Protein Inhibitor of Activated STAT-1 - CiteSeerX

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

An androgen receptor (AR) interacting protein was isolated from a HeLa cell cDNA library by twohybrid screening in yeast using the AR DNA1ligand 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 DNA1ligand binding domains was androgen dependent in the yeast liquid b-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 544–634) 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-terminal1DNA 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 25–30 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. (Molecular Endocrinology 14: 14–26, 2000)

INTRODUCTION The androgen receptor (AR) is expressed in Sertoli cells of the seminiferous epithelium, in peritubular myoid cells and in interstitial cells of Leydig (1–3), indicating that androgen stimulation of spermatogenesis (1, 4–6) 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 liganddependent nuclear receptors that form dimers in complex with specific nucleotide sequences (7, 8) to function as transcription factors (9–12). Nuclear receptors have conserved DNA- and ligand-binding domains with similar three-dimensional structures (13–18). Nterminal and ligand-binding domains contain transcriptional activation regions designated AF1 and AF2, respectively (19–25); 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,

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Androgen Receptor Coactivator in Testis

and general transcription factors (26–31). 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 (33–43). 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 g-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 1–6 (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

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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.

RESULTS Isolation of PIAS1/GBP To identify proteins that interact with human AR, yeast two-hybrid screening was performed using the AR DNA1ligand-binding domain peptide (amino acids 481–919) expressed in frame with the yeast Gal4 DNA-binding domain peptide. Approximately 5 3 106 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 b-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). AR-PIAS1 Interaction in Yeast Is Androgen Dependent Androgen dependency of the AR interaction with PIAS1 was analyzed in the yeast liquid b-galactosidase assay with increasing concentrations of DHT or testosterone (T). Yeast strain Y190 transformed with pBDGAL-AR 481–919 and pGADGH-PIAS1 was grown in selective medium with or without androgen. At 0.01 mM DHT, the AR-PIAS1 interaction stimulated a 5-fold increase in b-galactosidase activity over the control with no steroid added while 0.01 mM T stimulated a 2-fold increase (Fig. 2A). b-Galactosidase activities increased to a maximum 8-fold at 1 mM DHT and T. The results indicate that the ARPIAS1 interaction in yeast is androgen dependent. Ligand specificity of the AR-PIAS1 interaction in yeast was tested with different steroid hormones (Fig. 2 B). b-Galactosidase activity was increased about 2-fold at 1 mM progesterone (P), and at the same concentration estradiol (E2), dehydroepi-

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Fig. 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). F, 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.

androsterone (DHEA), dexamethasone (DEX), or hydroxyflutamide (OH-FL) did not increase b-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).

Androgen-dependent binding of full-length AR and PIAS1 (amino acids 5–651) was tested in the yeast reversed two-hybrid liquid b-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 mM) stimulated a 4.6-fold increase in b-galactosidase activity that did not increase further at higher concentrations of DHT up to 1.0 mM (Fig. 2 C). T (0.01 mM) stimulated a 3.4-fold increase in b-galactosidase activity that increased to 6.5-fold at 1.0 mM, indicating full-length AR


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Fig. 3. Direct Interaction of AR and PIAS1 in Vitro Binding of [35S]PIAS1 and PIAS1 peptide fragments to GST-AR amino acids 544–634 (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 1–651 (lanes 1 and 2), amino acids 1–318 (lanes 3 and 4), amino acids 318–651 (lanes 5 and 6), and amino acids 1–73 contiguous with 564–651 (lanes 7 and 8) GST control (2) GST-AR 544– 634 (1).

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 1–651) bound glutathione-S-transferase (GST)AR (amino acids 544–634) (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 1–318 had AR binding activity similar to that of full-length PIAS1, while binding of the C-terminal peptide 318–651 was not increased over the GST-glutathione-Sepharose control (Fig. 3). It should be noted that peptide fragment 1–318 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 1–73 contiguous with 564–651 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 acti-

Fig. 2. Androgen Dependence of PIAS1 Binding to AR Amino Acids 481–919 (DNA and Ligand Binding Domains) in a Yeast Two-Hybrid Liquid b-Galactosidase Assay

