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1996;157;5231-5239 J. Immunol.
Barth and TB Strom
WY Almawi, MS Saouda, AC Stevens, ML Lipman, CM
antiproliferative effects by lipocortins
Partial mediation of glucocorticoid
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists, Inc. All rights reserved.
Copyright ©1996 by The American Association of
Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc., 9650
is published twice each month byThe Journal of Immunology
on March 10, 2010 www.jimmunol.orgDownloaded from
Partial Mediation of Glucocorticoid Antiproliferative Effects
by
Lipocortins'
Wassim
Y.
AImawi,** Myrna
S.
Saouda,* Anthony C. Stevens,t Mark
1.
Lipman,+
Claudia
M.
Barth,t and Terry
B.
Stromt
The glucocorticoids (GCs) dexamethasone
(DEX)
and prednisolone (PRED), in a concentration-dependent fashion, profoundly
inhibit mitogen-induced proliferation of human peripheral blood mononuclear lymphocytes (PBML). This inhibition was specific
for GCs, as non-GC steroids were devoid of any antiproliferative capacity. GCs enhanced the mRNA (Northern blot) and protein
(Western blot) expression of
the
calcium and phospholipid binding proteins lipocortin
I,
II,
and
V.
As a consequence of
mitogenic stimulation, PBML secrete PGE, and leukotriene B, (LTB,). Antiproliferative concentrations of both
DEX
and PRED
as well as recombinant lipocortin
I
abolished PGE, and
LTB,
production, suggesting an involvement of lipocortins in GC-
mediated antiproliferative effects, possibly by inhibiting eicosanoid production and, consequently, mitogen-induced cellular
proliferation. Whereas
5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid mimicked
DEX
and
PRED
in inhibiting
PGE,
and LTB, production, neither 5,8,11,14-eicosatetraynoic acid nor nordihydroguaiaretic acid had any effect on mitogen-
induced PBML proliferation, indicating that the GC-mediated antiproliferative effect
is
separate from their effects on eicosanoid
release. Furthermore, neutralizing anti-lipocortin
I
and anti-lipocortin
II
mAb, while reversing the inhibitory activity of
DEX
and
PRED on PGE, and LTB, production, only partially reversed DEX- and PRED-mediated antiproliferative effects. This indicates
that the GC-mediated antiproliferative effect
is
not dependent on inhibition of eicosanoid release by lipocortins and suggests the
existence of lipocortin-dependent and lipocortin-independent pathways by which GCs mediate their antiproliferative effects.
The
Journal
of Immunology, 1996, 157: 5231-5239.
G
lucocorticoids (GCS),~ as anti-inflammatory and immu-
nosuppressive agents, are used in treating autoimmune
diseases and graft rejection episodes
(1,
2). However,
despite their wide-spread use, the precise mechanism by which
GCs
mediate their antiproliferative effects remains evasive due in
part to the myriad of biological effects mediated by the GCs.
Among the mechanisms postulated for GC-mediated antiprolifera-
tive effects include blockade of activation-associated increases in
transmembrane ionic fluxes
(3,
4),
alteration in membrane lipid
phospholipid profile (5,
6),
inhibition of cytokine gene expression
(7-9),
andor induction of the calcium and phospholipid binding
proteins, the lipocortins
(10,
11).
It is well documented that GCs induce lipocortin expression at
the mRNA and protein levels. Lipocortins, due to their capacity to
inhibit phospholipase
A,
(PLA,) activity
(12,
13),
block arachi-
donic acid (AA) release from membrane-bound stores, resulting in
*Department of Biochemistry, Faculty
of
Medicine, American University of
Medicine, Beth Israel Hospital and Harvard Medical School, Boston, MA 02215
Beirut, Beirut, Lebanon; and 'Division
of
Clinical Immunology, Department of
ber 5, 1996.
Received for publication November 30, 1995. Accepted for publication Septem-
The costs of publication of this article were defrayed in part by the payment of
page charges. This article
must
therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section
1
734 solely
to
indicate this fact.
'
This work
was
supported by grants from the National Institutes of Health, the
Kidney Foundation
of
Canada, and the American University
of
Beirut-M.P.P.
partment
of
Biochemistry, Faculty
of
Medicine, American University of Beirut,
Address correspondence and reprlnt requests
to
Dr. Wassim
Y.
