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INTRODUCTION |
Phospholipase A2s
(PLA2s)1 are a
group of enzymes that hydrolyze the ester bond of fatty acids from the
sn-2 position of glycerophospholipids. The release of
arachidonic acid (AA) from membranes by PLA2 and its
subsequent conversion into leukotrienes, prostaglandins, and other
eicosanoids plays an important role in inflammation (1-4). The
mammalian calcium-dependent PLA2s can be
grouped into major classes based on their molecular mass and cellular
distribution, including the low molecular mass (10-14 kDa) secreted
forms (sPLA2) and the structurally unrelated high molecular
mass (85 kDa) cytosolic PLA2 (cPLA2) (1, 3,
5).
To date, five different sPLA2 isozymes have been described
in mammalian cells. The 14-kDa sPLA2 enzyme from synovial
fluid and platelets (Group IIA) may be involved in the pathogenesis of
inflammatory reactions (3, 6, 7). The 14-kDa PLA2 lacks
apparent selectivity for the sn-2 fatty acids of
phospholipids and requires much higher Ca2+ concentrations
(millimolar) than normal intracellular Ca2+ levels
(nanomolar to micromolar) for activity. The 85-kDa high molecular mass
cPLA2 has higher selectivity to hydrolyze phospholipids containing AA esterified in the sn-2 position (1, 3, 5, 8-11). Its activity is regulated by phosphorylation, G-protein activation, and physiologically relevant concentrations of calcium. Because cPLA2 may play a central role in producing AA and
lysophospholipid for subsequent metabolism to prostaglandins,
leukotrienes, hydroxyeicosatetraenoic acids, and platelet-activating
factor, all potent lipid mediators of inflammation, the activation of
cPLA2 may play an important role in modulating the airway
inflammatory response (1, 3, 5).
S-100 proteins are a family of proteins first described by Moore (12)
who initially characterized a group of abundant low molecular weight
(10-12 kDa) acidic proteins in neural tissue. S-100 proteins are a
group of Ca2+-binding proteins that are expressed in a cell
type-dependent fashion. This family includes S-100
,
S-100
, and p11/calpactin light chain (13). p11 was described as a
member of the S-100 family of EF hand type Ca2+-binding
proteins but does not have the ability to bind Ca2+ ions
due to crucial amino acid deletions and substitutions in the two EF
hand loops of the protein (14, 15). p11 binds to and inhibits the
phosphorylation of a 36-kDa protein known as p36, also known as annexin
II as well as calpactin heavy chain (16, 17).
Glucocorticoids are effective in the treatment of immune and
inflammatory disorders affecting the lung and other organs. One mechanism of glucocorticoid modulation of the inflammatory response is
inhibition of the release of AA from cellular lipids (18, 19) and
inhibition of prostaglandin H synthase-2 synthase or cyclooxygenase-2
expression in a number of tissues (20-24). The rate of eicosanoid
synthesis may be regulated by the availability of free AA that can be
metabolized into prostanoids and leukotrienes via the cyclooxygenase
and lipoxygenase pathways. The decreased synthesis of bioactive
eicosanoids may represent an important mechanism of the
anti-inflammatory action of glucocorticoids. Glucocorticoids can induce
annexins which might inhibit sPLA2 activity in
vitro (25-30). A recent study has demonstrated that p11 can
directly interact with the COOH-terminal region of 85-kDa cPLA2 and inhibit cPLA2 enzyme activity (31).
Therefore, it was of interest to study whether p11 plays a role in
glucocorticoid induced changes in cellular arachidonate release.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
HeLa cells were obtained from the American
Type Culture Collection (Rockville, MD) and grown in DMEM medium with
10% fetal bovine serum. BEAS-2B cells, a human bronchial epithelial
cell line, were a gift from Curtis Harris and John Lechner, National Cancer Institute, Bethesda, MD. BEAS-B cells were grown in LHC-8 medium
(Biofluids, Rockville, MD) without hydrocortisone or serum. All
experiments were performed when cells were 80% confluent.
Immunoblot of p11 Protein--
HeLa or BEAS-2B cells were grown
on 175-cm2 flasks and treated with dexamethasone
(Calbiochem) (10
7, 10
9, and
10
11 M) for 24, 36 or 48 h. For time
course experiments, the culture medium was changed at the same time,
and all cells were harvested at the same time. Dexamethasone
(10
7 M) was added at the indicated times
prior to harvesting. At the indicated times treated and control cells
were rinsed three times with cold PBS. After washing, the cells were
transferred to 0.5 ml of homogenization buffer; 50 mM
Hepes, pH 8.0, 1 mM EDTA, 1 mM EGTA, 100 µM leupeptin, 1 mM dithiothereitol, 10 mM phenylmethylsulfonyl fluoride, 0.5 mM
soybean trypsin inhibitor, 15 mM aprotinin, and 0.5%
Triton X-100. Cells in homogenization buffer were sonicated for 15 s times three using a microprobe. Total protein was assayed by BCA
reagent (Pierce). Samples containing 20 µg of cell lysate protein
were separated on 18% Tris-glycine gels (Novex, San Diego, CA) using
Tris-glycine SDS running buffer. The separated proteins were
electrophoretically transferred onto a nitrocellulose membrane (Novex),
then blocked with 5% non-fat dry milk overnight. 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
as second antibody (Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA). The blot was developed using the ECL Western blotting
detection system (Amersham Pharmacia Biotech).
