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Effect of retinoic acid on chondrocyte glycosaminoglycan biosynthsis
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Effect of Retinoic
STANLEY Department of Biochemical
174,
(1976)
74-81
Acid on Chondrocyte Biosynthsis S. SHAPIRO Nutrition, Received
AND
JAMES
Glycosaminoglycan
P. POON
Roche Research Center, Nutley, New Jersey 07110 September
18, 1975
The effect of retinoic acid on glycosaminoglycan biosynthesis was investigated in rat costal cartilage chondrocytes in uitro. At levels of lo-!’ to lo-” M retinoic acid, s”SO, uptake into glycosaminoglycans was reduced 50%. At these low levels of retinoic acid there was no evidence of lysosomal enzyme release. The results are explained best in terms of modification of glycosaminoglycan synthesis, rather than accelerated degradation. Retinoic acid selectively modified the incorporation of YSO, or L’4Clglucosamine into individual glycosaminoglycans fractions under the conditions studied. The relative incorporation of radiolabeled precursor into heparan sulfate (and/or) heparin increased threeto fourfold. The relative incorporation of radiolabeled precursor remained constant for chondroitin &sulfate, whereas incorporation into chondroitin 4-sulfate and chondroitin (and/or) hyaluronic acid decreased. Under the conditions studied, retinoic acid did not appear to be cytotoxic and did exhibit selective control over glycosaminoglycan biosynthesis. It is suggested that the decreased incorporation of Y!jO, into glycosaminoglycans at hypervitaminosis A levels of retinol may be accounted for by the presence of low levels of retinoic acid, a naturally occurring metabolite.
The classical studies of Fell and Mellanby (1) reported that lo-15 IU/ml of retinol when added to the culture medium of 6- or 7-day-old embryonic chick limb cartilage resulted in limb disintegration and resorption. The matrix became soft, lacked glycosaminoglycans (GAG)’ and finally disappeared. The breakdown products of the cartilage, i.e., inorganic sulfate, hexosamine, and hydroxyproline appeared in the culture medium (2, 3). The retinol mediated resorption was shown to be due to labilization of the lysosomal membrane, and subsequent release of acid hydrolyases into, and outside, the chondrocyte (4-6). It was also reported by Weissman (7) that cartilage explants, when re-
moved from hypervitaminosis A rabbits released lysosomal enzymes into the culture medium. Known lysosomal membrane stabilizers neutralized the retinol-induced lysis both in vitro and in vivo. Cortisone administered in vivo protected rabbits against the cartilage lesions of hypervitaminosis A (8). In addition, less acid hydrolyases were released from cartilage of hypervitaminosis A animals protected with cortisone than by cartilage of animals given high dosesof the vitamin alone. Hydrocortisone was shown to delay cartilage resorption in embryonic chick cartilage in vitro (9). Chloroquine, like cortisone has been shown to antagonize the retinol effect on lysosomal membranes (8). Hypervitaminosis A levels of retinol were observed to initially inhibit ““SO, incorporation into cartilage prior to causing accelerated matrix breakdown (10). McElligott (11) reported that sulfate fixation by chondrocytes was reduced under conditions of hypervitaminosis A, and this was
’ Abbreviation used: GAG, glycosaminoglycans; CHase, chondroitinase ABC (EC 4.2.2.5); A-Di-4S, 2 acetamido-2-deoxy-3-O-(p-D-gluco-4-enepyranosyluronic acid)-4-O-sulfa-n-galatose; A-Di-GS, 2 acetamido-2.deoxy-3-O-(P-n-gluco-4-enepyranosyluronic acidW@sulfo-n-galactose; A-Di-OS, 2 acetamido-Zdeoxy-3-O-(P-n-gluco-4.enepyranosyluronic acid)-ngalactose; TCA, trichloroacetic acid. 74 Copyright All rights
0 1976 by Academc Press, Inc. of reproduction in any form reserved.
RETINOIC
ACID
AND
independent of any lysosomal effect. More recently, Solursh and Meier (12) reported a selective inhibition of GAG biosynthesis by lo-” M retinol without lysosomal release. We now report that retinoic acid, a naturally occurring metabolite of retinol at lo-!’ M modified GAG biosynthesis in chondrocytes in vitro. At this low level of retinoic acid, there was no detectable lysosomal membrane labilization, and there was no detectable decrease in cellular protein or DNA. METHODS Preparation of c!n~&rocytes. Costa1 cartilage from &day-old rats were trimmed and cleaned of connecting tissue. The cartilage was minced with an extra fine dissecting scalpel under a dissecting microscope. Finely minced cartilage (400-500 mgl was suspended in 8.0 ml of Pucks saline G containing 6000 units of collagenase (Type II, Worthington Bicchemicals) and gently stirred at 37°C for 3-4 h. The chondrocytes were separated from undigested debris by repeated sedimentaion in 50.0 ml of growth medium (F-10, 10% fetal calf serum and 100 units/ml of penicillin and streptomycin). Media (3.0 ml) containing 0.3-1.0 x 10” cells were placed in Falcon tissue culture flasks (25 cm> growth area). Retinoic acid in 0.02 ml of ethanol was added at this time. The flasks were incubated at 37°C under 95% air and 5% CO,. After 72 h, 20-40 WCi of HZ3”S0, or 1.5-2.0 $i of l’aC]glucosamine (New England Nuclear) was added, and the incubation was continued for an additional 16 h. Upon completion of the incubation, the medium was decanted from the cell layer. The l:“SJGAG found in the medium is referred to as the medium GAG in this text. The cell layer was washed twice with 3.0 ml of Pucks saline G and then 200 units of collagenase in 2.0 ml of Pucks saline G were added to each flask. After 3 h of incubation, the cells separated from the flasks and floated. Release of the cells from the flasks by crude collagenase resulted in the solubilization l:‘S]GAG which is referred to as matrix GAG in this text. The cells were separated from the collagenase solution by centrifugation and washed twice with Pucks saline G. Cell number was determined immediately. The remaining cells were saved for l”“SlGAG determination, DNA or protein determinations. The medium and matrix containing solutions were mixed with a papain solution to a final concentration of 0.10 M sodium acetate, pH 5.8, 5 mM EDTA, and 5 mM cysteine containing 1.0 mg of papain. The papain digestion was carried out at 55°C for 18 h. Determination of9 or 1% incY~rporateff into chonrlrocvtr glycosaminoglycans, 50% TCA was added to
GAG
BIOSYNTHESIS
75
