/. Embryol exp. Morph. Vol. 53, pp. 91-102, 1979
Printed in Great Britain © Company of Biologists Limited 1979
91
Reversible inhibition of chondrogenic expression
by certain hyaluronidase preparations
By JOHN P. PENNYPACKER 1 AND PAUL F. GOETINCK 2
From the Laboratory of Developmental Biology and Anomalies,
National Institute of Dental Research, Bethesda
and the Department of Animal Genetics, University of Connecticut
SUMMARY
Embryonic chick chondrocytes were cultured in the presence or absence of different
preparations of testicular hyaluronidase. This treatment inhibited the accumulation of
cartilage matrix as indicated by phase-contrast microscopy, by Alcian green staining, and by
accumulation of 35S-labeled material. In addition, most preparations of testicular hyaluronidase caused a conversion of the cells to a fibroblastic phenotype characterized by a faster
growth rate and the synthesis of type-1 collagen. This effect was found to be concentrationdependent and was not observed at the minimum concentration of hyaluronidase required to
inhibit matrix accumulation. Since two more highly purified hyaluronidase preparations
prevented matrix accumulation but did not cause the fibroblastic transformation, it is likely
that the conversion to a fibroblastic phenotype is caused by a contaminant in the other
hyaluronidase preparations.
INTRODUCTION
The extracellular matrix of cartilage is composed of three major macromolecular species: proteoglycan, hyaluronic acid, and a unique type of collagen
(Miller & Matukas, 1969; Trelstad, Kang, Igarashi & Gross, 1970; Hascall &
Heinegard, 1974). The proteoglycans of the matrix contain chondroitin-4-sulfate
and chondroitin-6-sulfate with lesser amounts of keratin sulfate linked to
a common core-protein (Hascall & Heinegard, 1975; Rosenberg et al. 1975;
Hascall, Oegema, Brown & Caplan (1976). The organization of the cartilage
matrix involves all of these components and together they provide cartilage with
its unique structural resilience.
More recent studies have suggested that matrix constituents may play other
active roles during chondrogenesis. For example, Kosher, Lash & Minor (1973)
have demonstrated that, in culture, somite chondrogenesis is greatly enhanced
by the addition of proteoglycans to the medium. Furthermore, there have been
several reports that proteoglycan synthesis by cultured chondrocytes is stimu1
Author's address: Laboratory of Developmental Biology and Anomalies, National
Institute of Dental Research, Building 30, Room 414, National Institutes of Health,
Bethesda, Md 20205, U.S.A.
2
Author's address: Department of Animal Genetics, University of Connecticut, Storrs,
Connecticut 06268, U.S.A.
92
J. P. PENNYPACKER AND P. F. GOETINCK
lated by exogenous proteoglycans (Nevo & Dorfman, 1972; Huang, 1974;
Schwartz & Dorfman, 1975). Hyaluronic acid has been found to inhibit proteoglycan synthesis when added to chondrocytes in suspension culture (Solursh,
Vaerewyck & Reiter, 1974; Wiebkin, Hardingham & Muir, 1975). Both the
inhibition by hyaluronic acid and the stimulation by proteoglycans are abolished
when the cells are pre-treated with trypsin, suggesting that these interactions
involve trypsin-labile binding sites on the cell surface.
In the experiments to be reported here, the relationship between chondrogenic
expression and the proteoglycan-rich matrix is further explored. Testicular
hyaluronidase, an enzyme which degrades chondroitin-6-sulfate, chondroitin4-sulfate, and hyaluronic acid, was added to the medium of cultured chondrocytes. The resulting effects on chondrogenic expression were characterized. A
preliminary report of this study has been published (Pennypacker & Goetinck,
19766; Goetinck & Pennypacker, 1977).
METHODS AND MATERIALS
Cell culture. Sterna of 14-day White Leghorn chick embryos were dissected
under sterile conditions, stripped of their perichondrium and incubated at
37 °C with gentle stirring in 0-1 % pronase, 0-2 % collagenase and 10 % chicken
serum in Tyrode's solution. After incubation for 45 min, the enzyme solution
was removed after centrifugation, and single cells were obtained by passing the
suspension through two layers of 20 fim mesh Nitex monofilament screen-cloth.