Immunoblot of cPLA2 Protein--
HeLa cells grown in
175-cm2 flasks were treated with dexamethasone
(10
7, 10
9, and 10
11
M) for 24, 36, or 48 h. At the indicated times, crude
cytosolic extracts of treated and control cells were prepared as
described above for the immunoblot of p11 protein. Samples containing
20 µg of cell lysate protein were separated on 8% Tris-glycine gels (Novex) using Tris-glysine SDS running buffer. cPLA2
protein expression was detected by using 1:1000 dilution
rabbit-anti-human cPLA2 polyclonal antibody (provided by
the Genetics Institute, Boston, MA) and 1:5000 dilution of
horseradish-peroxidase-conjugated goat-anti-rabbit IgG as a second
antibody (Jackson ImmunoResearch Laboratories, Inc.). The blot was
developed using the ECL Western blotting detection system.
Immunoprecipitation of Native p11 Protein from HeLa and BEAS-2B
Cells--
The HeLa or BEAS-2B cells grown on 175-cm2
culture flasks were washed with cold PBS, and the cells were lysed in
0.5 ml of homogenization buffer with protease inhibitors, but without
EGTA and EDTA. The crude cytosolic protein was isolated as described above for immunoblot for p11 protein. For immunoprecipitation, the
isolated crude cytosolic fraction (200 µl, 400 µg of protein) was
added to a microcentrifuge tube containing 1 ml HBSS (with calcium and
magnesium) and 10 µl of rabbit anti-human cPLA2 antibody. The samples were incubated at 4 °C for 30 min, 25 µl of Protein G
Plus/Protein A-agarose (Pierce) was then added to each sample, and the
mixture was incubated at 4 °C for 4 h, followed by
centrifugation in a microcentrifuge at 2500 rpm for 5 min at 4 °C.
The supernatant was aspirated, and the pellet was washed four times
with 1.0 ml cold PBSTDS (phosphate-buffered saline, 1% Triton X-100,
0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) with
repeated centrifugation. After four washings with PBSTDS, 20 µl of
protein loading buffer was added to the pellet and the sample was
boiled for 10 min before electrophoresis on 16% polyacrylamide gels
(Novex) using Tris-glycine/SDS buffer. The separated proteins were
electrophoretically transferred onto a nitrocellulose membrane blocked
with 5% non-fat milk and then probed with a 1:2000 dilution of mouse
anti-human p11 monoclonal antibody. The blots were then probed with a
1:5000 dilution of horseradish peroxidase-labeled donkey anti-mouse IgG and developed by using the ECL Western blotting detection system.
Ribonuclease Protection Assay (RPA) for cPLA2 and p11
mRNA Levels--
The HeLa cells were treated with dexamethasone
(10
7, 10
9, and 10
11
M) for 24 to 48 h. Total cellular RNA was extracted
from 175 cm2 culture flasks by the single step guanidinium
thiocyanate-phenol-chloroform extraction method (Tri-reagent, Molecular
Research Inc., Cincinnati, OH). The RNA pellet was precipitated with
isopropyl alcohol, washed with 70% ethanol, and redissolved in diethyl
pyrocarbonate water. To construct the probe for cPLA2
mRNA, a 306-bp product of cPLA2 cDNA was amplified
by polymerase chain reaction using the following sets of sense
and antisense primers: 5' primer,
5'-CTCACACCACAGAAAGTTAAAAGAT-3'(799-823); 3' primer,
5'-AAATAAGTCGGGAGCCATAAA-3' (1104-1084) (Biosynthesis Inc.,
Lewisville, TX). The product for cPLA2 gene was cloned into the TA cloning vector (Invitrogen, San Diego, CA). Orientation of the
insert was determined by DNA sequencing. To construct the probe for p11
mRNA, a 320-bp product of p11 cDNA was amplified by polymerase
chain reaction using the following sets of sense and antisense primers:
5' primer, 5'-ACCACACCAAAATGCCATCTC-3'(101-121); 3' primer,
5'-CTGCTCATTTCTGCCTACTT-3' (400-419) (Genosys Biotechnologies, Inc., The Woodlands, TX). The product for p11 was cloned into the
pGEM-T Easy Vector (Promega, Madison, WI). Orientation of the insert
was determined by DNA sequencing. The cPLA2 and
glyceradehyde 3-phosphate dehydrogenase (GAPDH) RNA probes were
prepared by in vitro transcription using T7 polymerase with
[
-32P]CTP. The p11 RNA probes was prepared by in
vitro transcription using SP6 polymerase with
[
-32P]CTP. An RPA assay kit (RPAII, Ambion, Austin
Texas) was used. Hybridization was performed at 45 °C for 16 h
and with 10 µg (for GAPDH) or 20 µg (for cPLA2) and 40 µg (for p11) of total RNA. 104 dpm (for GAPDH) and 2 × 104 dpm (for cPLA2 and p11) of
32P-labeled RNA probe were used. After hybridization, the
unhybridized RNA was digested by addition of 1:100 diluted RNaseA/T1
mix at 37 °C for 60 min. Digestion was terminated by the addition of RNase inactviation and precipitation mixture. The protected RNA fragment was analyzed by autoradiography after separation on 6% polyacrylamide, 8 M urea gels (Novex).
