ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 286, No. 1, April, pp. 99-106, 1991
Transforming Growth Factor-(31 Stimulates Synthesis of Proteoglycan Aggregates in Calf Articular Cartilage Organ Cultures Teresa I. Morales Bone Research Branch, National
Institute
of Dental Research, National
Received August 7, 1990, and in revised form November
of Health, Bethesda, Maryland
20892
14, 1990
Previous work showed that transforming growth factor-81 (TGF-Bl), added alone to bovine cartilage organ cultures, stimulated [36S]sulfate incorporation into macromolecular material but did not investigate the fidelity of the stimulated system to maintain synthesis of cartilage-type proteoglycans. This paper provides evidence that chondrocytes synthesize the appropriate proteoglycan matrix under TGF-@l stimulation: (i) there is a coordinated increase in hyaluronic acid and proteoglycan monomer synthesis, (ii) link-stable proteoglycan aggregates are assembled, (iii) the hybrid chondroitin sulfate/ keratan sulfate monomeric species is synthesized, and (iv) there is an increase in protein core synthesis. Some variation in glycosylation patterns was observed when proteoglycans synthesized under TGF-/I1 stimulation were compared to those synthesized under basal conditions. Thus comparing TGF-j31 to basal samples respectively, the monomers were larger (K,” on Sepharose CL2B = 0.29 vs 0.41), the chondroitin sulfate chains were longer by -3.5 kDa, the percentage of total glycosaminoglycan in keratan sulfate increased slightly from -4% and the unsulfated disaccharide de(basal) to -6%, creased from 28% (basal) to 12%. All of these variations are in the direction of a more anionic proteoglycan. Since the ability of proteoglycans to confer resiliency to the cartilage matrix is directly related to their anionic nature, these changes would presumably have a beneficial effect on tissue function. 0 1991 Academic Pros. 113.2.
Cartilage proteoglycans illustrate well the relationship between structure and function. An average of 100 chondroitin sulfate chains and as many as 120 shorter keratan sulfate chains are covalently attached to a core protein (1, 2). Moreover, up to 100 proteoglycan monomers bind to a single chain of hyaluronic acid forming immobilized complexes with molecular masses on the order of 100 million daltons. In tissues, the inextensible collagen network 0003-9861/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
Institutes
compresses these aggregates (l), increasing the electrostatic repulsion of the sulfated glycosaminoglycan chains and the ability of cartilage to oppose compressive forces in the joint. Resident chondrocytes regulate the resiliency of the tissue by assembly of the appropriate proteoglycans and by control of their concentration in the extracellular matrix. During degenerative disease, chondrocytes are unable to maintain homeostasis and there is a progressive, irreversible loss of proteoglycans from the cartilage surface resulting in diminished joint function (3). Experimentally, we have recently shown that addition of transforming growth factor-p1 (TGF-Pl)’ alone to bovine cartilage organ cultures prevents spontaneous proteoglycan losses and maintains relatively constant concentrations of proteoglycans in the matrix for several weeks. In these cultures, anabolism is increased and catabolism decreased relative to basal controls (4). In a separate publication we show that calf articular cartilage synthesizes TGF-fil and we have proposed that TGF-fl plays a predominant role in the intrinsic control of cartilage proteoglycan metabolism (5). In this paper I explore the effects of exogenous TGF-@l administration on proteoglycan aggregate formation and monomer structure. It is shown that TGF-/I increases synthesis of stable proteoglycan aggregates through a coordinated increase in hyaluronic acid and proteoglycan monomer synthesis. The monomers contain chondroitin sulfate and keratan sulfate in a proportion of 16 to 1, indicating a high fidelity of the TGF-P-stimulated system for maintenance of the hybrid species. Small variations in size and glycosylation patterns are observed when the proteogly-
r Abbreviations used: TGF-01, transforming growth factor-/31; DMEM, Dulbecco’s modified Eagle’s medium; BSA, bovine serum albumin; FCS, fetal calf serum; CHAPS, 3-[(cholamidopropyl) dimethylammoniol-lpropanesulfonate; CS, chondroitin sulfate; KS, keratan sulfate; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 99
Inc. reserved.
100
TERESA
cans from the stimulated synthesized under basal tions are in keeping with charged and presumably MATERIALS
AND
cultures are compared to those conditions, but all of the variathe synthesis of a more highly more resilient proteoglycan.
