Role of protein kinase C in the inhibition by fibroblast growth factor of apoptosis in serum-depleted endothelial cells

ROLE

OF PROTEIN FACTOR

KINASE

Satohiko

‘Department

INHIBITION

Yoshiya and Hiroshi

September

25,

GROWTH

ENDOTHELIAL

and 4Marine Chikusa-ku,

of Radiological Institute

FIBROBLAST

Simada2 Eazuhiko Hayashi4

of Molecular Biology Nagoya University,

Metropolitan

BY

IN SERUM-DEPLETED

Arakil,

Institute

‘Tokyo Received

C IN THE

OF APOPTOSIS

2 National

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1081- 1085

BIOCHEMICAL

Vol. 172, No. 3, 1990 November 15, 1990

Sciences,

Kaji3,

Biological Japan

Anagawa,

of Gerontology,

CELLS

Laboratory,

Chiba-shi,

Itabashi-ku,

Japan

Tokyo,

Japan

1990

Apopt sis in vascular endothelial cells is suppressed by fibroblast growth factor to investigate the signal transduction system that reguIates en(FGF) P. In order dothelial apoptosis, we studied the effects of several mitogenic factors. Apoptosis occurred in human vascular endothelial cells under serum-free conditions, and FGF inhibited apoptosis without a requirement of any cooperative factors, as distinct from the mitogenic response. Other mitogenic agents, such as epidermal growth factor, transferrin, transforming growth factor beta, and interleukin 1 etc., with the exception of dexamethasone, had no such inhibitory effects. The effect of FGF was mimicked by a phorbol ester and was prevented by an inhibitor of protein kinase C. The results suggest that the FGF and protein kinase C are important in endothelial apoptosis. 0 1990 AcademicPress, 1°C. Apoptosis

(or

lishment

of

is thought

programmed immune

to

be

stasis

and

cular

endothelial

has

some

cells6j7. tosis

the

presence tigated

diseases

this

--in

is worth of

problem due

.

Recently, by

effect

it

that a

independent using

cells

in growth, showed

the

Vascular

that

apoptosis expressed

pathway

effect

response of signal serum-free

of or

FGF

in vas-

cells,

it

transduction.

apop-

reflects

We have to

FGF

in endothelial

in inhibiting

whether

conditions

homeo-

occurs

is characteristically the

estab-

degeneration

carcinogenesis, In endothelial

whether

under

embryogenesis,

deprivationl.

mitogenic

avoid

the inves-

additional

in serum, MATERIALS

Reagents. MCDB-104

during

degeneration2p3. we

FGF

asking

such

to factors

occurs

tissue

as angiogenesis

vitro

result

of another

complexities

45*

mitogenic

Thus, is

death) and

as important cells

a specific

cell

tolerance,

FGF was extracted was purchased from

*Address correspondence to: Laboratory, School of Science, Mie-ken 517, Japan.

AND

METHODS

from bovine brains Kyokuto Pharmaceutical Hiroshi Hayashi, Nagoya University,

1081

by

the method of Industry, Tokyo,

Sugashima Marine 429-63 Sugashima-cho,

Lobb8. Japan.

Biological Toba-shi,

0006-291 X/90 $ I. 50 Copyright 0 IYYO by Academic Press. Inc. All righry of reproduction in an! form wserrvd.

Vol.

172,

No.

3, 1990

BIOCHEMICAL

AND

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RESEARCH

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Fetal bovine serum (FBS) was purchased from GIBCO, Grand Istand, NY, U. S. A.. Bovine recombinant basic FGF (bFGF), and porcine transforming growth factor beta 1 (TGF-beta) were purchased from R & D Systems Inc., Minneapolis, U. S. A. epidermal growth factor (EGF), transferrin, insulin, and dexamethasone were from Collaborative Research Inc., Bedford, MA. U. S. A.; cycloheximide, heparin, and l2-0-tetradecanoic 13-phorbol acetate (TPA) were from Sigma Chemical Company, St. Louis, MO, U. S. A. Human platelet derived growth facDGF) was purified from human platelets by the method of Raines and ;;Jrj with some modifications. Interleukin 1 (IL-I) was obtafned from Dainippon Pharmaceutical Company, Tokyo, Japan; tumor necrosis factor (TNF) was from Asahi Chemical Industries, Tokyo, Japan. H-7 was purchased from Seikagaku Kogyo Co., Tokyo, Japan. Cell cultures. Hump umbilical vein endothelial (HUVE) cells were obtained by the method of Jaffe . The cells were cultured on collagen-coated plastic dishes in a culture medium, MCDB-104, supplemented with 10% FBS and 70 rig/ml FGF (supplemented with 100 ug/ml heparin) at 37 C in 5% CO2 and 95% air. The cultures were stained by a treatment with antibodies against human factor VIII raised in rabbit to confirm that they were endothelial cells. For experiments, HUVE cells were grown until cultures reached confluence. After several days of confluent growth, the medium was replaced by the same medium without serum and FGF. The number of trypsinlzed or floating cells was determined with a Coulter counter at intervals. Throughout the experiments, we used cells with a population doubling1 level of 10 to 20. Fragmentation of DNA was analyzed as described previously . RESULTS In

order

to

perimental When was

serum much

process

estimate

the

conditions, and higher

of

FGF

alone

1).

was

perimental reagents tive

on apoptosis

since

the

under

effecters

FGF effect

the for

inhibited

effect

on the

inhibit

apoptosis we

kinase

C.

While

IL-1

was from

a serum-free alone.

fragmented

studies excluded

death

rate

However,

the

the

pat-

by deprivation

and

of

by cycloheximide our

experimental

culture of

exmedium.

