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The Journal of Immunology, 2000, 164: 5689-5697.
Copyright © 2000 by The American Association of Immunologists

Cooperation Among Stat1, Glucocorticoid Receptor, and PU.1 in Transcriptional Activation of the High-Affinity Fc{gamma} Receptor I in Monocytes1

Saara Aittomäki*, Marko Pesu*,{dagger}, Bernd Groner{ddagger}, Olli A. Jänne§, Jorma J. Palvimo§ and Olli Silvennoinen2,*,{dagger}

* Institute of Medical Technology, and Department of Medical Biochemistry, University of Tampere, and {dagger} Department of Clinical Microbiology, Tampere University Hospital, Tampere, Finland; {ddagger} Chemotherapeutisches Forschungsinstitut, Georg Speyer Haus, Frankfurt am Main, Germany; and § Institute of Biomedicine, Departments of Physiology and Clinical Chemistry, University of Helsinki, Helsinki, Finland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IFN-{gamma} and glucocorticoids regulate inflammatory and immune responses through Stat1 and glucocorticoid receptor (GR) transcription factors, respectively. The biological responses to these polypeptides are determined by integration of various signaling pathways in a cell-type and promoter-dependent manner. In this study we have characterized the molecular basis for the functional cooperation between IFN-{gamma} and dexamethasone (Dex) in the induction of the high-affinity Fc{gamma} receptor I (Fc{gamma}RI) in monocytes. Dex did not affect IFN-{gamma}-induced Stat1 DNA binding activity or induce novel DNA-binding complexes to the Fc{gamma}RI promoter. By using cell systems lacking functional GR or Stat1, we showed that GR stimulated Stat1-dependent transcription in a ligand-dependent manner, while Stat1 did not influence GR-dependent transcription. The cooperation required phosphorylation of Tyr701, DNA binding, and the trans-activation domain of Stat1, but did not involve Ser727 phosphorylation of Stat1 or physical interaction between GR and Stat1. The costimulatory effect of Dex was not dependent on a consensus glucocorticoid response element in the Stat1-responsive promoters, but required the DNA-binding and trans-activation functions of GR, and Dex-induced protein synthesis. GR activated the natural Fc{gamma}RI promoter construct, and this response required both Stat1 and the Ets family transcription factor PU.1. Previously, physical association between GR and Stat5 has been shown to enhance Stat5-dependent and suppress GR-dependent transcription. The results shown here demonstrate a distinct, indirect mechanism of cross-modulation between cytokine and steroid receptor signaling that integrates Stat1 and GR pathways with cell type-specific PU.1 transcription factor in the regulation of Fc{gamma}RI gene transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Monocytes and their terminally differentiated counterparts, macrophages, carry out a variety of functions in the first-line defense against foreign invaders such as phagocytosis and microbicidal activity against intracellular and extracellular microbes. Monocytes also serve as APC and critical activators of specific immune responses. During the immune response, activated T cells provide a reciprocal activation stimulus to monocytes by secretion of cytokines such as IFN-{gamma}, one of the most potent monocyte-activating agents (1). An important consequence of IFN-{gamma} stimulation in monocytes is up-regulated expression of the high-affinity receptor for IgG (Fc{gamma}RI, CD64) (2). Fc{gamma}RI is a 72-kDa glycoprotein expressed predominantly on monocytes. It plays an important role in endocytosis of immune complexes and opsonized microbes and in Ab-mediated cytotoxic reactions (3). Engagement of the Fc{gamma}RI promotes cellular signal transduction through Src family and Syk tyrosine kinases and results in functional activation of monocytes and modulation of cytokine production (4, 5, 6, 7).

In recent years the molecular mechanisms for IFN-{gamma} signal transduction have been elucidated (8, 9). IFN-{gamma} binds to a receptor complex consisting of IFN-{gamma}RI and the accessory chain IFN-{gamma}RII. Ligand-induced dimerization of the receptor chains results in activation of Jak1 and Jak2 tyrosine kinases and phosphorylation of the IFN-{gamma}RI on specific tyrosine residues that serve as docking sites for the Src homology 2 (SH2) domain of the latent cytoplasmic transcription factor Stat1. In the receptor complex Stat1 becomes tyrosine phosphorylated and forms dimers that are translocated to the nucleus and bind cognate promoter DNA sequences, referred to as IFN-{gamma}-activated site (GAS)3 (8). The Stat1 signaling pathway is essential for induction of the Fc{gamma}RI gene, and Stat1-deficient mice are unresponsive to IFN-{gamma}-induced Fc{gamma}RI expression (10). Characterization of the Fc{gamma}RI promoter has shown that the IFN-{gamma} regulatory region and regions determining myeloid cell-specific expression are conferred by two cis-elements within 190 nucleotides upstream of the translation initiation site. The IFN-{gamma}-inducible region is localized to an IFN-{gamma} response region (GRR), which contains a GAS-like Stat1 binding element (11, 12). The cell type-specific expression requires a downstream myeloid cell-activating transcription element, which binds the Ets family transcription factor PU.1/Spi-1 (12, 13).