Effect of RU486 on Dexamethasone-induced p11 Expression--
The
HeLa cells grown on 175-cm2 flasks were treated with
dexamethasone (10
7 M) with or without the
glucocorticoid receptor antagonist, RU486 (10
7,
10
8, 10
9, 10
10,
10
11, and 10
12 M) for 24 h. At the end of incubation time, crude cytosolic extracts of treated
and control cells were prepared and Western blots were done as
described in the experimental procedures section for immunoblot of p11 protein.
Arachidonic Acid Release from Dexamethasone-treated
Cells--
The HeLa cells grown on T-75-cm2 cultured
flasks were labeled for 18 h with 1 µCi/ml
[5,6,8,9,11,12,14,15-3H]arachidonic acid
([3H]AA)(214 Ci/mmol; Amersham Pharmacia Biotech) in DMEM
media with 10% fetal calf serum. Subsequently, some cultures were
treated with dexamethasone (10
7 M) for
24 h, while others were maintained as controls. Following 20-h
incubation with dexamethasone, all cells were relabeled with 1 µCi/ml
[3H]AA for 4 h before harvesting the cells. For
studies of AA release after calcium ionophore stimulation, following
three washes, 12 ml of calcium ionophore A23187 (10
6
M) (Calbiochem) in HBSS(+) with 0.5% BSA or HBSS(+) with
0.5% BSA alone were added to each flask, and the cells were incubated at 37 °C for 30 min. The supernatant was harvested for HPLC
analysis. The samples for HPLC analysis were extracted by
octadecylsilane C18 cartridges (Sep-Pak C18;
Waters Associates, Milford, MA) and chromatographed by reverse phase
HPLC. Individual Sep-Pak C18 cartridges were prepared with
15 ml of methanol followed by 5 ml of 5 mM EDTA and 10 ml
of water. Samples were loaded onto the cartridges washed with 10 ml of
water and eluted with 4 ml of methanol. The methanol fraction was
collected and evaporated to dryness under steady flow nitrogen gas and
resuspended in 200 µl of methanol for analysis by HPLC. An
ultrasphere C18 column (4.7 × 250 mm) (Beckman
Instruments) with 5-µm particle size was used. A gradient program was
used with mobile phase A, water/acetonitrile/phosphoric acid
(75:25:0.025), and mobile phase B,
methanol/acetonitrile/trifluoroacetic acid (60:40:0.0016), at a flow
rate of 1.5 ml/min. The AA fraction of HPLC elution was collected and
measured for radioactivity.
Stable Transfection of a p11 Antisense Plasmid in HeLa
Cells--
A 321-bp cDNA corresponding to bases 115-436 of the
p11 cDNA sequence was used to construct the antisense p11
expression plasmid, the insert was cloned into the mammalian expression
vector pcDNA3.1(+) (Invitrogen) in the antisense orientation,
giving rise to ASp11-pcDNA3.1(+). The identity and orientation of
construct was confirmed by DNA sequencing. HeLa cells grown in
175-cm2 flasks were exposed to 120 µl of LipofectAMINE
Reagent (Life Technologies, Inc.) with 20 µg of ASp11-pcDNA3.1(+)
plasmid after repeated washing with serum-free DMEM medium. Control
cells were transfected with pcDNA3.1(+) expression plasmid alone.
