266 Btochimtca et Btoplo'stca Aeta.

1051 I 1990l 266 275

I~lsevicr

BBAMCR 12642

Production of collagens, collagenase and collagenase inhibitor

during the dedifferentiation of articular chondrocytes
by serial subcultures
V6ronique Lefebvre, Chantal Peeters-Joris and Gilbert Vaes
Laboratoire de Chimie Physiologique (Connective Tissue Group), Uni~ersit~ de Louvain and lnternattonal Institute of Cellular
and Molecular Pathology, Brussels (Belgium)

(Received 26 June 1989)

Key words: Chondrocyte dedifferentiation; Collagen synthesis: Collagenase; lnterleukin l; Tissue inhibitor of metalloproteinase:
(Rabbit cell)

Rabbit articular chondrocytes were cultured in monolayer and the progressive loss of their differentiated phenotype was
monitored from passage to passage. The cell densities achieved in confluent cultures decreased abruptly between the
primoculture and the second or third subculture, and more slowly thereafter, reflecting parallel morphological changes.
The synthesis of collagen (but not that of other proteins) decreased sharply, and a smaller proportion of collagen was
incorporated into the matrix. Cells in primocuiture synthesized mainly the cartilage-specific collagens, types II and XI,
which were mostly deposited in the matrix, but no type I nor |II collagen. With increasing passages, the synthesis of
type II collagen decreased progressively while that of types I and III collagens increased, the latter being almost
completely released in the culture medium. Simultaneously, the production of type XI collagen was apparently switched
to that of type V. Fully differentiated confluent chondrocytes in primoculture produced the collagenase inhibitor TIMP
(tissue inhibitor of metalioproteinases) but no detectable procoilagenase; their production of procollagenase was,
however, induced by interleukin 1. The production of TIMP increased from passage to passage. A spontaneous
production of procollagenase was only occasionally observed in confluent cultures of dedifferentiated chondrocytes.
However, interleukin 1 induced an always higher production of procollagenase from dedifferentiated chondrocytes than
from cells in primocuiture.

Introduction phenotype modulation (often called dedifferentiation)

towards a fibroblastic phenotype [3,4]. Contrary to
Although normally responsible for synthesizing the non-activated chondrocytes [5], fibroblasts are a par-
matrix of articular cartilage, chondrocytes can become ticularly good source of collagenase and related tissue
activated to produce enzymes which degrade the col- metalloproteinases, such as proteoglycanase (stromely-
lagens and proteoglycans of this tissue (reviewed in Ref. sin) [6]. This suggests that the production of collagenase
1). This activation is thought to play a critical role in by chondrocytes could be linked to their dedifferentia-
the joint destruction that occurs in arthritis. Therefore, tion.
unravelling the conditions leading to or accompanying To investigate this hypothesis, we cultured rabbit
such activation is of importance. articular chondrocytes in monolayer for several pas-
In osteoarthritic cartilage, an increased production of sages, allowing the progressive loss of their differenti-
collagenase has been reported [2] as well as the ap- ated phenotype [7]. This was monitored by fluoro-
pearance of type I collagen, a sign of chondrocyte graphic visualization of the collagen type chains pro-
duced by the cells after their electrophoretic separation,
and by quantitative evaluation of the collagen and
non-collagenous proteins produced. In parallel, both the
Abbreviations: APMA, 4-aminophenylmercuric acetate; DMEM, spontaneous and IL-l-induced [8] chondrocyte produc-
Dulbecco's modified Eagle's medium; FCS, foetal calf serum; IL-1, tions of procollagenase were evaluated. Because of its
interleukin 1; TIMP, tissue inhibitor of metalloproteinases.
potentially important regulatory role on the activity of
Correspondence: G. Vaes, Laboratoire de Chimie Physiologique, UCL
collagenase [9], the production of the collagenase inhibi-
75.39, Avenue Hippocrate 75, B-1200 Bruxelles, Belgium. tor T I M P was also recorded. New assay procedures

0167-4889/90/$03.50 ,~5 1990 Elsevier Science Publishers B.V. (Biomedical Division)