Glucocorticoids have profound immunomodulatory effects. They are widely used as immunosuppressive and anti-inflammatory agents in autoimmune and allergic inflammatory diseases (14). Glucocorticoids modulate the growth, differentiation, and function of lymphocytes, neutrophils, eosinophils, mast cells, endothelial cells, and monocytes through activation of the glucocorticoid receptor (GR) (15). GR belongs to the nuclear hormone receptor superfamily and functions as a ligand-induced transcription factor. In unstimulated cells, GR exists in the cytoplasm in a complex with heat shock proteins and immunophilins, and ligand binding dissociates the complex and promotes nuclear transfer of GR. In the nucleus, GR homodimers bind to cognate DNA motifs known as glucocorticoid response elements (GREs). GR displays both stimulatory and inhibitory effects on transcription (15, 16). The molecular basis for the anti-inflammatory effects of glucocorticoids has been extensively studied in T cells and nonhemopoietic cells and has been shown to involve several mechanisms, including competition for limiting amounts of transcriptional coactivators (CBP/p300/NCoA-1/p/CIP), interference with AP-1-mediated transcription, and inhibition of NF-{kappa}B activation by direct protein-protein interactions as well as by induction of I{kappa}B{alpha} (17, 18, 19, 20, 21, 22). However, the molecular mechanisms of glucocorticoid effects on monocytes are not clearly defined.

Transcriptional responsiveness is controlled by transcription factors binding to promoter sequences and their interaction with transcriptional coactivators/corepressors and the basal transcription machinery. The mechanism by which Stat factors activate RNA polymerase II-dependent gene transcription is still largely uncharacterized, but Stat1, Stat2, Stat3, Stat5, and Stat6 have been shown to associate with the general coactivators CREB binding protein (CBP) and p300 (23, 24, 25, 26, 27). Regulation and specificity of Stat-mediated transcriptional responses are likely to be governed by combinatorial interactions and cross-talk between different transcription factors. Recent investigations have identified one such interaction. A steroid receptor, GR, cooperates with Stat3 in IL-6-stimulated induction of acute phase proteins and with prolactin-induced Stat5, in transcriptional activation of the ß-casein promoter (28, 29, 30, 31). In the case of Stat5, the cross-talk not only involves physical interaction with GR, but also results in inhibition of GR-dependent transcription (31, 32, 33).

The synthetic glucocorticoid dexamethasone (Dex) is a widely used immunosuppressive and anti-inflammatory agent. Previous studies have shown that IFN-{gamma} and Dex cooperatively induce Fc{gamma}RI expression in monocytes (34, 35). This study was aimed at delineating the mechanisms of action of Dex on the regulation of Fc{gamma}RI gene expression, and our results indicate that GR functions as a ligand-dependent costimulator of Stat1-mediated transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid constructs

The GAS-luc luciferase construct contains a GAS site from the IRF-1 gene promoter inserted upstream of the thymidine kinase (TK) promoter driving the firefly luciferase (luc) gene. Mut-GAS-luc has an insert containing the GAS site TTTCCCCGCCA from the IRF-1 promoter with an AA to CC substitution (underlined) (36). GRE-luc contains two copies of the rat tyrosine aminotransferase gene GRE inserted upstream of the TK promoter (37). Fc{gamma}RI-luc was constructed by cloning a fragment of Fc{gamma}RI promoter corresponding to nucleotides -189 to +1 (12) by PCR using human genomic DNA as a template and inserting it into luc vector without any heterologous promoter.

Stat1{alpha}, Stat1ß, Stat1Y701F, Stat1S727A, hGRwt, hGRD4x, rGR3–556, and rGR407–795, CBP, Stat5a, and EpoR have all been previously described (32, 33, 38, 39). The DNA-binding-deficient Stat1 (E428A,E429A) (40) was generated by PCR. The murine PU.1 cDNA (provided by Dr. R. Maki) was subcloned into the EcoRI site of pCIneo (Promega, Madison, WI) (41).

Cell culture and transfection assays

HepG2, COS-7, and RAW264.7 cells (from American Type Culture Collection, Manassas, VA) were cultured in DMEM plus 10% FCS, and THP-1 cells (from American Type Culture Collection) were cultured in RPMI 1640 medium plus 10% FCS, all from Life Technologies/BRL (Gaithersburg, MD). U3A cells, provided by Dr. I. Kerr, were grown in DMEM plus 10% Cosmic calf serum (HyClone, Logan, UT) (42). Human peripheral blood monocytes were isolated from leukocyte-enriched buffy coats using Optiprep (Nycomed Pharma, Norway) density gradient centrifugation according to the manufacturer’s instructions. After centrifugation monocytes were collected from the low density cell fraction and washed twice with medium. The monocyte fraction was analyzed with forward and side scatter and for CD64 expression using FACScan. The fraction contained ~60% monocytes; the rest of the cells were other mononuclear leukocytes. After isolation cells were maintained in RPMI 1640 medium plus 10% FCS.

Transfection of HepG2 and U3A cells was performed using the calcium phosphate precipitation method. Semiconfluent cells were transfected in 3.5-cm plates with 1 µg of luciferase reporter plasmids, 0.5 µg of pCMV-ßgal as internal transfection efficiency control, and different expression plasmids as indicated in the figure legends. Twenty-four hours after transfection the cells received fresh medium with 1% charcoal-stripped FCS. Cells were either left untreated or were treated with 10 ng/ml of human IFN-{gamma} (Immugenex, Los Angeles, CA), 5 µM dexamethasone (Dex; Oradexon, Organon, Oss, The Netherlands), or both for 16 h. In experiments with cycloheximide (CHX), HepG2 cells were first treated for 16 h with Dex in the presence or the absence of CHX (10 µg/ml), after which they were either washed or directly stimulated with IFN-{gamma} for 6 h. Cells were lysed into Promega’s Reporter lysis buffer, and luciferase activity was determined with reagents from Promega, using 1254 Luminova luminometer (Bio-Orbit). Values were normalized against ß-galactosidase activities.

