ANTISENSE). Conditions
were otherwise as in Fig. 6 . Similar results were obtained when 0.5
µg pSG-PIAS1 was used instead of pSG(l)-PIAS1. B, PIAS1 repressed
progesterone-dependent PR transactivation in transient cotransfection
assays. Each 6-cm dish contained 0.1 µg pSG5-hPRB [plusm] 10
nM progesterone and variable amounts (µg) of pSG-PIAS1
(right) compared with the same amounts of control pSG5
parent vector (left). Conditions were otherwise as
described in Fig. 6 and in Materials and Methods.
Similar results were obtained using 3 µg pSG(l)-PIAS1 compared with
the same amount of pSG antisense-PIAS1.
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DISCUSSION
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The results indicate PIAS1 can function as an AR or GR coactivator
and PR repressor. PIAS1 exhibited androgen-dependent binding to an AR
DNA+ligand binding domain peptide and to full-length AR in a yeast
two-hybrid assay. Full-length [35S]PIAS1 bound
a GST-AR DNA-binding domain fusion protein in affinity matrix assays.
AR binding activity was present in PIAS1 amino acids 1318, a region
containing three LXXLL motifs. PIAS1 enhanced the androgen-dependent
transcriptional activity of full-length AR and the constitutive
transcriptional activity of the AR N terminus+DNA-binding domain. These
results are consistent with an AR coactivator effect mediated through
interaction with the AR DNA-binding domain. However, interactions with
the N-terminal and C-terminal regions of AR were not excluded.
Similar enhancement of transactivation was observed with GR; however,
PR transactivation was repressed by PIAS1. The DNA-binding domain of GR
has 79% and PR 82% sequence identity with AR. Since the DNA binding
domain is relatively conserved throughout the nuclear receptor family,
PIAS1 may be a coregulator for other nuclear receptors in testis. In
addition to AR (1 2 3 4 5 6 ) and GR (60 ), receptors for thyroid hormone (61 ),
estrogen (62 63 ), retinoic acid (64 65 ), and vitamin D (66 ) are
involved in testicular development and/or function.
PIAS1 is co-expressed with AR in human Leydig and Sertoli cells, the
testis cells that mediate androgen control of spermatogenesis. Leydig
and Sertoli cell functions are also regulated by factors that activate
STAT-1 signaling. Thus, PIAS1 could be a integrator of STAT-1 and AR
signaling that enables one pathway to influence signaling by the other.
STATs are activated by a number of cytokines and growth factors
including interleukins, epidermal growth factor, platelet derived
growth factor, GH, and PRL (67 68 69 ). STAT-1 phosphorylation on Tyr 701
induces dimerization of monomers, nuclear translocation, and
sequence-specific DNA binding (48 49 50 ). The cytokines interleukin-1,
interleukin-6 (IL-6), and tumor necrosis factor-
are secreted by
testicular macrophages and modulate gonadotropin effects on Leydig
cells and Sertoli cells (70 ). Interleukin-1 is also produced in
immature rat germ cells (71 ) and stimulates stage-specific DNA
synthesis in seminiferous tubules in vitro (72 ).
Leukemia-inhibitory factor (LIF), a member of the gp130 cytokine
family, is structurally and functionally similar to IL-6 and is
produced in Leydig cells, Sertoli cells, and germ cells (68 69 ).
Studies of Jenab and Morris (73 74 75 ) indicate that LIF and IL-6
increase the expression of early response genes in Sertoli cells by way
of activated STAT-1 and STAT-3.
