Cur. J. Biochem. 205, 277-286 (1992) ,c' FEBS 1992

Transforming growth factor+ induces selective increase of proteoglycan production and changes in the copolymeric structure of dermatan sulphate in human skin fibroblasts Gunilla WESTERGREN-THORSSON', Artur SCHMIDTCHEN I , Bengt SARNSTRAND',*, Lars-Ake FRANSSON' and Anders MALMSTROM Department of Physiological Chemistry, University of Lund, Sweden ' Department of Pharmacology Preclinical R&D, Astra Draco AB, Lund, Sweden (Received December 27, 1991)

-

EJB 91 1402

Human embryonic skin fibroblasts were pretreated with transforming growth factor-p (TGF-4) for 6 h and then labeled with [35S]sulphate and ['H]leucine for 24 h. Radiolabeled proteoglycans from the culture medium and the cell layer were isolated and separated by isopycnic density-gradient centrifugation, followed by gel, ion-exchange and hydrophobic-interaction chromatography. The major proteoglycan species were examined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate before and after enzymatic degradation of the polysaccharide chains. The results showed that TGF-P increased the production of several different 35S-labelled proteoglycans. A large chondroitin/dermatan sulphate proteoglycan (with core proteins of approximately 400 - 500 kDa) increased 5 - 7-fold and a small dermatan sulphate proteoglycan (PG-S1, also termed biglycan, with a core protein of 43 kDa) increased 3 -4-fold both in the medium and in the cell layer. Only a small effect was observed on another dermatan sulphate proteoglycan, PG-S2 (also named decorin). These observations are generally in agreement with results of other studies using similar cell types. In addition, we have found that the major heparan sulphate proteoglycan of the cell layer (protein core approximately 350 kDa) was increased by TGF-fl treatment, whereas all the other smaller heparan sulphate proteoglycans with protein cores from 250 kDa to 30 kDa appeared unaffected. To investigate whether TGF-P also influences the glycosaminoglycan (GAG) chain-synthesizing machinery, we also characterized GAGs derived from proteoglycans synthesized by TGF-P-trcated cells. There was generally no increase in the size of the GAG chains. However, the dermatan sulphate chains on biglycan and decorin from TGF-fi treated cultures contained a larger proportion of Dglucuronosyl residues than those derived from untreated cultures. No effect was noted on the 4- and 6-sulphation of the GAG chains. By the use of p-nitrophenyl fi-D-xyloside (an initiator of GAG synthesis) it could be demonstrated that chain synthesis was also enhanced in TGF-P-treated cells (approximately twofold). Furthermore, the dermatan sulphate chains synthesized on the xyloside in TGF-fitreated fibroblasts contained a larger proportion of D-glucuronosyl residues than those of the control. These novel findings indicate that TGF-P affects proteoglycan synthesis both quantitatively and qualitatively and that it can also change the copolymeric structure of the GAG by affecting the GAGsynthesizing machinery. Altered proteoglycan structure and production may have profound effects on the properties of extracellular matrices, whch can affect cell growth and migration as well as organisation of matrix fibres.

Extracellular matrices consist of a variety of macromolecules such as collagens, fibronectin, hyaluronan and various proteoglycans. The latter are complex macromolecules consisting of a core protein substituted with glycosaminoglycans Correspondence to G. Westergren-Thorsson, Department of Physiological Chemistry, P.O. Box 94, S-221 00 Lund, Sweden, Fax C4646113417 ..lhbrri~iarinns.TGF-,B, transforming growth factor$; PG, proreoglycan; GAG, glycosaminoglycan; IdoA, L-iduronic acid; GlcA, D-glucuronic acid ; MalNEt, N-ethyl maleimide. Ertzymes. Chondroitin ABC lyase (EC 4.2.2.4), chondroitin AC-I lyasc (EC 4.2.2.5); heparan sulphate lyase (EC 4.2.2.8); heparin lyase (EC 4.2.2.7).

(GAG). There are several different types of GAGs, including chondroitin sulphate, dermatan sulphate and heparan sulphate. Primary cultures of human fibroblasts produce many different proteoglycans some secreted to the medium, others retained in the pericellular matrix or the plasma membrane (Carlstedt et al., 1981; Coster et al., 1984, 1986; Heremans et al., 1988; Lorieset al., 1989; Schmidtchenet al., 1990a). Major forms are: a large secreted chondroitin sulphate/dermatan sulphate proteoglycan related to versican (core proteins larger than 200 kDa) (Carlstedt et al., 1981; Zimmermann and Ruoslahti, 1989; Schmidtchen et al., 1990a), two small secreted chondroitin sulphate/dermatan sulphate proteoglycans, PG-S1 (or biglycan) and PG-S2 (or decorin) with similar but distinct core proteins of 43 kDa (Carlstedt et al., 2982;