the papain digestion to give a final TCA concentration of 5%. A minimum of four flasks per reaction condition were carried out. Two flasks per reaction condition were combined for GAG identification. The combined volume for the medium was 7.4 ml and that of the matrix solution 4.6 ml. The TCA supernatants were dialyzed for 2 days with frequent changes of deionized water. A 50-/*l aliquot of the dialyzed TCA supernatant solution containing the [3SlGAG was chromatographed on Whatman No. 3 paper in isobutyric: 0.5 M NH,OH (5:3, V/V) overnight (13). The origins containing the radioactive GAG were cut out, placed in 10 ml of Aquasol (New England Nuclear) and counted in a liquid scintillation spectrophotometer. D~gestiorz wzth c~hondroitinaw ABC. The digestion of I:‘SlGAG with chondroitinase .4BC was performed according to the method of Saito (14). Papain digested and dialyzed medium and matrix I’“SIGAG solutions (500 ~1) were evaporated to dryness under a stream of nitrogen. The samples were then redissolved in 20 ~1 of “enriched” Tris buffer and 30 ~1 of enzyme (0.15 units). The solutions were incubat,ed at 37°C for 4 h and stopped by streaking on Whatman No. 3 paper. The entire reaction was chromatographed in butanol: acetic acid: 1 M NH,OH (2:3:11 according to the method of Saito e/ al. (141. After drying, the standard dissaccharides were visualized under uv light. The chromatograms were cut into i/z-in. segments, placed in 10 ml of Aquasol, and counted in a liquid scintillation spectrophotometer. Chondroqvtu wll nurnh~~r d~tc~rmination. Chondrocytes harvested from the collagenase solutions by centrifugation were washed twice with Pucks saline G. The washed cell pellet was suspended in a small volume of Pucks saline G and cell number was determined with a hemocytometer. DNA and protcir~ tir~iprnlinntlorrs. Chondrocytes from above were collected by centrifugation. Twotenths milliliter 0.01 M Tris-HCl, pH 7.4, was added to the cell pellets. The cells were lysed by- rapidly freezing and thawing seven times in dry ice and a&one. DNA was determined according to Leyva and Kelley (151. Protein was determined according to Lowry ct ni. 116). Deternrinntiorl of N-sulfatd GAG corltc~nt. Nitrous acid degradation was carried out according to the method of Cifonelli as described by Conrad and Hart (17). Aliquots of the dialyzed TCA supernatant solutions were evaporated to dryness under a stream of nitrogen and redissolved in 10 ~1 of water. The samples were then mixed with 20 ~1 of 1 N HCl and 20 ~1 of freshly prepared 20% n-butyl nitrite IV/V) in absolute ethanol (20 ~1 of absolute ethanol without n-butyl nitrite was used in the reaction blank). The reaction was carried out at room temperature for 2 h with gentle shaking At completion. the reaction (80 ~1) was chromatographed on Whatman No. 3 paper in isobutyric acid: 0.5 M NH,OH (5:3. v/v) for 36 h
76
SHAPIRO
AND
(131. The heparan sulfateiheparin content was determined from the chromatogram as previously described by Shapiro and Sherman (18). Recovery of added [“%]GAG from culture medium. [““SlGAG synthesized by control chondrocyte was isolated in the following manner. Medium solutions from control experiments were pooled and treated with papain solution (as described above). TCA (50%) was added to the reaction mixture to a final concentration of 54:. The TCA supernatant was dialyzed extensively against water for 2 days. The dialysate was concentrated and passed through a Millipore 0.22-pm filter. l”“S]GAG (55,500 cpml was added to each flask along with retinoic acid at Day 1. The incubation was stopped at Day 4. GAG was determined as previously described. Effect of lysosomal stabilizers and retinoic acid on GAG synthesis. Lysosomal stabilizers, cortisone acetate (Merck; final concentrations, 1 and 5 *g/ml), chloroquine (Sterling-Winthrop; final concentration, 0.1 pg/ml) and e-amino caproic acid (Sigma; final concentration, 0.1 M) were added to chondrocyte cultures treated with 2 x 10 i and 2 x 10 ’ M retinoic acid at Day 1. The incubation was stopped at Day 4. GAG was isolated as previously described. RESULTS
Effect of Retinoic thesis
Acid
on GAG
Biosyn-
Chondrocytes grown in the presence of 2.0 x 10m9to 2.0 x 10m7M retinoic acid exhibited a dose dependent decrease in YSO, incorporation into GAG (Table I). Identical results were obtained if retinoic acid was added to the culture flasks 18 h after seeding and incubated for 72 h prior TABLE
EFFECT OF Retin$
I
RETINOIC ACID ON :Y30, INTO MATRIX GAG”
acid
CHase”
ii%]
’
INCORPORATION
SDi-68
.LDi-48
resistant
ftotal)
2.0 x lo-!’ 1.0 x 10-H 2.0 x 10-w 1.0
42,540 26,367 18,712 16,164 9,779 8,077
x IO-’
2.0 x 10-T
3,900 3,100 3,155 3,302 3,054 3,569
7,985 3,910 2,750 2,079 1,021 929
30,654 19,347 12,788 10,764 5,685 3,569
U The experiment was carried out as described under Methods; 40 x 10” cpm of carrier free H,:“SO, was added per flask. b Values are expressed in counts per minute and represent
two culture
a total
flasks
incorporation
per reaction
of a pooled
condition.
sample
of
POON
to the addition of radiolabeled GAG precursor. The [“S]GAG was digested with chondroitinase ABC and the products of the digestion identified. The A-Di-6S and A-Di-4S originating from chondroitin 6sulfate and chondroitin 4-sulfate, respectively, show a concomitant decrease in :?30, incorporated into GAG in the presence of increasing retinoic acid. The level of ““SO, incorporated into chondroitinase resistant material remained relatively constant (Table I). When [llC]glucosamine was utilized as a GAG precursor, slightly different results were obtained (Table II). Retinoic acid (2.0 x IO-!’ M) caused a 20% reduction in [ ‘“Clglucosamine incorporated. However, there was no further change in the total 1“C]glucosamine incorporated into GAG until levels of 1.0 x lo-’ M retinoic acid. The [ “C]glucosamine incorporated into chondroitinase resistant material increased with increasing doses of retinoic acid, and A-Di-OS (originating from chondroitin and/or hyaluronic acid) decreased significantly with increasing retinoic acid. Previous studies with this cell system (19) demonstrated the absence of dermatan sulfate. Comparison of Retinoic Acid dium and Matrix GAG
Effect on Me-
The relative incorporation of ““SO, in medium, matrix, and cellular GAG remaining after collagenase treatment was 1.0:0.4:0.0025. The low levels of cellular GAG did not change in the presence of retinoic acid. Due to the relatively low levels of radiolabeled GAG associated with the cellular fraction, this fraction was not routinely examined and was not subjected to chondroitinase ABC digestion. The results in Table III show that YSO, incorporation into GAG associated with both the matrix and the medium is reduced. The :“SO, incorporation into matrix GAG appeared to be reduced to a slightly greater extent than the medium. Using 1’“CJglucosamine as a GAG precursor (Table IV), one seesno reduction in the total incorporation of precursor in the medium. However, the matrix l”‘C]GAG was reduced at levels of retinoic acid of 2.5 x lo-!’ to 4.0 x 10mHM about 20%.
RETINOIC
ACID
AND
GAG
TABLE EFFECT
Retinoic
acid
2.0 1.0 2.0 1.0 2.0
ON
[“CIGAG” (total)
CM)
x x x x x
ACID
OF RETINOIC
INCORPORATION
~~~INTO MATRIX A-Di-4s”
A-Di-6s’
resist-
GAG”
~~~ &Di-OS”
ant
13,468 11,040 10,506 10,368 8,887 8,754
10 !’ 10~’ 10-H 10-y 10 7
II
[‘XJGLUCOSAMINE
CHase” ~
77
BIOSYNTHESIS
2,861 3,744 4,213 4,554 4,618 5,483
1,591 1,426 1,223 1,306 1,012 1,269
6,568 4,268 3,900 3,542 2,419 1,784
2,447 1,582 1,140 956 818 340
” The experiment was carried out as described under Methods; 4 x 10” cpm of I’% lglucosamine was added per flask. ’ Values are expressed in counts per minute and represent a total incorporation of a pooled sample of two culture flasks per reaction condition. TABLE EFFECT
OF RETINOIC
_ ~~~ Retinoic acid (M)
2.5 1.0 2.0 4.0 1.6
x x x x x
10 10 10 10 10
” n ’ ” ;
III
TABLE
ACID ON :‘“S INCORPORATION INTO GAG”
Medium [“:‘S]GAG ----~~~ (cpm x (5% I10 ,I) 171 138 109 105 70 37
Matrix [,“SIGAG (cpm x lo-.‘)
100 80 64 61 41 21
74 46 36 24 28 I4
EFFECT
ACID
INCORPORATION
Retinoic acid CM) ($%I 100 62 49 32 37 19
” The experiment was carried out as described under Methods; 40 x 10” cpm of carrier free H,““SO, was added; values in counts per minute are a combined value of two culture flasks per reaction condition.
