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INTRODUCTION |
The S-100 protein family is a multigenic family of low molecular
weight (9-11 kDa) calcium-binding proteins (1). S-100A10, known as p11
or calpactin I light chain, is a distinct member of S100 family because
its two EF-hands carry mutations that limit its ability to bind calcium
(2). p11 is a natural ligand of annexin II, forming an annexin
II2-p112 heterotetramer
(AIIt)1 (3-5). This complex
is implicated in the regulation of exocytosis and endocytosis by
reorganization of F-actin (6, 7), the formation of the cornified
envelope in epithelial keratinocytes (8), and the stimulation of
t-PA-dependent plasminogen activation and the activation of
procathepsin B (9, 10).
p11 also interacts with the C terminus of cytosolic phospholipase
A2 and inhibits cPLA2 activity resulting in
reduced arachidonic acid release (11). Antisense inhibition of p11
results in enhanced cPLA2 activity and increased AA
release, whereas p11 overexpression reduces cPLA2 activity
and AA release (12). Dexamethasone is known to reduce cPLA2
activity, and recent studies suggest that this effect may be mediated
by up-regulation of p11 (12). Recent data also suggest that retinoic
acid-increased arachidonic acid release may be in part mediated by the
reduction of p11 protein expression (13). Seung-Wook Kim et
al. (14) have reported that annexin I and annexin
II2-p112 but not annexin II inhibited cPLA2 activity. Taken together, these data suggest that p11
acts as a regulatory protein of cPLA2 and may play a role
in the regulation of cPLA2 activity and AA release.
IFN-
is a pro-inflammatory cytokine that is secreted by activated
T-lymphocytes and natural killer cells and regulates cellular antiviral, antitumor, and immunological responses (15). IFN-
has
been reported to regulate cyclooxygenase-2 (COX-2) expression and
induce prostaglandin formation, which may play a major role in the
induction of inflammatory processes (16). IFN-
also induces
intercellular adhesion molecule-1 (ICAM-1), which plays an important
role in the adherence and migration of leukocytes at sites of
inflammation in several cell types (17, 18). The binding of IFN-
to
its surface receptor activates the receptor-associated tyrosine
kinases, JAK1 and JAK2. JAKs tyrosine phosphorylate and activate the
latent cytosolic transcription factor STAT1, which then dimerizes,
translocates to the nucleus, and binds to the
-activated sequence
(GAS) elements of IFN-
response genes, resulting in gene activation
(19, 20). IFN-
stimulates an increase in PLA2 activity
in a variety of cell lines (21, 22). IFN-
also induces AA release,
as well as cPLA2 gene transcription (23). It was
of interest to study the effect of IFN-
on p11 expression. In this
study we investigate the effect of IFN-
on p11 transcription, steady-state levels of p11 mRNA and protein expression and the signal transduction pathway involved in human epithelial cells.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
HeLa cells (American Type Culture
Collection, Manassas, VA) were grown in Dulbecco's modified
Eagle's medium with 10% fetal bovine serum. A human bronchial
epithelial (BEAS-2B) cell line (American Type Culture Collection) was
grown in LHC-8 medium (Biofluids, Rockville, MD) without serum. All
experiments were performed when cells were 80-90% confluent.
Treatment--
For the time course experiments, after replacing
culture medium at the same time, the cells were treated with or without
300 units/ml of IFN-
at 4, 12, 24, or 48 h prior to harvest at
the 48-h time point. For the dose response experiments, cells were treated with or without IFN-
at the indicated dose for 24 h. For the inhibition experiment, cells were preincubated with AG-490 for
2 h before treatment with or without IFN-
. Inhibitors were maintained for the incubation period.
Immunoblot of p11 Protein--
BEAS-2B and HeLa cells were grown
on 175-cm2 collagen-coated tissue culture flasks (BD
PharMingen Labware, Franklin Lakes, NJ) and treated with IFN-
(Roche
Molecular Biochemicals, Indianapolis, IN). Cells were harvested with
trypsin (E-PET, Biofluids). After washing three times with cold
phosphate-buffered saline, cells were resuspended in 50 mM
HEPES buffer including Complete protease inhibitor (Roche Molecular
Biochemicals) and then sonicated three times for 15 s and
centrifuged at 14,000 rpm for 15 min. Total protein was assayed by BCA
reagent (Pierce). Ten micrograms of crude cell lysates were separated
on 16% Tris-glycine gels (Invitrogen) and electrophoretically
transferred onto a nitrocellulose membrane (Invitrogen). p11 protein
expression was detected by using 1:2000 dilution of mouse anti-p11
monoclonal antibody (Transduction Laboratories, Lexington, KY) and
1:5000 dilution horseradish peroxidase-conjugated donkey anti-mouse IgG
(Jackson ImmunoResearch Laboratory, Inc, West Grove, PA). The blot was
developed using the ECL Western blotting detection system (Amersham
Biosciences) and exposed to radiographic film.
Ribonuclease Protection Assay (RPA)--
BEAS-2B and HeLa cells
were treated with and without IFN-
. Total cellular RNA was extracted
using Tri-reagent (Molecular Research Inc. Cincinnati, OH) and
redissolved in diethyl pyrocarbonate (DEPC) water. To construct the
probe for p11 mRNA, a 319-bp product of p11 cDNA was amplified
by PCR using the following set of sense and antisense primers: 5'
primer: 5'-ACCACACCAAAATGCCATCT-3'(corresponding to bases 61-80 of the
human p11 cDNA sequence, GenBankTM accession number
M81457); 3' primer: 5'-CTGCTCATTTCTGCCTACTT-3' (corresponding to bases
361-379 of the p11 cDNA sequence). The PCR product was cloned into
the pGEM-T Easy vector (Promega, Madison, WI). Orientation of the
insert was determined by DNA sequencing. The p11 cRNA probe and
glyceradehyde-3-phosphate dehydrogenase (GAPDH) (Ambion, Austin, TX)
were radiolabeled using an in vitro transcription kit
(Ambion) with SP6 polymerase and [
-32P]UTP (800 Ci
(29.6TBq)/mmol) (PerkinElmer Life Sciences). An RPA assay kit (RPAII,
Ambion, Austin, TX) was used to quantitate target mRNA. Ten
micrograms of total RNA (for GAPDH) or 50 µg (for p11) were mixed
with 10,000 cpm (for GAPDH) or 20,000 cpm (for p11) of
-32P-labeled riboprobe, the mixture was hybridized at
45 °C overnight, and the unprotected RNA was digested by the
addition of 1:100 dilution RNase A/T1 at 37 °C for 60 min. Digestion
was terminated by the addition of RNase inactivation and precipitation
mixture. The protected fragments were separated on 6% polyacrylamide 8 M urea gels (Invitrogen) and visualized by autoradiography.
p11 Promoter Construct Preparation--
The p11 coding region
was released from p11-pET30a vector (Novagen, Madison, WI). Two
micrograms of purified p11 insert were sent to Genome Systems (St.
