Vol.
172,
No.
November
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
15, 1990
1022-1027
EFFECT OF TUMOR NECROSIS FACTOR/CACHECTIN ON THE ACTIVITY OF THE LOW DENSITY LIPOPROTEIN RECEPTOR ON HUMAN SKIN FIBROBLASTS Kenji
Harada,
Takanari
Hitoshi
Gotoda,
The Third
Shimano,
Natsuko
Department
Masanobu Kawakami,
Mori,
Fumimaro Takaku
of Internal
University
of Tokyo,
Medicine,
Shun Ishibashi,
and Nobuhiro
Faculty
Hongo, Tokyo,
Yamada*
of Medicine,
Japan,
113
Received September 12, 1990 We have necrosis low
investigated
factor
density
the
(TNF)/cachectin
lipoprotein
recombinant
effects on the
(LDL)
to
to
cultured
TNF was added
TNF also
stimulated
results
the
growth
indicate
that
which might be related 'J1990Acadrmlc mess,Inc. There coronary
is heart
its
plasma
the
cell
level
is
surface
(2).
(PDGF) (4) and epidermal LDL receptor
we observed
a stimulatory
(M-CSF)
macrophages
on LDL receptor and
suggested
metabolism
stimulates receptor
the growth of activity (4).
biologically secreted
by
*To whom all
factor
activity
and
of
0
of reproduction
rights
1990
When doubled
1.6.fold.
the
growth.
incidence
of
levels
(1).
the
LDL receptor
pathway
by
LDL receptor
activity
(EGF)
growth
and on
factor
(5) have been reported Recently cells.
target
monocyte-colony
stimulating
activity
in human monocyte-derived cytokines in involvement of
the in
atherogenesis
as well
smooth muscle cells in an atheromatous
(6).
PDGF
and their LDL lesion, many
active substances such as PDGF, M-CSF and TNF are the component cells and influence the process of TNF, which is identical to cachectin, is a should
0006-291X/90$1.50 All
between
on their
arterial Thus,
correspondence
Copyrighr
human
activity,
on cell
131, platelet-derived
effect
cholesterol
atherosclerosis.
mainly
growth
to stimulate factor
effect
LDL-cholesterol
via
Insulin
fibroblasts LDL-receptor
stimulatory
regulated
of
LDL binding
of human skin
by cells
binding
of TNF was dose-dependent and concentrations of l-10 rig/ml.
and plasma
utilized
human tumor
fibroblasts.
cells,
relationship
disease
Plasma LDL is
cellular
TNF increases
to its
a positive
recombinant
human skin
after 24 h of incubation. The effect its maximal effect was observed at These
of
by Academic in any
Press, form
Inc. reserved.
1022
be addressed.
Vol.
172,
No.
BIOCHEMICAL
3, 1990
monocyte/macrophage not
only
derived
cytostatic
and
AND
BIOPHYSICAL
It
protein. cytotoxic
is
RESEARCH
well
activity
interferon
in human fibroblasts
human fibroblasts of fibroblast, receptor
activity.
potential
role
(10). there
(9) and the
Although the
present
of TNF in the cellular
that
regard as
TNF has to
well, tells(8)
tumor
such as and Br-
growth-stimulation
TNF functions
have been no reports In
known
with
cells (7) but other biological activities induction of interleukin-1 in human endothelial
COMMUNICATIONS
of
study, uptake
of
as a growth its
effects
factor on LDL-
we investigated
the
of LDL.
