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Partial reversal by beta-d-xyloside of salicylate-induced inhibition of glycosaminoglycan synthesis in articular cartilage.

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1084
PARTIAL REVERSAL BY BETA-D-XYLOSIDE OF
SALICYLATE-INDUCED INHIBITION OF
GLYCOSAMINOGLYCAN SYNTHESIS IN
ARTICULAR CARTILAGE
MARSHALL J. PALMOSKI and KENNETH D. BRANDT
While net 35S-glycosaminoglycan synthesis in
normal canine articular cartilage was suppressed by
lO-jM sodium salicylate to about 70% of the control
value, addition of xyloside (10-6M-10-3M)
to the salicylate-treated cultures led to a concentration-dependent
increase in glycosaminoglycan synthesis, which rose to
120-237% of controls. Similar results were obtained
when 3H-glucosaminewas used to measure glycosaminoglycan synthesis, confirming that salicylate suppresses
and xyloside stimulates net glycosaminoglycan synthesis, and not merely sulfation. Salicylate (10d3M)did not
affect the activity of xylosyl or galactosyl transferase
prepared from canine knee cartilage, and net protein
synthesis was unaltered by either salicylate or xyloside.
The proportion of newly synthesized proteoglycans existing as aggregates when cartilage was cultured with
xyloside was similar to that in controls, although the
average hydrodynamic size of disaggregated proteoglycans and of sulfated glycosaminoglycans was diminished.
The extracellular matrix of normal articular
cartilage is composed of proteoglycan molecules interFrom the Rheumatology Division, Indiana University
School of Medicine, Indianapolis, IN.
Supported in part by grants from the National Institute of
Arthritis, Metabolism and Digestive Diseases (AM 20582 and AM
27075) and awards from the Arthritis Foundation and the Grace M.
Showalter Trust.
Marshall J. Palmoski, PhD: Associate Professor of Medicine and Anatomy; Kenneth D. Brandt, MD: Professor of Medicine
and Chief, Rheumatology Division, Indiana University School of
Medicine.
Address reprint requests to Marshall J. Palmoski, PhD,
Kheumatology Division, Indiana University School of Medicine,
1100 West Michigan Street, Indianapolis, IN 46223.
Submitted for publication August 3, 1981; accepted in
revised form March 9, 1982.
Arthritis and Rheumatism, Vol. 25, No. 9 (September 1982)
spersed in a network of collagen fibrils. The proteoglycans account for most of the elasticity ofjoint cartilage
and for its ability to withstand deformation (1). In
normal cartilage most proteoglycans exist as high
molecular weight aggregates, in which numerous individual proteoglycans are noncovalently associated
with hyaluronic acid in a reaction stabilized by “link”
glycoproteins (2).
In osteoarthritis, the proteoglycan content of
the diseased cartilage is diminished, and cartilage
matrix is eventually lost. Although some studies have
reported the net synthesis of sulfated glycosaminoglycan in osteoarthritic cartilage to be normal (3,4), in
several instances it has been found to be increased in
the early stages of the disease, before any depletion of
tissue is noted. This increase in matrix metabolism is
thought to reflect an attempt by the chondrocyte to
repair the lesion (5-8). It may be clinically relevant,
therefore, that salicylate, the drug most often used in
the treatment of osteoarthritis, inhibits glycosaminoglycan synthesis (9,lO). We have recently shown, in in
vitro studies, that 10-3M salicylate (which corresponds to a concentration of the drug readily achieved
in the serum of humans) reduced proteoglycan synthesis in normal canine articular cartilage by 30%, with no
effect on proteoglycan catabolism or aggregation ( 1 1).
Notably, under similar conditions the augmented glycosaminoglycan synthesis in osteoarthritic cartilage
was suppressed by up to 90% (8).
Since xylosides can initiate free glycosaminoglycan chain synthesis by substituting for the xylosylated protein core of the proteoglycan, they may be
used to assess the potential of a cell to synthesize
glycosaminoglycans independent of restrictions imposed on it by availability of the protein core (12-16).
GAG SYNTHESIS
In an attempt to elucidate the possible mechanism(s)
of action of salicylate on cartilage metabolism, we
examined the effect of beta-D-xyloside on articular
cartilage in which glycosaminoglycan synthesis was
suppressed by sodium salicylate.