Relutiue Distribution of GAG’s Acid-Treated Chondrocytes
OF RETINOIC
in Retinoic
A distinct change in the GAG profile was observed when the data in Table I were expressed as a percentage of total incorporation (Fig. 1). The fraction ofY30, incorporated into the chondroitinase-resistant material increased appreciably with increasing retinoic acid. The Y30, incorporated into A-Di-4S showed a relative decrease in the amount of :%SO.,incorporated in chondroitin 4-sulfate. The profile of LDi-6S shows little or no change in chondroitin 6-sulfate as a percentage of the entire labeled GAG pool. When the I‘“Clglucosamine data (Table II) were represented in a similar fashion, identical results were obtained (Fig. 2). Although the total J‘“CJglucosamine incorporated into
2.5 1.0 2.0 4.0 1.6
x x x x x
10 ” 10-x 10-x 10 * lo-’
IV ON I “CIGLUCOSAMINE INTO
GAG” Matrix I “CIGAG
Medium \“ClGAG (cpm X 10. :‘I
(%I
tcpm x 10-r’)
(r/r)
118 124 121 118 120 87
100 105 103 100 102 74
54 42 36 46 42 32
100 78 67 8ri 77 60
” The experiment was carried out as described under Methods; the values in counts per minute are a combined value of two culture flasks per reaction condition.
GAG did not change much, there was a significant change in the distribution of radiolabeled glucosamine. In agreement with Fig. 1, the chondroitinase-resistant material increased with increasing retinoic acid. Chondroitin 6-sulfate remained constant. Chondroitin 4-sulfate decreased, and chondroitin (and/or) hyaluronic acid decreased. Identification of the Chondroitinase-Resistant Material
The matrix JY3JGAG was treated with nitrous acid, and the amount of J”“S] released from polymeric GAG as inorganic sulfate and sulfate bound to oligosaccharides was determined. As seen in Table V, at every concentration of retinoic acid there was good agreement between the chondroitinase resistant [%]GAG and cal-
78
SHAPIRO
AND
POON
FIG. 1. Effect of retinoic acid on J”SO, incorporation into matrix GAG. Matrix 13”S]GAG was isolated from chondrocytes grown in the presence of increasing levels of retinoic acid. Total [WGAG was determined (Methods1 and then digested with chondroitinase ABC (Methods). The W fractions are represented as a percentage of the entire W incorporated (Table I).
pCI-GAG ,TOlOli
cware Rellslml
no,-65
AD,-4s
LIDI-OS
FIG. 2. Effect of retinoic acid on l’lClglucosamine incorporation into matrix GAG. Matrix l’Y!]GAG was isolated from chondrocytes grown in the presence of increasing levels of retinoic acid. Total [“CIGAG was determined (Methods) and then digested with chondroitinase ABC (Methods). The “‘C fractions are represented as a percentage of the entire “C incorporated (Table II). TABLE
V
IN NITROUS ACID DEGRADABLE WITH RETINOIC ACID”
INCREASE
Retinoic
2 2 2 2 2
acid
x x x x x
lo-“’ 10-k’ 10-x 10-7 10 Ii
(M)
I’WGAG
[W]CHase resistant (%I
[“S]Heparan sulfateiheparin” (%1
14.0 16.3 22.0 32.0 50.6
13.0 23.0 25.0 40.0 59.0
(i Nitrous acid degradation was performed as described under Methods. 0 Values represent ‘W found as oligosaccharides and inorganic srlfateiundegraded polysaccharides after nitrous acid treatment.
culated amounts of heparan sulfate (and/ or) heparin. Thus, it is concluded that all or nearly all of the chondroitinase-resistant material is heparin sulfate (and/or) heparin.
Effect of Lysosomal inoic Acid Effect
Stabilizers
on the Ret-
The possibility that retinoic acid was reducing [““SIGAG levels by accelerating breakdown due to increased lysosomal enzyme release was tested. Known lysosoma1 membrane stabilizers were tested as antagonists of retinoic acid (Table VI). At 2 x 10mx M retinoic acid ““SO, incorporation into GAG was 30% that of control cells. Cortisone acetate and chloroquine, two lysosomal membrane stabilizers, did not reverse the inhibition of sulfate incorporation. In addition, e-amino caproic acid, a cathepsin inhibitor (20), did not reverse the sulfate inhibition. These results are consistent with the fact that lysosomal enzymes were not responsible for the reduced levels of YS detected in GAG when cells
RETINOIC TABLE COMBINED RETINOIC
Retinoic acid
ACID
AND
GAG
TABLE
VI EFFECT
EFFECT
OF LYSOSOMAL STABILIZERS AND ACID ON :Y3 INCORPORATION INTO GAG” ~.~-ChloroE-Amino None Cortisone ace-
(M)
tate 5
lKf/
0 2 x 10-s 2 x 10 i
100 35 24
!4 Ill1
111 37 N.D.
97 40 31
quine, 0.1 I*gi
caproic acid (0.1
ml
Ml
97 30 26
Retinoic
acid
VII
[ %lGAG
(M)
__~~ 2 2 2 2
x x x x
” Reaction was Methods. IW]GAG flask on Day 1.
performed was added
recovered (96 cpm)
as described to the medium
under of each
were grown in the presence of retinoic acid. Recovery of Added cyte Medium
I”“S]GAG
-.
2.2 2.2 2.2 2.2 2.2
x x x x x
lo-” 10 !’ 10-n 10-T 10 ”
VIII ACID
[““SIGACI’
285 206 135 102 54 30
ON CHONDROCYTES Cell number
100 72 47 36 19 11
99.5 95.5 105.0 101.5 109.0 91.5 ~.- ~~
Protein
DNA (F&T)
56.6 50.0 61.6 61.6 65.1 51.6
3.1 2.5 4.3 3.6 3.6 2.8 ..-
” Experiment was performed as described under Methods. Values represent an average of two determinations; each determination represents two pooled flasks, or a total of four flasks per reaction condition.
OF
100 91 98 93 102
10 I” 10 ” IO-” 10 i
Retinoic acid /hi)
103 29 25
EFFECT OF RETINOIC ACID ON THE RECOVERY ADDED [W]GAG TO THE CULTURE MEDIUM”
OF RETINOIC
0
‘I Experiment was carried out as described under Methods; values represent :‘“S incorporated relative to the control reaction (100%).
