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INTRODUCTION |
Ubiquitin is the most familiar of the proteinaceous protein
modifiers, and the enzymology of its activation and transfer to target
molecules has been studied extensively. More recently, a
sizeable group of ubiquitin-related proteins have come to light; at least a dozen distinct ubiquitin-like proteins
(Ubls)1 similarly ubiquitin
form covalent attachments to other macromolecules (1). Ubls mediate an
impressive range of cellular functions, including cell-cycle
progression, DNA repair, and apoptosis, suggesting that covalent
posttranslational modification of proteins is a versatile principle of
determining the half-life, intracellular localization, and activity of
proteins (reviewed in Ref. 2). Ubiquitin cross-reactive protein, better
known as IFN-stimulated gene 15 (ISG15), was the
first example of a growing class of ubiquitin-like proteins that
includes SUMO-1, Nedd8, and FAT 10 (3). ISG15 is one of the
most strongly induced genes after interferon (IFN) treatment (4-6) and
is also significantly induced by influenza B virus (7),
lipopolysaccharide (LPS) (8), and genotoxic stress (9). It functions
intracellularly as a ubiquitin homolog and can form conjugates with
certain cellular proteins, a substantial amount of which are
colocalized with intermediate filaments of the cytoskeleton (10).
Conjugation of Ubls including ISG15 occurs by a mechanism similar but
distinct from ubiquitination (11). It involves a three-step mechanism
where specific enzymes (or enzyme complexes) activate and covalently
link Ubls to their substrates (12, 13). An ISG15-activating enzyme has
been recently identified as Ube1L (7). Interestingly, UBE1L was found
to be absent in 14 different lung cancer cell lines tested,
suggesting a possible link between block of ISG15 conjugation and
carcinogenesis (14). Monocytes and lymphocytes can release free ISG15
(15). Cytokine-like properties of ISG15, such as the induction of
IFN-
production and augmentation of natural
killer/lymphokine-activated killer cell proliferation and function
(16), suggest an important role of ISG15 in immunomodulation.
Modification of proteins by ubiquitin and Ubls is reversible.
Ubiquitin (or Ubl)-substrate deconjugation is performed by members of a
diverse group of specialized cysteine proteases called deubiquitinating enzymes or ubiquitin-specific proteases.
UBP43 (USP18), a member of the ubiquitin protease
family, has been cloned in our laboratory during the analysis of
differential gene expression in hematopoietic tissues of AML1-ETO
knock-in mice (17). UBP43 encodes a 43-kDa protein and
exhibits homology to catalytic domains of ubiquitin-specific proteases
(USPs) that function to release free ubiquitin from ubiquitin-protein
conjugates. Recently, we have demonstrated that UBP43 is a major
ISG15-specific protease and activity of this enzyme is crucial for
maintaining a proper balance of ISG15-conjugated proteins in cells
(18).
Vertebrates and invertebrates respond to bacterial invasion by
activation of a defense mechanism that is part of the innate immune
response (19). This response is mainly triggered by the recognition of
LPS, which are cell wall components of Gram-negative bacteria (20). In
mammals, it is primarily monocytes and macrophages that respond to LPS
by releasing cytokines and chemokines to provoke inflammatory responses
(21). After exposure to LPS, the macrophages undergo profound changes
in protein composition that include alteration of cell surface,
secreted, and intracellular products. The changes in LPS-stimulated
macrophages include transcriptional activation of multiple genes, which
may contribute to the natural immunity to microorganisms.
We previously reported that the highest level of murine
UBP43 expression was detected in thymus and peritoneal
macrophages of normal adult mice. Among various hematopoietic cell
lines tested, monocyte/macrophage lines also exhibited the highest
level of UBP43 expression (17). Macrophages are primary
effector cells in host defense, and high activity of the UBP43 may play
an important role in their function as well as have an affect on
overall immune response. These facts directed us to identify
transacting factors regulating the expression of this gene under normal
and stressed conditions. In this report we show that UBP43
is strongly up-regulated by LPS. Two interferon regulatory factor (IRF)
binding sites in the UBP43 promoter are important for the
basal and LPS-induced levels of UBP43 expression. We show
that IRF-3 (a transcription factor known to be involved in regulation
of defensive responses) is responsible for LPS-induction of
UBP43, whereas IRF-2 mediates the basal level of expression.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Culture--
The murine macrophage-like cell
line, RAW 264.7, was generously provided by Dr. M. Ostrowski (Ohio
State University, Columbus, OH) and was cultured in RPMI 1640 (Invitrogen) with 5% iron-supplemented bovine calf serum
(HyClone, Logan, UT) and 2 mM L-glutamine
(Invitrogen) at 37 °C with 7% CO2. RAW 264.7 cells were
maintained in cell culture between 1 × 105 and 1 × 106 cells/ml. IRF-1/IRF-2 double knock-out murine
embryonic fibroblasts (MEFs) were generously provided by Dr. Janet
Stein (University of Massachusetts Medical School, Worcester, MA) with
the permission from Dr. Tadatsugu Taniguchi and were maintained in
Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal
bovine serum and 2 mM L-glutamine. LPS
(Escherichia coli serotype O127:B8) was purchased
from Sigma.