MATERIALS AND METHODS
Tissues and culture conditions. The distal femurs
were removed aseptically from adult mongrel dogs (weighing
20-30 kg) immediately after the animals were killed with an
overdose of sodium pentothal. Cartilage slices less than 0.5
mm thick were shaved with a scalpel from the weightbearing surfaces of the femoral condyles. Posterior surfaces
of the patellas and the shavings from each animal (approximately 400 mg) were pooled separately and cultured in
Falcon tissue-culture dishes (60 x 16 mm). Each dish
contained 10-15 mg of cartilage in 5 ml of Ham’s F-12
nutrient mixture, pH 7.4, supplemented with 10% fetal calf
serum, streptomycin (50 pg/ml), and penicillin (SO unitdml)
(all obtained from Grand Island Biological Co., Grand Island, NY), and various concentrations of p-nitrophenylbeta-D-xylopranoside (Koch-Light Laboratory, Coinbrook,
Bucks, England) with or without 10-3M sodium salicylate.
For studies of glycosaminoglycan metabolism, cartilage slices were incubated with gentle rocking at 37°C for 18
hours under 5% COZ:95% air. Na235S04(20 pCi/mI; 825.9
mCi/mmol) or 10 pCi/ml of 6-3H-glucosamine hydrochloride
(20.7 Ci/mmole) (both from New England Nuclear Corp.,
Boston, MA) was then added, and the incubation was
continued for an additional 24 hours. The medium was then
removed, and the cartilage was washed twice with 3-ml
portions of 0.9% saline. Medium and wash were combined
and placed in Spectrapor membrane tubing No. 3 (approximate molecular weight cutoff = 3,500 daltons) (Spectrum
Medical Industries, Inc., Los Angeles, CA), and the samples
were dialyzed at 4°C for 24 hours against 200 volumes of
0.05M sodium acetate, pH 6.8, and then for 24 hours against
running distilled water. The sacs were then decanted and
washed with water, and retentate and wash were combined
in 15-ml graduated, conical centrifuge tubes. The volumes
were then recorded.
Determination of net glycosaminoglycan synthesis. After incubation of triplicate or quadruplicate samples with
Na235S04and recovery of the medium as described above,
the cartilage was suspended in 3 ml of 0.1M borate buffer,
pH 8.0, containing 0.02M calcium dichloride. One milligram
of pronase (Calbiochem, San Diego, CA) was added, and the
sample was digested under toluene for 24 hours at 55°C. The
digest was dialyzed overnight at 4°C against 400 volumes of
distilled water, and the retentate was then processed as
above. Net glycosaminoglycan synthesis was determined
from the sum of the nondialyzable 35Scounts per minute
(cpm) in 0.1-ml aliquots of the medium and the pronase
digest. These were then added separately to 10-ml portions
of Ready-Solve HP 60 (Beckman Instruments, Inc., Irvine,
CA) and counted in a Model LS 230 Beckman liquid scintillation spectrometer. Results were adjusted for differences in
wet weight of the cartilage.
In those experiments in which the cartilage was
1085
cultured in the presence of 3H-glucosamine hydrochloride,
the tissue was washed, after incubation, with 0.9% saline
and digested under toluene for 24 hours at 65°C in 3 ml of
0.2M sodium acetate buffer, pH 5.5, that contained 2 mM
cysteine hydrochloride, 5 mM ethylenediaminetetraacetate
(EDTA), and 0.5 mg of 2x crystallized papain (Worthington
Biochemical Corp., Freehold, N J). The glycosaminoglycans
were isolated from the digests by precipitation with 9aminoacridine hydrochloride (Sigma Chemical Co., St. Louis, MO) and converted to their sodium salts by ion exchange
with Bio Rad AG-50 (Na+) (Bio-Rad Lab., Richmond, CA).
The resin was removed by filtration, and 0. I-rnl portions of
the filtrates were counted as above. Culture medium and
wash were combined, dialyzed against the acetate buffer,
and digested with papain. The glycosaminoglycans were
then isolated and counted. Net 3H-glycosaminoglycan synthesis was determined from the sum of the nondialyzable 9aminoacridine precipitable radioactivity in the papain digests
of the tissue and medium.
Determination of net protein synthesis. Triplicate
samples of cartilage (approximately 10 mg each) were incubated as described above in 5 ml of medium that contained 5
pCi/ml of L-4, 5 3H(-N) leucine (60 Ci/mM: New England
Nuclear Corp., Boston, MA). The medium was then removed, and the cartilage was washed with cold saline and
suspended in 3 ml of 0.1M EDTA in 0.1M sodium acetate,
pH 5.1, containing 0.1% sodium dodecyl sulfate. The samples were homogenized, and 3H-leucine-labeled protein was
determined, as previously described (17).
