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Regulation of Class II Transactivator Promoter IV in Macrophages and Microglia: Involvement of the Suppressors of Cytokine Signaling-1 Protein
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated promoter (promoter IV), have provided
new opportunities to understand the molecular mechanisms of
IFN--induced class II MHC expression. Here, we investigated the
molecular regulation of IFN--induced murine CIITA promoter IV
activity in microglia/macrophages. In the macrophage cell line
RAW264.7, IFN- inducibility of CIITA promoter IV is dependent on an
IFN- activation sequence (GAS) element and adjacent E-Box, and an
IFN response factor (IRF) element, all within 196 bp of the
transcription start site. In both RAW cells and the microglia cell line
EOC20, two IFN--activated transcription factors, STAT-1
and
IRF-1, bind the GAS and IRF elements, respectively. The E-Box binds
upstream stimulating factor-1 (USF-1), a constitutively expressed
transcription factor. Functionally, the GAS, E-Box, and IRF elements
are each essential for IFN--induced CIITA promoter IV activity. The
effects of the suppressors of cytokine signaling-1 (SOCS-1) protein on
IFN--induced CIITA and class II MHC expression were examined.
Ectopic expression of SOCS-1 inhibits IFN--induced activation of
CIITA promoter IV and subsequent class II MHC protein expression.
Interestingly, SOCS-1 inhibits the constitutive expression of STAT-1
and its IFN--induced tyrosine phosphorylation and binding to the GAS
element in CIITA promoter IV. As well, IFN--induced expression of
IRF-1 and its binding to the IRF element is inhibited. These results
indicate that SOCS-1 may be responsible for attenuating IFN--induced
CIITA and class II MHC expression in
macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated microglia and macrophages express
class II MHC molecules, the costimulatory molecules B7 and CD40, and
serve as the major APCs in the CNS (4, 5, 6, 7, 8, 9, 10, 11). In CNS
diseases such as MS, Alzheimer’s disease and HIV-1-associated
dementia, prominent expression of class II MHC molecules has been
detected on microglia and macrophages (for review, see Refs. 12, 13). The expression of class II MHC and costimulatory molecules
by microglia and macrophages allows them to activate naive,
autoreactive CD4+ Th cells and restimulate memory
Th1 cells, leading to inflammation and demyelination
(14, 15, 16). Thus, microglia and macrophages are implicated
in both the initiation and perpetuation of inflammation in the
CNS.
Class II MHC molecules are constitutively expressed at high levels on
professional APCs such as thymic epithelium, dendritic cells, and B
cells. Class II MHC expression can be induced by IFN- on a number of
cells found in the CNS including infiltrating macrophages, astrocytes,
and microglia (for review, see Refs. 16, 17). Regulated
control of class II MHC gene expression is required to ensure that a
proper immune response can be initiated against pathogens. However,
aberrant expression of class II MHC Ags is thought to be involved in
the pathogenesis of a number of autoimmune disorders (for review, see
Ref. 18). The regulation of class II MHC gene expression
is primarily at the level of transcription, and the class II
transactivator (CIITA), a non-DNA-binding protein, is required for both
constitutive and IFN--inducible class II MHC expression
(19, 20, 21). CIITA, which is inducible by IFN-, acts as a
transcriptional integrator by interacting with DNA-bound transcription
factors, components of the general transcription machinery, and other
transcriptional coactivators to permit activation of class II MHC gene
expression (for review, see Refs. 22, 23, 24). Understanding
the regulation of class II MHC gene expression in microglia will
require understanding the regulation of CIITA expression.
Recently, it has been shown that expression of the CIITA
gene is controlled by the alternative usage of three distinct
promoters: constitutive expression in dendritic cells and in B
lymphocytes by promoters I and III, respectively, and IFN--inducible
expression in other cell types primarily by promoter IV
(25). Promoter III has also been shown to be IFN-
inducible in a number of cell types such as a fibrosarcoma cell line,
endothelial cells, and a murine macrophage cell line
(26, 27, 28). In a melanoma cell line and in primary rat
astrocytes, IFN- activation of the human CIITA promoter IV is
controlled by three cis-acting elements, an IFN-
activation sequence (GAS), an E-Box, and an IFN regulatory factor (IRF)
element that bind the transcription factors STAT-1, upstream
stimulating factor-1 (USF-1), and IRF-1, respectively
(28, 29, 30). Furthermore, specific IFN- activation of the
CIITA promoter IV is controlled by the cooperative interaction of
IFN--activated STAT-1 and constitutively expressed USF-1. The
regulatory elements and factors required for IFN--induced activation
of the mouse CIITA promoter IV are not known in macrophages or
microglia.
Cytokine activation of signal transduction pathways is transient,
indicating that mechanisms limiting the duration of cytokine signaling
are important for regulating their actions. Recently, a family of
SH2-containing proteins called suppressors of cytokine signaling (SOCS)
have been isolated and shown to negatively regulate cytokine signal
transduction pathways (for review, see Refs. 31, 32, 33). SOCS
proteins are induced by a variety of cytokines and attenuate signaling
either through a classic negative feedback loop or through
cross-inhibition of cytokine signaling pathways (34, 35, 36, 37).