TABLE
79
BIOSYNTHESIS
to Chondro-
To test further the hypothesis that breakdown was not the cause for reduced levels of [:‘“S]GAG detected, [““SIGAG was added back to chondrocyte cultures grown in the presence of retinoic acid. The IY3]GAG was prepared from control cells grown as described under methods (Table VII). The levels of retinoic acid used resulted in reduced sulfate incorporation into matrix and medium GAG (Table III). After 4 days in culture, the added [““S]GAG was quantitatively recovered, indicating the absence of GAG degrading enzymes in the medium at the levels of retinoic acid tested. In addition, there was no radiolabel detected in the matrix GAG of these cells, indicating no degradation of added [““SJGAG and reincorporation of Y30., into newly synthesized GAG.
Effect of Retinoic DNA Synthesis
Acid
on Protein
and
At levels of retinoic acid sufficient to reduce :‘“SO, incorporation into GAG, there was no decrease in DNA, cell number, or protein synthesis (Table VIII). Thus, retinoic acid was not operating as a general cytotoxic agent. DISCUSSION
There have been numerous reports on the effect of retinol on cartilage. These studies have documented that retinol at hypervitaminosis A levels (lo-15 IUlml) cause a release of lysosomal enzymes resulting in degradation and resorption of cartilage. There have also been reports that retinol inhibits sulfate fixation into cartilage prior to lysosomal release (lo12). Solursh reported decreased incorporation of Y30, into GAG at levels of retinol which did not result in lysosomal release 112). We have shown that retinoic acid, a naturally occurring metabolite of retinol, inhibits sulfate fixation into GAG by chondrocytes in vitro at 1OF’ M. Retinol at lo-’ M has been reported (12) and confirmed by us to inhibit sulfate incorporation in chondrocytes in uitro. There is no evidence available to us to indicate a role for lysosoma1 enzymes at the levels of retinoic acid tested (5, 7; Table VI). Thus, we conclude that retinoic acid at levels which do not affect lysosomal release inhibit sulfate incorporation into GAG in chondrocytes in
80
SHAPIRO
uitro. The use of [‘%]glucosamine as GAG precursor showed that the synthesis of the polymer is affected as well as the terminal sulfation (Fig. 2). We have not made any attempt to show conversion of glucosamine to galactosamine for incorporation into chondroitin 416 sulfate, nor have we attempted to quantitate the [ ‘%]glucosamine data by taking into account differences in the isotope pools of label [‘Y!]glucosamine or ]“C]galactosamine. The distinct possibility exists that nondialyzable glycolipids, glycopeptides, or glycogen were included in the GAG fraction. Any of these fractions containing radiolabe1from precursor [ ‘“Clglucosamine would have appeared as chondroitinase resistant material. This may explain why control levels of [ “C]glucosamine-labeled chondroitinase-resistant material was somewhat greater than the corresponding ““SO,-labeled material. However, the GAG changes expressed in Fig. 2 are not compromised by the increased control levels of [ ‘“Clchondroitinase-resistant material. The changes observed in the individual GAG fractions as a result of retinoic acid are identical to those observed using %O, as a GAG precursor (Fig. 21. Taken qualitatively, the [ ‘“Clglucosamine data (Fig. 2) were used to confirm the “SO, data (Fig. 1) and did indicate changes in GAG biosynthesis as well as changes in sulfation. In addition to a total reduction in sulfate incorporation into GAG, retinoic acid resulted in a change in the fractional composition of the total GAG pool (Figs. 1 and 2). The relative levels of N-sulfated GAG increased with retinoic acid (Table V, Figs. 1 and 2), and while chondroitin 4-sulfate and chondroitin (and/or) hyaluronic acid decreased. Thus, the retinoic acid effect is not a general inhibition, and in fact affects each GAG selectively. Protein and DNA synthesis (Table VIII) were not changed by retinoic acid. During the time course of this experiment (Days l-4), the cultures are in linear phase of growth. The DNA content of the cultures increased 3.5fold. :%O, incorporation into GAG increased twofold for each 48 h (equal ““SO, pulse conditions of 18 h) of culture. Thus, after 4 days of culture conditions in which the
AND
POON
cells were in a growth phase, retinoic had no measurable effect on total DNA or protein synthesis. In addition, levels of retinoic acid from 2 x 10-l” to 2 x lo+ M had no effect on the incorporation of L[ ‘“C]proline into collagen. This supports the hypothesis that the retinoic acid effect cannot be explained in terms of a general cytotoxic effect but rather a selective effect on GAG biosynthesis. The chondrocytes grown in the absence of retinoic acid appeared typically polygonal. At high levels of retinoic acid (lo-’ M) the cells appeared more fibroblastic. This observation is in agreement with Solursh and Meier, who reported similar effects with retinol at higher concentrations (12). We have shown that retinoic acid a naturally occurring metabolite of retinol, inhibits sulfation at levels much lower than retinol. Studies have been reported indicating a decrease in sulfate fixation into GAG with lo-15 IU/ml (lo-: M) retinol in vitro (10-12). At these levels of retinol any small degree of oxidation to retinoic acid either enzymatically or by nonbiochemical means, such as air oxidation, will obscure the results of the experiments. We suggest that future investigators using high levels of retinol in vitro be concerned with any low levels of retinoic acid present. ACKNOWLEDGMENTS We thank Dr. M. Brin, Dr. K. Gibson and Dr. L. Machlin for their suggestions and for reviewing the manuscript. REFERENCES 1. FELL, id. 2.
3. 4. 5. 6. 7.
H. B.. AND MELLANBY, E. (1952) J. Phys~London/ 116, 320-349. FELL, H. B., MELLANBY, E., AND PELC, S. R. (1954) Brit. Med. J. 2, 611. DINGLE, J. T., FELL, H. B., AND LUCY, J. A. (1966) Biochrvl. J. 98, 173-181. DE DUVE, C., WATTIAUX, R., AND WIBO, M. (1962) Biochi~rn. Phnrmacol. 9, 97-116. DINGLE, J. T., (1961)Biochurrz. J. 79, 509-512. LUCY, J. A., DINGLE, J. T., AND FELL, H. B. (1961) Bzoc~hrnz. J. 79, 500-508. WEISSMAN, G., AND THOMAS, L. (1963) I. Clin. Znurst. 42, 661-669. WEISSMAN, G. (1964) Fc&. Pnr. 23, 1038-1044.
8. 9. FELL,
H.
343-362.
B.,
THOMAS,
L. (1961)Biochem.
J.
114,
RETINOIC
ACID
AND
10. FELL, H. B., MELLANBY, E., AND PELC, S. R. (1958) J. Physiol. 133, 89. 11. MCELLIGOTT, T. F. (1962) J. Path. Bad. 83, 347353. 12. SOLURSH, M., AND MEIER, S. (1973) C&c. ‘I’issue Res. 13, 131-142. 13.
SUZUKI,
Bid.
14. 15.
S.,
AND
STROMINGER,
J.
L.
(1960)
Chem. 235, 257-266. SAITO, H., YAMAGATA, T., AND SUZUKI, S. (1968) J. Bd. Chcm. 243, 1536-1542. LEYVA, A., AND KELLEY. W. N. (1974) Anal.
J.