278 Krusius and Ruoslahti, 1986; Fisher et al., 1989; Schmidtchen et al., 1990a), and one large heparan sulphate proteoglycan (core protein 300-350 kDa) which is retained in the pericellular matrix (Heremans et al., 1988; Schmidtchen et al., 1990a). More recently Breuer et al. (1991) have identified another large dermatan sulphate proteoglycan with a protein core of 740 kDa. Minor proteoglycan forms include a secreted heparan sulphate proteoglycan (core protein 250 kDa) (Schmidtchen et al., 1990a), other heparan sulphate proteoglycans [core proteins of 130, 70 (glypican). 45 (fibroglycan) and 35 kDa] found both in the medium and in the cell layer (Lories et al., 1989; Marynen et al., 1989; David et al., 1990; Schmidtchen et al., 1990 a, b), and two exclusively membrane-bound proteoglycans (90-kDa core) with either heparan sulphate or chondroitin sulphate/dermatan sulphate chains (David et al., 1989; Schmidtchen et al., 1990a). Extracellular proteoglycans can regulate the organization of connective tissue matrices in various ways, e. g. the large chondroitin sulphate/dermatan sulphate proteoglycans interact with hyaluronan (Norling et al., 1978)or they self-associate (Coster et al., 1981) to form large aggregates which may contribute to the formation of a gel around cells and fibres. Moreover, the interaction between small chondroitin sulphate/dermatan sulphate proteoglycans and collagen may regulate fibre formation (Scott and Orford, 1981;Vogel et al., 1984). The GAG components of proteoglycans include many different variants that are important for the function of the proteoglycan. For example, GAG chains with high amounts of L-iduronic acid (IdoA) inhibit cell proliferation more than GAG chains containing high amounts of D-glucuronic (GlcA) (Westergren-Thorsson et al., 1991a). The mechanism regulating the formation of the co-polymeric structure of GAG chains is not fully understood. Transforming growth factor-fl (TGF-8) is a cytokine that enhances the synthesis of many extracellular matrix components including proteogiycans (Chen et al., 1987; Bassols and MassaguC, 1988; Westergren-Thorsson et al., 1990, 1991b) and hyaluronan (Heldin et al., 1989; WestergrenThorsson et al., 1990). It has been reported that, among the small proteoglycans, it is mainly the production of biglycan that is stimulated by TGF-fi in human embryonic skin and bovine sclera fibroblasts (Westergren-Thorsson et al., 1991 b), human embryonic lung fibroblasts (Romaris et al., 1991), osteosarcoma cells (Breuer et al., 1990) and mesangial cells (Border et al., 1990). However, KahBri et al. (1991) noticed a decreased production of decorin after 48 h treatment with TGF-/) in human adult skin and gingival fibroblasts and Border et al. (1990) reported an increased production of decorin in mesangial cells but to a lesser degree than that of biglycan. Kiihiri et al. (1991) have also observed an increased production of the large proteoglycan versican in skin and gingival fibroblasts. In epithelial cells, TGF-j3 promotes the addition of chondroitin sulphate chains to the cell-surface proteoglycan syndecan (Rapraeger, 1989). TGF-P also alters the expression of basement-membrane-type heparan sulphate proteoglycan in colon carcinoma cells (Dodge et al., 1990). TGF-fl appears to stimulate the production of various proteoglycans in different ways depending on the type of cell and the conditions used. The different responses may be due to modulation of TGF-fl activity via binding to decorin (Yamaguchi et al., 1990) and to the various TGF-P receptors, one of which is a proteoglycan, p-glycan (Cheifetz et al., 1988; Segarini and Seyedin, 1988). So far little information concerning the effects of TGF-P on GAG formation in these various proteoglycans has been

FIBROBLAST CULTURE CELL LAYER

MEDIUM

DEAE-cellulose ion-exchange chromatography

ISOPYCNIC DENSITY GRADIENT CENTRIFUGATION Highdensity material

Mediumdensity material

(HDI

(MDI

GEL CHROMATOGRAPHY Large proteoglycans

Small proteoglycans

(A)

(5)

ION-EXCHANGE CHROMATOGRAPHY ON MONO 0

HS proteoglycans

CS/OS proteoglycans II

I I

PURIFIED PROTEOGLY CAN FRACTIONS

Scheme 1. Flow chart for the isolation of proteoglycans

reported. This study was therefore undertaken to gain knowledge of possible effects of TGF-P on the GAG polymer-modification process during biosynthesis of various proteoglycans in human embryonic skin fibroblasts.

MATERIALS AND METHODS Materials TGF-P(l) from porcine platelets was purchased from R & D Systems, Inc. (Minneapolis, MN). The material was dissolved in 4 mM HCI as recommended by the manufacturers. Cell culture medium was obtained from Nord Vacc AB, Sweden. [35S]Sulphate and [3H]leucine were purchased from Amersham International (Amersham, UK). 4,h-Diamidine 2phenyl dihydrochloride for screening of mycoplasma was bought from Boehringer-Mannheim (Scandinavia AB). pNitrophenyl P-D-xyloside was obtained from Sigma Chemical Co. The heparan sulphate, heparin, chondroitin ABC and chondroitin AC-I lysases were products of Seikagaku Kogyo Co. (Tokyo, Japan). DEAE cellulose (DE-52) was from Whatman, Bio-Gel P6 from Bio-Rad and Sephacryl S-500 HR, Mono Q HR, Superose 6 , octyl-Sepharose C L d B and