EMSA

The GAS site from the murine IRF-1 gene (IRF-GAS, made by annealing the oligonucleotide 5'-CTAGAGCCTGATTTCCCCGAAATGATGAG-3' and its complement) and the GRR from the human Fc{gamma}RI gene (5'-GATATGAGCATGGGAAAAGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAGGAACATGATGAAAATG-3') were labeled with T4-polynucleotide kinase using [{gamma}-32P]ATP and were used as probes. The 190-bp insert from Fc{gamma}RI-luc plasmid was excised from the vector and labeled like the other probes. Cells were grown for 16 h in medium containing 1% charcoal-stripped FCS and were treated with IFN-{gamma} (100 ng/ml) for 15 min or with Dex (5 µM) for 30 min. Nuclear extracts were prepared as previously described (43). Extracts (10 µg protein) were incubated for 30 min on ice with 32P-labeled oligonucleotide, 0.1 µg/µl herring sperm DNA, and 1.5 µg/µl BSA in a total volume of 15 µl. For supershift analysis, nuclear extracts were incubated with 0.5 µg of anti-Stat1 Ab (N-terminal; Transduction Laboratories, Lexington, KY) for 30 min on ice before adding BSA, herring sperm DNA, and probe. Reactions were resolved by 4.5% PAGE in 2.2x TBE (175 V, 4°C). The gel was dried under vacuum and autoradiographed.

Flow cytometry

Human peripheral blood monocytes were suspended in RPMI 1640 and 10% human AB+ serum, stained with 1 µg of FITC-conjugated mouse anti-human-CD64 Ab (Immunotech, Marseilles, France) or with 1 µg of control Ab and isotype-matched FITC-conjugated mouse anti-keyhole limpet hemocyanin Ab (Becton Dickinson, Mountain View, CA) for 30 min at 4°C, and washed twice with PBS. CD64 expression was analyzed by FACScan (Becton Dickinson) from the monocyte population in which the contaminating cells had been gated out. The experiment was performed three times with similar results.

Immunoprecipitation and Western blot analysis

COS-7 cells were transfected as indicated with 3 µg of Stat5a or Stat1, 2 µg of EpoR, and 5 µg of GR expression plasmids by electroporation (Gene Pulser, Bio-Rad, Hercules, CA; 260 V, 960 µF). Forty-eight hours after transfection, the cells were treated with Dex (5 µM) for 30 min and either Epo (40 IU/ml) or IFN-{gamma} (100 ng/ml) for 15 min. Cells were lysed in Nonidet P-40 lysis buffer (50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.5% sodium deoxycholate, 20 mM NaF, 1% Nonidet P-40, 10% glycerol, 0.2 mM Na3VO4, 2 mM PMSF, and 20 µg/ml aprotinin). One milligram of protein was immunoprecipitated with anti-Stat5a Ab (13-3600, Zymed, San Francisco, CA), anti-Stat1 Ab (S21120, Transduction Laboratories), or anti-GR antiserum (31). Immunoprecipitates were resolved on SDS-PAGE and transferred to nitrocellulose membrane for subsequent immunoblotting.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dex enhances the IFN-{gamma}-induced expression of Fc{gamma}RI in monocytes but does not affect Stat1 DNA binding or Fc{gamma}RI promoter binding factors

IFN-{gamma} and Dex are both potent immunomodulatory agents, exerting pleiotropic effects on different cell types through activation of distinct nuclear signaling pathways mediated by Stat1 and GR, respectively. In monocytes, an important target for IFN-{gamma}-mediated gene regulation is the Fc{gamma}RI, and this response is further enhanced by Dex treatment (34, 35). These findings raised the possibility that Stat1 and GR signaling pathways cooperate functionally. In this study we have investigated the molecular mechanism for the costimulatory effect of Dex on Fc{gamma}RI expression.

Human peripheral blood monocytes were treated with optimal doses of either IFN-{gamma} and Dex alone or both agents together for 20 h, and the expression of Fc{gamma}RI was analyzed using FACS. In accordance with previous results, IFN-{gamma} increased the expression of Fc{gamma}RI (anti-CD64 staining mean fluorescence intensity, 64 vs 295; see Materials and Methods), which was further stimulated by the presence of Dex (mean fluorescence intensity, 412) (34, 35). Dex treatment alone did not influence Fc{gamma}RI expression. Pretreatment of monocytes with the GR antagonist RU486 abolished the effect of Dex on Fc{gamma}RI expression, but did not affect basal or IFN-{gamma}-induced expression. Thus, GR does not directly regulate the expression of Fc{gamma}RI, and the effect of Dex requires functional activation of both GR- and IFN-{gamma}-induced factors.