In addition to PIAS1 that inhibits STAT-1, another
member of the PIAS family, PIAS3, has been shown to be an inhibitor of
STAT-3 signaling (76 ). PIAS3 mRNA was also abundant in human testis,
but unlike PIAS1, it was expressed at similar levels in other organs
(76 ). PIAS1 and -3 appear to have similar mechanisms of action. They
inhibited binding of their respective STATs to response element DNA
through protein-protein interactions and prevented STAT
transactivation. PIAS1 did not interact with STAT-3 nor PIAS3 with
STAT-1. Other known members of the human PIAS family include PIASx
,
PIASxß, and PIASy. A mutant PIASxß with deletion of amino acids
1133 interacted with a homeobox DNA-binding protein, Msx2. This
mutant protein, referred to as Miz1, had sequence-specific DNA binding
activity and enhanced the DNA binding of Msx2 (48 77 ). Moilanen
et al. (58 ) reported recently the isolation of a rat testis
cDNA coding for a 64-kDa protein that interacted with the zinc finger
region of rat AR. This rat protein, which they named AR interacting
protein 3 (ARIP3), has sequence identity with human PIASx
. ARIP3 at
lower levels of expression enhanced rat AR transactivation but had less
activity at higher levels. Expression of ARIP3 mRNA was localized to
testis of rat and human by Northern hybridization, and an ARIP3
antibody recognized epitopes in rat Sertoli cells and spermatogenic
cells. However, the ARIP3 probes used in this study may have
cross-reacted with some other members of the PIAS family. Thus far,
PIAS1 and -3 are the only PIAS family members shown to interact with
STATs.
The binding of PIAS1/GBP to AR, to activated STAT-1, and to an RNA
helicase underscores its multifunctional potential. GBP interaction
with Gu RNA helicase II appeared specific in the yeast two-hybrid assay
in that other nuclear proteins failed to interact. Gu RNA helicase II
is a member of the Asp-Glu-Ala-Asp (D-E-A-D) box family of
ATP-dependent RNA helicases (52 78 ) that function in preribosomal RNA
processing, nuclear export of RNA, ribosomal assembly, and mRNA
translation (78 79 80 ). The helicase family consists of more than 200
proteins, all of which share the Walker box nucleoside
triphosphate-binding site (81 ). RNA helicase A mediated the association
of CBP (82 ) and the breast cancer tumor suppressor protein BRAC1 (83 )
with RNA polymerase II. Gu RNA helicase II was reported to have both
RNA unwinding and RNA folding activity, which introduces a
double-stranded region into a single-stranded RNA (52 ). GBP and Gu RNA
helicase II mRNAs were highly expressed in human testis. GBP was
present throughout the nucleoplasm (51 ), while Gu RNA helicase II was
localized predominantly in nucleoli (53 ). The function of GBP in
relation to Gu RNA helicase II is not well understood (51 ), and it
remains to be determined whether helicase binding of GBP influences GBP
interactions with nuclear receptors and STAT-1.
The discovery that certain DNA-binding domain mutations diminish
transactivation without altering DNA binding led to the suggestion that
transcriptional regulatory proteins interact with DNA-binding domains
(13 84 ). Structural analysis of several nuclear receptor DNA-binding
domains indicated a potential interface positioned to bind regulatory
proteins that would straddle the DNA response element (85 ). A number of
regulatory factors interact directly with the DNA-binding domain or
DNA-binding domain and hinge regions including POU domain transcription
factors and PCAF (41 86 87 88 89 90 ). In addition to ARIP3, Moilanen et
al. (45 58 90 ) reported two other coactivator proteins that
interacted with the AR DNA-binding domain. SNURF, a ring finger protein
that bound the DNA-binding domain and adjoining N-terminal half of the
hinge region was expressed in testis, brain, and other organs. ANPK, a
130-kDa Ser/Thr protein kinase, colocalized with AR in the nucleus and
was expressed in a wide variety of organs including testis. AR itself
was not a substrate for ANPK, suggesting the coactivator function of
ANPK may have resulted from phosphorylation of AR-associated regulatory
proteins. PIAS1 is a candidate substrate since it contains several
potential phosphorylation sites similar to those recognized by ANPK in
immune complex kinase assays.