GAG
BIOSYNTHESIS
81
Biochern. 62, 173-179. 16. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Bid. Chem. 193, 265-275. 17. CONRAD, G. W., AND HART, G. W., Res. Rid. (1975) 44, 253-269. 18. SHAPIRO, S. S., AND SHERMAN, M. I. (1974) Arch. Riwhem. Biophys. 162. 272-280. 19. SHAPIRO, S. S., AND POON, J. P. (1975) Biochrm. Hiophys. Acta 385, 221-231. 20. ALI, S. Y. (1964)Biochenz. J. 93, 611-618.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Effect of Retinoic
STANLEY Department of Biochemical
174,
(1976)
74-81
Acid on Chondrocyte Biosynthsis S. SHAPIRO Nutrition, Received
AND
JAMES
Glycosaminoglycan
P. POON
Roche Research Center, Nutley, New Jersey 07110 September
18, 1975
The effect of retinoic acid on glycosaminoglycan biosynthesis was investigated in rat costal cartilage chondrocytes in uitro. At levels of lo-!’ to lo-” M retinoic acid, s”SO, uptake into glycosaminoglycans was reduced 50%. At these low levels of retinoic acid there was no evidence of lysosomal enzyme release. The results are explained best in terms of modification of glycosaminoglycan synthesis, rather than accelerated degradation. Retinoic acid selectively modified the incorporation of YSO, or L’4Clglucosamine into individual glycosaminoglycans fractions under the conditions studied. The relative incorporation of radiolabeled precursor into heparan sulfate (and/or) heparin increased threeto fourfold. The relative incorporation of radiolabeled precursor remained constant for chondroitin &sulfate, whereas incorporation into chondroitin 4-sulfate and chondroitin (and/or) hyaluronic acid decreased. Under the conditions studied, retinoic acid did not appear to be cytotoxic and did exhibit selective control over glycosaminoglycan biosynthesis. It is suggested that the decreased incorporation of Y!jO, into glycosaminoglycans at hypervitaminosis A levels of retinol may be accounted for by the presence of low levels of retinoic acid, a naturally occurring metabolite.
The classical studies of Fell and Mellanby (1) reported that lo-15 IU/ml of retinol when added to the culture medium of 6- or 7-day-old embryonic chick limb cartilage resulted in limb disintegration and resorption. The matrix became soft, lacked glycosaminoglycans (GAG)’ and finally disappeared. The breakdown products of the cartilage, i.e., inorganic sulfate, hexosamine, and hydroxyproline appeared in the culture medium (2, 3). The retinol mediated resorption was shown to be due to labilization of the lysosomal membrane, and subsequent release of acid hydrolyases into, and outside, the chondrocyte (4-6). It was also reported by Weissman (7) that cartilage explants, when re-
moved from hypervitaminosis A rabbits released lysosomal enzymes into the culture medium. Known lysosomal membrane stabilizers neutralized the retinol-induced lysis both in vitro and in vivo. Cortisone administered in vivo protected rabbits against the cartilage lesions of hypervitaminosis A (8). In addition, less acid hydrolyases were released from cartilage of hypervitaminosis A animals protected with cortisone than by cartilage of animals given high dosesof the vitamin alone. Hydrocortisone was shown to delay cartilage resorption in embryonic chick cartilage in vitro (9). Chloroquine, like cortisone has been shown to antagonize the retinol effect on lysosomal membranes (8). Hypervitaminosis A levels of retinol were observed to initially inhibit ““SO, incorporation into cartilage prior to causing accelerated matrix breakdown (10). McElligott (11) reported that sulfate fixation by chondrocytes was reduced under conditions of hypervitaminosis A, and this was
’ Abbreviation used: GAG, glycosaminoglycans; CHase, chondroitinase ABC (EC 4.2.2.5); A-Di-4S, 2 acetamido-2-deoxy-3-O-(p-D-gluco-4-enepyranosyluronic acid)-4-O-sulfa-n-galatose; A-Di-GS, 2 acetamido-2.deoxy-3-O-(P-n-gluco-4-enepyranosyluronic acidW@sulfo-n-galactose; A-Di-OS, 2 acetamido-Zdeoxy-3-O-(P-n-gluco-4.enepyranosyluronic acid)-ngalactose; TCA, trichloroacetic acid. 74 Copyright All rights
0 1976 by Academc Press, Inc. of reproduction in any form reserved.
RETINOIC
ACID
AND
independent of any lysosomal effect. More recently, Solursh and Meier (12) reported a selective inhibition of GAG biosynthesis by lo-” M retinol without lysosomal release. We now report that retinoic acid, a naturally occurring metabolite of retinol at lo-!’ M modified GAG biosynthesis in chondrocytes in vitro. At this low level of retinoic acid, there was no detectable lysosomal membrane labilization, and there was no detectable decrease in cellular protein or DNA. METHODS Preparation of c!n~&rocytes. Costa1 cartilage from &day-old rats were trimmed and cleaned of connecting tissue. The cartilage was minced with an extra fine dissecting scalpel under a dissecting microscope. Finely minced cartilage (400-500 mgl was suspended in 8.0 ml of Pucks saline G containing 6000 units of collagenase (Type II, Worthington Bicchemicals) and gently stirred at 37°C for 3-4 h. The chondrocytes were separated from undigested debris by repeated sedimentaion in 50.0 ml of growth medium (F-10, 10% fetal calf serum and 100 units/ml of penicillin and streptomycin). Media (3.0 ml) containing 0.3-1.0 x 10” cells were placed in Falcon tissue culture flasks (25 cm> growth area). Retinoic acid in 0.02 ml of ethanol was added at this time. The flasks were incubated at 37°C under 95% air and 5% CO,. After 72 h, 20-40 WCi of HZ3”S0, or 1.5-2.0 $i of l’aC]glucosamine (New England Nuclear) was added, and the incubation was continued for an additional 16 h. Upon completion of the incubation, the medium was decanted from the cell layer. The l:“SJGAG found in the medium is referred to as the medium GAG in this text. The cell layer was washed twice with 3.0 ml of Pucks saline G and then 200 units of collagenase in 2.0 ml of Pucks saline G were added to each flask. After 3 h of incubation, the cells separated from the flasks and floated. Release of the cells from the flasks by crude collagenase resulted in the solubilization l:‘S]GAG which is referred to as matrix GAG in this text. The cells were separated from the collagenase solution by centrifugation and washed twice with Pucks saline G. Cell number was determined immediately. The remaining cells were saved for l”“SlGAG determination, DNA or protein determinations. The medium and matrix containing solutions were mixed with a papain solution to a final concentration of 0.10 M sodium acetate, pH 5.8, 5 mM EDTA, and 5 mM cysteine containing 1.0 mg of papain. The papain digestion was carried out at 55°C for 18 h. Determination of9 or 1% incY~rporateff into chonrlrocvtr glycosaminoglycans, 50% TCA was added to
GAG
BIOSYNTHESIS
75