Louis, MO), for screening of a human genomic P1 library. Two positive
clones were obtained and amplified by company protocol. These clones
were confirmed by Northern blot using 32P-endlabeled
oligonucleotide 5'-GGCAGGACGGCCGGGTTCTT-3', corresponding to
bases 36-55 within the first exon of M77483, the reported sequence of
human p11 (24). A positive band at a size of ~1500 base pairs was
obtained from one of the clones. The insert from the P1 plasmid was
cloned into pBluescript II SK+ (Stratagene, La Jolla, CA). Plasmid DNA
was fully sequenced bi-directionally in triplicate to obtain the
sequence of the p11 5'-flanking promoter region from
1498 relative to
the transcription start site to +89 (GenBankTM accession
number AL450992). To create a p11-SEAP construct, p11
promoter region was cloned into pSEAP2-Basic vector
(Clontech, Palo Alto, CA) by blunt ligation, and
sequences were confirmed by bi-directional DNA sequencing. The
designated name for this construct is SEAP-1498+89. To create a
p11-pCAT construct, the p11 promoter region was cloned into
pCAT basic vector (Promega). The designated name of this construct is
pCAT-1498+89.
Deletion and Mutation Constructs--
5' deletion and mutation
constructs were generated by PCR using a FailSafe PCR System (Epicentre
Technology, Madison, WI) and ligated into the pCAT basic vector.
The following sense and antisense primers were used:
5'-GGGGGTACCTACTGCCTTGGAAACTTAGT-3' and 5'-TAAGAGCTCACCTTGGCCGA-3' (to
obtain the p11 promoter region from
1048 to +89) and
5'-GGGGGTACCGCGAGGCCTCTGCGA-3' and 5'-TAAGAGCTCACCTTGGCCGA-3' (to
obtain the p11 promoter region from
188 to +89) to
generate deletion plasmids pCAT-1048+89 and pCAT-188+89. pCAT-1498+89
plasmid was cut with BglII and religated into pCAT basic
vector, which also cut with BglII to create the deletion
construct pCAT-1434+89. The site-directed mutagenesis constructs were
generated by ligation of two pieces of PCR product produced from
backbone pCAT-1498+89. The reporter construct mutated at the GAS-2 site
(TTCCAGAAA mutated to CTCGAGAGA)
was created using the following two primer sets: 5'-GGGGGTACCGAGATTTCCT-3' and
5'-AAGAATTCTCTCGAGTAAATTTAGA-3' to
produce the first PCR product from
1498 to
1073; and
5'-CTCTAAATTTACTCGAGAGAATTCTTCT-3' and 5'-TAAGAGCTCACCTTGGCCGA-3' to produce the second PCR product from
1093 to +89. Both PCR products were cut with EcoRI and
ligated with T4 ligase. The ligation reaction was used as a template to create the GAS-2 mutation construct from
1498 to +89 with the primers
5'-GGGGGTACCGAGATTTCCT-3' and 5'-TAAGAGCTCACCTTGGCCGA-3'. The mutation
construct, GAS-3, (TTCCACCAA mutated to
CTCGAGCAA) was created using the
following two primer sets: 5'-GGGGGTACCGAGATTTCCT-3' and
5'-CAGCTGTTGCTCGAGAAGAGCCCAAT-3' to
create a PCR product from
1498 to
1204 and
5'-ATTGGGCTCTTCTCGAGCAACAGCT-3' and
5'-TAAGAGCTCACCTTGGCCGA-3' to create a PCR product from
1231 to +89.
Both PCR products were cut with XhoI and religated. The
ligation mixture was used as a template to create the GAS-3 mutation
construct from
1498 to +89 using the primers
5'-GGGGGTACCGAGATTTCCT-3' and 5'-TAAGAGCTCACCTTGGCCGA-3'. The
designated names for the mutation constructs are mGAS-2 and mGAS-3. The
sequences of the deletion and mutation constructs were confirmed by
bi-directional DNA sequencing.
Transient Transfection Assay--
HeLa cells were maintained in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The
day before transfection, 5 × 105 cells were seeded
into 6-well plates. 1.8 µg of p11 promoter constructs were
cotransfected with 0.2 µg of the pCMV/
-gal construct (Clontech) into serum-free cells using 6 µl of
the LipofectAMINE reagent (Invitrogen) in each well. After 5 h of
transfection, the cells were allowed to recover overnight in fresh
Dulbecco's modified Eagle's medium with 10% fetal bovine serum. For
time course experiments, the SEAP-1498+89 construct was used. At the indicated time point, SEAP activity was measured in 25-µl aliquots of
culture medium with a SEAP reporter assay kit according to the
manufacturer (Tropix, Bedford, MA) in a luminometer (Packard Bioscience, Meriden, CN). At the final time point, the transfected cells were lysed and were centrifuged for 10 min.
-galactosidase activity was measured with a
-galactosidase ELISA kit (Roche Molecular Biochemicals). Data are expressed as a ratio of SEAP activity
to
-galactosidase activity. For dose response experiments, SEAP-1498+89-transfected cells were treated with different
concentrations of IFN-
for 24 h. SEAP and
-galactosidase
activity were measured as described above. For deletion and mutation
experiments, pCAT p11 promoter constructs were used. Cells
were treated with 300 units/ml of IFN-
for 24 h and then
harvested. pCAT and
-galactosidase activity were assayed separately
using pCAT and
-galactosidase ELISA kits according to the
manufacturer's instructions (Roche Molecular Biochemicals). The pCAT
activity was normalized to
-galactosidase activity to represent
relative pCAT activity. For comparison between different constructs,
the activities of the deletion and mutation constructs were expressed
as a percentage of the full-length pCAT-1498+89 promoter construct that
was set at 100%.