279 CELLAYER

High density from CsCl CONTR 0L

A

A

0

m

Medium density from CsCl

-

CONTROL

A

-

CONTROL

15

0

A

0

10

>

-

CONTROL

A

B

5

0

15

ii

10

5

FRACTION NUMBER

FRACTION NUMBER

Fig. 1. Gel chromatography on Sephacryl S-500 HR of fractions obtained after CsCl density-gradientcentrifugation. High dcnsity (HD) and medium-density (MD) proteoglycan material were prepared by CsCl density-gradientcentrifugation and subjected to gel chromatography as described in Materials and Methods. The chromatograms obtained from the H D material of the cell layer are shown in (A) and (B) and thosc from HD material of the culture medium in (E) and (F). MD material from the cell layer is shown in (C) and (D) and MD material from the medium in ( G ) and (H). Material from untreated cells is in the top row and material from TGF-p-treatcd cells in the bottom row. Fractions were pooled (sce bars), into A which contains large proteoglycans and B which contains small proteoglycans. (-) [3]Leucinc; (- . . -) 3 5 S 0 4 Recovery was 70% or better.

Sephadex G-50 were from Pharmacia, Sweden. Stock solutions (approx. 8 M) of guanidinium chloride from Fluka Chemie AG (practical grade) were treated with activated charcoal before use. Stock solutions of urea (8 M) were passed through a bed of ion-exchange resin before use. Chaps was from Fluka Chemie AG. Mulgophene (tridecyloxypolyethyleneoxy-ethanol) was purchased from GFA Corp., (Johanneshov. Sweden). Other chemicals were obtained from sources listed previously (Coster et al., 1984). Cell culture Fibroblast cultures were established from human embryonic skin (Carlstedt ct al., 1981) and used for experiments between passages 5 and 20. The cells were maintained in Eagle's minimal essential medium, supplemented with 10% new-born calf serum. The cultures were checked for mycoplasma by the fluorescent dye 4,s-diamidine 2-phenyl dihydrochloride. Experiments were performed on cells that were grown in 75-cm2 plastic dishes. After confluence, the serum concentration was decreased from 10% to 5 % , from 5% to 0.4% and from 0.4% to 0% at 12 h intervals. In the presence of serum, TGF-p has no effect on proteoglycan production (Westergren-Thorsson et al., 1990) probably due to its binding to a2-macroglobulin (Huang et al., 1988). Furthermore, proteoglycan production is qualitatively the same

up to 76 h after serum starvation (Schmidtchen, A., unpublished data). Sulphate-poor medium containing 0.1 1 mM SO:- and 10 ng/ml TGF-fi was then added. The same volume of 4 mM HC1 was added to the controls (Westergren-Thorsson et al., 1990). After 6 h of incubation with TGF-fi, the medium was changed and fresh sulphate-poor medium without TGFp was addcd to the cultures and the cells were incubated with 50 kCi/ml of [35S]sulphateand 20 pCi/ml of [3H]leucine for an additional 24-h period. Some cultures were treated with TGF-,!3 as described above and then p-nitrophenyl p-u-xyloside was added, at concentrations ranging over 5 - 1000 pM, together with 50 pCi/ml [35S]sulphatefor an additional 24 h. Extraction procedures

After incubation with [35S]sulphate and ['H]leucine thc medium was decanted and di-isopropylphosphorofluoridate was added to a final concentration of 1 mM. Proteoglycans of the medium were recovered by passage over columns (0.7 x 4 cm) of DEAE-cellulose (DE-52), which had been equilibrated in 6 M urea, 50 mM sodium acetate, pH 8.5, 5 mM MalNEt, and 5 pg/ml ovalbumin. The columns were washed with 60 bed volumes of the same buffer and then with 6 bed volumes of the same solvent containing 0.4 M sodium acetate. Finally proteoglycans were eluted with 6 bed volumes of 4 M guanidinium chloride, 50 mM sodium acetate, pH 5.8,

280 MEDIUM

CELL LAYER Fraction A from S-500 CONTROL(

Froction A from S-500

Fraction B from S-500

c

Froction B f-om 5-500

CONTROL^

E

CONTROL

G

CONTRO

1.2

12

30

0.9 8

15 4

o

h

0.0

c

’”,’ 1.2

12 3c

0.9

a

I

u

=

n

%

U

1: 4

i

0.3

o -0

20

40

60

80

100

20

40

60

80

c

100

FRACTION NUMBER

0.0

20

40

60

80

100

20

40

60

80

i

100

FRACTION NUMBER

Fig. 2. Ion-exchange FPLC on Mono Q of proteoglycans isolated after density gradient centrifugation followed by gel chromatography. Proteoglycans of‘ high density (HD) were separated into large (A) and small (B) protcoglycans by gel chromatography (Fig.1). These materials wcrc thcn subjected to ion-exchange FPLC. Results obtained with proteoglycans derived from the cell layer or the medium are shown in ( A D) and (E-H), rcspectively. Large proteoglycans are in A, B, E and F and small proteoglycans are in C, D, G and H, respectively. Protcoglycans from untreated cells are in the top row and those from treated cells in the bottom row. The origin of thc various protcoglycan preparations is also indicated in the panels. Peak I contains mainly heparan sulphate proteoglycans whereas peak I1 contains mostly chondroitin sulpliatcidermatan sulphate proteoglycans. (- - - -) NaCI gradient; (-) [3H]leucine; (- . . -) 3 5 S 0 4 .