Extraction of proteoglycans. In a separate experiment, samples of cartilage (approximately 50 mg) from a
single dog were cultured as described above in 10 ml of
medium containing NaZ3%04and either salicylate ( lOP3hf),
xyloside (10-4M), or salicylate plus xyloside. After the
medium was removed, the cartilage was stirred for 48 hours
at 4°C in 10 ml of 4M guanidinium chloride in O.OSM sodium
acetate, pH 5.8, containing 0.01M EDTA, 0.1M 6-aminohexanoic acid, and 0.05M benzamidine hydrochloride (18). The
cartilage residue was digested completely with pronase, as
described above, and radioactivities of the medium, 4M
guanidinium hydrochloride extract, and the pronase digest
were determined, as described, and adjusted for differences
in wet weight of the tissue.
Isolation and purification of aggregated proteoglycans.
Proteoglycans in the medium and 4M guanidinium chloride
extract were isolated by equilibrium density gradient centrifugation under associative conditions that favored formation
of proteoglycan aggregates (19). The bottom two-fifths of the
gradient (d 2 1.76 grn/ml), which contains proteoglycan
aggregates, was recovered and dialyzed against several
changes of 0.05M sodium acetate, pH 6.9. Portions were
digested with hyaluronic acid beta 1+3 hydrolase (EC
3.2.1.36) (Biotrics, Inc., Arlington, MA), as described (20).
The digests were chromatographed on a Sepharose 2B
column, and elution profiles of digested and undigested
samples were compared. The lack of activity of the enzyme
against purified disaggregated proteoglycans (20) was confirmed by its failure to alter their elution profile after they
had been treated in the same fashion.
Gel chromatography of proteoglycans and glycosaminoglycans. Portions (0.5 ml) of the proteoglycan aggregate
1086
fractions from the medium and from the guanidinium chloride extract in 0.5M sodium acetate, pH 6.9 (see above) were
applied to a column (95 x 1.O cm) of Sepharose 2B (Pharmacia Fine Chemicals, Piscataway, NJ) and eluted with the
same buffer at a rate of 2 ml/hour; the radioactivity (35S)of 1ml effluent fractions was then determined.
The average hydrodynamic size of glycosaminoglycans was assessed by chromatography on a column (95 x 1.O
cm) of Sephadex (3-200 (Pharmacia Fine Chemicals, Piscataway, NJ) after the 35S-labeled cartilage, medium, or
proteoglycan aggregate fractions were digested with papain,
as described above. Portions (1 ml) of the digests were
applied to the column and eluted with 0.025M sodium
chloride; the radioactivity of effluent fractions (1 ml) was
then determined.
The partition coefficients (KD's) of the proteoglycan
and glycosaminoglycan samples were calculated from the
formula: KD = (V,-Vo)/(Vt-V,,). V, was taken as the peak
fraction in the elution diagram, V, as the void volume, and
V, as the total column volume.
Extraction of glycosyltransferasesfrom articular cartilage. Cartilage from the femoral condyles of 4 normal adult
mongrel dogs was suspended ( 2 gm/lO ml) in 0.05M 2, (Nmorpholino) ethanesulfonic acid (Mes) buffer, pH 6.5, containing 0.05M potassium chloride (KCI). The samples were
immersed in ice and homogenized at top speed in a Polytron
homogenizer (Brinkman Instruments, Westbury, NY) for six
30-second intervals when the temperature did not exceed
5°C. After the homogenate was centrifuged at 10,OOOg for 10
minutes, enzyme assays (see below) were performed on the
supernatant. Protein concentration in the supernatants was
measured by the method of Lowry et al (21).
Enzyme assays. Xylosyltransferase activity in the
homogenates was measured, according to the method described by Schwartz (14), in 0.05M Mes buffer, pH 6.5,
containing 0.05M KCI, 0.003M manganese dichloride, and
0.012M magnesium dichloride (MgC12). Incubation mixtures
contained 0.75 rnmole of potassium fluoride (KF) in Mes
buffer (0.I ml), 0.37 nmole of uridine diphosphate (UDP)-I4C
xylose (specific activity, 267 pCilpmole) (0.01 ml), and 0.1
ml of the homogenate in Mes buffer (see above). Salicylate
was added in 0.01 ml of Mes buffer to give a final concentration of 10p3M; an equal volume of buffer was added to the
controls. After incubation for 30 minutes at 37"C, 0.05 ml of
1% bovine serum albumin and 0.3 ml of 10% trichloroacetic
acid (TCA): 4% phosphotungstic acid were added. Precipitated protein was recovered by centrifugation, washed
twice with 5% TCA, and dissolved in 0.1 ml of 1M sodium
hydroxide for liquid scintillation counting.
Galactosyltransferase activity in the cartilage homogenate was assayed in an incubation mixture containing 2
pmole of xylose (0.1 ml), 0.73 nmole of UDP-I4C galactose
(267 pCi/mole) (0.01 ml), 1.0 pmole of MgClz (0.1 ml), and
0.1 ml of the tissue homogenate in 0.05M Tris acetate buffer,
pH 5.5, containing 0.05M KCI and 0.001M EDTA (14).
