ARIP3 (Androgen Receptor-Interacting Protein 3) and Other PIAS (Protein Inhibitor of Activated STAT) Proteins Differ in Their Ability to Modulate Steroid Receptor-Dependent Transcriptional Activation

Noora Kotaja, Saara Aittomäki, Olli Silvennoinen, Jorma J. Palvimo and Olli A. Jänne

Department of Physiology (N.K., J.J.P., O.A.J.) Institute of Biomedicine University of Helsinki FIN-00014 Helsinki, Finland
Department of Clinical Chemistry (O.A.J.) University of Helsinki FIN-00290 Helsinki, Finland
Department of Medical Biochemistry (S.A., O.S.) University of Tampere and Tampere University Hospital FIN-33014 Tampere, Finland


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid receptors mediate their actions by using various coregulatory proteins. We have recently characterized ARIP3/PIASx{alpha} as an androgen receptor (AR)-interacting protein (ARIP) that belongs to the PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] protein family implicated in the inhibition of cytokine signaling. We have analyzed herein the roles that four different PIAS proteins (ARIP3/PIASx{alpha}, Miz1/PIASxß, GBP/PIAS1, and PIAS3) play in the regulation of steroid receptor- or STAT-mediated transcriptional activation. All PIAS proteins are able to coactivate steroid receptor-dependent transcription but to a differential degree, depending on the receptor, the promoter, and the cell type. Miz1 and PIAS1 are more potent than ARIP3 in activating AR function on minimal promoters. With the natural probasin promoter, PIAS proteins influence AR function more divergently, in that ARIP3 represses, but Miz1 and PIAS1 activate it. Miz1 and PIAS1 possess inherent transcription activating function, whereas ARIP3 and PIAS3 are devoid of this feature. ARIP3 enhances glucocorticoid receptor-dependent transcription more efficiently than Miz1 or PIAS1, and all PIAS proteins also activate estrogen receptor- and progesterone receptor-dependent transcription but to a dissimilar degree. The same amounts of PIAS proteins that modulate steroid receptor-dependent transcription influence only marginally transactivation mediated by various STAT proteins. It remains to be established whether the PIAS proteins play a more significant physiological role in steroid receptor than in cytokine signaling.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The transcriptional activity of steroid receptors relies not only on their ability to enter the nucleus and bind DNA but also on their interactions with other transcription factors and a number of coregulator protein complexes (1, 2, 3). Coregulators encompass coactivators (e.g. Refs. 4, 5, 6, 7, 8, 9, 10, 11, 12), corepressors (e.g. Refs. 13, 14, 15), cointegrators (e.g. Refs. 16, 17), and mediator protein complexes (e.g. Refs. 18, 19). In view of the central role of steroid receptors in the regulation of cell growth, differentiation, and homeostasis, this large number of coregulatory proteins is perhaps not surprising. Most of the auxiliary proteins may interact with multiple signaling systems, and the same coregulators can be used by diverse classes of signal-inducible transcription factors.

ARIP3 (androgen receptor-interacting protein 3) is a steroid receptor coregulator found in a yeast two-hybrid screen with the androgen receptor (AR) zinc finger region (ZFR) as a bait (20). ARIP3 belongs to a novel family of nuclear proteins that also includes Miz1 (Msx-interacting zinc finger), GBP (Gu/RNA helicase II-binding protein), PIAS1 (protein inhibitor of activated Stat1) and PIAS3. These proteins are reported to modulate functions of very different transcription factors. Mouse Miz1 interacts with homeodomain-containing Msx2 protein and may enhance its DNA binding (21). PIAS1 and PIAS3 bind to Stat1 (signal transducer and activator of transcription 1) and Stat3, respectively, and inhibit STAT-mediated signaling by perturbing with DNA binding of Stat1 and Stat3 (22, 23). Human GBP, which is almost identical to PIAS1, was isolated in a yeast two-hybrid screen with Gu/RNA helicase II as a bait (24). Recently, Tan et al. (25) identified PIAS1 as a steroid receptor coregulator through an approach similar to the approach that we used for ARIP3. It is worth pointing out in this context that, similar to ARIP3, the expression of PIAS1 was mainly confined to the testis (25). Additional PIAS sequences (PIASx{alpha}, PIASxß, and PIASy) were found in a cDNA library screen with PIAS1 cDNA (22). Human PIASx{alpha} corresponds to rat ARIP3, and PIASxß is the human counterpart of mouse Miz1.

Even though members of the PIAS protein family were identified through interaction with very dissimilar signaling molecules, their high sequence conservation predicts similar functions. In view of this, we have compared the ability of different PIAS proteins to influence the transactivation mediated by AR, glucocorticoid receptor (GR), progesterone receptor (PR), and estrogen receptor {alpha} (ER{alpha}) and ß (ERß). We report herein that the PIAS family members do indeed interact with steroid receptors and modulate (i.e. activate or repress) their function in a fashion that is dependent on the promoter and the cell type. Under the experimental conditions used in our studies, the effects of PIAS proteins on transcriptional activation mediated by different STAT proteins were minor in comparison with those on steroid receptors.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PIAS Proteins Modulate AR-Dependent Transactivation in a Distinct Fashion
Sequence comparison of ARIP3 and PIAS family members examined in this study is shown in Fig. 1AGo. The relative levels of FLAG-tagged proteins encoded by ARIP3 and PIAS expression vectors were similar in HeLa and HepG2 cells (Fig. 1Go, B and C). The effects of ARIP3, Miz1, PIAS3, and PIAS1 on AR-dependent transactivation were first studied in HeLa cells by cotransfecting a constant amount of AR expression construct along with increasing amounts (2, 10, and 20 ng) of expression vectors encoding the PIAS proteins. When a reporter gene driven by two androgen response elements (AREs) in front of E1b TATA sequence (ARE2TATA-LUC) was used, low amounts of ARIP3 activated AR-dependent transcription up to approximately 3-fold, but the effect vanished with increasing amounts of ARIP3 (Fig. 2AGo). Even though ARIP3 and Miz1 differ only in their very C-terminal 22 and 71 amino acids, respectively, the two proteins displayed distinct actions on AR function. Whereas ARIP3 activated the ARE2TATA promoter approximately 3-fold, Miz1 enhanced AR-dependent transcription up to about 8-fold in a dose-dependent fashion. The effect of PIAS1 on AR-mediated transactivation was comparable to that of Miz1, and PIAS3 displayed a dose-response curve similar to that of ARIP3. Comparable results were obtained in COS-1 cells. ARIP3 or PIAS proteins did not influence the reporter gene activity in the absence of hormone or alter the amount of AR in transfected cells (data not shown).