and 5 pg/ml ovalbumin. In this way unincorporated radioactive precursors were removed and medium-derived proteoglycans were conveniently concentrated and brought into a solvent suitable for density gradient centrifugation. The cell layer was washed with phosphate-buffered saline and extracted with 6 ml ice-cold 4 M guanidinium chloride, 50 mM sodium acetate pH 5.8, 10 mM EDTA, 10 mM MalNEt, l mM di-isopropylphosphorofluoridate,1 YOTriton X-100 and 5 pg/ in1 ovalbumin. Medium proteoglycan preparations and celllayer extracts were kept frozen until further analyses. Density gradient centrifugation in CsCl

The crude proteoglycan material from the medium and the cell-layer extracts were brought to a density of 1.35 g/ml in 4 M guanidinium chloride, 1% Triton X-100 by adding solid CsC1, Triton X-100 and water (Carlstedt et al., 1981). Centrifugation was performed at 36000 rev./min for 65 h at 15 C, using a Beckman model L8 60 M ultracentrifuge and a Ti 50.2 rotor. The tubes were emptied from the bottom and fractions of 2 ml werc collected. The density was determined by weighing aliquots (100 pl) of the fractions. Proteoglycans were pooled as two major fractions, one with high density (density > 1.40 g/ml) and one with medium density (1.40 g/ ml > density > 1.30 g/ml). Fractions were then diluted 25fold with 50 mM sodium acetate, pH 5.8, containing 6 M urea, and concentrated on columns of DEAE-cellulose which were eluted stepwise as described above.

500HR. The column was eluted with 4 M guanidinium chloride, 50 mM sodium acetate pH 5.8, 0.1% Triton X-1 00 and 5 pg/ml ovalbumin at a rate of 13 ml/h. Ion-exchange FPLC on Mono Q HR

After dialysis against 7 M urea, 10 mM Tris pH 8.0, 0.1 O h Mulgophene, material was subjected to ion-exchange chromatography, which was performed on a Mono Q HR 515 column connected to an FPLC system (LKB). Samples were eluted by a gradient of NaCl (0.3 - 1.2 M ) as described by Lindblom et al. (1989). Appropriate fractions were pooled and material was recovered by passage through a DEAEcellulose column as described above. Purified proteoglycans were stored in 4 M guanidinium chloride at - 20 C. Hydrophobic interaction chromatography

Material was first concentrated by passage over DEAEcellulose as described above and eluted with 4 M guanidinium chloride, 50 mM sodium acetate, pH 5.8. Samples were then subjected to hydrophobic interaction chromatography (Choi et al., 3989) on octyl-Sepharose CL-4B (0.5 x 10-cm Omnifit column) by using an FPLC system (LKB). The column was equilibrated with 4 M guanidinium chloride and eluted with a linear gradient of Chaps (0- 1.5%) at a flow rate of 0.1 mll min; fractions of 0.5 ml were collected.

Gel chromatography on Sephacryl S-500 HR

Degradative methods

Proteoglycans were separated by gel chromatography at room temperature on a column (2.5 x 100 cm) of Sephacryl S-

Digestions with heparan sulphate, heparin and chondroitin ABC and chondroitin AC-I lyases were performed in the

281 Table 1. The effect of TGF-P on proteoglycan production. Cultures were treated as described in Materials and Methods; proteoglycans wcre isolated and separated by isopycnic density gradient centrifugation into onc high-density (HD) and one medium-density (MD) fraction. Thc material was further separated into large (A) and small (B) proteoglycans by gel chromatography on Sephacryl S-500 HR. Such experiments were carried out five times. Representative proteoglycans werc finally separated into pool I and I 1 by ion-exchange chromatography to determine whether thcy contained heparan sulphate (HS) or chondroitin sulphate/dermatan sulphate (CS/DS) proteoglycans. The effect of TGF-fi on proteoglycan production is given as a percentage or control. "S and 3H values were obtained after ion-exchange FPLC on Mono Q and werc corrected for the recovery of "S in the final step. Yields of the various proteoglycan pools are expressed as the percentage of total proteoglycan material from each plate (medium and cell layer) obtaincd after ion-exchange chromatography on Mono Q. The recovcry in cach step was typically around 60% or better. Only the major proteoglycan fraction of medium density, MD.AI, is shown as the other fractions only conpained minute amounts of proteoglycan material. Plate

Fraction

Molecular mass of dominating core protein

'Type of dominating glycosaminoglycan

3H

nd 400, 500 90.70.45, 35 43 350 nd 400,500 90, 70, 45, 35 43 350 250 400,500 130, 70, 45, 35 43 250 400,500 130, 70, 45, 35 43