PIAS1 has the potential to function as a modulator of multiple cellular
regulatory mechanisms in the testis that include transactivation by
STAT-1, AR, and other nuclear receptors and to mediate cross-talk
between STAT-1 and AR signaling. As a helicase- binding protein, it may
influence a transcription-related process such as chromatin remodeling
or recruitment of transcription factors. Since activated STAT-1
utilizes CBP and other acetyltransferase coactivators shared by nuclear
receptors (91 ), inhibition of STAT-1 might make these coactivators more
available to AR or other nuclear receptors. In this role, PIAS1 could
serve as an integrator of STAT-1 and nuclear receptor signaling.
 |
MATERIALS AND METHODS
|
---|
Plasmid Construction
AR Fusion Protein for Two-Hybrid Screening
Human AR expression vector pCMVhAR-Exo (19 92 ) with the internal
EcoRI site mutated was the template for PCR amplification of
DNA-ligand-binding domain codons 481919 using primers
5'-GGCCGAATTCGGCTACACTCGGCCCCCTCA-3', and
5'-GGCCGAATTCTCACTGTGTGTGGAAATAGATGGGC-3'; the PCR fragment was
digested with EcoRI and cloned into the yeast
ß-galactosidase DNA-binding domain vector pBDGAL4 CAM
(Stratagene, La Jolla, CA) to create the plasmid
pBDGAL-481919.
Mammalian Cell Expression Vectors
pSG5 (Stratagene) was modified by insertion of a
double-stranded oligonucleotide:
5'AATTATGAATTCACTAGTGATATCGGATCCGGTACCTCGAGA-3' containing
restriction sites for
EcoRI-SpeI-EcoR5-BamHI-KpnI-XhoI.
Since pGADGH-PIAS1 from the two hybrid screen lacked the four
NH2-terminal codons, full-length cDNAs were
obtained from a human testis cDNA library constructed in Lambda ZAP.
Primers containing EcoRI restriction sites at the 5'-ends
were used to PCR amplify PIAS1 cDNA and bands of 2.0 kb and 1.5 kb were
obtained. The 2-kb product corresponded to full-length PIAS1, whereas
the smaller band contained an in-frame 588 bp internal deletion. These
two cDNAs were cloned into pSG5 in both sense and antisense
orientations. Full-length PIAS1 was also cloned into the
EcoR5 site of the pSG5-linker vector, referred to as pSG(l),
which introduced an extra base at the cloning site upstream of the
natural translation initiation site, ATGG. pSG(l)-PIAS1 was a less
efficient in expressing the 76-kDa protein in COS cells than was
pSG5-PIAS1 and also expressed a smaller form of PIAS1 probably
initiated from an internal translation start site.
A mutant with deletion of amino acids 1318 was created in pSG5-PIAS1
by digestion with BamHI and another with deletion of
amino acids 318651 by PCR. The expression vector pCMVAR (amino
acids 1660) that expresses the constitutively active AR N-terminal +
DNA-binding domain (93 ) was provided by Elizabeth M. Wilson, University
of North Carolina School of Medicine, Chapel Hill.
All constructs were verified by automated sequencing using an ABI PRISM
Model 377 DNA Sequencer (PE Applied Biosystems, Norwalk,
CT) using the ABI Prism Big Dye Terminator Cycle Sequencing
Ready Reaction Kit (ABI, Foster City, CA) with AmpliTaq DNA Polymerase
FS. Primers were synthesized on an automated PE Applied Biosystems DNA synthesizer Model 394 using standard cyanoethyl
phosphoramidite chemistry.
Yeast Two-Hybrid Screening
The vector pBDGAL4 CAM expressing GAL4 DNA-binding
domain fusion with human AR amino acids 481919 was used as
bait to screen a HeLa cell cDNA library cloned into the GAL4 activation
domain (gift from Dr. Yue Xiong, University of North Carolina, Chapel
Hill). Yeast strain Hf7c was transformed with bait plus cDNA library
and plated on synthetic medium lacking Leu, Trp, and His with the
addition of 1 µM DHT and 5 mM 3-amino-1,2,4
triazole (94 ). Yeast colonies were assayed for ß-galactosidase
activity (blue-white assay). From colonies that showed blue color
consistently, cDNAs were rescued using standard procedures. Yeast cells
were transformed with the expression vectors containing rescued cDNAs
or a control cDNA expressing lamin together with the AR bait vector to
test the specificity of the protein-protein interaction and its
dependency on DHT (1 µM). To confirm the androgen
dependence of the interaction with AR, liquid ß-galactosidase assays
were performed with selected clones.