Almawi, De-
850 Third Ave., New York, NY 10022-6222,
Abbreviations used in this paper: GCs, glucocorticoids; PLA,, phospholipase
A,; LTB,, leukotriene B,;
DEX,
dexamethasone; PRED, prednisolone; PBML, pe-
rlpheral blood mononuclear lymphocytes; ETYA, eicosatetraynoic acid; NDGA,
nordihydroguaiaretic acid; AA, arachldonic acid.
Copyright
0
1996
by
The American Association
of
Immunologists
the inhibition of PC and leukotriene (LT) production
(14,
15). This
prompted the conclusion that GCs exert their effects through
li-
pocortin induction, which, in turn, blocks
AA
release and, conse-
quently,
PC
and LT production
(IO,
121, resulting in the suppres-
sion of select elements of the signal transduction pathway(s)
(I
3).
Other reports challenged this conclusion by presenting data show-
ing that GC-mediated suppression may be a separate event from
classical lipocortin induction, assessed by the inhibition of eico-
sanoid production (15-17). This did not rule out a possible in-
volvement of lipocortins in transducing GC-mediated effects via
an, as yet, undisclosed mechanism
(16,
18).
Previously, we demonstrated that GC-mediated antiproliferative ef-
fects do not involve altering the generation
of
second messenger sys-
tems (rise in intracellular calcium, activation and translocation of
protein kinase C from cytosolic to membrane-bound compart-
ments) that operate as a consequence of cellular activation
(19).
GCs mediate their antiproliferative effects by inhibiting cytokine
expression
as
1) antiproliferative concentrations of dexamethasone
(DEX) and prednisolone (PRED) blocked steady state IL-I
(9),
IL-2
(7),
and IFN-y
(8)
mRNA expression; and 2) the combination
of IL-1,
IL-6,
and IFN-y completely abrogated GC-mediated an-
tiproliferative effects (7). Here we show that lipocortins mediate in
part GC-mediated antiproliferative effects, indicating the existence
of a lipocortin-dependent and independent pathways by which
GCs mediate their effects.
Materials and Methods
Preparation
of
PBML
Venous blood from healthy volunteers was diluted 1/2 in saline, layered on
Hypaque-Ficoll
(S.G.
1.077, Pharmacia Fine Chemicals, Piscataway,
NJ),
and centrifuged for 20 min at 2000
X
g. The
interphase containing
PBML
was washed three times in saline and was resuspended at
IO6
cells/ml
in
0022-1 767/96/$02.00
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5232
PARTIAL MEDIATION
OF
GLUCOCORTICOID EFFECTS
BY
LIPOCORTINS
RPMI 1640 culture medium (M. A. Bioproducts, Bethesda, MD)
supple-
mented with 50 pM @ME (Sigma Chemical Co.,
St.
Louis, MO), 2 mM
L-glutamine (Life Technologies, Grand Island, NY), penicillin-streptomy-
cin (Life Technologies) at 100 IU/ml and
100
pg/ml, respectively, and
10%
(v/v)
human type AB serum (M. A. Bioproducts), referred to hereafter
as
complete medium.
Reagents and Abs
PMA and leupeptin were purchased from Sigma Chemical Co., and PHA
was obtained from Difco Laboratories (Detroit, MI). Stock solutions of
DEX, PRED, AA,
5,8,11,14-eicosatetraynoic
acid (ETYA), nordihydrogui-
aretic acid (NDGA), cholesterol, /3-estradiol, and aldosterone were made in
70% ethanol and stored at -20°C until used (all obtained from Sigma
Chemical Co.). All other steroids used in the study were supplied as oil-
based injections from the Beth Israel Hospital Pharmacy (Boston, MA).
Anti-lipoconin
I
(lA),
11,
and
V
mAb were obtained from Biogen, Inc.
(Cambridge, MA), courtesy of Dr.
J.
Browning. Gold-conjugated goat anti-
mouse Abs were obtained from Bio-Rad (Mississauga, Ontario, Canada),
and mouse IgGl was purchased from Ortho Pharmaceutical Corp.
(Raritan, NJ).
Proliferation assays
For
mitogen-induced proliferation PBML (5
X
IO5
cells/ml) were cultured
in 96-well, flat-bottom microtiter plates (Nunc, Burlington, Ontario, Can-
ada) and were stimulated with PHA (5 pg/ml) and PMA (5 ng/ml) or with
Con A
(10
pg/ml).
The
cells were incubated for 72 h at 37°C in a
5%
CO,
humidified atmosphere.