METHODS
General procedures. Articular cartilage slices were dissected from the metacarpalphalangeal joint of young bovines, approximately 3-4 months old, and -2 g of cartilage (wet weight) was cultured in 15 vol (ml/g) of Dulbecco’s modified Eagle’s medium containing 4.5 g/liter glucose and 0.58 g/liter L-glutamine (DMEM) for 5-7 days as described before (6). The tissue was then distributed into 24-well Costar plates in portions of -100 mg per well. Treatments were initiated as follows: (i) the basal samples were cultured in DMEM containing 0.1% bovine serum albumin (BSA), (ii) the experimental samples were cultured in the same medium as above supplemented with 5 rig/ml TGF-PI, and (iii) additional samples were in DMEM + 20% FCS. All cultures were maintained in 1.5 ml medium with daily changes. TGF-/31 was from porcine platelets (R & D Systems, Inc). Aliquots of TGF$l, 300 rig/ml in 4 mM HCl + 0.1% BSA were added daily to fresh culture medium. Following treatment and radiolabeling of the cultures (described below), the tissues were sequentially extracted in 4 M guanidine HCl buffer and 0.5 N NaOH. Unless otherwise indicated, biosynthesis of proteoglycans was estimated from the amount of 35S label in macromolecular material following removal of unincorporated isotope on Pharmacia PDlO columns. The macromolecular 35S activities in the guanidine HCI and NaOH extracts were added in the calculations. Rates were expressed as dpm [?S]sulfate incorporated/pg hydroxyproline; previous experiments showed that the collagen content of the cultures is constant for prolonged periods of time under the experimental conditions described in this paper (4). Unless otherwise specified, the dimensions of all columns were 0.7 X 100 cm. Buffers and solutions used. Guanidine HCl buffer (4 M or 0.5 M): 0.05 M Tris HCl, pH 7.0, containing 4 M (or 0.5 M) guanidine hydrochloride and 0.5% (w/v) 3-[(3-cholamidopropyl) dimethylammoniol-lpropanesulfonate (Chaps). Proteinuse inhibitor mix: 1 X lo-” M each pepstatin A and leupeptin, 1 mM each phenylmethylsulfonyl fluoride, iodoacetamide (or 5 mM N-ethylmaleimide), and o-phenanthroline. Urea buffer:0.05 M sodium acetate, pH 6.0, containing 0.15 M sodium chloride and 8 M urea. Bicarbonate buffer: 0.15 M sodium bicarbonate buffer, pH 8.0. Acetate buffer: 0.05 M sodium acetate, pH 6.0, containing 5 mM EDTA, 0.15 M NaCl and 0.5% Chaps. Pyridine buffer: 0.5 M pyridinium acetate, pH 5.0. 2’ri.s acetate bufec0.1 M Tris acetate, pH 7.6, containing 0.05 M EDTA. Ammonium acetate bu/fer:0.05 M ammonium acetate, pH 6.0. Guanidine HCl buffer (1 M): 0.1 M Tris maleate, pH 6.8, containing 1 M guanidine HCl and 0.5% Chaps. Collugenase buffer: 0.025 M Tris HCI buffer, pH 7.5, containing 0.005 M calcium chloride and inhibitor mix without o-phenanthroline. Tris buffer: 0.05 M Tris buffer, pH 8.0, containing 0.05 M sodium fluoride and 0.25% Chaps. 2% borate buffer: 0.5 M Tris-HCl, 0.1 M boric acid, 0.0036 M sulfuric acid, pH 8.0, in 52% acetonitrile and 12% methanol. Proteoglycan monomer and glycosaminoglycan analysis. Cartilage batches (500 mg wet wt in 15 vol of medium) were cultured under the appropriate experimental treatment for 6 days and then double labeled with 60 &i/ml [3H]serine and 20 &i/ml [36S]sulfate for 4 h. The tissues were rinsed with Hank’s balanced salt solution containing proteinase inhibitor mix and frozen in blocks of distilled water. Slices of 30 pm were prepared with a freezing microtome and extracted in 4 M guanidine HCl buffer + proteinase inhibitors. The extracts were chromatographed on lo-ml Sephadex G-50 columns for equilibration into urea buffer and removal of unincorporated isotope. The V, fractions of each sample were pooled and applied on lo-ml columns of DEAE-Sephacel in urea buffer. These were washed with 3 column vol of buffer and the bound fractions eluted by a linear sodium chloride gradient, 0.15 to 1.5 M. The peaks eluting with -0.7 M sodium chloride were dialyzed against bi-
