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Receptor I in Monocytes1
*
Institute of Medical Technology, and Department of Medical Biochemistry, University of Tampere, and
Department of Clinical Microbiology, Tampere University Hospital, Tampere, Finland;
Chemotherapeutisches Forschungsinstitut, Georg Speyer Haus, Frankfurt am Main, Germany; and
§
Institute of Biomedicine, Departments of Physiology and Clinical Chemistry, University of Helsinki, Helsinki, Finland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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- and dexamethasone (Dex) in the induction of the
high-affinity Fc receptor I (FcRI) in monocytes. Dex did not
affect IFN--induced Stat1 DNA binding activity or induce novel
DNA-binding complexes to the FcRI 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 FcRI 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 FcRI gene
transcription. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, one of the most
potent monocyte-activating agents (1). An important
consequence of IFN- stimulation in monocytes is up-regulated
expression of the high-affinity receptor for IgG (FcRI, CD64)
(2). FcRI 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 FcRI 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- signal
transduction have been elucidated (8, 9). IFN- binds to
a receptor complex consisting of IFN-RI and the accessory chain
IFN-RII. Ligand-induced dimerization of the receptor chains results
in activation of Jak1 and Jak2 tyrosine kinases and phosphorylation of
the IFN-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--activated site
(GAS)3
(8). The Stat1 signaling pathway is essential for
induction of the FcRI gene, and Stat1-deficient mice are
unresponsive to IFN--induced FcRI expression (10).
Characterization of the FcRI promoter has shown that the IFN-
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--inducible region is localized to an IFN- 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-
B activation
by direct protein-protein interactions as well as by induction of
IB
(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- and Dex cooperatively induce FcRI expression
in monocytes (34, 35). This study was aimed at delineating
the mechanisms of action of Dex on the regulation of FcRI gene
expression, and our results indicate that GR functions as a
ligand-dependent costimulator of Stat1-mediated transcription.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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). FcRI-luc
was constructed by cloning a fragment of FcRI 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, 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- (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- 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 FcRI gene
(5'-GATATGAGCATGGGAAAAGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAGGAACATGATGAAAATG-3')
were labeled with T4-polynucleotide kinase using
[-32P]ATP and were used as probes. The
190-bp insert from FcRI-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- (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- (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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced expression of FcRI in monocytes
but does not affect Stat1 DNA binding or FcRI promoter binding
factors
IFN- 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--mediated
gene regulation is the FcRI, 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 FcRI
expression.
Human peripheral blood monocytes were treated with optimal doses of
either IFN- and Dex alone or both agents together for 20 h, and
the expression of FcRI was analyzed using FACS. In accordance with
previous results, IFN- increased the expression of FcRI
(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 FcRI expression.
Pretreatment of monocytes with the GR antagonist RU486 abolished the
effect of Dex on FcRI expression, but did not affect basal or
IFN--induced expression. Thus, GR does not directly regulate the
expression of FcRI, and the effect of Dex requires functional
activation of both GR- and IFN--induced factors.
Because Stat1 is essential for IFN--induced expression of FcRI
(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-, 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 FcRI promoter, and the proximal promoter
region (nucleotides -189 to +1) of FcRI (12).
Treatment of monocytes with IFN- 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- stimulation (Fig. 1
). Pretreatment of the lysate with
anti-Stat1 Ab supershifted the GAS binding complex. IFN- induced
two binding complexes to the GRR oligonucleotide, and the FcRI
binding complexes contained both constitutive components and
IFN--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
FcRI 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--induced complexes to GAS, GRR, or FcRI
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- signaling and Stat1 DNA binding.
Furthermore, GR does not appear to bind directly or induce novel
FcRI promoter binding complexes in monocytes.
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We examined the possibility that GR regulates the IFN--induced
transcriptional activation and used a heterologous HepG2 cell system
for this purpose. HepG2 cells are fully responsive to IFN-, 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- readily stimulated the GAS-luc
activity, and Dex treatment did not influence basal or IFN--induced
reporter activity (Fig. 2A).
HepG2 cells were transfected with increasing amounts of GR expression
plasmid. Coexpression of GR did not affect basal or IFN--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- 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-, or their combination did not influence Mut-GAS-luc
reporter activity (Fig. 2B), indicating that GR specifically
enhances the Stat1-dependent transcription.
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Stat1 is expressed as two alternatively spliced variants, Stat1
and Stat1ß. The Stat1ß isoform lacks the 38 C-terminal residues
(38). IFN- stimulates the tyrosine phosphorylation and
DNA binding of both isoforms, but only Stat1 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 or Stat1ß.
Transfection of Stat1 rendered the cells responsive to
IFN--induced activation of the GAS-luc reporter, and cotransfection
of GR with Stat1 resulted in synergistic activation of the reporter
in response to IFN- and Dex stimulation (Fig. 2
C). 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- 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- 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- (40). As expected, the Stat1E428A,E429A mutant did
not induce IFN--dependent reporter activation and also failed to
cooperate with GR (Fig. 2C). 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-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. 3). The
induction was further enhanced by ectopic expression of GR, and, as
expected, the reporter activity was not affected by IFN- treatment.
Cotransfection of GR with Stat1 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- stimulation. Similar results were obtained in HepG2
cells in which activation of GRE-luc required transfected GR, but
costimulation with IFN-, and cotransfection of Stat1 or different
Stat1 variants, did not affect the Dex-induced activation of GRE-luc
reporter (data not shown).
