Vol. 171, No. 1, 1990 August

COMMUNICATIONS

BlOCHEMlCALANDBlOPHYSlCALRESEARCH

Pages 20-25

31, 1990

INHIBITION

Herman

OF BASIC CALCIUM PHOSPHATE CRYSTAL-INDUCED MITOGENESIS BY PHOSPHOCITRATE

S Cheung*,

D Sallis+,

Peter

G Mitchell*,

and Janine

A Struve*

of Rheumatology, Dept. of Medicine, Medical College of Wisconsin, 8700 W. Wisconsin Ave, Milwaukee, WI 53226 of Biochemistry, University of Tasmania, G.P.O. Box 252C Hobart, Tasmania 7001, Australia

*Division +Department

Received

John

June

29,

1990

Basic calcium phosphate crystals control the traverse of cells from the Go/G1 to S-phase of the cell cycle and initiate proliferation by rendering fibroblasts competent to respond to insulin-like growth factors in plasma. Simultaneous addition of phosphocitrate [a powerful inhibitor of hydroxyapatite crystallization] to cells exposed to basic calcium phosphate crystals caused a dose-dependent inhibition of crystal-induced DNA synthesis and C-&I= transcription. This inhibition was specific for crystal-induced mitogenesis, since similar concentrations of phosphocitrate had no effects on either PDGF or 10% calf serum-induced thymidine incorporation and c-m transcription. 01990 Academic Press, Inc.

Synovial basic

hyperplasia

calcium

phosphate

and tricalcium cultured

fashion

incorporation behind mitogenic

effect nor

[BCPI

crystals

Cl]).

crystal

were

control

the

onset

addition particles

such for

via

phospholipase

the

phospholipase

C and inositol

collagenase and neutral the

proto-oncogenes

both

BCP crystals

transcription crystals

[c-b

synthesis are

0 1990 by Academic Press, Inc. in any form reserved.

[7,8]. delays

proto-oncogenes

20

of

in a doseby 2 to 3 hours

cultures

urate,or

not

beads

factor

crystals

of PGE? C51,activation

161,and

induction

does

not induced

block

of of

We also demonstrated with similar kinetics but

[2].

[PDGF] as

and PDGF exert

as stimulation pathway

were

latex

growth

or DNA synthesis

and PDGF [9].

of reproduction

as sodium

induced

lag

these

hydrolysis

0006-291X/90$1.50 Copyright All rights

from

cells,such

and PDGF. &interferon

of these

cells

C41. Moreover,BCP

and cq~xl

mitosis

fibroblasts

A2/cyclo-oxygenase

protease

phosphate,

stimulates

platelet-derived

phospholipid

with

peak of thymidine

to quiescent

a competence growth factor in vitro similar biologic effects on cultured production

synovial

media

associated

octacalcium

crystals

and the

Conditioned

can substitute

synovitis

(hydroxyapatite,

or canine

Both

of serum.

BCP crystals

of chronic

Each of these

fibroblasts [2,3].

after

the

a feature

phosphate

human skin

dependent

is

the

by BCP

that by

Vol.

171, No. 1, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Phorphecitrate [PC], a powerful inhibitor of hydroxyapatite crystallization [lO,ll] has been shown to prevent crystal-induced membranolysis

of polymorphonuclear

can be attributed

to the

some cellular

influence cultured

aortic

hypercholesterol protection

cellular

events

surface e.g.

rats

by PC led

to the

be influenced.

Modified

Eagle's

penicillin

Stock medium

at 37'C. For all

confluence

in culture

[Multiwell,Gibcol by removing

the

platelet-poor before

in medium

containing

[PPl,and

incorporation

that

method [lo0

crystals

used

were RNA Blot

chloride

calf

in Dulbecco serum,

in humidified

10%

were

to

were

grown

rendered

in this

isothiocyanate. as described

from

[171.

plasmid

to a specific [16].

The fos

quiescent 2% human

medium

from

the

by heating

The suspensions

were

published

Unless

for

24 h

gels

et al

sequences

plasmid

from

100 mm

by disruption

of

in 4 M

RNA (5 ug/lane)

was

to nitrocellulose

of the

desired

10' cpm/ug in this

of

experiments.

precipitated

hybridized

used as a probe

work,we

specified,BCP

cells

were than

In earlier

followed

and transferred

The filters

with gene. using

work

filters

inserts The the

inserts random

was a 1.0

were primer

Kb PstI

v-

[19].

and PC : BCP crystals were prepared as and sieved to yield lo-20 urn aggregates,

at 200°C for sonicated

all

[IS].

incubated

maximum induction

otherwise

saline

were

[3H]-thymidine

[2]. the

by scraping

by Cathala

greater

earlier

earlier

The RNA was then

carrying

pa-1

plates for

throughout

activity insert

culture

produced

buffered

Preoaration of B@ Crystals described 141. They were crushed

procedure

and then

published ug/mll

RNA was prepared

previously

fragment

sterilized

the

16 mm in diameter

incubating

concentration

on formaldehyde

as described

method

grown

1 ml of DMEM containing

19,151.

in physiological

electrophoresed

fos

in this

in guanidinium

purified

serum

each

24 h and processed

cells

Analysis:

washing

for

the

in Balb/c-3T3

labeled

were

procedures,cells

in multi-well

BCP crystal

DNA synthesis

lithium

examined

and mitogenesis.