Transient Transfection with Wild-type STAT1 Expression Plasmid
and Mutant STAT1 Dominant Negative Expression Plasmid--
Wild-type
STAT1 and mutant STAT1 dominant negative expression plasmids were
utilized. The wild-type STAT1 plasmid contains the human STAT1 cDNA
(encoding the 750 amino acid STAT1 protein), the STAT1 dominant
negative plasmid is mutated at amino acid 701 (the JAK1/2
phosphorylation site), pcDNA3.1 with no insert was used as a
control vector. All three plasmids have a potent CMV promoter. HeLa
cells were transfected with 1 µg of the p11 promoter construct pCAT-1498+89 alone or cotransfected with 1 µg of wild-type STAT1, mutant STAT1, or control vector pcDNA3.1 individually. Controls for transfection efficiency were performed using
cotransfection with 0.2 µg of pCMV/
-gal. Transient transfection
was performed as described above. After transfection, some cells were
exposed into IFN-
(300 units/ml) for 12 h. Cells were
harvested, lysed, and pCAT and
-galactosidase activity were assayed
by ELISA.
Effect of AG-490 on IFN-
-induced p11 Promoter Activity and
p11 Protein Levels--
HeLa cells were seeded into 6-well plates and
cotransfected with the SEAP-1498+89 plasmid and pCMV/
-gal control
plasmid as described above. Cells were pretreated with the JAK-2 kinase
inhibitor AG-490 (Calbiochem, San Diego, CA) (50 µM or
100 µM) for 2 h, and then treated with or without
IFN-
(300 units/ml) for 24 h. SEAP and
-galactosidase
activities were detected as described above. For p11 Western blot
studies, HeLa cells were grown on T-75-cm2 flasks until
80% confluent. Cells were pretreated with AG-490 (100 µM) for 2 h and then incubated with IFN-
(300 units/ml) for 12 h and lysed. Western blotting for p11 protein was
performed as described above.
Electrophoretic Mobility Shift Assay and Immunoblot of STAT1
Protein--
HeLa cells were incubated with and without IFN-
(300 units/ml) at 10 min, 30 min, 2, 4, 6, or 24 h prior to harvest at
the 24-h time point, and nuclear extracts were prepared using a nuclear extraction kit according to the manufacturer's directions (Sigma). DNA
binding was performed by incubating 2 µg of nuclear protein in a
total volume of 10 µl of binding buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.5 mM dithiothreitol, 0.5 mM EDTA, 1 mM MgCl2, 0.05 µg/µl, poly(dI-dC)·poly(dI-dC), 15% glycerol) and 10,000 cpm of
32P-labeled double-stranded GAS-2 oligonucleotide
5'-TTATTCCAGAAAATTCTT-3' for 20 min at room temperature. Protein-DNA
complexes were resolved on a 6% DNA retardation gel (Invitrogen) in
0.5× Tris borate-EDTA buffer at 250 V for 30 min. The dried gel was
exposed to x-ray film (Kodak, Rochester, NY) with an intensifying
screen at
70 °C overnight or until adequate signal was developed.
For competition analysis, 50, 100, or 200× molar excess of the cold
double-stranded oligonucleotide GAS-2 or nonspecific double-stranded
oligonucleotide SP-1 consensus sequence was preincubated with nuclear
extracts for 20 min at room temperature followed by an additional
incubation for 15 min at room temperature with the labeled probe. To
identify bands containing specific STAT1 protein, the sample was
incubated with anti-STAT1
(provided by Dr. Jahar Haque) antibody for
1 h on ice before the DNA binding reaction was performed. For the immunoblot of STAT1 protein, 10 µg of crude cell lysate or nuclear extracts from HeLa cells were separated using 8% Tris-glycine gel, and
Western blotting was performed as above with 1:1000 dilution of
anti-phospho-STAT1 or anti-STAT1 polyclonal antibody (Cell Signaling
Technology, Beverly, MA) and 1:5000 dilution horseradish peroxidase-conjugated goat anti-rabbit IgG as second antibody.
Quantification of Autoradiographs--
A Molecular Dynamics 301 computing densitometer (Molecular Dynamics, Sunnyvale, CA) was used to
digitalize images. The optical density of bands was analyzed with
background subtraction using Image Quant software (Molecular Dynamics).
Transfection of HeLa Cells with Small Inhibitory Antisense RNAs
(iRNAs)--
iRNAs were prepared by IDT (Coralville, IA) and targeted
bases 4-24 of the p11 coding sequence. Untemplated TT
were added to the 3'-end of each strand. The iRNA sequences were
5'-CCACACCAAAAUGCCAUCTT-3' and 5'-GAUGGCAUUUUGGUGUGGTT-3'. The
single-stranded iRNAs were annealed by incubating a 100 µM concentration of each single strand in annealing
buffer (100 mM potassium acetate, 30 mM HEPES,
pH 7.4, 2 mM magnesium acetate) for 2 min at 90 °C.
Cells grown in 6-well plates were transfected with 100 nM
iRNA duplexes using LipofectAMINE reagent (Invitrogen) (5 µl in 1 ml
of culture media) for 5 h. After transfection, media was changed,
and cells were labeled with [3H]AA (1 µCi/ml of culture
media, Amersham Biosciences) for 16 h. Cells were then treated
with or without IFN (300 units/ml) for 24 h. The effect of
treatment of cells with iRNA duplexes on p11 protein levels was
determined by immunoblotting of cell lysates. The effect of iRNA
treatment on arachidonate release was determined by scintillation
counting of media from labeled cells after treatment with the calcium
ionophor A23187 or vehicle (Me2SO) for 30 min. Media was
harvested and after centrifugation at 750 × g for 10 min, [3H]AA release was measured in a scintillation
counter (Beckman, Fullerton, CA).
Statistical Analysis--
The dose-related effects were analyzed
with one-way ANOVA. For other experiments, comparisons with control
were performed using a two-tailed unpaired Student's t
test. Values of p < 0.05 were considered statistically significant.
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RESULTS |
IFN-
Increases the Level of the p11 Protein in BEAS-2B
Cells--
Western blot studies demonstrated that p11 is
constitutively expressed in BEAS-2B cells. IFN-
(300 units/ml)
increased p11 protein expression in BEAS-2B cells over a 4-48-h
period, with a maximum effect observed at 12-24 h (Fig.
1, A and B).
Treatment of cells with 10, 100, 300, 1000 units/ml of IFN-
for
24 h resulted in a dose-dependent increase in p11
protein levels (Fig. 1, C and D).

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Fig. 1.