IFN- induces SOCS-1 mRNA in a variety of tissues and cell types
(34, 37), and SOCS-1 has been shown to inhibit
IFN--activated STAT-1 phosphorylation in the human cell lines
HeLa and MCF-7 (38). SOCS-1, which contains a centrally
located SH2 domain, has been shown to bind to tyrosine-phosphorylated
Janus kinase (JAK) 2 and inhibit its kinase activity (35, 39).
In this study we determined the molecular mechanism of IFN--induced
CIITA promoter IV expression in murine microglia and macrophages. In
addition, we studied the influence of the SOCS-1 protein on IFN-
activation of the JAK/STAT pathway in macrophages and on
IFN--induced CIITA and class II MHC gene expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse rIFN- was purchased from R&D Systems (Minneapolis, MN).
Polyclonal antiserum to STAT-1 and monoclonal antiserum to
phosphotyrosine (4G10) were purchased from Upstate Biotechnology (Lake
Placid, NY), and antisera against STAT-3, STAT-6, USF-1, IRF-1, and
IRF-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Monoclonal antiserum to c-myc (9E10) was purchased from
Calbiochem (San Diego, CA).
Cells
The microglial cell line EOC20 was derived from C3H/HeJ CH-2k mice using a nonviral immortalization procedure. This cell line is B7-1 positive, Mac-1 positive, and class I MHC positive, as well as phagocytic (40). The EOC20 cells were maintained in DMEM complete medium (2 mM glutamine and 10% heat-inactivated FBS) with 20% conditioned medium from LADMAC cells as a source of CSF-1. Experiments described in this study were performed on the EOC20 line at passages 7–20. The murine macrophage cell line RAW264.7 was purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM complete medium. Cells were seeded at 1 x 106 cells/dish and grown to confluence in 100- or 60-mm dishes. For passage, monolayers were scraped and replated at a 1:5 dilution.
RNA isolation, riboprobes, and RNase protection assays (RPAs)
Total cellular RNA was isolated from confluent monolayers of RAW
cells that were stimulated with IFN- for various time periods. The
mouse CIITA, class II MHC, and GAPDH riboprobes have been previously
described (41). A pGEM-4Z vector containing a fragment of
the mouse IRF-1 cDNA (corresponding to bp 1–314) inserted at the
polylinker sites XbaI/SalI was linearized with
NdeI. In vitro transcription of this fragment with T7 RNA
polymerase generates a 302-bp antisense RNA probe.
RPAs were conducted with an RPA kit according to the manufacturer’s instructions (Ambion, Austin, TX). Total RNA (20 µg) from RAW cells was hybridized with CIITA, class II MHC, IRF-1, and GAPDH riboprobes (25 x 103 cpm) at 42°C overnight in 20 µl of 40 mM PIPES pH 6.4, 80% deionized formamide, 400 mM NaOAc, and 1 mM EDTA. The hybridized mixture was then treated with RNase A/T1 (1:500 dilution in 200 µl of the RNase digestion buffer) at room temperature for 1 h, ethanol precipitated, and analyzed by 5% denaturing (8 M urea) polyacrylamide gel electrophoresis. After drying, the gels were exposed to PhosphorImager screens (Molecular Dynamics, Sunnyvale, CA) and then x-ray film for varying periods of time. The protected fragments of the CIITA, class II MHC, IRF-1, and GAPDH riboprobes are 429, 307, 261, and 212 nt in length, respectively. The protected RNA fragments were quantitated by scanning with the PhosphorImager, which can quantitate varying intensities of protected RNA fragments. Values for CIITA, class II, and IRF-1 mRNA expression were normalized to GAPDH mRNA levels for each experimental condition. GAPDH mRNA is a housekeeping gene whose levels are not affected by cytokine treatment.
CIITA promoter and SOCS constructs
The primers used to PCR amplify a 1487-bp DNA fragment of the type IV promoter of the mouse CIITA gene were derived from the sequence of the type III and IV promoters of the mouse CIITA gene (25). The sense primer is located at the 3' end of the type III promoter and has the sequence 5'-TGCCTGGTTCCTGGCCCTTCTG-3', and the antisense primer is located at the 3' end of the type IV promoter and has the sequence 5'-CGCGGCTGTGGCTGTGCCCCGTGCT-3'. The resulting 1487-bp fragment was gel purified and ligated into a linearized pCRII vector (Invitrogen, Carlsbad, CA). The complete sequence of the insert was obtained by automatic sequencing, which was performed by the University of Alabama at Birmingham Center for AIDS Research Molecular Biology Core Facility. The 1487-bp insert was released from pCRII by digestion with the restriction enzymes KpnI/XhoI and gel purified. The fragment was ligated into the KpnI/XhoI site of the pGL3-basic vector (Promega, Madison, WI), which contains the gene for luciferase as a reporter. The designated name for this construct is mCIITAp1.4. Plasmid constructs containing 5' deletions were prepared by digestion of mCIITAp1.4 with KpnI/NheI, KpnI/BglII, KpnI/Tth111I, and KpnI/Eco72I. Appropriate fragments were gel purified, blunt-ended with the Klenow fragment of DNA polymerase I according to the manufacturer (Promega), and religated to generate mCIITApD1-mCIITApD4. The deletion construct mCIITApD5 was generated by religating a 129-bp (-46 to +83) PCR fragment into a MluI/XhoI digestion of pGL3-Basic. The site-directed mutation constructs mCIITA-GAS, mCIITA-E-Box, mCIITA-IRF, mCIITA-GAS + E-box, mCIITA-GAS + IRF, and mCIITA-GAS + E-Box + IRF were generated on the mCIITAp1.4 plasmid backbone using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) following the manufacturer’s instructions, and were confirmed by sequencing. A pcDNA3 expression vector containing an N-terminal myc-tagged cDNA of mouse SOCS-1 was a generous gift from Professor Akihiko Yoshimura (Kurume University, Kurume, Japan) (35, 39).