I. MORALES carbonate buffer + proteinase inhibitor mix, dried, and resuspended in 4 M guanidine HCl buffer + inhibitors for chromatography on a Sepharose CL-2B equilibrated in the same buffer. Following chromatography the fractions containing the large proteoglycans were combined into three pools representing the ascending shoulder, middle portion, and descending portion of the peak (Fig. 6, top panels). The peak containing the small proteoglycans was pooled separately. All pools were dialyzed against ammonium acetate buffer and the portions dried. The samples, resuspended in 300 ~1 Tris acetate buffer, received papain (activated by 5 mM dithiothreitol, 10 min, 25°C) to 0.05 mg/ml. Following incubation at 65’C for 3 h, papain was inactivated by heating at 100°C for 5 min and the samples were subdivided in two portions. Chondroitin ABC lyase (ICN Immunobiologicals), 0.5 milliunits/pg chondroitin sulfate, was added to both halves. Keratanase (ICN Immunobiologicals), 0.5 milliunits/pg, was added to only one of the portions. Samples were incubated for 18 h at 37°C and analyzed by elution from a Sephacryl S200 column in 1 M goanidine HCl buffer. In other samples, the papainreleased glycosaminoglycan chains were precipitated with 4 vol of ethanol, resuspended in pyridine buffer, and eluted from a 1 X 30-cm Superose 6 column calibrated with chondroitin sulfate standards (7). Proteoglycan aggregate analysis. Cartilage samples were cultured for 9 or 18 days and then double labeled with 60 &i/ml [3H]glucosamine and [s5S]sulfate in the experimental medium for 16 h. The labeling medium was removed, and the cultures were washed with DMEM (3X at 25”C, 1 h at 37°C) to remove unincorporated isotope and then chased for 48 h at 37’C in DMEM + 20% FCS to allow for stabilization of newly synthesized aggregates.2 The cartilage samples were washed with collagenase buffer, and replicates from each treatment were pooled, transferred to plastic blocks containing distilled water, and frozen on dry ice. Thirty-micrometer slices were prepared from each sample and the frozen tissue slices transfered to a vial with buffer. Purified Clostridium histolyticum collagenase (type III, Biofactures Co.) was added to all samples (1100 BTC nominal units/gram wet weight; one BTC unit releases 1.0 nmol of leucine equivalent per minute from bovine tendon collagen at 37’C, pH 7.2). Digestion was at 37°C for 5 h. Guanidine HCl and o-phenanthroline were then added to a final concentration of 0.5 M and 1 mM, respectively, and the samples were gently homogenized and centrifuged (9). The aggregates were purified as described before (9, 10). Disaccharide analysis. The purified aggregates were dialyzed against acetate buffer and digested with 0.05 mg/ml papain in the presence of 1 mM dithiothreitol at 60°C for 5 h. The reaction was stopped by iodoacetamide, 5 mM, and the samples were concentrated by speed vacuum centrifugation and dialyzed against Tris buffer. Following digestion with Chondroitin ABC lyase, (5 munits/pg, 37”C, 5 h) 4 vol of ethanol was added and the samples were stored overnight at -2OOC. After removal of a small pellet containing the enzyme and undigested material, supernatants were eluted from a partisil 5 PAC column equilibrated in Tris borate buffer. Oligosaccharide competition experiments. Ten milligrams of umbilical cord hyaluronate (Sigma)/ml in acetate buffer was digested for 2 h at 37°C with 150 pg/ml bovine testicular hyaluronidase (chromatographically purified, Sigma) and eluted from a Bio-Gel PlO column in bicarbonate buffer (not shown). This enzyme produces a mixture of products, including tetra to decasaccharides (11). The column was calibrated with tetra- and hexasaccharides obtained by a limit digestion of hyaluronate with another enzyme, Streptomyces hyaluronidase. The decasaccharide fraction of the testicular hyaluronidase digest was dried and resuspended in distilled water. The hexuronate content was measured by the method of Bitter and Muir (12) and the decasaccharide mixture added to crude
* A time-dependent maturation of newly synthesized been described during which the affinity of the monomer acid increases; it is believed that this process involves a hyaluronic acid binding region of the proteoglycan with disulfide bonds [reviewed in Ref. (8)].
monomers has for hyaluronic refolding of the reformation of
TRANSFORMING
GROWTH
FACTOR-01
STIMULATION
cartilage supernatants in a 3% proportion to the endogenous hyaluronate. Following incubation at 37”C, 18 h, the samples were eluted from a CL2B column in 0.5 M guanidine HCl buffer or purified by velocity sedimentation and equilibrium density gradient centrifugation as described above.