Cells were exposed to the mixture of LipofectAMINE and plasmid for
4 h. Following removal of the transfection reagent, fresh DMEM
with 10% serum and 1000 µg/ml Geneticin (G418 sulfate) (Calbiochem)
was added to each flask. Subsequent cultures of selected HeLa cells
were routinely grown in the presence of selective pressure. Transfected HeLa cells were cloned by limiting dilution and clones used for Western
blot and AA release.
For [3H]AA release studies, equal numbers of cells
transfected with pcDNA3.1(+) vector alone as control, and the cells
transfected with the p11 antisense plasmid ASp11-pcDNA3.1(+) were
grown in T-75-cm2 culture flasks. Cells were labeled for
18 h with 1 µCi/ml [3H]AA in DMEM medium with 10%
fetal calf serum and 1000 µg/ml Geneticin. Following repeated washing
with media, 12 ml of fresh medium with 10% serum and 1000 µg/ml
Geneticin were added to each flask. For studies of AA release after
calcium ionophore stimulation, following repeated washing with HBSS(+)
with 0.5% BSA for three times, 12 ml of calcium ionophore A23187
(10
6 M) in HBSS(+) with 0.5% BSA or HBSS
with 0.5% BSA without A23187 were added to each flask, and the cells
were incubated at 37 °C for 30 min. The supernatants were extracted
by Sep-Pak C18 cartridges and chromatographed by reverse
phase HPLC as described above. The AA fraction of HPLC elution was
collected and measured for radioactivity.
Stable Transfection of a p11 Expression Plasmid in HeLa
Cells--
A cDNA containing the coding region of the p11 gene was
cloned into the mammalian expression vector pcDNA3.1(+)
(Invitrogen) to create p11-pcDNA3.1(+). The identity and
orientation of construct was confirmed by DNA sequencing. The
pcNDA3.1(+) vector carries the human cytomegalovirus immediate early
enhancer-promoter sequences to promote constitutive expression of the
cloned p11 insert in mammalian cells. The HeLa cells grown in
175-cm2 flasks were exposed to 120 µl of LipofectAMINE
Reagent (Life Technologies, Inc.) with 20 µg of p11-pcDNA3.1(+)
plasmid after repeated washing with serum-free DMEM medium. Control
cells were transfected with pcDNA3.1(+) expression plasmid alone.
Cells were exposed to the mixture of LipofectAMINE and plasmid for
4 h. Following removal of the transfection reagent, fresh DMEM
with 10% serum and 1000 µg/ml Geneticin (G418 sulfate) (Calbiochem)
was added to HeLa cells. Subsequent cultures of selected HeLa cells
were routinely grown in the presence of selective pressure. Transfected HeLa cells were cloned by limiting dilution and clones were used for
Western blot and AA release after four passages.
For [3H]AA release, equal numbers of cells transfected
with pcDNA3.1(+) vector alone as control and the cells transfected
with the p11 expression plasmid p11-pcDNA3.1(+) were grown in
T-75-cm2 culture flasks. Cells were labeled for 18 h
with 1 µCi/ml [3H]AA in DMEM medium with 10% fetal
calf serum with 1000 µg/ml Geneticin. For studies of AA release after
calcium ionophore stimulation, following three washes with HBSS(+) with
0.5% BSA, 12 ml of calcium ionophore A23187 (10
6
M) in HBSS(+) with 0.5% BSA or HBSS(+) with 0.5% BSA
alone were added to each flask, and the cells were incubated at
37 °C for 30 min. The supernatants were collected, extracted on
Sep-Pak C18 cartridges and chromatographed on reverse phase
HPLC as described above. The AA fraction of HPLC elution was collected
and measured for radioactivity.
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RESULTS |
Dexamethasone Increases p11 Protein Levels in Human HeLa Cells and
BEAS-2B Cells--
The effect of dexamethasone treatment on human
epithelial cell expression of p11 was studied by Western blot of two
different epithelial cell lines, HeLa cells and BEAS-2B cells. Fig.
1A demonstrates the effect of
dexamethasone treatment of HeLa cells on cellular p11 accumulation.
Treatment of cells with dexamethasone (10
7 M)
for 24-48 h resulted in a significant increase in p11 protein expression in cell lysates. In addition, treatment of cells with 10
7, 10
9, and 10
11
M dexamethasone for 24 h resulted in a dose-related
increase cellular p11 protein levels (Fig. 1B). Treatment of
BEAS-2B cells with dexamethasone (10
7 M) for
24-48 h also resulted in a significant increase in p11 protein
expression in cell lysates (Fig. 1C).

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Fig. 1.
The effect of dexamethasone on p11 protein
levels. A, the effect of dexamethasone on p11 protein
levels in HeLa cells. Cells were grown to near confluence and then
treated with dexamethasone (10 7 M) for 24-48
h. Cell lysates from treated and untreated cells were processed as
described under "Experimental Procedures," and 20 µg of total
protein was subjected to gel electrophoresis and immunoblotting.
