J. Embryol exp. Morph. Vol. 63, pp. 181-191, 1981
Printed in Great Britain © Company of Biologists Limited 1981
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Proteoglycan synthesis by sternal chondrocytes
perturbed with vitamin A
By N. S. VASAN 1
From the Department of Anatomy, CMDNJ-New Jersey Medical School
SUMMARY
Proteoglycan synthesis by sternal chondrocytes was studied in the presence of excess
vitamin A (10 i.u./ml). Proteoglycans synthesized by the treated cells were smaller, and
had larger amounts of chondroitinase ABC-resistant materials than control cells. Vitamin
A-pretreated cells, when provided with normal feeding medium, failed to revert back to
normal morphology and synthetic processes. Chrondrocyte cultures prelabelled with [35S]sulphate, when maintained in the presence of excess of vitamin A, showed: (1) increased
release of labelled proteoglycans into the medium, and (2) increased (19%) degradation of
the proteoglycans. The proteoglycans synthesized by the vitamin A-treated chondrocytes are
also incapable of binding with exogenous large molecular weight hyaluronic acid. Thus, high
levels of vitamin A modulate the differentiation of chondrocytes by altering cellular synthetic
processes.
INTRODUCTION
Vitamin A has a well-documented effect upon cartilage, both in vivo and in
vitro. Vitamin A in excess causes resorption of cartilage (Wolbach, 1947), loss
of extracellular glycosaminoglycans (Fell & Mellanby, 1952; Goodman, Smith,
Hembry & Dingle, 1974), and increases the synthesis and release of lyzosomal
enzymes (Lucy, Dingle & Fell, 1961; Fell & Dingle, 1963).
Recently, Solursh & Meier (1973) have reported that vitamin A in excess
inhibits mucopolysaccharide synthesis and enhances the degradation of
cartilage matrix in chondrocyte cultures. In an earlier investigation we have
reported that vitamin A in excess depresses the synthesis and increases the rate
of degradation of mucopolysaccharide, the greater effect being on the synthesis
of mucopolysaccharide (Vasan & Lash, 1975). Studying the effect of various
levels of retinoic acid on chondrocytes, Shapiro & Poon (1976) concluded that
the effect could best be explained in terms of modification of mucopolysaccharide synthesis, rather than accelerated degradation. It has been reported that
while the proteoglycans synthesized by vitamin A-treated limb mesenchymal
cell culture were smaller, the mannosylation of a specific fraction of glycopeptide was stimulated (Pennypacker, Lewis & Hassell, 1978). Vitamin Atreated cells also retained a 240000 dalton surface protein and retarded the
1
Author's address: Department of Anatomy, CMDNJ-New Jersey Medical School,
100 Bergen Street, Newark, New Jersey 07103, U.S.A.
182
N. S. VASAN
appearance of an 80000 dalton surface protein (Lewis, Pratt, Pennypacker &
Hassell, 1978). In this report, using sternal chondrocytes, the effect of excess
vitamin A on the synthesis of proteoglycan molecules has been extended further.
MATERIALS AND METHODS
Chondrocytes were obtained from 13-day-old embryonic chick sterna by the
previously described method (Vasan & Lash, 1975). The freshly liberated
chondrocytes were plated at an initial density of 5 x 104 cells per 60 mm Falcon
tissue culture dish, containing 5 ml of the F12X nutrient medium (Marzullo &
Lash, 1970). The medium was supplemented with 7 % foetal calf serum (GIBCO)
which contained 1 % penicillin-streptomycin and 1 % kanamycin.
Hypervitaminosis A chondrocytes were supplied with F12X feeding medium
containing 10 i.u./ml vitamin A (retinyl acetate) (kindly supplied by Dr. Stanley
Shapiro, Roche Research Center, Hoffman-La Roche, Nutley, N.J.). Vitamin A
was added at zero time, just before plating. The possibility of initial damage was
excluded by the fact that after 24 h of culture both the control and the experimental showed similar plating efficiencies (51 ±3%) (Vasan & Lash, 1975).
Similar results were also obtained by Kochhar, Dingle & Lucy (1968) using
vitamin A-treated fibroblasts. The cultures were fed daily by replacing half the
medium with fresh medium. All cultures were maintained at 38 °C in a humidified atmosphere of 95 % air/5 % CO2 for the experimental periods.
In some experiments, cultures were pre-labelled to study the effect of vitamin
A by exposing 4-day cultures to [35S]sulphate (Na2[35S]O4-carrier free) overnight (16 h) and then washed three times with normal feeding medium, before
adding the medium containing vitamin A (10 i.u./ml). The media collected
after 72 h were frozen at - 7 0 °C until further analyses were made.