In all experiments, the chondrocytes were plated at a density of 250 cells per
plate in 60 mm Falcon plastic culture dishes. The cultures were maintained at
37 °C in an atmosphere of 5 % CO 2 :95 % air, and the medium was replaced on
the 4th, 7th, 9th and 1 lth days in culture with 50 % Ham's F-10 (Grand Island
Biological Co., GIBCO) with 2 x the vitamins and 4 x the amino acid concentration; 38 % Hanks' balanced salt solution (HBSS); 10 % fetal calf serum
(GIBCO); 0-5 % (w/v) bovine serum albumin; 50 ^g/ml ascorbic acid (Fisher);
and antibiotics (Cahn, Coon & Cahn, 1967). Cultures were usually terminated
after 13 days of incubation and, in some cases, were stained with Alcian green
and metanil yellow (Ham & Sattler, 1968).
Proteoglycan analysis. The total amount of cell-associated sulfated proteoglycan was determined by adding 1 /id of Na35SO4 (New England Nuclear:
NEN) per ml to the culture medium on days 7, 9, and 11. Day 7 was chosen as
the time for the initial exposure as it precedes the period of cartilage matrix
deposition. The isotope incorporated into the cell-associated proteoglycans was
determined by measuring hyaluronidase-sensitive radioactivity. For this purpose,
the 35S-labeled 13-day cultures were cooled to 4 °C, the medium was aspirated
off, and the cultures were washed twice with cold HBSS. The cell material was
then removed from the culture dish into a known volume of cold HBSS using
a Teflon scraper. The samples were placed in a boiling water bath for 5 min to
Reversible inhibition of chondrogenic expression
93
destroy any enzymic activity and homogenized by sonication. From this
sample an aliquot was taken for DNA determination (Burton, 1956). Two
additional aliquots were taken. Testicular hyaluronidase (Calbiochem) was
added to one aliquot at a final concentration of 1 mg/ml in 0 1 M sodium
acetate, 0 1 M-NaCl, pH 50, and buffer alone was added to the other. After
incubation for 24 h at 37 °C, pronase (Calbiochem) was added to each sample
to a final concentration of 1 mg/ml in 0-2 M Tris-HCl, pH 8-2. The samples
were incubated 16 h at 55 °C, and then the radioactive glycosaminoglycans
were precipitated by addition of three volumes of 80 % ethanol containing 5 %
potassium acetate. The precipitates were collected by nitration, dissolved in
water, and the radioactivity was measured by liquid scintillation spectrometry.
The hyaluronidase-sensitive CPM, representing chondroitin-6-sulfate and
chondroitin-4-sulfate, were obtained by subtraction of the CPM after the
hyaluronidase treatment from the total CPM.
Collagen analysis. The relative concentration of collagen in the cultures was
estimated by measuring hydroxyproline content according to the procedure of
Woessner (1961). In addition, the cultures were incubated with 15/*Ci/ml of
2,3-[3H]proline (specificactivity 33-8 Ci/m-mole; NEN) or 2-[3H]glycine (specific
activity 11-4 Ci/m-mole; NEN) to label newly synthesized protein. When
[3H]proline was used, the culture medium was Dulbecco-modified Eagle's
medium supplemented with 10 % fetal calf serum (GIBCO), 50 /*g/ml /?-aminopropionitrile (Aldrich Chemical Co.) and 100/*g/ml ascorbic acid (Fisher
Chemical). With [3H]glycine, Eagle's minimal essential medium was supplemented with 10 % fetal calf serum (GIBCO), 50/*g/ml /?-aminopropiontrile
and 100/tg/ml ascorbic acid. In both cases, cultures were labeled on day-11
for 48 h. Extraction, purification and carboxymethylcellulose (CMC; CM 32,
Reeve Angel) chromatography of the labeled collagen was carried out on
a 1-6 x 7-0 cm jacketed column as described by Linsenmeyer, Trelstad, Toole &
Gross (1973). Carrier collagen was prepared from lathyritic chicks as described
by Kang, Nagai, Piez & Gross (1966).
Liquid scintillation spectrometry. All measurements of radioactive samples
were made by liquid scintillation counting with a Nuclear Chicago (Searle
Analytic) Mark I scintillation spectrometer. The counting solution used was
prepared by mixing two volumes of toluene containing 4-0 g/1 of 2,5-diphenyloxazole and 0-1 g/1 of l,4-di[2-phenyloxazoly])] benzene with one volume of
Triton X-100.
RESULTS
The effect of testicular hyaluronidase preparations on chondrogenic expression
was determined by replacing the normal medium with medium plus enzyme on
days 7, 9, and 11 of culture. Different results were obtained, depending upon the
source of the enzyme employed. In all cases, the presence of added hyaluronidase
prevented the accumulation of a cartilage matrix as judged both by phase
7
EMB53
94
J. P. PENNYPACKER AND P. F. GOETINCK
contrast microscopy and staining with Alcian green. Most preparations of
testicular hyaluronidase also altered the morphology of the cells. Under these
conditions, the chondrocytes became fibroblast-like (Fig. la, b) and formed
dense clones which were two to three times the diameter of the control clones.