Salicylate was added in 0.01 ml of the above Tris acetate
buffer to give a final concentration of 10-3M, and an equal
volume of the buffer was added to the controls. After
incubation for 30 minutes at 37"C, the mixture was precipitated and counted as above.
PALMOSKI AND BRANDT
RESULTS
Based on the total nondialyzable 3sSradioactivity in the media and pronase digests of the cartilage,
beta-D-xylopyranoside caused a concentration-dependent increase in newly synthesized glycosaminoglycans (Table 1). Consistent with our recently reported
observations (1 I), 10p3M salicylate diminished net
3sS-glycosaminoglycan synthesis to 59-73% of the
control level (Table 2) in cartilage from 5 dogs. In
contrast, when xyloside was present in the incubation
medium in addition to salicylate, net glycosaminoglycan synthesis was 121-236% greater than control values and 168-323% greater than glycosaminoglycan
synthesis in cultures incubated with salicylate alone
(Table 2).
Measures of net synthesis based upon incorporation of 3H-glucosamine into glycosaminoglycans isolated and purified by precipitation with 9-aminoacridine were similar to estimates based on incorporation
of 35S04into nondialyzable material (Figure 1). Thus,
in the presence of 10-3M salicylate, 3H-glycosaminoglycan synthesis was 80% of the control; with lOP4M
xyloside, it was 155% of controls. Although glycosaminoglycan synthesis was inhibited by salicylate and
stimulated by xyloside, net 3H-leucine incorporation
into protein was unaffected by either compound at the
concentrations used (Figure 1).
While salicylate did not affect the distribution of
3sS-glycosaminoglycans between the culture medium
and cartilage, at xyloside concentrations of 10-4M and
10p3M,the proportion of the total 35S-glycosaminoglycans recovered from the medium was about 6-fold
greater than that in controls (Tables 1 and 3). Salicylate did not reverse the alteration in distribution of 3sSglycosaminoglycans caused by xyloside (Table 3).
Based on Sephadex G-200 chromatography,
glycosaminoglycans isolated after papain digestion of
the medium of control cultures (Table 4) were somewhat smaller in average hydrodynamic size than those
obtained from the cartilage (KD = 0.32, 0.23, respectively). The size of the glycosarninoglycans in the
xyloside-treated cultures varied with the concentration of the additive. With 10p4Mxyloside, the elution
profile of the 3sS-glycosaminoglycans in the medium
and cartilage was similar to that of controls (Table 4).
Their chain length was greater, therefore, than that of
glycosaminoglycans in the medium (KD = 30) and in
the cartilage (KD = 0.36) after incubation with 10-3M
xyloside. Salicylate, alone or with xyloside, did not
GAG SYNTHESIS
I087
Table 1. Effects of beta-D-xylopyranoside on net glycosaminoglycan synthesis by normal canine
articular cartilage*
Xyloside
concentration
Control
X IO.'M
1 X lWSM
1 x IO-~M
1 X 10-'M
I
Net "Sgl ycosaminoglycan
synthesis
(7% of control)
'?c of total '5S-gIycosaminogIycan~
Culture medium
Cartilage
14.2 2 1.4
13.2 2 2.3
15.5 1.9
14.3 t 1.2
85.9 & 1.8
85.8 ? 1.4
87.8 ? 2.3
84.5
1.9
26.7 I1.2
14.1 ? 1.8
-
121 _t 5
121 t 7
210 ? 10
245 ? 9
*
*
* Net 3~S-glycosaminoglycansynthesis was derived from the sum of the nondialyzable "S counts perminute in 0.1-ml samples of the medium plus pronase digest of the tissue (see text for details). Data
represent mean t SEM of quadruplicate cultures from I dog.
alter the size of glycosarninoglycans in either medium
or tissue (Table 4).
Table 5 shows the relative proportions of 35Slabeled proteoglycans in the culture medium, 4M
guanidinium chloride extract, and pronase digest of
the cartilage residue. In control and salicylate-treated
cultures, the medium contained about 13-16% of the
total, and the guanidium extract about 60%. In marked
contrast, in cultures treated with 10-4M xyloside,
whether or not salicylate was also present, the medium
contained 79-84% of the total nondialyzable 35S-labeled material, while the guanidiniurn extracts contained only 12-16%.