 267

were used, established [10] to improve the recovery of Synthesis of collagen and non-collagenous proteins
TIMP and the validity of the enzymatic assay of procol- The proteins synthesized by confluent chondrocytes
lagenase in crude culture media which contain both at successive passages were labelled for 24 h with L-[2,3-
procollagenase and TIMP. Our study provides an over- H3]proline (15 /~Ci/ml) in fresh DMEM supplemented
view of both quantitative and qualitative changes occur- with 10% FCS, 4-aminopropionitrile fumarate (100
ring in collagen production during the chondrocyte /xg/ml) and ascorbic acid (100 /~g/ml). The culture
dedifferentiation. It shows that these changes are medium was then removed and centrifuged to sediment
accompanied by enhanced production of TIMP and by the floating cells. The matrix and cell layer was dis-
increased IL-l-induced production of procollagenase. solved in 0.25 M NH4OH and combined with the
floating cells.
Materials and Methods The production of collagen and non-collagenous pro-
teins was quantified according to Peterkofsky et al. [13].
Materials After extensive dialysis against water, the samples were
Recombinant human IL-la (specific activity 108 incubated for 90 min at 37°C in a 50 mM Tris-HC1
U/mg) was a gift from Dr. P.T. Lomedico (Hoffmann- buffer (pH 7.5) containing 150 mM NaCI, 5 mM CaC12,
LaRoche, Nutley, N J, U.S.A.). Mouse bone-conditioned 0.2 mg of NaN3/ml, 0.05% (v/v) Triton X-100 and 3
culture medium containing latent tissue procollagenase mM N-ethyl-maleimide, and with or without 20 /zg of
was kindly donated by J.M. Delaiss6 from our labora- purified bacterial collagenase/ml. Undigested proteins
tory (see Ref. 10). DAPI (4',6-diamidino-2-phenylin- were precipitated by 10% (w/v) cold trichloroacetic
dole) was from Serva Feinbiochemica (Heidelberg, acid in the presence of 1 mg of bovine serum
F.R.G.); L-[-2,3-3H]proline and 14C-methylated pro- albumin/ml. After centrifugation (35000 g-min) the
teins, from Amersham Belgium (Brussels); 4-amino- pellets were dissolved in 0.2 M NaOH, and the amount
propionitrile fumarate, from Janssen Chimica (Beerse, of radioactivity was determined in the pellets and in the
Belgium); calf thymus DNA, N-ethylmaleimide and supernatants. Collagenase sensitive and unsensitive pro-
purified bacterial collagenase (type VII), from Sigma teins were considered as collagen and non-collagenous
Chemical, MO, U.S.A.); pepsin, from Boehringer, proteins respectively.
Mannheim (F.R.G.); z-ascorbic acid and ammonium The types of collagen were determined from samples
hydroxide, from Merck (Darmstadt, F.R.G.); X-ray treated with pepsin (0.2 mg/ml in 0.5 M acetic acid for
films, from Fuji Photo Film Company (Japan). Other 3 h at 15°C, followed by extensive dialysis against
special chemicals, culture plates and culture media were water). To facilitate the identification of the collagens
from suppliers previously mentioned [10]. or of their subtypes, aliquots of the samples were fur-
ther digested either by purified bacterial collagenase (as
Articular chondrocyte cultures described above), or by tissue collagenase (5 U / m l for 4
Cartilage pieces from rabbit humeri, femurs, patellae h at 25°C, in the same buffer as that used for the
and tibiae were freed from adhering other tissues by bacterial collagenase digestion). Types V [14] and XI
successive digestions at 37 °C with hyaluronidase (0.05% collagens [15] are indeed resistant to the action of tissue
(w/v) for 10 min) and trypsin (0.25% (w/v) for 30 min) collagenase while types I, II and III [16], as well as type
in phosphate-buffered saline. The pieces were then I trimer [17], are cleaved into characteristic 3/4-1/4
digested overnight with crude bacterial collagenase fragments. Procollagenase-containing mouse bone-con-
(0.15% (w/v) in DMEM supplemented with 10%, v/v, ditioned culture medium was used as a source of tissue
FCS). The dissociated chondrocytes were washed and collagenase, after the activation of procollagenase by
cultured following procedures described for synovial trypsin [18]. Collagenase-digested and non-digested
fibroblasts [11], except that the trypsin solution used to aliquots were concentrated 25-fold by dialysis and
disperse the cells at each passage was supplemented lyophilization, resuspended in electrophoresis buffer
with 0.05% (w/v) bacterial collagenase. The chondro- with or without dithiothreitol, and denatured at 100 °C
cytes were plated at (0.8-1.0)- 105 cells per cm2 culture for 2 min. Sodium dodecyl sulfate-polyacrylamide gel
dish and cultured in DMEM supplemented with 10% electrophoreses [19] were performed on 5-8% (w/v)
FCS; the medium was renewed once or twice a week. polyacrylamide linear gradient gels. Fluorographs [20]
Subcultures were done at 7-day intervals and at a split were exposed for 2 or 8 days at - 8 0 ° C . Type I
ratio of 1 : 2 in order to allow the progressive dediffer- guinea-pig skin collagen, partially digested by animal
entiation of the chondrocytes [7]. collagenase, and 14C-methylated proteins were used re-
Cell densities were determined at the end of each spectively as collagen and protein standards. Individual
experiment by assaying the DNA content of the cul- collagen bands resolved from the samples were quanti-
tures with the DAPI fluorimetric method [12]. Calf tated by scanning densitometry of the fluorographs.
thymus DNA (50 pg/ml) was used as a standard and it
was assumed that 1 0 6 cells contain 6 #g of DNA.
268