OF PROTEOGLYCAN
ANABOLISM
Velocity Sedimentation 1A.
101
Equilibrium Centrifugation
1
I \c.
RESULTS
For each experiment described in this paper, articular cartilage was dissected from the two metacarpalphalangeal joints of one calf. The slices were pooled, sorted into individual portions, and maintained as organ cultures for I the duration of the experimental period (see Materials I I I --c-----land Methods). All samples were first cultured for 5-7 days Aggregate Monomer Other Aggregate Membrane Bound under basal conditions (DMEM containing 0.1% BSA). Components During this period, the rates of incorporation of [35S]sulfate into macromolecules declined, generally to - 4 FIG. 2. Velocity sedimentation and equilibrium centrifugation of cartilage extracts. Cartilage extracts prepared under nondenaturing of values on Day 0 (5) and then stayed relatively constant. conditions (i.e., containing native proteoglycan aggregates) were subAfter 5-7 days, the cartilage slices were sorted into ex- jected to velocity sedimentation on preformed cesium sulfate gradients perimental groups as follows: (i) continuation of basal (0.15-0.5 M, 20,000 rpm, 6 h, 8°C) and the ?S radioactivity in the eluted fractions is shown in the figure. A and B show samples treated for 18 conditions (ii) TGF$l treatment in basal medium + 5 ng/ days with TGF-(31 and FCS, respectively. The bottom of the gradient ml TGF-/31, and (iii) DMEM + 20% FCS. The experitubes is illustrated in the left hand side of the panels. The aggregate mental treatments were carried out for 7 to 18 days, as samples were pooled as indicated by the horizontal bar and subjected indicated. to equilibrium density gradient centrifugation (38,000 rpm, 48 h, 8°C). TGF-Bl treatment for 6 days stimulated [35S]sulfate C and D illustrate the fractionation of TGF-fll- and FCS-treated samples respectively. incorporation an average of 4.5-fold f 1.2, n = 3 (*SD, n = number of experiments using different animals). This confirms results previously published in a communication (4). Stimulation by 20% FCS was 3.84 + 1.0, n = 2 cursor pool is derived almost exclusively from environ(+ range). We previously showed that >95% of the 35S mental sulfate ( 13).3,4 activity is incorporated into proteoglycans in this calf organ culture system (6). Previous work also demonstrated Analysis of Native Proteoglycan Aggregates that in various cell cultures, including chondrocytes, inCartilage organ cultures were maintained for 9 or 18 corporation of this label into proteoglycans is independent days and double labeled with [3H]glucosamine and of cellular metabolic changes since the intracellular pre[35S]sulfate. The anabolic rates of these cultures, measured as [35S]sulfate incorporated/pg hydroxyproline are shown in Fig. 1. A threefold increase in sulfate incorpoTotal %-Incorporation in Explants ration was seen after 9 days and maintained for 18 days in the TGF-pl-stimulated cultures. The stimulation by 20% FCS was comparable. Following 18 days of treatment, the TGF-fil- and FCS-treated cultures contained 18 and Ej Basal 20 yg chondroitin sulfate/mg wet wt, while the basal cul0 FCS tures had a diminished chondroitin sulfate content, only e OTGF P I 7 I.cg/mg. This supports our previous observation that 2 TGF-Pl prevents spontaneous proteoglycan loss from ” 600 bovine cartilage organ cultures (4). % Assessment of proteoglycan aggregate synthesis re8 quired extraction of the complexes from cartilage in nondenaturing buffers. Purified bacterial collagenase was used to open the collagen network of the tissue so that native aggregates could be extracted in nondissociative buffers
a
Day 9
Day 1E
FIG. 1. Total 3’s incorporation in explants maintained up to 18 days under various treatments. Sulfate incorporation was determined by analysis of [3SS]sulfate incorporated into macromolecular components (see Materials and Methods).