Transfections and luciferase assay
For deletion and site-specific mutation studies of CIITA
promoter IV, 2 µg of the mCIITA promoter IV constructs (full-length,
deletion, or mutant constructs) were cotransfected with 0.5 µg of the
pCMV-
-galactosidase construct into 0.5 x
106 RAW cells in six-well plates using the
Lipofectamine Plus method according to the manufacturer (Life
Technologies, Rockville, MD). After transfection, the cells were
treated with IFN- for 12 h, which we previously determined to
be optimal for IFN--induced CIITA promoter IV activity (29, 30). The medium was aspirated, and cells were lysed with 250
µl of lysis buffer containing 25 mM trisphosphate (pH 7.8), 2 mM DTT,
2 mM diaminocyclohexane tetraacetic acid, 10% glycerol, and 1% Triton
X-100. Extracts were assayed in triplicate for luciferase activity in a
volume of 130 µl containing 30 µl cell extract, 20 mM tricine, 0.1
mM EDTA, 1 mM magnesium carbonate, 2.67 mM MgSO4,
33.3 mM DDT, 0.27 mM coenzyme A, 0.47 mM luciferin, and 0.53 mM ATP,
and light intensity was measured with a luminometer (Promega).
Luciferase activity was integrated over a 10-s time period. Extracts
were also assayed in triplicate for -galactosidase activity, and the
luciferase activity of each sample was normalized to -galactosidase
activity to calculate relative luciferase activity (RLA). Fold
induction was calculated by dividing the RLA of the IFN--treated
samples by the RLA of those samples incubated in medium alone.
Stable transfection of SOCS-1
SOCS-1 stable transfectants were created by transfecting RAW cells with the pcDNA3 expression vector containing N-terminal myc-tagged cDNA of mouse SOCS-1 (35, 39) using the Lipofectamine Plus method according to the manufacturer (Life Technologies). Mock cDNA, which has only the pcDNA3 plasmid, was used as a negative control. Cells were selected in G418 sulfate (100 µg/ml) and screened for SOCS-1 expression by immunoblotting for c-myc expression.
Nuclear extracts and EMSA
EOC20 and RAW cells were grown in 100-mm dishes and then
stimulated in medium containing 1% FBS with or without IFN- for
1–2 h. The cells were then washed with cold PBS, harvested by
scraping, and pelleted. Cells were resuspended in 0.5 ml buffer A (10
mM KCl, 20 mM HEPES, 1 mM MgCl2, 1 mM DTT, 0.4 mM
PMSF, 1 mM NaF, 1 mM
Na3VO4), incubated on ice
for 10 min, and pelleted at 1000 x g for 10 min.
Pellets were resuspended in 0.25 ml of buffer A plus 0.1% Nonidet
P-40, incubated on ice for 10 min, and centrifuged at 3000 x
g for 10 min. The nuclear pellet was resuspended in 0.25 ml
of buffer B (10 mM HEPES, 400 mM NaCl, 0.1 mM EDTA, 1 mM
MgCl2, 1 mM DTT, 0.4 mM PMSF, 15% glycerol, 1 mM
NaF, 1 mM Na3VO4) and
incubated for 30 min at 4°C with constant gentle mixing. Nuclei were
then pelleted at 14,000 x g for 15 min, and extracts
were dialyzed for 2 h at 4°C against 1 liter of buffer C (20 mM
HEPES, 200 mM KCl, 1 mM MgCl2, 0.1 mM EDTA, 1 mM
DTT, 0.4 mM PMSF, 15% glycerol, 1 mM NaF, 1 mM
Na3VO4). Extracts were
cleared by centrifugation at 14,000 x g for 15 min at
4°C. Protein concentrations were determined using a Bio-Rad protein
assay. EMSA was performed using the following oligonucleotides as
probes and/or competitors: the oligonucleotide CIITA-GAS + E-box
corresponds to the type IV CIITA promoter sequence -178 to -140, and
the CIITA-IRF-1 oligonucleotide corresponds to the type IV CIITA
promoter sequence -81 to -43, as previously published
(25). 32P-labeled oligonucleotide
(0.2 ng, 20,000 cpm) was incubated for 30 min at room temperature with
10 µg of nuclear extract in a volume of 20 µl containing 50 mM KCl,
2.5 mM MgCl2, 1 mM EDTA, 1 mM DTT, 10 mM Tris-Cl
(pH 7.5), 12% glycerol, 1 µg salmon sperm DNA and 1 µg
poly(dI:dC). For supershift analysis, 1 µl of Ab was incubated with
the nuclear extracts at 4°C for 30 min in binding buffer, followed by
an additional incubation for 30 min at room temperature with labeled
oligonucleotide. For competition experiments, unlabeled DNA was
incubated with the nuclear extracts at 4°C for 20 min before addition
of labeled probe. Bound and free DNA were resolved by electrophoresis
through a 6% polyacrylamide gel at 250 V in 1x TGE (50 mM Tris-Cl,
380 mM glycine, 2 mM EDTA). Dried gels were exposed to Kodak XAR-5 film
at -70°C with intensifying screens. Four different preparations of
nuclear extracts were tested by EMSA.