35s

9'0 control

kDa Ccll layer, control HD.AI HD.AII HD.BI HD.BII MD.AI Cell layer. TGFB HD.AI HD.AII HD.BI HD.BII MD.AI Medium, control HD.AI HD.AIT HD.BI HD.BII Mcdium, TGF-fi HD.AI HD.AII HD.BI HD.BII

Amount of

-__

HS CS/DS HS DS HS HS CS/DS HS DS HS HS CS/DS HS DS HS CS/DS HS DS

presence of proteinase inhibitors as described by Lindblom et al. (1989). Purified proteoglycans (10- 100 pl) were precipitated with a ninefold excess of 95% ethanol at 4°C overnight and recovered by centrifugation at 7000 rev./min for 60 inin in the JS 7.5 swing-out rotor of the Beckman JA 20 centrifuge. The pellets were air-dried for 30 min and then solubilized in 300 p1 of the appropriate digestion buffer. Digestions were performed at 37 "C for 3 - 4 h or overnight. GAG chains were released from the core protein by alkaline-borohydride treatment (Carlson, 1968). Heparan sulphate was degraded by HNOz as described (Shively and Conrad, 1976). SDS-polyacrylamide gel electrophoresis Electrophoresis was performed on 3 - 12% polyacrylamide gradient gels (T/C = 30/0.8), with a 3% stacking gel using the buffer system of Laemmli (1970) as described by Lindblom et al. (1989). Digested samples were precipitatcd with a ninefold excess of 95% ethanol, centrifuged and dried. Samples were dissolved in 60 p1 5%) (massjvol.) SDS, 20% (by vol.) glycerol, 4 mM EDTA, 0.04% bromphenol blue, 125 mM Tris, pH 5.8. To reduce disulphide bonds, 2inercaptoethanol was added to a final concentration of 10% (by vol.). Samples were boiled for 3 min and electrophoresed for approximately 30 h at a constant current of 10 mA. Approximately 30000 -40000 dpm of 3H-leucine-labeled material was applied in each lane. After electrophoresis, gels were stained with 0.25% Coomassie brilliant blue R-250, destained and soaked in sodium salicylate (Chamberlain, 1979). Fluorography was performed at - 60 "C for 3 - 7 days using Kodak XAR-5 film.

Yield ~.

311

35s

total

ZOO 100

100 100

100 100 100 133 300 84 338 272 100 100 100 100 86 359 112 543

100

100 100 142 522 115 510 27 1 100 100 100 100 132 759 104 543

3.5 8.7 11.4 1.8 12.2 1.8 10.1 3.7 2.4 12.8 5.7 18.1 8.9 4.1 1.9 25.2 3.8 8.6

5.0 12.4 17.6 2.4 7.0 2.0 18.8 5.9 3.5 5.5 5.8 20.4 14.8 3.0 2.2 44.9 4.5 4.8

Gel chromatography on Superose 6 GAG chains were chromatographed on a column (0.25 x 25 cm) of Superose 6, eluted with 4 M guanidinium chloride, 50 mM sodium acetate, pH 5.8 and 0.1% Triton X100. The column was operated with an LKB FPLC system at a flow rate of 0.25 ml/min. Lichrosorb-NH2 The amount of 4-sulphated and 6-sulphated disaccharides of the GAG chains was determined after degradation with chondroitin ABC lyase followed by separation of the degradation products on a Lichrosorb-NH, column (4mm x 250 mm; Merck connected to an FPLC system (LKB) (Hjerpe et al., 1979). After digestion, 4 vol. ethanol was added to the digest. After 2 h, this was centrifuged and supernatants were evaporated to dryness in a Speed Vac concentrator (Savant). The residues were dissolved in 0.1 M sodium acetate, pH 5.0. Gel chromatography on Bio Gel P6 After digestion of the GAGS with chondroitin AC-I lyasc, the products were chromatographed on a column (1 x 100 cm) of Bio-Gel P6, eluted with 0.5 M NH,HC03. The column was operated with a flow rate of 4 ml/h. The amount of GlcA was calculated as described by Malmstrom et al, (1975). Statistical methods Mean values & standard error of the mean were calculated.

282

Fig. 3. SDS/PAGE of the large and small proteoglycans from the medium before and after digestion with chondroitinABC lyase. The proteoglycans werc isolated from untreated and TGF-/&treatedcells using CsCl density-gradient centrifugation (HD) followed by gel chromatography (fraction A and B) and ion-exchange FPLC (fractions I and 11). Proteoglycans from fraction TI were subjccted to electrophoresis beforc ( - ) and after (+) treatment with chondroitin ABC lyase. CS, chondroitin sulphate; DS, dermatan sulphate. The migration position of the protein standards are indicated on thc left. S = start and F = front. It should be pointed out that the 35S-labeledheparan sulphate proteoglycans arc vcry much over-represented comparcd to the 3H-labeledcore proteins of dermatan sulphate proteoglycan.