B, the dose effect of dexamethasone on p11 protein levels in
HeLa cells. Cells were grown to near confluence and then treated with
dexamethasone (10 7 to 10 11 M)
for 24 h. Cell lysates from treated and untreated cells were
processed as described under "Experimental Procedures" and 20 µg
of total protein was subjected to gel electrophoresis and
immunoblotting. C, the effect of dexamethasone on p11
protein levels in BEAS-2B cells. Cells were grown to near confluence
and then treated with dexamethasone (10 7 M)
for 24-48 h. Cell lysates from treated and untreated cells were
processed as described under "Experimental Procedures," and 20 µg
of total protein was subjected to gel electrophoresis and
immunoblotting.
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Effect of Dexamethasone on Steady State Levels of p11
mRNA--
Steady state levels of mRNA for p11 were measured by
RPA of total cellular RNA extracted from HeLa cells that were incubated without or with dexamethasone (10
7 M) for
24-48 h. As shown in Fig. 2A,
these cells produce p11 mRNA and the steady state level of p11
mRNA was increased by dexamethasone treatment over 24-48 h. In
addition, dexamethasone in concentrations of 10
7
to 10
11 M induced a dose-related
change in p11 mRNA levels (Fig. 2B).

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Fig. 2.
The effect of dexamethasone on p11 mRNA
levels in HeLa cells. A, the effect of dexamethasone on
p11 mRNA levels. HeLa cells were treated with dexamethasone
(10 7 M) for 24, 36, and 48 h before
total RNA was extracted. Ten µg and 40 µg of the total RNA were
hybridized to GAPDH and p11-specific radiolabeled cRNA probes,
respectively, and assayed by RPA. The protected fragments of p11 (320 bp) and GAPDH were visualized by autoradiography. The result shown is
representative of three separate experiments demonstrating the same
result. B, the dose effect of dexamethasone on p11 mRNA
levels. The HeLa cells were treated with dexamethasone
(10 7, 10 9, and 10 11
M) for 24 h before total RNA was extracted. Ten µg
or 40 µg of the total RNA were hybridized to GAPDH and p11-specific
radiolabeled RNA probes and assayed by RPA. The protected fragments of
p11 (320 bp) were visualized by autoradiography. The result shown is
representative of three separate experiments.
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Effect of Dexamethasone on cPLA2 Protein and mRNA
Levels in HeLa Cells--
The effect of dexamethasone treatment on
human epithelial cell expression of cPLA2 was studied by
Western blot of cell lysates. Treatment of cells with dexamethasone
(10
7 M) for 24-48 h had no effect on
cPLA2 protein expression (Fig. 3A). In addition, treatment of
cells with 10
7, 10
9, and 10
11
M dexamethasone for 24 h did not result in a change in
cPLA2 protein levels (Fig. 3B). The effect of
dexamethasone treatment on human epithelial cell expression of
cPLA2 was also studied by RPA of HeLa cells treated with
dexamethasone. Fig. 3C demonstrates the effect of
dexamethasone treatment of HeLa cells on steady state levels of
cPLA2 mRNA. Treatment of cells with dexamethasone (10
7 M) for 24-48 h had no clear effect on
levels of cPLA2 mRNA.

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Fig. 3.
The effect of dexamethasone on
cPLA2 protein and steady state mRNA levels.
A, the effect of dexamethasone on cPLA2 protein
levels in HeLa cells. Cells were grown to near confluence and then
treated with dexamethasone (10 7 M) for 24-48
h. Cell lysates from treated and untreated cells were processed as
described under "Experimental Procedures," and 20 µg of total
protein was subjected to gel electrophoresis and immunoblotting.
B, the dose effect of dexamethasone on cPLA2
protein levels in HeLa cells. Cells were grown to near confluence and
then treated with dexamethasone (10 7 to
10 11 M) for 24 h. Cell lysates from
treated and untreated cells were processed as described under
"Experimental Procedures," and 20 µg of total protein was
subjected to gel electrophoresis and immunoblotting. C, the
effect of dexamethasone on cPLA2 mRNA levels. HeLa
cells were treated with dexamethasone (10 7 M)
for 24, 36, and 48 h before total RNA was extracted. Ten µg and
20 µg of the total RNA were hybridized to GAPDH and
cPLA2-specific radiolabeled cRNA probes, respectively, and
assayed by RPA. The protected fragments of cPLA2 (306 bp)
and GAPDH were visualized by autoradiography. The result shown is
representative of three separate experiments.