Because Stat1 is essential for IFN-{gamma}-induced expression of Fc{gamma}RI (10, 11), we tested the possibility that Dex regulates activation events of Stat1. Human peripheral blood monocytes were left untreated or were treated with IFN-{gamma}, Dex, or both agents for different time periods. Functional activation of Stat1 was examined by EMSA using nuclear extracts with three different probes, namely the prototype Stat1-binding GAS oligonucleotide, the Stat1 binding site containing GRR from the Fc{gamma}RI promoter, and the proximal promoter region (nucleotides -189 to +1) of Fc{gamma}RI (12). Treatment of monocytes with IFN-{gamma} for 15 min induced DNA binding complexes to all three probes, and the intensities of the retarded bands were reduced after 20 h of IFN-{gamma} stimulation (Fig. 1Go). Pretreatment of the lysate with anti-Stat1 Ab supershifted the GAS binding complex. IFN-{gamma} induced two binding complexes to the GRR oligonucleotide, and the Fc{gamma}RI binding complexes contained both constitutive components and IFN-{gamma}-induced complexes, which is consistent with previous reports (11, 12, 13). Dex treatment alone, either short term (30 min) or long term (20 h), did not induce novel binding complexes to any of the oligonucleotides. In accordance with this result, analysis of the Fc{gamma}RI promoter region failed to reveal the presence of a consensus GRE motif. Furthermore, Dex treatment did not affect the intensities or mobilities of the IFN-{gamma}-induced complexes to GAS, GRR, or Fc{gamma}RI oligonucleotides. Similar results were obtained with the mouse macrophage cell line RAW264.7 and the human monocytic cell line THP-1 (data not shown). These results indicate that the costimulatory effect of Dex is not due to enhanced IFN-{gamma} signaling and Stat1 DNA binding. Furthermore, GR does not appear to bind directly or induce novel Fc{gamma}RI promoter binding complexes in monocytes.



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  		FIGURE 1. Dex treatment does not influence Stat1 DNA binding in monocytes. Human peripheral blood monocytes were treated as indicated with IFN-{gamma} (100 ng/ml) and Dex (5 µM). The treatments were 15 min for IFN-{gamma} and 30 min for Dex (A) and 20 h for both IFN-{gamma} and Dex (B). Nuclear lysates were prepared and analyzed by EMSA with 32P-labeled IRF-GAS (A, lanes 1–5; B, lanes 1–4), GRR (A, lanes 6–9; B, lanes 5–8), or Fc{gamma}RI (A, lanes 10–13; B, lanes 9–12) probes. The arrows indicate the positions of IFN-{gamma}-induced complexes. IFN-{gamma}-inducible complex was supershifted with specific anti-Stat1-Ab (A, lane 5). The constitutive Fc{gamma}RI binding complexes composed of PU.1 migrate close to the free probe.

 
GR enhances Stat1-dependent transcription

We examined the possibility that GR regulates the IFN-{gamma}-induced transcriptional activation and used a heterologous HepG2 cell system for this purpose. HepG2 cells are fully responsive to IFN-{gamma}, but express very low levels of GR and are unresponsive to Dex stimulation (see below). HepG2 cells were transfected with a luciferase reporter construct containing a GAS element placed upstream of a minimal heterologous promoter (GAS-luc). IFN-{gamma} readily stimulated the GAS-luc activity, and Dex treatment did not influence basal or IFN-{gamma}-induced reporter activity (Fig. 2GoA). HepG2 cells were transfected with increasing amounts of GR expression plasmid. Coexpression of GR did not affect basal or IFN-{gamma}-induced reporter activity, and stimulation of the GR-transfected cells with Dex alone had no effect. However, when GR-transfected cells were treated simultaneously with both IFN-{gamma} and Dex, a strong synergistic increase in GAS-luc reporter activity was observed. This increase in reporter activity was proportional to the amount of transfected GR. To confirm that GR directly up-regulates the Stat1-mediated transcription, HepG2 cells were cotransfected with GR and a reporter construct in which the Stat1-binding site was mutated (Mut-GAS). Treatment of the cells with Dex, IFN-{gamma}, or their combination did not influence Mut-GAS-luc reporter activity (Fig. 2GoB), indicating that GR specifically enhances the Stat1-dependent transcription.



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  		FIGURE 2. GR stimulates Stat1-dependent reporter gene activation. A, HepG2 cells were transfected with 1 µg of GAS-luc reporter construct and 0, 0.1, 0.5, or 1.5 µg of GR expression plasmid. The amount of transfected DNA was kept constant in different experiments by adding empty expression vector when appropriate. Transfection efficiencies were monitored by cotransfection of pCMV-ß-gal plasmid. Cells were treated with IFN-{gamma} (10 ng/ml), Dex (5 µM), or both or were left untreated for 16 h before measuring the luciferase activity. B, HepG2 cells were transfected with GAS-luc or a mutated Stat1 binding site containing construct (Mut-GAS) alone or with 0.5 µg of GR expression plasmid. Cells were treated as described in A. C, U3A cells were transfected with 1 µg of GAS-luc and as indicated with 0.4 µg of various Stat1 expression plasmids (Stat1{alpha}, Stat1ß, Stat1S727A, Stat1 DB Mut (Stat1E428A, E429A)) and GR (0.05 µg) expression plasmid and treated as described in A. Three experiments were performed. The luciferase values were normalized to ß-galactosidase activity and are presented as relative luciferase units (RLU).

