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

REFERENCES 2. 3. 4. 5. 6.

7. 8.

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important

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1.

of

atherosclerosis,

enhanced

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play

receptors

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.

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172,

No.

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

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