However, two preparations of enzyme (Sigma, bovine type IV and VI) did not
affect the chondrocyte morphology (Fig. 1 c), although the matrix was eliminated.
The hyaluronidase preparations tested in this study did not cause detachment
of the chondrocytes from the substratum. A list of the enzyme preparations
tested is shown in Table 1.
The enzyme preparations that caused no morphological changes were also
the most purified hyaluronidases suggesting that the morphological changes
were due to a contaminant in the preparations. This possibility was considered
by testing lower concentrations of a preparation (Calbiochem, Grade B) to
determine if the morphological effects could be eliminated while still preventing
matrix deposition. Matrix accumulation was measured by Na235SO4 incorporation during the period of active proteoglycan synthesis (days 7-13) as well
as by Alcian green staining. Between 00125-0-5jug hyaluronidase per ml of
medium there is a gradual reduction in accumulated sulfated proteoglycans, and
at concentrations of 0-5 /tg/ml and higher, all cell-associated sulfated proteoglycan is eliminated (Fig. 2). Similar results were obtained by staining with
Alcian green and metanil yellow. In both cases, at the minimum enzyme concentration required for inhibition of matrix accumulation (0-5 /tg/ml), there
was no effect on the chondrocyte morphology. Concentrations greater than
0-5 /ig were required to bring about the morphological transition to fibroblastlike cells.
The amount as well as type of collagen synthesized by the cells was determined
at three concentrations of hyaluronidase. Table 2 shows that at 0-025 /tg/ml and
0-5 /*g/ml, there is no difference in hydroxyproline accumulation per /*g DNA.
However, in cultures treated with a concentration (125/*g/ml) sufficient to
bring about morphological changes, the hydroxyproline content is reduced to
less than half the amounts accumulated in control cultures. The type of collagen
synthesized in hyaluronidase-treated cultures was characterized by chromatographing radioactively-labeled collagen on CMC. Collagen extracted
from control cultures (Fig. 3), or from cultures treated with 0-5/tg of
hyaluronidase per ml or less (not shown) co-eluted from the CMC with carrier
skin al(I). The absence of a radioactive a2 peak suggests that all of the collagen
is [al(II)] 3 (Miller & Matukas, 1969). Labeled collagen from cultures treated
with 125 fig hyaluronidase/ml, however, co-eluted with both al and al carrier
collagen peaks in a ratio of approximately 4:1 (Fig. 4). The presence of a2
indicates that the cells were synthesizing type I collagen.
Concentrations of'hyaluronidase' that caused the fibroblastic transformation
also increased the amount of DNA per plate, while lower concentrations did
not change the DNA content (Table 2). Between 5-125 fig hyaluronidase/ml,
Reversible inhibition of chondrogenic expression
95
Fig. 1. Phase contrast micrographs (xl97) of day-13 control cultures (A), and
day-13 cultures treated with 100/*g/ml of Calbiochem, Grade B testicular hyaluronidase (B) or Sigma, type IV testicular hyaluronidase (C).
the DNA content per dish on day 13 increased in a linear relationship with
increasing enzyme concentration (not shown). As expected from this observation
the growth rate of cultures exposed to hyaluronidase (125 /*g/ml) was greater
than control cultures (Fig. 5).
The transforming activity in the hyaluronidase preparations caused chondrocytes to assume a fibroblastic appearance within 24 h. The ability of the cells
7-2
96
J. P. PENNYPACKER AND P. F. GOETINCK
Table 1. Hyaluronidase preparations tested
Source
Calbiochem, Grade B
Lot 200943
Sigma, type I
Lot 116C-0259
Sigma, typs III
Lot 446-0890
Sigma, type IV
Lot 76V-0342
Sigma, type VI
Lot 16C-O176
Activity
Test concentrations
300 i.u.* (u./mg)
100/tg/ml
300 n.f.f (u./mg)
100/tg/ml
610 n.f. (u./mg)
1090 n.f. (u./ml)
57 n.f. (u./mg)
100/fg/ml (61 u./ml)
66/ig/ml (66 u./ml)
100/tg/ml (100 u./ml)
50 units/ml)
* i.u., international units.
t n.f., national formulary units.