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Dexamethasone Increases p11 Bound to
cPLA2--
The above results demonstrated that
dexamethasone treatment increased p11 expression, but had little or no
effect on cPLA2 expression. To further investigate the
interaction between p11 and cPLA2 in human epithelial
cells, immunoprecipitation of the p11·cPLA2 complex from
HeLa cells and BEAS-2B cells was performed. As shown in Fig.
4, A and B, p11 was
precipitated from the HeLa cell and BEAS-2B cell lysates by rabbit
anti-human cPLA2 polyclonal antibody followed by the
addition of Protein G Plus/Protein A-agarose. Immunoblots of the
purified complex were developed for p11 protein. There was more p11
coprecipitated with cPLA2 after dexamethasone treatment.
This result demonstrated that dexamethasone treatment resulted not only
in an increase in cellular p11 protein but also in an increase in p11
bound to cPLA2.

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Fig. 4.
Immunoprecipitation of
p11·cPLA2 complex. A, immunoprecipitation
of the p11·cPLA2 complex from HeLa cells. Cell lysates
from untreated cells and cells treated with dexamethasone
(10 7 M) for 24, 36, and 48 h were
incubated with rabbit anti-human cPLA2, 25 µl of Protein
G Plus/Protein A-agarose beads was then added to each sample for
further incubation. The beads were collected, washed, and subjected to
SDS-polyacrylamide gel electrophoresis. The precipitated p11 protein
was then detected by Western blotting analysis as described under
"Experimental Procedures." The position of p11 protein is
indicated. B, immunoprecipitation of the
p11·cPLA2 complex from BEAS-2B cells. Lysates from
untreated cells and cells treated with dexamethasone (10 7
M) for 24, 36, and 48 h were incubated with rabbit
anti-human cPLA2, and 25 µl of Protein G Plus/Protein
A-agarose beads was then added to each sample for further incubation.
The beads were collected, washed, and subjected to SDS-polyacrylamide
gel electrophoresis. The precipitated p11 protein was then detected by
Western blotting analysis as described under "Experimental
Procedures." The position of p11 protein is indicated.
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RU486 Inhibits Dexamethasone-induced p11 Protein
Increases--
In an attempt to determine if the effect of
dexamethasone on p11 protein levels is mediated via a glucocorticoid
receptor interaction, RU486 (10
7 to
10
12 M) was incubated with cells prior to and
concomitant with the dexamethasone treatment. Treatment with RU 486 resulted in a dose-dependent inhibition of the
dexamethasone-induced increases in p11 protein levels. Fig.
5 shows the effect of RU486
(10
10-10
12 M) on
dexamethasone-induced p11 protein levels.

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Fig. 5.
The effect of RU486 treatment on
dexamethasone-induced p11 protein levels. Cells were grown to near
confluence. RU486 was incubated with cells prior to and concomitant
with or without dexamethasone (10 10 to 10 12
M) for 24 h. Cell lysates from treated and untreated
cells were processed as described under "Experimental Procedures,"
and 20 µg of total protein was subjected to gel electrophoresis and
immunoblotting.
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Dexamethasone Inhibits AA Release from the HeLa Cells--
The
results from RPA and Western blot studies indicated that dexamethasone
treatment had an effect on p11 mRNA levels and protein production
but little or no effect on the mRNA expression or protein level of
cPLA2. In these cells, dexamethasone treatment does alter
the release of 3H-labeled AA both at base line and after
exposure to the calcium ionophore A23187. Fig.
6 demonstrates that labeled AA release from dexamethasone-treated HeLa cells (HD) is lower than
that from untreated HeLa cells (HC). After treatment with
A23187, the release of labeled AA from dexamethasone-treated HeLa cells
(HD+A) is significantly decreased compared with untreated
control cells (HC+A).

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Fig. 6.
[3H]AA release from
dexamethasone treated HeLa cells. The cells grown in
T-75-cm2 flasks were labeled for 18 h with 1 µCi/ml
[3H]AA in 12 ml of DMEM medium and then treated with
10 7 M dexamethasone for 24 h. After
repeated washing, the cells were then incubated with 10 6
M ionophore A23187 in 12 ml of HBSS (containing 1.3 mM Ca2+) for 30 min, and the supernatants were
extracted by Sep-Pak C18 cartridges and chromatographed by
HPLC as described under "Experimental Procedures." Data were
expressed as AA release measured separately from 11-12 individual
flasks from two separate sets of experiments. HC = HeLa
control cells; HD = HeLa cells treated with
dexamethasone; A = treatment with A23187.
p < 0.001 for HC versus HD;
p < 0.001 for HC+A versus HD+A.