For
MLR, PBML
(lo6
cells/ml) were cocultured
with mitomycin C-treated (25 pg/ml; Sigma Chemical
Co.)
allogeneic cells
(lo6 celldml) in complete medium containing 50 pM
@ME
(Sigma Chem-
ical Co.) for 5 days at 37°C. [3H]TdR (1 pCi/well; New England Nuclear,
Boston, MA) was added during the last 4 h of the culture period, and
proliferation was determined by measuring the cellular uptake of [3H]TdR
by liquid scintillation.
Determination of
LTB,
and
PGE,
PBMC were cultured in 24-well, flat-bottom plates (Falcon, Lincoln Park,
NJ) at 5
X
lo5 cells/ml in complete medium.
The
cells were pretreated with
the indicated agents for 4 h at 37T, followed by stimulation with PHA plus
PMA. Cellfree supernatants of cultured and treated PBML were assayed
for LTB, and
PGE
by RIA using a commercially available kit from Am-
ersham Cop (Arlington Heights,
IL).
All determinations were performed
in duplicate.
Cellular fractionation
PBML
(lo6
cellslml) were stimulated with PHA (5 pg/ml) and PMA (5
ng/ml), cultured for 24 h at 37°C with
or
without test drugs, washed
twice in HBSS (Life Technologies, Grand Island, NY), and resuspended
at lo7 cells/ml in extraction buffer (20 mM Tris-C1, pH 7.2;
2
mM
EDTA; 50 mM 2-ME; and 100 pg/ml leupeptin). The cells were then
sonicated for 20
s
and centrifuged at
50,000
X
g
for
1
h at 4°C. Cy-
tosolic lipocortin-containing fractions were lyophilized at stored at
-20°C until assayed.
Western blot analysis
Cytosolic lipocortin-containing fractions were subjected to 7.5% SDS-
PAGE according
to
the method of Laemmli (20). Proteins were transferred
to nitrocellulose membranes, and the membranes were incubated with the
primary Ab for 2 h at room temperature. Gold-conjugated goat anti-mouse
mAb was added to washed membranes, and the membranes were incubated
for
18
to 20 h at room temperature.
Northern blot analysis
Total cellular RNA was extracted by the guanidium isothiocyanateLiC1
method under strict RNase-free conditions (21).
RNA
(10 pgfiane) was
electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde (22)
and electrotransferred onto Hybond N+ membranes (Amersham). The
membranes were prehybridized at 42°C for
4
h in a prehybridization
so-
lution containing 50%
(v/v)
deionized formamide,
l
X
Denhardt's solution,
I%
SDS,
1
mM NaCI,
5
mM
Tris-CI (pH 7.4), and
10%
dextran sulfate
(Sigma Chemical Co.). [32P]dCTP-labeled (New England Nuclear) cDNA
probes were then added, and the membranes were hybridized at 42°C for
12 to 18 h. After washing twice in
2X
SSC/O.l%
SDS
at 42°C and twice
Concentration
(M)
FIGURE
1.
Specificity
of
the GC-mediated antiproliferative effect.
The proliferation of PBML stimulated with PHA
(5
pghnl)
and PMA
(5
ng/ml) and treated with culture medium (positive control), ethanol (ve-
hicle control), or the indicated steroid at the indicated concentration.
Proliferation was determined
72
h post-culture initiation; control
values were: background cpm,
781
t
124;
positive control cpm,
92,715
-+
9,877;
and ethanol control cpm,
86,552
2
9,140.
Data
points indicate the percent suppression, calculated
as:(l
-
[(test
cpm
-
background cpm)/(control cpm
-
background cpm)l)
x
100%
in
0.1
X
SSC/O.l% SDS at 75°C for
20
min, and the membranes were
exposed to Kodak x-ray film (Eastman Kodak, Rochester, NY) at -70°C
for 24 to 72 h.
