New collagen markers of ‘derepression’ synthesized by rabbit articular chondrocytes in culture

BIOCHEMICAL

Vol. 68, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NEW COLLAGEN MARKERS OF 'DEREPRESSION' BY RABBIT Herman

S. Cheung,

ARTICULAR

Wilson

SYNTHESIZED

CHONDROCYTES IN CULTURE

Harvey,

Paul

D. Benya

and Marcel

E. Nimni

Rheumatic Disease Section Departments of Medicine 6 Biochemistry School of Medicine University of Southern California Los Angeles, California 90033 December 17, 1975

Received

SUMMARY: Chondrocytes isolated from rabbit articular cartilage undergo a progressive 'derepression' in culture and synthesize Type I, Type III and an unidentified collagen designated Peak 'Xl. In contrast, cartilage slices synthesize predominantly Type II collagen with increasing prolonged culture. amounts of Peak 'Xl during Four far

in

distinct

different

Types

marked

Type tissue

tures

(l-4)

Type

triple

These

have been

being

and Type of tissues

III

slices

Type

II

collagen

exclusively;

this

tissue

from

rabbit

articular

however,

I collagen,

tissue-specificity

of

collagen

synthesis

by increased that

resemble

accelerated chondrocytes

DNA synthesis fibroblasts.

by adding in

culture

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

cartilage

found

(15).

These

"transformed"

from

a change A similar

bromodeoxyuridine (16,17)

produce isolated

reflecting

and "transformation"

1371 Copyright All rights

However have been

chondrocytes

Type

to chick

struc-

respectively.

synthesize

extract

show

to cartilagenous

also

is

con-

(7-14).

culture,

in phenotype

(al),

[al(I

[al(III)],

so

chains or

IV collagen

(5,6),

collagen

found

designated

[al(I)],a2

restricted

membranes

been

polypeptide

of the

3 and Type

In

the

units

11 [al(

and basement

in a variety

have

of different

stranded

specificity

I [al(I)],a2

molecules

tissues.

IV and consist

into

formation.

of collagen

vertebrate

I through

combined

types

change

and embryo

and is accompaniel into cells

motile cease

in

cells to

Vol. 68, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL

accumulate

extracellular

matrix

or altered

chondroblasts

(18-20).

and have been

MATERIALS For each organ thick)

were

week old flasks

isolated

male

supplemented

Eagle

slices (DMEM),

labeled

ml),

B-aminopropionitrile

acid

(lOOng/ml).

to their

The pepsin M Tris

into

this

in

(pH 7.4) salt

Three

the

sample

Collagen and the

precipitate

time ducted

the

of enzyme at room

F12 medium,

pared

at weekly

rat

secondary

the

to half

Primary

cultures

and subsequent

and resuspension with

in DMEM. a split

1372

ratio

in

48 hours

5ml cold

was extracted gentle

was dissolved

by centrifugation. salt-precipitation

against from

and Sokoloff

back

and lyophilized.

collagen

released

was reduced

for

at 4'C with

and acid

(SOuCi/

and L-ascorbic

collagen

extensively

(21)

3H-proline

sample

skin

Modified

and added

(0.5N)

environ-

and radio-

by shaking

was removed

by neutral

temperature.

intervals

pulverized

GIBCO,

and strepto-

231ug/ml)

24 hours

residue

of Green

treatment

while

by trypsinization

for

from

outgrowth,

and the

were enzymatically

technique

obtained

5ml of Dulbecco's

acid

1 M NaCl,

was dialyzed

Chondrocytes using

then

plastic

in

(BAPN;

in acetic

solution

purified

Falcon

a 10% CO2- 90% air

any cell

mg of acid-soluble

was then

were

was digested

and insoluble

of two eight

in

by dissolving

with

joints

Sml DMEM containing

were

(lmg/ml)

neutral

shaking.

in

The mixture

was inactivated

0.05

from

(O.l-0.2mm

0.1% penicillin

were maintained

24 hours

dedifferentiated

slices

25cm'

media

serum,

fumarate

pepsin

in

at pH 7.2

The slices

medium.