Northern Blot Analysis--
Total RNA from mouse brain and
thymus tissues was prepared by the guanidine isothiocyanate extraction
followed by cesium chloride gradient purification (22). Total RNA from
peritoneal macrophages and RAW 264.7 cells was isolated using RNazol B
reagent according to the manufacturer's instructions (Tel-Test Inc.,
Friendswood, TX). Ten µg of total RNA from each mouse tissue or time
point was separated in an agarose/formaldehyde (0.22 M)
gel, blotted on Hybond N+ membrane (Amersham Biosciences),
and probed with either a full-length UBP43 cDNA (17) or
a full-length ISG15 cDNA (GenBankTM accession no.
U58202) that has been amplified by PCR from mouse
cytomegalovirus-infected cells.
Immunoblotting--
Rabbit polyclonal IgGs against human ISG15
were kindly provided by Dr. E. Borden (Cleveland Clinic Foundation) and
were used at final concentration of 0.5 µg/ml (23). The production of anti-UBP43 antibodies has been previously described
(18). For Western blotting, anti-UBP43
antibodies were used at a final concentration of 0.2 µg/ml. Cell
lysates were resolved by SDS-PAGE, transferred to nitrocellulose
membrane, and immunoblotted using standard procedures (24). Generation
of UBP43 knockout (UBP43
/) mouse model in which UBP43 gene has been deleted by homologous
recombination will be published
elsewhere.2 Absence of
UBP43 expression in UBP43/ mice was confirmed
by Western blot analysis using anti-UBP43 antibodies as described above.
RNase Protection Assay--
RNase-protection assay was performed
with a RiboquantTM kit (BD PharMingen, San Diego, CA) according to the
manufacturer's instructions. The labeled 284-nucleotide riboprobe
extended from 197 to +71 of the murine UBP43 upstream
region plus 16 nucleotides transcribed from pBluescript II KS()
(Stratagene, La Jolla, CA). Ten µg of control yeast tRNA or 10 µg
of total RNA isolated from mouse thymus or brain were used in each reaction.
Reporter Plasmids--
The murine UBP43 promoter was
isolated from 129SV murine genomic DNA library (Stratagene). The
isolated 3.5-kb promoter fragment including a part of the first exon
was cloned into pBluescript II KS() and sequenced. The promoter was
then subcloned into the luciferase reporter plasmid pXP2 (25),
resulting in p3KUBP43-luc. To generate a series of 5'-region deletions,
p3KUBP43-luc was digested with HindIII/SpeI,
HindIII/NheI, or
HindIII/HindIII, blunt ended with T4 DNA
polymerase, and re-ligated. The resulting plasmids (p1.5KUBP43-luc,
p0.7KUBP43-luc, and p0.1KUBP43-luc) were named by indicating the length
of 5'-flanking region in each construct, respectively. To create
p0.2KUBP43-luc, 240 bp of the UBP43 promoter was amplified
by PCR using 5'-gtgtcctggtctagacgactggactg-3' and
5'-gcgaagaccgagctccatctgcaaag-3' as the upstream and downstream primers, respectively. The PCR product was sequenced and then inserted
into pXP2. Mutant constructs p0.7KUBP43 (IRFE1m)-luc, p0.7KUBP43
(IRFE2m)-luc, and p0.7KUBP43 (IRFE1/2m)-luc were created by PCR-based
mutagenesis with oligonucleotide pairs of an upstream primer
(5'-acatctgtaaggatccagcaagcattt-3') and one of three downstream primers
(5'-gtccaagcttaagttttcc-3' (IRFE1m),
5'-gtccaagctttcgttttcccctagatccaaagggcagcgagactcaggc-3' (IRFE2m), or
5'-gtccaagcttaagttttcccctagatccaaagggcagcgagactcaggc-3' (IRFE1/2m))
using the wild-type promoter as a template. They were then sequenced
and inserted into the BamHI/HindIII sites of
p0.1KUBP43-luc. Expression constructs for IRF-1 (pCMVIRF1) and IRF-2
(pCMVIRF2) were generously provided by Dr. Alan McLachlan (Scripps
Research Institute, La Jolla, CA) with the permission of Dr. John
Hiscott (McGill University, Montreal, Canada), and the
dominant-negative IRF-3 expression construct was generously provided by
Dr. Michael David (University of California at San Diego, La Jolla, CA).
Transient Transfections--
Transfection of RAW 264.7 cells was
performed by electroporation (260 V, 975 microfarads) using a Gene
Pulser II (Bio-Rad) equipped with a capacitance extender. UBP43-luc
constructs (1 pmol/transfection) and an internal control for
transfection efficiency, the promoterless Renilla luciferase
expression construct, pRL-null (0.03 pmol/transfection), were
co-transfected into 2.5 × 106 cells in 0.2 ml of
complete RPMI in a 0.4-cm cuvette (Bio-Rad). The total amount of DNA
was adjusted to 7 µg with pBluescript II KS(). Cells from three
electroporations were pooled together to eliminate differences between
individual transfections. The mixture was then equally divided into
three wells of a six-well plate (Corning Inc., Corning, NY). They were
next allowed to adhere for 2 h before the medium was changed.