100
90
% 70
2
60
1 50
2 40
I 30
1 20
2 10
C
o
c
00125
0-025
005
0125
0-25
Hyaluronidase concentration (j/g/ml)
0-5
Fig. 2. Effect of testicular hyaluronidase (Calbiochem, Grade B) on the accumulation of cell-associated sulfated proteoglycans. Each point is based upon the mean
of at least four determinations.
to recover from the enzyme treatment was determined by exposing 7-day
cultures to hyaluronidase (125 /*g/ml) for 24,48, 72, or 96 h, and then maintaining them in medium without hyaluronidase. After 24 or 48 h, the cells regained
a polygonal morphology and stained with Alcian green at day 13. Beyond the
48 h exposure, the clones of cells were larger in diameter, and there was an
increase in those that did not recover from the treatment. After 72 h, only
a few clones recovered, even when the recovery period was extended. The effects
of the 96 h exposure were irreversible. However, when cells from cultures
exposed to hyaluronidase for at least 96 h were subcultured at low density, they
regained the chondrocyte morphology and deposited an Alcian green staining
Reversible inhibition of chondrogenic expression
97
O-6—i
25
0-4-
20 ?
o
15 Z
o-: -
10 &
20
Fraction number
Fig. 3. Elution profile of collagen extracted from control cultures and chromatographed on carboxymethylcellulose. The chondrocytes were labeled with [3H]proline during the final 48 h in culture.
Table 2. Effect of hyaluronidase on collagen accumulation
Number Hypro/
of
plate
cultures
C"g)
0025/tg hyaluronidase/ml medium
Control
Hyaluronidase
0-5 /tg hyaluronidase/ml medium
Control
Hyaluronidase
125-0 /tg hyaluronidase/ml medium
Control
Hyaluronidase
DNA/plate
Hypro/DNA
0»g)
7
9
0-641
0-542
2-729 ±0-746
2-765 ±0-739
0-239 ±0057
0-192 ±0042
5
6
0-605
0-577
4-610±0-886
4-414±0-735
0-134±0-22
0134±0035
10
10
0-612
1083
2-316±0-682
8-724 ±0-953*
0-261 ±0071
0124±0014*
Values represent mean±s.D. * Significant difference at P < 0001.
matrix. Furthermore, the collagen synthesized was type II, as judged by CMC
chromatography (not shown).
Previous studies on various cell lines have suggested that protease treatment
can stimulate cell division (Burger, 1970; Noonan & Burger, 1973). Proteases
also cause the release of matrix glycosaminoglycans from cartilage rudiments
(Bosmann, 1968). One 'hyaluronidase' preparation (Calbiochem Grade B) was
tested for its ability to degrade protein and proteoglycans under normal culture
conditions. For this purpose purified 35SO4-labeled proteoglycans or [3H]tryptophan-labeled embryo protein (Peterkofsky & Diegelmann, 1971) were
solubilized in culture medium and maintained in the presence or absence of
testicular hyaluronidase in a CO2 incubator for 24 h. Over 90 % of the 35SO4-,
labeled proteoglycans were degraded by the hyaluronidase treatment while
there was no effect on the [3H]tryptophan-labeled protein (Table 3). This
98
J. P. PENNYPACKER AND P. F. GOETINCK
0-6
-
0-4
0-3-
0-2-
01 -
15
T
25
Fraction number
Fig. 4. Elution profile of collagen extracted from cultures treated with 125 fig
hyaluronidase (Calbiochem, Grade B) per ml and chromatographed on carboxymethylcellulose. The chondrocytes were labeled with [3H]proline during the final
48 h in culture.
11
12
Days in culture
13
14
15
Fig. 5. Growth curves of control ( D — • and 125 fig/m\ hyaluronidase (Calbiochem,
Grade B)-treated cultures ( • — • ) . In this experiment, hyaluronidase treatment
was initiated on day 2 of culture. Each point represents the cell number per plate
based upon the cell count of three cultures.
Reversible inhibition of chondrogenic expression
99
Table 3. Enzymic activity in hyaluronidase preparations
CPM/sample
3
[ H]Tryptophan embryo protein*
Control
Hyaluronidase (125 /tg/ml)
35
S-SO4 Proteoglycanf
Control
Hyaluronidase (125 /tg/ml)
1549±136
1528± 33
36507
3673
* CPM/sample precipitated with 10 % trichloracetic acid.
t CPM/sample precipitated with 80 % ethanol containing 5 % potassium acetate.
indicates that hyaluronidase is active under normal culture conditions and
suggests that the transforming activity may not be a protease. The latter
conclusion is supported by the observation that trypsin (0-01 % or 0-001 %) or
pronase (0001 %) alone or in combination with a hyaluronidase preparation
lacking the transforming activity (Sigma, type IV) do not cause the morphological changes. Higher concentrations of either protease cause the chondrocytes
to detach. However, this does not rule out the possibility that a protease with
a different specificity is involved.