at 4°C with

calf

separated

for

and knee

culture

and maintained

Medium

actively

shoulder,

All

10% fetal

Cartilage

cartilage

and cultured

flask).

with

(GIBCO),

ment,

hip,

rabbits,

termed

AND METHODS

experiment,

from

white

(70mg per

mycin

culture

RESEARCH COMMUNICATIONS

0.5

con-

in Ham's

were prepared

Subcultures of 1:2

The

digestion

were grown

acid.

slices

(22).

and the

cultures

N acetic

cartilage

et al.

(3)

were pre-

and medium

was

Vol. 68, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 1: SDS-PAGE profiles of 3H-proline labeled collagen synthesized in organ and cell cultures. (a) Rabbit articular cartilage slices, first day of culture; (b) chondrocytes , primary culture; (c) chondrocytes, fifth subculture; (d) adult rabbit skin fibroblasts, primary culture. changed of

in

all

cultures

chondrocytes

described

for

Samples taining 2 M urea, phoresis continuous

every

three

days.

it

was labeled

was confluent, the were

20 mM Tris

cartilage

slice

lyophilized, borate

and incubated

cultures

dissolved (pH 8.6),

for

1 hour

When each serial and processed but in

without

a buffer

0.5% sodium at

55°C.

(PAGE) was performed

with

5% acrylamide

0.1 M Tris-borate

buffer

(pH 8.6)

1373

as

pulverization. solution

dodecyl

con-

sulphate

Polyacrylamide

gel

- 0.1% bis containing

culture

gels

(SDS), electro in a

0.1% SDS (23).

Vol. 68, No. 4, 1976

Gels

10.4cm

slices

in

(24)

at 55'C

BIOCHEMICAL

length

were placed

overnight.

shown in

were identified

of collagen 1C.

it

helical-region

disulfide in

of 0.4

Nuclear)

N NaOH

containing

in a Beckman

of

SDS-PAGE system.

a different

SDS-PAGE

[al(III)],,

al

collagens

300,000

Type

III

by CMC

Type after

the

presence

collagen+

have

and in

trimeric

form

420,000,

in

of

behaves

chains

al(II1)

col-

reduction

ol(IV)

than

III

but

Since

mobility

and a2

obtained

reflecting

Standard

(6,25),

the

The radioactive

al-chains,

bonds.

of 140,000

have

standard

approximately

like

with

labeled

chromatography.

migrates

this

weight

should

and 1 mm

0.2ml

obtained

The peaks with

weight

and alkylation

they

with

to counting

profile Figure

has a molecular

molecular

prior

4 DISCUSSION

cellulose)

identically

3% hours,

(New England

RESULTS

by co-migration

(carboxymethyl lagen

for

"Biovials"

sample

RESEARCH COMMUNICATIONS

counter.

A representative is

165 volts

in Beckman

to each

scintillation

system

run at

Two ml of Aquasol

0.6% HCl was added liquid

were

AND BIOPHYSICAL

a

a SDS-PAGE

system. Peak both

'Xl,

pepsin

is neither

synthesized

digestion

of 1.03 is

gels

bacterial molecular

insensitive that is

peak

IX'

is

of

BAPN,

by its

disappearance

collagenase

(27).

chain not

Peak

and its

ol(IV).

'Xl

to

that

isolated

it

from ratio

(26).

nature

Its

upon

collagenous

treatment peak

appearance

low level

Further

resistant

hydroxyproline/proline

Although its

to al(IV),

is

suggesting

a B-component.

chromatography

weight

single

presence

gave a radioactive

indicated

purified similar

nor

by Dowex column

further

the

and reduction-alkylation,

procollagen

polyacrylamide

in

'Xl

with

highly

has a

as a sulphydryl-

of hydroxylation

characterization

suggest of this

peak

in progress. Figure

cartilage

1A shows a profile slices

during

the

of first

the

collagen

24 hours

1374

in

synthesized culture.

by This

radio-

Vol. 68, No. 4, 1976

Figure

2:

active

BIOCHEMICAL

Rabbit

material

yielded

PAGE identical only

articular

with

cells

a cyanogen

authentic

cartilage

was produced.