Western blots demonstrate that
IFN- treatment increases p11 protein levels in
BEAS-2B cells. After treatment with IFN- , cells were lysed and
10 µg of total protein lysates were examined by Western blot analysis
using anti-annexin II light chain monoclonal antibody. A,
time course (4-48 h) effect of IFN- (300 units/ml)-treated and
untreated BEAS-2B cells showing an increased p11 protein level at
4 h, with peak levels at 12-24 h. The result shown is
representative of four separate experiments. B, densitometry
measurements from four time course experiments of IFN- (300 units/ml) treated and control BEAS-2B cells demonstrating increased p11
protein levels at 4-48 h (data presented as mean ± S.E., *,
p < 0.05 compared with control). C, IFN-
(10, 100, 300, 1000 units/ml) treatment for 24 h induces a
dose-dependent increase in p11 protein level. The result
shown is representative of three separate experiments. D,
densitometry measurements from three dose response (0-1000 units/ml)
experiments demonstrating a dose-related increased p11 protein levels
(data presented as mean ± S.E., p < 0.001 for
dose effect by one-way ANOVA).
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The Effect of IFN-
on p11 Protein Levels in HeLa
Cells--
As IFN-
increased p11 protein levels in a human airway
epithelial cell line, BEAS-2B cells, we further investigated the effect of IFN-
on p11 expression in another epithelial cell line, HeLa cells. HeLa cells were treated with or without 300 units/ml of IFN-
for 4-48 h or with IFN-
(10, 100, 300, 1000 units/ml) for 24 h
and p11 protein expression was determined by Western blot analysis.
Fig. 2, A-D show that IFN-
also induced a time and dose-dependent increase in p11
protein in HeLa cells.

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Fig. 2.
Western blots demonstrate that
IFN- treatment increases p11 protein levels in
HeLa cells. HeLa cells were grown to near confluence and treated
with and without IFN- . Ten micrograms of total crude cell lysates
were subjected to gel electrophoresis and immunoblotting with
anti-annexin II light chain monoclonal antibody. A, time
course (4-48 h) of IFN- (300 units/ml)-treated and untreated
control HeLa cells showing increased p11 protein levels at 4 h,
with the greatest increase at 12-24 h. The result shown is
representative of four separate experiments. B, mean
densitometry measurements from four time course experiments of IFN-
(300 units/ml)-treated HeLa cells demonstrating increased p11 protein
levels at 4-48 h (data presented as mean ± S.E., *,
p < 0.05 compared with control values). C,
IFN- (10, 100, 300, 1000 units/ml) treatment for 24 h induces a
significant dose-dependent increase in p11 protein levels.
The result shown is representative of three separate experiments.
D, mean densitometry measurements from three dose response
(0-1000 units/ml) experiments demonstrating a dose-related p11 protein
level increase (data presented as mean ± S.E., p < 0.001 for dose effect by one-way ANOVA).
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IFN-
Increases p11 Steady-state mRNA Levels in BEAS-2B
Cells--
To determine if the observed increases in p11 protein
levels correlate with changes in p11 mRNA expression, BEAS-2B cells were incubated with or without IFN-
(300 units/ml) for 4-48 h or
IFN-
(10, 300, 1000 units/ml) for 24 h. Total RNA was isolated and steady-state p11 mRNA levels were studied by RPA. As shown in
Fig. 3, A and
B, IFN-
treatment stimulated increased p11 mRNA levels over 4-48 h, compared with the untreated control cells. BEAS-2B
cells treated with 10, 300, 1000 units/ml of IFN-
for 24 h
demonstrated a significant dose-related increase in steady-state p11
mRNA levels as shown in Fig. 3, C and D.
GAPDH mRNA levels are presented as controls for equivalent RNA
loading and to normalize for p11 mRNA expression.

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Fig. 3.
IFN- treatment
increases p11 steady-state mRNA levels in BEAS-2B cells. Cells
were incubated in the presence or absence of IFN- at the indicated
time and dose, total cellular RNA was extracted. Ten and fifty
micrograms of total cellular RNA were hybridized to GAPDH and
p11-specific radiolabeled cRNAs, respectively, and subjected to RPA.
The protected fragments of p11 (319 bp) and GAPDH were visualized by
autoradiography. A, IFN- (300 units/ml) treatment for 4-48 h resulted in increased p11 steady-state mRNA
expression in BEAS-2B cells. GAPDH mRNA levels are presented as an
internal control to evaluate RNA loading. The results are
representative of three different experiments. B,
densitometry measurements from three time courses of IFN- (300 units/ml)-untreated or treated experiments showing a
time-dependent increase in steady-state p11 mRNA levels
with greatest expression at 12 h (data presented as mean ± S.E.; *, p < 0.05 compared with control values).
C, IFN- (10, 300, 1000 units/ml) treatment for 24 h
results in a dose-dependent effect on p11 mRNA levels
in BEAS-2B cells. The results are representative of three different
experiments. D, densitometry measurements from three dose
response experiments of IFN- -treated cells demonstrates a
significant dose-related increase in p11 steady-state mRNA levels
(data presented as mean ± S.E., p < 0.001 for
dose effect by one-way ANOVA)
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IFN-
Induces p11 mRNA Expression in HeLa Cells--
To
further investigate if IFN-
induced p11 mRNA expression was
increased in HeLa cells, cells were incubated with or without IFN-
(10, 300, 1000 units/ml) for 24 h, and the steady-state levels of
p11 mRNA were measured by RPA. Untreated HeLa cells constitutively
produced p11 mRNA, and IFN-
treatment induced the steady-state
p11 mRNA expression in a dose-dependent manner (Fig.
4, A and B).

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Fig. 4.
Dose-dependent effect of
IFN- on p11 steady-state mRNA in HeLa
cells. A, IFN- (10-1000 units/ml) treatment for
24 h in HeLa cells showing an increased p11 steady-state mRNA
expression with a dose response manner. The data are representative of
three different experiments. B, densitometry measurement
from three dose response experiments of IFN- (10, 300, 1000 units/ml) treatment for 24 h demonstrating a significant dose
effect on p11 mRNA expression in HeLa cells (data presented as
mean ± S.E., p < 0.001 for dose effect by
one-way ANOVA)
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|
IFN-
Induces p11 Expression at the Transcriptional Level in HeLa
Cells--
To further investigate if the observed increase in
steady-state p11 mRNA levels reflects an IFN-
induced increase
in p11 gene transcription, a reporter gene construct
containing a 1498-bp sequence of the 5'-flanking region of the
p11 promoter in the SEAP vector was created (SEAP-1498+89)
and transient transfection of this p11 promoter construct
into HeLa cells was accomplished as described under "Experimental
Procedures." The results are shown in Fig.