Production of collagenase and collagenase inhibitor

(TIMP)
Collagenase and TIMP were evaluated as described
[10] in conditioned culture media obtained (unless
mE
o
20,
1.6
1.2
6

2 5
otherwise indicated) at cell confluence, in DMEM with 08
or without 10% FCS, 10% NU serum or IL-1, as speci- ~o 0.4

fied in the figure legends. Briefly, free TIMP was as- 0.0 5

sayed by measuring the inhibition of the trypsin- 1 I I I I I

0 2 4 6 8 10
activated collagenase present in a crude skin fibroblast-
passage number
conditioned medium. This assay underestimates the total
Fig. l. Evolution of the cell densities achieved by confluent chondro-
amount of TIMP present because it does not consider
cytes during their dedifferentiation by serial monolayer subcultures.
the proportion of T I M P that binds to other metal- Six different chondrocyte cell lines (referred to as lines l to VI in Figs.
loproteinases present in the fibroblast medium. There- 2, 3, 5 and 6) were cultured on plastic for 6 to 10 passages to allow'
fore, all parallel assays were done using the same their progressive dedifferentiation. The cellular densities achieved
fibroblast medium. TIMP bound to metalloproteinases after 9 to 10 days of culture at various passages were evaluated by
measuring the DNA content of the cultures; the results, expressed in
was measured after destruction of the enzymes and
10 5 cells/cm 2, are the means of evaluations done on 1 to 6 of the
dissociation of the complexes at pH 2 and 100°C for 30 different cell lines, as indicated by the corresponding figures on the
min. Prior to the activation of procollagenase by either graph; vertical bars correspond to + 1 S.D.
trypsin or APMA, the free TIMP present in the condi-
tioned media was neutralized whenever needed (see the
figure legends) by the binding of other tissue metal-
loproteinases (i.e., the proteoglycanase and gelatinase loss, is observed between the primoculture and either
present in the culture medium of rabbit bone marrow- the second or third culture passage, depending upon the
derived macrophages [21]). Collagenase was then as- experiment. This is followed by a slower decrease there-
sayed at 2 5 ° C using soluble [3H]acetylated guinea-pig after (Fig. 1).
skin collagen as a substrate. The procollagenase values
presented have been corrected for the stimulation ( × 1.5) Relative production of collagen and non-collagenous pro-
exerted on collagenase activity by the macrophage-con- teins
ditioned medium [10]. 1 U of collagenase degrades 1 ~g The chondrocyte synthesis of collagen and non-col-
of collagen per min at 25 ° C and 1 U of TIMP inhibits lagenous proteins was characterized during the progres-
2 U of collagenase by 50%. sive dedifferentiation of several cell lines by serial
monolayer subcultures. The results of a representative
Cultures of chondrocytes on collagen/proteoglycan-coated experiment, obtained with cell line I (see Figs. 1, 3, 5
plates and 6), are illustrated in Fig. 2 for the relative produc-
The capability of chondrocytes, at various passages, tion of collagen and non-collagenous proteins, and in
to degrade type I or type II collagen and cartilage Fig. 3, for the collagen types produced.
proteoglycans was assayed by culturing the cells on the Collagen is actively synthesized in primoculture and
surface of [14C]collagen/[3H]proteoglycan films [11]. first subculture where it is mainly incorporated in the
Type II collagen was prepared from calf articular carti- matrix and cell layer. A sharp reduction of collagen
lage [16] and 14C-acetylated [22]. synthesis occurs in subsequent passages, along with a
decreased incorporation into the matrix and cell layers
Results and a slightly increased release into the media (Fig. 2A
and B). Inversely, the production of non-collagenous
Morphological aspects and cell densities of confluent proteins increases steadily from the primoculture to the
cultures 10th passage. Most of these proteins are located in the
Observed at low magnification (not shown), matrix and cell layers but with increasing passages the
chondrocytes in primoculture appear as small polygonal amounts released into the media increase more, relative
cells surrounded by an abundant matrix. In some areas, to those incorporated in the matrix and cell layers (Fig.
the cells pile up in small multilayer 'cartilaginous nod- 2A and B). Consequently, the ratio of newly synthesized
ules'. This morphology changes progressively with in- collagen to newly synthesized total proteins decreases
creasing passages. The abundance of extracellular ma- sharply during the first two passages and more slowly
trix decreases and confluent cells appear larger and thereafter (Fig. 2C). The ratio of newly synthesized
flattened, although still polygonal in shape, and they no collagen incorporated into the matrix and cell layers
longer pile up. In agreement with this evolution, the cell relative to the total of newly synthesized collagen de-
densities achieved in confluent cultures decrease from creases progressively with increasing cell passages (Fig.
passage to passage. An abrupt diminution, approx. 50% 2D).
 269

A B

0.8 0.4 _

0.6 0.3

0, 02
0.2 0.1 0
o.o [- -~- o.o
I I l I I Ic I I l I I Dl

~6 - 80 ~ .