3 The primary evidence for this is that cysteine and methionine, the only known sources of metabolic sulfate, contribute
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 286, No. 1, April, pp. 99-106, 1991
Transforming Growth Factor-(31 Stimulates Synthesis of Proteoglycan Aggregates in Calf Articular Cartilage Organ Cultures Teresa I. Morales Bone Research Branch, National
Institute
of Dental Research, National
Received August 7, 1990, and in revised form November
of Health, Bethesda, Maryland
20892
14, 1990
Previous work showed that transforming growth factor-81 (TGF-Bl), added alone to bovine cartilage organ cultures, stimulated [36S]sulfate incorporation into macromolecular material but did not investigate the fidelity of the stimulated system to maintain synthesis of cartilage-type proteoglycans. This paper provides evidence that chondrocytes synthesize the appropriate proteoglycan matrix under TGF-@l stimulation: (i) there is a coordinated increase in hyaluronic acid and proteoglycan monomer synthesis, (ii) link-stable proteoglycan aggregates are assembled, (iii) the hybrid chondroitin sulfate/ keratan sulfate monomeric species is synthesized, and (iv) there is an increase in protein core synthesis. Some variation in glycosylation patterns was observed when proteoglycans synthesized under TGF-/I1 stimulation were compared to those synthesized under basal conditions. Thus comparing TGF-j31 to basal samples respectively, the monomers were larger (K,” on Sepharose CL2B = 0.29 vs 0.41), the chondroitin sulfate chains were longer by -3.5 kDa, the percentage of total glycosaminoglycan in keratan sulfate increased slightly from -4% and the unsulfated disaccharide de(basal) to -6%, creased from 28% (basal) to 12%. All of these variations are in the direction of a more anionic proteoglycan. Since the ability of proteoglycans to confer resiliency to the cartilage matrix is directly related to their anionic nature, these changes would presumably have a beneficial effect on tissue function. 0 1991 Academic Pros. 113.2.
Cartilage proteoglycans illustrate well the relationship between structure and function. An average of 100 chondroitin sulfate chains and as many as 120 shorter keratan sulfate chains are covalently attached to a core protein (1, 2). Moreover, up to 100 proteoglycan monomers bind to a single chain of hyaluronic acid forming immobilized complexes with molecular masses on the order of 100 million daltons. In tissues, the inextensible collagen network 0003-9861/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
Institutes
compresses these aggregates (l), increasing the electrostatic repulsion of the sulfated glycosaminoglycan chains and the ability of cartilage to oppose compressive forces in the joint. Resident chondrocytes regulate the resiliency of the tissue by assembly of the appropriate proteoglycans and by control of their concentration in the extracellular matrix. During degenerative disease, chondrocytes are unable to maintain homeostasis and there is a progressive, irreversible loss of proteoglycans from the cartilage surface resulting in diminished joint function (3). Experimentally, we have recently shown that addition of transforming growth factor-p1 (TGF-Pl)’ alone to bovine cartilage organ cultures prevents spontaneous proteoglycan losses and maintains relatively constant concentrations of proteoglycans in the matrix for several weeks. In these cultures, anabolism is increased and catabolism decreased relative to basal controls (4). In a separate publication we show that calf articular cartilage synthesizes TGF-fil and we have proposed that TGF-fl plays a predominant role in the intrinsic control of cartilage proteoglycan metabolism (5). In this paper I explore the effects of exogenous TGF-@l administration on proteoglycan aggregate formation and monomer structure. It is shown that TGF-/I increases synthesis of stable proteoglycan aggregates through a coordinated increase in hyaluronic acid and proteoglycan monomer synthesis. The monomers contain chondroitin sulfate and keratan sulfate in a proportion of 16 to 1, indicating a high fidelity of the TGF-P-stimulated system for maintenance of the hybrid species. Small variations in size and glycosylation patterns are observed when the proteogly-
r Abbreviations used: TGF-01, transforming growth factor-/31; DMEM, Dulbecco’s modified Eagle’s medium; BSA, bovine serum albumin; FCS, fetal calf serum; CHAPS, 3-[(cholamidopropyl) dimethylammoniol-lpropanesulfonate; CS, chondroitin sulfate; KS, keratan sulfate; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 99
Inc. reserved.
100
TERESA
cans from the stimulated synthesized under basal tions are in keeping with charged and presumably MATERIALS
AND
cultures are compared to those conditions, but all of the variathe synthesis of a more highly more resilient proteoglycan.