The proteoglycan aggregate fraction accounted
for 81% and 83.1%, respectively, of the total nondialyzable 35S cpm in the 4M guanidinium extracts of
control and salicylate-treated cultures. In cultures that
contained xyloside, with or without salicylate, this
Table 2. Effects of beta-D-xylopyranoside and sodium salicylate on net glycoaaminoglycan synthe5iS
in normal canine articular cartilage
Cartilage
source
Dog 1
Dog 2
Dog 3
Dog 4
Dog 5
Additions to culture
medium
Salicylate
Xyloside
Salicylate
Salicylate
Xyloside
Salicylate
Salicylate
Xyloside
Salicylate
Salicylate
Xyloside
Salicylate
Salicylate
Xyloside
Salicylate
+ xyloside
+ xyloside
X yloside
concentration
55 of
control*
Net "Sglycosaminoglycan
synthesis in
medium containing
salicylate +
xyloside (% of
saiicylate alone)
10-3M
13 t 3
217 t 10
151 + 6
207
13 t 5
352
12
236 t 1 1
323
70 2 2
235 t 10
154 2 6
220
12 t 3
142 ? 7
121 t 5
I68
59 -+ 7
206 t 8
160 ? 7
271
10-3M
-
+ xyloside
10- 'M
-
+ xyloside
+ xyloside
10-~,w
IO-~M
*
* "S-glycosaminoglycan synthesis was derived from the sum of the nondialyzable ' 5 S counts per
minute in the medium plus pronase digest of the tissue (see text for details). Salicylate was used in the
incubation medium at a concentration of 10 'Ad. Data represent mean + SEM of triplicate samples of
cartilage from each dog.
PALMOSKI AND BRANDT
180
-J
0
e
160
Z
0
U
LI
140
-
Table 4. Effects of beta-D-xylopyranoside and sodium salicylate
on the average hydrodynamic size of glycosaminoglycans in the
culture medium and cartilage*
-
Sephadex (3-200 KD
of
glycosaminogl y can
0
s
Additions to culture
Control
Salicylate
Xyloside
Salicylate
Xyloside
Salicylate
Xyloside
concentration
+ xyloside
+ xyloside
Culture
medium
Cartilage
0.32
0.29
0.80
0.78
0.32
0.35
0.23
0.25
0.36
0.34
0.28
0.26
* Samples (0.5 ml) of the 35S-glycosaminoglycans obtained after
papain digestion of the culture medium or cartilage slices (see text
for details) were applied to a column of Sephadex (3-200 (95 x I .O
cm) and eluted with 0.025M sodium chloride at a rate of 2 mlihour.
Radioactivities (3sS) of 1-ml effluent fractions were determined, and
the partition coefficients (KD) of the glycosaminoglycans were
calculated as described in the text. Salicylate was used in the
incubation medium at a concentration of 10-3M.
Salicylate
Xyloside
Salicylate +
Xyloside
ADDITIONS T O CULTURE
Figure 1. Net synthesis of "S-labeled glycosaniinoglycans (clear
bar), lH-glucosamine-labeled glycosaminoglycans (solid bar), and
3H-leucine-labeled protein (diagonally marked bar), by cartilage
cultured in the presence of salicylate (10-3M),xyloside (10-4M),or
salicylate ( 1 0 - 3 M ) plus xyloside ( 10-4121). Results represent mean
counts per minute (cpm)/lO mg wet weight of cartilage, t SEM.
fraction accounted for a slightly lower proportion of
the 4M guanidinium extract (74.1%, 72.8%, respectively).
Sepharose 2B elution profiles of proteoglycan
aggregate fractions from the guanidinium extracts
showed that, in all cases, about 30% of the sample
eluted at V, (Figure 2). Proteoglycans from control
and salicylate-treated cultures that were retarded by
the gel eluted in a single broad peak (KD = 0.20, 0.25,
respectively). In cultures exposed to xyloside, alone
or with salicylate, about 88% of the 35S-labeledmaterial that was retarded by the gel also eluted with a KD of
0.20-0.25. In addition, however, some of the 35S cpm
eluted in a peak near Vt, (KD = 0.90>, similar to 35Sglycosaminoglycans isolated from the cartilage and
chromatographed on the same column.
Incubation of the proteoglycan aggregate fraction of the guanidinium extract with hyaluronic acid
beta 1-3 hydrolase, which specifically degrades hyaluronic acid, abolished the void volume peak and
reduced the average hydrodynamic size of the retarded proteoglycans, as indicated by a shift in their KD
from 0.20-0.25 to about 0.50 (Figure 2). No change
was seen in the minor peak (KD = 0.90) in the
Table 3. Effects of beta-D-xylopyranoside and sodium salicylate on the distribution of "Sglycosaminoglycans between the culture medium and pronase digest of cartilage*
Additions to
culture medium
Control
Salicylate
Xyloside
Salicylate
Xyloside
Salicylate
Xyloside
concentration
-
+ xyloside
+ xyloside
1 0 - 3 ~
~o-~M
IO-~M
IO-~M
% of total '5S-glycosaminoglycans
Culture medium
13.3
12.5
93.0
94.5
69.6
72.7
t 1.2
2
2
2
t
2
3.0
1.5
1.1
2.1
2.3
Cartilage
86.7
87.5
8.0
5.5
30.4
27.3
1.2
3.0
1.5
2 1.1
t 3.1
t 1.5
f
2
2
* 3SS-glycosaminoglycanswere derived from the sum of the nondialyzable 35Scounts per minute in the
medium plus digest of the tissue (see text for details). Salicylate was used in the incubation medium at
a concentration of 10-3M. Data represent mean 2 SEM of triplicate samples of cartilage from 1 dog.