MATERIALS AND METHODS Sodium [1"1]-iodine was purchased from ICN Materials [3H]-thymidine was purchased from Radiochemicals (Irvine, CA). Recombinant human TNF/cachectin New England Nuclear (Boston, MA). was at least 99.9% pure based on HPLC, was formulated in an aqueous and had no measurable endotoxin on the limulus amebocyte buffer, assay (11). Lipoproteins LDL (d = 1.019 1.063 g/ml) were prepared from human plasma containing 0.1 % EDTA, 0.02 % sodium azide and 0.5 mg/ml benzamidine, of fasted normolipidemic volunteers and isolated by sequential ultracentrifugation in a 50.2 Ti rotor (Beckman Instrument, Palo Alto, CA) as described previously (12). High density lipoproteins were removed by ultracentrifugation at d=1.21, and the resulting non-lipoprotein fraction was extensively dialyzed against a buffer containing 2 mM sodium phosphate and 0.15 M NaCl, pH 7.4 and used as lipoprotein deficient serum (LPDS). The LDL was recentrifuged at d=1.063 g/ml and dialyzed twice against 2 x 5 L of buffer containing 2 mM sodium phosphate, 150 m&l NaCl and 0.01 % (w/v) EDTA, pH 7.4, at 4OC. Lipoproteins were radioiodinated with Na lz51 using the iodine monochloride method (13). Bindino of 1iDoDroteins bv cells In the binding assay, human skin fibroblasts were preincubated with MEM containing 5 mg/ml of LPDS for 24 h (14). To measure 4OC binding, the cells were precooled at 4OC for 30 min. The cells were then washed with ice-cold PBS and incubated with ice-cold radioiodinated lipoproteins with and without excess unlabeled lipoproteins dissolved in binding buffer (MEM containing 5 mg/ml of BSA and 10 mM HEPES at pH 7.4). After 2-3 h on a rocking platform on ice, the medium was removed and the cells were washed with buffer A (150 mM NaCl, 2 mM CaCl?, 2 mg/ml BSA, and 10 mM Tris-HCl at pH 7.4) rapidly three times, 10 min twice, and finally rapidly rinsed with buffer A without BSA. The cells were dissolved in 1 ml of 0.1 N NaOH in order to measure their radioactivity. Their protein concentrations were measured by the Lowry method(l5). Incorooration of ['Hlthvmidine into DNA At various times after incubation with TNF, ['HI -thymidine incorporation into DNA was measured by the method described previously (16). Cells were incubated with F3H] -thy&dine at 1 yCi/ml for 24 h at 37OC. RESULTS To determine fibroblasts,
the
effects
of TNF on LDL binding
TNF was added to the culture 1023
at various
to human skin times
prior
to
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
Time
RESEARCH
COMMUNICATIONS
(day)
Fig. 1. Time-related effect of TNF on the specific binding of The cells were preincubated with 10 LDL to human skin fibroblasts. ng TNF/ml for the indicated periods at 37% prior to the LDL24 h before the assay, binding assay. culture medium was replaced by medium containing LPDS. Thereafter, the cells were incubated with ice-cold radioiodinated LDL (1 Fig/ml) with or without excess unlabeled lipoproteins at 4oc. This assay was performed in triplicate. Values are expressed as the mean + SD.
the binding iodinated measured
assay
of
LDL
at
4OC.
LDL was added and the by
subtracting
TNF
specific
non-specific
11d 10
oncentrations
assay,
bind.ng
binding
from
of
1 pg/ml
of
LDL was then
total
binding.
I
I
0
In the binding
(n
ml)
0
3
I Time
2 (day)
Dose-related effect of TNF on the specific binding of Fig. 2. The cells were preincubated with LDL to human skin fibroblasts. various amounts of TNF for 48 h at 37OC prior to the LDL-binding 24 h before the assay, culture medium was replaced by assay. medium containing LPDS. Thereafter, the cells were incubated with with or without excess ice-cold radioiodinated LDL (1 ug/ml) This assay was performed in unlabeled lipoproteins at 4-C. triplicate. Values are expressed as the mean i SD. At various times Effect of TNF on cell proliferation. Fig.3. incorporation into DNA after incubation with TNF, ['HI -thymidine Cells were incubated with [ Hl~thymidlne at 1 pCi/ml was measured. for 24 h at 37OC. This assay was performed in triplicate. 1024
Vol.
As
172,
No.
shown
which
in
had
stimulated was to
BIOCHEMICAL
3, 1990
incubated
2-fold
after
from
Next,
various
culture
2 with
assay.
of
of
1 ug/ml of
binding
in
1 rig/ml
of
TNF
stimulated
of
TNF
and
for
on cells 12 h and
thereafter
1 and
(0.1,
lipoprotein-free
medium
One and 10 rig/ml LDL to
experiment
the
was
effect
10 rig/ml)
for
were
a 48 h prior
of TNF stimulated
fibroblasts (Fig.
