Increased levels of c-fos and c-myc mRNA in ATP-stimulated endothelial cells

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 172, No. 1,1990

Pages 306-312

O~ober 15,1990

INCREASED

LEVELS

OF C-FOS AND C-MYC m R N A

IN A T P - S T I M U L A T E D

ENDOTHELIAL

O. B o u t h e r i n - F a l s o n ,

S. Reuse,

and J.M.

Boeynaems

Institute

J.E.

of I n t e r d i s c i p l i n a r y

Universit6 Campus

Dumont

Research,

Libre de Brussels,

Erasme,

B-1070

CELLS

808 route de Lennik

Brussels,

Belgium

Received August 27, 1990 In b o v i n e a o r t i c e n d o t h e l i a l cells, A T P i n d u c e d a t r a n s i e n t a n d s e q u e n t i a l a c c u m u l a t i o n of c - f o s a n d c - m y c mRNA, w h i c h w a s d e t e c t e d a f t e r 1 h o u r a n d 3 hours, r e s p e c t i v e l y . The e f f e c t of A T P o n c - f o s m R N A w a s s t r o n g e r t h a n t h a t of T N F a n d bFGF. B o t h A T P a n d b F G F i n c r e a s e d c-myc m R N A a f t e r a 3 h o u r t r e a t m e n t , w h e r e a s TNF d i d not. If n o n e of the 3 a g o n i s t s t e s t e d i n d u c e d a s e l e c t i v e e x p r e s s i o n of c-fos or c-myc, e a c h of t h e m w a s a s s o c i a t e d w i t h a d i f f e r e n t q u a n t i t a t i v e c o m b i n a t i o n of t h e 2 s i g n a l s , w h i c h m i g h t be r e l a t e d to the d i s t i n c t r e s p o n s e s t h a t t h e y t r i g g e r in e n d o t h e l i a l cells. ©199UAcad. . . . P..... Inc.

ATP

induces

cells:

a rapid

potent

inhibitor

release muscle These

rich

In e n d o t h e l i a l

coupled

in the

cells,

bisphosphate

(3).

platelets

by P2y-receptors,

sustained

between

smooth

(i, 2). are

P2y receptors

hydrolyzes

The effect

likely

platelets,

a n d the b l o o d v e s s e l

as e l s e w h e r e , C which

(PGI2) , a

the v a s c u l a r

interaction

nucleotides,

to a p h o s p h o l i p a s e

inositol

and a more

relaxes

PGI 2 to i n h i b i t

are m e d i a t e d

role

of a d e n i n e

which

endothelial

of p r o s t a c y c l i n

aggregation,

(NO),

with

which

an important

source

(2).

oxide

in v a s c u l a r

release

of p l a t e l e t

synergizes

responses,

to p l a y

responses

and transient

of n i t r i c and

acute

wall are

phosphatidyl-

of A T P on PGI 2 and NO

ABBREVIATIONS BAEC

: bovine

growth tumor

factor; necrosis

aortic bFGF

endothelial : basic

cells;

fibroblast

factor.

0006-291X/9O $I.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any/brm reserved.

306

aFGF

: acidic

growth

factor;

fibroblast TNF

:

a

Vol. 172, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

r e l e a s e is m e d i a t e d by a rapid rise of cytosolic Ca 2+ (4, 5).

So

far little is known about the late consequences of the i n t e r a c t i o n b e t w e e n ATP and its receptors on endothelial cells. Several b i o c h e m i c a l responses c l a s s i c a l l y associated with the action of mitogens have been detected in A T P - s t i m u l a t e d endothelial cells:

a c t i v a t i o n of Na+/H + antiport

28 kD heat shock proteins choline m e t a b o l i s m

(6), p h o s p h o r y l a t i o n of

(7, 8) and stimulation of p h o s p h a t i d y l -

(9, 10).

It was shown recently that,

in

aortic e n d o t h e l i a l cells, ATP induces a mitogenic response, m e a s u r e d by an increase in the number of nuclei

(ii).