Other experiments were conducted to investigate whether the effect of
vitamin A treatment is reversible. For this, cultures were grown in a medium
containing vitamin A (10 i.u./ml) for four days. Then the cell layer was washed
five times with Sims balanced salt solution and the feeding medium before replacing with normal feeding medium, and cultured an additional 72 h. Vitamin
A-treated cultures were also trypsinized (as suggested by Dr P. F. Goetinck,
Storrs, Conn.), replated at the initial density and provided with normal medium
for 5 days.
In all the experiments the cultures were exposed to media containing 10 /tCi/
ml of Na2[35S]O4 (carrier free - New England Nuclear, Boston, Ma.).
Proteoglycan extraction and analysis
The medium was removed, the cultures were washed twice with 2 ml of saline,
and the washes were pooled with the medium. Proteoglycan was precipitated
using cold ethanol (final concentration 70%) containing 1-3% potassium
acetate (Kimata, Okayama, Oohira & Suzuki, 1974). The precipitate was dis-
Proteoglycan synthesis by vitamin A-treated chondrocytes 183
solved in 4-0 M guanidinium chloride (GuHCl) containing proteolytic inhibitors
(Oegema, Hascall, Dziewiatkowski, 1975) to solubilize the proteoglycans. The
insoluble residue was removed by centrifugation and the extract was dialysed
against 0-5 M-NaCl before being subjected to column chromatography.
The proteoglycan from the cell layer was extracted using 4-0 M-GUHC1
solution buffered with 0-05 M sodium acetate, pH 5-8, containing proteolytic
inhibitors, at 5 °C for 24 h. The insoluble material was removed by centrifugation and the extract was subjected to various analyses after 48 h of dialysis.
Characterization of [35S]sulphate-labelled glycosaminoglycans
The relative amounts of chondroitin 4-sulphate and chondroitin 6-sulphate
synthesized by cell cultures incubated in the presence of [35S]sulphate were
determined by the enzymatic method of Saito, Yamagata & Suzuki (1968) using
chondroitinase ABC and AC (Miles Laboratories, Inc.).
Molecular sieve chromatography of proteoglycans
Column chromatography was performed using controlled-pore glass beads
(CPG-10-2500; Electronucleonics, Fairfield, N.J.) according to the procedure
described earlier (Vasan & Lash, 1979). The elution salt, 0-5 M-NaCl contained
0 0 2 % sodium azide. The eluate was collected in 1-0 ml fractions and 35S.
incorporation measured by liquid scintillation counter. In some experiments an
aliquot was counted and the remainder used for other analyses.
Preparation of Ax-Dx proteoglycan monomers
Proteoglycan monomers (A^-D]) were obtained by the method of Hascall &
Heinegard (1974). Proteoglycan extracted with 4-0 M-GUHC1 was first dialysed
against 100 volumes of 0-5 M-GUHC1 buffer, pH 5-8, (containing proteolytic
inhibitors) for 24 h at 4 °C. An aggregate (Ax) fraction was then prepared with
associative caesium chloride density gradient centrifugation (initial density
1-65 g/ml, 40000 rev./min, 15 °C, 48 h in a fixed angle rotor). The Ax fraction
was subsequently partioned in a dissociative gradient (initial density 1-52 g/ml,
40000 rev./min, 15 °C, 48 h, fixed angle rotor) to separate monomeric proteoglycans (Ai-D]). This monomer was subjected to extensive dialysis and
lyophilized.
Interaction ofA1~D1 Fractions with hyaluronic acid
[ S]sulphate-labelled proteoglycan monomer (A^Di obtained as described
above) in 4 0 M-GUHC1 was mixed with 1 mg of unlabelled proteoglycan
monomer obtained from 14-day embryonic chick sterna. To this, hyaluronic
acid (1 mg/ml in 4-0 M-GuHCl) was added. (High-molecular-weight cockscomb
hyaluronic acid was kindly provided by Dr E. A. Balaz, New York, U.S.A.).
The mixtures, as well as controls (A^Di alone) without hyaluronic acid, were
set at room temperature for 2 h. They were dialysed against 0-5 M-NaCl at 4 °C
35
184
N. S. VASAN
(with two changes of dialysing solution, solvent conditions which facilitate the
interaction of proteoglycans monomer with hyaluronic acid (Hardingham &
Muir, 1972; Vasan & Lash, 1978, 1979). The samples were chromatographed
on a CPG-10-2500 Column and the eluted fractions analysed for radioactivity.