METHODS
General procedures. Articular cartilage slices were dissected from the metacarpalphalangeal joint of young bovines, approximately 3-4 months old, and -2 g of cartilage (wet weight) was cultured in 15 vol (ml/g) of Dulbecco’s modified Eagle’s medium containing 4.5 g/liter glucose and 0.58 g/liter L-glutamine (DMEM) for 5-7 days as described before (6). The tissue was then distributed into 24-well Costar plates in portions of -100 mg per well. Treatments were initiated as follows: (i) the basal samples were cultured in DMEM containing 0.1% bovine serum albumin (BSA), (ii) the experimental samples were cultured in the same medium as above supplemented with 5 rig/ml TGF-PI, and (iii) additional samples were in DMEM + 20% FCS. All cultures were maintained in 1.5 ml medium with daily changes. TGF-/31 was from porcine platelets (R & D Systems, Inc). Aliquots of TGF$l, 300 rig/ml in 4 mM HCl + 0.1% BSA were added daily to fresh culture medium. Following treatment and radiolabeling of the cultures (described below), the tissues were sequentially extracted in 4 M guanidine HCl buffer and 0.5 N NaOH. Unless otherwise indicated, biosynthesis of proteoglycans was estimated from the amount of 35S label in macromolecular material following removal of unincorporated isotope on Pharmacia PDlO columns. The macromolecular 35S activities in the guanidine HCI and NaOH extracts were added in the calculations. Rates were expressed as dpm [?S]sulfate incorporated/pg hydroxyproline; previous experiments showed that the collagen content of the cultures is constant for prolonged periods of time under the experimental conditions described in this paper (4). Unless otherwise specified, the dimensions of all columns were 0.7 X 100 cm. Buffers and solutions used. Guanidine HCl buffer (4 M or 0.5 M): 0.05 M Tris HCl, pH 7.0, containing 4 M (or 0.5 M) guanidine hydrochloride and 0.5% (w/v) 3-[(3-cholamidopropyl) dimethylammoniol-lpropanesulfonate (Chaps). Proteinuse inhibitor mix: 1 X lo-” M each pepstatin A and leupeptin, 1 mM each phenylmethylsulfonyl fluoride, iodoacetamide (or 5 mM N-ethylmaleimide), and o-phenanthroline. Urea buffer:0.05 M sodium acetate, pH 6.0, containing 0.15 M sodium chloride and 8 M urea. Bicarbonate buffer: 0.15 M sodium bicarbonate buffer, pH 8.0. Acetate buffer: 0.05 M sodium acetate, pH 6.0, containing 5 mM EDTA, 0.15 M NaCl and 0.5% Chaps. Pyridine buffer: 0.5 M pyridinium acetate, pH 5.0. 2’ri.s acetate bufec0.1 M Tris acetate, pH 7.6, containing 0.05 M EDTA. Ammonium acetate bu/fer:0.05 M ammonium acetate, pH 6.0. Guanidine HCl buffer (1 M): 0.1 M Tris maleate, pH 6.8, containing 1 M guanidine HCl and 0.5% Chaps. Collugenase buffer: 0.025 M Tris HCI buffer, pH 7.5, containing 0.005 M calcium chloride and inhibitor mix without o-phenanthroline. Tris buffer: 0.05 M Tris buffer, pH 8.0, containing 0.05 M sodium fluoride and 0.25% Chaps. 2% borate buffer: 0.5 M Tris-HCl, 0.1 M boric acid, 0.0036 M sulfuric acid, pH 8.0, in 52% acetonitrile and 12% methanol. Proteoglycan monomer and glycosaminoglycan analysis. Cartilage batches (500 mg wet wt in 15 vol of medium) were cultured under the appropriate experimental treatment for 6 days and then double labeled with 60 &i/ml [3H]serine and 20 &i/ml [36S]sulfate for 4 h. The tissues were rinsed with Hank’s balanced salt solution containing proteinase inhibitor mix and frozen in blocks of distilled water. Slices of 30 pm were prepared with a freezing microtome and extracted in 4 M guanidine HCl buffer + proteinase inhibitors. The extracts were chromatographed on lo-ml Sephadex G-50 columns for equilibration into urea buffer and removal of unincorporated isotope. The V, fractions of each sample were pooled and applied on lo-ml columns of DEAE-Sephacel in urea buffer. These were washed with 3 column vol of buffer and the bound fractions eluted by a linear sodium chloride gradient, 0.15 to 1.5 M. The peaks eluting with -0.7 M sodium chloride were dialyzed against bi-