 
DNA binding and the trans-activation domain of Stat1 are required for cooperation with GR

Stat1 is expressed as two alternatively spliced variants, Stat1{alpha} and Stat1ß. The Stat1ß isoform lacks the 38 C-terminal residues (38). IFN-{gamma} stimulates the tyrosine phosphorylation and DNA binding of both isoforms, but only Stat1{alpha} is transcriptionally active. To study the structural requirements of Stat1 for cooperation with GR, we used the Stat1-deficient U3A fibrosarcoma cell line (42). U3A cells were transfected with either the GAS-luc reporter alone or together with GR and Stat1{alpha} or Stat1ß. Transfection of Stat1{alpha} rendered the cells responsive to IFN-{gamma}-induced activation of the GAS-luc reporter, and cotransfection of GR with Stat1{alpha} resulted in synergistic activation of the reporter in response to IFN-{gamma} and Dex stimulation (Fig. 2GoC). In GR-transfected U3A cells, Dex induced some basal, Stat1-independent reporter activity. Stat1ß was not able to mediate activation of GAS-luc, and cotransfection of GR and Stat1ß and simultaneous exposure to Dex and IFN-{gamma} had no significant effect on the reporter activity compared with Dex treatment alone. The different Stat1 constructs used in these experiments were expressed at similar levels (data not shown).

Dimerization, nuclear localization, and DNA binding of Stat1 are critically dependent on phosphorylation of a single C-terminal Tyr701 residue (38). We next investigated whether the nuclear localization and DNA binding of Stat1 are required for cooperation with GR. Cotransfection of GR with the Stat1Y701F mutant and stimulation with IFN-{gamma} and Dex resulted in only basal activation of the reporter, and the cooperation between Stat1 and GR was abolished (data not shown). Because the Stat1Y701F mutant does not translocate to the nucleus, the lack of cooperation could result from the cytoplasmic localization of Stat1. Therefore, we tested the DNA-binding-deficient mutant of Stat1 (E428A,E429A), which becomes tyrosine phosphorylated and translocates to the nucleus in response to IFN-{gamma} (40). As expected, the Stat1E428A,E429A mutant did not induce IFN-{gamma}-dependent reporter activation and also failed to cooperate with GR (Fig. 2GoC). The C-terminus of Stat1 contains also a serine residue (Ser727), which is phosphorylated upon cytokine stimulation and is required for Stat1 association with nuclear MCM5 coactivator (38, 44). However, activation of GR in both Stat1S727A and Stat1{alpha}-transfected cells resulted in a 2-fold increase in reporter activity. These results indicate that nuclear translocation and DNA binding of Stat1, but not its phosphorylation on Ser727, are required for functional cooperation with GR. In addition, GR is not able to substitute for the function of the trans-activation domain of Stat1 in reporter gene activation.

Stat1 does not affect GR-dependent transcription

To investigate whether Stat1 would reciprocally regulate GR-dependent transcription, activation of a GRE-containing reporter construct (GRE-luc) was analyzed in U3A cells. Dex treatment activated GRE-luc in U3A cells (Fig. 3Go). The induction was further enhanced by ectopic expression of GR, and, as expected, the reporter activity was not affected by IFN-{gamma} treatment. Cotransfection of GR with Stat1{alpha} or the different Stat1 variants (Stat1ß, Stat1Y701F, Stat1S727A) had no effect on Dex-induced activation of the GRE-luc reporter in either the presence or the absence of IFN-{gamma} stimulation. Similar results were obtained in HepG2 cells in which activation of GRE-luc required transfected GR, but costimulation with IFN-{gamma}, and cotransfection of Stat1{alpha} or different Stat1 variants, did not affect the Dex-induced activation of GRE-luc reporter (data not shown).



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  		FIGURE 3. Stat1 does not regulate GR-dependent gene activation. U3A cells were transfected with 1.5 µg of GRE-luc, 0.5 µg of GR expression vector, and 1 µg of different Stat1 mutants (Stat1{alpha}, Stat1ß, Stat1S727A, Stat1Y701F) as indicated. The cells were treated with IFN-{gamma} (10 ng/ml) and Dex (5 µM) as indicated for 16 h, and the normalized luciferase values of three experiments with SDs are shown.

 
GR interacts with Stat5, but not with Stat1

To gain further insight into the mechanism of cooperation between Stat1 and GR, we investigated whether the proteins physically interact. Another Stat family member, Stat5, is previously shown to associate with GR, and this interaction occurs in the cytoplasm in a prolactin-independent manner (32). To study the association between Stat1 and GR, coimmunoprecipitation experiments were performed from COS-7 cells transfected with expression vectors encoding Stat1 and GR. Immunoblotting of the Stat1 immunocomplex with anti-GR Ab failed to detect any coprecipitated GR (Fig. 4GoA). Likewise, immunoblotting of GR immunoprecipitates with anti-Stat1 Ab did not detect any coimmunoprecipitation of the two proteins (Fig. 4GoB). We also tested the possibility that ligand-induced post-translational regulation, e.g., phosphorylation, would be required for association, but stimulation with Dex and IFN-{gamma} did not induce complex formation between Stat1 and GR (Fig. 4GoA). Stat5 is activated by multiple cytokines, including Epo. As a control, COS-7 cells were transfected with EpoR, Stat5, and GR expression vectors, and Stat5 and GR were found to coimmunoprecipitate in a ligand-independent manner (Fig. 4Go). These results indicate that the functional cooperation between Stat1 and GR does not involve direct protein-protein interaction, and imply that GR cooperates with different Stat proteins by mechanisms that are distinct.