..j
.~
Z
+ 8
--, OOio
40
0
0 ~ 4 20
0 0

0 2 4 6 8 10 0 2 4 6 8 10
passage number passage number

Fig. 2. Production of collagen and non-collagenous proteins by dedifferentiating chondrocytes in monolayer culture. The chondrocyte cell line I
(see Figs. 1, 3, 5 and 6) was characterized for its synthesis of collagen (m) and non-collagenous proteins (©) during its progressive dedifferentiation
by serial monolayer subcultures. A and B present the accumulation of these newly synthesized proteins in either the matrix and cell layer (A) or the
culture medium (B) as a function of the cell passage number. Results are expressed in cpm incorporated into protein per cell during the labelling
period with [3H]proline (see Materials and Methods). Each point is the mean of three determinations; vertical bars correspond to _+1 S.D. (shown
only if its magnitude exceeds the size of the symbol used for the experimental points). C shows the progressive reduction in the percent ratio of
newly synthesized collagen to newly synthesized total proteins with increasing passage number. D shows the parallel reduction in the percentage of
newly synthesized total collagen deposited in the matrix and cell layer, The former ratio only (C) was calculated on cpm values corrected according
to Peterkofsky et al. [13] in order to include in the collagen determinations the collagenase-resistant propeptides of procollagens otherwise included
in the non-collagenous proteins and to take account of the higher proline and hydroxyproline content of collagen relative to non-collagenous
proteins. (COLL, collagen; NCP, non-collagenous proteins).

Collagen types produced chains [lower band, migrating together with the al(II)
The matrix and cell layers as well as the culture chains] of type XI collagen. This is another product of
media of the chondrocyte cell line presented above (Fig. differentiated chondrocytes existing predominantly un-
2) were monitored from the primoculture to the seventh der a heterotrimer [al(XI).a2(XI).a3(XI)] form [23].
subculture to determine the types of collagen synthe-
sized (Fig. 3) and their relative proportions (Table I). TABLE I
In primoculture, the most abundant neosynthesized
Relative proportions of collagen types synthesized by chondrocytes during
collagen found in the medium (Fig. 3A) and, in consid- their progressive dedifferentiation in monolayer culture
erably larger amounts, in the matrix and cell layer (Fig.
The table was established by compiling the data obtained from
3B), has a migration similar to that of the al(I) chain densitometry of fluorographs presented in Fig. 3. Type III collagen
standard. No a2(I) chains are detected. This indicates was considered to correspond to band A of the culture medium, since
the absence of type I collagen with a chain composition the contribution of types I and II collagens to this band, visualized
of [al(I)]2.a2(I), but the presence of s o m e [al(I)] 3 can- after dithiothreitol reduction (not shown), was found to be minimal.
All other collagen types were evaluated from both the medium and
not be excluded. Thus, the major collagen synthesized
the cell and matrix compartment: type I collagen [(al)2.a2 ] was
appears to be type II, a typical product of differentiated estimated equal to 3-fold the values of bands F; type XI, to 3-fold the
chondrocytes, consisting of three al(II) chains which values of bands D; type V, to 1.5-fold the difference between bands C
are transformed by a crude preparation of tissue col- and D; type II (and type 1 trimer), to the difference between bands E
lagenase (Fig. 3C and D) into fragments of the size of and the values estimated for the participation of types I, V and XI
collagens to these bands.
the 3 / 4 ~1 and 3 / 4 a2 standards or smaller (discussed
below). This material is followed by two bands, visible Cell Relative proportions (%) of collagen type
almost exclusively in the matrix layer, which migrate passage
I II III V XI
between the 3 / 4 fl(I) and the al(I) standards. These number
t w o b a n d s p e r s i s t a f t e r d i g e s t i o n w i t h c r u d e t i s s u e col-
0 0 59.2 0 10.9 29.9
l a g e n a s e as d o e s a m i n o r c o m p o n e n t o f t h e b a n d 1 0 62.1 0.5 11.2 26.2
m i g r a t i n g a t t h e a l ( I ) level. T h e s e t i s s u e c o l l a g e n a s e - r e - 2 19.1 41.8 7.3 19.8 12.0
sistant b a n d s a p p e a r to be c o l l a g e n o u s by their c o m - 3 23.2 36.0 9.7 31.1 0
plete removal by bacterial collagenase (not shown). 5 48.9 10.0 5.1 36.0 0
7 57.0 0.7 9.2 33.1 0
T h e y c o n t a i n m a i n l y t h e a l ( u p p e r ) , c~2 ( c e n t r e ) a n d a 3
 270

A 0 1 2 3 5 7 gs B '~ o- 0 1 2 3 5 7 gs

~ ~ A
%r
34/"