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Figure 1. Sequence Comparison of the PIAS Family Members

A, Comparison of rat ARIP3 (PIASx{alpha}), mouse Miz1 (PIASxß), mouse PIAS1, and mouse PIAS3 amino acid sequences. Gaps in the sequence are shown by dashes. Black boxes and gray shadings depict amino acids that are identical or conserved among the sequences, respectively. B, Immunoblot analysis of proteins encoded by the following expression vectors in HeLa cells: empty pFLAG-CMV2 (lane 1), pFLAG-ARIP3 (lane 2), pFLAG-Miz1 (lane 3), pFLAG-PIAS3 (lane 4), and pFLAG-PIAS1 (lane 5). HeLa cells were transfected by the FuGene reagent with expression vectors (200 ng DNA/well, 12-well plate) and cultured for 48 h. Whole-cell extracts were resolved by SDS-PAGE and immunoblotted using monoclonal M2 antibody against the FLAG epitope. C, Corresponding immunoblot analysis of proteins expressed in HepG2 cells.

 


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Figure 2. PIAS Family Members Differ in Their Ability to Potentiate AR-Dependent Transcription

A, Modulation of transactivation from minimal ARE2TATA promoter. HeLa cells cultured on 12-well plates were transfected with 200 ng of pARE2TATA-LUC reporter, 20 ng of pSG5-rAR, 20 ng of pCMVß, and increasing amounts (2 ng, 10 ng, and 20 ng) of pFLAG-ARIP3, pFLAG-Miz1, pFLAG-PIAS3, or pFLAG-PIAS1 in the presence (+) or absence (-) of 100 nM testosterone (T). Total amount of DNA was kept constant by adding empty pFLAG-CMV2 as needed. After normalization for transfection efficiency using ß-galactosidase activity, reporter gene activities are expressed relative to those of rAR + T without a coregulator (=1.0). B, Modulation of transactivation from the natural probasin (PB) promoter. The experimental conditions were the same as in panel A, except that pPB(-285/+32)-LUC reporter was used. C and D, Effects of PIAS proteins on AR-dependent transcriptional activation in HepG2 cells from the ARE2TATA promoter and the probasin promoter, respectively. HepG2 cells were cultured on 12-well plates and transfected with same amounts of plasmids as described in panel A. The values represent means ± SD from three to six independent experiments.

 
Immunoblot analysis showed that ARIP3 is expressed to a level somewhat higher than that of Miz1 or PIAS1 (Fig. 1BGo). Since low amounts of ARIP3 activated AR function but higher levels were inhibitory (Fig. 2AGo), experiments were also performed with lower amounts of expression vectors; 0.5 ng of ARIP3 plasmid enhanced AR-dependent transcription by 1.5-fold, 1 ng of ARIP3 led to an approximately 2.5-fold increase, and 5 ng of ARIP3 attenuated the maximal activation. In the case of Miz1, 0.5 and 1 ng of Miz1 plasmid exhibited marginal effects, and 5 ng stimulated AR-dependent transcription by about 4-fold (data not shown).

With the more complex probasin promoter, cotransfection with 10 ng and 20 ng of ARIP3 repressed AR-dependent transactivation, whereas Miz1 and PIAS1 enhanced it by approximately 2.5- to 3-fold (Fig. 2BGo). PIAS3 behaved in a fashion similar to that of ARIP3, in that it repressed the transcription at the highest dose. ARIP3 or PIAS proteins did not influence probasin promoter activity in the absence of hormone (data not shown).

The effects of PIAS proteins on AR-dependent transactivation were also studied in HepG2 cells (Fig. 2CGo). Interestingly, the PIAS proteins activated AR function on the minimal ARE2TATA promoter to a similar degree; maximal induction was 4- to 5-fold by ARIP3, Miz1, and PIAS1, and about 7-fold by PIAS3. In contrast to HeLa or COS-1 cells, ARIP3 or other PIAS proteins failed to repress the probasin promoter in HepG2 cells; rather, they all activated AR-dependent transcription and PIAS3 was the most potent activator (~6-fold activation) (Fig. 2DGo). As shown in Fig. 1Go, relative expression levels of the PIAS proteins in HepG2 cells were comparable to those in HeLa cells, and therefore, cell line-dependent differences in their activities are not due to different protein levels. In sum, the PIAS proteins modulate AR-dependent transcription in a cell line- and promoter-dependent fashion.

Miz1 and PIAS1 Possess Intrinsic Transcription-Activating Functions
To examine whether the ability of PIAS proteins to enhance AR-dependent transcription is explainable by differences in their intrinsic transcription-activating functions, ARIP3, Miz1, PIAS3, and PIAS1 were fused to Gal4 DNA-binding domain (Gal4) and transfected to HeLa cells with a reporter construct driven by five Gal4-binding sites (G5-LUC) (Fig. 3AGo). Gal4-Miz1 activated the reporter gene by 23-fold and Gal4-PIAS1 by 10-fold [compared with the activity of Gal4 DNA-binding domain (DBD) alone], indicating the presence of transcription-activating regions in these proteins. If anything, Gal4-ARIP3 and Gal4-PIAS3 fusion proteins repressed the promoter activity. In HepG2 cells, the relative activities of Gal4-Miz1 and Gal4-PIAS1 were lower than in HeLa cells, and Gal4-ARIP3 and Gal4-PIAS3 again repressed Gal4 DBD activity by 40% and 70%, respectively (Fig. 3BGo). The use of lower or higher amounts (100–250 ng) of expression plasmids yielded essentially identical results, in that only PIAS1 and Miz1 exhibited intrinsic transcription-activating function (data not shown). This presence of transcription activation regions in PIAS1 and Miz1 may, at least in part, explain their differential ability to stimulate AR-dependent transcription in HeLa cells, but it does not apply to HepG2 cells.