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  		FIGURE 4. Physical association of GR with Stat5a, but not with Stat1. COS-7 cells were transfected (Tx) as indicated with Stat5a (1 µg), Epo receptor (2 µg), Stat1 (1 µg), and GR (5 µg) expression vectors. Where indicated, the cells were treated with Epo (40 IU/ml) or IFN-{gamma} (100 ng/ml) for 15 min and with Dex (5 µM) for 30 min. A, One milligram of whole cell extract was immunoprecipitated with anti-Stat5a, anti-Stat1, or control (contr.) Ab; separated on SDS-PAGE; and immunoblotted with anti-GR antiserum. B, Similarly, 1 mg of cell extract was immunoprecipitated with anti-GR antiserum and probed with either anti-Stat5a or anti-Stat1 Abs. Twenty micrograms of total cell lysates (TCL) were included as controls. The arrows indicate the positions of the GR, Stat1, and Stat5a proteins.

 
The DNA binding domain of GR is required for functional cooperation with Stat1

GR has a modular structure composed of three functional domains; a constitutively active N-terminal activation function domain (AF1), a central DNA binding domain (DBD), and a C-terminal ligand binding domain that harbors trans-activation function (AF2) and recruits coactivator proteins (15, 17). We delineated the functional domains of GR that were required for stimulation of Stat1-dependent transcription by using various GR mutants in HepG2 cells. Expression and function of the GR mutants were confirmed by immunoblotting and GRE-luc reporter assays (data not shown). We analyzed whether the DBD of GR was required for stimulation of Stat1-dependent transcription. Mutant GRD4x, containing four amino acid substitutions in the DBD (N454D/A458T/R460D/D462C) failed to enhance Stat1-dependent reporter activity in HepG2 cells after IFN-{gamma} and Dex stimulation (Fig. 5Go). Similar results were obtained with another DBD mutant (A458T; data not shown). The ability of the N-terminal deletion mutant GR407–795, which lacks the AF1 domain, was tested for its cooperation with Stat1. In cells transfected with GR407–795, the IFN-{gamma}-induced reporter activity was less efficiently increased by Dex treatment compared with wild-type GR, indicating that the AF1 domain of GR is needed for optimal stimulation of Stat1-dependent transcription (Fig. 5Go). Deletion of the GR ligand binding domain, which also mediates association of the latent GR to 90-kDa heat shock protein, results in a constitutively active GR variant. The C-terminal deletion mutant GR3–556 stimulated the IFN-{gamma}-induced reporter activity in the absence of Dex as efficiently as ligand-stimulated wild-type GR.



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  		FIGURE 5. DNA binding and trans-activation activity of GR are required for cooperation with Stat1. A, HepG2 cells were transfected with 1 µg of GAS-luc and either empty vector or 0.5 µg of different GR mutants (GR wt, GRD4x, GR407–795, GR3–556) and treated with 10 ng/ml IFN-{gamma} and 5 µM Dex as indicated for 16 h. The normalized luciferase values of three independent experiments with SDs are shown as relative luciferase units (RLU). B, HepG2 cells were transfected with 1 µg of GAS-luc reporter construct and 0.5 µg of GR expression plasmid as indicated. Cells were treated with CHX (10 µg/ml), Dex (5 µM), or both or were left untreated for 16 h. Thereafter, cells were either washed with PBS, and new medium was added, or the cells were left unwashed. Cells were then treated with IFN-{gamma} (10 ng/ml) for 6 h, where indicated, before measuring luciferase activity.

 
These results indicated that stimulation of Stat1-dependent activity requires specific regions of GR that are important for the receptor’s own trans-activation ability. However, the GAS-luc reporter and the Fc{gamma}RI promoter lack consensus GRE motifs, thus GR does not appear to interact directly with the Stat1-dependent promoter. To investigate this issue further, we studied whether the GR effect required de novo protein synthesis. HepG2 cells were sequentially treated first with Dex for 16 h in the presence or the absence of the protein synthesis inhibitor CHX, after which the cells were either washed or directly stimulated with IFN-{gamma} for 6 h. The results presented in Fig. 5GoB show that inhibition of protein synthesis during Dex treatment abrogated the stimulatory effect of GR on Stat1-dependent transcription. It should be noted that CHX pretreatment alone did not affect the IFN-{gamma} response when the inhibitor was washed away. Taken together, these results strongly suggest that GR is enhancing the Stat1-dependent gene activation by inducing the synthesis of transcriptional coregulators.

GR action on Fc{gamma}RI promoter is dependent on Stat1 and PU.1

In the Fc{gamma}RI promoter, the IFN-{gamma}-inducible region is conferred by GRR, and the cell-type specific expression requires a downstream myeloid cell-activating transcription element, which binds the Ets family transcription factor PU.1/Spi-1 (11, 13). To study directly the regulation of the natural Fc{gamma}RI promoter, a luciferase reporter driven by the regulatory region of the native Fc{gamma}RI promoter (nucleotides -189 to +1) was constructed (Fc{gamma}RI-luc) (12). HepG2 cells allowed the analysis of individual transcription factors in regulation of Fc{gamma}RI-luc expression, because these cells lack endogenous PU.1 and express very low levels of GR. Transfection of Fc{gamma}RI-luc into HepG2 cells, either alone or together with GR, did not result in any reporter activity after IFN-{gamma} and Dex stimulations (Fig. 6Go). Transfection of PU.1 into HepG2 cells stimulated Fc{gamma}RI-luc activity, which is in accordance with the basal expression of Fc{gamma}RI in monocytes. The presence of PU.1 was also absolutely required for the IFN-{gamma} induction of Fc{gamma}RI-luc reporter. When both PU.1 and GR were coexpressed in HepG2 cells, Dex increased the IFN-{gamma}-dependent activation of Fc{gamma}RI-luc reporter to a comparable degree to that observed in monocytes. Together these results demonstrate that the stimulatory effect of GR on Fc{gamma}RI is dependent on both Stat1 and PU.1.