Results
Comparative effects
of
GC
and non-GC steroids on mitogen-
induced
T
cell proliferation
The effect of GC and non-GC steroids on mitogen-induced PBML
proliferation was assessed by adding the steroids (and ethanol), at
lo-'
to 10"o M, to PBML cultures stimulated with PHA
(5
fig/
ml) and PMA
(5
ng/ml; referred to thereafter as PHA-PMA) at
culture initiation; proliferation was determined by measuring the
cellular uptake
of
[3H]TdR
72
h postactivation. The results pre-
sented in Figure
1
show that, of all the steroids tested, only DEX
(EC,,
=
5
X
lo-'
M), betamethasone (EC,,
=
7.5
X
lo-'
M),
hydrocortisone (EC,,
=
5
X
10"
M), and PRED (EC,,
=
5
X
M) inhibited mitogen-induced cellular proliferation (EC,,
=
drug concentration yielding
50%
suppression
of
PHA-PMA re-
sponses). In contrast, the non-GC steroids aldosterone, andros-
terone, cholesterol, diethylstilbesterol, /3-estradiol, nandrolone,
pregnenolone, progesterone, and testosterone were devoid of an-
tiproliferative capacity at all concentrations tested, as the prolifer-
ative responses of mitogen-stimulated PBML cultures treated with
non-GC steroids were not statistically different from those
of
either
positive or ethanol control cultures (data not shown).
Suppression
of
mitogen- and alloantigen-induced
T
cell
proliferation by GCs
The antiproliferative capacity of GCs was further investigated by
adding ethanol, DEX, or PRED, at
10"'
to
IO"
M, to PBML
cultures stimulated with Con
A
(10 pg/ml; Fig.
2.4)
or with mit-
omycin C-treated allogeneic cells (MLR; Fig.
2B);
cellular prolif-
eration was determined
3
days
(Con A) or
5
days (MLR) post-
culture initiation. Both DEX and PRED, in a concentration-
dependent fashion, inhibited Con A-induced and MLR-driven T
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The
Journal
of
Immunology
5233
FIGURE
2.
inhibition by
GCs
of mitogen-
and alloantigen-induced proliferation.
A,
PBML
(5
X
1
O4
cells) were stimulated with
Con
A
(1
o
pg/mI);
6,
PBML
(I
.5
x
1
o5
cells)
were stimulated with mitomycin C-treated
allogeneic cells
(1.5
x
lo5
cells). Both
groups of cells were treated with ethanol
(vehicle control),
DEX,
or PRED. Prolifera-
tion was assessed
3
days
(A)
or
5
days
(B)
postculture initiation. Proliferation values
for
Con
A:
background cpm,
1,123
5
258;
positive control cpm,
108,849
?
11,624;
for
MLR:
background cpm,
51 3
2
126;
positive
control cpm,
74,083
rt
6,466.
Data points
are the mean of eight individually performed
experiments and indicate the percent sup-
pression calculated as follows:
(1
-
[(test
cpm
-
background cpm)/(control cpm
-
background cpm)])
X
100%.
80
-
60
-
40
-
20
-
DEX’
MP’
Concentration
(M)
DEX
MP
..
1
10”O
I
o-~
1
o-8
10”
1
o-6
1
o-~
cell proliferation (Fig.
2,
A
and
B).
Furthermore, DEX was
50-
to
100-fold more potent than PRED in inhibiting both T cell prolif-
erative responses, assessed by comparing
the
EC,, values for both
agents.
GCs
induce lipocortin expression
The effect of GCs on lipocortin
I,
11,
and
V
expression was first
assessed by examining the effect of DEX and PRED on lipocortin
I
and
I1
steady state mRNA expression. Total cellular RNA, ex-
tracted from DEX-treated and mitogen-stimulated cultures, was
subjected to Northern blot analysis using 32P-labeled lipocortin
I
and lipocortin
I1
cDNA probes.
PBML stimulation with PHA-PMA resulted in a reproducible
induction
of
lipocortin
I,
but not lipocortin
11,
mRNA expression.
DEX, in a concentration-dependent fashion, up-regulated lipocor-
tin
I
and lipocortin
I1
mRNA expression, with the maximal re-
Concentration
(M)
sponse seen at to
10”
M (Fig.
3).
Similarly, lipocortin
I
and
I1
mRNA levels were up-regulated in PRED-treated and mitogen-
stimulated PBML cultures (data not shown).
We next assessed whether GC-induced up-regulation in lipocor-
tin
I
and lipocortin
I1
steady state mRNA levels resulted in parallel
increases in lipocortin
I,
lipocortin
11,
and lipocortin
V
protein
secretion. Cytosolic fractions of GC-treated and mitogen-stimu-
lated PBML cultures were subjected to SDS-PAGEmestern blot
analysis, using specific anti-lipocortin
I, 11,
and
V
mAbs. Parallel
to its effect on lipocortin
I
mRNA, stimulation of PBML with
PHA-PMA resulted in the induction of lipocortin
I,
but not lipocor-
tin
I1
or lipocortin
V,
protein secretion (Fig.
