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
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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
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