RESULTS
Morphology and matrix accumulation
Chondrocytes supplied with normal medium exhibited the typical polygonal
morphology and accumulated precocious amounts of hyaline matrix after 48 h
of culture (Fig. 1 a). Chondrocytes exposed to vitamin A became flattened and
stellate within the first 24 h of culture (Fig. 1 b) and failed to accumulate extracellular matrix. After 4 days of vitamin A exposure, chondrocytes failed to
return to normal morphology even after 48 h of feeding with control medium
(Fig. 1 c). There was also an inhibition of [35S]sulphate incorporation into
cetylpyridinium chloride-precipitable glycosaminoglycans and an increase in the
release of glycosaminoglycans into the medium due to vitamin A exposure.
Trypsinization and replating of vitamin A-treated cells, provided with normal
feeding medium neither changed the cellular morphology to normal polygonal
shape nor resulted in the accumulation of hyaline matrix (Fig. 1 d).
Molecular sieve chromatography of proteoglycans
To determine more accurately the molecular size distribution during chondrogenic expression, molecular sieve chromatography was used to characterize the
proteoglycans. Using the technique of separation by means of controlled pore
glass beads (Vasan & Lash, 1978, 1979; Lash, Ovadia & Vasan, 1978) it is now
possible to resolve proteoglycans into an excluded large aggregate fraction
(PG-A), an included intermediate size fraction (PG-1) and a monomer fraction
eluted close to the total volume (PG-M). Proteoglycans extracted from the
control culture showed the presence of large proteoglycan aggregate (PG-A) and
an intermediate size population (PG-I) included in the column which eluted
close to the void volume (Fig. 2). On the contrary proteoglycans extracted from
vitamin A-treated culture resolved into only one included peak eluted close to
the total volume (PG-M) (Fig. 2).
Fig. 1. Phase-contrast micrographs showing the morphology of chondrocytes
grown (a) 6 days in F12X medium; (b) F12X supplied with 10 i.u./ml of vitamin A,
(c) and cultures pretreated with vitamin A for 4 days and again cultured in normal
F12X medium for over 48 h. Control chondrocytes show a typical polygonal
morphology with surrounding hyaline matrix (a) while the vitamin A-treated
culture (b) becomes fibroblastic with very little matrix. Vitamin A-treated cultures
when provided with normal feeding medium (c) or trypsin treated and then provided
with normal feeding medium (d) did not reverse the cellular morphology to normal
polygonal shape, x 240.
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i
Fig. 1. For legend see opposite.
oo
O
1
1
I
5
3"
186
N. S. VASAN
0
10
:0
f
30
40
50
60
^0 |
SO
Fig. 2. Molecular sieve chromatographic profile of proteoglycans on a CPG-10-2500
column. Proteoglycans from control (solid line) and vitamin A-treated (dotted line)
cell layer were extracted with 4 0 M-GUHC1 as described in the methods and chromatographed. The bars on the top of the graph showing proteoglycan aggregates (PG-A),
proteoglycan intermediates (PG-I) and proteoglycan monomers (PG-M) are the
characteristic of any mature embryonic cartilage.
Table 1. Relative amounts of[35S]suIphated acid mucopolysaccharides present in
the various proteoglycan fractions resolved by CPG-10-2500 column chromatography
Vitamin A ( %)
Control (%)
Type of
mucopolysaccharide
Chondroitin 6-sulphate*
Chondroitin 4-sulphatef
Resistant material!
A
A
PG-A
PG-I
PG-M
PG-A
PG-I
PG-M
32
60
8
33
48
19
—
—
—
—
—
22
43
35
* Determined as the relative amount of radioactivity associated with 6-sulphated disaccharide after chondroitinase enzyme treatment.
t Relative amount of radioactivity associated with 4-sulphated disaccharide.
X Labelled material not degraded by chondroitinase enzyme.
The proteoglycans isolated by molecular sieve chromatography were subjected to chondroitinases AC and MBC digestion and the resultant disaccharides
were separated on a descending paper chromatography. Table 1 represents the
various amounts of [35S]sulphated acid mucopolysaccharide in these fractions.
The small molecular size fraction (PG-M) obtained from vitamin A-treated
culture was composed mainly of chondroitin 4-sulphate and enzyme-resistant
material. A major proportion of this enzyme-resistant material was reported as
heparan sulfate (Pennypacker et ah 1978). Proteoglycan fractions (PG-A and
PG-I) obtained from control cultures showed a higher proportion of chondroitin 4- and 6-sulphate while the enzyme-resistant material was low (Table 1).
