Inhibition of cellular cholesterol esterification can decrease low density lipoprotein receptor number in human fibroblasts

Vol. 145, No. 1, 1987

BIOCHEMICAL

AND BIOPHYSICAL

CHOLESTEROL

ESTERIFICATION

RESEARCH COMMUNICATIONS Pages 350-356

May 29,1987

INHIBITION

OF CELLULAR LIPOPROTEIN

RECEPTOR

NUMBER

Bruce Dept.

Received

of

Biochemistry, Queen’s Medical

January

20,

CAN

IN HUMAN

DECREASE

LOW

DENSITY

FIEROBLASTS

Middleton of Nottingham Medical School, Nottingham NG7 2UH, U.K.

University Centre,

1987

In fibroblasts deprived of exogenous cholesterol to induce low density lipoprotein receptors there is a continuing flux of cholesterol esterification. The structurally unrelated inhibitors of acyl-CoA: cholesterol acyl-transferase, progesterone, trimethylcyclohexanyl mandelate and 3Cdecyldimethylsilyll-N-C2-~4-methylphenyl~-l-phenylethyll propanamide, (58035), could all inhibit this basal rate of esterification within lh of addition. Exposure of cholesterol-deprived fibroblasts for 17h to progesterone or trimethylcyclohexanyl mandelate caused decreased specific binding and metabolism of low density lipoprotein. The effect was not a direct inhibition of 1 ipoprotein binding; it was time dependent and followed from the reversible inhibition of cholesterol esterification by these two compounds. The irreversible inhibition of esterification by 58035 left the receptor number unaffected. The results indicate that down regulation of low density lipoprotein receptors is initiated by accumulation of cholesterol in a specific intracellular pool. Inhibition of cholesterol esterification by progesterone and trimethylcyclohexanyl mandelate causes accumulation of cholesterol in this pool but 58035 does not. B 1987 Academic Press, Inc.

Human

of their density

skin

fibroblasts

cholesterol

normal

metabolism.

cholesterol

synthesis.

Agents

similarly include

repress

LDL

inhibitors

of

cellular

could binding

can regulate de

endogenous

novo

free

and cholesterol

synthesis.

cholesterol

acyl-transferase

acyl-CoA:

cholesterol

Cl1 have

Brown

inhibit

and

increase

show exquisitely

and

cholesterol

esterification which

individuals

Goldstein

lipoprotein(LDL)-derived

stimulate

that

from

esterification

occurred

fine shown

Copyright All rights

$1.50

0 1987 by Academic Press, of reproduction in any form

Inc. reserved.

350

low

how

LDL receptor

number,

cholesterol cholesterol Such agents (ACAT)

at a significant

should could provided rate

Abbreviations: LDL, low density lipoprotein; ACAT, acyl-CoA: cholesterol acyl-transferase; 58035, 3-Cdecyldimethylsilyll-N-C2-(4-methylphenylj-lphenylethyll propanamide; TMCM, trimethylcyclohexanyl mandelate; LPDS, lipoprotein-deficient serum; FEE, foetal bovine serum; ICso, concentration inhibitor giving SO% inhibition; HDL, high density lipoprotein. 0006-291X/87

control

in

of

BIOCHEMICAL

Vol. 145, No. 1, 1987 relation it

to processes

has been

receptor not

generating

reported

that

down-regulation

found

intracellular

but

of cellular

LDL receptors

free

ACAT inhibition that

in human fibroblasts.

inhibitors

AND BIOPHYSICAL

in J774

the

effect

The present

cholesterol

in human skin

RESEARCH COMMUNICATIONS cholesterol.