5A. IFN-
(300 units/ml)
treatment of HeLa cells resulted in a significant increase in
p11 promoter activity (3-4-fold) over a 3-24 h time
course. HeLa cells treated with IFN-
(0, 30, 300 units/ml) for
24 h demonstrated a significant dose-related effect in relative
p11 promoter activity as shown in Fig. 5B.
-galactosidase activity was used to evaluate the transfection efficiency. These results demonstrate that IFN-
induces p11
expression at the transcriptional level.

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Fig. 5.
IFN- regulates
p11 gene expression at the transcriptional level.
HeLa cells were transfected with the p11 SEAP-1498+89 reporter gene
construct containing 1498 bp of the p11 5' promoter region and with the
pCMV/ -gal plasmid as a transfection control. After overnight
incubation in fresh Dulbecco's modified Eagle's medium with 10%
fetal bovine serum, the cells were treated with or without IFN- for
the indicated dose and time. Twenty-five microliters of culture medium
was collected for SEAP p11 promoter activity assay. Cell
lysates were used to detect -galactosidase activity. The relative
p11 promoter activity was determined by the ratio of SEAP
and -galactosidase activities. A, IFN- (300 units/ml)
induced a significantly increased p11 reporter gene activity with the
greatest effect at 12 and 24 h compared with control
(p < 0.001). B, IFN- (30, 300 units/ml)
treatment for 24 h demonstrates a dose-dependent
effect on p11 promoter activity in HeLa cells
(p < 0.001 for dose effect by one-way ANOVA). Values
represent the mean ± S.E. of three different experiments.
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AG-490 Inhibits IFN-
-induced p11 Promoter Activity--
To
further understand the mechanism of IFN-
induction of p11
expression, HeLa cells transfected with pSEAP-1498+89 promoter construct were pretreated with the JAK-2 tyrosine kinase inhibitor, AG-490 (50 or 100 µM) for 2 h and then treated with
or without IFN-
(300 units/ml) for 24 h. The results are
presented in Fig. 6 and demonstrate that
AG-490 significantly inhibited IFN-
induced p11 promoter
activity in a dose-related manner (p < 0.001, n = 6). AG-490 alone had no effect on p11
promoter activity, indicating that it is not cytotoxic at the
concentration used in this experiment.

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Fig. 6.
AG-490 inhibits
IFN- -induced p11 promoter
activity. HeLa cells were transfected with the p11 SEAP-1498+89
construct and pCMV/ -gal plasmid as described under the
"Experimental Procedures." After pretreatment with AG-490 (50 or
100 µM) for 2 h, cells were incubated in the
presence or absence of INF- (300 units/ml) for 24 h.
p11 relative promoter activities were measured. AG-490
inhibited IFN- induced changes in p11 reporter gene expression in a
dose-related manner (p < 0.001 by ANOVA). The data
presented are from one of three experiments each showing similar
results.
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AG-490 Inhibited the Effect of IFN-
on p11 Protein Level in HeLa
Cells--
To confirm the pathway of IFN-
induced p11 expression,
HeLa cells were preincubated with AG-490 (100 µM) for
2 h and then treated with or without IFN-
for 12 h.
IFN-
significantly increased p11 expression whereas in the cells
pretreated with AG-490, this effect was substantially diminished as
shown in Fig. 7,. Therefore, IFN-
induced the expression of p11 was blocked by JAK-2 tyrosine kinase
inhibitor.

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Fig. 7.
AG-490 inhibits
IFN- -induced p11 protein expression. HeLa
cells were pretreated with 100 µM of AG-490 for 2 h
and then incubated with 300 units/ml IFN- for 12 h. The cells
were lysed, and Western blots for p11 were processed as described under
"Experimental Procedures." Data presented are the mean ± S.E.
from three separate experiments. p < 0.05 as compared
with IFN- -treated cells versus AG490 plus IFN- -treated
cells.
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Identification of IFN-
Response Elements in the p11
Promoter--
In order to further study the transcriptional regulation
of p11 promoter by IFN-
, we characterized putative
IFN-
response elements in the p11 promoter. The sequence
analysis of 1498 bp of the p11 5'-flanking promoter region for putative
IFN-
response elements is shown in Fig.
8A. There are three interferon
GAS and one IFN-
response elements (
-IRE) located separately at
positions
215 (GAS-1),
1090 (GAS-2),
1219 (GAS-3), and
1448
(
-IRE) in the p11 promoter region. To determine the
region responsible for the IFN-
induced effect on p11 expression in
the p11 promoter, p11 promoter deletion
constructs were generated including pCAT-1498+89, pCAT-1434+89,
pCAT-1048+89, pCAT-188+89. The deletion constructs (1.8 µg) were
cotransfected with the pCMV/
-gal plasmid into HeLa cells. Cells were
then treated with and without 300 units/ml of IFN-
and harvested at
24 h. pCAT and
-galactosidase activities were assayed. The
activity of the deletion constructs was evaluated as a percentage of
1498+89 (100%) construct activity. Compared with the p11
promoter activity in the SEAP vector, IFN-
also induced a similar
activation (3-4-fold) of the full-length p11 promoter in
the pCAT vector (Fig. 8B). The removal of a 64-bp fragment
(pCAT-1434+89), which includes a
-IRE element did not have any
effect on IFN-
-induced p11 promoter activity. Further deletion to
1048 resulted in a reduced IFN-
-induced p11
promoter activity, suggesting that the fragment between
1434 and
1048 containing GAS-2 and GAS-3 sites is important for
IFN-
-induced p11 promoter activity. The deletion
construct pCAT-1048+89, which contains the GAS-1 element did not
exhibit a significant increase p11 promoter activity in
response to IFN-
activity. These data suggest that the region
between
1434 and
1048 of the p11 promoter, containing
two potential GAS elements, plays a critical role in the IFN-
induction of p11 expression.

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Fig. 8.
The effect of IFN-
on promoter activity of the p11 5'-deletion reporter constructs.