RESULTS General strategy Cultures of human skin fibroblasts were treated with 10 ng/ml of TGF-fi for an induction period of 6 h which results in maximal stimulation of proteoglycan production and does not change the number of cells (Westergren-Thorsson et al., 1990). It has been demonstrated previously that the effect on proteoglycan production remains for a t least 48 h after removal of TGF-fl. Furthermore, incorporation of [35S]-su1phate into proteoglycan is linear for more than 48 h in both treated and untreated cultures (Westergren-Thorsson et al., 1990). After removal of TGF-fl, treated and untreated cultures were incubated with radiolabeled precursors (sulphate and leucine) for 24 h. Proteoglycans from the medium and from a guanidinium chloride/Triton X-I 00 extract of the cell layer were isolated and fractionated by density gradient centrifugation in CsCI, by gel chromatography on Sephacryl S-500 and by ion-exchange FPLC on Mono Q. In our experience, the use of density gradient ccntrifugation (under dissociative conditions) as the first step is important to facilitate removal of non-proteoglycan proteins, especially in cell-derived material. Furthermore, analysis of proteoglycan core proteins of partially and completely purified preparations has not revealed any significant sclcctive losses of proteoglycans in the various steps (Carlstedt el al., 1981; Coster et al., 1984, 1986; Schmidtchen et al., 1990 a). Major proteoglycan fractions were characterized by SDSjPAGE before and after removal of the GAG chains. The small chondroitin sulphate/dermatan sulphate proteoglycans biglycan and decorin were separated by hydrophobic interaction chromatography on octyl-Sepharose CL-4B (Choi et al., 1989; see also Scheme 1). Finally, the

GAG chains were released from individual proteoglycans and their copolymeric structure was examined. Proteoglycan purification The first purification step was isopycnic density gradient centrifugation. Proteoglycans from the medium were mainly in the high-density fraction (density > 1.40 g/ml). TGF-Ptreated cultures yielded more radiolabeled proteoglycans in this fraction (93%) than did untreated ones (78%; results not shown). Proteoglycans from the cell layer appeared both in the high-density and in the medium-density fractions (I .40 g/ ml > density > 1.30 g/ml, 65% in TGF-P-treated cultures and 60% in untreated ones). The low-density fraction (density < 1.30 g/ml), which mainly contained proteins, was not studied. The high- and medium-density proteoglycan fractions from both the cell layer and the medium were then further analyzed (see Scheme 1). The next purification/separation step was gel chromatography on Sephacryl S-500 HR (Fig. 3 ) . The high-density material separated into two peaks (Fig. 1) consisting of large proteoglycans (fraction A), small proteoglycans and some free glycan chains (fraction B), (Schmidtchen et al., 1990a). Treatment with TGF-P resulted in a 3.1 5 0.4-fold increase (n = 5 ) in the production of large proteoglycans (fraction A) and a 2.1 f 0.4-fold increase (n = 5) in the production of small proteoglycans (fraction B) from the medium and approximately the same was seen in the cell-layer-derived fractions (35S-labeled material, cf. Fig. 1A and E with 1 B and F). Medium-density proteoglycans were also separated into large and small forms by gel chromatography. Fraction MD-A from the cell layer, which mainly contains the large heparan

28 3 1.2

1.2

A

CONTROL

- 1.5 0.8

0.8

- 1.0 0.4

0.4

0.0

0.0 0

10

20

30

40

50

0

10

20

30

40

- 0.5 -lo

50

Fig. 4. Hydrophobic interaction chromatography (A, B) and SDS/PAGE (C, D) of the small proteoglycan population from the medium. The small proteoglycans were prepared from the culture medium of (A) untreated and (B) TGF-P-treated cells and separated as describcd in Figs 1 and 2 but without prior CsCl gradient centrifugation. They were then separated by hydrophobic interaction chromatography, heparan sulphate proteoglycan and frec glycan chains (GAG) did not bind to the gel whereas PG-S2 and PG-Sl were retarded. Different fractions from hydrophobic interaction chromatography were then identified by electrophoresis. (- - - -) Chaps gradient; (-) 35S04.In (C) and (D) the migration positions of the protein standards are indicated on the left.

sulphate proteoglycans (Schmidtchen et al., 1990a) appeared to increase after TGF-fl treatment (Fig. 1C and D). Representative proteoglycans were finally purified and separated into heparan sulphate (pool I) and chondroitin sulphate/dermatan sulphate types (pool 11) by ion-exchange FPLC on Mono Q (Fig. 2). To identify the core proteins of these proteoglycans, they were subjected to SDS/PAGE after degradation of their glycan side chains.

exchange FPLC on Mono Q (Fig. 2). This proteoglycan yielded two core proteins of 400 and 500 kDa after digestion with chondroitin ABC lyase both before and after treatment with TGF-8 (Fig. 3). This indicates that this proteoglycan is a large chondroitin sulphate/dermatan sulphate proteoglycan.Trcatment with TGF-P increased production of this proteoglycan 5-fold in the cell layer and 7.6-fold in the medium, expressed as 3sSincorporation (cf. Fig. 2A and E with 2B and F; see also fractions HD.A.11 in Table 1).

Effect of TGF-/? on the large chondroitin sulphate/dermatan sulphate proteoglycan

Effect of TGF-P on the small chondroitin sulphate/dermatan sulphate proteoglycans These high-density proteoglycans are found both in the cell layer and in the medium and they are obtained in fraction

A major high-density proteoglycan both from the medium and the cell layer was obtained in fraction A I1 after ion-

284 CONTROL

----

TGF-8

I A.