Cells were harvested 48 h after electroporation and assayed for
luciferase activity. For LPS treatment, cells were cultured in fresh
medium for 36 h after electroporation. LPS (1 µg/ml) was added
for 7 h, and cells were then lysed and assayed for firefly and
Renilla luciferase activities with the Dual Luciferase assay
system (Promega, Madison, WI) using a Monolight 3010 luminometer (BD
PharMingen). The firefly luciferase activity was normalized based on
Renilla luciferase activity. All data were reported as a
mean -fold induction, which was calculated by dividing the normalized
reporter activity of each stimulated sample by that of the
corresponding unstimulated control sample. From the mean values of
three independent experiments, the overall (average) mean and its
standard deviation were presented. When the effect of IRF-1 and IRF-2
on UBP43 promoter activity was studied, 0.5 µg of the
respective expression construct was co-transfected with 2 µg of
either p0.7KUBP43-luc or p0.7KUBP43 (IRFE1/2m)-luc into
IRF-1
/IRF-2/ double knock-out MEFs.
Transfection of MEFs was performed using Superfect reagent (Qiagen,
Valencia, CA) according to the manufacturer's instructions.
Dose-dependent inhibitory action of dominant-negative IRF-3
mutant was analyzed in RAW264.7 cells by co-transfection of wild-type
(p0.7KUBP43-luc) or mutated version (p0.7KUBP43(IRFE1/2m)-luc) of
UBP43 promoter (1 pmol/transfection) and dominant-negative mutant of IRF-3 (
nIRF-3) (0.3, 0.5, and 1 pmol/transfection, respectively).
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared by a previously described method (26) with minor
modifications. Nuclear proteins were extracted from unstimulated cells
and from cells stimulated with 1 µg/ml LPS for seven h. Cells were
washed in cold phosphate-buffered saline and pelleted. Pellets from
1-5 × 107 cells were resuspended in 400 µl of
Buffer A (10 mM Hepes, pH 7.9, 10 mM KCI, 10 mM NaF, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, 2 mM benzamidine, 2 µg/ml leupeptin, 2 µg/ml
antipain, 2 µg/ml chymostatin). After incubation on ice for 5-15
min, 25 µl of 10% Nonidet P-40 was added and lysates were vortexed
for 10 min. Pelleted nuclei were resuspended in 150 µl of Buffer B
(10 mM Hepes, pH 7.9, 400 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 1 mM
EGTA, and protease inhibitors as in Buffer A) and incubated for 15 min
on ice with occasional shaking. The nuclear lysates were cleared by
centrifugation, frozen in aliquots in liquid nitrogen, and stored at
80 °C. The double-stranded oligonucleotides were end-labeled with
[-32P]ATP using T4 polynucleotide kinase and purified
on polyacrylamide gels. EMSA reactions were performed in 20 µl of
EMSA buffer (10 mM Hepes, pH 7.9, 100 mM NaCl,
1 mM dithiothreitol, 0.1 mM EDTA, 0.1 mM EGTA, 5% glycerol, and 100 ng/µl poly d(I-C). Ten
µg of nuclear extracts were incubated in EMSA buffer with 1 µl of
labeled oligonucleotide (5000-10,000 cpm; 5-10 fmol) for 20 min at
room temperature. In competition analysis, 1 µl of unlabeled
competitors (1 pmol) were added to the reaction mixtures. For
supershift analysis, 2 µg of respective antibodies (anti-IRF-l
(M-20), anti-IRF-2 (C-19), anti-p48/ISGF3 (C-20) (Santa Cruz
Biotechnology, Santa Cruz, CA) or anti-IRF-3 (Ref. 27)) were incubated
with the extract in EMSA buffer for 10 min at room temperature before
the labeled oligonucleotide was added. The EMSA reactions were
separated on 7% or 4% polyacrylamide gels in 0.5× TBE (45 mM Tris borate, 1 mM EDTA, pH 8.0) for 2 h
at 50 V. The gels were vacuum dried, and the signals were detected by autoradiography.
| |
RESULTS |
LPS Activates UBP43 Expression--
To investigate the
transcriptional regulation of UBP43 in macrophages, we
characterized the effect of LPS on UBP43 expression. As
shown by Northern blot analysis (Fig.
1A), the level of
UBP43 mRNA was significantly increased in thymi and
macrophages of LPS-challenged mice. When RAW 264.7 macrophage-like
cells were stimulated with LPS, an increase of UBP43
mRNA was detectable 2 h after stimulation, reaching a maximum
expression at ~10 h (Fig. 1B). As expected, peritoneal
macrophages as well as RAW 264.7 cells showed a significant up-regulation of ISG15 mRNA in response to LPS (Fig. 1,
A and B). As indicated on the Western blot (Fig.
1C), up-regulation of UBP43 expression on the
transcriptional level was paralleled by changes in the level of UBP43
protein in LPS-stimulated RAW 264.7 cells. Unstimulated RAW 264.7 cells
showed very low level of UBP43 protein; however, LPS treatment led to a
significant increase in the production of UBP43 protein. These results
indicate that LPS signaling strongly increases the expression of
UBP43.
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Fig. 1.
Activation of UBP43
expression by LPS. A, total RNA from peritoneal
macrophages, brain, and thymus was collected from mice with and without
LPS treatment and analyzed by Northern blot hybridization with
UBP43 or ISG15 cDNA. Ethidium bromide-stained
28 S rRNA is shown to indicate the relative RNA loading. B,
total RNA was collected from RAW 264.7 cells without (control) or with
LPS stimulation for 0, 2, 4, 10, and 24 h and analyzed by Northern
blot hybridization with UBP43 and ISG15 cDNA.