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Figure 3. Miz1 and PIAS1 Contain Intrinsic Transcription-Activating Functions

A, Activation of transcription by Gal4 DBD fusion proteins in HeLa cells. Cells were transfected with 200 ng of pG5-LUC reporter (containing five Gal4-binding sites in front of the minimal TATA box), 20 ng of pCMVß, and 150 ng of Gal4 DBD (Gal4) fusion constructs Gal4-ARIP3, Gal4-Miz1, Gal4-PIAS3, or Gal4-PIAS1. After normalization for transfection efficiency using ß-galactosidase activity, reporter gene activities are expressed relative to that of Gal4 DBD alone (=1.0). B, Activation of transcription by Gal4 DBD fusion proteins in HepG2 cells. The experimental conditions were the same as those described in panel A. The values are means ± SD from three independent experiments.

 
PIAS Proteins Activate and Repress GR Function in a Promoter-Dependent Fashion
HeLa and HepG2 cells were used to study the influence of PIAS proteins on GR-dependent transcription. Hormone response elements in the ARE2TATA promoter also mediate GR-dependent signaling (26). Coexpression of ARIP3 with GR enhanced GR-dependent transcription >=30-fold from the minimal promoter in HeLa cells (Fig. 4AGo), which is 10 times more than that with AR. On the other hand, Miz1 or PIAS1 stimulated transcriptional activity of GR to the same extent as that of AR. In contrast to AR, PIAS3 was as efficient as PIAS1 or Miz1 in coactivating the function of GR. Interestingly, when GR-dependent transcription from the minimal promoter was examined in HepG2 cells, the differences between PIAS proteins diminished, and they all activated GR function by 5- to 6-fold, which is comparable to that of AR in the same cells (c.f., Figs. 2CGo and 4BGo). Thus, PIAS proteins possess steroid receptor selectivity in their coregulatory properties, but this selectivity is dependent on the cell context.



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Figure 4. Modulation of GR-Dependent Transcription by PIAS Proteins

A and B, Effects of cotransfection of PIAS proteins with GR (pSG5-hGR) on the ARE2TATA promoter activity in HeLa cells (A) and in HepG2 cells (B). Experimental conditions were same as those in Fig. 2AGo, except that GR-dependent transcription was activated by exposure to 100 nM dexamethasone (DEX). C, The same experiment was performed in HeLa cells using pHH-LUC reporter, which contains region -203/+105 of the mouse mammary tumor virus promoter in front of the LUC gene. The values represent means ± SD from three to six independent experiments.

 
When HH-LUC containing the mouse mammary tumor virus promoter was used as the reporter, both ARIP3 and PIAS3 caused a dose-dependent repression of GR-dependent transactivation in HeLa cells (Fig. 4CGo). These findings resemble those of ARIP3 and PIAS3 on AR with the probasin promoter. By contrast, Miz1 and PIAS1 did not modulate GR-dependent transcription from HH-LUC. The effect of GR on this latter promoter in another cell type, CV-1 cells, has been reported to be activated 2-fold by coexpression of PIAS1 (25).

ARIP3 was originally identified by an interaction screen using AR ZFR (AR DBD plus one-third of the hinge region) as the bait (20). Since the influence of PIAS proteins differed on AR- and GR-dependent transcription, it was pertinent to determine whether the DBDs were mainly responsible for their dissimilar responses. To study this possibility, receptor chimeras GAG (GR DBD is replaced with AR DBD) and AGA (AR containing GR DBD) (27) were examined in cotransfections with ARIP3 and Miz1 along with ARE2TATA-LUC reporter. As shown in Fig. 5Go, ARIP3 and Miz1 influenced chimeric GAG receptor function in a fashion identical with that of wild-type GR. Likewise, AR and AGA responded to coexpressed PIAS proteins in a comparable manner. Thus, the receptor selectivity in the action of these two PIAS proteins appears to require receptor regions outside the DBDs.



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Figure 5. Effects of ARIP3 and Miz1 on Chimeric Forms of AR and GR

A, Cotransfections of ARIP3 and Miz1 with rGR. B, Cotransfections with the GAG chimera (rGR DBD is replaced with that of AR). C, Cotransfections of ARIP3 and Miz1 with mAR. D, Cotransfections with the AGA chimera (=mAR containing GR DBD). HeLa cells were transfected with 200 ng of pARE2TATA-LUC, 20 ng of pCMV5-rGR, pCMV5-GAG, pCMV5-mAR, or pCMV5-AGA (panels A–D, respectively), 20 ng of pCMVß, and increasing amounts (2 ng, 10 ng, and 20 ng) of pFLAG-ARIP3 or pFLAG-Miz1, in the presence (+) or absence (-) of 100 nM testosterone (T) or dexamethasone (DEX) under the experimental conditions described in Fig. 2Go. Mean ± SD values from three independent experiments are shown.

