Vol.

174,

January

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 758-766

31, 1991

PROSTAGLANDINSANTAGONIZE FIBROBLAST PROLIFERATION TUMOR NECROSiS FACTOR Takamitsu

STIMULATED

BY

Hori, Yoichi Yamanaka, Makio Hayakawa, Sayumi Shibamoto, Masafumi Tsujimoto*, Naoto Oku, and Fumiaki Ito

Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-01,JAPAN *Suntory Institute for Biomedical Research, Mishima, Osaka 618, .JAPAN Received December 3, 1990

Tumor necrosis factor (TNF) is known to be a mitogen for human diploid FS-4 fibroblasts. We have shown in an earlier study (Hori et al. (1989)J.Cell.Physiol.l4l,Z75-280) that indomethacin further enhances the cell proliferation stimulated by TNF. Since indomethacin inhibits the activity of cyclooxygenase, the role of prostaglandins in TNF-stimulated cell growth was examined. Cell growth stimulated by TNF and indomethacin was inhibited by exogenously added prostaglandins (PGEz, PGFza , and of cell growth. PGDz), amongwhich PGE:! caused the greatest inhibition Treatment of FS-4 cells with 10 rig/ml TNF resulted in the release of prostaglandins (PGE2, 6-keto-PGFla , PGA2. PGD2, and PGFza) 2 to 4 fold over that of untreated cells. The amount of all these prostaglandins increased in a time-dependent manner over 6 h after treatment. In both TNF-treated and control cells, PGEz was released as the predominant when PGE2production and DNA synthesis were prostaglandin. Furthermore, determined in FS-4 cells treated with increasing doses of indomethacin, these two cellular responses were inversely affected by indomethacin. induced by TNF antagonize growth These data show that prostaglandins stimulatory action of TNF. 0 1991Academic P1e.55, Inc.

Tumor necrosis factor factor

eliciting

to stimulate cytokines

which was originally

identified

a hemorrhagic necrosis of some tumors in vivo,

the growth of somenormal human fibroblasts

and growth factors,

in various cells that

(TNF),

TNF also stimulates

and organ systems (3-5).

prostaglandins

affect

cellular

A

(1,2).

prostaglandin

as a is known

Like other synthesis

number of studies have shown

proliferation

and

play an important

Abbreviations used: TNF, tumor necrosis factor: IL-l, interleukin 1; PG, prostaglandin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum. 0006-291X/91 $1.50 Copyright 0 I991 by Academic Press. Inc. All rights of reproduction in any form reserved.

758

Vol.

174,

role

No.

2, 1991

in the

control

f ibroblasts, the

BIOCHEMICAL

of human

vascular

In a previous

paper,

by

mitogenic

action

suggests

show

inhibiting

that

prostaglandins

t.o

of TNF in

the

that

(6).

antagonize

OR

smooth

muscle

we showed

cells

that

synthesis

normal

the

the the

other

produced

through

hand,

antagonize

the

the

mitogenic

to TNF (7). which

fibroblasts

(8).

prostaglandin

effect

BALB/c3T3

growth

of prostaglandins,

human

mitogenic

In

cell

indomethacin,

TNF stimulates

are

COMMUNICATIONS

of cells.

1 (IL-l)

growth-promoting

prostaglandins

RESEARCH

stimulate

interleukin

the

a possibility

antagonizes

reported

by

BIOPHYSICAL

in a variety

prostaglandins

induced

activity

then

has been of

prostaglandins response

of proliferation

EGF

production

AND

of TNF.