Yeast Liquid ß-Galactosidase Assay
Y190 yeast cells transformed with the AR bait and cDNA library
expression vectors or bait alone were incubated overnight at 30 C in 2
ml selective medium containing various steroids at the concentrations
indicated. Selective media either lacked Trp and Leu or, in assays with
AR bait alone, lacked Trp only (95 ). After incubation for 1 day, YPD
medium (8 ml) containing the same amount of steroid was added and
incubations continued for 3 h at the same temperature. The liquid
ß-galactosidase assay was performed according to instructions
(CLONTECH Laboratories, Inc.).
GST-AR Binding of [35S] PIAS1 in
Affinity Matrix Assay
GST-AR DNA-binding domain fusion protein was expressed in
Escherichia coli. Bacteria transformed with pGST-AR-DNA
binding domain (AR amino acids 544634) and cultured overnight at 37 C
were diluted 1:10 in fresh LB medium and incubated with shaking. After
2 h, isopropyl-ß-thiogalactopyranoside was added to a
final concentration of 1 mM and incubation
continued at 30 C for 3 h. Bacteria cells were collected by
centrifugation and the fusion protein was extracted three times by
sonicating in buffer (20 mM Tris, pH 7.5, 50
mM NaCl, 0.1 mM EDTA, 10%
glycerol). Full-length PIAS1 was cloned into pSG5 and
[35S]PIAS1 synthesized using the TNT Quick
Coupled Transcription/Translation kit (Promega Corp.,
Madison, WI). Extracts containing equal amounts of GST-AR DNA binding
domain or GST control proteins, determined by immunoblotting using GST
antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA),
were incubated 1 h at 4 C with 20 µl glutathione-Sepharose beads
(Pharmacia Biotech, Piscataway, NJ) in PBS, pH 7.5,
containing 1 mg/ml BSA and 0.02% NP40. Beads were washed three times,
each with 1 ml of the same buffer. [35S]PIAS1,
20
, was mixed with washed beads and incubated 1 h at 4 C and
again washed three times with PBS buffer. SDS buffer (30 µl) was
added and boiled 5 min. Supernatant proteins were separated by PAGE in
10% SDS gels. Gels were dried and autoradiography performed with
BioMax MS film (Eastman Kodak Co., Rochester, NY) at
-80 C.
Northern Hybridization
Total RNA was isolated using a modification of the method of
Chirgwin et al. (96 ). RNA suspended in sterile
H20 was glyoxylated, fractionated through 1%
agarose gels, and transferred to a Biotrans nylon membrane (ICN Biomedicals, Inc., Aurora, OH). Membranes were stained with
methylene blue to ensure equal loading of RNA samples. cDNA probes were
labeled with [32P]dCTP (Amersham Pharmacia Biotech, Arlington Heights, IL) using the Prime-a-Gene
System (Promega Corp.). Membranes were hybridized in
aqueous solution (5x SSC, 5x Denhardts solution, 1% SDS, and 100
µg/ml salmon sperm DNA) overnight at 68 C. After washing at 50 C for
1 h in 0.1x SSC containing 0.1% SDS, the membranes were placed
in a cassette with intensifying screen to expose X-OMAT film
(Eastman Kodak Co.) at -80 C.
Immunoblotting
Anti-PIAS1 polyclonal antibody against a glutathione
transferase-PIAS1 fusion protein containing 102 C-terminal amino acids
(549650) of PIAS1 was raised in rabbit (J. Liao and K. Shuai,
unpublished). To test the specificity of the antibody, PIAS1, PIASx
,
PIASxß, and PIASy were expressed in COS cells. Protein extracts were
analyzed by Western blotting and detected by enhanced chemiluminescence
(ECL). Only PIAS1 (76 kDa) was detected by the C-terminal antibody
(data not shown). Expression of the other proteins was confirmed by
staining with Flag antibody. Efficiencies of expression and protein
products of the vectors pSG(l)-PIAS1 and pSG-PIAS1 were analyzed in COS
cells by immunoblotting of cell lysates.
