iNOS protein in the cytosol was identified by Western blot using affinity-pu- rifled anti-iNOS antibody. As shown in Figure. 4, iNOS. 80. 728. Journal of Leukocyte.
Activation of inducible nitric oxide synthase gene in murine macrophages requires protein phosphatases 1 and 2A activities Zhongyun
Dong,
Department
ofCell
Abstract: The tify phosphatase
purpose activities
of nitric oxide lipopolysaccharide
in
(LPP), vitro
and
mouse
treatment
Xiulan
Yang,
Biology,
The
of these required
murine (LPS),
University
Xie, Shin-Hun
of Texas
M.D.
macrophages synthetic
exposed lipopeptide (IFN-’y).
macrophages
with
IFN-y
FP2B,
in
selected inhibitor-i
LPS
Norma
Cancer
Center,
identified, all been categorized
to
The and
Juang,
Anderson
been have
studies was to idenfor the production
interferon-ganuna of
Keping
and
tion
Llansa, Houston,
according
major
to their
“hallmark” substrates, and inhibitor-2, and
[ii,
12].
protein
FF1
and
phosphatase
J. Fidler
Texas
serine/threonine into four
FF2C)
and Isaiah
phosphatases (FF1, PP2A,
in vitro
specificity
for
sensitivity to thermostable cation requirement for ac-
PF2A,
the
(FPase)
protein classes
predominant
present
in
types
eukaryotic
of
cells,
or IFN-yand LPP resulted in production ofNO, which was inhibited by addition of the specific phosphatase 1 and 2A (PP1/2A) inhibitors okadaic acid (OA), calyculin A, and cantharidin (but not the nonactive analogues okadaic acid tetraacetate and 1 ,4-dimethylendothall). OA suppressed the accumulation of steady-state inducible NO synthase (iNOS) mRNA and
proliferation and differentiation [1 1, 12]. Activation of macrophages to produce a variety of biologically active molecules or to lyse tumor cells has been shown
iNOS
nase
protein (without alteration The cytosol and nuclei of control tamed large amounts of PP1/2A inhibited by OA in a dose-dependent together, are sion
these
protein
indicate
that
involved in the regulation in murine macrophages.
of
PPi/2A
activities
iNOS gene expresLeukoc. Biol. 58:
J.
1995.
725-732; Key
data
of their stability). macrophages conactivities that were manner. Taken
Words:
play
a key
.
inducible
nitric
oxide
synthase
phosphatases
to require pendent
tumoricidal
and
microbicidal
activities,
isoforms
the
gamma
(IFN-y)
charide
(LFS)
Treatment phosphorylation
of inducible
nitric
oxide
synthases
(iNOS).
NOS of endothelial or neuronal cells, iNOS is of Ca2+ and calmodulin and is expressed only
cells
of signals
are
exposed
to cytokines
or bacterial
products
such such
as interferonas lipopolysac-
[5-10]. of cells
kinase kinases
C (PKC), [13-15],
with
IFN-y
and
LPS triggers
resulting
in
lead
to
ine/threonine could
calcium-calmodulin-deand protein tyrosine
mitogen-activated
(serine/threonine protein existence of “cross-talk” play
an
[20,
21]
active
of PFi/2A (IL-i)
(MAP)
activities tyrosine
[16-19]. kinases and
suggests
role
that
in this
activities [22], tumor
ki-
protein kinases) between
kinases
also
inhibition interleukin-i
kinase The ser-
phosphatases
process.
Indeed,
the
has been shown to induce necrosis factor (TNF) [23],
in human monocytes of kinase and
and macrophosphatase
activities expression
in the induction of monokines suggests that the of the iNOS gene in macrophages may also be
regulated
by PF1/2A. we incubated
To
determine
munne
Abbreviations:
reversible
conformational
changes that are responsible for signaling [ii, 12]. Protein phosphorylation is controlled by the activity of both kinases and phosphatases, which catalyze the interconversion process [ii, 12]. Although numerous different kinases have
CaA,
whether
macrophages
calyculin
Eagle’s
minimal
essential
Hanks’
balanced
salt
mouse
A; DME, medium;
solution;
it is,
with
in this
IFN-y,
absence [okadaic
LPS,
or presence acid (OA),
LPS,
1,4-dimethylendothall;
FBS,
IL-i,
iNOS,
interferon-gamma;
lipopeptide;
fetal
interleukin
inducible
lipopolysaccharide;
EMEM,
bovine
serum;
1; IFN-’y, nitric
MAP,
oxide
acid;
OA(OAc)4
lamide
gel
protein
phosphatase
electrophoresis;
exudate
kinase; tumor
SDS,
sodium
necrosis
factor.