C, Western blot analysis of UBP43 expression in
RAW 264.7 cells with or without LPS treatment for 7 h.
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Effects of UBP43 Expression on the Level of Intracellular ISG15
Conjugates--
To identify biochemical changes related to
UBP43 expression in control and LPS-stimulated macrophages,
we analyzed the overall protein ISG15ylation status in extracts from
wild-type (UBP43+/+) and UBP43-deficient
(UBP43/) cells. Although ISG15 conjugates
were undetectable in normal (wild-type macrophages, not treated with
LPS) peritoneal exudate macrophages, LPS treatment resulted in a
substantial increase of intracellular ISG15 conjugates (Fig.
2, first and third
lanes from left). Significantly, the absence of UBP43
in UBP43-deficient macrophages led to an increase in total cell protein
ISG15ylation even without LPS treatment (Fig. 2, second lane
from left). Upon LPS stimulation,
UBP43/ macrophages showed substantially
higher levels of conjugates when compared with that of wild-type cells
(Fig. 2, fourth lane from left). These data
suggest that UBP43 is essential in the regulation of the level of ISG15
conjugates in control and LPS-treated macrophages.
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Fig. 2.
Effects of UBP43 expression
on the level of intracellular ISG15 conjugates in mouse peritoneal
macrophages. Protein extracts were prepared from
thioglycolate-elicited peritoneal macrophages derived from
UBP43+/+ and UBP43/
mice either left untreated (control) or treated with 1 µg/ml LPS
(LPS) for 12 h. Proteins (15 µg/lane) were separated on an
8-14% gradient SDS-PAGE gel and electroblotted to a nitrocellulose
membrane. The membrane was probed with anti-ISG15 antibodies. The
position of molecular size markers is indicated on the
right.
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Identification of the Transcription Initiation Site for the UBP43
Gene--
To further study the molecular mechanism of UBP43
activation by LPS, a 3.5-kb fragment containing the 5'-flanking region
and part of the first exon of UBP43 was isolated by
screening a murine genomic library using UBP43 cDNA as a
probe. The sequence of this 3.5 kb has been deposited in GenBankTM
under accession number: AF388669. To identify the transcription
initiation site of UBP43, RNase protection assays were
performed. The riboprobe was 284 nucleotides long and contained 268 nucleotides that correlated to UBP43 DNA sequence from bp
197 to +71. When this probe was hybridized to total RNA prepared from
the thymus of LPS-treated mice and digested with ribonucleases, it
generated a major band and three minor bands. Such protected bands were
not detectable when brain RNA or yeast tRNA were used as negative
controls (Fig. 3). With the calculation
based on the molecular weight marker, the results demonstrated that the
major UBP43 transcription initiation site is 111 bp upstream
of the 3'-end of exon 1 (Fig.
4A). No TATA box was
identified around the transcription initiation site of the
UBP43 gene. However, there are two GC box consensus
sequences in the nearest upstream region.
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Fig. 3.
Identification of UBP43
transcription initiation site. Ten µg of total RNA
prepared from brain or thymus of LPS-treated mice or 10 µg of yeast
tRNA were hybridized with a 32P-labeled 284-nucleotide
riboprobe. After treatment with ribonucleases A and T1, the protected
products were separated on a 6% polyacrylamide sequencing gel.
Brackets indicate the positions of the protected products.
The major product is marked with an asterisk. An
arrow shows the position of undigested riboprobe. Labeled
HinfI-digested  174 DNA was used as the molecular size
marker. The length assigned to each protected band was estimated to be
8% larger than that predicted from DNA size markers, based on the
relative mobility of the undigested riboprobe and a DNA marker of the
same size.
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Fig. 4.
Sequence and deletion analysis of
UBP43 promoter. A, sequence (200 bp)
of the UBP43 proximal promoter region and part of the
5'-untranslated region of the UBP43 gene is presented. The
major transcription initiation site is marked with an arrow
and numbered as +1. Potential IRFE sites are shown in bold.
GC boxes are underlined. The translation start codon of
UBP43 is shown in italic bold. These
sequence data are available from GenBankTM/EMBL/DDBJ under accession
no. AF388669. B, LPS induction of UBP43 promoter.
Schematic representations of UBP43 promoter-luciferase
constructs are shown on the left. These constructs and the
promoterless pXP2 vector were transfected into RAW 264.7 cells. The
number in the name of each construct indicates the length of the
5'-flanking region of UBP43 included in the construct. Half
of the transfected cells were treated with LPS for 7 h, and the
other half were cultured under normal conditions. The cells were
harvested and assayed for luciferase activity as described under
"Experimental Procedures." The data are expressed as -fold increase
in relative luciferase activity in LPS-stimulated cells over the
untreated cells. The data represent the mean -fold of induction of
three independent experiments ± S.D. of the mean.