macrophages

was specific study

esterification

Recently

shows

caused

C21

LDL

to macrophages that

can cause

only

and

some

down-regulation

of

fibroblasts. NATERIFILS CIND METHODS

Trimethylcyclohexanyl mandelate (Cyclospasmol “‘) was a gift from GistBrocades n.v., The Netherlands. Progesterone was obtained from Sigma (U.K.1 Ltd. 3-CDecyldimethylsilyll-N-E2-~4-methylphenyl~-l-phenylethyll propanamide (58035) was a gift from Sandoz Inc. U.S.A. Growth media and foetal bovine serum (FBS) were from Imperial Ltd. U.K. C9,10-%I-Oleic acid, [*‘Clcholesterol and [“HI-i-i& were from Amersham International Plc. U.K. C1‘%lCholesteryl oleate was synthesised as described in C31. Lipoprotein-deficient serum (LPDS) and LDL were isolated from serum of normal individuals. C1esIlLDL was prepared as described in E41 and was kindly given by Dr FI.N.Salter of this department. Skin fibroblasts were from individuals with normal cholesterol metabolism and were grown in Eagle’s MEN (alpha modification) supplemented with penicillin (100 units per ml) , streptomycin (1OOpg per ml), glutamine (2mN) and FBS (lO%,v/v) in an atmosphere of CO= and air (10:9O,v/v). Lipoprotein-deficient(LPD) medium contained human LPDS (5mg per ml) in place of FBS. LDL receptors were induced in near confluent monolayers by culture for 2x24h in 2 lots of LPD-medium. Specific binding of LDL to high affinity receptors was then determined at 4O and 37O and metabolism of LDL was measured at 37O; all as described in C41. Esterification of cellular cholesterol was measured after 4Bh in LPD-medium (low flux) or 17h after the addition of LDL (1001(g per ml) to cells precultured for 31h in LPD-medium (high flux). Monolayers were then incubated with 1001N C9,10-WI-oleate bound to defatted bovine serum albumin (0.6 mg per ml) for lh at 37O. The incubation was terminated, cholesteryl esters extracted, purified and counted for radioactivity exactly as described in C4l.cIll inhibitors were added to growth media in ethanol solution to give final ethanol concentrations of O.~%(V/V). Monolayers were freed of inhibitor-containing medium by washing with growth medium for 15 min at 37O before adding the appropriate assay medium. Use of C3Hl-H&l showed that this procedure removed more than 99% of cell associated counts. Protein was measured by the Folin reagent CSI. RESULTS This

study

progesterone acylamide, propanamide of cholesterol 10pN Cbl; CBI.

These

used E61;

three

ACAT inhibitors

an ester,

of differing

trimethylcyclohexanyl

structure: mandelate

a steroid, (TNCN)

E71 ; an N-

3-Cdecyldimethylsilyll-N-C2-~4-methylphenyl~-l-phenylethyll (58035)

C81. These

in intact

cells

compounds with

TNCN, ICSO 15KN CB.Niddleton, results

were

all

obtained

exert

different

their potencies:

unpublished at high

351

effects

rates

data];

on esterification

progesterone, 58035,

of cholesterol

ICII&9

O.lpN

Vol.

145,

No.

Table

BIOCHEMICAL

1, 1987

1.

of and

Inhibition

rates

AND

cholesterol its reversal

RESEARCH

at

esterification

in

Cholesterol

Low

BIOPHYSICAL

human

low

High

(2frM)

Progesterone

(100pM)

(100~M)

flux

High

flux removed)

that

The

the

basal

deprived

of

inhibitable

by

65”

46

?

5**

349

+

2t3**

4512

II? 466

50

+

396

2

66*’

4150

5

to

control

13"

with

inhibition

within

deficient

medium

deprived were

raised

were

specific

on

1 over

by

indicates

that

inhibition

of

cells the

then binding.

58035

the exposed

to Table

in

progesterone

effect

of

when

washing This

differ

cholesterolrates

11.