DISCUSSION
The stability of the cartilage phenotype in culture is important because it
provides a model system for developmental studies. The cell morphology,
growth in culture, and extracellular matrix constituents have been well characterized and provide phenotypic markers. A variety of treatments, such as embryo
extract, 5-bromo-2'-deoxyuridine, and repeated subculture cause the transition
to a fibroblast-like morphology (Coon, 1966; Mayne, Vail & Miller, 1965;
Mayne, Vail, Mayne & Miller, 1976; Mayne, Vail & Miller, 1976). In general
this transition to a fibroblastic morphology is accompanied by decreased
proteoglycan synthesis and the accumulation of [al(I)]2a2 and [al(I)] 3 collagen.
In this study, higher concentrations of certain hyaluronidase preparations
inhibited chondrogenic expression. The cells became bi-polar, and in many
areas of the colonies, were organized in parallel arrays. Accompanying these
morphological changes, there was an increase in the growth rate, and the
density to which the cells grew. Analysis of the collagen by CMC chromatography indicated a 4:1 ratio of radioactivity in the al and a2 peaks. Although
the collagen was not further characterized in this study, a similar ratio was
observed after embryo extract and 5-bromo-2'-deoxyuridine treatment, suggesting the accumulation of type I trimer (Mayne et al. 1975, 1976).
The fibroblastic transition was found to be reversible after subculture at low
density. While the effect of embryo extract is also reversible after subculture
100
J. P. PENNYPACKER AND P. F. GOETINCK
5-bromo-2'-deoxyuridine is not readily reversible (Mayne, Abbott & Holtzer,
1973). Furthermore, the morphology, organization and growth characteristics
of embryo extract and 'hyaluronidase-' (125/^g/ml; Calbiochem, B grade)
treated cultures are similar (unpublished observations). However, 5-bromo-2'deoxyuridine-treated chondrocytes are more flattened and have a lower confluent density (Daniel, Kosher, Lash & Hertz, 1973). Therefore, similarities
exist between the embryo extract and 'hyaluronidase' effects and their mechanism of action may be similar.
Two hyaluronidase preparations did not cause the fibroblastic transition.
This finding indicates that the effect is due to a contaminant. Several studies
have suggested that the growth of fibroblasts can be stimulated by mild treatment
with proteases (Burger, 1970; Noonan & Burger, 1973). However, neither
trypsin nor pronase alone or in combination with inactive hyaluronidase
preparations caused morphological changes. Furthermore, testicular hyaluronidase (Calbiochem, B grade) had no effect on [3H]tryptophan-labeled embryo
protein. This observation suggests that if the contaminant that caused the
transformation is a protease, it is highly specific.
It is also of interest that at the minimum concentration of hyaluronidase
required to prevent matrix accumulation, the chondrocytes retained a normal
morphology, and synthesized type II collagen. This would suggest that chondrocytes can maintain their normal phenotype in the absence of cell-associated
proteoglycans. This conclusion is supported by studies on chick embryos
homozygous for the autosomal recessive gene, nanomelia (Laundauer, 1965).
In this mutant, the proteoglycan content is 10% or less of normal amounts
(Mathews, 1967). In other respects, the cartilage of the mutant is indistinguishable from normal cartilage (Pennypacker & Goetinck, 1976a). Other studies
have also demonstrated that normal chondrocytes can respond to such a change
in their microenvironment by enhanced synthesis of proteoglycans (Thomas,
1956; Sheldon, Robinson & Afzelius, 1960; Bosmann, 1968; Jackson, 1970).
Studies are in progress to determine what further changes resulting from treatment with crude hyaluronidase preparations initiate altered cellular function
by chondrocytes.
This work was supported by Grant HD 09174 from the N.I.C.H.H.D. and by training
Grant 5-T01-GM00317 to first author, from the N.T.G.M.S. This work was also supported
by NCI CA 14733. This paper is Scientific Contribution no. 672 of the Storrs Agricultural
Experiment Section, the University of Connecticut. The authors wish to thank Drs Claudia
Caputo, Vincent Hascall and George Martin for reviewing the manuscript.
Reversible inhibition of chondrogenic expression
101
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{Received 9 October 1978, revised 22 March 1979)