4,
A
and
B).
DEX
(Fig.
4A)
and PRED (Fig.
4B)
in a concentration-dependent fash-
ion, enhanced the cytosolic accumulation of lipocortin
I,
li-
pocortin
11,
and lipocortin
V
proteins. Smaller amounts of li-
pocortin
I
and lipocortin
I1
were also detected in the membrane
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5234
28s
-
18S4
LC
I
-
LC
II-
PHA
PARTIAL MEDIATION
OF
GLUCOCORTICOID EFFECTS
BY
LIPOCORTINS
togen-stimulated PBML cultures were treated with the inhibitors
of AA release, ETYA and NDGA, and their supernatants were
assayed for PGE, and LTB,. NDGA and ETYA,
in
a concentra-
tion-dependent manner, inhibited PGE, (Fig. 7A) and LTB, (Fig.
7B) production
in
a manner analogous to DEX, PRED, and
li-
pocortin
I.
In
contrast to DEX, which inhibited mitogen-induced
proliferation at concentrations as low as M, NDGA and
ETYA, at all concentrations tested, failed to inhibit mitogen-in-
duced T cell proliferation (Fig.
8).
To rule out the possibility that the GC-mediated antiproliferative
effect was due to inhibition of AA release, DEX-treated and mi-
togen-stimulated PBML cultures were reconstituted with exoge-
nous AA, and proliferation was determined 72
h
postincubation.
Fw
The results presented
in
Figure 9 demonstrate that, at all concen-
trations tested, AA did not alter DEX-mediated antiproliferative
0
effects (Fig. 9).
Effect of anti-lipocortin /-neutralizing mAb
on
DEX-induced
0
inhibition of eicosanoid production and cellular proliferation
‘0
To determine whether DEX-induced inhibition of eicosanoid pro-
duction and cellular proliferation was mediated via lipocortins, the
effects of neutralizing anti-lipocortin I and anti-lipocortin I1 mAb
on DEX-mediated blockade of PGE, and LTB, production and on
DEX-induced inhibition of mitogen-induced cellular proliferation
vidually and
in
combination, totally abrogated DEX-induced
inhi-
-
+++
++
were assessed. Anti-lipocortin
I
and anti-lipocortin I1 mAb, indi-
DEX
(M)
-
-
10-5 10-6
10-7
10-8
FIGURE
3.
Northern blot analysis
of
total cellular
RNA.
Total cellu-
lar
RNA
(10
&lane) was extracted from
PBML
stimulated with
PHA-
PMA
and treated with
DEX
at
to
lo-’
M.
Top,
Ethidium
bromide
staining
of
total
RNA
electrophoresed on
a
1%
agarose denaturing
gel.
Following Northern transfer, the membranes were probed with 32P-
labeled lipocortin
I
(middle)
and
lipocortin
II
(bottom)
cDNA and ex-
posed to Kodak x-ray
film
for
24
h
at
-70°C.
fractions of GC-treated and mitogen-stimulated cultures (data
not shown).
Inhibition of eicosanoid production and T cell proliferation
by lipocortin
I
and
GCs
We next investigated whether induction
of
lipocortins by PRED
and DEX results
in
inhibition of AA release and, subsequently,
blockade of mitogen-induced cellular proliferation. Mitogen-stim-
ulated cultures were treated with DEX, PRED, and lipocortin I,
and their supernatants were assayed for PGE, and LTB, by RIA.
DEX, PRED, and lipocortin I,
in
a concentration-dependent fash-
ion, inhibited PGE, (Fig.
5A)
and LTB, (Fig.
5B)
production. The
addition of
50
to
100
pM AA reversed DEX-, PRED-, and
li-
pocortin I-induced blockade
of
PGE, production, indicating that
all three agents inhibit PGE, (and LTB,) production by blocking
the release of AA (data not shown).
We then assessed the effect of lipocortin I on mitogen-induced
cellular proliferation by adding lipocortin I and DEX, at to
M, to mitogen-stimulated cultures. Lipocortin I,
in
a concen-
tration-dependent fashion, inhibited mitogen-induced T cell pro-
liferation (Fig. 6). By comparison to lipocortin I, DEX was
>15-
fold more potent
in
inhibiting T cell proliferation, assessed by
comparing the EC,, values for both agents.