GAG SYNTHESIS
1089
Table 5. Effects of beta-D-xylopyranoside and sodium salicylate on the distribution of proteoglycans in culture medium, 4M guanidinium
chloride extract, and pronase digest of normal dog articular cartilage*
Additions to culture
Control
Fraction
Culture medium
4M guanidinium
chloride extract
Pronase digest
Total
Radioactivity
79,771
Salicylate
% of
total
CPm
Xyloside
% of
16.3
Radioactivity
42,382
total
CPm
12.8
Radioactivity
782,208
287,756
122,345
58.7
25.0
201,316
87,414
60.8
26.4
489,872
100.0
331 , I 12
100.0
Salicylate
% of
total
CPm
+ xyloside
% of
83.6
Radioactivity
6 4 5 3 18
total
CPm
79.3
109,471
43,977
11.7
4.7
132,685
35,827
16.3
4.4
935,656
100.0
814,020
100.0
* The radioactivity values represent nondialyzable 35Scounts per minute (cpm)/lO mg wet weight of cartilage. Salicylate was present in the
incubation medium at a concentration of 10-3M, and xyloside at a concentration of 10-4M.
chromatographs of samples from xyloside-treated cartilage (see above). After digestion of the aggregate
fraction with the hyaluronidase, 3sS-material from the
xyloside-treated cartilage was somewhat smaller in
hydrodynamic size than that from control or salicylate-treated cultures (KD = 0.55 and 0.45, respectively). This may have been caused by the presence of
shorter glycosaminoglycan chains on the proteoglycans from xyloside-treated cultures, since Sephadex
G-200 elution profiles of the glycosaminoglycans isolated after papain digestion of the aggregate fraction
from control cartilage had a KD of 0.30, whereas the
KD of the corresponding glycosaminoglycans from
cartilage treated with xyloside was 0.40 (Figure 3).
The proteoglycan aggregate fractions from the
medium of control and salicylate-treated cartilage represented 55% and 52%, respectively, of all the nondialyzable 35S cpm in the medium, and were wholly
retarded by Sepharose 2B (KD = 0.55) (Table 6). This
material was larger in average hydrodynamic size,
therefore, than purified chondroitin sulfate, for which
the KD was 0.90. Digestion with hyaluronic acid beta
1+3 hydrolase did not alter the elution profile of the
proteoglycan aggregate fraction prepared from medium of control or salicylate-treated cultures. In contrast, proteoglycans in the aggregate fraction from the
medium of cultures containing xyloside, with or without salicylate, represented nearly 90% of all the nondialyzable 35Scpm in the medium, and exhibited a KDon
a Sepharose 2B of 0.90. On Sephadex G-200 chromatography, this material had a KD of 0.35, indicating
that its size was essentially the same as that of purified
chondroitin sulfate (Table 6).
The aggregate fraction of the guanidinium extract of salicylate-treated cartilage contained 78% as
much 3sS-proteoglycans/mgwet weight cartilage as the
control; by contrast, the aggregate fraction from the
guanidinium extract of the xyloside-treated cultures
represented only about 30-35% of control. This represented essentially all of the proteoglycan synthesized
by xyloside-treated cartilage, since the medium from
these cultures contained only glycosaminoglycans (see
above), and the cartilage residue accounted for only
5% of the total nondialyzable 35Scpm (Table 5).
