Activation of inducible nitric oxide synthase gene ... - Semantic Scholar

1 downloads 0 Views 2MB Size Report
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