A, the putative IFN- response elements located in the
p11 promoter. Potential elements include sequences at
position 215 (GAS-1), 1090 (GAS-2), 1219 (GAS-3), and 1448
( -IRE), as well as other cis-acting elements in the p11
full-length promoter. B, each deletion construct was
cotransfected with the pCMV/ -gal plasmid into HeLa cells. The
transfected cells were treated with or without 300 units/ml IFN- for
24 h, and cell lysates were assayed with pCAT and
-galactosidase ELISA. The ratio of pCAT/ -gal is presented as
relative p11 promoter activity. Values of each construct are
calculated as percentage of the wild type pCAT-1498+89 promoter, which
is set at 100%. p11 deletion construct promoter activity suggested
that GAS-2 and GAS-3 sites located between 1434 and 1048 contribute
to the effect of IFN- on p11 expression. Data presented as mean ± S.E. of five experiments, p < 0.001 compared with
the activity of pCAT-1498+89 construct.
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|
GAS-2 and GAS-3 Elements Are Involved in IFN-
Induction of the p11 Promoter Activity--
To ascertain the role of
the GAS-2 and GAS-3 elements in IFN-
-induced p11 promoter activity
in the context of the full-length reporter construct, two full-length
mutation constructs (mGAS-2, mGAS-3) were created (Fig.
9A). HeLa cells were
transfected with the wild-type pCAT-1498+89, mGAS-2 or mGAS-3 promoter
construct. As shown in Fig. 9B, mutation of the GAS-2 site
significantly diminished IFN-
-induced p11 promoter
activity, with a reduction about ~73% of p11 promoter
activity compared with that of the full-length wild-type pCAT-1498+89
promoter construct. Mutation of the GAS-3 site inhibited to a lesser
degree (about 26% reduction) the IFN-
-induced p11
promoter activity of the wild-type pCAT-1498+89 promoter construct.
These data suggest that the GAS-2 site has an important role in
IFN-
-induced p11 expression whereas the GAS-3 site also
makes a contribution to this response.

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Fig. 9.
GAS-2 and GAS-3 elements are
important for IFN- induction of p11
promoter activity. Wild type pCAT-1498 + 89, mGAS-2, or
mGAS-3 plasmids were created and each was cotransfected with
pCMV/ -gal plasmid into HeLa cells as described under "Experimental
Procedures." The transfected cells were incubated with IFN- (300 units/ml) for 24 h and then analyzed for pCAT and
-galactosidase activity. Values of each construct are plotted as
percentage of the wild type pCAT-1498+89 promoter, which is set at
100%. A, wild type and mutation constructs with the mutated
sequence described in the text box. B, mutation at the GAS-2
or GAS- 3 site significantly inhibited IFN- -induced p11
promoter activity. Data are presented as mean ± S.E. of five
experiments. Activity of the wild type construct after IFN-
treatment was significantly greater than that of mGAS-2
(p < 0.001) or mGAS-3 (p < 0.05).
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STAT1 Dominant Negative Mutant Vector Blocked IFN-
-induced p11
Promoter Activity--
To further investigate the pathway of IFN-
induced p11 expression, the expression plasmid encoding wild-type STAT1
or mutant STAT1 (Tyr-701) were cotransfected with the p11
promoter construct pCAT-1499+89 (Fig.
10). IFN-
treatment significantly
increased pCAT-1498+89 activity in cells cotransfected with wild-type
STAT1 expression vector or empty vector, pcDNA3.1. However, in
cells cotransfected with the dominant negative vector, STAT1 (Tyr-701), IFN-
treatment had substantially less effect on the activity of the
p11 reporter gene.

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Fig. 10.
STAT1 mutation at Tyr-701 abolished the
effect of IFN- on p11
promoter activity. A, Hela cells were transfected
with 1 µg of p11 promoter construct pCAT-1499 + 89 or
cotransfected 1 µg of pCAT-1499+89 construct with 1 µg of
wild-STAT1, mu-STAT1-tyr or pcDNA3.1 control vector for 6 h.
The medium was replaced with fresh medium overnight, and cells were
then treated with or without IFN- (300 units/ml) for an additional
12 h. The cells were lysed, and pCAT and -galactosidase
activity were measured. Control cells are hatched bars and
IFN- -treated cells are solid bars, n = 9-12. B, representation of the data as the percentage
increase of p11 promoter activity of IFN- -treated cells
compared with untreated cells. Values are expressed as mean ± S.E. from three separate experiments. p < 0.001 comparing mu-STAT1-Tyr with pcDNA3.1 control vector.
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IFN-
Induces a Rapid Tyrosine Phosphorylation and Nuclear
Translocation of STAT1 Protein--
To determine the time course of
IFN-
induction of tyrosine phosphorylation of STAT1 protein in these
cells, HeLa cells were treated with IFN-
(300 units/ml) for 10 min,
30 min, 2 h or 4 h and crude cell lysates were prepared.
Western blot analysis with anti-phospho-STAT1 and anti-STAT1 were
performed. As shown in Fig. 11,
A and B, IFN-
induces a rapid tyrosine
phosphorylation of STAT1, starting at 10 min whereas no change was
observed in total STAT1 protein levels. Next, to determine whether
tyrosine phosphorylation of STAT1 in response to IFN-
was
accompanied by translocation into the nucleus, nuclear protein
extracted from HeLa cells was studied by Western blot. As shown in Fig.
11C, STAT1 rapidly translocated to the nucleus following
treatment with 300 units/ml of IFN-
for 10 min, 30 min, 2 h,
and 4 h. These data suggest that IFN-
treatment of these cells
causes a rapid tyrosine phosphorylation and nuclear translocation of
STAT1 protein in response to IFN-
.

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Fig. 11.
IFN- induces
tyrosine phosphorylation and nuclear translocation of STAT1
protein. Hela cells were treated with IFN- (300 units/ml) for
the indicated time and crude cell lysates or nuclear extracts were
prepared. A and B, ten micrograms of crude cell
lysates were subjected to 8% Tris-glycine gel electrophoresis and
Western blot analysis was performed using anti-phospho-STAT1 or
anti-STAT1 antibody. IFN- induced a rapid tyrosine phosphorylation
of STAT1. Total cellular STAT1 protein did not change. C,
nuclear protein from control cells or from cells treated with IFN-
was separated on 8% Tris-glycine gels, and a Western blot was
performed using anti-STAT1 antibody. IFN- induced a rapid nuclear
translocation of STAT1 protein. The results are representative of three
experiments with similar results.