200 ,5

C
D
~1 E
I )2 F
4111 G
34(~ 92.5 341~2
H
~iiiiiiiiiiii!i~~ -

69

46

cs 1 2 3 5 gs
C r~ cs 0* 0 1 2 3 5 gs
I'
~,,~' I"

/; 200

(t2
34,h 92.5
34(t2

99

Fig. 3. Analysis of the collagen types synthesized by chondrocytes during their dedifferentiation in monolayer culture. The chondrocyte cell line l
(see Figs. 1, 2, 5 and 6) was characterized at several passages for the collagen types it produced. The newly synthesized proteins labelled with
[3H]proline and accumulated in the culture medium (A and C) or in the cell and matrix layer (B and D) were submitted to a limited pepsin
digestion following which aliquots were treated with crude tissue collagenase. Fluorographs of the denatured proteins were prepared after their
separation by electrophoresis. Identical sample volumes were deposited in each well without correction for cell density variation with increasing celt
passages. Type I collagen partially digested by animal collagenase was used as a collagen standard (well cs): "~, [~, M and c~2 symbols correspond
respectively to trimers, dimers and monomers of the typical M(I) and ~2(l)chains: their 3 / 4 equivalents are the 3 / 4 fragments generated by animal
collagenase. ]4C-Methylated proteins were used as globular protein standards (gs); their M r- 10 3 are indicated on the right. The pepsin-resistant
(A D) and animal collagenase-resistant (C and D) collagens and peptides are fractionated in the wells 0 *, 0. 1, 2, 3, 5 and 7, indicating cell
passage number. Fluorographs of 0 * wells were exposed for 2 days and those of all other samples for 8 days. They display different bands (bands
A M) characteristic of the following peptides or collagen chains: A='((I,II,II1); B=/~(I,II); C = M ( V , X I ) ; D = ~ 2 ( X I ) ; E = c d ( l . [ I ) , c~2(V),
c~3(XI); F = c~2(I); G, H and I = respectively 108, 83 and 63 kDa peptides: J = ~2(g), c~3(XI); K = 3 / 4 M(I,II,III), [1/4 cd(lll)]3; L = 3 / 4 ~2(ll
and fragments from M(II); M = fragments from al(II). The M, of the peptides migrating in the G. ft and 1 bands were estimated by comparison
with the globular protein standards. Band H disappeared after reduction by dithiothreitol (not shown).

The presence of some newly synthesized cd(V) and With increasing cell passages, the pattern of collagen
c~2(V) chains, comigrating respectively with the M(XI) types synthesized changes progressively. Chondrocytes
and the ~3(XI) chains is possible, because the ratio of in the first passage still produce types II and Xl (possi-
M(XI) to ~2(XI) or c~3(XI) to ~2(XI) chains is greater bly also type V) collagens but no type I. They have
than 1:1. The absence of reducible "},-components in initiated synthesis of type Ili collagen, visible almost
the gels indicates that type III collagen is not synthe- exclusively in the media as y-components transformed
sized by the cells in primoculture. Identical collagen by dithiothreitol reduction (not shown) into al compo-
bands were observed in gels run under reducing condi- nents comigrating with the M(I) chains. With further
tions (not shown). passages, the synthesis of type III collagen increases
 271

progressively as well as that of type I collagen, demon- dedifferentiated in the first passage and we occasionally
strated by the presence of ct2(I) chains in both matrix saw chondrocyte cultures that remained essentially dif-
layers and media. Conversely, the synthesis of type II ferentiated in the second passage.
collagen decreases progressively. Types I and III col-
lagens are digested by a crude mouse bone collagenase Production of active collagenase and collagenase inhibitor
preparation but, in contrast to type II collagen, they Free active collagenase was not detectable in the
produce only typical 3 / 4 ct fragments. Interestingly, the medium from non-stimulated chondrocytes (not shown).
3 / 4 etl fragments of (pepsinized) type II collagen are The fact that no active collagenase (nor proteo-
further degraded into smaller pieces by either the col- glycanase) was produced by these cultures, was ascer-
lagenase or, more likely, by contaminating proteinases tained by culturing chondrocytes from different pas-
such as proteoglycanase (stromelysin) or gelatinase, sages on 14C-labelled type II or type I collagen/
known to be present in the crude collagenase prepara- [3H]proteoglycan-coated plates. Regardless of passage
tion [24]. The disappearance of these smaller pieces number, the cells did not degrade either substrate (Fig.
after the fifth passage provides evidence for the virtual 4A), although they all did after stimulation with IL-1
switching off of type II collagen synthesis after this (shown for first-passage cells in Fig. 4B). In a unique
passage. A progressive reduction in type XI collagen case (not shown), we observed degradation of type II
synthesis occurs in parallel, shown by the disappearance collagen by non-stimulated chondrocytes in the 9th
of the a2(XI) chains after the fifth passage as well. The passage, but not by chondrocytes in the 2nd passage,
simultaneous disappearance of al and ct3 chains of type when the cells were cultured in the same experiment on
XI collagen is likely but presumably masked by a type II collagen-coated plates.
progressive increase in the synthesis of type V collagen. The absence of active collagenase in the medium can
Several unidentified peptides, resistant to bacterial col- be related to the presence of the inhibitor TIMP pro-
lagenase (not shown) as well as to pepsin, are produced duced by the chondrocytes [10]. This production was
by chondrocytes after the second passage in parallel always higher in cultures with serum, either FCS or NU
with their dedifferentiation (Fig. 3, bands G, H and I). serum, than in its absence (compare cell lines I and III
It should be noted that, from one experiment to to the others in Fig. 5). Regardless of the culture media,
another, the dedifferentiation of chondrocytes may be the production of TIMP increased steadily from passage
initiated at slightly different passages. We frequently to passage (Fig. 5I-VI). The inhibitor was almost com-
observed chondrocyte populations which were half- pletely in the free form as its amount did not increase