Effects of NDGA and ETYA
on
cellular proliferation and
eicosanoid production
To
assess whether GC-induced antiproliferative effects was due
to
blockade of AA release (resulting from lipocortin induction), mi-
bition of PGE, and LTB, production by mitogen-stimulated
PBML cultures (Fig.
10).
The addition of anti-lipocortin I mAb
or
anti-lipocortin I1 mAb
to DEX-treated and PHA-PMA-stimulated cultures resulted
in
par-
tial abrogation
of
DEX-induced inhibition of PBML proliferation,
which did not exceed 45% of the control values (Fig.
11).
Fur-
thermore, the addition of both anti-lipocortin
I
and anti-lipocortin
I1 mAb did not result
in
any additive or synergistic effect, hence
demonstrating that the GC-mediated antiproliferative effect is par-
tially mediated by lipocortins. Taken together, these results sug-
gest that GC-mediated antiproliferative effects follow lipocortin-
dependent and lipocortin-independent pathways via a mechanism
distinct from the inhibition of AA release.
Discussion
The GCs DEX and PRED, in a concentration-dependent fashion,
profoundly inhibit the proliferation
of
human PBML cultures
in-
duced by mitogenic
or
allogeneic stimuli, and up-regulate lipocor-
tin
mRNA and protein expression. In view of the inducibility of
lipocortins by GCs and the mimicry of GC effects by lipocortin
I,
we investigated whether GC-mediated antiproliferative effects are
mediated by lipocortins.
Insofar as
GCs
are known to suppress mitogen and Ag-induced
cellular proliferation, we tested the capacity of non-GC steroids to
inhibit mitogen-induced cellular proliferation. Immunosuppression
was specific for the GCs, as non-GC steroids did not affect mito-
gen-elicited
or
OKT3-stimulated (7, 19) cellular proliferation. This
is
in
contrast to published reports claiming that P-estradiol (23,
24), cholesterol (25), diethylstilbestrol (26), progesterone (27,28),
testosterone (29). and other non-GC steroids
(30,
31) are immu-
nosuppressive. It should be noted that
in
the studies quoted, ste-
roid-mediated immunosuppression was associated with conditions
such as arthritis (23), cancer (26), pregnancy (27, 28), and adult
thymectomy (29). predisposing factors that may have contributed
to the decreased immunity reported. In contrast and similar to
our
finding, non-GC immunosuppression was either lacking (16,32) or
seen only at toxic concentrations (33) in normal human subjects.
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The
Journal
of
Immunology
5235
*&
F?
IRRELEVENT
3'
ANTIBODY
I
!
LIPOCORTIN
I
B
PHA+PMA
-
+
+
+
+
PREDNISOLONE
-
-
+
+
+
CONCENTRATION
(M)
10-7
10-6
10-5
Control Antibody
LIPOCORTIN
I
LIPOCORTIN
I1
LIPOCORTIN
V
FIGURE
4.
Western blot analysis. Western blot analysis
of
cytoplasmic preparations
of
unstimulated
PBML
(UNSTIMULATED),
of
PBML
stim-
ulated with
PHA-PMA
(PHNPMA), and
of
PBML
stimulated with
PHA-PMA
and treated with DEX
(A)
or
with PRED
(B)
at
the
indicated con-
centrations. Membranes were hybridized with irrelevant
lgGl
or
with anti-lipocortin I, It, and
V
Abs, and protein-antibody interactions were
visualized by gold staining.
PHA-PMA stimulation was associated with a reproducible in-
togen-stimulated cultures. DEX and PRED, in a concentration-
duction of lipocortin
I,
but not lipocortin
11,
mRNA expression.
dependent fashion, up-regulated lipocortin
I
and induced lipocortin
Insofar as
GCs
up-regulate lipocortin mRNA expression at the
I1
steady state mRNA expression, with maximal effects seen at
transcriptional and post-transcriptional levels
(13,34-38),
here we
IO-"
M. The decline in mRNA expression seen at concentrations
demonstrate that antiproliferative concentrations of DEX and
higher than
IO"
M
is
most likely due to mRNA degradation, as
PRED up-regulate lipocortin mRNA and protein expression in mi-
previously suggested
(IO,
39).
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5236
PARTIAL MEDIATION
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GLUCOCORTICOID EFFECTS
BY
LIPOCORTINS
PGE
Production
c
C
Q,
-1 1
-10
-9
-8
-7
-6
-5
log
Concentration (M)
LTB
Production
,!':iB,
60
-
40
Lr,
Q,
C
L
v
a"
20
0
-1
1
-1
0
-9
-8 -7
-6
-5
log
Concentration
(M)
FIGURE
5.