411

formation: the

no

and

monolayers

in

effect of

progesterone (Table

in

58035

compounds

conditions

TMCM

difference from

in

(Table

by

the

lipoprotein-

esterification

under

exerted

to

62%

ester

synthesis

by

to

medium

cholesterol

unaffected.

and

>90%

the

inhibition

reversed

46%

evidence

significant

exposed

from to

protein

The

thus

caused

except SD.

11 while

reversibility their

mechanism

of

ACAT.

inhibitors added

24h.

remained

TMCM

determine were

or

and

previously

LDL of

fibroblasts

they

flux

?

in

that

265

JSSJ~

observed

1 shows

33"

or

seek

activity

2

(low

to

ACAT

cells

of

was

to

ranged

inhibition

completely

due

increased

phospholipid

and

inhibition

to

Inhibition

of

investigation

Table

inclusion

their

periods

rapidly

To

by

in

was

inhibition

43-fold

this

415

in LPD-medium alone rates). .4CAT inhibitors present during the means of 3 experiments

4Sh flux were are

esterification

addition

48h.

This

observed

Table

of

for

cells.

of

compounds. lh

respect

cultured LDL (high assay and Results

cholesterol

these

I? 390

+

cholesterol

exogenous

4240

235

object

of

204

1.5*-

first

rate

?

2

were determined in cells or after addition of were added lh before reversibility was tested.

esterification.

of

protein)

35

**P(O.Ol Rates rates), ethanol where

3961

4

935

58035

was

flux

(Inhibitor

Control

was

high

(pmol/h/mg

rate

Inhibitor

TMCM

and

fibroblasts

esterification

flux

COMMUNICATIONS

to to

the

inhibition

on

lipoprotein-deficient allow

medium

2 shows

ACAT

full 17h

that

medium

expression or

2h

exposure

before of 352

of

LDL

subsequent up-regulated

expression for

of at

least

receptors.

31h

receptors, in

Inhibitors

measurement cells

LDL

to

of TMCM

absence were

LDL or

Vol.

145,

No.

Table

The

2.

BIOCHEMICAL

1, 1987

effect

of

ACAT

AND

inhlbltors human

BIOPHYSICAL

on

expression

LDL

of

LDL

bIndIng

(ng

with

LDL/mg

17h (Inhibitor

Contl-01 58035

(2pM) (lOOtiN)

r

18

107

127 nd

2

10

104+ 129

+

22

40

k

40 to

2 9** control;

respect

removed) 122r

4

8

nd nd

6”

43

+

53 not

2

nd

=

culture at

in LPD-medium prior 4O. Inhibitors or ethanol measurement of LDL binding assay.

Results

are

Ei*El** determined.

to were

added

and, of

means

unless

3

? SD.

progesterone

17h

for

due

effect

to

a

was

direct

seen

inhibition the

58035

effect when

the

reduction

of

inhibition

of

LDL

3.

The

4 o by

of

inhibitors

metabolism

of

2). at

TNCN

and

subsequent

Control 58035

(2~l.l)

(1001-IM)

Progesterone

(100rM)

high

because

before after

a)

the

17h

the

NAT

concentrations

and

was

at

but

inhibitor 2).

The

in

mirrored

Cxes17-LDL

b)

exposure

(Table

progesterone

was

no

C*‘YI-LDL

the

contrast,

of

This.

37*

their

(Table

effect.

of LDL

by

cholesterol fibroblasts metabolism

Internal (ng

LDL/mg)

esterification

on

human

and

Binding trig

By

binding.

LDL

binding

out

metabolism

BInding

Inhibltor

2h

washed

even

of

LDL

added

were

binding

without

50-60X

on

(Table

again

effect

of

were drugs

at

1055

compounds

CIESII-LDL

and was

the

the

binding

binding

the

compounds

LDL

58035

compound

Table

of

inhibit

not

of

when

addition

did

in

resulted

persisted

before

TMCM

20

9

with

were Induced by 48h of CxEsIl-LDL binding medium 17h or 2h before were present during the

indicated, experiments

It

6

124+ “P