its

enhanced

the

This

production, In this

by TNF treatment response

exerts

of human

report, and that

fibroblasts

result which we these to

TNF. MATERIALS

AND METHODS

Materials Recombinant human TNF was produced in Escherichia coli and purified to homogeneity (298%), as judged by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (9). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Nissui Pharmaceutical Co.,LTD.(Tokyo, Japan); from Filtron PAY LTD.(Victoria, Australia). and fetal calf serum (FCS), Prostaglandins and indomethacin were obtained from Sigma Co.(St.Louis, MO). All other reagents were of analytical grade. Cell cultures The human diploid FS-4 fibroblast line, isolated from --~foreskin tissue, was a gift from Dr.J.Vilzek of New York University Medical Center. Confluent FS-4 cells grown in 35-mm plastic dishes were cultured in DMEM containing 0.2 % FCS for 2 days as described previously (10). These Gl-arrested FS-4 cells were used for assays of cell growth and prostaglandin release. Assay of cell growt, Gl-arrested FS-4 cells were incubated in 2% FCSsupplemented DMEM containing TNF and other factors. After incubation for 5 cell growth was determined by staining of the cells with 0.5% days, crystal violet as previously described(l1). For evaluation of the level of DNA synthesis, Gl-arrested FS-4 cells were treated with 10 rig/ml TNF and varying concentrations of indomethacin in the presence of 2% FCS. L3HlThymidine incorporation was measured during the last 3 h of 30 h culture as previously reported(8). Assays of prostaglandin release Gl-arrested FS-4 cells were cultured with 10 rig/ml TNF in 2 ml of 2 % FCS-supplemented DMEM. After the indicated periods of time, prostaglandins weie extracted from the culture medium by the use of a Sep-Pak C-18 cartridge (Waters; Milford, MA) according to the The prostaglandin fraction eluted by ethyl acetate method of Powell (12). was analyzed by high-performance liquid chromatography (HPLC) or [12511PGEz radioimmunoassay kit(New England Nuclear; Boston, MA). In the case of HPLC analysis, FS-4 cells were labeled with l.OLCi of [14C]-arachidonic acid (50 mCi/mmol; American Radiolabeled Chemicals Inc.) for 24 h prior to the TNF treatment. Prostaglandins extracted from the culture medium were separated by reversed phase HPLC on a TSK gel ODS-80TM (TOSOH; 4.6x15Omrn) in a Shimadzu HPLC system. Fractions from the column were collected and 759

Vol.

174,

No.

2, 1991

BIOCHEMICAL

radioactivities (LX-3500).

their counter

were

AND

measured

BIOPHYSICAL

with

RESEARCH

an Aluka

COMMUNICATIONS

liquid

scintillation

RESULTS Effect

of

exogenously

It

added

has been

Pibroblasts

demonstrated

(1,Z).

promoting

effect

Further, is

induced

by TNF (8).

ability

to induce

the

However, the

the

induced

cell

stimulated

growth. PGDz ,

of

growth

and

concentration

of

.

we had not

To explore

exogenously

added

was

10e5M,

it

caused fully

not of

the

are

TNF has the inhibitory

on

W-I by

was

greatest

antagonized

inhibit

the

effect

for

we first the

cells the

any significant

indomethacin

growth-

simultaneously

prostaglandins

enhanced have

its

possibility,

of confluent

further

did

this

some human

whether

that

growth.

for that

determined

of prostaglandins

effect PGEz

mitogen

prostaglandin

yet

Growth

cell

suggested

by endogenous

itself

stimulatory PGFza

previously

TNF-stimulated

a potent

growth.

TNF

which

This

we

(Fig.1).

by 10 rig/ml

indomethacin,

TNF is

production cell

effects

on

that

antagonized

TNF-stimulated

examined

prostaglandins

maximally addit

effect reversed ion;

TNF-

ion

on cell by

and

PGE2, at the

of indomethacin.

PGA, PGD, PGF,< PGE,

01:

n -6

-5.5

-5

Prostaglandin

Fia.l Effect of exogenous prostaglandins on TNF-stimulated cell growth in FS-4 cells were incubated with or without the presence of indomethacin. of 10 "g/ml TNF and lo-sM various prostaglandins in the presence The following other cultures were also indomethacin(0) for 5 days. of TNF only(A), or addition of examined: control(O), addition indomethacin only(A). Then the cells were stained with 0.5% crystal

violet

as described

in MATERIALS AND METHODS. 760

of

Vol.

174,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

x103(dpm) 2.0

-

7 I

I Control

too(%)

/’ I’

LO---__>r c .-> z

_______

_ _____

_________

____-_-----

----

1

4.0

E

50

0

6 (c-,

0

-/’

--.-------

1

AI

0

I

-

TNF

:

.-0 2

3.0

-

2.0

-

PGE,

is s

6-keto-PGF,,

PGD,

“GF,,

PGA,

\,iJ

j

1.0 0 i

I

1

I

I

0

10

20

30

40

Retention

50

(min)

Time

QQ

Reversed phase-HPLC of arachidonate metabolites produced by FS-3 Cells were prelabeled with [ 14C]-arachidonic acid (1 fiCi/ml) for 2,1 h and further incubated with or without 10 rig/ml TNF for 4 h. The supernatants were collected and analyzed as described in MATERIALS AND METHODS. Prostaglandins were identified by comparison of their retention times with those of authentic standards. cells.