~ ~oo 100
"6 (~ 80 80
60
== 8 ,o 40
~ 2o 20
,,, 0 0
"r
I I I l l I I I I I l l I I
o
z lOO - -
- 100
a
Lu z 80 80
GO uJ
< (-9 60 60
"'
rr" ~0 40 40
in"" o 20
~lt~ .
~ ~-- , ~- . 20
s t.lr-- ~lr I_lr
m m
0
~0 I I I I I I l I I I l I l l
0 2 4 6 8 10 12 0 2 4 6 8 10 12

TIME IN CULTURE ( days )

Fig. 4. Degradation of collagen and proteoglycans by chondrocytes at different passages and after IL-1 stimulation. 50000 chondrocytes were
cultured for the time indicated in 2 ml of serum-free medium [11] on []4C] (type II) collagen/[3H]proteoglycan - (experiment A) or [14C] (type I)
collagen/[3H]proteoglycan-coated plates (experiment B). Substrate degradations were evaluated by the release of soluble 14C- or 3H-labelled
materials from the plates and expressed as a percentage of the total amount of substrate initially present in the wells. Each point is the mean of
three cultures; vertical bars correspond to ___S.D. and are shown only if their magnitude exceeds the size of the symbol used for the experimental
points. Arrows show the maximal release of 14C-soluble products achieved by trypsin (50 t.tg/well). In experiment A, chondrocytes were cultured
without stimulant at passages 1 (o), 2 (zx), 4 (tz) and 8 (=); in experiment B, first-passage chondrocytes were stimulated with 0 (m), 0.3 (~), 3 (O) or 30
(A) U of IL-1/ml.
 272

12 - II 1.2
10 10 _~
8 08 ~
Go
~ 6 0.6 ~
4 0.4 E
2 - 0.2
- 0.0
I I I I I I . I I I I I I

5 - III 10 - 1.0
4 ,0 - 0.8
GO 3 - 06 '-°o
• .0
2 -04
E E
- 02
- 00
I I 1 I 1 I . I I I l I I

1,0 - IJI -05

/
0.8 -- 04

~°o 0.6 -- 0.3 Go

0.4 --02
E E
D 0.2 .-01
z3
0.0 - 0.0
I I I I I I I I I I I I
0 2 4 6 8 10 0 2 4 6 8 10
passage number passage number

Fig. 5. Production of procollagenase and TIMP by chondrocytes during their dedifferentiation in monolayer culture. The 6 chondrocyte cell lines
(Fig. 1) were considered independently for their procollagenase and collagenase inhibitor production (I VI). In 1, conditioned media were produced
from the 2nd to the 9th day of culture in DMEM with 10% FCS; before assays, they were freezed and thawed several times in order to denature
their a2-macroglobulin content. In II to VI, conditioned media were produced at or near confluence for 2 days in DMEM without (full lines) or
with (dotted lines) 10% NU serum (a serum devoid of active collagenase inhibitors). Free TIMP was measured in all media without other treatment
(o) or after optimal prometalloproteinase activation ( o ) with APMA alone (IV to VI) or APMA and trypsin (I). In II to VI, procollagenase (~) was
measured after neutralization of the free inhibitor present by binding to active tissue metalloproteinases added to the media prior to activation of
the proenzyme with trypsin. Results are expressed as a function of cell passage number, in I, in U / m l , as the cell numbers were increasing during
conditioned media production, and in II to VI, in U / m l per 106 cells, as the latter cell lines were studied at or near confluence.