[3H] t h y m i d i n e - l a b e l e d

In order to understand the link between the acute

responses of e n d o t h e l i a l cells to ATP and their late mitogenic response,

we have studied the effect of ATP on the e x p r e s s i o n of

c-fos and c-myc protooncogenes. MATERIALS

AND

METHODS

Cell c u l t u r e Bovine aortic e n d o t h e l i a l cells (BAEC) were obtained by collagenase d i g e s t i o n of a bovine aorta, as p r e v i o u s l y described (3). The cells were seeded on 100 mm Petri dishes and incubated at 37°C in a 5% CO 2 h u m i d i f i e d incubator, in the following medium: M E M - D - v a l i n e (80% v/v), fetal calf serum (FCS: 20% v/v), 2 m M glutamine, 100 U/ml penicillin, i00 ~g/ml streptomycin, 2.5 ~g/ml a m p h o t e r i c i n B. M E M - D - v a l i n e was used to prevent the p r o l i f e r a tion of any c o n t a m i n a t i n g smooth muscle cells. The m e d i u m was c h a n g e d the following day and renewed twice a week. After 4 or 5 days, the p r i m a r y cultures formed confluent monolayers and could be subcultured. The cells were detached by a 5 min incubation in a Ca ++ - and Mg + - free Hanks buffer containing trypsin (10 mg/ml) and E D T A (i mM). Thereafter, the cells were m a i n t a i n e d and s u b c u l t u r e d in the following medium: DMEM (60% v/v), Ham's F12 (20%. v/v), FCS (20% v/v), with the same concentrations of penicillin, s t r e p t o m y c i n and amphotericin B, as m e n t i o n e d above. ~@ii s t i m u l a t i o n BAEC (3rd - 5th passage) werw seeded on 35 mm Petri dishes, at a d e n s i t y of ± 15.000 c e l l s / c m z, in culture m e d i u m c o n t a i n i n g 2.5% FCS. A f t e r 4 hours, this m e d i u m was removed and replaced by a s e r u m - f r e e medium. 24 hours later, ATP, Tumor Necrosis Factor-a (TNF) and basic Fibroblast Growth Factor (bFGF) were added and the i n c u b a t i o n was c o n t i n u e d for 30 min up to 6 hours. In most experiments, c y c l o h e x i m i d e (i0 ~g/ml) was added for the last hour of the incubation. ~A

purification

At the time of harvest, the cells were rapidly scraped from the dish in 4M g u a n i d i n i u m m o n o t h y o c y a n a t e (1.9 ml for three 100 m m dishes) (12). A f t e r the addition of 840 mg CsCI 2 to the homogenate, each sample was layered on top of a 1.2 m l - c u s h i o n of 5.7 M CsCI 2 in 0.i M EDTA. S e p a r a t i o n of total RNA was p e r f o r m e d by c e n t r i f u g a t i o n at 15°C for 17 h at 36,000 rpm in a Beckman SW56 307

Vol. 172, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

rotor. The R N A was p r e c i p i t a t e d in 2 M LiCI and then treated w i t h p r o t e i n a s e K (60~g/ml) in NaCL 0.3 M-SDS 0.1%. After classical phenol c h l o r o f o r m extractions, the amount of total RNA was d e t e r m i n e d spectrophotometrically. N o r t h e r n blot analysis A f t e r d e n a t u r a t i o n using glyoxal according to the procedure of M a c M a s t e r and Carmichael (13) equal aliquots (4 to i0 ~g per lane) were f r a c t i o n a t e d by electrophoresis on a 1% agarose gel in 10 m M p h o s p h a t e pH 7.0. A c r i d i n e staining of independent lanes r e v e a l e d that the amounts of RNA were equal in all samples. As size markers, phage lambda cleaved by EcoRI and HindIII, was t r e a t e d similarly. G l y o x y l a t e d RNA were t r a n s f e r r e d by d i f f u s i o n b l o t t i n g to a nylon membrane (Pall Biodyne A) using 20 x SSC (i x SSC = 0.15 M NaCl/0.015 M Na Citrate) as d e s c r i b e d (14). After baking, the blots were p r e h y b r i d i z e d overnight at 42°C in a b u f f e r c o n s i s t i n g of 50% formamide (v/v), 5 x Denhardt solution (100 x solution: 2% Ficoll, 2% polyvinylpyrrolidone), 5 x SSPE (20 x solution: 3.6 M NaCl, 0.2 M Na phosphate pH 8.3, 20 mM EDTA), 0.3% SDS, 250 ~g/ml denaturated salmon testes DNA, 200 ~g/ml BSA. E s s e n t i a l l y the third exon of the human c-myc gene (1.398 bp Clal fragment of PKH47 human c-myc obtained from Dr Saule), and v-fos (1o400 bp Bgl II f r a g m e n ~ o f pFBJ-neo o b t a i n e d from Dr R. M u l l e r EMBL) D N A probes were [a~ZP] labeled by r a n d o m p r i m i n g e x t e n s i o n to a specific activity of a p p r o x i m a t e l y 2 x 108 c p m / ~ g (15). H y b r i d i z a t i o n s were carried out for 48 h at 42°C in the same b u f f e r as for prehybridization, but c o n t a i n i n g in addition 10% sulfate (W/V) and the heat denatured probe. Filters were w a s h e d in 2 x SSC - 0.1% SDS at room temperature, four times ten minutes, and then in 0.1 x SSC - 0.1% SDS at 60-65°C, four times t h i r t y minutes. Filters were a u t o r a d i o g r a p h e d at -70°C using Kodak XAR-5 films (Eastman Kodak, R o c h e s t e r N.Y.) in the presence of i n t e n s i f y i n g screens. The same filter was probed s u c c e s s i v e l y with c-myc and v-fos. Probe was stripped from the paper by incubating for 1 h at 65°C in 50% formamide, 10 m M Na phosphate pH 6.5, and then by w a s h i n g for 15 minutes at room temperature in 2 x SSC, 0.1% SDS. As this study was not carried out with a cell line, the levels of i n d u c t i o n of m R N A was different from one culture to another, but the e x p e r i m e n t s have been repeated 2 or 3 times with similar data being obtained.