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Identification of LPS Response Region in the UBP43
Promoter--
To determine whether the isolated 3.5 kb upstream
sequence of the UBP43 gene confers inducibility by LPS, we made a
luciferase-reporter construct, in which the 3.5-kb upstream sequence of
UBP43 was inserted into promoterless luciferase reporter,
pXP2, to form p3KUBP43-luc. Promoter activity was readily detectable
when p3KUBP43-luc was transfected into RAW 264.7 cells. The luciferase
activity of p3KUBP43-luc was ~200-fold greater than the promoterless
pXP2 construct (data not shown), indicating a strong promoter activity of this fragment. Upon LPS treatment, p3KUBP43-luc showed a 5-fold increase in promoter activity (Fig. 4B). To identify the
region in the UBP43 promoter that is critical for LPS
response, a series of deletion constructs were created and their
activities were examined using transient transfection assays in
RAW264.7 cells. Deletion of the UBP43 promoter to 200 bp
(p0.2KUBP43-luc) did not significantly reduce LPS-induced
UBP43 promoter activity. A further deletion of the promoter
to 100 bp (p0.1KUBP43-luc) completely abolished LPS induction of the
reporter gene (Fig. 4B). These results indicate that the LPS
response element of the UBP43 promoter is located between bp
200 and bp 100.
Two Sequences Homologous to Interferon Regulatory Factor Binding
Element (IRFE) Are Important for Constitutive and LPS-inducible UBP43
Promoter Activity--
To identify the regulatory elements in the
promoter of UBP43 responsible for LPS induction, we first
examined the proximal 200-bp sequence of the UBP43 promoter
using the TRANSFAC database (transfac.gbf.de/TRANSFAC/). Two putative
IRF binding sites were identified. They were designated as IRFE-1 and
IRFE-2 (Fig. 4A). Both sites were located between the bp
95 to 130 region of the UBP43 promoter. The sequence of
the IRFE-1 site (bp 95 to 104) closely resembles the IRFE (28). The
second site IRFE-2 (bp 118 to 130) is closely related to the
interferon-stimulated response element (29). To delineate whether the
IRFE-1 and IRFE-2 sites were important for regulation of the
UBP43 promoter in RAW 264.7 cells, point mutations were
introduced at either the IRFE-1 or IRFE-2 site, or at both sites of the
UBP43 promoter-luciferase reporter gene construct
(p0.7KUBP43-luc) to disrupt the consensus binding sites of the
respective elements (Fig. 5A).
Transfection analysis of IRFE-mutated promoter constructs into RAW
264.7 cells demonstrated that these mutations affected the
UBP43 basal promoter activity (Fig. 5B). Mutation
at either one of the two IRFEs alone decreased the promoter activity to
50% of the activity in the control. Mutation at both sites together
decreased the promoter activity to 12% of the control level,
indicating that these two IRFEs are important for the basal
UBP43 promoter activity. We also studied the effect of these
mutations on LPS-induced UBP43 promoter activity. Loss of
IRFE-1 in the UBP43 promoter (p0.7KUBP43(IRFE1m)-luc) caused
only a slight decrease of LPS induced promoter activation, whereas
mutation of the IRFE-2 site (p0.7KUBP43(IRFE2m)-luc) decreased promoter
inducibility to 48% of the control (Fig. 5C). When both sites were mutated simultaneously (p0.7KUBP43(IRFE1/2m)-luc), it
essentially abolished the response to LPS, reducing the inducible promoter activity to the level of the control vector alone (pXP2). These data indicate that an intact IRFE region is required for LPS
induction of UBP43 gene expression as well as for its basal promoter activity in RAW 264.7 cells. Furthermore, the IRFE-2 site is
more capable of mediating LPS inducibility of the UBP43 promoter. Nevertheless, both cis-acting elements were required to
provide optimal responsiveness to LPS.
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Fig. 5.
IRFEs are important for basal promoter
activity and for the induction of UBP43 expression by
LPS. A, sequences of oligonucleotides of wild-type and
mutant IRFE-1 and IRFE-2 used in EMSA (Figs. 6 and 8) and sequences
of oligonucleotides of an IRFE in ISG15 promoter and a PU.1 binding
site in promoter of human M-CSF receptor (55). The consensus sequence
for IRF binding site is shown at the bottom of the
panel. B, promoterless luciferase vector pXP2,
the wild-type UBP43 promoter-luciferase construct
p0.7KUBP43-luc, the single IRFE mutation constructs
p0.7KUBP43(IRFE1m)-luc and p0.7KUBP43(IRFE2m)-luc, and the
double IRFE site mutation construct p0.7KUBP43(IRFE1/2m)-luc were
transfected into RAW 264.7 cells. C, the transfection was
performed as described in panel B. Fortyeight h after
transfection, half of the cells were treated with LPS for 7 h and
the other half were cultured under normal conditions. Luciferase
activities were measured, normalized, and presented as -fold increase
of relative luciferase activity in LPS-treated cells over the untreated
cells. The average promoter activity was generated from three separate
experiments. The error bars indicate the S.D. of the mean.
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IRF-2 Is the Major Constitutive Binding Protein on UBP43
IRFEs--
The transfection experiments showed that IRFE-1 and IRFE-2
are functional elements for both constitutive and LPS-induced
UBP43 promoter activity in macrophages. To identify the
transcription factors interacting with these IRFEs, double-stranded
oligonucleotides encompassing IRFE-1 and IRFE-2 sites were synthesized
as shown in Fig. 5A. When incubated with nuclear extracts
from RAW 264.7 cells treated with LPS, both IRFE probes displayed
similar binding patterns in EMSA (Fig.