Inhibition of eicosanoid production by DEX, PRED, and
lipocortin
I.
PGE,
(A) and
LTB,
(5)
levels were determined by
RIA
in
the culture supernatants of
PBML
stimulated with
PHA-PMA
and
treated with
DEX
(circles), lipocortin
1
(squares), and
PRED
(triangles).
Data points represent the mean of five individually performed exper-
iments,
and
indicate suppression, calculated as:
(1
-
[(test conc.
-
background conc.)/(control conc.
-
background conc.)])
X
100%.
DEX and PRED also enhanced the cytosolic accumulation
of
lipocortin
I,
lipococortin
11,
and lipocortin
V
proteins. Similar to
earlier reports, lipocortins
I
and I1 migrated on SDS-PAGE as two
bands with apparent molecular masses of 37 and 33 kDa, respec-
tively (36,40). In our hands, a third band migrating at 31 kDa was
consistently observed only in DEX-treated cultures; the nature and
significance of this band have yet to be determined.
Lipocortin
I
shares with DEX and PRED the capacity to block
PGE, and LTB, synthesis and to inhibit mitogen-induced cellular
proliferation. In view of the inducibility of lipocortins by GCs (34,
36), and the effect of lipocortins as PLA, inhibitors on blocking
AA release and subsequently PG and LT synthesis (13, 41), we
tested whether the GC-mediated antiproliferative effect is due to
the induction of lipocortin expression, which, in turn, is associated
with inhibition of AA release and metabolism. While the PG and
LT inhibitors NDGA and ETYA (42, 43) share with GCs and
lipocortin
I
the capacity to inhibit PGE, and LTB, production,
both NDGA and ETYA failed to inhibit mitogen-induced cellular
proliferation, indicating that GC- and lipocortin 1-mediated
antiproliferative effects were not the result of inhibition of AA
release and metabolism.
Lipocortin I was reported to mediate several GC effects, includ-
ing superoxide generation by A23 187-stimulated macrophages
-
C
Q,
100
-
80
-
60
-
40
-
20
-
DEX
+
LC1
0
,:.1'1
'
"',';-l'o'
'",'b-o
'
'
,';-E
'
"',
b-7
1
b-6
70-5
Concentration
(M)
FIGURE
6.
Inhibition
by
DEX
and lipocortin
1
of mitogen-induced
PBML
proliferation.
PBML
were stimulated with
PHA-PMA
and
treated with
DEX
(closed triangles)
or
lipocortin
1
(LC
I; open tri-
angles). Proliferation was assessed 3 days after culture initiation;
control cpm values were: background cpm, 404
?
103;
positive
control cpm, 82,679
f
8,629; and ethanol control cpm, 69,844
?
8,379. Data points are the mean of
six
individually performed ex-
periments and indicate the percent suppression, calculated as de-
scribed
in
Fig. 2.
(44), inhibition of IL-I-induced neutrophil migration (43, inhibi-
tion of cellular proliferation (37), and induction of cellular differ-
entiation (36, 46). Here we showed that lipocortin I also inhibits
mitogen-induced and OKT3-stimulated (7, 19) cellular prolifera-
tion. Our results clearly demonstrate that GC-mediated inhibition
of eicosanoid production was the result
of
induction of lipocortins,
as has also been reported in bronchial alveolar lavage cells (47), in
A549 lung adenocarcinoma cells (37, 48), and in differentiated
U-937 cells (34). However, GC-mediated antiproliferative effects
were not exclusively due to enhanced lipocortin expression, since
anti-lipocortin I and anti-lipocortin
II
mAb, while totally abrogating
the GC-mediated inhibition of eicosanoid production, only partially
antagonized GC-mediated antiproliferative effects.
In
essence, the
GC-mediated inhibition of eicosanoid production appears to be
lipocortin dependent (47, 48), while the antiproliferative effects
follow both lipocortin-dependent and lipocortin-independent
pathways (48, 49).