I. MORALES carbonate buffer + proteinase inhibitor mix, dried, and resuspended in 4 M guanidine HCl buffer + inhibitors for chromatography on a Sepharose CL-2B equilibrated in the same buffer. Following chromatography the fractions containing the large proteoglycans were combined into three pools representing the ascending shoulder, middle portion, and descending portion of the peak (Fig. 6, top panels). The peak containing the small proteoglycans was pooled separately. All pools were dialyzed against ammonium acetate buffer and the portions dried. The samples, resuspended in 300 ~1 Tris acetate buffer, received papain (activated by 5 mM dithiothreitol, 10 min, 25°C) to 0.05 mg/ml. Following incubation at 65’C for 3 h, papain was inactivated by heating at 100°C for 5 min and the samples were subdivided in two portions. Chondroitin ABC lyase (ICN Immunobiologicals), 0.5 milliunits/pg chondroitin sulfate, was added to both halves. Keratanase (ICN Immunobiologicals), 0.5 milliunits/pg, was added to only one of the portions. Samples were incubated for 18 h at 37°C and analyzed by elution from a Sephacryl S200 column in 1 M goanidine HCl buffer. In other samples, the papainreleased glycosaminoglycan chains were precipitated with 4 vol of ethanol, resuspended in pyridine buffer, and eluted from a 1 X 30-cm Superose 6 column calibrated with chondroitin sulfate standards (7). Proteoglycan aggregate analysis. Cartilage samples were cultured for 9 or 18 days and then double labeled with 60 &i/ml [3H]glucosamine and [s5S]sulfate in the experimental medium for 16 h. The labeling medium was removed, and the cultures were washed with DMEM (3X at 25”C, 1 h at 37°C) to remove unincorporated isotope and then chased for 48 h at 37’C in DMEM + 20% FCS to allow for stabilization of newly synthesized aggregates.2 The cartilage samples were washed with collagenase buffer, and replicates from each treatment were pooled, transferred to plastic blocks containing distilled water, and frozen on dry ice. Thirty-micrometer slices were prepared from each sample and the frozen tissue slices transfered to a vial with buffer. Purified Clostridium histolyticum collagenase (type III, Biofactures Co.) was added to all samples (1100 BTC nominal units/gram wet weight; one BTC unit releases 1.0 nmol of leucine equivalent per minute from bovine tendon collagen at 37’C, pH 7.2). Digestion was at 37°C for 5 h. Guanidine HCl and o-phenanthroline were then added to a final concentration of 0.5 M and 1 mM, respectively, and the samples were gently homogenized and centrifuged (9). The aggregates were purified as described before (9, 10). Disaccharide analysis. The purified aggregates were dialyzed against acetate buffer and digested with 0.05 mg/ml papain in the presence of 1 mM dithiothreitol at 60°C for 5 h. The reaction was stopped by iodoacetamide, 5 mM, and the samples were concentrated by speed vacuum centrifugation and dialyzed against Tris buffer. Following digestion with Chondroitin ABC lyase, (5 munits/pg, 37”C, 5 h) 4 vol of ethanol was added and the samples were stored overnight at -2OOC. After removal of a small pellet containing the enzyme and undigested material, supernatants were eluted from a partisil 5 PAC column equilibrated in Tris borate buffer. Oligosaccharide competition experiments. Ten milligrams of umbilical cord hyaluronate (Sigma)/ml in acetate buffer was digested for 2 h at 37°C with 150 pg/ml bovine testicular hyaluronidase (chromatographically purified, Sigma) and eluted from a Bio-Gel PlO column in bicarbonate buffer (not shown). This enzyme produces a mixture of products, including tetra to decasaccharides (11). The column was calibrated with tetra- and hexasaccharides obtained by a limit digestion of hyaluronate with another enzyme, Streptomyces hyaluronidase. The decasaccharide fraction of the testicular hyaluronidase digest was dried and resuspended in distilled water. The hexuronate content was measured by the method of Bitter and Muir (12) and the decasaccharide mixture added to crude
* A time-dependent maturation of newly synthesized been described during which the affinity of the monomer acid increases; it is believed that this process involves a hyaluronic acid binding region of the proteoglycan with disulfide bonds [reviewed in Ref. (8)].
monomers has for hyaluronic refolding of the reformation of
TRANSFORMING
GROWTH
FACTOR-01
STIMULATION
cartilage supernatants in a 3% proportion to the endogenous hyaluronate. Following incubation at 37”C, 18 h, the samples were eluted from a CL2B column in 0.5 M guanidine HCl buffer or purified by velocity sedimentation and equilibrium density gradient centrifugation as described above.