 
PIAS Proteins Modulate PR-, ER{alpha}-, and ERß-Dependent Transactivation
To study whether PIAS proteins modulate steroid receptor function more generally, PR, ER{alpha}, and ERß were coexpressed with PIAS proteins in HeLa cells, and the activities of their cognate minimal promoters were monitored. All PIAS family members were able to enhance ligand-dependent transactivation by PR, but they modulated PR function in a manner clearly different from that of GR; ARIP3 and Miz1 activated PR function to a similar degree (~6-fold increase) that exceeded the effect of PIAS1 or PIAS3 (max. ~3-fold stimulation) (Fig. 6Go). It is of note that with a more complex promoter (the mouse mammary tumor virus promoter), Tan et al. (25) found PIAS1 to repress PR-dependent transactivation. In the case of ER{alpha}, the activities of PIAS proteins were similar, and they all elicited 2- to 3-fold maximal stimulation of transcription, whereas with ERß, Miz1 activity exceeded that of other PIAS proteins (Fig. 6Go, B and C). Modulatory effects of PIAS proteins on thyroid hormone receptor-dependent transcription were minor in comparison to those of the five steroid receptors examined in this study (data not shown).



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Figure 6. PIAS Proteins Influence the Transactivation Mediated by PR, ER{alpha}, and ERß

A, Effects of increasing amounts (2, 10, and 20 ng) of the PIAS family members on progesterone-dependent transcription mediated by PR (pSG5-hPR) from the ARE2TATA promoter. B and C, Influence of the PIAS proteins on estradiol-induced transcription mediated by ER{alpha} (pSG5-hER{alpha}) and ERß (pSG5-hERß), respectively, from the ERE2TATA promoter. Experimental conditions and the amounts of expression plasmids used were the same as in the experiments with AR described in the legend to Fig. 2Go, except that the cells were treated with 100 nM progesterone (P) or 10 nM estradiol (E) as indicated. Mean ± SD values from three independent experiments are shown.

 
PIAS Proteins Interact with Steroid Receptors in Vitro and in Vivo
Physical interaction of steroid receptors with PIAS proteins was examined by glutathione S-transferase (GST) pull-down experiments. GST-ARIP3 and GST-Miz1 bound to glutathione sepharose were incubated with [35S]methionine-labeled receptors synthesized by translation in vitro. All receptors studied were capable of interacting with GST-Miz1 and GST-ARIP3 in vitro (Fig. 7Go, A and B, and data not shown). The interactions were specific, as the receptors failed to adhere markedly to GST alone, and no binding of a control protein, luciferase, was observed under the conditions used. The interactions were largely hormone independent, as comparable amounts of proteins were bound without the cognate ligand, as illustrated for rAR-Miz1 and ER{alpha}-Miz1 interactions in Fig. 7CGo. Other steroid receptors behaved the same way with Miz1 and ARIP3 (data not shown). This was in contrast to the interaction of AR and ER{alpha} with amino acid residues 563–1,121 of glucocorticoid receptor interacting protein 1 (GRIP1) (GRIP1b) fused to GST, which was clearly ligand-enhanced under the same in vitro conditions (Fig. 7CGo). Overall, the interactions of ARIP3 and Miz1 with the five steroid receptors were quite similar; ER{alpha} and ERß bound somewhat more efficiently to GST-Miz1 and GST-ARIP3 than the other receptors. In any event, receptor selectivity of the PIAS proteins in transactivation assays does not seem to be caused by their markedly dissimilar in vitro binding affinities for PIAS proteins.



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Figure 7. Miz1 and ARIP3 Interact with Steroid Receptors in Vitro

A, AR, GR, PR, ER{alpha}, and ERß were labeled with [35S]methionine by translation in vitro and incubated with Glutathione Sepharose-bound GST or GST-Miz1. Bound proteins were eluted with the SDS sample buffer, resolved by SDS PAGE, and visualized by fluorography. Lane 1 of each panel represents 5% of the labeled protein incubated with the matrix. Testosterone, dexamethasone, progesterone, or estradiol (each 1 µM) was included in binding reactions with AR, GR, PR, or ER, respectively. B, The corresponding pull-down experiments were performed by using GST-ARIP3 fusion protein except that hPR was not included. C, [35S]Methionine-labeled AR and ER{alpha} were synthesized by translation in vitro and incubated with Glutathione Sepharose-bound GST, GST-Miz1, or GST-GRIP1b with (+) or without (-) 1 µM testosterone (T) or estradiol (E) as indicated. The conditions were otherwise identical with those in panel A. Lane 1 represents 5% of the input.

 
Interactions of the PIAS proteins with AR were also compared by using a mammalian two-hybrid system in HeLa cells (Fig. 8Go). Full-length ARIP3 and PIAS proteins fused to VP16 activation domain (VP16) were cotransfected with expression vector encoding Gal4 DBD-AR (Gal4-AR) and G5-LUC reporter. All PIAS proteins interacted with AR in this assay, in that they increased reporter gene activity over that of Gal4-AR and polyoma virus coat protein fused to VP16 (VP16-CP). ARIP3 and Miz1 were more active than PIAS3 and PIAS1. Direct comparison of their potencies, however, is hampered by dissimilar expression levels of the VP16 fusion proteins; VP16-PIAS1 and VP16-PIAS3 were expressed to levels lower than those of VP16-ARIP3 and VP16-Miz1 fusions (data not shown). ARIP3-AR interaction was detectable already in the absence of hormone, but the presence of androgen increased it markedly (Fig. 8Go). Miz1-apo-AR interaction was minor compared with that of ARIP3 with apo-AR. It was, however, greatly enhanced by the hormone (>=30-fold induction), whereas the effect of androgen was intermediate on other PIAS proteins. Thus, in contrast to cell-free conditions, the interaction of AR with PIAS proteins in intact cells is highly hormone dependent.