A, Androgen dependence of PIAS1 binding to AR amino acids 481–919. B, Steroid specificity of AR 481–919 activation for PIAS1 binding. C, Androgen dependence of PIAS1 binding to full-length AR in a reverse two-hybrid liquid b-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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vation 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 318–651 to GST-glutathione-Sepharose may have obscured a low level of binding to AR 544–634 in this system. 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, PIASxa, PIASxb, 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 1587– 2101. 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

Fig. 4. Northern Hybridization of PIAS1 mRNA in Primate And Rodent Male Reproductive Tract Using a Specific 32PLabeled PIAS1 Probe (Nucleotides 1587–2101) A, Poly (A) RNA (5 mg per lane) isolated from human testis (58-yr-old) and epididymis (62-yr-old). B, Total RNA (10 mg 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 mg per lane) from testes of Sprague Dawley rats 20–75 days of age.

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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). Expression of PIAS1 protein was localized in human and mouse testis by immunostaining using antiPIAS1-specific antibodies raised against the C-terminal region of PIAS1 (amino acids 549–650) (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. 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 mg) (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 1–5 mg 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 mg and was lower at 1.0 mg/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 PIASxa, another member of the PIAS family, that AR coactivator function decreased with transfection of larger amounts of the PIASxa expression vector (48, 58). In the process of screening a human testis cDNA library for full-length PIAS1 cDNAs, a mutant fulllength PIAS1 containing an in-frame 588-bp internal deletion (codons 341–536) was isolated and cloned into pSG5 as described in Materials and Methods. This


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Fig. 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 549–650 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: 3143. 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: 3143. C, In the human testis, AR antibody labeled nuclei of Sertoli cells (arrowheads), Leydig cells, peritubular myoid cells and vascular smooth muscle cells. Magnification: 3550. D, PIAS1-immunoreactivity in within the human seminiferous tubule is present in nuclei of Sertoli cells (arrowheads), spermatocytes, and round spermatids. Magnification: 3550.

mutant did not enhance the DHT-dependent AR increase in luciferase activity (Fig. 7A). 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 Nterminal and DNA-binding domain fragment (amino acids 1–660). Because it lacks the ligand-binding domain, this AR fragment is constitutively active in tran-

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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 progesteroneinduced PR transactivation (Fig. 8B). In the same assay system using 0.1 mg PR expression vector (pRSVhPRB) 2/1 10 nM progesterone, ligand-dependent PR stimulation of luciferase activity in the presence of pSG-PIAS1 (0.1–1.0 mg) was 6–10 times lower than the control with PR and empty pSG5 parent vector.

DISCUSSION Fig. 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 mg, and MMTV-luciferase, 2.5 mg/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.

sient cotransfection assays. We tested the effect of PIAS1 on the transcriptional activity of AR (1–660) 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 mg) but was unchanged by an equal amount of the PIAS1 antisense vector (Fig. 7C). Since PIAS1 interacted with the AR DNA and ligandbinding 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) transactivation using the MMTV-luciferase reporter. PIAS1 1 pSGhGR in the presence of DEX (10 nM) induced a

The results indicate PIAS1 can function as an AR or GR coactivator and PR repressor. PIAS1 exhibited androgen-dependent binding to an AR DNA1ligand 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 1–318, 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 terminus1DNA-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 Cterminal 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 (1–6) 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 (67–69). STAT-1 phosphorylation on Tyr 701 induces dimerization of monomers, nuclear translocation, and sequence-specific DNA binding (48–50). The cytokines interleukin-1, interleukin-6 (IL-6), and tumor necrosis factor-a are secreted by testicular macrophages and modulate gonadotropin effects on Leydig cells and Sertoli cells


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(70). Interleukin-1 is also produced in immature rat germ cells (71) and stimulates stage-specific DNA synthesis in seminiferous tubules in vitro (72). Leukemiainhibitory 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 (73–75) 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 proteinprotein 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 PIASxa, PIASxb, and PIASy. A mutant PIASxb with deletion of amino acids 1–133 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 PIASxa. 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.

