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.
1378
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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