The possibility that salicylate inhibited the endogenous xylosyl transferase in the cartilage was not
excluded by the above studies since the experimental
design provided an exogenous source of xylose for
chondroitin sulfate biosynthesis. Therefore, xylosyl
transferase activity was measured directly by incubation of a 10,OOOg supernatant from a cartilage homogenate with 14C-UDP xylose in the presence or absence
of salicylate. Galactosyl transferase activity was similarly measured, with the use of I4C-UDP galactose as
precursor. Salicylate ( 10--3iqldid not suppress activity
of either xylosyl or galactosyl transferase in these
studies (104%, 110% of control, respectively) (Table
7) -
DISCUSSION
These results show that beta-D-xylopyranoside
can overcome the inhibition of glycosaminoglycan
synthesis produced by salicylate in cultures of normal
articular cartilage. Including xyloside in the culture
medium stimulated chondroitin sulfate biosynthesis as
much as 3 times over control levels, and the degree of
stimulation was directly proportional to the xyloside
concentration (Tables 1,2). In the xyloside-treated
cultures, approximately 70-90% of the newly synthe-
PALMOSKI AND BRANDT
1090
smaller than that in control or salicylate-treated cultures. This was not true at lOP4M, even though net
glycosaminoglycan synthesis was significantly increased by xyloside (Table 4). A decrease in hydrodynamic size of glycosaminoglycans synthesized in the
presence of xyloside has been noted previously
(22,23). It may result from a molar excess of 1) the
xylose acceptor, relative to the tissue content of
enzymes required for chain elongation, 2) the precursor monosaccharides, or 3) energy supply (24). It is
clear that xyloside stimulated biosynthesis of the glycosaminoglycans and not only sulfation, because
when 3H-glucosamine was used as a precursor, results
were similar to those obtained with 35S04(Figure 2).
While 10-3M salicylate reduced proteoglycan
synthesis to about 70% of the control level, it did not
affect the proportion of proteoglycans existing in aggregates (Figure 2). Beta-D-xylopyranoside (
20
VO
30
40
50
60
70
vt
I"
80
EFFLUENT VOLUME (ml)
Figure 2. Sepharose 2B gel chromatography of 35S-labeled proteoglycans extracted with 4M guanidinium hydrochloride and purified
), and of the same fraction
under associative conditions -(
after digestion with hyaluronic acid beta 1-3 hydrolase (-----------)
(see text for details). A = control; B = cartilage cultured in presence
of 10-3M salicylate; C = cartilage cultured in presence of 10-4M
xyloside; D = cartilage cultured in presence of 10-3M salicylate plus
10-4M xyloside.
X
C
2
V
A
":I
m
m
sized glycosaminoglycans diffused into the medium
(Table l), whereas in control cultures most of the 35Sglycosaminoglycans were retained in the cartilage.
This was not unexpected, since xyloside, serving as a
receptor for glycosaminoglycan chain biosynthesis
(16), decreased the availability of precursor sugars for
biosynthesis of 3SS-proteoglycans,whose size would
have restricted their diffusion out of the tissue. Nonetheless, some free glycosaminoglycans remained in the
cartilage and were present in the 4M guanidinium
chloride extracts, in which they eluted near V, on the
Sepharose 2B column (Figure 2).
When xyloside was used at a concentration of
10-3M, the average hydrodynamic size of glycosaminoglycans synthesized by xyloside-treated cartilage was
1 -
D
3 2 1 -
I
I
20
vo
30
40
50
60
70
vt
80
EFFLUENT VOLUME (ml)
Figure 3. Sephadex G-200 chromatography of 35S-labeled glycosaminoglycans obtained after papain digestion of the proteoglycan
aggregate fraction (guanidinium chloride) (see text for details). See
Figure 2 legend for descriptions of A, B, C, and D.
GAG SYNTHESIS
1091
hyaluronic acid beta 1-3 hydrolase (Figure 2) was not
caused by digestion of the glycosaminoglycan chains,
since the enzyme did not affect elution profiles or
purified disaggregated proteoglycans, or by digestion
of chondroitin sulfate. The elution profiles of those
35S-proteoglycans in the aggregate fraction (guanidinium chloride) that eluted somewhat after the void
volume were also shifted after digestion with hyaluronidase, suggesting that these proteoglycans also
were associated with hyaluronic acid. We have previously shown that proteoglycan aggregates digested
with the hyaluronidase preparation used in these studies are similar in size to proteoglycan monomers
prepared by dissociative density gradient centrifugation (20). The size of proteoglycan aggregates from
nasal and tracheal cartilage has been shown to be
proportional to the length of the hyaluronic acid molecule in the aggregate (27). Since the dissociated proteoglycans in the present study were essentially unimodal with respect to average hydrodynamic size
(Figure 2), the hyaluronate extracted from the cartilage by 4M guanidiniurn was presumably polydisperse
or the proteoglycan :hyaluronic acid ratio was diminished.