6A). Nuclear extracts prepared
from RAW 264.7 cells without LPS treatment showed identical binding
patterns (data not shown). The major shifted band can be competed with unlabeled self-oligonucleotide, an oligonucleotide with another IRFE,
and an oligonucleotide containing the consensus IRF binding site
(ISG15/IRFE) from the ISG15 gene promoter (30). However, oligonucleotides with IRFE-1 and IRFE-2 mutations or containing a
non-IRF related PU.1 transcription factor binding site were not able to
efficiently compete for binding. Complexes designated with asterisks
(*) are shifted bands likely generated with either nonspecific complex or degraded proteins. We could not reproducibly observe these bands even in seemingly identical runs with the same
nuclear protein preparation. These results (shown in Fig. 6A) suggested that a similar DNA-nuclear protein complex was
formed independently of LPS treatment with both IRFE-1 and IRFE-2
oligonucleotides and the protein in the complex was probably an IRF
family member. To identify this protein, specific antibodies against
either IRF-1 or IRF-2 were used in supershift assays. As shown in Fig.
6B, the majority of IRF complex was abrogated by IRF-2
antibody. The addition of IRF-1 antibody did not affect the complexes
formed with IRFE-1 or IRFE-2 probes. These data demonstrated that IRF-2 was the major transcription factor constitutively bound to a critical region of the UBP43 promoter.
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Fig. 6.
Identification of IRF binding to the
UBP43 promoter by EMSA. A, the same
IRF family members interact with both IRFE-1 and IRFE-2 of the
UBP43 promoter. The double-stranded UBP43
promoter bp 109 to 89 (IRFE-1) and bp 135 to 111 (IRFE-2) were
32P-labeled and incubated with 1 µg of double-stranded
poly(dI-dC) in the absence and the presence of nuclear proteins
prepared from LPS-treated RAW264.7 cells (RAW264.7 NE). Unlabeled
oligonucleotides of wild-type IRFE-1 (IRFE wt)
and IRFE-2 (IRFE wt), mutant IRFE-1
(IRFE1 mut) and IRFE-2 (IRFE2
mut), IRF binding oligonucleotide from the ISG15 promoter
(ISG15/IRFE), and an IRFE-unrelated PU.1 binding site
containing oligonucleotide (PU.1) were added at a 100-fold
molar excess over the probe oligonucleotide in competition assays.
IRF points to the complexes formed between oligonucleotides
and full-length IRF proteins. Asterisks (*) mark either the
complexes formed between the oligonucleotide probe and degraded IRF
protein or nonspecific complexes. B, IRF-2 is the major
protein from RAW 264.7 nuclear extracts that binds to IRFE sites of
UBP43 promoter. 32P-Labeled IRFE1 and IRFE2
oligonucleotides were incubated with or without nuclear proteins
prepared from LPS-treated RAW 264.7 cells. Two µg of antibodies
against either IRF-1 ( -IRF1) or IRF-2
(-IRF2) were added to the reaction for supershift assays.
The gel was electrophoresed longer than the gel presented in
panels A and B. The asterisk (*) marks
a newly detected complex that is discussed in Fig. 8.
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IRF-2 Is a Positive Regulator of the UBP43 Promoter--
The data
presented above demonstrate the ability of IRF-2 to bind both IRFEs of
UBP43 promoter in the presence or absence of LPS stimulation
in RAW 264.7 cells. This leads to a hypothesis that IRF-2 directs
constitutive expression of the UBP43 gene, a role opposite
to the transcriptional repression activity associated with this factor
(31, 32). To directly test this hypothesis, transactivation experiments
using IRF-1 and IRF-2 expression plasmids and a UBP43
promoter-luciferase construct were performed. Because RAW 264.7 cells
contain a high level of endogenous IRF-2, mouse embryonic fibroblasts
with an IRF-1 and IRF-2 double knockout were used in the
transactivation assays. As shown in Fig.
7, both IRF-1 and IRF-2 activated the
UBP43 promoter specifically via the IRF binding site because
the UBP43 promoter with IRFE-1 and IRFE-2 mutations did not
show any significant activation. Furthermore, IRF-2 is a stronger
activator than IRF-1 (8-fold versus 3-fold activation).
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Fig. 7.
IRF-2 is positive regulator of
UBP43 expression. Luciferase reporter gene
constructs containing either wild-type (p0.7KUBP43-luc) or both IRFE
sites mutated (p0.7KUBP43(IRFE1/2m)-luc) of UBP43 promoter
was co-transfected with IRF-1, IRF-2, or empty vector pcDNA3
expression constructs into IRF-1/
IRF-2/ double knockout MEFs. Co-transfected
Renilla luciferase construct was used to normalize the
transfection efficiency. The data represent the mean -fold induction of
three independent experiments. The error bars
indicate the S.D. of the mean.
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IRF-3 Plays an Important Role in LPS Induction of UBP43
Expression--
We noticed an additional slower migrating band in EMSA
when samples were electrophoresed for a longer time (Fig.
6B). To analyze this complex, nuclear extracts prepared from
RAW 264.7 cell with or without LPS treatment were used in EMSA. The
slower migrating band was only visible with nuclear extracts from
LPS-treated cells (Fig. 8A)
and was more abundant when IRFE-2 oligonucleotide was used as a probe.