Materials C o l l a g e n a s e type IA was p u r c h a s e d from Cooper Biochemical (Worthington Biochemical Corporation). DMEM, Ham's FI2, MEM-DVal, FCS, penicillin, streptomycin, a m p h o t e r i c i n B and glutamine were from Gibco laboratories. Trypsin was o b t a i n e d from Flow Laboratories. ATP and c y c l o h e x i m i d e were obtained from Sigma Chem. Co. R e c o m b i n a n t human TNF was a gift of Dr W. Fiers. R e c o m b i n a n t bovine bFGF was purchased from Amersham.

RESULTS

ATP i n d u c e d the e x p r e s s i o n of c-fos m R N A in BAEC

(Fig.

1 and 2).

A l t h o u g h this effect could be detected in the absence of cyclo-

308

Vol. 172, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

o ~ ~

m

A

..;

,J

o

:,1

~

~

~

Figure 1

Effects of ATP, TNF and bFGF on the levels of c-fos and c-myc mRNA in BAEC. BAEC, cultured as described in Methods, were incubated with ATP (50 gM), TNF (200 U/ml) or bFGF (20 ng/ml) for 1 hour (panel A: c-fos) or 3 hours (panel B: c-myc). Cycloheximide (i0 gg/ml) was present throughout the incubation (A) or during the last hour (B). C: control. Figure 2

Increased level of c-fos mRNA in stimulated endothelial cells: comparison between ATP, bradykinin, TNF and bFGF. BAEC, cultured as described in Methods, were incubated for 1 hour in the presence of the various agonists and of cycloheximide (10 ~g/ml). C: control.

heximide

(not shown),

heximide-treated to A T P w a s longer mRNA

3 hours

a greater

TNF or bFGF producing was

increases

After

3 hours, bFGF was

stimulatory

effective

ATP

than

of c - f o s

after

of A T P o n c - f o s

the m a x i m a l

At concentrations in i n o s i t o l

the

level whereas

be d e t e c t e d

might

following

of A T P

be related

to t h e

deprivation

had a characteristic

309

a n d PGI2, c-fos

(Fig.

T N F h a d no effect.

The constitutive

a 24 h o u r s

of a g o n i s t s

of c - m y c m R N A

stimulatory,

experiments

of e i t h e r

in i n c r e a s i n g

of A T P c o u l d 3).

1 h o u r b u t no

effect

phosphates

also

(Fig.

in r e s p o n s e

The effect

than bradykinin

increased

mRNA

detectable

effect

cycloheximide

action

magnitude

similar

2).

quiescent

phenomenon,

(not shown).

1 a n d 2).

(Fig.

these

Accumulation

(Fig.

also more

3).

cells.

a transient

after

had

the signal was much more intense in cyclo-

in t h e

absence

expression

ATP mRNA

1 and The of

of c - m y c

fact t h a t B A E C w e r e of s e r u m

time course

(Fig.

in not

(ii).