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Electrophoretic Mobility Gel Shift of the GAS-2 Sequence--
To
confirm that IFN-
induces p11 expression through binding to a
specific GAS site, an electrophoretic mobility gel shift assay was
used. Since the GAS-2 site appears to play a pivotal role in
IFN-
-induced p11 expression, we synthesized an 18-bp double-stranded
oligonucleotide GAS-2 probe. Nuclear protein extracted from HeLa cells
was used for the electrophoretic mobility gel shift assays. To confirm
the binding specificity, excess of unlabeled GAS-2 or an irrelevent
consensus oligonucleotide, SP-1, was used as a competitor. Fig.
12A shows that there is
complex formation with the GAS-2 probe and this complex can be
partially inhibited by 50× excess of the cold GAS-2 probe and
completely inhibited by 200× excess of the cold probe; whereas
incubation with cold SP-1 oligonucleotide did not affect the complex
formation. These data suggest that this complex is specific for the
GAS-2 probe. Preincubation with anti-STAT1
antibody completely
abolished complex formation suggesting the presence of STAT1 protein in
this complex (Fig. 12B). IFN-
treatment for 10 min, 30 min, and 2, 4, 6, and 24 h significantly increased GAS-2 binding
with maximum observed at 30 min and 2 h as shown in Fig.
13. These data suggest that STAT1
protein mediates IFN-
induction of p11 expression through binding to
the GAS-2 element in the p11 promoter.

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Fig. 12.
IFN- induces
binding of STAT1 to the p11 GAS-2 sequence. Nuclear protein was
extracted from HeLa cells. 2 µg of nuclear extract were incubated
with 10,000 cpm of 32P-labeled double-stranded
oligonucleotide encoding the GAS-2 binding sequence from the
p11 promoter. A, to assess the specificity of
complex formation, nuclear extracts were preincubated with 50, 100, or
200× excess of non-labeled cold specific GAS-2 probe or 200× excess
of the nonspecific SP-1 oligonucleotide for 30 min before adding hot
probe. B, to identify the protein components of the IFN-
induced binding complex, nuclear extracts were preincubated with 1:20,
1:30, 1:50, and 1:100 of anti-STAT1 antibody for 1 h on ice
before adding the hot probe. The arrow indicated the complex
formation.
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Fig. 13.
IFN- induces a
rapid STAT1 binding to the GAS-2 site in the p11
promoter. HeLa cells were incubated with IFN- (300 units/ml) for 10 min, 30 min and 2, 4, 6, and 24 h. Cells were
lysed, and nuclear protein was extracted as described under
"Experimental Procedures." Two micrograms of nuclear protein were
incubated with the 32P-labeled double-stranded GAS-2
oligonucleotide probe. EMSA studies demonstrated that IFN- induces
STAT1 binding (arrow) to the GAS-2 site, with greatest
binding observed at 30 min and 2 h. The autoradiography is
representative of three experiments with similar results.
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Effect of Inhibition of p11 Protein Expression--
IFN-
treatment of cells may result in a variety of effects including an
increase in gene expression of cPLA2 as well as an increase
in cPLA2 activity (23, 25). In order to determine if
IFN-
induced changes in cellular p11 protein levels have an effect
on cellular function, cells were treated with iRNA directed against the
p11 coding sequence. iRNA treatment of HeLa cells resulted in a
reduction of cellular p11 protein levels(Fig.
14A). [3H]Arachidonic acid release HeLa cells was studied with
and without stimulation with the calcium ionophor, A23187. A23187
stimulated release of arachidonic acid was similar in control cells
treated with or without IFN-
. Calcium ionophor stimulated
arachidonic acid release from iRNA treated cells was increased compared
with control cells. Further, calcium ionophor-stimulated arachidonic acid release from iRNA-treated cells was significantly greater with
IFN-
-treated cells than iRNA-treated cells not treated with IFN-
or from control cells treated with IFN-
(Fig. 14B).
Therefore, IFN-induced changes in cellular p11 protein levels
subsequently may serve to down-regulate IFN-
-induced changes in
PLA2 activity.

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Fig. 14.
iRNA inhibition of p11 protein production
reveals that IFN- treatment results in an
increase in calcium ionophor-induced arachidonate release. HeLa
cells were transfected with double-stranded iRNA directed against
codons 2-7 of the p11 coding sequence with LipofectAMINE
for 5 h as described under "Experimental Procedures." Control
cells were treated with LipofectAMINE alone. A, Western blot
for p11 protein. After transfection for 5 or 16 h of incubation in
media, and treatment with or without IFN (300 units/ml) for 24 h,
cells were harvested and cell lysates prepared. The immunoblotting was
performed as described under "Experimental Procedures." In control
cells, IFN- treatment resulted in an increase in cellular p11
protein levels compared with control cells treated with vehicle alone.
iRNA treatment resulted in a substantial decrease in cellular p11
protein levels compared with control cells. The blot presented is
representative of three separate experiments each with similar results.
B, cellular [3H]arachidonic acid release from
HeLa cells transfected with iRNA against p11 or treated with
LipofectAMINE alone (control cells). After transfection, cells were
labeled with [3H]AA for 16 h. Media were changed,
and cells were treated with IFN- (300 units/ml) for 24 h. Media
was changed, and cells were treated with the calcium ionophor, A23187
(10 6 M) or vehicle for 30 min. The media were
collected and centrifuged and an aliquot taken for scintillation
counting. iRNA treatment resulted in an increase in calcium ionophor
induced arachidonate release compared with control cells. IFN-
treatment resulted in an increase in calcium ionophor-induced
arachidonate release from iRNA-treated cells compared with
interferon-treated control cells suggesting that IFN- induced
changes in phospholipase A2 are not inhibited by p11 in the
iRNA-treated cells. Data presented are mean ± S.E.,
(n = 3). * indicates p < 0.01 by
Student's t test.
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 |
DISCUSSION |
p11 is a unique member of the S-100 family of calcium-binding
proteins. p11 is present in a variety of cells separately or as a
heterotetramer with annexin II (p36). The heterotetramer is composed of
two copies of 36-kDa heavy chain, annexin II subunits and two copies of
11-kDa light chain, p11 subunits as (p36)2
(p11)2 (4). Recently, Waisman and co-workers (9, 10, 26,
27) (reported that the annexin II tetramer serves as a receptor to interact not only with tPA and plasminogen, but also with procathepsin B. The C-terminal lysine residues of the p11 subunit of the annexin II
tetramer bind plasminogen and participate in the stimulation of
t-PA-dependent plasminogen activation. Recombinant p11
subunit stimulates t-PA dependent plasminogen activation (27). These data provide evidence that p11, acting as a regulatory protein, modulates the activity of the p36 subunit and stimulates the protease activity by AIIt.
p11 also interacts with the C-terminal region of cPLA2 and
results in decreased cPLA2 activity and arachidonic acid
release (11). Annexin I and annexin II2-p112
but not annexin II inhibits cPLA2 activity (14). Yao
et al. (12) have shown that dexamethasone treatment of cells
reduced cPLA2 activity by up-regulation of p11 expression.