Furthermore, similar to the IRF-2 complex, it was specifically competed
with unlabeled IRFE-1, IRFE-2, and ISG15/IRFE consensus
oligonucleotides, but not by mutant oligonucleotides or PU.1
oligonucleotides. These EMSA results indicated that the slower
migrating complex is specific and might recruit another IRF family
member. Because the IRFE-2-mutant version of the UBP43 promoter (p0.7KUBP43(IRFE2 m)-luc) demonstrated a measurable difference in inducibility by LPS relative to the wild-type promoter (Fig. 5C), we suspected that the transcriptional factors involved
in the formation of this complex were likely to mediate LPS induction of UBP43. Recently, a new member of the IRF family, IRF-3,
has been identified (33). It has been shown that LPS stimulation is
able to induce phosphorylation, nuclear translocation, and subsequent
DNA binding of IRF-3 (27). To determine whether IRF-3 participates in
the formation of LPS-inducible complex, we performed supershift
experiments with antibodies against IRF-3. As shown in Fig.
8B, IRF-3 antibodies caused a specific supershift of the slower migrating complex, whereas no effect was observed on the mobility of the IRF-2 complex. Neither p48/ISGF3 and Stat1 (data not
shown), as the components of ISGF3 complex, nor IRF-1 and IRF-2 (Fig.
6B) proteins were present in this complex. To test whether
IRF-3 is functionally important for the induction of the UBP43 promoter by LPS, the level of UBP43
inducibility was analyzed in the presence of expression constructs of
either an empty vector or a dominant negative form of IRF-3 (Fig.
8C). The expression of the dominant negative form of IRF-3
clearly decreased the induction of UBP43 promoter activity
by LPS. These results indicate that IRF-3 is critical for the LPS
induction of UBP43 expression.
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Fig. 8.
IRF-3 mediates LPS induction of
UBP43 promoter activity. A, the slower
migrating complex is specifically induced upon LPS treatment. Nuclear
extracts were prepared from RAW 264.7 cells with or without LPS
stimulation (+LPS and LPS, respectively). EMSA
were performed using 32P-labeled double-stranded
oligonucleotides that correspond to bp 135 to 111 of IRFE-2 in the
UBP43 promoter. The added competitors indicated on the
top and other components of the reactions are as described
in Fig. 6. The slower migrating band is marked with an
asterisk. B, IRF-3 is involved in the formation
of slower migrating complex. For supershift assays, nuclear proteins
were pre-incubated for 15 min with 2 µg of antibodies against either
IRF-3 (-IRF3) or anti-p48/ISGF3
(-ISGF3) prior to the addition of
32P-labeled double-stranded oligonucleotide.
Arrows with IRF-2 and IRF-3 mark specific complexes formed
between UBP43 IRFE-2 and IRF-2 or IRF-3 proteins,
respectively. The arrow with SS marks the band
supershifted by IRF-3 antibodies. C,
dose-dependent inhibition of UBP43 promoter
activation by expression of dominant-negative form of IRF-3. RAW 264.7 cells were co-transfected with luciferase reporter gene constructs
containing either wild-type (p0.7KUBP43-luc) or mutated IRFE sites
(p0.7KUBP43(IRFE1/2m)-luc) of UBP43 promoter and empty
pcDNA3 vector or dominant-negative mutant of IRF-3 (nIRF-3).
Triangle indicates an increasing amount of nIRF-3 used in
transfection (described under "Experimental Procedures"). LPS was
applied 48 h after transfection for 7 h, and then cells were
harvested and assayed for luciferase activity. A co-transfected
Renilla luciferase construct was used to normalize the
transfection efficiency. The data are expressed as -fold increase in
relative luciferase activity in LPS-treated cells over the untreated
cells. The data represent the mean -fold induction of three independent
experiments. The S.D. of the mean is indicated by the error
bars.
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DISCUSSION |
In the present study, we assessed the transcriptional regulation
of an ISG15 specific protease, UBP43, in macrophages. We demonstrate
that LPS treatment stimulates the expression of UBP43. We
also show that LPS increases the level of intracellular ISG15 conjugates. Massive accumulation of ISG15 conjugates observed in
LPS-stimulated UBP43/ macrophages confers
the crucial role of UBP43 in maintaining the proper balance of
ISG15-conjugated proteins in cells. We describe the isolation and
functional characterization of the promoter region of the
UBP43 gene and the identification of sequence elements and
trans-acting factors involved in the regulation of its expression by LPS.
The proximal UBP43 promoter does not contain classical
initiation elements, such as a TATA box, CAAT box, or consensus
initiation element (Inr). However, it possesses several GC-rich motifs
(Fig. 4A), which can functionally substitute a TATA box for
directing transcription initiation with multiple transcription start
sites (34-36). Successive 5' end deletion of the UBP43
promoter led us to define a 100-bp region necessary and sufficient to
promote maximal induction of transcription by LPS. This region contains tandem IRFEs, located 100 bp upstream of the transcription start site.
We also found that these sites are indispensable for the basal
transcription of the UBP43 gene because mutation of either IRFE decreases the level of UBP43 promoter activity in
unstimulated cells.