The

obtained

under the

well-defined

died.

inhibited

occurring

for

was that

FGF

conditions

above their

mentioned

ability

cells. (Fig.

Other

TNF,

to inhibit

medium.

the

any

effect

(Fig. condiThe

of

interference

ex-

individual by coopera-

we

apoptosis. also

examined a variety of FGF and recombinant

inhibited

apoptosis,

however,

than FGF in repeated experiments (data not no effect on apoptosis in spite of its mitogenic

mitogenic

factors

tested

had

no or

little

ability

to

2).

studied the effect TPA, an activator and

condition,

Dexamethasone

apoptosis.

was always smaller By contrast, EGF had

shown).

Next,

the

DNA

was

suitable

in cells

of FGF

from death

and

under

in the serum.

cells

mitogenic basic

apoptosis

were

effecters

since

cell

serum

conditions

Using

medium,

by deprivation

apoptosis

that of

factors

apoptosis

the

indistinguishable

Furthermore,

Thus, it appeared 2). tions in the absence

various

from

obtained

reflected

of

endothelial

absent

that

fragmentation (Fig.

studied

were

than

unequivocally

tern

we

FGF

effects

which

of drugs of protein

have

been

known to affect the kinase C, inhibited

reported1’*18 1082

to activate

activity of protein apoptosis (Fig. 3). protein

kinase

C

Vol.

BIOCHEMICAL

172, No. 3, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

- 4.0 - 2.0

-

1.0

l-l

- 0.5

02

01

Fig. 1. Degradation of DNA in endothelial cells. DNA was extracted from cells and then analyzed by agarose gel electrophoresis. Lane 1, DNA from living ceils; Lane 2, DNA from dead cells collected from medium 8 h after FGF deprivation; Lane 3, DNA from dead cells collected from the medium 8 h after serum and FGF deprivation. Molecular weights are expressed in terms of kilobase-pairs. Fig. 2. Inhibition of apoptosis by mitogenic factors. The number of cells that remained on dishes after a J-day incubation in the absence of serum and FGF was counted. At the start of the J-day period, 1 ug/ml cycloheximide (CHX), 70 rig/ml FGF, 10 ng/mI PDGF, 5 rig/ml TGF-beta, 5 rig/ml bFGF, 5 ug/ml insulin (ins.), 5 ug/ml transferrin (trans.), 25 rig/ml EGF, or 50 rig/ml dexamethasone (DEX) was added. The batched bar represents the number of cells before deprivation of FGF and serum. no, no additions. Each result represents the average of two determlnatlons.

in

the

cell,

activation effect

had of

of

no effect.

protein

FGF.

kinase Indeed,

If FGF C,

exerts

inhibitors

as shown

O6Tiz-z CHX( uglml)

L(0

01

of

in

1

10

its

protein

Figure

103

TPA (nglml)

0

inhibitory 4,

10

1allmlIm

IL-1 (U./ml)

effect

kinase H-7,

C should

an

0

I

on apoptosis

inhibitor

10

cancel of

the

protein

103lcco

TNF(U.lml)

Fig. 3. Dose-response curves for the inhibition of apoptosis. The number of cells remaining on dishes after a J-day incubation in the absence of serum and FGF was counted. Abbreviations are explained in the text. (0) represents the number of cells before deprivation of FGF and serum. 1083

via

Vol.

172,

No.

3, 1990

BIOCHEMICAL

0

AND

2

BIOPHYSICAL

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RESEARCH

6

COMMUNICATIONS

8

Time(h)

Fig. 4. Effects of H-7 on inhibition of apoptosis. The rate of cell death after cells were deprived of serum and FGF was measured in terms of the number of cells detached from each dish. Cells were washed with fresh medium free of serum and FGF at tlme 0, and Immediately after that either 70 rig/ml FGF or 50 uM H-7 or both was added. At intervals of 2 hours, the medium was removed and the cells floating In It were counted. Each point represents the number of cells that died during the 2 hours prior to the measurement. The symbols are: ( A ), control; ( 0 ), +FGF; ( A ), +H-7; ( l ), +FGF and H-7.

kinase the

C,

idea

overcame that

tivation

the

inhibition

of protein

effect of

kinase

of FGF

apoptosis

(Fig.

4).

The

in endothelial

results

are

is

mediated

cells

consistent by

with the

ac-

C. DISCUSSION

It

has

dothelial ways

been cells17.

mimic FGF

other

activates

activation

protein

of protein

kinase

kinase

C

in

C by TPA

vascular

en-

does

al-

not

to

belong

to

simpler

cases,

such

as synthesis

of

l5

protease

or

proteins”. and

that

they

TNF

in our

several

TNF

did

activates

not

inhibit

may

apoptosis.

Even

though

kinase

C 1op18,

the

absence

be related

to the

fact

that

protein

system

it

has

been

of any effect protein

kinase

reported

of IL-1

and

C consists

of

dependent

on

subtypes”.

Recently, led

activation protein

experiments to the of

kinase

ferentiated nals

l-lowever,

FGF

responses in endothelial cells, as demonstrated in the cases of 11,lZ effect or prostaglandin synthesis 13p14. The inhibition of apoptosis

seems

IL-l

IL-Z,

that

FGF

mitogenic by

suggested

with

suggestion

protein

kinase

C mediates

cell

to connect

a cell

that

inhibition

C2’. signal

line

of

of apoptosis

There

may

transduction

different

lymphocyte

(but

for specific

apoptosis for

ACKNOWLEDGMENT Mitsuyoshi

Matsuo

for

his helpful 1084

by IL-2

be a general

to apoptosis.

\Ve thank

that

discussions.

are

is associated

mechanism in

a given

many type

with which

by

types

of

of cell)

difsig-

Vol.

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RESEARCH

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