10 III 5

/
8 4

GO 6 3 Go

4 2
E
2
0
1 I I I 1 1 I 1 I I I I

5 -- ~ I0 _ ? IJl 12 5
4 1O0

/
Go 3 0.75 G o
2 - 0.50
E
3 1 - 0.25 D
- 0.00
- I I I I I I t 1 I I I I I
0 2 4 6 8 10 0 2 4 6 8 10
passage number passage number

Fig. 6. Production of procollagenase by chondrocytes stimulated with IL-1 during their dedifferentiation in monolayer culture. In parallel with the
experiments reported for non-stimulated cells in Fig. 5, the chondrocyte cell lines I1, II1, IV and VI, cultured at or near confluence in DMEM, were
stimulated for 2 days with 100 U of lL-1/ml. Procollagenase was assayed in the conditioned media as done for the corresponding non-stimulated
cells. Results are expressed as a function of cell passage number in U / m l per 106 cells.
 273

by heating the media at acid pH (not shown). The fragments from type IX collagen [29] were not identifia-
absence of bound TIMP suggests again that only ble in our fluorographs, unless sometimes after long
minimal or no active collagenase ( a n d / o r other tissue exposure (not shown). This absence was apparently not
metalloproteinases) was formed in the cultures. due to the technical procedure used (unpublished data).
It is therefore likely that our chondrocytes did not
Spontaneous production of procollagenase and response to synthesize significant amounts of type IX collagen, at
IL-1 least during the labelling period with [3H]proline. Dif-
Treatment of media with APMA a n d / o r trypsin ferentiated chondrocytes simultaneously produced other,
(which activates the proenzymes of collagenase and non-collagenous proteins among which the collagenase
other tissue metalloproteinases under conditions where inhibitor TIMP, but no or very low levels of procol-
it does not inactivate TIMP [10]) did not elicit measura- lagenase. Thus, their metabolic behaviour is oriented
ble collagenase activity in either differentiated primo- towards the synthesis and preservation of the cartilage
cultures or dedifferentiated cultures from later passages. matrix collagen.
The inactivation of free TIMP by complex formation As expected [26,30,31], the subsequent monolayer
with an exogenous metalloproteinase, before activation cultures of chondrocytes caused their progressive dedif-
of procollagenase by trypsin, did not elicit collagenase ferentiation. This was manifested by changes in the
activity in media from differentiated primocultures collagen types synthesized, as described previously by
analyzed at or near confluence (Fig. 5II-VI). With others [4,7,26,27,30,31]. Furthermore, it involved a re-
increasing passage numbers and cell dedifferentiation, duction in the ratio of newly synthesized collagen to
collagenase became detectable, after this treatment, in newly synthesized non-collagenous proteins. This reduc-
only two (lines II and III) out of the five cell lines tested tion is explained by the progressive disappearance of
at or near confluence. However, preliminary evidence the abundant synthesis of types II and XI collagens and
(not shown) suggests that low levels of collagenase can its replacement by only low productions of types I, III
be produced transiently by differentiated cells, and in and V collagens. Moreover, the ratio of newly synthe-
relatively larger quantities by dedifferentiated cells, be- sized collagen incorporated into the matrix to that
fore approaching confluence. released into the culture medium decreased in parallel.
When collagenase was detected (as for cell lines II This decrease manifested principally the shifts from
and III of Fig. 5), its activity remained lower than that types II and XI collagens, mainly deposited in the
of the free TIMP present in the same medium. In the matrix and cell layers, to types I and V collagens, more
four other cell lines (Fig. 5I, IV, V and VI), treatment of soluble in the culture medium, and to type III collagen,
the media with APMA, followed or not by trypsin, almost exclusively present in the medium. Nevertheless,
decreased the amount of free TIMP measurable in late the proportions,of type II plus type I collagens and of
passage cultures but not in primocultures. type XI plus type V collagens remained constant at all
Production of procollagenase could be induced by passages, the former representing about 60% of the total
IL-1 in all chondrocyte cultures; it was optimal using an collagen produced and the latter about 30-40%. The
IL-1 concentration of 100 U / m l regardless of the cell proportion of type III collagen never exceeded 10% of
passage number (not shown). However, the procol- the total collagen synthesized.
lagenase response of differentiated chondrocytes in Changes in production of collagenase inhibitor and
primoculture was always lower than that of dedifferenti- procollagenase and shifts in collagen synthesis were
ated cells in later passage cultures (Fig. 6). The evaluated in the same chondrocyte cultures while they
chondrocyte production of TIMP was unaffected by progressively dedifferentiated throughout subcultures.
IL-1 [25] and usually lower (in U / m l ) than that of Articular chondrocytes have been previously shown to
procollagenase. produce TIMP [10,32,33] but they produce only very
low or undetectable levels of collagenase, unless
Discussion activated by cytokines (most notably, IL-1) or other
agents [1,5,8,25,33,34]. However, in most of these stud-
Serial subcultures allowed us to establish a parallel ies, the phenotype of the chondrocytes was not con-
between the loss of the differentiated phenotype of trolled, although the shift from a differentiated to a
rabbit articular chondrocytes, as expressed by their modulated phenotype was sometimes suspected [34].
production of collagens, and the modulation of their Also, problems arising in enzymatic assays of col-
production of collagenase inhibitor and procollagenase. lagenase or TIMP [10], due to the possible simultaneous
The differentiated phenotype expressed by chondro- presence of (pro)collagenase and inhibitor in the culture
cytes in primoculture (Table I) was characterized by medium, were generally not discussed.
abundant synthesis of types II and XI collagens [26]. Using modified assay procedures established to
Apparently some type V collagen was also produced, in improve the recovery of both TIMP and procollagenase
agreement with other reports [27,28]. Pepsin-resistant in medium where they co-exist [10], we have shown here
274