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  		FIGURE 6. GR activation enhances IFN-{gamma}-induced transcription from the natural Fc{gamma}RI promoter. One microgram of Fc{gamma}RI-luc reporter containing 190 bp from the Fc{gamma}RI promoter was transfected into HepG2 cells with empty vector, 0.05 µg PU.1, and 0.5 µg GR expression plasmids as indicated. The cells were treated with IFN-{gamma}, Dex, or both for 16 h. Shown are the mean normalized luciferase values of three experiments with SDs.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IFN-{gamma} and glucocorticoids display profound immunomodulatory effects, and the final biological responses are determined by integration of different signaling pathways and cell type- and promoter-specific factors. In this study we have investigated the molecular mechanism by which Dex and IFN-{gamma} regulate Fc{gamma}RI expression in monocytes. Our results demonstrate that the stimulatory effect of Dex on Fc{gamma}RI expression is mediated by functional cross-talk among three distinct transcription factors: GR, Stat1, and PU.1.

A possible mechanism for the costimulatory effect of Dex on Fc{gamma}RI expression is the enhancement of IFN-{gamma}-dependent signaling events through inducing the expression of the IFN-{gamma} receptor or other critical signaling proteins, such as Jak kinases or Stat1, or alternatively, through inducing Fc{gamma}RI promoter-binding transcription factors. In monocytes, Dex treatment, either short or long term, did not modulate the IFN-{gamma}-induced Stat1 DNA binding activity or protein expression (data not shown) or induce novel DNA binding complexes to the Fc{gamma}RI promoter. Thus, Dex does not appear to directly regulate the immediate IFN-{gamma} signaling events or Fc{gamma}RI promoter-binding factors.

With the use of heterologous cell systems devoid of functional GR or Stat1, we demonstrated that GR stimulated in a ligand-dependent manner activation of a minimal Stat1-dependent reporter as well as the natural Fc{gamma}RI promoter. GR stimulated the GAS reporter more efficiently than the natural Fc{gamma}RI promoter, suggesting that the costimulus provided by GR activates Stat1-dependent transcription more efficiently in the context of the TK minimal promoter than in the natural promoter. The magnitude of GR-mediated enhancement of the natural Fc{gamma}RI promoter activity was similar to the level of enhancement on Fc{gamma}RI surface expression in monocytes after Dex treatment. The cooperation between GR and Stat1 required tyrosine phosphorylation and DNA binding of Stat1 as well as Stat1-dependent transcriptional activity, but was not dependent on Ser727 phosphorylation. Our results also revealed that the AF1 and AF2 domains of GR are not able to substitute for the trans-activation domain of Stat1 for induction of transcription. Stat5 mutants lacking the C-terminal trans-activation domain were previously found to synergize with GR in ß-casein induction (25, 33), indicating that GR cooperates differently with Stat1 and Stat5.

Glucocorticoids regulate gene transcription through several mechanisms. In addition to GR-GRE interaction, GR can modulate gene responses by a mechanism independent of DNA binding, involving direct protein-protein interactions between GR and other transcription factors and coactivators (17, 18, 19, 20, 21, 22, 45). In cytokine receptor signaling, GR has been shown to cooperate with prolactin-induced Stat5 and with IL-6 in acute phase protein synthesis through cooperation with C/EBPß and Stat3 (28, 29, 30, 31). The functional cooperation between GR and Stat5 is well established in the induction of ß-casein gene expression, and it involves both direct protein-protein interaction as well as DNA binding (31, 32, 33, 46). In accordance with these findings, Stat5 was found in this work to coimmunoprecipitate with GR in an Epo-independent manner. In contrast, we could not detect cellular association between Stat1 and GR in coimmunoprecipitation or EMSA experiments. Furthermore, Stat5 has been shown to suppress the GR-mediated transcription (31, 33), but Stat1 did not have any effect on GR-dependent transcriptional activation. Taken together, these results indicate that GR is regulating cytokine-induced gene activation by distinct mechanisms, depending on which Stat is activated. In the case of Stat5-mediated gene responses, the cooperation occurs through direct protein-protein interaction, whereas stimulation of Stat1-dependent responses does not involve physical interaction between GR and Stat1. It is possible that GR modulates Stat3-dependent responses through a similar mechanism as with Stat1, because the costimulatory effect of Dex on IL-6-induced haptoglobin expression has been shown to depend on the trans-activation domain of Stat3 (30).

The precise mechanisms of Stat1-mediated transcriptional activation are currently unknown and an important subject for future studies. The stimulatory effect of GR on Fc{gamma}RI activity required both activated Stat1 and the presence of PU.1. This result is in accordance with previous studies showing that the myeloid cell-specific expression of Fc{gamma}RI and IFN-{gamma} responsiveness are dependent on PU.1 (12, 13, 47) and demonstrate that all other transcription factors required for Fc{gamma}RI expression are also present in nonhemopoietic cells. PU.1 contains both acidic and glutamine-rich activation domains, which may individually regulate specific gene responses (41). Our results indicate that the trans-activation domains of GR cannot replace the function of PU.1 in Fc{gamma}RI expression. We also tested the possibility that PU.1 would be a target for GR regulation, but Dex did not influence PU.1 protein levels or DNA binding in monocytes (data not shown).