Fig. 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 mg/6-cm dish, was used, and similar results were obtained with the pSG-PIAS1 vector, 0.5 mg/6 cm dish. A, Deletion of PIAS1 amino acids 341–536 abolished enhancement of AR transactivation. CONTROL: 0.1 mg pSGAR, 2.5 mg MMTV-luciferase, 60.1 nM DHT. ANTISENSE: control 1 3 mg pSG-antisense PIAS1. SENSE: control 1 3 mg

pSG(l)-PIAS1. MUTANT: control 1 3 mg 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 6E2, P, DEX, or OH-FL 2. ANTISENSE: control 1 3 mg pSG-antisense PIAS1. 3. SENSE: control 1 3 mg pSG(l)-PIAS1. C, PIAS1 enhanced the constitutive transcriptional activity of AR N-terminal and DNA binding domains (amino acids 1–660). CONTROL: 10 ng pCMV-AR amino acids 1–660, 2.5 mg MMTV-luciferase, 60.1 nM DHT. ANTISENSE: control 1 3 mg pSG-antisense PIAS1. SENSE: control 1 3 mg PIAS1. Numbers in parentheses indicate the fold increase over background in the absense of DHT.

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Fig. 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 mg pSG-hGR [plusm] 10 nM DEX and 3 mg 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 mg pSG-PIAS1 was used instead of pSG(l)-PIAS1. B, PIAS1 repressed progesteronedependent PR transactivation in transient cotransfection assays. Each 6-cm dish contained 0.1 mg pSG5-hPRB [plusm] 10 nM progesterone and variable amounts (mg) 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 mg pSG(l)-PIAS1 compared with the same amount of pSG antisense-PIAS1.

Gu RNA helicase II is a member of the Asp-Glu-AlaAsp (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 (78–80). 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

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(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, 86–90). 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 481–919 using primers 59-GGCCGAATTCGGCTACACTCGGCCCCCTCA-39, and 59GGCCGAATTCTCACTGTGTGTGGAAATAGATGGGC-39; the PCR fragment was digested with EcoRI and cloned into the yeast b-galactosidase DNA-binding domain vector pBDGAL4 CAM (Stratagene, La Jolla, CA) to create the plasmid pBDGAL-481–919. Mammalian Cell Expression Vectors pSG5 (Stratagene) was modified by insertion of a double-stranded oligonucleotide: 59AATTATGAATTCACTAGTGATATCGGATCCGGTACCTCGAGA-39 containing restriction sites for EcoRI-SpeIEcoR5-BamHI-KpnI-XhoI. Since pGADGH-PIAS1 from the two hybrid screen lacked the four NH2-terminal codons, fulllength cDNAs were obtained from a human testis cDNA library constructed in Lambda ZAP. Primers containing EcoRI restriction sites at the 59-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 1–318 was created in pSG5-PIAS1 by digestion with BamHI and another with deletion of amino acids 318–651 by PCR. The expression vector pCMVAR (amino acids 1–660) that expresses the constitutively active AR N-terminal 1 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 481–919 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 mM DHT and 5 mM 3-amino-1,2,4 triazole (94). Yeast colonies were assayed for b-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 mM). To confirm the androgen dependence of the interaction with AR, liquid b-galactosidase assays were performed with selected clones. Yeast Liquid b-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,

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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 b-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 544–634) and cultured overnight at 37 C were diluted 1:10 in fresh LB medium and incubated with shaking. After 2 h, isopropyl-b-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 ml 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, 20l, was mixed with washed beads and incubated 1 h at 4 C and again washed three times with PBS buffer. SDS buffer (30 ml) 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 280 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 (53 SSC, 53 Denhardt’s solution, 1% SDS, and 100 mg/ml salmon sperm DNA) overnight at 68 C. After washing at 50 C for 1 h in 0.13 SSC containing 0.1% SDS, the membranes were placed in a cassette with intensifying screen to expose X-OMAT film (Eastman Kodak Co.) at 280 C. Immunoblotting Anti-PIAS1 polyclonal antibody against a glutathione transferase-PIAS1 fusion protein containing 102 C-terminal amino acids (549–650) of PIAS1 was raised in rabbit (J. Liao and K. Shuai, unpublished). To test the specificity of the antibody, PIAS1, PIASxa, PIASxb, 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

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and testis from an adult mouse were processed similarly. The human subject had received no therapy before orchiectomy. Testicular tissue was fixed in Bouin’s fluid and embedded in paraffin using standard procedures. Sections 8 mm thick were cut and mounted on glass slides. Before immunostaining, endogenous peroxidase was blocked (methanol 1 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 549– 650 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 mg/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 mg MMTV long terminal repeat-luciferase vector (MMTV-LUC), 0.1 mg 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 75–80% 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 6 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.

Received July 14, 1999. Re-revision received September 25, 1999. Accepted September 29, 1999. 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.

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