Immunofluorescence flow cytometry
RAW cells were plated at 5 x 105
cells/well into six-well (35-mm) plates (Costar, Cambridge, MA), and
wells were either untreated or treated with IFN- for 36 h. The
cells were scraped; then Fc receptors were blocked with 10% normal
mouse serum, stained for class II MHC using anti-class II MHC Abs
(1:10 dilution), and analyzed on the FACStar (Becton Dickinson,
Mountain View, CA). Negative controls were incubated with IgG
isotype-matched Abs. Ten thousand cells were analyzed for each sample.
Class II MHC expression is presented as total class II MHC, which is
calculated as the percentage of positive cells x mean
fluorescence intensity (MFI). This is calculated as such because both
parameters are affected by cytokine treatment (41).
Immunoprecipitation and Western blotting
For detection of IRF-1 and c-myc-tagged SOCS-1, 50
µg of cell lysates were boiled in sample buffer, separated on 10%
SDS-PAGE, transferred to nitrocellulose membrane, and probed with
anti-IRF-1 or anti-c-myc Ab. For
immunoprecipitation, cells were treated with medium or IFN- (100
U/ml) for 30 min, and cell lysates were prepared as described
previously (7). Total protein (0.5 mg) was incubated with
polyclonal antisera against STAT-1, STAT-3, or STAT-6 (5 µl).
Protein G-agarose (50 µl) was added for 2 h at 4°C. The
immunoprecipitates were washed five times with lysis buffer, eluted
from the agarose beads by boiling in 2x SDS-sample buffer, and
subjected to 6% SDS-PAGE. Proteins were then transferred to
nitrocellulose and probed with monoclonal anti-phosphotyrosine Ab
4G10 (1 µg/ml), anti-STAT-3 Ab, or anti-STAT-6 Ab. Membranes
were stripped at 50°C in buffer containing 100 mM 2-ME, 2% SDS, 62.5
mM Tris-HCl (pH 6.7) with occasional shaking, and reprobed for
STAT-1 protein. ECL was used for detection of bound Ab.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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response elements in the murine CIITA
promoter IV
To investigate the molecular mechanism of IFN--induced CIITA
expression, we cloned the full-length murine CIITA promoter IV by
genomic PCR using primers with a forward sequence located in the 3' end
of the mouse CIITA promoter III and a reverse sequence located in the
3' end of the mouse CIITA promoter IV, as previously published
(25). Analysis of this 1487-bp sequence using the MAT
Inspector program (42) confirmed the presence of a GAS, an
E-Box, and an IRF element, as well as two putative AP-1 elements and a
NF-GMa element (Fig. 1
). The 1487-bp
fragment was inserted into the pGL3-basic vector containing the
luciferase gene. This full-length CIITA promoter IV/luciferase reporter
construct is called mCIITAp1.4. We initially attempted to perform the
functional analysis of this promoter in microglia, but were unable to
transfect either the EOC20 cell line or primary microglia with any
currently available transfection protocol. The macrophage cell line
RAW264.7 has been reported to be highly transfectable
(43). IFN- is a potent inducer of IRF-1, CIITA, class
II MHC mRNA (Fig. 2), and class II MHC
surface expression (data not shown) in the RAW cells, similar to what
we have previously observed in microglia (41). Thus, all
transfection studies were performed in the RAW264.7 macrophage
cell line.
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-induced CIITA gene
activation in macrophages, a series of 5' deletions to the CIITA
promoter IV were made, designated as mCIITApD1-mCIITApD5 (Fig. 1
). Each
of the constructs was transiently transfected into RAW cells with a
pCMV--galactosidase construct used to normalize luciferase activity
for transfection efficiency. The cells were then stimulated with
IFN- (100 U/ml) for 12 h, which we have previously determined
to be the optimal time point for IFN--induced CIITA promoter IV
activity (29, 30). The empty vector was not modulated by
IFN- treatment (data not shown). A very low basal level of
luciferase activity for the full-length construct, mCIITAp1.4, was
detected, and a 7.9-fold induction in RLA was seen after stimulation
with IFN- (Fig. 1). Deletion constructs mCIITApD1, mCIITApD2, and
mCIITApD3 showed slightly lower IFN--induced fold induction levels
of 5.9, 6.4, and 5.6, respectively. These data indicate that the
sequence from -1404 to -196, which contains the putative AP-1 sites,
is not required for IFN--induced activity of the murine CIITA
promoter IV. Further deletion of the NF-GMa, GAS, and E-Box elements
(mCIITApD4) led to a 70% loss in IFN--induced reporter activity
when compared with the full-length mCIITAp1.4. Finally, deletion of the
IRF element in construct mCIITApD5 essentially abolished
IFN--induced CIITA promoter IV activity. These results collectively
demonstrate that IFN--induced activation of the CIITA
gene in the RAW cell line is dependent on elements contained in a
196-bp fragment of the murine CIITA promoter IV.