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Figure 8. Interaction of AR with PIAS Proteins in Mammalian Cells

Interaction of AR with ARIP3 and other PIAS proteins was examined by a two-hybrid system in HeLa cells. The cells were transfected using the FuGene reagent with 200 ng of pG5-LUC, 20 ng of pCMVß, and 100 ng of Gal4 DBD fusion of AR (Gal4-AR) together with VP16 AD fusion of ARIP3 (VP16-ARIP3), Miz1 (VP16-Miz1), PIAS3 (VP16-PIAS3), and PIAS1 (VP16-PIAS1), or polyoma virus coat protein (VP16-CP) as indicated. Twenty hours after transfection, the medium was changed to one containing charcoal-stripped 2% FBS with (+) or without (-) 100 nM testosterone, and the cells were incubated for an additional 28 h. After normalization for transfection efficiency using ß-galactosidase activity, the reporter gene activities are expressed relative to that of Gal4 DBD alone (=1.0). Mean ± SD values from three independent experiments are shown.

 
Effects of PIAS Proteins on Transcriptional Activation Mediated by STATs
PIAS1 and PIAS3 are reported to function as specific inhibitors of Stat1 and Stat3 signaling, respectively (22, 23). To study whether ARIP3/PIAS proteins are involved in the regulation of STATs and cytokine signaling more generally, we examined the effects of ARIP3, PIAS1, and PIAS3 on STAT-dependent transcriptional activation by using the same or higher amounts of PIAS expression vectors than those in experiments on steroid receptor-dependent signaling.

The three PIAS proteins were first tested for their ability to regulate interferon-{gamma} (IFN-{gamma})-activated Stat1. HeLa cells were transfected with GAS-LUC reporter containing a Stat1-binding site from the IRF-1 promoter in front of the minimal tk promoter (28) and increasing amounts (2 ng, 10 ng, and 20 ng) of ARIP3, PIAS1, or PIAS3 expression vectors. The cells were treated with IFN-{gamma} or left untreated. Ectopic expression of ARIP3, PIAS1, or PIAS3, in the amounts used in the preceding studies on steroid receptor function, minimally influenced IFN-{gamma}-induced activation of GAS-LUC in HeLa cells (Table 1Go). However, when higher amounts of PIAS1 expression plasmid (30–90 ng) were used in HepG2 cells, a 20–30% decrease in IFN-{gamma}-induced activation of GAS-LUC reporter was observed (Fig. 9AGo). By contrast, even the higher amounts of ectopically expressed PIAS1 failed to perturb with the function of endogenous Stat1 in HeLa cells (Fig. 9BGo).


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Table 1. Influence of ARIP3, PIAS1, and PIAS3 on Transcriptional Activation Mediated by Stat1, Stat5, and Stat6 in HeLa Cells

 


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Figure 9. Influence of Ectopic Expression of PIAS1 on Stat1- and Stat5-Dependent Transcriptional Activation in HeLa and HepG2 Cells

HeLa and HepG2 cells were transfected using the FuGene reagent on 12-well plates with 200 ng of the indicated LUC reporter, 20 ng of pCMVß, and 20 ng of Stat5 expression vector together with the amounts of PIAS1 expression plasmid shown (in nanograms). With GAS-LUC, no exogenous Stat1 was transfected, and 30 ng of EpoR expression vector were cotransfected in Stat5 experiments. Twenty-four hours after transfection, the cells were cultured in the absence (-) or presence (+) of cytokines (10 ng/ml IFN-{gamma} or 4 U/ml Epo) for 24 h. Reporter gene activities were normalized by the ß-galactosidase activity and are expressed relative to that of the corresponding cytokine alone (=100, black bars). The values are means ± SD from three separate experiments.

 
We also tested whether ARIP3, PIAS1, and PIAS3 affect transcriptional activation brought about by ectopically expressed Stat5 or Stat6. HeLa cells were cotransfected with a Stat5-responsive Spi-LUC reporter driven by six repeats of a Stat5-binding site from the serine protease inhibitor 2.1 gene (29) and expression vectors encoding erythropoietin receptor (EpoR) and Stat5, as well as ARIP3, PIAS1, or PIAS3, and cells were treated with erythropoietin (Epo) or left untreated. Alternatively, HeLa cells were transfected with a Stat6-responsive fN{epsilon}N4-LUC reporter containing four repeats of a Stat6-binding site in front of the c-fos minimal promoter (30) and expression vectors for Stat6, as well as ARIP3, PIAS1, or PIAS3, and cells were exposed to interleukin-4 (IL-4) or left untreated. PIAS1 or PIAS3 had negligible effects on Epo- or IL-4-induced transcriptional activation mediated by Stat5 and Stat6, respectively (Table 1Go). ARIP3, on the other hand, up-regulated Stat6-mediated transactivation to a modest degree. Ectopic Miz1 expression with different STAT-responsive reporters did not influence STAT activities in HeLa cells (data not shown). Likewise, higher amounts (30 and 90 ng) of PIAS1 did not alter Stat5-dependent transactivation in HeLa or HepG2 cells (Fig. 9Go, C and D).

There is cross-talk between cytokine and glucocorticoid signaling (31), as exemplified by the synergistic activation of the Spi promoter by glucocorticoids and Epo (32). Even though ARIP3 is a powerful coactivator of GR function on glucocorticoid-dependent promoters (Fig. 4Go) and interacts with GR in vitro (Fig. 7Go), ectopically expressed ARIP3 did not influence significantly the synergism between Stat5 and GR in the activation of Spi-LUC reporter in HeLa cells (S. Aittomäki and O. Silvennoinen, unpublished).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The receptors for androgens, glucocorticoids, mineralocorticoids, and progesterone may recognize the same or similar DNA response elements and yet they regulate distinct target genes in vivo (33). In addition to the obvious ways to achieve steroid-specific gene activation, including cell-specific expression of the receptor protein and differential availability of the ligand, coregulatory proteins provide an additional level of control to ensure that appropriate responses to hormones are achieved. Although most coregulatory proteins identified to date are recognized by the conserved activation function-2 (AF-2) located in the C terminus of the ligand-binding domain (1, 2, 3, 4) and, in the case of AR, by the less well conserved AF-1 in the N-terminal region (34, 35), recent data indicate that also the DBD and hinge region of nuclear receptors present important interaction interfaces for coregulatory proteins. SNURF (36), Ubc9 (37), ANPK (38), TLS/FUS (39), HET/SAF-B (40), PCAF (11), N-CoR (15), SMRT (14), and also POU domain-containing proteins Oct-1/2 and Brn-3a/3b (41, 42) are among the proteins that can interact with the ZFRs and/or hinge regions of nuclear receptors. All of them are not necessarily involved in direct transcriptional control; instead, they may mediate other processes, such as nuclear targeting and intranuclear compartmentalization. In this regard, GR ZFR is shown to be required for the interaction with nuclear matrix (43), and SNURF is capable of modulating nuclear trafficking of AR (44).