requests:
University
Boulevard,
2A;
synthase;
protein;
dodecyl Isaiah
Houston, April
TX 25,
sulfate; J. Fidler,
M.D.
bromide;
tetraacetate;
protein
protein
Anderson
LPP,
mitogen-activated PAGE,
phosphate-buffered
PPase,
PKC,
of Texas
Received
acid
PBS,
1 and
macrophages;
Reprinf The
,okadaic
HBSS,
recombinant
MIT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium okadaic
neal
of proteins,
that
as well
as the regulation of some immune responses, can be mediated by macrophage production and release of nitric oxide [i-4]. The formation of NO from the terminal guanidino nitrogen of L-arginine and molecular oxygen is catalyzed by
after
transduction
or synthetic lipopeptide (LPP) in the of three specific PF1/2A inhibitors
INTRODUCTION
Unlike the independent
protein protein
and
study
several
in the
and prostaglandin E2 [24] phages. The interrelationship
macrophages
Macrophage
role
cell
saline;
phosphatase;
kinase TCA,
C; P1K,
Department
of Cell
Cancer
Center,
PP1/2A,
PEM, protein
trichloroacetic
OA,
polyacryperitotyrosine acid;
Biology 1515
TNF, (173),
Holcombe
77030.
1995;
revised
August
3, 1995;
accepted
August
4,
1995.
Journal
of
Leukocyte
Biology
Volume
58,
December
1995
725
calyculin between
A (CaA), the level
and cantharidin]. of FPi/2A activities
A direct correlation and expression
of
iNOS at the mRNA and protein levels, as well as production of NO, is shown, suggesting that FF1/2A activities are required to induce iNOS gene expression.
In vitro TNF
activation
Purified
cultures
CMEM
containing
incubation
vested
AND
METHODS
Eagle’s tion
minimal and
Bioproducts tivity, San
essential
(HBSS),
bovine
(Walkersville,
x
5.2
106
Francisco,
(Grand
Island,
(DME)
were
acid
medium
fetal U/mg
Mouse
protein)
was CaA
were
NY).
OA,
cantharidin,
Sodium
purchased
tetraacetate,
from
OA
(OAc)4,
salt
solu-
from
M.A.
against
recombinant
IFN-y
(specific
ac-
a generous
CA). OA and
against
l-actin
Laboratories
was
purchased
from
(Costa
in rabbits
to amino
acids
from
a generous
College, LPS
were
lysate
free
assay
of Cape
York,
of NY)
[25].
of endotoxin
(sensitivity
Cod
kit
All
reagents
used
determined
by
ng/ml)
antibody
iNOS
was
(Cornell
was
purchased
in tissue
Limulus
murine
tures
in
transformed
EMEM
nonessential
supplemented
were
with
amino acids,
vitamin
L-glutamine,
solution,
and
5%
The
1 mM U/ml was
were
tm
nitrocellulose
1%
OVA
tg/ml
( Molecular
Specific pathogen-free female C57B116 Jackson Laboratory, Bar Harbor, ME. weeks of age. Animals were maintained American
Association
in accordance Department ices,
for
with
and of Health.
mice in
purchased were
and
when
by
Animal of the
of Health
and
and
cultivation
and
RNA
isolation
Human
Total
exudate
Serv-
RNA
macro-
phages Peritoneal lavage
exudate from
collate
broth
given
before
free
phosphate-buffered
wells into
harvest
and
1
were macrophage
and
Journal
PEMs
washed
(PBS) ml
as described
of
and
resuspended were
was
criteria.