Contribution of the GAS, E-Box, and IRF elements to IFN--induced
CIITA promoter IV activity
To assess the importance of the GAS, E-Box, and IRF elements in
IFN--induced CIITA promoter IV activity, mCIITAp1.4 constructs with
mutations in each of these elements individually or in combination were
generated. Mutation of the GAS element resulted in 
70% reduction of
IFN--induced CIITA promoter IV activity, whereas mutation of the
E-Box or IRF element individually resulted in 50% reduction of
IFN--induced activity (Fig. 3).
Mutations in both the GAS and IRF elements or in both the E-Box and IRF
elements essentially abolished IFN--induced CIITA promoter IV
activation, as did mutations in all three elements (Fig. 3). Mutation
of the GAS and E-Box together showed a 70% inhibition of
IFN--induction of CIITA promoter IV activity (data not shown),
similar to that seen with the mutation of the GAS element alone (Fig. 3). Thus, in the RAW264.7 cell line, IFN- induction of CIITA
promoter IV activity requires all three regulatory elements.
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induction of
CIITA promoter IV (29). In RAW cells, mutation of the
NF-GMa element resulted in an 12-fold induction in RLA following
IFN- treatment (data not shown), which is 1.5 times that of the
wild-type construct (7.9-fold induction). These results indicate that
the NF-GMa element also negatively controls IFN- induction of the
mouse CIITA promoter IV in the RAW264.7 macrophage cell line. DNA-protein complex formation over the GAS and E-Box elements in CIITA promoter IV
To identify the protein(s) that bind to the GAS and E-Box elements
in CIITA promoter IV, nuclear extracts were prepared from EOC20 and RAW
cells, and EMSA was performed with 32P-labeled
oligonucleotides spanning both the GAS and E-Box elements. Using
extracts from unstimulated EOC20 cells, two DNA-protein complexes were
detected: complexes 2 and 3 (Fig. 4, lane 2). Extracts from EOC20 cells stimulated with IFN-
for 1 h contained an additional complex, complex 1, a doublet with
slower electrophoretic mobility (lane 3). Using an
excess of unlabeled GAS + E-Box oligonucleotides, all three complexes
from cells stimulated with IFN- were competed away
(lane 4). To determine the identities of the proteins
in the three complexes, supershift experiments were performed using Abs
against USF-1, STAT-1, and STAT-3. The IFN--induced complex 1 is
supershifted in the presence of STAT-1 antisera (lane
9), but not STAT-3 (lane 10). These results
indicate complex 1 is composed of STAT-1. Complexes 2 and 3 present
in extracts from unstimulated cells were supershifted by USF-1 antisera
(lane 6), as were all three complexes observed in
IFN--stimulated cells (lane 8). These results
indicate that complexes 1, 2, and 3 all contain USF-1. Identical
DNA-protein complexes were seen in unstimulated and IFN--stimulated
RAW cells, and Ab supershift experiments showed that complex 1
contained STAT-1 and that all three complexes contained USF-1 (data
not shown).
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We next analyzed protein(s) binding to the IRF element in CIITA
promoter IV. Nuclear extracts from EOC20 cells that were unstimulated
or stimulated with IFN- for 2 h were incubated with a
32P-labeled oligonucleotide containing the IRF
element in CIITA promoter IV. Extracts from unstimulated EOC20 cells
showed several DNA-protein complexes (Fig. 5, lane 1). All of the
complexes could be competed away by an excess of the unlabeled IRF
oligonucleotide (data not shown). Only the complexes designated A and B
were consistently present in all of the extracts tested, whereas the
appearance of the other complexes varied between extracts. The identity
of the proteins was analyzed by supershift experiments using Abs to
IRF-1, IRF-2, and STAT-1. Complex B was completely supershifted by
antisera to IRF-1 (lane 3), whereas complex A was
completely supershifted by antisera to IRF-2 (lane
4). The inclusion of antisera to STAT-1 did not affect binding
of complex A or B in unstimulated EOC20 cells (lane
5). These results indicate that complex A and B contain IRF-2 and
IRF-1, respectively, and that they are constitutively bound to the IRF
element in CIITA promoter IV in the EOC20 cells. In extracts from cells
stimulated with IFN- there was a pronounced enhancement of complex B
binding (lane 6), which was again supershifted by
antisera to IRF-1 (lane 8). No enhancement in binding
of complex A containing IRF-2 after IFN- stimulation was observed.