ARIP3, a rat counterpart of human PIASx{alpha}, is among the proteins that interact with the AR ZFR/hinge region in vitro and in vivo and modulate AR-dependent transactivation in intact cells (20). It is predominantly expressed in testis, albeit lower ARIP3 mRNA levels are found in other tissues as well. Likewise, another family member, PIAS1, which has recently been reported to activate AR function, shows the highest expression in testis (25). In contrast to ARIP3 and PIAS1, PIAS3 is reported to be ubiquitously expressed (23). The PIAS proteins are relatively well conserved, as Drosophila genome contains a gene termed zimp that encodes a homolog of the PIAS family (45). Zimp is an essential gene for Drosophila development. It is expressed as three alternatively spliced forms, two of which are detected only in adult flies. The zimp transcripts encode proteins of 544 and 522 amino acids that share an N-terminal 515-amino acid region and differ in their C termini. The splice variants of zimp resemble ARIP3/PIASx{alpha} and Miz1/PIASxß, in that residues 1–550 of these latter proteins are identical, and they differ only in their C termini (ARIP3 residues 551–572, Miz1 residues 551–621). The presence of a PIAS homolog, but not relatives of the p160 gene family of nuclear receptor coactivators in the Drosophila genome (46), suggests that the PIAS proteins serve a function different from, and perhaps more ancient than, that of the p160 coactivators in steroid receptor signaling.

Since the PIAS family members are highly homologous, with regions exhibiting amino acid sequence identities of 60–80% (Ref. 23 and Fig. 1Go), it was pertinent to compare their ability to modulate steroid receptor-dependent transcription. Each PIAS family member activated steroid receptor function from simple promoters, and none of them influenced basal transcription in the absence of ligand. However, the proteins behaved in a receptor-selective fashion, in that their ability to modulate transcription mediated by different steroid receptors varied substantially. There were also interesting differences in their cell line-specific functions. On more complex promoters, such as probasin and mouse mammary tumor virus promoters, PIAS3 and ARIP3 acted predominantly as corepressors of AR and GR function in HeLa and COS-1 cells, whereas in HepG2 cells, all PIAS proteins activated AR-dependent transcription. Moreover, the differences among the PIAS protein activities on the minimal promoter were diminished in HepG2 cells, in that they all activated the function of GR and AR to a similar degree.

Miz1 and PIAS1 exhibited intrinsic transcription-activating function in both HeLa and HepG2 cells when fused to Gal4 DBD, whereas ARIP3 and PIAS3 were devoid of this feature. This intrinsic transcription-activating function of PIAS1 and Miz1 was in line with their more robust activity on AR in HeLa cells but, surprisingly, not in HepG2 cells or with other steroid receptors in either HeLa or HepG2 cells. Since Miz1 and ARIP3 differ merely in their very C-terminal 71 and 22 amino acids, respectively, it is likely that the Ser/Thr-rich extension in the Miz1 C terminus contributes to the activating function. In this regard, ARIP3 and Miz1 resemble the C-terminal SRC-1 variants, SRC-1a and SRC-1e, which differ in their ability to potentiate transcription by ER in a promoter context-dependent fashion, and their functional differences relate to an activation domain present only in the SRC-1e isoform (47).

Inhibition of STAT-DNA interaction is the postulated mechanism underlying the down-regulation of STAT signaling by PIAS1 and PIAS3 (22, 23). The contrasting effects of PIAS proteins on steroid receptor function in different cell lines imply that their action on steroid receptors is hardly based on the interference with receptor-DNA interaction, i.e. a mechanism suggested for Stat1 and Stat3. Experiments with chimeric AR and GR forms also showed that the receptor-selective effects of PIAS proteins are dependent on regions other than AR or GR DBD. Moreover, our previous work indicated that ARIP3 does not influence significantly the interaction of AR with ARE (20). Differences in steroid receptor-PIAS interactions also failed to provide a mechanistic explanation for the dissimilar effects of PIAS proteins on steroid receptor function. It is likely that PIAS proteins form complexes with other coregulatory proteins, perhaps simultaneously with steroid receptors. Dissimilar amounts of these yet-to-be-identified PIAS-interacting proteins might form the basis for the cell- and promoter-specific actions of PIAS proteins on steroid receptor-dependent transcriptional activation.

ARIP3 contains two LXXLL motifs starting at residues 18 and 304 that are conserved in mammalian PIAS proteins, but not in Zimp. However, these putative nuclear receptor boxes do not seem to play an important role in the ability of the PIAS proteins to modulate steroid receptor function (N. Kotaja, O. A. Jänne, and J. J. Palvimo, in preparation). Mammalian PIAS proteins, Zimp, and the predicted proteins in Caenorhabditis elegans (Ce 1523698), and Saccharomyces cerevisiae Nfi-1 (2104683), all share a well conserved region comprising one His and five Cys residues that may form a zinc-binding motif. The possibility that this region serves as an interaction interface for steroid receptors will be addressed in our future experiments. Interestingly, this region is not essential for the interaction of PIAS1 with Stat1 (48).