The role of lipocortins in GC-mediated antiproliferative effects
remains to be determined. Lipocortins, which are phosphorylated
on tyrosine, serine, and threonine residues by src-like kinases (50-
52),
may block the action of certain elements in the signaling path-
way that operate as a consequence
of
cellular stimulation, hence
leading to decreased cellular proliferation. In addition to partially
acting via lipocortins, GCs may exert their antiproliferative effects
directly by binding their cytosolic receptor, which, when translo-
cating to the nucleus, binds the promoter region of several cytohne
genes on specific sites collectively referred to as GC response el-
ements (53-55). Binding
of
GC receptor complex to GC response
element DNA sites inhibits cellular proliferation through blockade
of cytokine gene expression at the transcriptional and transcrip-
tional levels in
cis-
or
trans-acting fashions, as previously re-
ported (54, 56).
In conclusion, the demonstration that lipocortins, while mediat-
ing GC effects in inhibiting the release of AA release and eico-
sanoid production, only partially mediate GC-associated antipro-
liferative effects is in line with our earlier thesis (7, 19, 53) that
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Journal
of
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'''1
A
5237
FIGURE
7.
Inhibition of eicosanoid pro-
duction by NDCA and ETYA. RIA of PCE,
(A)
and LTB,
(6)
levels in the culture super-
natants of PBML stimulated with PHA-PMA
and treated with NDGA (closed circles) or
ETYA (open circles). Data points represent
the mean of five individually performed ex-
periments and indicate the percent suppres-
sion, calculated as described in Figure
5.
4-
C
Q,
Q,
2
n
1001
80-
60-
100
80
60
40
20
Concentration
(M)
log
Concentration
(M)
FIGURE
8.
Failure of NDCA and ETYA to inhibit mitogen-induced
proliferation. PBML were stimulated with PHA-PMA and treated with
DEX
(solid squares), ETYA (open squares), or NDCA (open circles).
Data points represent the mean of four individually performed exper-
iments; control cpm values were: background cpm,
621
t
142;
pos-
itive control cpm,
99,552
2
12,241;
and ethanol control cpm,
93,162
t
16,211.
Percent suppression was calculated as described in
Figure
2.
61
H
80
-
60
40
-
20
-
0
~-~;~~~;.b-;~~~o-~o~..ll"~~~~~"'
'
""""
'
"""'
'
-
10.~
10"
Concentration
(MI
FIGURE
9.
Failure of
AA
to abrogate the GC-mediated antiproliferative
effect. PBML were treated with ethanol (open triangle) or
M
DEX
(closed triangle), reconstituted with AA at the indicated concentrations,
stimulated with PHA-PMA, and cultured for
72
h at
37T.
Data points
represent the mean of six individually performed experiments; control
values were: background cpm,
928
?
180;
positive control cpm,
80,774
2
13,473;
and ethanol control cpm,
84,188
?
12,729.
The per-
cent response was calculated according to: [(test cpm
-
background
cpm)/(control cpm
-
background cpm)l
X
100%.
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PARTIAL MEDIATION
OF
GLUCOCORTICOID EFFECTS
BY
LIPOCORTINS
5238
100
-
8
(0
c
0
g
60
-
Q)
80
-
a
Q)
40
2
c
C
a
20
-
0
PGE
LTB4
FIGURE
10.
Abrogation of DEX-induced suppression
of
eicosanoid
production
by
anti-lipocortin
I
Ab. PGE, (left) and LTB, (right) levels
were determined by RIA in the culture supernatants of PBML stimu-
lated with PHA-PMA and treated with
DEX
at
10”
M
(DEX), DEX
plus
control lgGl
(IgC
Control),
DEX
plus anti-lipocortin
I
Ab
(1
pLg/ml;
Anti-LC
I),
DEX
plus anti-lipocortin
II
Ab (Anti-LC
II),
or
DEX
plus
anti-lipocortin
I
and anti-lipocortin
II
Abs (Anti-LC
1/11),
Data points
represent the mean
of
five individually performed experiments and
indicate the percent response, calculated according to: [(test conc.
-
background conc.)/(control conc.
-
background conc.)]
X
100%.
GCs exert their antiproliferative effects principally by directly tar-
geting cytokine genes. The role
of
lipocortins in partially mediat-
ing the effects
of
GC remains to be determined.
Acknowledgments
The authors thank Dr. Barbara
P.
Wallner and Dr. Jeoffrey Browning at
Biogen (Cambridge, MA)
for
providing lipocortin
1
and
2
cDNA probes
and anti-lipocortin
I,
11,
and V mAb. The expert technical assistance
of
Edward T. Hadro, Joumana W. Assi, and Dagmara
M.
Chudzik is greatly
appreciated.
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