Again, antisera to STAT-1 had no effect on the mobility of any of
the DNA-protein complexes from EOC20 cells stimulated with IFN-
(lane 10). Identical DNA-protein complexes were
observed using extracts from unstimulated and IFN--stimulated RAW
cells (data not shown).
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-induced CIITA
promoter IV activity and class II MHC expression
Because little is known about how IFN- signaling and class II
MHC expression is negatively regulated in macrophages, we investigated
the effect of the SOCS-1 protein on IFN- activation of the JAK/STAT
pathway, and on IFN--induced CIITA promoter IV activity and class II
MHC gene expression in RAW cells. SOCS-1 stable transfectants in the
RAW264.7 cell line were generated. Clonal populations were screened for
ectopic SOCS-1 expression by immunoblotting for the c-myc
epitope tagged to the murine SOCS-1 cDNA (44). No SOCS-1
expression was detected in mock transfected cells carrying the empty
pcDNA3 expression plasmid (Fig. 6, lane 1). Two clones with strong SOCS-1 expression
(lanes 2 and 3) were selected for further
analysis.
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-induced class II MHC surface expression in the SOCS-1 stable
transfectants was analyzed using flow cytometry. Treatment with IFN-
led to a 14-fold increase in class II MHC surface expression in the
mock transfected cells (Table I).
However, in both clones overexpressing SOCS-1, IFN--induced class II
MHC surface expression was inhibited by 88% (Table I). To determine
whether SOCS-1 expression could inhibit IFN--induced CIITA promoter
IV activation, the SOCS-1 stable transfectants were transiently
transfected with the mCIITAp1.4 construct, treated with IFN-, and
then promoter activity was determined. IFN- treatment led to a
nearly 8-fold increase in CIITA promoter IV activity in the mock
transfected cells, and SOCS-1 expression resulted in a 70–81%
inhibition in IFN--induced CIITA promoter IV activity (Table I). We
also analyzed whether IFN--induced CIITA mRNA expression was
inhibited by SOCS-1 expression. As shown in Fig. 7, IFN- treatment led to strong
expression of CIITA mRNA after 6 and 12 h (13- and 23-fold
induction, respectively, lanes 2 and 3). SOCS-1
expression attenuated this effect, resulting in a complete loss of
IFN--induced CIITA mRNA expression (lanes 5 and
6). These data indicate that ectopic expression of SOCS-1
inhibits IFN--induced class II MHC expression in macrophages, and
that this inhibition correlates with SOCS-1 inhibition of
IFN--induced CIITA mRNA expression and activation of CIITA
promoter IV.
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constitutive expression, IFN--induced
STAT-1 phosphorylation, and binding to the GAS element
SOCS-1 has been shown to inhibit IFN--induced STAT-1
phosphorylation in several human cell lines (38). We have
demonstrated that IFN- induction of CIITA promoter IV requires
STAT-1 binding to the GAS element (Figs. 3 and 4). Accordingly, we
next examined IFN--induced STAT-1 phosphorylation in the SOCS-1
stable transfectants. STAT-1 was immunoprecipitated from cells that
were untreated or treated with IFN- for 30 min, and then Western
blotted for tyrosine phosphorylation. In the SOCS-1 transfectants,
IFN--induced STAT-1 phosphorylation was completely inhibited
(Fig. 8A, top,
lanes 4 and 6). Interestingly, when compared with
the mock transfected cells, STAT-1 total protein levels were
noticeably reduced in the two SOCS-1 transfectants (Fig. 8A,
middle, compare lanes 1 and 2 to
lanes 3–6). However, SOCS-1 expression had no effect on
STAT-6 or STAT-3 total protein expression (Fig. 8A, lower
two panels). We next examined IFN--induced binding of STAT-1 to
the GAS element in CIITA promoter IV. In the mock transfected cells,
IFN- induced strong binding of STAT-1 to the GAS element of CIITA
promoter IV (Fig. 8B, lane 2). SOCS-1
overexpression led to almost complete inhibition of IFN--induced
binding of STAT-1 (Fig. 8B, lanes 4 and
6). SOCS-1 expression had no effect on the binding of USF-1
to the E-Box element (Fig. 8B, lanes 3–6).
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-induced IRF-1
expression and IRF-1 binding to the IRF element
We have shown that IFN--induced CIITA promoter IV activity in
RAW cells also requires IRF-1 binding to the IRF element (Figs. 3 and 5). The IRF-1 gene is an immediate-early gene in the IFN-
signaling pathway, and its promoter contains a single GAS element that
binds tyrosine-phosphorylated STAT-1 in response to IFN-
(45). If SOCS-1 expression leads to a general inhibition
of STAT-1 expression and its IFN--induced tyrosine
phosphorylation, we would also expect to see inhibition of
IFN--induced IRF-1 expression in the SOCS-1 transfectants. To
confirm this, IFN--induced IRF-1 mRNA and protein expression in the
SOCS-1 stable transfectants was examined. RNA was isolated from cells
untreated or treated with IFN- for 2 h, and then analyzed by
RPA. In the SOCS-1 transfectants, IFN--induced IRF-1 mRNA expression
was markedly reduced (Fig. 9A,
compare lane 2 with lanes 4 and 6).