Analysis

of prostaglandins We next

control acid

medium major

examined

cells for

24 h were was then

incubated

analyzed

FS-4 for

for

was increased minor

which

also

enhanced Next,

HPLC (Fig.3). observed

prostaglandin

(PGAz,

to a similar the

time

course

Au increase as

early

identified

extent

released

(see

PGFza

the

,

culture

and

cultures,

the

90 % of the

and

total

medium.

PGEz

release

the

of

PGFl a )

6-keto

was

Fig.3). production

of PGEz, addition

increased 761

and the culture

In both

more than into

and

[i4C]-arachidonic

TNF,

by TNF treatment PGD2,

TNF-treated

with

(Fig.2).

comprised

in production

in Fig.1

both

or without

of prostaglandin

as 2 h after

in

prelabeled

4 h with

2 to 4 times

prostaglandins

cells

prostaglandins

was PGEr,

cells.

production

of prostaglandins

other

was

HPLC.

prostaglandin

by FS-4

prostaglandin

using

radioactivity release

produced

PGAz,

of TNF. in

a

was determined and

6-keto

The content

time-dependent

by PGFra

of each manner

Vol.

174,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

(dpm)

6000

-

600

t-E

400 200 lLL 0

2

4

6

Time

0

2

4

6

Time

Time-course cells were incubated indicated periods of by HPLC as described PGDz; Panel D, PGFza

6

11 after

prostaglandin

at

Relationship

the any

start

time

between

negative

regulator

thymidine

incorporation of

the culture reversed

prostaglandins

As

by the addition

of DNA synthesis. induced

mitogenesis,

prostaglandin

indomethacin

with

shown in Fig.3,

we

the

the

of indomethacin.

by 5 to 7 ,uM indomethacin,

required to induce complete inhibition 762

[3H]-

blocked

regulating

which

by the

was

was monitored

the by

the amount of PGEz in

level.

Half-maximal whereas

of

of PGEz,

TNF increased

over the control

was

view

in FS-4 cells,

by TNF act as a determined

production

Production

prostaglandin

medium about 4-fold

was obtained

and PGEz was the dominant

B h.

prostaglandins.

most inhibitory

radioimmunoassay.

this

TNF-induced when

concentrations

and

during

endogenous

of

amount of endogenous major

of TNF treatment,

the amount of PGEI and inhibition

To examine whether

varying

(h)

of TNF-stimulated prostaglandin production. FS-4 with(@) or without(O) 10 rig/ml TNF for the time. Production of each prostaglandin was determined in Fig.2. Panel A, PGEz; Panel B, PGAz; Panel C, ; Panel E, 6-keto-PGFla .

Fig.3

over

(h)

This

decrease

approximately

of PGEz production

increase in

was PGEz

30 film was

(Fig.4).

On the

Vol.

174,

No.

BIOCHEMICAL

2, 1991

AND

BIOPHYSICAL

RESEARCH

(ng)l

,

COMMUNICATIONS

x104 (dpm)

Fig.4 Relationship between PGEz production and inhibition of DNA synthesis. FS-4 cells were incubated with 10 rig/ml TNF and indicated concentrations of indomethacin. Levels of PGEz released into the culture medium during a 4-h incubation was determined by radioimmunoassay. For the estimation of DNA synthesis, the cultures were treated with TNF and indomethacin for 24 h and washed, after which 2 ml of DMEM containing 0.5~ Ci/ml [sH]-thymidine was added to each culture. Following a further incubation for 3 h, each culture was washed again and the TCA-insoluble fraction was then counted as described in MATERIALS AND METHODS.

other

DNA

hand,

synthesis

enhanced

by increasing

response

was achieved

nearly

equal

concentration

the

to EC50 for

production

the

cells

PGEz production.