The supershift results suggest that IRF-2 binds both IRFEs in
unstimulated RAW 264.7 cells. Furthermore, IRF-2 has a higher potential
to activate the UBP43 promoter compared with IRF-1. IRF-2
was originally regarded as a transcriptional repressor that antagonizes
IRF-1 activity by competing for binding to the IRFEs of interferon and
IFN-inducible genes (28, 31, 37). However, recent evidence indicates
that IRF-2 is a dual-function transcription factor, as it activates the
transcription of EBNA-1 (38), histone H4 (39, 40), VCAM-1 (41),
gp91phox (42), and CIITA (43). The latent
transactivation domain located in the central region of IRF-2 possibly
accounts for the transactivating capability of IRF-2 (32). Our findings
provide yet another example of IRF-2 being an activator. Binding of
IRF-2 to UBP43 IRFEs was not affected by LPS treatment,
suggesting therefore that IRF-2 confers a basal transcriptional
activity to the UBP43 promoter.
As demonstrated in transfection experiments by mutating the IRFE bases
generally known to be indispensable for the binding of IRF family
members, an intact IRFE region is required for the induction of
UBP43 gene expression by LPS (Fig. 5). Our results suggest
that IRF-3 mediates the LPS induction of UBP43 in the RAW
264.7 macrophage-like cell line by binding to the IRFEs of the
UBP43 promoter.
Among the IRF family members, IRF-3 is of particular interest, because
its activation appears to have a direct role in the induction of
defensive responses. Recently, viral infection (33, 44-46), LPS
stimulation (27), as well as general genotoxic stress (47) were shown
to induce the phosphorylation, nuclear translocation, and subsequent
IRFE binding of IRF-3. However, it was suggested that IRF-3 has no
intrinsic transactivation capabilities and it may instead require
assembly with other co-activators, such as CBP and/or p300, to induce
gene expression (44, 46, 48). LPS activates IRF-3 phosphorylation via a
p38 MAPK dependent pathway (27). Our data show a strong increase of
UBP43 transcription upon LPS stimulation (Fig. 1), and such
induction can be reduced significantly by a p38 MAP kinase inhibitor
SB203580 or by co-transfection with a dominant negative p38 MAPK
expression construct (data not shown). The anti-IRF-3 antibodies
supershifted the IRFE-specific LPS-inducible complex in nuclear
extracts from RAW 264.7 cells. Together with the repression of
LPS-mediated activation of the UBP43 gene by a
dominant-negative IRF-3 mutant (Fig. 8C), these results
demonstrate that IRF-3 plays a primary role in the LPS-induced activation of the UBP43 gene.
The family of IRFs is involved in a wide range of host defense
mechanisms (reviewed in Refs. 37 and 49)). IRF proteins stimulate the
expression of many genes with antiviral, antiproliferative, apoptotic,
and immunomodulatory functions. The cloning of UBP43 has
recently been reported by three other groups using differential expression analyses (50-52). In addition to LPS induction as shown in
this report, UBP43 expression is also up-regulated by
porcine reproductive and respiratory syndrome virus infection (50) and interferon treatment (51, 52).
Based on experimental evidence of several laboratories, Taniguchi
and co-workers (53) categorized IFN and IFN-inducible genes into four
distinct groups in terms of activation. Group one, or "ISGF3 only"
group, is totally dependent on the IFN/
-activated transcription
factor ISGF3. Second, the "ISGF3/IRF-3" group, can be activated by
both virus and virus-induced interferon as well as bacteria and general
genotoxic stresses. Groups three and four include IFN genes themselves
whose transcription depends on "IRF-3/IRF-7" (IFN) or "IRF-7
only" (IFN). Based on the data presented here and on work of Kang
et al. (52), UBP43 belongs to the second, ISGF3/IRF-3 group
to which ISG15 has also been assigned. These genes have acquired
regulatory mechanism, which ensures gene induction even in the absence
of IFN/ signaling, to exert their function in the host defense
against extracellular pathogens. Coordinated induction of ISG15 and
UBP43 suggests that ISG15 conjugation is a dynamic process and critical
balance of ISG15 modification should be maintained at all times. Unlike
ubiquitination of proteins, which mostly are destined to degradation,
modification by Ubls mediates specific functions depending on the type
of Ubls. In this regard, the reversible Ubl modification resembles the
phosphorylation and dephosphorylation reaction of proteins, and
probably serves the same functions, which are to modulate the
structure, activity, or localization of the target proteins.
It is not known whether linkage of ISG15 to its target proteins results
in their degradation or rather, as is the case for other ubiquitin-like
proteins such as SUMO-1 and Nedd8 (1, 54), this linkage modifies the
biological activities of the targeted proteins. Because the proteins
that are targeted by ISG15 have not been yet identified, the exact
function of ISG15 modification remains to be elucidated. The direct
identification of UBP43 substrates and the study of cellular response
to bacterial infection in the absence of UBP43 expression in
the future will provide valuable information regarding the importance
of UBP43 and ISG15 modification in innate immunity.
TOP
ABSTRACT
INTRODUCTION
Leukemia and Lymphoma Society Scholar. To whom correspondence
should be addressed: MEM-L51, Scripps Research Inst., 10550 N. Torrey
Pines Rd., La Jolla, CA 92037. Tel.: 858-784-9558; Fax: 858-784-9593; E-mail: dzhang@scripps.edu.