Proteoglycan synthesis by vitamin A-treated chondrocytes 187
0
10
20 1
30
40
50
60
70 f
•o
80
>',
Fractions
Fig. 3. Elution pattern of proteoglycans by CPG-10-2500 Column. Chondrocytes
were grown in medium containing 10 i.u./ml vitamin A for 4 days. Then the cell
layer was washed five times with normal medium and the chondrocyte recovery was
studied. There was no morphological recovery (Fig. 1 c). Cultures were exposed to
radioactive sulphate for 16 h and the proteoglycans in the cell layer analysed after
24 h (A), 48 h ( • ) and 72 h (O).
6
Pi; A
Pu 1
Pi; M
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5
-
4
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i
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Fraction!
Fig. 4. Cultures were grown in normal medium, prelabelled with radioactive
sulphate and then maintained in the presence of 10 i.u./ml of vitamin A for 72 h.
The proteoglycans in the medium were then subjected to potassium acetate-ethanol
precipitation. The elution profile on pattern of control (solid line) and vitamin Atreated (dotted line) are shown in this figure.
Additional experiments were carried out to determine whether the alterations
noticed in proteoglycan synthesis due to exposure of the chondrocytes to
vitamin A could be reversed. In the 72 h period with normal medium there was
no reversal of phenotypic expression observed (Fig. lc). At the same time the
type of proteoglycan synthesized, after 24, 48, and 72 h feeding with normal
188
N. S. VASAN
:o f
30
70 t
V,
Fig. 5. CPG-10-2500 Column Chromatography of proteoglycan monomers (A^DO
from control (dotted line) and vitamin A treated (dotted line with open circle (O)).
Upon the addition of hyaluronic acid, there is significant interaction, and larger
aggregates are formed in control culture (solid line) while there is no change in the
vitamin A-treated culture (solid line with solid circle (•)).
medium did not result in any appreciable change in the distribution of molecular
sizes (Fig. 3).
Investigation was further extended to probe whether the small molecular size
proteoglycans observed due to vitamin A treatment were the result of degradation of chondrocyte proteoglycans or a defect in the synthetic mechanism.
Normally grown, prelabelled (35S) chondrocytes were exposed to vitamin Acontaining medium for 72 h and the proteoglycan in the medium was analyzed.
In addition to the increase in the release of pre-labelled proteoglycans, vitamin A
also caused a certain amount of degradation (19 %), resulting in the small
molecules eluted close to the total volume (Fig. 4).
The decrease in the size of proteoglycans synthesized by the vitamin Atreated chondrocyte culture could be due to the lack of ability to form large
aggregates. One of the reasons for such failure could be due to the failure of the
synthesis of proper core protein which possesses the important hyaluronic acidbinding region. To test this hypothesis, the monomeric fraction ( A J - D J ) obtained after CsCl-GuHCl gradient centrifugation was mixed with cockscomb
hyaluronic acid and subjected to column chromatography. Proteoglycan
monomer (A^Dj) obtained from vitamin A-treated chondrocyte culture failed
to show appreciable affinity for hyaluronic acid while the control A ^ D i
fractions showed hyaluronic acid-binding capacity (Fig. 5).
DISCUSSION
Inhibition of chondrogenic differentiation using excess of vitamin A has been
well documented (Solursh & Meier, 1973; Vasan & Lash, 1975; Pennypacker
Proteoglycan synthesis by vitamin A-treated chondrocytes 189
et al. 1978; Lewis et al. 1978). Treated cells in cultures did not develop cartilage
nodules with predominant quantities of hyaline matrix. Instead of polygonal
morphology (Fig. 1 a) they became flattened and stellate (Fig. 1 b). Vitamin Apretreated cultures when postcultured with normal feeding medium for 72 h,
or trypsinized and cultured in normal medium, did not show any change in
morphology (Fig. 1 c, d). When well differentiated colonies of chondrocytes were
picked from the primary cultures and subcultured in the presence of vitamin A,
there were alterations in the morphological and biochemical synthetic mechanisms as seen in the present study (work in progress) (Solursh & Meier, 1973).
Hence the observations made in this report may not be due to the selection of
chondrocytes by the normal medium and non-chondrocytes by the Vitamin A
medium. Recently, Jetten, Jetten & Sherman (1979) showed that retinoic acid
causes phenotypic changes in embryonal carcinoma cells (an undifferentiated
stem cell of teratocarcinomas). The observed phenotypic alteration induced by
retinoic acid is maintained, even several generations after retinoic acid is removed
from the culture medium. The absence of a cartilagenous matrix is mainly due to
the inhibition in the synthesis and not due to an extraordinary degradation (Vasan
& Lash, 1975). The alteration in the cellular morphology seen in the treated
culture could be due to the retention of high molecular weight glycoprotein
involved in cell-cell adhesion and, therefore, the maintenance of close cell
contacts (Lewis et al. 1978; Hassell, Pennypacker, Yamada & Pratt, 1978).