Leukocyte
These
(CMEM)
with
of MEM
EMEM
by
of 2.0
peritoneal
plates.
>98%
and in
plated
PEMs
nonadherent pure,
cultures containing
according were
Mg-
then
macrophage
58,
December
calculated
mM
transferred
con-
Tween
using
20
with
0.45-
3%
pH
BSA,
7.5,
in TBS),
of goat
150 (1
incubated
anti-rabbit
ECL
protein
anti-
Western
blot-
expression
was
a Personal
Densitometer
ImageQuant ratio
(15-20 onto
antibodies
by an
as the
pro-
dithiothreibol
anti-l-actin
on
leupeptin, in sample
Ti-is-HC1,
iNOS
Triton 2 mM
proteins
blocked
mM
film
tM
and
or
The
20
The
visualized
CA)
software.
of the
peak
Each
areas
of the
protein.
Northern
using
the
blot
ultraviolet
analyses
me
plated
was
For 1%
with
probes
mM
used
were
cDNA
quantitated
as
90
mm,
calculated
as
cells.
The
GAPDH
transcript.
to
mor-
given
5%
In vitro
the
3 mM
PP1/2A
sodium
washed citrate,
[27]. of
The above.
the
areas
Each for
pH
fragment iNOS sample
the
iNOS
MA),
cDNA
probes
two or three 7.2,
times
0.1%
SDS.
corresponding
(GAPDH)
steady-state
elec-
Boston,
a UV Stratalinker
using
were
PstI cDNA
a 1.3-kb
described ratio
Hybridizations
of total
gels, Pont,
of to the
10 jtg
using
dehydrogenase
fragment
cultures
according
analyses, (Du
pJ/cm2
Filters
glyceraldehyde-3-phosphate
iNOS
blot
membrane
CA).
NaCl,
treated kit
formaldehyde-agarose
120,000
[27].
and
isolation
Northern
nylon
Jolla,
control
RNA
denaturing
Screen
La 30
from
reagent
as described
at 50-60#{176}C with to rat
TRI
on
(Stratagene,
performed DNA
extracted
instructions. fractioned
cross-linked
and
mRNA
a mur-
expression
measurement transcript
was and
the
assay
activators The
Volume
PBS
X-100-soluble 100
F(ab’)2 and
Sunnyvale,
was
and
The
below.
Biology
20
(0.1%
of the
RNA
to Gene
38-mm2
also
After
with
boiled. were
anti-iNOS
-actin
trotransferred
were 4
serum-free into
were
cells/dish.
to remove
MD)
Ca2t
was
1,800
ml of thiogly-
Cockeysville,
of 2 x i0
monolayer medium
collected
washed
III
density with
phagocytic
were
Microtest
at the
FBS-supplemented
were injection
Laboratories,
in 0.2
flat-bottom
phologic
726
cells
dishes
reagents
The saline
i0
resultant
or other
(PEMs)
intraperitoneal Biological
[8].
x
of 96-well 100-mm
the cultures
an
(Baltimore
days
EMEM,
macrophages
mice
at was
to 1 mg/ml
SDS,
and
(TBS;
range
and
cellular
macrophages,
of peritoneal
washed
diluted
filters
(Amersham).
the
absorbance
concentration
States
manufacturer’s
Collection
The
in TTBS
was
re-
dihydrochlo-
The
(PMSF),
SDS-PAGE
rabbit
linear
and
50-tl
of Griess
the
Care
United
protein
mm. Nitrite
Triton
2.3%
blue]
in TTBS
measurement
iNOS
6.8,
7.5%
C)
Dynamics,
sample
the 8-12
approved
standards
Department
from
used
facilities
of Laboratory
regulations
Institutes
were
The
Accreditation
current
of Agriculture
National
mice
with
in the
10
later, and
saline
system
determined
Briefly,
diamine
fluoride
peroxidase-conjugated
detection
[26]. volume
were
minutes pH
on
(Amersham)
quantitated
Mice
human
Neither LPS activity of TNF
was
al.