The ability of PIAS proteins to interact with steroid receptors and, depending on the promoter and cell type context, to play both positive and negative regulatory roles is intriguingly similar to the behavior of Zac1b (49). Like ARIP3, Zac1 (zinc finger protein that regulates apoptosis and cell cycle arrest) is a member of a larger protein family, the PLAG (pleomorphic adenoma gene) family (50). In addition to nuclear receptors, Zac1b may also bind to the C-terminal activation domain of GRIP1 and interact with CREB-binding protein (CBP) and p300 (49). Similar to ARIP3 and Miz1, Zac1 also interacts with nuclear receptors in a hormone-independent manner in vitro. Altered Zac1 expression has been associated with cancer, and its expression is repressed in ovarian cancer cell lines (51, 52). In this regard, it is of interest that PIAS1 expression is severely repressed in HRAS-transformed fibroblasts and the repression is blocked by a mitogen-activated protein (MAP) kinase inhibitor (53).

Taken together, the PIAS proteins modulate transcriptional activity of steroid receptors and, depending on the cell and promoter context, they either activate or repress transcription dependent on steroid receptors. The biological functions of this protein family are obviously not restricted to the inhibition of STAT signaling. It is currently unknown which of the functions of the PIAS proteins, i.e. the modulation of steroid receptor action or the inhibition of STAT-mediated signaling, is biologically more important. These actions do not have to be mutually exclusive, and they may well be dependent on the concentration of individual PIAS proteins and their interaction partners in a given cell type. ARIP3 and PIAS1 are predominantly expressed in the testis (20, 25) which is a target for cytokine regulation through STAT proteins (54, 55) and for steroid hormone action. Testis may thus represent a tissue where the cross-talk between steroids and cytokines is governed by the function of PIAS proteins, such as ARIP3 and PIAS1.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
pARE2TATA-LUC reporter containing two AREs (from the first intron of the rat C3 gene) in front of minimal TATA sequence and pPB(-285/+32)-LUC containing nucleotides -285 to +32 of the rat probasin promoter driving luciferase coding region have been described (36, 56). ERE2TATA-LUC was constructed in the same way except that the inserted 45-bp oligomer contained two estrogen response elements in lieu of AREs (57). Mouse mammary tumor virus promoter LUC construct (pHH-LUC, containing region -203/+105 of the promoter) was obtained from American Type Culture Collection (ATCC, Manassas, VA). pG5-LUC has five Gal4-binding sites in front of the minimal TATA box sequence driving the LUC gene (Promega Corp., Madison, WI). pSG5-rAR expression vector was constructed as previously described (58). pM-rAR was also described previously (36).

pSG5-hPR1 was gift from Dr. Pierre Chambon. pSG5-hGR was created as described previously (36). pCMV5-hER{alpha} and pCMV5-hERß were from Drs. Benita S. Katzenellenbogen and Jan-Åke Gustafsson, respectively. pSG5-ER{alpha} was created by ligating ER{alpha} digested with EcoRI and BamHI into the pSG5 (Stratagene, La Jolla, CA). pSG5-hERß was constructed by first inserting ERß C terminus as an EcoRI/BamHI fragment into pSG5 and by subsequently cloning the N terminus as an EcoRI fragment. pCMV5-AGA derived from the full-length mouse AR by swapping its DBD (amino acid residues 575–634) for that of the rat GR, the corresponding pCMV5-GAG derived from full-length rat GR, and their wild-type counterparts pCMV5-mAR and pCMV5-rGR were gifts from Dr. Diane M. Robins (27). PIAS3 and PIAS1 cDNAs were from Dr. K. Shuai, and Miz1 cDNA was a gift from Dr. Rob Maxon. The following mammalian two-hybrid vectors were used (from CLONTECH Laboratories, Inc., Palo Alto, CA): pM for expressing the DBD of the Saccharomyces cerevisiae Gal4 protein (residues 1–147), pVP16 for expressing the transcriptional activation domain (VP16 AD) of the herpes simplex virus VP16 protein (amino acid residues 411–456), and VP16-CP for expressing a fusion of VP16 AD to the polyoma virus coat protein. The ß-galactosidase expression plasmid pCMVß was purchased from CLONTECH Laboratories, Inc. Luciferase reporter constructs GAS-LUC, Spi-LUC, and fN{epsilon}N4-LUC and plasmids encoding EpoR, Stat1, Stat5, and Stat6 have been described previously (28, 29, 30, 32). Testosterone was from Makor Chemicals (Jerusalem, Israel), progesterone, estradiol, and dexamethasone were from Sigma (St. Louis, MO), and IFN-{gamma} was from Immugenex (Los Angeles, CA). Luciferase assay reagent was purchased from Promega Corp.. Restriction endonucleases, DNA-modifying enzymes, and [35S]methionine were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL).

Plasmid Construction
pFLAG-ARIP3 was constructed by cloning PCR-generated cDNA fragments into pFLAG-CMV2 (Kodak IBI, Rochester, NY) as described previously (20). Full-length Miz1 was constructed by digesting the ARIP3 N terminus from pFLAG-ARIP3 with KpnI and SpeI and ligating it to the Miz1 C terminus digested from pBluescript IIKS-Miz1. pM2-ARIP3 was cloned by digesting full-length ARIP3 from pFLAG-ARIP3 with EcoRI and ligating the insert into pM2 vector. To construct pM2-Miz1, EcoRI and XbaI were used to digest Miz1 cDNA from pFLAG-Miz1, and the insert was then ligated into pM2 vector. pVP16-ARIP3 was created by first cloning the N-terminal PCR-generated EcoRI/BamHI fragment to pVP16 vector and then inserting the C-terminal BamHI fragment downstream of the BamHI site. To construct pVP16-Miz1, Miz1 C terminus was digested from pFLAG-Miz1 with BamHI and then inserted to pVP16-ARIP3(1–103) cut with the same enzyme. pM-PIAS1 and pVP16-PIAS1 were generated by inserting full-length PIAS1 cleaved from pCMV5-FLAG-PIAS1 with BglII and HindIII into pM or pVP16 vectors digested with BamHI and HindIII. pM-PIAS3 and pVP16-PIAS3 were constructed by cloning the full-length PIAS3 from pFLAG-PIAS3 to pM and pVP16 with SalI and HindIII. pGEX4T3-ARIP3 was obtained by transferring full-length ARIP3 from pFLAG-ARIP3 to the pGEX-4T3 vector as an EcoRI fragment. To create pGEX-5X1-Miz1, Miz1 was cleaved with EcoRV and XhoI from pFLAG-Miz1 and inserted into the SmaI/XhoI sites of pGEX-5X1. pGEX-5X1-GRIP1b (amino acids 563–1,121) was constructed by digesting pM-GRIP1(563–1,121) (a gift from Dr. Michael Stallcup) with EcoRI and SalI and transferring the insert into the corresponding site of pGEX-5X1 vector.