Control

-12

- 09 -

PG-S1

06

-03

-

-

I

I

Y

ic

,

-

I D

'.* 2

- 09

z

PG-S2

- 0.6 23

45

60

80

20

60

40

- 03

80

Fraction num3er

Fig. 5. Gel chromatography on Bio-Gel P6 of 35S-labelled dermatan sulphate chains derived from PG-S1 and PG-S2. Biglycan and decorin were prepared from the medium as described in Fig. 4. The protcoglycans were then treated with alkali to release the GAG chains from the core protein. The chains were recovered by ion-cxchange chromatography and then digested with chondroitin AC-I lyase and chromatographed on Bio-Gel P6. PG-S1 is shown in (A, B), PG-S2 i n (C. D) from untreated (A, C) and TGF-/3 treated cells (13, D). The elution positions of standard oligosaccharides are indicated in the chrornatograms; 2 , 4 , 6 , 8 ,di-, tetra-, hexa-, oeta- and deca-saccharidc 35S04. respcctivcly. (-)

5

e

Fig. 7.Ion-exchange FPLC on Mono-Q of proteoglycans isolated from medium-density fractions after gradient centrifugation and gel chromatography. Proteoglycans of medium density from the cell layer were separated into large (A) and small (B) proteoglycans by gel chromatography (Fig 1 C and D). The large proteoglycans were subjected to ion-exchange FPLC. Proteoglycans from untreated cells are shown in (A) and those from TGF-/3-treated cells in (B). (- - - -) NaCl gradient; (-) [3H]leucine; (- - - -) 35S04.The inset shows an SDSjPAGE electrophoretogram of the heparan sulphate material, pooled from peak I of TGF-/3-treated cells (B). Lane 1, undigested (-); lane 2 (+), heparan sulphate/heparin lyase-treatcd proteoglycan.

1000

iu I-

a

0

d

Fraction Number

1

A

h

-

500

0

40

20

60

80

100

FRACTION NUMBER

Fig.6. Gel chromatography on (A) Superose 6 of proteoglycans and xyloside-initiated GAGs and (B) on Bio-Gel P6 of xyloside-initiated GAGs treated with chondroitinase AC-I lyase. Cultures were preincubated with TGF-8 and then with p-nitrophenyl 8-D-xylosidc (50 pM) and "SO,. Proteoglycan and xylosidc-initiated GAG were isolated using ion-exchange chromatography and gel chromatography (see inscrt). (-)Control and (-) TGF-8-treated cells. The xylosidebound GAGs wcrc pooled as indicated and subjected to chondroitin AC-I lyase digestion and gel chromatography (see B). In (B) GAGs from uiitreatcd (-) or TGF-8-treated cells (- - -) are shown. For further details see Fig. 5. -

~

B. 1I (together with some heparan sulphate proteoglycan) after ion-exchange FPLC of the high-density fraction (Fig. 2). After treatmcnt with TGF-/3, this fraction increased approximately 5 - 7-fold both in the cell laycr and in the medium, expressed as "S incorporation (cf. Fig. 2C and G with 2 D and H ; see also fractions HD.B.11 in Table 1). These proteoglycan

fractions were then treated with chondroitin ABC lyase to identify their core proteins. The results shown in Fig. 3 (right) indicate that TGF-/3 induces a considerable increase in the production of medium chondroitin sulphate/dermatan suiphate proteoglycans with 43-kDa core proteins. Similar results were obtained with the proteoglycan from the cell layer (results not shown). In a separate experiment small chondroitin sulphatei dermatan sulphate proteoglycans (HD.B.11) from the medium were prepared as described in Materials and Methods (with the exception of CsCl gradient centrifugation) and then subjected to hydrophobic interaction chromatography on octylSepharose. As shown in Fig. 4A and B there were three components, one that was not bound at all, and two that were retarded. The three components were analyzed by SDSjPAGE as shown in Fig. 4 C and D. The two retarded components were of different size and should correspond to PG-S2 or decorin (approx. 300 kDa) and PG-S1 or biglycan (approx. 200 kDa), respectively (Morgelin et al., 1989). They accounted for 46Y0 and 15%, respectively, of the 3sS-labeled material in the untreated cultures, whereas corresponding figures were 37% and 28% in the TGF-P-treated ones. Hence, production of PG-S1 increased 3.3 & 0.9 fold (n = 3), whereas that of PG-S2 only increased 1.4 f 0.2 fold ( n = 3). The non-retarded components which amounted to 39% and 37% of the 3sSlabeled material from untreated and treated cultures, respectively, consisted of chondroitin sulphate/dermatan sulphate chains (sensitive to chondroitin ABC lyase) and small heparan sulphate proteoglycans in approximately equal amounts.