Alkiba et al. reported that priming of RGM1 cells with TGF
alone or both TGF
and IL-1
inhibits A23187-induced cPLA2 catalyzed AA release and concurrently increased p11
and cPLA2 but not annexin II heavy chain protein
expression, suggesting that p11 expression might be involved in the
impairment of the hydrolytic activity of cPLA2 (29, 30).
These data provide evidence that p11 may have a physiological role in
the modulation of cPLA2 activity and subsequent arachidonic
acid release.
p11 also has been shown to interact with BAD to attenuate the
pro-apoptotic effect of BAD and overexpression of p11 dampens the
proapoptotic activity of BAD in transfected cells (31). Overexpression
of p11 induces PC12 cell morphology changes and enhances cell survival
(32). Gonadotropin stimulates increased p11 mRNA expression in
granulosa cells of preovulatory follicles (33). These data provide
evidence that p11, as an anti-apoptosis gene, may regulate BAD activity
to mediate the survival of cells.
IFN-
is known to modulate the expression of a variety of genes in
different cell types. IFN-
has been reported to induce intercellular
adhesion molecule-1 (ICAM-1) expression and vascular cell adhesion
molecule-1 (VCAM) expression at the transcriptional and
post-transcriptional level (17, 18, 34). Matsuura et al. (16) reported that IFN-
induces COX-2 mRNA expression
at the transcriptional level and increases PGE2 synthesis
in normal human epithelial keratinocytes cells. However, IFN-
may
also down-regulate gene expression. IFN-
-primed macrophages
demonstrate decreased COX-2 gene expression in response to
IL-1
stimulation (35). IFN-
inhibits IL-4-induced 15-lipoxygenase
mRNA and protein expression in cultured human monocytes (36). The
binding of IFN-
to its receptor leads to the recruitment and
activation of STAT1 protein that, after being activated by tyrosine
phosphorylation, translocates to the nucleus and mediates the
transcription of IFN-
response genes (19, 20, 37). STAT1 activation
involves specific members of JAK family that are associated with
receptors. IFN-
treatment activates JAK1, JAK2, and STAT1 (38). In
this study, we demonstrated that IFN-
up-regulates p11 expression in
human epithelial cells. This effect was confirmed using two different
cell lines, BEAS-2B cells and HeLa cells. Studies in two different cell
lines demonstrate that IFN-
induces steady-state p11 mRNA
expression and p11 protein production in human epithelial cells.
Transient transfection studies showed that IFN-
induces p11
expression at the transcriptional level. p11 has been reported to be
regulated in different cell types. Nerve growth factor (NGF) increases
p11 mRNA expression in rat pheochromocytoma (PC12) cells (39). Munz
et al. (40) reported that wound-derived growth factors
(TGF-
1, EGF, and KGF) differentially regulate p11 and annexin II
expression in cultured keratinocytes during skin injury and modify the
ratio between p11 and annexin II (39). Dexamethasone treatment of
BEAS-2B cells or of HeLa cells increases steady-state levels of p11
mRNA and p11 protein levels and results in a reduction of
cPLA2 activity (12). TGF-
or the mixture of TGF-
and
IL-1
induces an increase in p11 protein expression in rat gastric
epithelial cells (29, 30). However, little is known about the mechanism of regulation of p11 expression. A recent report has shown that retinoic acid reduces p11 protein levels by a post-translational mechanism. In this study, we demonstrated that IFN-
induced p11 expression is mediated by STAT1 binding to GAS in the p11
promoter. Several lines of evidence confirmed this effect. First,
AG-490, a specific JAK-2 kinase inhibitor abolished the effect of
IFN-
on p11 promoter and protein level, suggesting that
JAK-2 tyrosine kinase is required for activation of the p11
promoter. Second, functional analysis of the p11 promoter,
which contains four potential IFN-
cis-acting elements, one

IRE, and three GAS elements, indicated that two elements are
important for the induction by IFN-
of p11 expression. Third,
cotransfection of a STAT1 dominant negative plasmid and p11
promoter construct abolished the effect of IFN-
on p11
promoter activity. Fourth, IFN-
induces a rapid tyrosine
phosphorylation and nuclear translocation of STAT1 protein, indicating
that STAT1 is involved in the induction of IFN-
in p11 expression.
Fifth, electrophoretic mobility shift assays demonstrate that IFN-
induces a time-dependent STAT1 binding to the GAS-2 site of
the p11 promoter. Taken together, we believe that IFN-
induces activated STAT1 binding to GAS elements resulting in
p11 gene and protein expression.
STAT1-deficient mice show complete unresponsiveness to IFN-
, but
still respond normally to several other cytokines that also activate
STAT1, illustrating the critical role of STAT1 in IFN-
-induced gene
expression (41). IFN-
induced expression of the Class II
transactivator, ICAM, is mediated through STAT1 binding to one or two
of the GAS elements in the promoters of these genes (42, 43). IFN-
has also been reported to induce CD40 expression through STAT1 binding
to one of three GAS sites, another GAS element also is important to
IFN-
-induced CD40 gene expression, but maximal induction
of CD40 requires additional cis-acting elements (44). In this study we
have shown that both GAS-2 and GAS-3 sites are involved in
IFN-
-induced p11 expression. STAT1 has been shown to cooperate with
other transcriptional factors to induce gene expression. Therefore,
whether other transcriptional factors are involved in IFN-
induced
p11 expression remains to be explored.
Given the effect of IFN-
on cPLA2 gene
expression and activity, we studied the effect of IFN-
induced
changes in p11 expression using small inhibitory RNAs to inhibit p11
protein expression in cells treated with or without IFN-
. iRNA
treatment resulted in a reduction in cellular p11 protein levels and in
an increase in calcium ionophor-induced arachidonic acid release. The
arachidonic acid release from iRNA-treated cells subsequently treated
with the calcium ionophor, A23187, was significantly greater than IFN-
-treated control cells suggesting that the induced p11 may play
a counterregulatory role in this process. In summary, our data suggest
that IFN-
induces p11 expression in human epithelial cells mediated
by STAT1 binding to GAS elements in the p11 promoter.