The

PGEz

induced

the

up to 10m6 M gave

of control

cells.

increase

in

by TNF antagonize

inverse

DNA the

which

indomethacin,

in blocking

was

Half-maximal

at concentrations

on DNA synthesis and

incorporation

indomethacin.

5 to 1 flM

indomethacin

effect

prostaglandins

of

at approximately

indomethacin

contrast,

by [aHI-thymidine

measured

In

no significant

relationship

synthesis

mitogenic

was

between

indicates response

that of

FS-4

to TNF.

DISCUSSIO_N Studies mitogen this

for

of normal

mitogenic

acetylsalicylic observation endogenous growth

several

investigators

fibroblasts action

acid, suggests

of

TNF

that

play

further

mitogenic a role

by TNF. 763

that

as

TNF is

report,

enhanced

of prostaglandin

TNF-induced that

shown

In a previous

was

an inhibitor

prostaglandins

stimulation

(1,2).

have

we showed

by indomethacin

production response a

a potent

(8).

that or This

is

antagonized

by

negat ive

regu lator

in

Vol.

174, No. 2, 1991

To t.est two

the

for

following

treatment

BIOCHEMICAL

this

possible

quest.ions: in FS-4

cells?

;

in PGE:, production

cells

study,

(3,4),

t.hat

human foreskin to TNF.

variety

prostaglandins

of other

Production

of these

treatment

and

cant rol

from

demonstrated

the

increased

(8,il).

activity

of

increased

To examine inhibitory

in FS-4 two

indomethacin,

cells

cellular

regulator

supported

by the

prostaglandins

we with

responses

were

evidence of

result

PGE2,

cell

on

in

lea.ding

because

previously

we

is

TNF-treated

KS-4

suppressed

TNF enhanced

acid,

where that

PGEz

the

full

TNF induces

(unpublished

the

to be the

amount

determined

&

data).

Thus,

result

of the

induced This

shows

the

Since

and

production of

affected

PGEz

growth.

PGEz

doses

inversely

which

of

PGEz

increasing

764

by TNF

process

indicating

proliferation.

cell

extent

and PGH synthase.

that

of Fig.1,

a

u ).

One possible

and that

appears

between

treated

providing

negative

exogenous

of

of

processes

from

arachidonic

phospholipase

correlation

activity

synthesis These

the

TNF

production

of both

synthesis

cycloheximide

(PGH synthase)

prostaglandin activities

that

expressed,

PGEz

was sjmilar

certain

by TNF.

by

In

to produce

to a similar

of arachidonate

of excess is

the

(5). of

stimulated

prostaglandins that

observed

reported

PGF;!a , and 6-keto-PGFl

phospholipid,

of cyclooxygenase

TNF-stimulated

also

PGDz ,

stimulated

cyclooxygenase

synthesis

were

suggesting

we

presence

radioimmunoassay

of

release

production in the

inhibit

and macrophages

and

was enhanced

membrane

Further,

prostaglandin production

(PG.42,

seem t.o be activated

release

T&F

to

has been

TNF stimulated

composition

arachidonate

cells

(3),

fibroblasts

ed cells,

production

the

after act

TNE’ treatment

analysis

In addition,

the

and TNF-treat

PGHz

after

prostaglandins

thus

stimulated

prostaglandin

fibroblasts

HPLC

PCEz by exposure

asked

of TNF?

dermal

reverse-phase

we here

production

TNF-induced

An increase

revealed

m

does

RESEARCH COMMUNICATIONS

of prostaglandins,

Is prostaglandin

effect

this

to

role

growth-stimulating

in synovial

AND BIOPHYSICAL

growth and DNA

indomethacin.

by the addition by

TNF

conclusion inhibitory Libby

et al.

acts was effect have

of as

a

also of shown

Vol.

174,

No.

that

2, 1991

IL-la

smooth

BIOCHEMICAL

and IL-lp

muscle

negative

markedly

cells

regulation

common feature The activity

presence

of cell

growth

molecular. cell

by which

prostaglandin

remains

when PGEz was exogenously much higher to

inhibit

PGE2 suggests can

culture

and

whether of

wound

by stimulating

must le

and

of

prostaglandins

prostaglandins

in in

cytokines

the

studies various regulation

such

t.hese the

lymphocy t,es,

Further

infectious

repair,

immune function

prostaglandins.

its

be

a

negative of

was 0.1~

culture

PGEa

M at. most.

medium

of

FS-4

(3 to 10~ M) was concentrations

produced

with

to

concentration

in these

prostaglandins

associated

pathogens,

cells,

that.

exerts

the prostaglandin

The disparity

t,he

intracel

their

of lularly

receptors

than

to the medium.

of a variety

inflammation

of

(71,

cytokines.