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Antisense Inhibition of p11 Increases AA Release--
We have
shown that dexamethasone increases p11 expression and inhibits
PLA2 activity in vitro. It has been reported
that p11 can bind to cPLA2 and inhibit cPLA2
activity in vitro. In order to study whether dexamethasone
might alter cPLA2 activity in part by increasing p11
expression in human cells, we performed two studies. First, we
constructed a p11 antisense plasmid and then stably transfected HeLa
cells to examine the AA release in these cells. Western blot studies of
cloned transformed cells showed that p11 protein production was
decreased in HeLa cells which were transfected with
ASp11-pcDNA3.1(+) plasmid compared with HeLa cells, which were
transfected with pcDNA3.1(+) plasmid alone (Fig.
7A). There was no change in
cPLA2 expression in these cells (Fig. 7B).
[3H]AA release from the HeLa cells that were permanently
transfected with p11 antisense plasmid was increased both at base line
and after exposure to the calcium ionophore A23187 compared with
control cells (Fig. 8).

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Fig. 7.
The p11 and cPLA2 protein levels
in HeLa cells transfected with a p11 antisense plasmid.
A, cell lysates from cells transfected with the p11
antisense plasmid or cells transfected with a control vector were
processed as described under "Experimental Procedures," and 20 µg
of cell lysate protein was subjected to gel electrophoresis and
immunoblotting for p11 protein. Three different samples of cell lysates
of p11 antisense cells and control cells were processed. B, protein
levels of cPLA2 in HeLa cells transfected with a p11
antisense plasmid. Cell lysates from cells transfected with the p11
antisense plasmid or cells transfected with a control vector were
processed as described under "Experimental Procedures," and 20 µg
of cell lysate protein was subjected to gel electrophoresis and
immunoblotting for cPLA2.
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Fig. 8.
[3H]AA release from HeLa cells
transfected with a p11 antisense plasmid. The cells grown in
T-75-cm2 flasks were labeled for 18 h with 1 µCi/ml
[3H]AA in 12 ml of DMEM with Geneticin. After repeated
washing, some cells were then incubated with 10 6
M ionophore A23187 in 12 ml of HBSS (containing 1.3 mM Ca2+) or with HBSS without A23187 for 30 min, and the supernatants were extracted by Sep-Pak C18
cartridges and chromatographed by HPLC as described under
"Experimental Procedures." Data were expressed as AA release
measured separately from 10 to 12 individual flasks in each group.
VC = vector control cells; ASp11 = HeLa
cells transfected with a plasmid expressing p11 antisense mRNA;
A = HeLa cells treated with A23187. p < 0.001 for VC versus ASp11; p < 0.05 for
VC+A versus ASp11+A.
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Increased p11 Expression Inhibits AA Release--
In order to
determine whether dexamethasone inhibition of cellular PLA2
activity might be related in part to increasing p11 expression in human
epithelial cells, we constructed a p11 expression plasmid and stably
transfected HeLa cells to examine the effect of increased p11
expression on AA release in these cells. The effect of the p11
expression plasmid on cellular p11 protein is demonstrated in Fig.
9A. Western blot results showed that p11 protein production was increased in HeLa cells that were transfected with p11-pcDNA3.1(+) plasmid compared with HeLa cells that were transfected with pcDNA3.1(+) plasmid alone. There was no change in
cPLA2 expression (Fig. 9B). AA release from the
HeLa cells which were permanently transfected with p11 expression
plasmid was decreased both at base line and after exposure to the
calcium ionophore A23187 (Fig. 10).
Therefore, dexamethasone treatment increases p11 protein expression and
reduces cellular arachidonate release. Furthermore, increasing cellular
p11 protein production independent of dexamethasone treatment reduces
cellular AA release as well.

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Fig. 9.
The p11 and cPLA2 protein levels
in HeLa cells transfected with a p11 expression plasmid.
A, cell lysates from cells transfected with the p11
expression vector or cells transfected with control vector were
processed as described under "Experimental Procedures," and 20 µg
of total protein was subjected to gel electrophoresis and
immunoblotting for p11 protein. Two cell lysates from cells transfected
with the p11 expression vector and cells transfected with the control
vector were processed. B, protein levels of
cPLA2 in HeLa cells transfected with a p11 expression
vector or cells transfected with a control vector. Cell lysates from
cells transfected with the p11 expression or cells transfected with a
control vector were processed as described under "Experimental
Procedures," and 20 µg of cell lysate protein was subjected to gel
electrophoresis and immunoblotting for cPLA2.
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Fig. 10.
