- Email: [email protected]
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.
Vane, J.R., Gryglewski, R.J., Botting, R.M. (1987) TIPS 8, 491-496. Boeynaems, J.M., Pearson, J.D. (1990) TIPS ii, 34-37. Pirotton, S., Raspe, E., Demolle, D., Erneux, C., Boeynaems, J.M. (1987) J. Biol. Chem. 262, 17461-17469. Carter, T.D., Hallam, T.J., Cusack, N.J., Pearson, J.D. (1988) Br. J. Pharmacol. 95, 1181-1190. Lfickoff, A., Pohl, V., Mulsch, A., Busse, R. (1988) Br. J. Pharmacol. 95, 189-196. Kitazono, T., Takeshige, K., Cragoe, E.J., Minakami, S. (1988) Biochem. Biophys; Res. Commun. 152, 1304-1309. Demolle, D., Lecomte, M., Boeynaems, J.M. (1998) J. Biol. Chem. 263, 18459-18465. Robaye, B., Hepburn, A., Lecocq, R., Fiers, W., Boeynaems, J.M., Dumont, J.E. (1989) Biochem. Biophys. Res. Commun. 163, 301-308. 311
Vol, 172, No. 1, 1990
9. i0 ii 12 13 14 15 16 17. 18. 19. 20. 21. 22.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Martin, T.W., Michaelis, K. (1989) J. Biol. Chem. 264, 88478856. Pirotton, S., Robaye, B., Lagneau, C., Boeynaems, J.M. (1990) J. Cell. Physiol. 142, 449-457. Van Coevorden, A., Roger, P.P., Boeynaems, J.M. (1989) Thromb. Haemostasis 62, 190. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Biochemistry 18, 5294-5300. McMaster, G.K. and Carmichael, G.G. (1977) Proc. Natl. Acad. Sci. USA 74, 4835-4838. Thomas, P.S. (1980) Proc. Natl. Acad. Sci. USA 77, 52015205. Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. Colotta, F., Lampugnani, M.G., Polentarutti, N., Dejana, E., Mantovani, A. (1988) Biochem. Biophys. Res. Commun. 152, 1104-1110. Lampugnani, M.G., Polentarutti, N., Pedenovi, M., Mantovani, A., Dejana, E., Colotta, F. (1990) Exp. Cell. Res. 186, 381384. Pober, J.S., Cotran, R.S. (1990) Physiol. Rev. 70, 427-451. Frater-Schr~der, M., Risau, W., Hallman, R., Gautschi, P., Bohlen, P. (1987) Proc. Natl. Acad. Sci. USA 84, 5277-5281. Weksler, B. (1990) J. Cell. Physiol. 142, 514-522. Presta, M., Moscatelli, D., Joseph-Silverstein, J., Refkin, D.B. (1986) Mol. Cell. Biol. 6, 4060-4066. Herschman, H.R. (1989) TIBS 14, 455-458.
312
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
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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
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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.
Vane, J.R., Gryglewski, R.J., Botting, R.M. (1987) TIPS 8, 491-496. Boeynaems, J.M., Pearson, J.D. (1990) TIPS ii, 34-37. Pirotton, S., Raspe, E., Demolle, D., Erneux, C., Boeynaems, J.M. (1987) J. Biol. Chem. 262, 17461-17469. Carter, T.D., Hallam, T.J., Cusack, N.J., Pearson, J.D. (1988) Br. J. Pharmacol. 95, 1181-1190. Lfickoff, A., Pohl, V., Mulsch, A., Busse, R. (1988) Br. J. Pharmacol. 95, 189-196. Kitazono, T., Takeshige, K., Cragoe, E.J., Minakami, S. (1988) Biochem. Biophys; Res. Commun. 152, 1304-1309. Demolle, D., Lecomte, M., Boeynaems, J.M. (1998) J. Biol. Chem. 263, 18459-18465. Robaye, B., Hepburn, A., Lecocq, R., Fiers, W., Boeynaems, J.M., Dumont, J.E. (1989) Biochem. Biophys. Res. Commun. 163, 301-308. 311
Vol, 172, No. 1, 1990
9. i0 ii 12 13 14 15 16 17. 18. 19. 20. 21. 22.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Martin, T.W., Michaelis, K. (1989) J. Biol. Chem. 264, 88478856. Pirotton, S., Robaye, B., Lagneau, C., Boeynaems, J.M. (1990) J. Cell. Physiol. 142, 449-457. Van Coevorden, A., Roger, P.P., Boeynaems, J.M. (1989) Thromb. Haemostasis 62, 190. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Biochemistry 18, 5294-5300. McMaster, G.K. and Carmichael, G.G. (1977) Proc. Natl. Acad. Sci. USA 74, 4835-4838. Thomas, P.S. (1980) Proc. Natl. Acad. Sci. USA 77, 52015205. Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. Colotta, F., Lampugnani, M.G., Polentarutti, N., Dejana, E., Mantovani, A. (1988) Biochem. Biophys. Res. Commun. 152, 1104-1110. Lampugnani, M.G., Polentarutti, N., Pedenovi, M., Mantovani, A., Dejana, E., Colotta, F. (1990) Exp. Cell. Res. 186, 381384. Pober, J.S., Cotran, R.S. (1990) Physiol. Rev. 70, 427-451. Frater-Schr~der, M., Risau, W., Hallman, R., Gautschi, P., Bohlen, P. (1987) Proc. Natl. Acad. Sci. USA 84, 5277-5281. Weksler, B. (1990) J. Cell. Physiol. 142, 514-522. Presta, M., Moscatelli, D., Joseph-Silverstein, J., Refkin, D.B. (1986) Mol. Cell. Biol. 6, 4060-4066. Herschman, H.R. (1989) TIBS 14, 455-458.
312