that fully differentiated, non-proliferating chondrocytes zymogens were not significantly activated by the
in primoculture secrete some T I M P but no detectable chondrocytes in culture and that they did not 'auto-
procollagenase. Interestingly, preliminary evidence sug- activate' in the media (a fact confirmed in nonreported
gests that low levels of procollagenase are produced by experiments). Moreover, in non-stimulated subcultures.
proliferating chondrocytes before they approach con- the level of free collagenase inhibitor recovered was
fluence, but the significance of this observation remains always higher than that of procollagenase when the
to be established. The production of procollagenase by latter was found. It is thus not surprising that, even
differentiated cells can, however, be induced by IL-1. In after full activation of the proenzyme, no collagenase
parallel with their dedifferentiation, the chondrocytes activity could be detected in the media unless we had
progressively increase their production of TIMP. Dedif- neutralized the inhibitor before the activation of procol-
ferentiated chondrocytes also respond to IL-1 by greater lagenase. It is interesting that non-stimulated chondro-
production of procollagenase than differentiated cells, cytes from various passages, cultured on [14C]collagen-
suggesting an increased sensitivity to the cytokine. A coated plates, did not degrade collagen, as would be
'spontaneous' (i.e., not induced by exogenous IL-1) expected if T I M P was in excess over the potential
induction of procollagenase production was observed in collagenase activity produced by the cells. Thus, the
some cell lines after the first or second subculture and balance between production of procollagenase, its rate
this production increased with further dedifferentiation. of activation into collagenase (a problem not investi-
This could result from an autocrine stimulation of the gated in the present study) and the production of TIMP,
cells by IL-1, since this cytokine can be produced by is obviously important for maintaining the collagen
chondrocytes [35]. Indirect evidence was obtained sug- matrix surrounding cells. We observed here that non-
gesting that, even in the absence of a detectable 'spon- stimulated dedifferentiated chondrocytes can sometimes
taneous' induction of procollagenase, the synthesis of increase their production of procollagenase, although
other latent tissue metalloproteinases can occur in they usually keep the balance in favour of preserving
dedifferentiated chondrocyte cultures. Treatment of the surrounding matrix by maintaining the production
their medium with A P M A followed (or not) by trypsin, of inhibitor at a higher level than that of procol-
under conditions where these agents do not destroy lagenase. It was only through IL-I stimulation that
T I M P but activate the zymogens of tissue metallopro- more proenzymes than T I M P were produced [25] and
teinases [10], caused a reduction in the amount of free that active metalloproteinases appeared and directly
TIMP. This effect, which was not observed with medium degraded the [14C]collagen and [3H]proteoglycans
from differentiated primocultures, is likely due to the coated on the culture plates.
activation of some tissue metalloproteinases (such as
proteoglycanase or gelatinase) that upon activation form
Acknowledgements
irreversible complexes with free T I M P [10]. Due to the
excess of free T I M P present, it was, however, not possi-
ble to assay these enzymes in the medium after the This work was supported by the Fund for Medical
activation of their latent proenzymes, because the exog- Scientific Research (Belgium) and by the Belgian State-
enous metalloproteinase preparation used to neutralize Prime Minister's Office Science Policy Programming
T I M P prior to the assay of procollagenase is itself active (interuniversity attraction poles, grant No. 7bis and
concerted actions, grant No, 88/93-122). V.L. was a
on proteoglycans and gelatin [21].
Research Fellow of the Institut pour l'Encouragement
Taking evidences altogether, it appears that dedif-
de la Recherche Scientifique dans l'Industrie et l'Agri-
ferentiated chondrocytes are more likely to switch to-
culture ' I R S I A ' . We gratefully acknowledge the expert
wards a 'catabolic' behaviour characterized by enhanced
technical assistance of F. Chenut and B. Dieudonn6 and
production of procollagenase in response to IL-1, possi-
the skillful secretarial work of Y. Marchand. Our thanks
bly even by a spontaneous induction of zymogen pro-
also go to Dr. K. Willard-Gallo for checking the
duction. This could be relevant to the pathological
manuscript.
events occurring in osteoarthritic cartilage, where foci of
type I collagen-producing dedifferentiated chondrocytes
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