Cell Culture and Transfections
HeLa (American Type Culture Collection) cells were maintained in DMEM containing penicillin (25 U/ml), streptomycin (25 U/ml), 10% (vol/vol) FBS, and nonessential amino acids. HepG2 cells were maintained in DMEM containing penicillin, streptomycin, 10% FBS, and sodium pyruvate. Cells were seeded onto 12-well plates and transfected 24 h later by FuGene transfection method (Roche Molecular Biochemicals, Indianapolis, IN). In brief, each well received 200 ng of the luciferase reporter plasmid, 20 ng of ß-galactosidase (pCMVß) internal control plasmid, and 20 ng of different steroid receptor or STAT expression vectors, and indicated amounts of ARIP3/PIAS expression vectors. Four hours before transfection, the medium was changed to one containing 10% charcoal-stripped FBS. Twenty hours after transfection, the cells received fresh medium containing 2% charcoal-stripped FBS with or without 100 nM steroid hormone or, for STAT experiments, with 10 ng/ml IFN-{gamma}, 4 U/ml Epo, or 10 ng/ml IL-4. Forty-eight hours after transfection, the cells were harvested, lysed in Reporter Lysis Buffer (Promega Corp., Madison, WI) and the cleared supernatants were used for luciferase measurements with reagents from Promega Corp. using a Luminoskan RT reader (Labsystems, Helsinki, Finland) and for ß-galactosidase assays as described previously (34, 59). Independent transfection experiments were conducted using triplicate dishes three to six times, and at least two different plasmid batches were used for each set of experiments.

Immunoblotting
Whole-cell extracts from HeLa and HepG2 cells were resolved by electrophoresis on 12% polyacrylamide gels (PAGE) under denaturing conditions. Proteins were electroblotted onto Hybond ECL membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). Membranes were incubated with M2 monoclonal antibody against FLAG epitope (Kodak, Rochester, NY) and horseradish peroxidase-conjugated goat antimouse IgG antibody (Zymed Laboratories, Inc., South San Francisco, CA), and immunocomplexes were visualized using ECL Western blotting detection reagents from Amersham Pharmacia Biotech according to the manufacturer’s instructions.

Protein-Protein Interaction in Vitro
GST-ARIP3, GST-Miz1, and GST-GRIP1b were produced in Epicurian coli BL21-CodonPlus bacteria (Stratagene, La Jolla, CA) and purified with Glutathione Sepharose 4B (Amersham Pharmacia Biotech) as previously described (60). Lysis buffer containing 50 mM Tris-HCl (pH 7.8), 150 mM KCl, 0.1% Nonidet P-40, 0.1% Triton-X 100, 0.5 mM EDTA, 10% glycerol, 5 mM MgCl2, and 1:200 protease inhibitor cocktail (Sigma, St. Louis, MO) was used. AR, GR, PR, ER{alpha}, and ERß were translated in vitro using the TNT-coupled transcription/translation system (Promega Corp.) in the presence of [35S]methionine. Protein-protein affinity chromatography with purified GST fusion proteins bound to Glutathione Sepharose and 10 µl of [35S]methionine-labeled in vitro translated protein was carried out at 4 C for 2 h, with or without the cognate hormone (1 µM), in binding buffer containing 4 mM Tris-HCl (pH 8.0), 40 mM NaCl, 10% glycerol, 0.5 mM EDTA, 0.4% Nonidet P-40, 0.1% Triton-X 100, 5 mM MgCl2, 50 µM ZnCl2, 20 µg/ml BSA, and 1:200 protease inhibitor cocktail in a total volume of 500 µl. The resin was washed four times with 1 ml of binding buffer. Bound proteins were released by boiling in SDS-PAGE sample buffer. After electrophoresis, the gels were fixed in methanol (45%)-acetic acid (10%), treated with Amplify (Amersham Pharmacia Biotech) and dried, and radioactive proteins were visualized by fluorography.


    ACKNOWLEDGMENTS
 
The excellent technical assistance of Kati Saastamoinen, Leena Pietilä, and Seija Mäki is gratefully acknowledged. We thank Pierre Chambon, Jan-Åke Gustafsson, Benita Katzenellenbogen, Rob Maxon, Diane Robins, Laura Seikku, Ke Shuai, and Michael Stallcup for plasmids.


    FOOTNOTES
 
Address requests for reprints to: Olli A. Jänne, M.D., Ph.D., Institute of Biomedicine, Department of Physiology, University of Helsinki, P.O. Box 9 (Siltavuorenpenger 20 J), FIN-00014 Helsinki, FINLAND. E-mail olli.janne{at}helsinki.fi

This work was supported by grants from the Academy of Finland, the Finnish Foundation for Cancer Research, the Sigrid Jusélius Foundation, Biocentrum Helsinki, Helsinki University Central Hospital, and CaP CURE.

Received for publication August 2, 2000. Revision received September 6, 2000. Accepted for publication September 7, 2000.


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