The

3).

After

1

Vol. 172, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

C .

ATP

.

.

.

C

.

ATP

)

Figure 3 T i m e c o u r s e o f c - m y c m R N A a c c u m u l a t i o n in A T P - s t i m u l a t e d B A E C . B A E C , c u l t u r e d as d e s c r i b e d i n M e t h o d s , w e r e i n c u b a t e d w i t h A T P

(50 ~M) for the indicated times. Left and right panels correspond to distinct experiments, which were performed respectively in presence and in absence of cycloheximide Ng/ml). C: control without ATP.

hour

the

level

increase after

was

of c - m y c

m R N A was

transient:

it w a s

actually

observed

decreased.

at 3 hours,

(i0

The

later

b u t no l o n g e r

6 hours.

DISCUSSION TWO

studies

of c - f o s

endothelial

cells

and c-myc

have been

Colotta

et al.

induced

an accumulation

A coordinated and aFGF

ned

observed

and c-myc

similar

indeed

detected

1 hour

of t r e a t m e n t

sequential

accumulation

ATP,

and bFGF

The release

mRNA

induce

of ATP,

whereas

rized,

except

(ii).

In t h e c a s e nor the

have been

identified cells

towards

study,

cells.

1 and obtai-

We have

cells

stimulated

after

by bFGF

and that

a

it is a

in e n d o t h e l i a l

identified have

neither

induces

as a m a j o r

not y e t b e e n

rapid

cells. acute

characte-

of a m i t o g e n i c

involved

a procoagulant

310

at

T N F o r bFGF.

evidence

a n d FGF,

TNF

by serum

we h a v e

bFGF-stimulated

responses

second messengers so far.

1 hour.

shown that ATP also produces

late responses

of T N F

responses

after

induced

and c-myc mRNA

for t h e p r e l i m i n a r y

and TNF

the accumulation

endothelial

than either distinct

vein

17).

with peak values

in c e l l s

we have

of PGI 2 a n d N O has b e e n

effect

endothelial

aortic

of c - f o s

of c - f o s

peaked

and c-myc was

in T N F - a n d

and c-myc

In a d d i t i o n ,

inducer

interleukin-i

In the p r e s e n t

in b o v i n e mRNA

(16,

o r in c o m b i n a t i o n : transient,

(17).

c-fos

stronger TNF

of c - f o s

mRNA was

results

for 3 h o u r s .

that both

separately

respectively

in h u m a n u m b i l i c a l

previously

of c - f o s m R N A w h i c h

expression

or bFGF,

of c - f o s 2 hours

(16)

expression

reported

action

functional

in t h e

late actions

a reprogramming

of

and proinflammatory

Vol. 172, No. 1, 1990

surface (19).

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(18) and inhibits FGF is mitogenic

some effects, synthase

cell proliferation

such as the down-regulation

endothelial agonists

to those of TNF.

of prostaglandin

of c-fos.

induction was actually not unexpected of c-myc

in m a n y cell types,

thelial

cells,

signals,

in view of the bewilstimulation

link between c-myc

has not been firmly established. of c-myc mRNA,

did not. expression

whereas

In endo-

TNF, an inhibitor

If none of the agonists of either c-fos or c-myc,

with a different

quantitative

tested indueach of them

combination

which might be related to the distinct responses

endothelial

(22).

bFGF and ATP, which increase cell proliferation,

of proliferation, was associated

of

these 3

in which c-fos is expressed

although a direct causal

induced an accumulation ced a selective

(21),

This promis-

is an early response to mitogenic

and cell p r o l i f e r a t i o n

H

Although the responses

the expression

dering array of cells responses Expression

activator

cells to ATP, TNF and bFGF are so different,

all stimulated

in vitro

cells and produces

(20) and the secretion of plasminogen

which are opposite

cuous

endothelial

for the endothelial

of the 2 of

cells.

ACKNOWLEDGMENTS

This work was supported by grants Politique

Scientifique"

"Fonds de la Recherche

from the

"Minist~re de la

(Sciences de la Vie BI0/04) Scientifique

M~dicale"

Falson was supported by the European Communities Stimulation

ST2000440).

S. Reuse is a research

and the

O. Boutherin(Action de fellow of the Van

Buuren Foundation.

REFERENCES

i. 2. 3. 4. 5. 6. 7. 8.

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