Immunostaining of PIAS1 in Testis
Human testis obtained from an 81-yr-old patient who underwent
orchiectomy for treatment of advanced prostate cancer and testis from
an adult mouse were processed similarly. The human subject had received
no therapy before orchiectomy. Testicular tissue was fixed in Bouins
fluid and embedded in paraffin using standard procedures. Sections 8
µm thick were cut and mounted on glass slides. Before immunostaining,
endogenous peroxidase was blocked (methanol + 5%
H2O2, 30 min at room
temperature) and antigen retrieval was performed by microwave treatment
in 0.01 M (pH 6.0) citrate buffer. Sections were
immunostained according to the double PAP procedure as described by
Ordronneau et al. (97 ). Goat antirabbit IgG serum absorbed
against human proteins was obtained from Antibodies Inc. (Davis, CA),
rabbit peroxidase-antiperoxidase complex from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), and
diaminobenzidine from Aldrich (Milwaukee, WI). PIAS1
antiserum was raised in rabbit using purified GST-PIAS1 amino acids
549650 as antigen and tested for specificity by immunoblotting as
described above. The optimal dilution of antiserum was 1:1000.
Immunohistochemical controls included serial dilutions of the primary
antiserum and preabsorption of the antiserum with purified antigen.
Rabbit antibody to the AR (optimal dilution 2 µg/ml) was a generous
gift from Dr.Gail Prins, University of Chicago, Chicago, IL.
Transient Cotransfection Assay
Cotransfection assays using monkey kidney CV1 cells were
performed as previously described (98 ). In brief, 2.5 µg MMTV long
terminal repeat-luciferase vector (MMTV-LUC), 0.1 µg human AR
expression vector (pSGhAR), human glucocorticoid receptor vector
(pSG5GR), or human progesterone receptor (pSG5hPRB) and various amounts
of PIAS1 or other vectors were cotransfected into 7580% confluent
CV1 cells in 6-cm culture dishes using the CaPO4
method. After 15% glycerol shock for 4 min, cells were incubated in
DMEM-H without phenol red and serum in the presence or absence of
steroid for 40 h. Cells were harvested in lysis buffer
(Ligand Pharmaceuticals, Inc., San Diego, CA), and
luciferase activity was measured in a luminometer (54 ). Luciferase
activity is expressed as mean ± SE light units of three
replicates and as fold increase in the presence of hormone over
background in the absence of hormone. Assay results shown in each
figure are representative of three or more experiments.
 |
ACKNOWLEDGMENTS
|
---|
Cell culture and cotransfections were performed in the
Tissue Culture Core of the Laboratories for Reproductive Biology (LRB)
with the excellent technical assistance of De-Ying Zang and Michelle
Cobb. Immunohistochemical assays were performed in the Immunotechnology
Core of the Laboratories for Reproductive Biology. Thanks to Elizabeth
M. Wilson for reagents, valuable discussions, and critical reading of
the manuscript. We thank Ronald Evans for the MMTV-luciferase reporter
vector, Pierre Chambon for the progesterone receptor expression vector,
hPRB, and Gail Prins for the AR antibody.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Frank S. French M.D., Laboratories for Reproductive Biology, University of North Carolina School of Medicine, Campus Box 7500, Chapel Hill, North Carolina 27599-7500.
This work was supported by NIH Grants R37 HD-04466 (F.S.F.), T32
HD-07315 (J.-A.T.), AI 43438 (K.S.), the Andrew W. Mellon Foundation,
and by NICHD/NIH through cooperative agreeement U54 HD-35041 as part of
the Specialized Cooperative Centers Program in Reproduction Research,
K.S. is a V Foundation scholar.
Received for publication July 14, 1999.
Revision received September 25, 1999.
Accepted for publication September 29, 1999.
 |
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