the papain digestion to give a final TCA concentration of 5%. A minimum of four flasks per reaction condition were carried out. Two flasks per reaction condition were combined for GAG identification. The combined volume for the medium was 7.4 ml and that of the matrix solution 4.6 ml. The TCA supernatants were dialyzed for 2 days with frequent changes of deionized water. A 50-/*l aliquot of the dialyzed TCA supernatant solution containing the [3SlGAG was chromatographed on Whatman No. 3 paper in isobutyric: 0.5 M NH,OH (5:3, V/V) overnight (13). The origins containing the radioactive GAG were cut out, placed in 10 ml of Aquasol (New England Nuclear) and counted in a liquid scintillation spectrophotometer. D~gestiorz wzth c~hondroitinaw ABC. The digestion of I:‘SlGAG with chondroitinase .4BC was performed according to the method of Saito (14). Papain digested and dialyzed medium and matrix I’“SIGAG solutions (500 ~1) were evaporated to dryness under a stream of nitrogen. The samples were then redissolved in 20 ~1 of “enriched” Tris buffer and 30 ~1 of enzyme (0.15 units). The solutions were incubat,ed at 37°C for 4 h and stopped by streaking on Whatman No. 3 paper. The entire reaction was chromatographed in butanol: acetic acid: 1 M NH,OH (2:3:11 according to the method of Saito e/ al. (141. After drying, the standard dissaccharides were visualized under uv light. The chromatograms were cut into i/z-in. segments, placed in 10 ml of Aquasol, and counted in a liquid scintillation spectrophotometer. Chondroqvtu wll nurnh~~r d~tc~rmination. Chondrocytes harvested from the collagenase solutions by centrifugation were washed twice with Pucks saline G. The washed cell pellet was suspended in a small volume of Pucks saline G and cell number was determined with a hemocytometer. DNA and protcir~ tir~iprnlinntlorrs. Chondrocytes from above were collected by centrifugation. Twotenths milliliter 0.01 M Tris-HCl, pH 7.4, was added to the cell pellets. The cells were lysed by- rapidly freezing and thawing seven times in dry ice and a&one. DNA was determined according to Leyva and Kelley (151. Protein was determined according to Lowry ct ni. 116). Deternrinntiorl of N-sulfatd GAG corltc~nt. Nitrous acid degradation was carried out according to the method of Cifonelli as described by Conrad and Hart (17). Aliquots of the dialyzed TCA supernatant solutions were evaporated to dryness under a stream of nitrogen and redissolved in 10 ~1 of water. The samples were then mixed with 20 ~1 of 1 N HCl and 20 ~1 of freshly prepared 20% n-butyl nitrite IV/V) in absolute ethanol (20 ~1 of absolute ethanol without n-butyl nitrite was used in the reaction blank). The reaction was carried out at room temperature for 2 h with gentle shaking At completion. the reaction (80 ~1) was chromatographed on Whatman No. 3 paper in isobutyric acid: 0.5 M NH,OH (5:3. v/v) for 36 h
76
SHAPIRO
AND
(131. The heparan sulfateiheparin content was determined from the chromatogram as previously described by Shapiro and Sherman (18). Recovery of added [“%]GAG from culture medium. [““SlGAG synthesized by control chondrocyte was isolated in the following manner. Medium solutions from control experiments were pooled and treated with papain solution (as described above). TCA (50%) was added to the reaction mixture to a final concentration of 54:. The TCA supernatant was dialyzed extensively against water for 2 days. The dialysate was concentrated and passed through a Millipore 0.22-pm filter. l”“S]GAG (55,500 cpml was added to each flask along with retinoic acid at Day 1. The incubation was stopped at Day 4. GAG was determined as previously described. Effect of lysosomal stabilizers and retinoic acid on GAG synthesis. Lysosomal stabilizers, cortisone acetate (Merck; final concentrations, 1 and 5 *g/ml), chloroquine (Sterling-Winthrop; final concentration, 0.1 pg/ml) and e-amino caproic acid (Sigma; final concentration, 0.1 M) were added to chondrocyte cultures treated with 2 x 10 i and 2 x 10 ’ M retinoic acid at Day 1. The incubation was stopped at Day 4. GAG was isolated as previously described. RESULTS
Effect of Retinoic thesis
Acid
on GAG
Biosyn-
Chondrocytes grown in the presence of 2.0 x 10m9to 2.0 x 10m7M retinoic acid exhibited a dose dependent decrease in YSO, incorporation into GAG (Table I). Identical results were obtained if retinoic acid was added to the culture flasks 18 h after seeding and incubated for 72 h prior TABLE
EFFECT OF Retin$
I
RETINOIC ACID ON :Y30, INTO MATRIX GAG”
acid
CHase”
ii%]
’
INCORPORATION
SDi-68
.LDi-48
resistant
ftotal)
2.0 x lo-!’ 1.0 x 10-H 2.0 x 10-w 1.0
42,540 26,367 18,712 16,164 9,779 8,077
x IO-’
2.0 x 10-T
3,900 3,100 3,155 3,302 3,054 3,569
7,985 3,910 2,750 2,079 1,021 929
30,654 19,347 12,788 10,764 5,685 3,569
U The experiment was carried out as described under Methods; 40 x 10” cpm of carrier free H,:“SO, was added per flask. b Values are expressed in counts per minute and represent
two culture
a total
flasks
incorporation
per reaction
of a pooled
condition.
sample
of
POON
to the addition of radiolabeled GAG precursor. The [“S]GAG was digested with chondroitinase ABC and the products of the digestion identified. The A-Di-6S and A-Di-4S originating from chondroitin 6sulfate and chondroitin 4-sulfate, respectively, show a concomitant decrease in :?30, incorporated into GAG in the presence of increasing retinoic acid. The level of ““SO, incorporated into chondroitinase resistant material remained relatively constant (Table I). When [llC]glucosamine was utilized as a GAG precursor, slightly different results were obtained (Table II). Retinoic acid (2.0 x IO-!’ M) caused a 20% reduction in [ ‘“Clglucosamine incorporated. However, there was no further change in the total 1“C]glucosamine incorporated into GAG until levels of 1.0 x lo-’ M retinoic acid. The [ “C]glucosamine incorporated into chondroitinase resistant material increased with increasing doses of retinoic acid, and A-Di-OS (originating from chondroitin and/or hyaluronic acid) decreased significantly with increasing retinoic acid. Previous studies with this cell system (19) demonstrated the absence of dermatan sulfate. Comparison of Retinoic Acid dium and Matrix GAG
Effect on Me-
The relative incorporation of ““SO, in medium, matrix, and cellular GAG remaining after collagenase treatment was 1.0:0.4:0.0025. The low levels of cellular GAG did not change in the presence of retinoic acid. Due to the relatively low levels of radiolabeled GAG associated with the cellular fraction, this fraction was not routinely examined and was not subjected to chondroitinase ABC digestion. The results in Table III show that YSO, incorporation into GAG associated with both the matrix and the medium is reduced. The :“SO, incorporation into matrix GAG appeared to be reduced to a slightly greater extent than the medium. Using 1’“CJglucosamine as a GAG precursor (Table IV), one seesno reduction in the total incorporation of precursor in the medium. However, the matrix l”‘C]GAG was reduced at levels of retinoic acid of 2.5 x lo-!’ to 4.0 x 10mHM about 20%.
RETINOIC
ACID
AND
GAG
TABLE EFFECT
Retinoic
acid
2.0 1.0 2.0 1.0 2.0
ON
[“CIGAG” (total)
CM)
x x x x x
ACID
OF RETINOIC
INCORPORATION
~~~INTO MATRIX A-Di-4s”
A-Di-6s’
resist-
GAG”
~~~ &Di-OS”
ant
13,468 11,040 10,506 10,368 8,887 8,754
10 !’ 10~’ 10-H 10-y 10 7
II
[‘XJGLUCOSAMINE
CHase” ~
77
BIOSYNTHESIS
2,861 3,744 4,213 4,554 4,618 5,483
1,591 1,426 1,223 1,306 1,012 1,269
6,568 4,268 3,900 3,542 2,419 1,784
2,447 1,582 1,140 956 818 340
” The experiment was carried out as described under Methods; 4 x 10” cpm of I’% lglucosamine was added per flask. ’ Values are expressed in counts per minute and represent a total incorporation of a pooled sample of two culture flasks per reaction condition. TABLE EFFECT
OF RETINOIC
_ ~~~ Retinoic acid (M)
2.5 1.0 2.0 4.0 1.6
x x x x x
10 10 10 10 10
” n ’ ” ;
III
TABLE
ACID ON :‘“S INCORPORATION INTO GAG”
Medium [“:‘S]GAG ----~~~ (cpm x (5% I10 ,I) 171 138 109 105 70 37
Matrix [,“SIGAG (cpm x lo-.‘)
100 80 64 61 41 21
74 46 36 24 28 I4
EFFECT
ACID
INCORPORATION
Retinoic acid CM) ($%I 100 62 49 32 37 19
” The experiment was carried out as described under Methods; 40 x 10” cpm of carrier free H,““SO, was added; values in counts per minute are a combined value of two culture flasks per reaction condition.