The

to the

vascular

appears

TNF-treatment

added

growth.

closely

added

Invasive

after

concentration cell

more

prostaglandins

production

medium

the possi bi 1 it)

become

unclear.

human

inhibitors

prustaglandins

mechanism

However,

required

by

COMMUNICATIONS

of

of cyclooxygenase

to certain

into

a

growth

response

released

cells,

the

RESEARCH

mitogenic

growth the

BIOPHYSICAL

increased

in the

in the

on

AND

or neoplastic, as

cytokines

proliferation Khich are

action

in progress

cells of cell

and the

IL-1

and

could of

TNF

augment

induce

the

(13).

In

repair

and

f ibroblasts,

smooth

may he counterbalanced to analyze physiological

hy

the production role

of

these

growth.

ACKNOWLEDGMENTS We are grateful to Mr.Moriya Hatta and Mr.Kentaro Iida for their assistance. in part by a Grant-in-Aid for Scientific This work was support.ed Research from the Ministry of Education, Science, and Culture of Japan. technical

REFERENCES ---1. 2. 3. 1.

Sugarman. B. Palladino,M.Jr., VilFek,J., Feinman,R., Dayer,.J .M., 2168 Gitter,B.D., Immunology

J. ,

Aggarwxl,B.B., Hass,P.E;. , Figari,I.S., and Shepard,H.M. (1985) Science 230, 943-945 Pallombella,V.J., Henrikson-DeStefano,D., Swenson, C . , Hirai,M., and Tsujimoto,M. (1986) J.Exp.Med. 163, 632-63-1 2163Beut ler,B., and Cerami ,A. ( 1985) J.Exp.Med. 162,

66,

Labus,.l.M., 196-200

Lees,b.L.,

765

and

Scheetz,M.E.

(1989)

Vol.

174,

5. 6. 7. 8. 9. 10. 11. 12. 13.

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Bachwich,P.R., Chensue,S.W., Larrick,J.W., and Kunke1,S.L. (1986) Biochem.Biophys.Res.Commun. 136, 94-101 Nolan,R.D., Danilowicz,R.M., and Eling,T.E. (1988) Mol.Phrmacol. 33, 650-656 Libby,P., Stephen,J.C.W., and Friedman,G.B. (1988) J.Clin.Invest. 81, 487-498 Hori,T., Kashiyama,S., Hayakawa,M., Shibamoto,S., Tsujimoto,M., Oku,N., and Ito,F. (1989) J.Cell.Physiol. 141, 275-280 Tsujimoto,M., Tanaka,S., Sakuragawa,Y., Tsuruoka,N., Funakoshi,K., Butsugan,T., Nakazato,H., Nishihara,T., Noguchi,T., and Vil$ek,J. (1987) J.Biochem. 101, 915-925 Hori,T., Yamanaka,Y., Hayakawa,M., Shibamoto,S., Oku,N., and Ito,F. (1990) Biochem.Biophys.Res.Commun. 169, 959-965 Hori,T., Kashiyama,S., Hayakawa,M., Shibamoto,S., Tsujimoto,M., Oku,N., and 1to.F. (1989) Exp.Cell.Res. 185, 41-49 Powell,W.S.(1982) in "Methods in Enzymology," Vol.86 (Lands,W.E.M., and Smith,W.L., eds) Academic Press, New York, pp.467-477 Arai,K., Lee,F., Miyajima,A., Miyatake,M., Arai,N., and Yokota,T. (1990) Annu.Rev.Biochem. 59, 783-836

766

Prostaglandins antagonize fibroblast proliferation stimulated by tumor necrosis factor.

Tumor necrosis factor (TNF) is known to be a mitogen for human diploid FS-4 fibroblasts. We have shown in an earlier study (Hori et al. (1989) J. Cell...
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