10% (v/v)

24 wells

subsequently

(1 uCi/ml)

according

demonstrated

cells

cells

at 50 ug/ml

once with

Cells

Assay:

13Hl-,thymidine

plates,

study,we

of c-a

some underlying

experiments.

DNA Synthesis with

present

with

experimental

washing

that

Balb/c-3T3

streptomycin containing

plasma

the

of

dishes

medium,

by in

by PC. The apparent

hypothesis

In the

[DMEM] supplemented

C02/90% air

adhesion

and Methods

cultures

at 50 units/ml,and

uptake

monocyte

transcription

Materials Culture:

lipoprotein

aortic

can be restricted

of PC on BCP crystal-induced

Cell

low density

[13],and

[I43

afforded may well

[12].

binding

both

cells

muscle

ischemic

membrane effects

crystal events

smooth

While some responses to PC phenomena,PC is also known to

leukocytes

90 minutes,weighed,and

before

use.

[20]. 21

suspended

PC was synthesized

in DMEM.

according

to a

Vol.

171,

No.

All

BIOCHEMICAL

1, 1990

analysis

Student's

of

T test.

least

All

AND

BIOPHYSICAL

statistical

significance

experiments

were

RESEARCH

was

run

in

COMMUNICATIONS

performed

by

quadruplicate,

applying

and

repeated

at

twice. Highly

purified

[o-32P]-dCTP

PDGF

were

were

randomly

cell

culture

from

was

prepared

Amersham

primed

with

supplies

described

Corporation

kits

are

as

from

[Arlington

Boehringer

products

of

1211.

Heights,ILl.

and Probes

[Indianapolis,IN].

Mannheim

Gibco

L3H]-thymidine

Laboratory

[Grand

All

Island,N.V.l.

Results To

define

3T3

cells,we

BCP

crystal-induced

lo-'M

was

dish.

the first

toxic

the

the

caused

to

incubated

in

on

10%

calf

in

the

basal

This

all

10T3M

subsequent

F,

50%

of

of

BCP

DNA

for PC had

BCP no

BCP

containing 2% PP

synthesis of

in PC did

control

cells

on PC at

crystal-induced

either a mitogenic

lOO(

10-3

M

IO-4

Concentrations

M

10-J

of

M

10-7

M

PC

Relationspip between PC concentrations and inhibition of BCP crystalinduced [ HI-thymidine incorporation into DNA of Salb/c-313 cells. Cells were grown and maintained in 24multiwell culture plate until confluence. They were then fed with OHEH and 2% PP containing one of the following : BCP crystals ClDDug/mll, PDGF [5Ong/mll, control [no crystal] and calf seru [lo%] in the presence or absence of various f concentrations of PC. H-thymidine incorporation was estimated 24 hours after stimulation as described in the M terial and Method. P Values are expressed as the means ,+ SD, [n=4]. [HI-thymidine incorporation is expressed in cpm. 0 PXPP; =BCP crystals; mlO%CS; -PDGF. 22

not

crystal-induced

effects

[Fig.l].

the

the

culture

and

concentrations

specific

inhibit

the

medium

the

PC on

experiments.

control

Fig.1.

from

of

of

a concentration

[100ug/mll

incorporation to

of

crystal-induced

of

proliferation

PC at

to

crystals

incorporation was

h.

cells

Similar

concentrations

used

24

detached

Balb/c-3T3

thymidine

was

cells

BCP

the

concentrations at

incubation.

thymidine

affect

various

PC,and

inhibition

serum-induced of

of

h of

similar

concentration effect

24

2% PP.

mitogenesis,since

to

inhibition

after

of

quiescent

lo-'Ml

a dose-dependent

effect

Up

of

[10-3M

harvested any

effect

PC may

incorporation

cells.

Otherwise,exposure

cells

or

examined

to

which

under

[3H]thymidine

concentrations

have

conditions

PDGF-

Vol.

171, No. 1, 1990

BIOCHEMICAL

-24h

AND BIDPHYSICAL

oh

3h

Time Ffg.2.

block

stimulated

entry

into

with

level cells

time

be added

of PC

cells

with

either

c-fos PC for

of PC

at which the

of PC to

were

again

able to stimulate

level

of PDGF-induced

CFig.21. expression

by BCP crystals

Stimulation

BCP crystals

of density-

or PDGF resulted

stimulation.

c-fos

In the

transcription

c-a,thus

BCP crystal-induced

the cells

suppress

supporting cellular

in maximal

presence

mRNA at 30 min was non-detectable. l/2 hour,then the PC was washed

on the

can be reversed

cycle

ability

of c-fos

time

as 3 h after

3 a & b respectively.

mRNA 30 min after

inhibits

cell

as late

decreased

induction

in Fig

of the

in the the

[P