Primary

cultures

of chondrocytes

produced

the profile

thus precluding

‘Type

collagen

II.1

collagens

1D) , it

(Figure buted

synthesized

to the

(Figure

1B).

are not is

identifiable

also

the

collagens This

of Drs.

by primary

supported chondrocytes

that

products.

1B.

on SDS-

indicating

that

such cartilage

fibroblasts

1A with

of rabbit could

in the primary in this

culture

skin

1375

of the fibroblasts

have contri-

chondrocyte Type

were

in these

that

not

‘X’

Miller.

III

The absence of fibroblast

2).

peak

no a2 chains

E. Chung and E. J.

cultures

(1 week).

pattern

Type III,

by the homogeneous population (Figure

culture

of Type I collagen

in Figure

synthesized

indicates

fibroblast

that

from

synthesized;

synthesis

the profile

is apparent

collagen,

derived

collagens

was a gift

By comparing

cultures.

peptide

shown in Figure

were the only

observed,

in primary

bromide

collagen

and al chains

tion

cartilage

Type II

slices

‘Xl

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

culture and peak contamina-

of morphologically

Vol. 68, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TAEILE1 DISTRIBUTION

OF

SUBCULTURED

RADIOACTIVELY

LAEELLED

CHONDROCYTES,

EXPRESSED

COLLAGEN AS

CULTURE

x1(1

SYNTHESIZED

PERCENTAGES

OF

& II)],

THE

BY

SERIALLY

TOTAL

a2

c%1:a2

PRIMARY DAY

1

DAY

3 7

DAY

0 '84,O 80,4

cc m

2"

69,9

518

12,o

3"

63,l

589

11,5

4"

C4,8

681

10‘6

5"

59,6

6,9

8#6

Secondary chains

and subsequent

in addition

(Figure chains

1).

However, is

Thus it

from chondrocytes,

and continue

In contrast cartilage

The appearance a chondrocyte

synthesized the

stable with

fibroblast

chondrocytes,

by a progressive IX’

appearance

in organ

product.

1376

culture

a2

culture of a2

overgrowth increase

or cells Type III

in

derived and

Type I collagen,

change

change during

produced

proportion

progressive

a rapid,

phenotypic

cells

in primary

to change by synthesizing

showed minimal

of peak

that

these

to change by synthesizing

to the rapid

slices

characterized

expect

appears

begin

of

inconsistent

where one would

et2 synthesis,

IX’

collagens

1, 2’-5’)

(Table

cultures

to the

1C and Table

a situation

peak

---

in cell

culture,

prolonged

culture,

of Peak ‘X’ emphasizes

(Table that

2). it

is

BIOCHEMICAL

Vol. 68, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 2 DISTRIBUTION

OF

CARTILAGE

TIME

RADIOACTIVELY

SLICES

(DAYS:

IN

PERCENTAGES

'X'

al

cx2

BY

OF

THE

99,o

0

m

7

0

587

94,3

0

a,

14

0

688

93,2

0

02

21

2,l

816

87,7

1.6

55

35

la7

8,l

87,9

2,3

43

DIFFERENT

EXPERIMENTS

OF

3

of al to a2 chains

detected

authors

cultures using

producibly

detect

to monitor

the

this

early

In summary,

a variety

however,

method.