2).
added to the
the binding
l.&fold,
and
the
The stimulation
of
LDL-
was approximately
to the
same degree
as
1.
To evaluate measured thymidine 1 ng TNF/ml.
the effect of incorporation Incorporation
by TNF-treatment
no additive
COMMUNICATIONS
1.6-fold.
iodinated
this
RESEARCH
slightly
incubation
TNF was dose-related
shown in Fig.
fold
to
BIOPHYSICAL
was
with 1 day
amounts
LDL-binding effect
LDL binding
been
reduced the
1,
Fig.
AND
effect
for
TNF on cellular proliferation, into cell DNA after treatment
of
[3H]-thymidine
15 h, but
on cell
longer
proliferation
we with
was increased
incubation
(Fig.
with
1.6
TNF had
3).
XtSCJSSION Receptor-dependent of
regulation cholesterol cell for
uptake
both
plasma
content
surface
(2) _
is adjusted
cholesterol.
of LDL is
the
cholesterol
Expression
When cells
are
levels of
in response
central
the
to the
mechanism and
cultured
cellular
LDL receptor
requirement with
of
of
on the the cell
lipoprotein-free
the numbers of LDL receptors on the cell surface are serum, increased. Whereas its expression is suppressed by exposure of the The expression of LDL cells to LDL-cholesterol in culture medium. receptors
is
regulated
in vitro
PDGF (4) and EGF (5) (3), receptor number on fibroblasts LDL-receptor cells
_
pathway These
factors
on the
stimulates
by several
have
been
enabling
as a source
results
suggest
LDL receptor.
LDL-receptor
of
known factors.
reported cholesterol cell
activity
in
increase supply
structure
a generalized
Recently
to
Insulin
effect
we demonstrated monocyte-derived
in
LDL
via
the
growing
of
growth
that
M-CSF
macrophages
(6).
TNF is known to stimulate the growth of human fibroblasts and increase the number of EGF receptors on human fibroblasts suggesting that of EGF receptor. growth
the mitogenic action of TNF stimulates the In the current study, maximal stimulation
was observed
at
a concentration
of
LDL binding to fibroblasts reached its incubation with TNF and thereafter slightly 1025
l-10
ng of
(10) (17), function of cell
TNF/ml,
and
maximum 1 day after declined. A similar
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
result obtained PDGF was added
from a time-course to cultures of arterial
previous study of cholesterol
(4). It as well
the
experiment
cholesterol
and,
TNF on the
to
a lesser
in
uptake
was
fully
expressed
on cell
medium,
LDL binding
for
cholesterol.
demand results
LDL via
the
a significant
cells
role
of
in
such
and in
as
the
its
TNF
subendothelial
as
the
in
the cells. factor for
and
M-CSF space
after
of
TNF
TNF
creating
interaction products
cellular of
from
uptake
plays as
it
cholesterol
well
appears
an LDL as that
receptora scavenger cytokines
metabolism a
significant
in
the
role
in
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play
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Recently we showed that growth and differentiation
Thus,
may
LDL
Monocyte-macrophages
M-CSF
modulate and
modify
[3H] changes
possible
lineage,
(6).
and
fibroblasts
progression
pathways
PDGF but
deprivation
Secretory
in
cells
Since
pathway.
metabolism.
target
synthesis phase of
of
binding
action
TNF.
monocyte-macrophage
independent
when in the
time-related
to
a
TNF can
receptor
ester stimulating
cholesterol
dependent pathway
LDL role
storing cholesterol CSF, a specific of
and
such
that
cholesterol
demonstrated
receptor
monocyte-macrophages play
after
a mitogenic
related
current
cholesterol
surfaces in
cellular the
quite
to
additional between
also
LDL
and the similar.
an increase
may
the early
to
both
extent,
be
treatment The
similar
TNF stimulated to a similar
stimulations
was observed muscle cells
that PDGF stimulated LDL receptor in the
may be very
these the
experiment smooth
COMMUNICATIONS
an increased content of cellular LDL-receptor synthesis (4) _ The effect
LDL receptor extent.
thymidine are
later,
down-regulated
of
in
appears as the
RESEARCH
and
Vol.
9.
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172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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