The costimulatory effect of Dex on Stat1-dependent reporter activity required GR-mediated induction of protein synthesis and the DBD and trans-activation domains of GR, but was not dependent on GRE in the promoter. It should be noted that the cooperation between Stat5 and GR depends only on the trans-activation domain of GR. The GR antagonist RU486, which blocks the ligand- and AF2-dependent trans-activation of GR, but not the nuclear translocation, abolished the enhancing effect of Dex on Fc{gamma}RI expression in monocytes. These findings are consistent with a model where GR induces transcription of a coactivator involved in Stat1-dependent activation of RNA polymerase II-mediated transcription. However, recruitment of an auxiliary protein through interaction with GR cannot be formally ruled out. The transcriptional coactivators CBP and p300 function as integrators for several nuclear signaling pathways, including Stats and steroid receptors, by stimulating transcription and catalyzing histone acetylation (18, 23, 45, 48). We also investigated the possibility that the enhancing effect of GR on Fc{gamma}RI promoter activation was mediated by the coactivator CBP. Ectopic expression of CBP in HepG2 cells did not enhance the IFN-{gamma}-induced Fc{gamma}RI-luc activity (data not shown), indicating that CBP was not a rate-limiting component in this response. Furthermore, MHC class II expression is stimulated in monocytes by IFN-{gamma} through Stat1 and class II trans-activator factors, and GR inhibits this response by squelching of CBP (49). Thus, the differential effect of Dex on Fc{gamma}RI and MHC class II expression, and the findings that Fc{gamma}RI does not contain GRE, and that Stat1 and GR are not physically associating argue against a role for CBP in the costimulatory action of GR. However, several other transcriptional coactivators exist (17, 50), and more are likely to be identified, and it will be important to determine their contributions to Stat1-mediated gene responses and regulation by GR.

Dex displays diverse and sometimes even opposite effects on various cell types, and Fc{gamma}RI is a good example of cell type-specific regulation of GR. In immature myeloid leukemia cell lines Dex treatment results in inhibition of IFN-{gamma}-induced Fc{gamma}RI expression (34, 35). We also analyzed the effect of Dex treatment on myeloid leukemic HL-60 cells, and our results excluded activation or DNA binding of Stat1 as well as the expression of GR or PU.1 proteins as targets for the inhibitory effect of Dex (data not shown). The inhibitory effect of Dex was not directly related to the differentiation stage of the cells, because induction of macrophage differentiation of HL-60 cells by 12-O-tetradecanoyl-phorbol-13-acetate treatment (48 h) did not alter the Dex-mediated repression on Fc{gamma}RI expression. Therefore, it seems likely that the difference in Dex effect between normal monocytes and transformed leukemia cells is due to inherent properties of the cells. At least two possible mechanisms can be envisioned for the inhibitory effect of Dex in HL-60 cells: GR is either inducing a repressive factor and/or the inhibition is due to competition of transcriptional coactivators.

T cells are an important target for the immunomodulatory effects of glucocorticoids. Despite the overall suppressive effect on T cell cytokine production, glucocorticoid treatment has been shown to result in increased IgE production in vivo (6, 51). The effects of glucocorticoids on accessory monocytes may provide insight into this seemingly paradoxical response. Glucocorticoids modulate cytokine production in monocytes, which leads to enhanced IL-10 synthesis and suppression of IL-12 synthesis, thereby promoting an anti-inflammatory response and Th2-type cytokine and IgE synthesis (6). Interestingly, ligation of Fc{gamma}RI on monocytes results in a similar cytokine response (4), and increased expression of Fc{gamma}RI by Dex may be a regulatory mechanism for this immunomodulatory response.

In this study we have analyzed the mechanisms of Dex in regulation of Fc{gamma}RI, which plays an important role in monocyte functions. Detailed understanding of the molecular mechanisms of glucocorticoids in different cell types is crucial for optimizing their clinical use and allow the development of derivatives with improved therapeutic value targeted specifically to a desired function.


   Acknowledgments
 
We thank Paula Kosonen for technical assistance and Drs A. Cato, J. E. Darnell, Jr., R. Goodman, O. Heikinheimo, I. Kerr, A. Lagerstedt, and R. Maki for kindly providing reagents.


   Footnotes
 
1 This work was supported by the Academy of Finland, the Sigrid Juselius Foundation, and the Medical Research Fund of Tampere University Hospital. Back

2 Address correspondence and reprint requests to Dr. Olli Silvennoinen, Department of Medical Biochemistry, University of Tampere, Lenkkeilijankatu 6, FIN-33014 Tampere, Finland. Back

3 Abbreviations used in this paper: GAS, IFN-{gamma}-activated site; AF, activation function domain; CREB, cAMP response element binding protein; CBP, CREB binding protein; IRF, IFN regulatory factor; CHX, cycloheximide; DBD, DNA binding domain; Epo, erythropoietin; EpoR, Epo receptor; Fc{gamma}RI, Fc receptor I for IgG; GR, glucocorticoid receptor; GRE, glucocorticoid response element; GRR, IFN-{gamma} response region; Dex, dexamethasone; RLU, relative luciferase unit; TK, thymidine kinase. Back

Received for publication October 14, 1999. Accepted for publication March 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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