Since

stages

of

of

culture

synthesize

it

isolated

a rapid,

chondrocytes

progressive

molecules

‘X’

were

and reis

now possible

of

the types

release

culture. 1377

cultured

produced

collagen

that

cartilage

‘derepression’

The chondrocyte

enzymatic

demonstrate

predominantly

to changes in the cellular

subsequent

and peak

of chondrocytes

clearly

fore

during

II

these markers,

origin.

be amplified

of Type I collagen

we can accurately

of mesenchymal sensitive

has

in a

conditions. experiments

of collagen

Type

‘derepression’

these

undergo

by CMC chromatography

the presence

(17,lS);

In contrast,

11) .

conditions

to detect

the appearance

of environmental

in organ

obtained

TOTAL*

al:a2

1,o

in chondrocyte

(Type

AS

SYNTHESIZED

0

been used by other

slices

EXPRESSED

[a1(111)1,

The ratio

variety

CULTURE,

COLLAGEN

1

*AVERAGE

not

LABELLED

collagen under

of chondrocytes

similar

and synthesize by other

phenotype

environment

cartilage

which

is

cells there-

may

and their

Vol. 68, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENTS We are grateful to Dr. Leon Sokoloff for his suggestions regarding the cell culture techniques. We also want to thank Drs. Endy Chung and Ted Miller for the Type III collagen used as a standard. This research was supported by a post-doctoral fellowship (H.C.) (AM 02036), and grants (AM 10358, AM 16404 $ DE 02848) from the National Institutes of Health; and The Robert E. and May R. Wright Foundation. References 1. 2. i: 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. ii:

19. 20. 21. 22. 23.

24. 25. 26. 27.

Miller, E. J. and Matukas, V. J. (1969) Proc. Natl. Acad. Sci. USA 64, 1264. Miller, E. J. (1971) Biochemistry 10, 1652. Strawich, E. and Nimni, M. E. (197n Biochemistry 10, 3905. Miller, E. J. (1971) Biochemistry 10, 3030. Kefalides, N. A. (1973) Int. Rev. Ennect. Tissue Res. 6, 63. Daniels, J. R. and Chu, G. H. (1974) J. Biol. Chem. 2, 3531. Miller, E. J., Epstein, E. H. and Piez, K. A. (1971) Blochem. Biophys. Res. Commun. 42, 1024. Chung, E. and Miller, E;- J. (1974) Science 183, 1200. E. M. and Miller, E. J. (m4) Biochemistry Chung, E., Keele, 13, 3459. Eij-stein, E. H. (1974) J. Biochem. 249, 3225. Byers, P. H., McKenney, K. H., Lichenstein, J. R. and Martin, G. R. (1974) Biochemistry 13, 5243. Trelstad, R. L. (1974) BioTTiem. Biophys. Res. Commun. 57, 717. Crystal, R. G. (1974) Fed. Proc. 33, 22. Miller, E. J. and Matukas, V. J. 7iQ74) Fed. Proc. 33, 1197. Layman, D. L., Sokoloff, L. and Miller, E. J. (1972)Exp. Cell Res. 73, 107. Schiltz, J. R., Mayne, R. and Holtzer, H. (1973) Differentiation 1, 97. M. S. and Miller, E. J. (1975) Fed. Proc. 34, 562. Flayne, R., Vail, Coon, H. G. (1966) Proc. Nat. Acad. Sci. USA 55, 66. Abbott, J., Holtzer, H. (1968) Proc. Natl. Aca. Sci. USA 59, 1144. Marzullo, G. and Lash, J. W. (1970) Devel. Biol. 22, 638. Green, W. T., Jr. (1971) Clin. Orth. 75, 248. Sokoloff, L., Malemud, C. J. and Green, W. R., Jr. (1970) Arthritis and Rheumatism 13, 118. A;- J. (1971) Biochem. Biophys. Res. Sykes. B. C. and Bailey, Cbmmun. 43, 340. . Matsumura, T. and Noda, H. (1973) Analytical Biochem. 56, 571. J. D., Kefalides, N. A. and Prockop, D. Grant, M. E., Schofield, (1973) J. Biol. Chem. 248, 7432. Klein, L. in Standard Methods of Clinical Chemistry (1970) vol. i6, pp. 41. Benya, P. D., Berger, K. and Schneir, M. (1973) Analytical Biochem. 53, 313.

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