equal
of macrophages
membranes.
horseradish
antibody ting
with
as a standard.
bromophenol
immunoglobulin
with
free
in target
scraped into ice-cold lysis buffer A (1% pH 8.0, 137 mM NaCl, 10% glycerol,
Tris-HCI,
probed
an for
centrifugation
resolved
et
reader.
nitrite
in Tris-buffered
mM NaCl),
with
sulfonyl
by
mM
were
[A
formula TNF-a.
plotted
naphthylethylene
Twenty
separated 0.005%
pyruvate,
har-
cpm
supernatants
temperature
phenylmethyl
aprotinin).
and
cells
cell
and
or recombinant
Ding
microplate
cultures
[62.5
Mycoplasma.
from
or treated
5 mM EDTA and 20 mM Tris-HC1,
(DTT),
sodium
FBS.
by the
B
and
curves
culture
mixed
at room
blotting
buffer
cul-
cells
by
0.1%
Western
Associates
as monolayer
and
sodium
amebocyte
from
maintained
H3P04)
was
tg/lane)
fibroblasts
harvested
by using
tein
filtered L929
washed
as pg/105 macrophages. increase the cytotoxic
in the
determined
except
calculated
standard
described
sulfanilamide,
2.5% nm
EDTA,
Cell line L929
(1%
of nitrite assay
with
0.15
was target
a the
cells.
monitored
Medical
Hole, MA).
(Woods
were
X-100,
cDNA
culture
purchased
samples
taming
from
iNOS
microplate
Control
(DLKEEK-
University the
ICN
corresponding
Full-length
Nathan
of 0.125
of
reagent) OH).
Carl as
limit
from
peptide
cDNA
(St.
Amersham
purchased
anti-iNOS
(TRI
Co.
from
were
supernatants from
and expressed nor did they
concentration
540
macrophage
calculated
L929
a
ride,
rabbit
Chemical
was
the
(Cincinnati,
Dr.
Sigma
and
in control
collected,
[3H]TdR-labeled
cultures
For
After
analysis
agent
3-(4,5-di-
(MU),
a synthetic
from
isolation
gift
New
with
Center
Research
LPS,
purchased
Affinity-purified
deduced
17-31
RNA
Molecular
CA).
by immunization
DINNNVKKT). was
Mesa,
Calbiochem-Novabio-
bromide
purchased
Okadaic
cpm
with
The
h with
or LPP. alone.
were
onto
[19].
LPS,
in CMEM
supernatants added
of
at 37#{176}C for 8-10
of IFN-y,
cytotoxicity
A
was
the
The by
MA).
and
TNF-cz was toxic,
Nitrite
(South
Technologies
1,4-dimethylendothall
Salmonella
MO). [a-32P]ATP (3000 Ci/mmol) was (Arlington Heights, IL). [3H]TdR (2 Ci/mmol) Biomedicals
Life
(Woburn,
Louis,
raised
of Genentech
from and
LC
were
balanced
gift
purchased
chem (San Diego, CA). Phenol-extracted methylthiazol-2-yl)-2,5-diphenyltetrazolium antibody
incubated activity
purchased
Hanks’
culture
Percent
cells
were
(FBS)
the
production
incubated
macrophages
filter,
h later. where
were
incubated
cells/wells)
100
and
concentrations
(10
TNF
recombinant nor LPP
(EMEM), serum
MD).
72
x
- B]/A
Reagents
we
a 0.2-jim
monolayers
PEMs
different
period,
through
MATERIALS
of mouse
control,
negative
of macrophages
1995
extraction
and
assay
for
PP1/2A
were
performed
as
previously
de-
scribed
[28].