Relutiue Distribution of GAG’s Acid-Treated Chondrocytes
OF RETINOIC
in Retinoic
A distinct change in the GAG profile was observed when the data in Table I were expressed as a percentage of total incorporation (Fig. 1). The fraction ofY30, incorporated into the chondroitinase-resistant material increased appreciably with increasing retinoic acid. The Y30, incorporated into A-Di-4S showed a relative decrease in the amount of :%SO.,incorporated in chondroitin 4-sulfate. The profile of LDi-6S shows little or no change in chondroitin 6-sulfate as a percentage of the entire labeled GAG pool. When the I‘“Clglucosamine data (Table II) were represented in a similar fashion, identical results were obtained (Fig. 2). Although the total J‘“CJglucosamine incorporated into
2.5 1.0 2.0 4.0 1.6
x x x x x
10 ” 10-x 10-x 10 * lo-’
IV ON I “CIGLUCOSAMINE INTO
GAG” Matrix I “CIGAG
Medium \“ClGAG (cpm X 10. :‘I
(%I
tcpm x 10-r’)
(r/r)
118 124 121 118 120 87
100 105 103 100 102 74
54 42 36 46 42 32
100 78 67 8ri 77 60
” The experiment was carried out as described under Methods; the values in counts per minute are a combined value of two culture flasks per reaction condition.
GAG did not change much, there was a significant change in the distribution of radiolabeled glucosamine. In agreement with Fig. 1, the chondroitinase-resistant material increased with increasing retinoic acid. Chondroitin 6-sulfate remained constant. Chondroitin 4-sulfate decreased, and chondroitin (and/or) hyaluronic acid decreased. Identification of the Chondroitinase-Resistant Material
The matrix JY3JGAG was treated with nitrous acid, and the amount of J”“S] released from polymeric GAG as inorganic sulfate and sulfate bound to oligosaccharides was determined. As seen in Table V, at every concentration of retinoic acid there was good agreement between the chondroitinase resistant [%]GAG and cal-
78
SHAPIRO
AND
POON
FIG. 1. Effect of retinoic acid on J”SO, incorporation into matrix GAG. Matrix 13”S]GAG was isolated from chondrocytes grown in the presence of increasing levels of retinoic acid. Total [WGAG was determined (Methods1 and then digested with chondroitinase ABC (Methods). The W fractions are represented as a percentage of the entire W incorporated (Table I).
pCI-GAG ,TOlOli
cware Rellslml
no,-65
AD,-4s
LIDI-OS
FIG. 2. Effect of retinoic acid on l’lClglucosamine incorporation into matrix GAG. Matrix l’Y!]GAG was isolated from chondrocytes grown in the presence of increasing levels of retinoic acid. Total [“CIGAG was determined (Methods) and then digested with chondroitinase ABC (Methods). The “‘C fractions are represented as a percentage of the entire “C incorporated (Table II). TABLE
V
IN NITROUS ACID DEGRADABLE WITH RETINOIC ACID”
INCREASE
Retinoic
2 2 2 2 2
acid
x x x x x
lo-“’ 10-k’ 10-x 10-7 10 Ii
(M)
I’WGAG
[W]CHase resistant (%I
[“S]Heparan sulfateiheparin” (%1
14.0 16.3 22.0 32.0 50.6
13.0 23.0 25.0 40.0 59.0
(i Nitrous acid degradation was performed as described under Methods. 0 Values represent ‘W found as oligosaccharides and inorganic srlfateiundegraded polysaccharides after nitrous acid treatment.
culated amounts of heparan sulfate (and/ or) heparin. Thus, it is concluded that all or nearly all of the chondroitinase-resistant material is heparin sulfate (and/or) heparin.
Effect of Lysosomal inoic Acid Effect
Stabilizers
on the Ret-
The possibility that retinoic acid was reducing [““SIGAG levels by accelerating breakdown due to increased lysosomal enzyme release was tested. Known lysosoma1 membrane stabilizers were tested as antagonists of retinoic acid (Table VI). At 2 x 10mx M retinoic acid ““SO, incorporation into GAG was 30% that of control cells. Cortisone acetate and chloroquine, two lysosomal membrane stabilizers, did not reverse the inhibition of sulfate incorporation. In addition, e-amino caproic acid, a cathepsin inhibitor (20), did not reverse the sulfate inhibition. These results are consistent with the fact that lysosomal enzymes were not responsible for the reduced levels of YS detected in GAG when cells
RETINOIC TABLE COMBINED RETINOIC
Retinoic acid
ACID
AND
GAG
TABLE
VI EFFECT
EFFECT
OF LYSOSOMAL STABILIZERS AND ACID ON :Y3 INCORPORATION INTO GAG” ~.~-ChloroE-Amino None Cortisone ace-
(M)
tate 5
lKf/
0 2 x 10-s 2 x 10 i
100 35 24
!4 Ill1
111 37 N.D.
97 40 31
quine, 0.1 I*gi
caproic acid (0.1
ml
Ml
97 30 26
Retinoic
acid
VII
[ %lGAG
(M)
__~~ 2 2 2 2
x x x x
” Reaction was Methods. IW]GAG flask on Day 1.
performed was added
recovered (96 cpm)
as described to the medium
under of each
were grown in the presence of retinoic acid. Recovery of Added cyte Medium
I”“S]GAG
-.
2.2 2.2 2.2 2.2 2.2
x x x x x
lo-” 10 !’ 10-n 10-T 10 ”
VIII ACID
[““SIGACI’
285 206 135 102 54 30
ON CHONDROCYTES Cell number
100 72 47 36 19 11
99.5 95.5 105.0 101.5 109.0 91.5 ~.- ~~
Protein
DNA (F&T)
56.6 50.0 61.6 61.6 65.1 51.6
3.1 2.5 4.3 3.6 3.6 2.8 ..-
” Experiment was performed as described under Methods. Values represent an average of two determinations; each determination represents two pooled flasks, or a total of four flasks per reaction condition.
OF
100 91 98 93 102
10 I” 10 ” IO-” 10 i
Retinoic acid /hi)
103 29 25
EFFECT OF RETINOIC ACID ON THE RECOVERY ADDED [W]GAG TO THE CULTURE MEDIUM”
OF RETINOIC
0
‘I Experiment was carried out as described under Methods; values represent :‘“S incorporated relative to the control reaction (100%).