Similarly, when lysates were prepared from cells untreated or treated
with IFN- for 3 h and then analyzed by Western blotting, SOCS-1
expression reduced IFN--induced IRF-1 protein expression (Fig. 9B, compare lane 2 with lanes 4 and
6). We next examined IFN--induced binding of IRF-1 to the
IRF element in CIITA promoter IV. In the mock transfected cells,
IFN--induced strong binding of IRF-1 (Fig. 9C, lane
2), whereas SOCS-1 expression led to almost complete inhibition of
IFN--induced binding of IRF-1 to the IRF probe (lanes
4 and 6). SOCS-1 overexpression had no effect on the
constitutive binding of IRF-2 to the IRF element (Fig. 9C,
lanes 3–6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activation of CIITA promoter IV in macrophages and microglia.
The effect of SOCS-1 expression on IFN- signaling and class II MHC
expression in macrophages was also examined. Our results demonstrate
that the GAS, E-Box, and IRF elements found within 196 bp of the
transcriptional start site in promoter IV each contribute to IFN-
inducibility of the promoter in macrophages. In macrophages and
microglia, USF-1, as well as IRF-1 and IRF-2, constitutively occupied
the E-Box and IRF elements, respectively. IFN- induced the binding
of STAT-1 to the GAS element and markedly augmented the binding of
IRF-1 to the IRF element. Ectopic expression of SOCS-1 attenuated
IFN--induced CIITA mRNA expression, CIITA promoter IV activation,
and class II MHC expression in macrophages. SOCS-1 expression reduced
constitutive STAT-1 protein expression, inhibited IFN--induced
tyrosine phosphorylation of STAT-1, and prevented its binding to the
GAS element of promoter IV. In addition, SOCS-1 expression inhibited
IFN--induced IRF-1 expression and IRF-1 binding to the IRF element
of CIITA promoter IV.
Our functional analysis of the murine CIITA promoter IV in macrophages
is essentially in agreement with previously published reports
demonstrating that the GAS, E-Box, and IRF elements are each required
for full IFN- activation of the human CIITA promoter IV
(28, 29, 30). In addition, it has been shown that binding of
STAT-1 to the GAS element requires the presence of the
constitutively expressed USF-1 on the adjacent E-Box element (29, 30). This is also the case in EOC20 and RAW264.7 cells (data not
shown). However, our results differ from those reports that have
demonstrated a more critical role for the IRF element in IFN-
activation of the human CIITA promoter IV (27, 28, 30).
Nikcevich et al. (27) reported that the GAS element did
not contribute to IFN- activation of the human CIITA promoter IV in
primary rat astrocytes. Our group (30) and Piskurich et
al. (28) have previously shown that the GAS element
partially contributes to IFN--induced CIITA promoter IV activity in
astrocytes and the human fibrosarcoma cell line 2fTGH, respectively,
whereas the IRF element is essential for promoter IV activation in
these cell types. In contrast, in RAW cells, our results demonstrate
that the GAS and IRF elements are each essential for IFN- induction
of the murine CIITA promoter IV (see Fig. 3). These differences in the
relative importance of the GAS and IRF elements in IFN- activation
of CIITA promoter IV may be due to differences in the murine and human
promoter, and/or to different CIITA activation pathways in the
different cell types. As noted above, we have observed that IRF-1 and
IRF-2 constitutively bind the IRF element of the murine CIITA promoter
IV in EOC20 and RAW cells (Fig. 5). In the studies using astrocytes and
2fTGH cells, the IRF element in the human CIITA promoter IV was not
constitutively occupied by either IRF-1 or IRF-2 (28, 30).
Thus, in cell types such as the macrophage that have IRF-1
constitutively bound to promoter IV, full activation of the promoter by
IFN- may be dependent on STAT-1 binding the GAS element, followed by
IFN--induced IRF-1 binding the IRF element.
A recent report has shown that IRF-1 and IRF-2 co-occupy the IRF
element in the human CIITA promoter IV, and the proteins
synergistically activate CIITA promoter IV in a pancreatic tumor cell
line (46). Our data show that IRF-1 and IRF-2 are
constitutively bound to the IRF element of the murine CIITA promoter
IV, but only IRF-1 binding is enhanced following IFN- treatment
(Fig. 5). IRF-1 functions as an activator of IFN-induced genes, whereas
IRF-2, which is induced by IFN- through IRF-1, generally antagonizes
IRF-1 activity by competing with IRF-1 for binding to IRF elements
(47, 48, 49). In addition, it has been reported that IL-4
augments IFN--induced IRF-2 expression in a mouse macrophage cell
line, possibly accounting for IL-4 inhibition of IFN--induced iNOS
gene expression (50). We have not attempted to directly
determine the role of IRF-2 in regulating CIITA promoter IV activity in
macrophages or microglia, but we have observed that IFN- treatment
extending out to 6 h does not enhance the binding of IRF-2 to the
IRF element of the promoter (data not shown). We have also not seen any
differences in IRF-2 occupancy of the IRF element in CIITA promoter IV
following treatment of cells with IFN- plus IL-4, IL-10, or TGF-
(data not shown), cytokines that we have previously shown to inhibit
IFN--induced CIITA expression in microglia (41). Thus,
at present, our results do not support a role for IRF-2 in regulating
CIITA promoter IV activity in macrophages/microglia.