Control
and
mm
lysed
for
250
mM
sucrose,
25%
mM
-mercaptocthanol,
MgCl2,
30
tg/ml
5
treated
and
leupeptin,
tions
were
washed
1%
twice
and
2.5
mM
at
to the
manufacturer’s
of
MgC12,
PPases
to
inhibitors
OA
for
phosphorylase
Cell
or
The in
and
extracts
Life
Technologies)
EDTA.
the
1 mM
frac-
7.0,
10
PEMs
were in
z
plated
at a density
medium or
determined
DME
by the
The
or
for MTT
cultures
were
medium
according activities
To evaluate
the
sensitivity
incubated
24
h. Cytotoxic
assay
measuring
washed
and
cells/38
mm2
containing
OA,
effects the
with
wells
0
lysed
in
at 0.42
mg/ml,
dimethyl
metabolically reader
at
the
sulfoxide.
viable
cells
nm
(Dynatech,
570
calculated
using
the
A is the absorbance
incubated
medium
of 96-well
inhibitors
50
were
activity
The [29]
conversion
was
monitored
Chantilly,
formula
VA).
cells
and
40
MTI’
to
by
a 96-well
Percent
con-
B is the absorbance
and
cells
were
formazan
30
N
z
was
100,
where
of the
control
X
were
Student’s
analyzed
for
their
statistical
-h--.
IFN-y/LPS
10 0 10
20
30
OA(OAc)4
results
two-tailed
IFN-’y LPS
20
analysis
experimental
by the
-0--
microplate
0
Statistical
MED
-0--
0
by
cytotoxicity
[1 - (A/B)]
-a--
of cells
the
of
(%)
cytotoxicity
of treated
removed,
40
CA,
for 2 h in medium
was
30
f}-+-----I
B
cells.
The
10
OA (nM)
,
MTT
20
0
the
OA(OAc)4,
of the
IFN-y/LPP
10
substrate
metabolic
:
determined
of [y-32P]ATP-labeled
of 1 x 10
(control)
IFN-y/LPS
20
60
cantharidin,
[29].
-
0
A.
plates
-o--LPP .-.*--
30
viability
taming
-
-
40
25%
PP2C
were
-u--IFN-y
was
immediately
was
-a--MED
A
50
10
and
extracts
addition
mM
PMSF,
were
the
2.5
fraction pH
60
7.0,
nuclear
Tris-HCI,
samples
PBS
pH
aprotinin,
nuclear
of PP2B
of 10 mM the
10 tgJml
mM
Suppression
before
EDTA,
[-mercaptoethanol,
OA(OAc)4,
mm
15
(20
activity
addition
mM
The
with
Tris-HCI,
cytoplasmic
(GIBCO-BRL
protocol.
by the
PMSF,
leupeptin).
system
2
at 1000g.
mM
washed
mM
KCI,
The
in buffer
PP1/2A
assay
achieved
X-100).
30
(20
mM
1 mM
10 tg/ml
-70#{176}C.The
a PPase
20
resuspended and
using was
glycerol,
were
buffer
by centrifugation
aprotinin,
frozen
macrophages
ml of lysis
Triton
separated
glycerol, j.tg/ml
in 0.5
significance
40
(aM)
60
1-test. 50
40
RESULTS
30
Effects
of PP1/2A
Inhibitors
IFN-’y/LPP-induced In the
first
set
FP1/2A-specific ridin
[31,
NO and
TNF
of experiments, could
OA
affect
[30],
iNOS
CaA
10 U/ml
IFN-yplus
0.1
0
z
whether [30],
activity
jigjml
LFS,
and
the
CMEM
alone
? LPS, tion
(Fig.
or IFN-y oxidation or CMEM
and LPP products containing
or LFF) was ineffective 1). OA (Fig. iA),
(Fig. 2A) inhibitors
did not blocked
macrophages
treated
produced of NO
CaA
0
were incubated containing 10 O.i j.tg/ml LPP or 10 U/ml
with
IFN-?
35 (Figs.
a single in generating (Fig. 1C),
induce production the induction of and
IFN-y
stimulus
0.5
1 CaA
Fig. 1 . Inhibition of 96-well plate) 10
absence or CaA seven
ofNO were
U/ml
(C). The
production incubated
IFN-y,
or presence
as described
0.1
of different
amount
and
2.5
2
by OA or CaA. Macrophages for 24 h with medium ig.ml
LPS,
and/or
concentrations
of N02
in Materials
independent
1.5 (aM)
in the culture Methods.
experiments.
*P