The significant augmentation of glycosaminoglycan synthesis produced by xyloside in the presence
of salicylate suggests that the latter drug did not inhibit
the galactosyl, glucosyl, or N-acetyl galactosamine
transferases essential for chondroitin sulfate chain
elongation, or the enzymes required for sulfation of
the molecule. The possibility that salicylate inhibits
xylosyl transferase in cartilage is also unlikely since
the drug had no effect on xylosyl transferase activity in
vitro (Table 7). Suppression by salicylate of synthesis
of the proteoglycan core protein is not excluded by the
Table 6 . Effects of beta-D-xylopyranoside and sodium salicylate
on the average hydrodynamic size of 35S-labeled material in the
proteoglycan aggregate fraction (medium) from cultures of adult
canine articular cartilage*
Sepharose 2B
Additions to culture
Control
Salicylate
Xyloside
Salicylate
+ xyloside
Sephadex (3-200
% in Vo
K,,
% in Vo
KD
0
0
0
0
0.56
0.53
0.89
0.90
0
0
0.37
0.35
* Samples (0.5 ml), obtained from the bottom two-fifths of a cesium
chloride density gradient of the medium following incubation (see
text for details), were applied to a column of Sepharose 2B (95 x 1 .O
cm) or of Sephadex (3-200 (95 x 1.0 cm) and eluted with O.OSM
sodium acetate or 0.025M sodium chloride, respectively, at a rate of
2 ml/hour. The radioactivity (35S)of I-rnl effluent fractions was
determined, and the partition coefficients (K,,) of the "S-labeled
material were calculated as described in the text. Salicylate was
present in the incubation medium at a concentration of 10 - 3 M , and
xyloside at a concentration of 10-4M. Vo = void volume.
which reduced proteoglycan synthesis to approximately 31% of control levels, also had no effect on aggregation. Thus, neither compound appeared to affect the
hyaluronate-binding region of the proteoglycan core
protein, nor the availability of sufficient high molecular weight hyaluronate to enable aggregation. In chick
chondrocyte cultures, beta-D-xyloside has been
shown to reduce 35S-proteoglycan synthesis without
altering synthesis of core protein (25). In the present
experiments, although synthesis of core protein was
not specifically examined, neither salicylate nor xyloside affected net protein synthesis (Figure 1). Other
researchers have shown that a large proportion of the
proteoglycans synthesized by chondrocytes that were
derived from chick limb bud mesenchyme grown in the
presence of xyloside were capable of aggregating (26).
In the present study the proportion of proteoglycans present in aggregates was similar in all cases,
i.e., about 30% eluted in the Sepharose 2B void
volume. However, the percentage of the total cpm in
the 4M guanidinium extract that was accounted for by
the aggregate fraction was somewhat lower (73%) in
xyloside-treated cultures than in salicylate-treated cartilage or controls (82% in each case). Thus, proteoglycans synthesized in the presence of xyloside may have
contained fewer glycosaminoglycan chains per molecule, or shorter chains (Figure 3), causing them to
equilibrate in the cesium chloride gradient at a buoyant
density less than that at which the proteoglycan aggregate fraction equilibrated.
Disappearance of the void-volume peak after
incubation of the proteoglycan aggregate fraction with
Table 7. Effect of salicylate on xylosyl transferase and galactosyl
transferase activity in canine articular cartilage*
~
~~
~
~
Xylosyl transferase
activity
Addition
to culture
medium
I4C-UDP
xylose cpm/
mg protein
Control
Salicylate
2,785
2,905
?
?
156
125
% of
control
-
104
Galactosyl transferase
activity
''C-UDP
galactose cpm/
mg protein
10,470 5 533
11,517 rt 921
% of
control
110
* Enzyme activity was measured by incubation of 30,OOOg supernatant fraction of a cartilage homogenate with I4C-uridine diphosphate
(UDP) xylose or I4C-UDP galactose in the presence or absence of
10-3M salicylate. Activity was measured as I4C counts per minute
(cpm) precipitable by trichloroacetic acid after a 30-minute incubation at 37°C. Data represent mean ? SEM of quadruplicate determinations.
PALMOSKI AND BRANDT
1092
present data, although this would be unlikely since net
protein synthesis was not diminished in salicylatetreated cultures (Figure 1).
The results are consistent with the observation
that salicylate inhibits the activity of UDP-glucose
dehydrogenase (28), which catalyzes the conversion of
UDP-glucose to UDP-glucuronic acid and is thus
essential for chondroitin sulfate biosynthesis. Inhibition of this enzyme would limit the quantity of glucuronic acid available for glycosaminoglycan formation, and the deficit would be accentuated under
conditions in which glycosaminoglycan biosynthesis
was stimulated, e.g., by xyloside. This could account
for our observation that the level of glycosaminoglycan synthesis was not as great when salicylate and
xyloside were present in combination as when xyloside, alone, was present. If salicylates did indeed
inhibit UDP-glucose dehydrogenase, however, this
inhibition could have been partial, since 35S-glycosaminoglycan synthesis with salicylate and xyloside averaged 64% greater than that in control cartilage
cultured in the absence of either additive (Table 2 ) .
ACKNOWLEDGMENT
Jeffrey Wilson provided excellent technical assistance.
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