OF PROTEOGLYCAN
ANABOLISM
Velocity Sedimentation 1A.
101
Equilibrium Centrifugation
1
I \c.
RESULTS
For each experiment described in this paper, articular cartilage was dissected from the two metacarpalphalangeal joints of one calf. The slices were pooled, sorted into individual portions, and maintained as organ cultures for I the duration of the experimental period (see Materials I I I --c-----land Methods). All samples were first cultured for 5-7 days Aggregate Monomer Other Aggregate Membrane Bound under basal conditions (DMEM containing 0.1% BSA). Components During this period, the rates of incorporation of [35S]sulfate into macromolecules declined, generally to - 4 FIG. 2. Velocity sedimentation and equilibrium centrifugation of cartilage extracts. Cartilage extracts prepared under nondenaturing of values on Day 0 (5) and then stayed relatively constant. conditions (i.e., containing native proteoglycan aggregates) were subAfter 5-7 days, the cartilage slices were sorted into ex- jected to velocity sedimentation on preformed cesium sulfate gradients perimental groups as follows: (i) continuation of basal (0.15-0.5 M, 20,000 rpm, 6 h, 8°C) and the ?S radioactivity in the eluted fractions is shown in the figure. A and B show samples treated for 18 conditions (ii) TGF$l treatment in basal medium + 5 ng/ days with TGF-(31 and FCS, respectively. The bottom of the gradient ml TGF-/31, and (iii) DMEM + 20% FCS. The experitubes is illustrated in the left hand side of the panels. The aggregate mental treatments were carried out for 7 to 18 days, as samples were pooled as indicated by the horizontal bar and subjected indicated. to equilibrium density gradient centrifugation (38,000 rpm, 48 h, 8°C). TGF-Bl treatment for 6 days stimulated [35S]sulfate C and D illustrate the fractionation of TGF-fll- and FCS-treated samples respectively. incorporation an average of 4.5-fold f 1.2, n = 3 (*SD, n = number of experiments using different animals). This confirms results previously published in a communication (4). Stimulation by 20% FCS was 3.84 + 1.0, n = 2 cursor pool is derived almost exclusively from environ(+ range). We previously showed that >95% of the 35S mental sulfate ( 13).3,4 activity is incorporated into proteoglycans in this calf organ culture system (6). Previous work also demonstrated Analysis of Native Proteoglycan Aggregates that in various cell cultures, including chondrocytes, inCartilage organ cultures were maintained for 9 or 18 corporation of this label into proteoglycans is independent days and double labeled with [3H]glucosamine and of cellular metabolic changes since the intracellular pre[35S]sulfate. The anabolic rates of these cultures, measured as [35S]sulfate incorporated/pg hydroxyproline are shown in Fig. 1. A threefold increase in sulfate incorpoTotal %-Incorporation in Explants ration was seen after 9 days and maintained for 18 days in the TGF-pl-stimulated cultures. The stimulation by 20% FCS was comparable. Following 18 days of treatment, the TGF-fil- and FCS-treated cultures contained 18 and Ej Basal 20 yg chondroitin sulfate/mg wet wt, while the basal cul0 FCS tures had a diminished chondroitin sulfate content, only e OTGF P I 7 I.cg/mg. This supports our previous observation that 2 TGF-Pl prevents spontaneous proteoglycan loss from ” 600 bovine cartilage organ cultures (4). % Assessment of proteoglycan aggregate synthesis re8 quired extraction of the complexes from cartilage in nondenaturing buffers. Purified bacterial collagenase was used to open the collagen network of the tissue so that native aggregates could be extracted in nondissociative buffers
a
Day 9
Day 1E
FIG. 1. Total 3’s incorporation in explants maintained up to 18 days under various treatments. Sulfate incorporation was determined by analysis of [3SS]sulfate incorporated into macromolecular components (see Materials and Methods).
3 The primary evidence for this is that cysteine and methionine, the only known sources of metabolic sulfate, contribute