[3H]AA release from HeLa cells
transfected with a p11 expression plasmid. The cells grown in
T-75-cm2 flasks were labeled for 18 h with 1 µCi/ml
[3H]AA in 12 ml of DMEM with Geneticin. After repeated
washing, the cells were then incubated with or without
10 6 M ionophore A23187 in 12 ml of HBSS
(containing 1.3 mM Ca2+) for 30 min, and the
supernatants were extracted by Sep-Pak C18 cartridges and
chromatographed by HPLC as described under "Experimental
Procedures." Data were expressed as AA release measured separately
from nine individual flasks in each group. VC = HeLa
cells transfected with control vector; p11 = HeLa cells
transfected with a p11 expression vector; A = HeLa
cells treated with A23187. p < 0.001 for VC
versus p11; p < 0.001 for VC+A
versus p11+A.
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DISCUSSION |
p11, or calpactin light chain, is a member of the S-100 family
small calcium binding proteins; however, it has several unique features. S-100 proteins contain two EF hands that function as calcium
binding domains (13). p11 does not have the ability to bind
Ca2+ ions due to amino acid deletions and substitutions in
the two EF hand motifs (14, 15). Instead, p11 is present in a variety of cells separately or as a heterotetramer binding to annexin II. The
heterotetramer is composed of two copies of the 36-kDa heavy chain,
annexin II subunits and two copies of 11-kDa light chain, p11 subunits
as (p36)2(p11)2 (32, 33).
Glucocorticosteroids are potent anti-inflammatory agents. This
anti-inflammatory effect may be produced via a variety of mechanisms. A
group of structurally related, calcium-dependent
phospholipid-binding proteins, annexins, which were formerly known as
lipocortins or calpactins, had been shown to be inducible by
glucocorticoids. Annexin I has been reported to inhibit
sPLA2 activity in vitro (25-30). These
observations led to the hypothesis that the inhibition of
sPLA2 by annexins is the mechanism of the anti-inflammatory action of glucocorticoids. Subsequent studies failed to show a direct
interaction between the 14-kDa PLA2 and annexins. Instead, this inhibition may be dependent on the concentration of substrate (34,
35), the extent of inhibition being more closely related to the
inhibitor:substrate rather than the inhibitor:enzyme ratio. In
addition, glucocorticoid treatment suppresses the induction of Group II
sPLA2 expression in a variety of cells (36-40).
cPLA2 selectively hydrolyzes AA from the
sn-2-ester bond of membrane phospholipids. cPLA2
may play an important role in the production of free fatty acids and
lysophospholipids, precursors of eicosanoids and PAF, all of which may
function as intracellular second messengers or potent inflammatory
mediators (1, 3, 5). It has been reported that dexamethasone treatment
reduces changes in cPLA2 protein and mRNA levels
induced by TNF treatment of HeLa cells (41). Dexamethasone may have
other effects on AA metabolism and at earlier time points, including
effects perhaps not requiring transcription such as inhibition of
phosphorylation of cPLA2 (42). We did not document an
effect of dexamethasone on unstimulated expression of
cPLA2; however, we did note an effect of dexamethasone on
cellular p11 protein and mRNA levels. Because it has been
demonstrated that p11 can directly interact with the carboxyl region of
cPLA2 and inhibit its activity in vitro (31), we
hypothesized that a part of the effect of dexamethasone on cellular AA
release might be mediated by a dexamethasone-induced change in p11
protein levels.
Four lines of evidence suggest that dexamethasone may alter cellular
arachidonate release in part by induction of p11 protein expression.
First, studies in two different cell lines demonstrate that
dexamethasone induces human epithelial cell p11 gene expression and
protein production. This effect was not associated with a reduction of
cPLA2 expression in HeLa cells. RU486, an antagonist that
competes with glucocorticoids for binding to the glucocorticoid receptor (43, 44), blocked the stimulatory effect of dexamethasone on
p11 protein production, suggesting that dexamethasone-induced p11 gene
expression and subsequent protein synthesis occurs via a glucocorticoid
receptor-mediated pathway. Second, in dexamethasone-treated cells,
there was increased p11 binding to cPLA2 as evidenced by p11 which was precipitated by anti-cPLA2 antibody as a
p11·cPLA2 complex. Third, cells stably transfected with a
plasmid that expresses p11 antisense mRNA and that subsequently
express less p11 protein have enhanced release of prelabeled AA both at
base line and after stimulation with the ionophore A23187. Fourth, we
studied the effect of p11 on AA release in the setting of
overexpression of p11 protein in a human epithelial cell line, Hela
cells. The release of prelabeled AA from cells that overexpressed p11
was significantly lower than that from control cells. Therefore,
overexpression of p11 inhibits PLA2 activity and reduces
the release of AA from [3H]AA-prelabeled cells. Thus,
manipulation of p11 levels independent of corticosteroid therapy also
alters AA release from permanently transfected cells.
AA release from cell membranes may be a complex process affected by a
variety of stimuli and involving multiple enzymes and regulatory
proteins. We suggest that one of these effects may be related to
modulation of p11 protein production and binding to
cPLA2.