Vitamin A-treated chondrocyte cultures failed to elaborate large aggregated
proteoglycan molecules. Chromatographic separation of proteoglycan extracted
from the treated chondrocytes showed radioactive sulphate incorporation into
small size molecules (Fig. 2). This is similar to the proteoglycan molecule isolated
and fractionated from limb mesenchymal cells (Pennypacker et al. 1978),
stage-18 and -24 limb buds (Vasan & Lash, 1979), tendon fibroblast and myoblast cultures (Vasan, unpublished observations) and virus transformed somite
cultures (Yoshimurae/ al. 1980). Chondrocyte cultures pretreated with Vitamin
A, then supplied with normal medium failed to show any appreciable changes
in their phenotypic (Fig. 1 c) or genotypic expression up to 12 h (Fig. 3).
Small-molecular-size proteoglycan obtained after vitamin A treatment
resembled that of the mesenchymal or non-chondrogenic cells. This may be the
result of changes in synthetic processes or increased degradation. Vitamin A
causes an enhancement in the release of proteoglycan into the medium (Vasan
& Lash, 1975; Pennypacker et al. 1978). When prelabelled (35S) chondrocytes
were exposed to vitamin A for 72 h, the released 35S-labelled proteoglycan
showed some small-size proteoglycans eluted close to the total volume (Fig. 4).
At least 19 % of the small-size proteoglycans could be due to the degradation of
the molecules by the lyzosomal enzymes (Lucy et al. 1961; Fell & Dingle, 1963;
Vasan & Lash, 1975). This is in contrast to the recent report of Pennypacker
et al. (1978) where a 48 h exposure to vitamin A of prelabelled culture did not
show any degradation. This discrepancy may be possible because Pennypacker
7
EMB 63
190
N. S. VASAN
et ah (1978) used only the Aj fraction for column chromatography, and the lowmolecular-weight molecules ( A ^ D ^ were still in the low-bouyant-density
fractions that were not included for chromatography. In the present study, total
proteoglycan was used without partitioning by gradient centrifugation.
Proteoglycans synthesized by mesenchymal tissues (stage-18 and -24 limb
buds) were predominantly small molecular size and failed to show any appreciable amount of hyaluronic-acid-binding capacity (Vasan & Lash, 1979). It has
been recognized, that hyaluronic-acid-binding by proteoglycan monomers is
necessary for the large aggregate formation. The decrease in hydrodynamic size,
and the elution close to the total volume are typical features of the proteoglycans obtained from vitamin A-treated culture. This could be due to a decrease
in the length of core protein. The decrease in the length of core protein could be
either at the hyaluronic-acid-binding region or chondroitin-sulphate-rich region.
Dissociated proteoglycan molecules (A^-D^ obtained from vitamin A-treated
chondrocytes showed a decrease in the hyaluronic-acid-binding capacity
(Fig. 5). This could be due to the enzymatic cleavage of the protein region or due
to the failure of the synthesis of the hyaluronic-acid-binding region. Thus the
vitamin A-treated culture produces proteoglycans which are not only incapable
of forming large aggregates (as the non-aggregating proteoglycans described by
Muir and coworkers), but chromatographically resemble the non-chondrogenic
type of proteoglycans synthesized by transformed chondrocytes, muscle cells and
tendon fibroblasts (unpublished observations). Work is in progress to gain more
knowledge on the chemical nature of these smaller proteoglycan molecules
obtained from different tissues and chondrocytes grown under various conditions. The result of the present investigation shows, that Vitamin A, in
addition to causing a definite increase in the degradation of proteoglycans, also
inhibited the differentiation of chondrocytes mostly by altering the synthetic
mechanism.
I am grateful to Dr Stanley Shapiro and Dr Peddrick Weis for the critical evaluation of the
manuscript and Dr Paul Goetinck for experimental suggestions. I like to thank Mr Markus
Meyenhofer for assistance in photography. This investigation was supported by a grant
from National Institute of Health Biomedical Research Support 5 S07 RR05393 to the
College of Medicine and Dentistry of New Jersey, New Jersey Medical School.
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(Received 11 August 1980, revised 28 November 1980)
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