SOCS proteins are distinguished by a novel carboxyl-terminal domain
called the SOCS box and a centrally located SH2 domain that is required
for their inhibitory effect (51). A divergent N-terminal
domain may also function as a protein interaction domain. The SH2
domain binds the tyrosine-phosphorylated JH1 domain of JAK2 and
inhibits its kinase activity, perhaps by steric hindrance (35, 39, 52). The SOCS box of SOCS-1 is proposed to interact with
elongins B and C, and couple SOCS-1 and perhaps its substrates to the
proteosomal protein degradation pathway (53). The results
of our studies on the effects of SOCS-1 on IFN- activation of the
JAK/STAT pathway in macrophages demonstrate that ectopic expression of
SOCS-1 can attenuate IFN- signaling in macrophages by inhibiting
tyrosine phosphorylation of STAT-1 (Fig. 8). This result is in
agreement with previous findings in other cell types where SOCS-1
inhibits IFN--induced responses by interacting with activated JAKs
and prevents phosphorylation of STAT-1 (37, 38, 54). We
have also made the novel observation that SOCS-1 overexpression reduces
constitutive STAT-1 protein expression (Fig. 8). We have observed
that STAT-1 mRNA is constitutively expressed in RAW cells, and in the
SOCS-1 transfectants, constitutive STAT-1 mRNA expression is reduced by
30% (data not shown), which may account for the reduction in
STAT-1 protein levels. Surprisingly little is known about the
regulation of the STAT-1 gene. Recently, it has been shown
that unphosphorylated STAT-1 can mediate the constitutive expression of
a number of genes (55). It would be of interest to
understand, first, the mechanism for constitutive STAT-1
gene expression and, second, how SOCS-1 expression leads to its
inhibition. One possibility is that SOCS-1 binds unphosphorylated
STAT-1, perhaps at a protein interaction motif in the N-terminal of
SOCS-1, and targets STAT-1 for proteolytic destruction.
We have observed that IFN- induces strong expression of SOCS-1 mRNA
in both RAW and EOC20 cells. In both cell types, IFN--induced SOCS-1
mRNA can be detected in as early as 30 min, reaches maximum levels
between 2 and 8 h, and is still detectable at 16 h (data not
shown). Our attempts to study endogenous SOCS-1 protein expression and
function in macrophages and microglia have been hampered by the
inability to detect the protein with any of the commercially available
SOCS-1 Abs. Recent reports indicate that SOCS-1 protein expression is
inhibited by rapid degradation and translational repression (52, 53, 56). One of these groups has also reported that SOCS-1 mRNA
transcripts are readily detectable, yet SOCS-1 protein could only be
detected after immunoprecipitation from a large amount of total protein
(9 mg) (56). Although IFN--induced SOCS-1 mRNA peaks
between 2 and 8 h (data not shown), which is before the peak of
IFN--induced CIITA mRNA at 16 h (Fig. 2), the kinetics and
duration of SOCS-1 protein expression are not known. Thus, at the
present time, we do not know the functional significance of
IFN--induced SOCS-1 protein expression on IFN--induced CIITA
expression in microglia/macrophages. However, studies of
SOCS-1-deficient mice indicate that endogenously expressed SOCS-1 has a
critical role in regulating IFN- responses. Two reports described a
perinatal lethality that leads to death within 3 wk (57, 58). Lymphocytes from the SOCS-1-deficient mice underwent
accelerated apoptosis that was associated with the increased expression
of the pro-apoptotic protein Bax (58). Lethality was
speculated to be due to an increased sensitivity to IFN-. A more
recent study of SOCS-1-deficient mice has provided evidence for
dysregulation of IFN- signal transduction pathways, including
constitutive binding of STAT-1 to an oligonucleotide containing a GAS
element, elevated expression of IRF-1 mRNA in the brain, and elevated
expression of class I MHC on bone marrow cells (59).
Interestingly, the defects were eliminated in mice deficient in both
SOCS-1 and IFN-. Collectively, these studies demonstrate that SOCS-1
is a critical regulator of cellular sensitivity to IFN-. It would be
of interest to examine CIITA and class II MHC expression in
SOCS-1-deficient mice, which would help determine the physiological
function of SOCS-1 in regulating IFN--induced CIITA and class II MHC
expression.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Etty N. Benveniste, Department of Cell Biology, MCLM 350, University of Alabama at Birmingham, 1918 University Boulevard, Birmingham, AL 35294-0005.
3 Abbreviations used in this paper: MS, multiple sclerosis; CIITA, class II transactivator; GAS, IFN- activation sequence; IRF, IFN regulatory factor; RLA, relative luciferase activity; SOCS, suppressors of cytokine signaling; USF-1, upstream stimulating factor-1; RPA, RNase protection assay; JAK, Janus kinase.
Received for publication August 21, 2000. Accepted for publication November 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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