TABLE
79
BIOSYNTHESIS
to Chondro-
To test further the hypothesis that breakdown was not the cause for reduced levels of [:‘“S]GAG detected, [““SIGAG was added back to chondrocyte cultures grown in the presence of retinoic acid. The IY3]GAG was prepared from control cells grown as described under methods (Table VII). The levels of retinoic acid used resulted in reduced sulfate incorporation into matrix and medium GAG (Table III). After 4 days in culture, the added [““S]GAG was quantitatively recovered, indicating the absence of GAG degrading enzymes in the medium at the levels of retinoic acid tested. In addition, there was no radiolabel detected in the matrix GAG of these cells, indicating no degradation of added [““SJGAG and reincorporation of Y30., into newly synthesized GAG.
Effect of Retinoic DNA Synthesis
Acid
on Protein
and
At levels of retinoic acid sufficient to reduce :‘“SO, incorporation into GAG, there was no decrease in DNA, cell number, or protein synthesis (Table VIII). Thus, retinoic acid was not operating as a general cytotoxic agent. DISCUSSION
There have been numerous reports on the effect of retinol on cartilage. These studies have documented that retinol at hypervitaminosis A levels (lo-15 IUlml) cause a release of lysosomal enzymes resulting in degradation and resorption of cartilage. There have also been reports that retinol inhibits sulfate fixation into cartilage prior to lysosomal release (lo12). Solursh reported decreased incorporation of Y30, into GAG at levels of retinol which did not result in lysosomal release 112). We have shown that retinoic acid, a naturally occurring metabolite of retinol, inhibits sulfate fixation into GAG by chondrocytes in vitro at 1OF’ M. Retinol at lo-’ M has been reported (12) and confirmed by us to inhibit sulfate incorporation in chondrocytes in uitro. There is no evidence available to us to indicate a role for lysosoma1 enzymes at the levels of retinoic acid tested (5, 7; Table VI). Thus, we conclude that retinoic acid at levels which do not affect lysosomal release inhibit sulfate incorporation into GAG in chondrocytes in
80
SHAPIRO
uitro. The use of [‘%]glucosamine as GAG precursor showed that the synthesis of the polymer is affected as well as the terminal sulfation (Fig. 2). We have not made any attempt to show conversion of glucosamine to galactosamine for incorporation into chondroitin 416 sulfate, nor have we attempted to quantitate the [ ‘%]glucosamine data by taking into account differences in the isotope pools of label [‘Y!]glucosamine or ]“C]galactosamine. The distinct possibility exists that nondialyzable glycolipids, glycopeptides, or glycogen were included in the GAG fraction. Any of these fractions containing radiolabe1from precursor [ ‘“Clglucosamine would have appeared as chondroitinase resistant material. This may explain why control levels of [ “C]glucosamine-labeled chondroitinase-resistant material was somewhat greater than the corresponding ““SO,-labeled material. However, the GAG changes expressed in Fig. 2 are not compromised by the increased control levels of [ ‘“Clchondroitinase-resistant material. The changes observed in the individual GAG fractions as a result of retinoic acid are identical to those observed using %O, as a GAG precursor (Fig. 21. Taken qualitatively, the [ ‘“Clglucosamine data (Fig. 2) were used to confirm the “SO, data (Fig. 1) and did indicate changes in GAG biosynthesis as well as changes in sulfation. In addition to a total reduction in sulfate incorporation into GAG, retinoic acid resulted in a change in the fractional composition of the total GAG pool (Figs. 1 and 2). The relative levels of N-sulfated GAG increased with retinoic acid (Table V, Figs. 1 and 2), and while chondroitin 4-sulfate and chondroitin (and/or) hyaluronic acid decreased. Thus, the retinoic acid effect is not a general inhibition, and in fact affects each GAG selectively. Protein and DNA synthesis (Table VIII) were not changed by retinoic acid. During the time course of this experiment (Days l-4), the cultures are in linear phase of growth. The DNA content of the cultures increased 3.5fold. :%O, incorporation into GAG increased twofold for each 48 h (equal ““SO, pulse conditions of 18 h) of culture. Thus, after 4 days of culture conditions in which the
AND
POON
cells were in a growth phase, retinoic had no measurable effect on total DNA or protein synthesis. In addition, levels of retinoic acid from 2 x 10-l” to 2 x lo+ M had no effect on the incorporation of L[ ‘“C]proline into collagen. This supports the hypothesis that the retinoic acid effect cannot be explained in terms of a general cytotoxic effect but rather a selective effect on GAG biosynthesis. The chondrocytes grown in the absence of retinoic acid appeared typically polygonal. At high levels of retinoic acid (lo-’ M) the cells appeared more fibroblastic. This observation is in agreement with Solursh and Meier, who reported similar effects with retinol at higher concentrations (12). We have shown that retinoic acid a naturally occurring metabolite of retinol, inhibits sulfation at levels much lower than retinol. Studies have been reported indicating a decrease in sulfate fixation into GAG with lo-15 IU/ml (lo-: M) retinol in vitro (10-12). At these levels of retinol any small degree of oxidation to retinoic acid either enzymatically or by nonbiochemical means, such as air oxidation, will obscure the results of the experiments. We suggest that future investigators using high levels of retinol in vitro be concerned with any low levels of retinoic acid present. ACKNOWLEDGMENTS We thank Dr. M. Brin, Dr. K. Gibson and Dr. L. Machlin for their suggestions and for reviewing the manuscript. REFERENCES 1. FELL, id. 2.
3. 4. 5. 6. 7.
H. B.. AND MELLANBY, E. (1952) J. Phys~London/ 116, 320-349. FELL, H. B., MELLANBY, E., AND PELC, S. R. (1954) Brit. Med. J. 2, 611. DINGLE, J. T., FELL, H. B., AND LUCY, J. A. (1966) Biochrvl. J. 98, 173-181. DE DUVE, C., WATTIAUX, R., AND WIBO, M. (1962) Biochi~rn. Phnrmacol. 9, 97-116. DINGLE, J. T., (1961)Biochurrz. J. 79, 509-512. LUCY, J. A., DINGLE, J. T., AND FELL, H. B. (1961) Bzoc~hrnz. J. 79, 500-508. WEISSMAN, G., AND THOMAS, L. (1963) I. Clin. Znurst. 42, 661-669. WEISSMAN, G. (1964) Fc&. Pnr. 23, 1038-1044.
8. 9. FELL,
H.
343-362.
B.,
THOMAS,
L. (1961)Biochem.
J.
114,
RETINOIC
ACID
AND
10. FELL, H. B., MELLANBY, E., AND PELC, S. R. (1958) J. Physiol. 133, 89. 11. MCELLIGOTT, T. F. (1962) J. Path. Bad. 83, 347353. 12. SOLURSH, M., AND MEIER, S. (1973) C&c. ‘I’issue Res. 13, 131-142. 13.
SUZUKI,
Bid.
14. 15.
S.,
AND
STROMINGER,
J.
L.
(1960)
Chem. 235, 257-266. SAITO, H., YAMAGATA, T., AND SUZUKI, S. (1968) J. Bd. Chcm. 243, 1536-1542. LEYVA, A., AND KELLEY. W. N. (1974) Anal.
J.
GAG
BIOSYNTHESIS
81
Biochern. 62, 173-179. 16. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Bid. Chem. 193, 265-275. 17. CONRAD, G. W., AND HART, G. W., Res. Rid. (1975) 44, 253-269. 18. SHAPIRO, S. S., AND SHERMAN, M. I. (1974) Arch. Riwhem. Biophys. 162. 272-280. 19. SHAPIRO, S. S., AND POON, J. P. (1975) Biochrm. Hiophys. Acta 385, 221-231. 20. ALI, S. Y. (1964)Biochenz. J. 93, 611-618.