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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. 4A). Likewise, immunoblotting
of GR immunoprecipitates with anti-Stat1 Ab did not detect any
coimmunoprecipitation of the two proteins (Fig. 4B). 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- did not induce complex formation
between Stat1 and GR (Fig. 4A). 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. 4). 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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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- and Dex
stimulation (Fig. 5). 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--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. 5). 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--induced reporter activity in the absence of Dex
as efficiently as ligand-stimulated wild-type GR.
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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- for 6 h. The results
presented in Fig. 5B 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- 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 FcRI promoter is dependent on Stat1 and PU.1
In the FcRI promoter, the IFN--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 FcRI promoter, a luciferase
reporter driven by the regulatory region of the native FcRI promoter
(nucleotides -189 to +1) was constructed (FcRI-luc)
(12). HepG2 cells allowed the analysis of individual
transcription factors in regulation of FcRI-luc expression, because
these cells lack endogenous PU.1 and express very low levels of GR.
Transfection of FcRI-luc into HepG2 cells, either alone or together
with GR, did not result in any reporter activity after IFN- and Dex
stimulations (Fig. 6). Transfection of
PU.1 into HepG2 cells stimulated FcRI-luc activity, which is in
accordance with the basal expression of FcRI in monocytes. The
presence of PU.1 was also absolutely required for the IFN- induction
of FcRI-luc reporter. When both PU.1 and GR were coexpressed in
HepG2 cells, Dex increased the IFN--dependent activation of
FcRI-luc reporter to a comparable degree to that observed in
monocytes. Together these results demonstrate that the stimulatory
effect of GR on FcRI is dependent on both Stat1 and PU.1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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- regulate FcRI
expression in monocytes. Our results demonstrate that the stimulatory
effect of Dex on FcRI 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 FcRI
expression is the enhancement of IFN--dependent signaling events
through inducing the expression of the IFN- receptor or other
critical signaling proteins, such as Jak kinases or Stat1, or
alternatively, through inducing FcRI promoter-binding transcription
factors. In monocytes, Dex treatment, either short or long term, did
not modulate the IFN--induced Stat1 DNA binding activity or protein
expression (data not shown) or induce novel DNA binding complexes to
the FcRI promoter. Thus, Dex does not appear to directly regulate
the immediate IFN- signaling events or FcRI 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
FcRI promoter. GR stimulated the GAS reporter more efficiently than
the natural FcRI 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 FcRI promoter
activity was similar to the level of enhancement on FcRI 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 FcRI 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
FcRI and IFN- responsiveness are dependent on PU.1 (12, 13, 47) and demonstrate that all other transcription factors
required for FcRI 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 FcRI 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 FcRI 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 FcRI promoter
activation was mediated by the coactivator CBP. Ectopic expression of
CBP in HepG2 cells did not enhance the IFN--induced FcRI-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- 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 FcRI and MHC class II expression, and the findings that
FcRI 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 FcRI is a good example of cell type-specific
regulation of GR. In immature myeloid leukemia cell lines Dex treatment
results in inhibition of IFN--induced FcRI 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 FcRI 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 FcRI on monocytes
results in a similar cytokine response (4), and increased
expression of FcRI by Dex may be a regulatory mechanism for this
immunomodulatory response.
In this study we have analyzed the mechanisms of Dex in regulation of
FcRI, 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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Olli Silvennoinen, Department of Medical Biochemistry, University of Tampere, Lenkkeilijankatu 6, FIN-33014 Tampere, Finland.
3 Abbreviations used in this paper: GAS, IFN--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; FcRI, Fc receptor I for IgG; GR, glucocorticoid receptor; GRE, glucocorticoid response element; GRR, IFN- response region; Dex, dexamethasone; RLU, relative luciferase unit; TK, thymidine kinase.
Received for publication October 14, 1999. Accepted for publication March 22, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptor type I. J. Exp. Med. 188:217.RI receptor signals through the activation of hck and MAP kinase. J. Immunol. 154:4039.[Abstract]
receptor signaling in macrophages and neutrophils. Mol. Cell. Biol. 18:4209.-induced transcription of the high-affinity Fc receptor for IgG requires assembly of a complex that includes the 91-kDa subunit of transcription factor ISGF3. Proc. Natl. Acad. Sci. USA 90:4314. responsiveness and basal expression of the myeloid human FcR1b gene is mediated by a functional PU.1 site and a transcription initiator consensus. J. Exp. Med. 179:1985. receptor. Mol. Cell. Biol. 14:5023.B activity through induction of IB synthesis. Science 270:286.B and the glucocorticoid receptor. Proc. Natl. Acad. Sci. USA 91:752.B-dependent transcription involves direct interference with transactivation. Proc. Natl. Acad. Sci. USA 94:13504.. Nature 383:344.[Medline]
-interferon augmentation of human monocyte immunoglobulin G Fc receptor expression. J. Immunol. 138:3235.[Abstract]
RI mRNA by IFN- and dexamethasone in human monocytes, neutrophils, and U-937 cells. J. Immunol. 145:267.[Abstract]
and IFN, and is likely to autoregulate the p91 gene. EMBO J. 13:158.[Medline]
and - signal transduction pathways. EMBO J. 12:4221.[Medline]
. Blood 90:4341. in IFN--induced transcriptional activation. EMBO J. 17:6963.[Medline]
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