285 1reatment with T(;F-/j increased the yield of the non-rctarded components 2.6 0.6-fold (11 = 3). Electrophoresis of thc non-retarded material (Fig. 4 A and R) indicated that chondroitin sulphate/dcrmatan sulphate chains (faster moving) iiccounted for the miijor part of this increase (results not shown). Effect of TCF-fl on chondroitin sulphate/dcrmatan sulphate chain length and structure The large as well as the small chondroitin sulphnte/ dcrmiitan sulphatc proteoglyciins were treated with alkali to rclease the GAG chains. 'I'hesc were then sub,iectetl to H N 0 2 degradation (to rcniovc any contaminating heparan sulphate) and finall) chromatographed on Supcrosc 6. .TGF-P had no effect on the s i x ofthc GAG chains in any of the proteoglycan preparations (results not shown). Ilermatan sulphatc chains of PG-S1 iind PG-S2 were further analjsed for the proportion of IdoA and GlcA residues by digestion with chondroitin ACI lyase followed by Chromatography on Bio-Gel 1'6 (Fig. 5). Dcrmatan sulphatc c h i n s on 1'G-SI and PG-S? from control ciilturcs contained 31 "1"and 37% GlcA respectively (Fig. 5 A and ( ' ) whcreas dcrmatan sulphate chains on PG-S1 and PGS2 from I'GI.'-j-treatcd cultures contained 44OA and 51%0 (ilcA respectively (Fig. 5 H and D). Hencc there was an incrcasc in CilcA content of approximately 1.5-fold in dcrmatan sulph;ite chains of both P(i-S1 and P(i-S1. Effect of 'I'GF-/l an xylosidcinitiated GAGS

I n 'TGF-P-treated cultures therc wits rcproducibly a 1 .S2-fold (11 = 2) incrcasc in the production ol'xylosidr-initiatcd GAG (Fig. 6A). The GAGS produced were complctel! sensitive to chondroitin ARC lywse (results not shown). TGI:-P also changed the structure of the dermatan sulphate produccd on the xyloside primer. I n untrc;ited cullurcs the xylosideinitiated dcrmatan sulphatc contained 43% GlcA whcreas in I'GF-/I trciited culturcs the dermatan sulphate contiiincd 6 I "/o GlcA (Fig. 6H). However no effect was noted with regard to the degree of 4-and 6-sulphation. nor could any disulphated disacchiirides bc detected. Effect of TGF-/I on heparan sulphate proteoglycan

TGF-/1 only increased the production of the ma,ior medium-density heparan sulphate proteoglycan produccd by the cells (see iilso Table 1). This protcoglycan has a core protein of350 kD:i and is principally located in the periccllular matrix. It IS obtained i n the medium-density fraction from the cell layer where i t constitutes approximately %'YO o f the ."Slabellctl protcoglycanq. After separation on Mono-Q of fr:iction MD.A.1, it is seen that TGF$ increascd the production ol'thib heparan sulphitte protcogljcan 2 - 3-fold (Fig. 7. peak I). Thc idcntity of this proteoplycan w a s confirinrd hy SDSI PAGL (Fig 7. inscrt) which showed a core protein of 350 k l h (see also Schniidtchen c t al.. 1090;i). Yo changc of incorporation into the minor fractions of medium density was noted (d'ita not hhoHn) n o r in the other high-density I.ractions as shown in I:ig. ? and l'iiblc 1.

The present study shows that T(;F-/I induces increased production 01' at least threc diffcrcnt "S-labeled protcoglyxns in human emhryonic skin fibroblasts: ( a ) 5 . 7-I'old

increase of the largc high-density chondroitin sulph;ite' dcrmatan sulphatc proteoglycan with core proteins larger than 200 kDa (see iilso Kihiiri et A..1991): (b) 5-fold incrcase of small chondroitin sulphatc/dcrmatan sulphatc proteoglyciins (43-klla core protein) with the greatcst effect on PC-Sl (see also Hreucr ct at.. 1990; Westergrcn-'Thorsson et i d . , 1991 b. Komaris ct al., 1991 : Kiihari et al.. 1991) and (c) 3-fold increase of an hcpardn sulphatc proteoglycan with a 350-klla core protein possibly of the basement mcmbrane type (also noted with colon carcinoma cells: Dodge et al.. 1990). Although thesc findings are i n gcneral agreement with other reports using similar cells. our data do not confirm the find~ngs of Kihari ct at. (1991) who reported a decrease of decorin and no change in heparan sulphate protcoglycans. The dccrcase of decorin can possibly be explaiiicd by different time lengths of the TGF-/? cxposurc. The eflect of TGF-B appeared greater when estiinatcd from the yield 01' the purified proteoglycans or their core protcins iis compared to the total pools of large and small proteoglycans obtained after gel chromatography. I t is therefore possible that the level of othcr minor protcoglycans is decrcascd as a result of TGF-P treatment. The increased proteoglycan production in cell cultures trcatcd with I ' G F - j can be explained by an up-rcgulation of the mRNA message of PG-S1 and PGL (WestcrgrenI'horsson ct al., 1991 b; KihCri et al., 1991). I t does not sccm to be due to :in increased cell proliferation (WestcrgrcnI'horsson et at.. 1990). Inhibition of reccptor-mediated endocytosis of small dermatan sulphate proteoglycan may also contribute to cxtr;icellular accumulation of this material (Schmidt et al., 1990). TGF-/j could cause this effcct. Further work must be performed to disclose the detailed mechanism behind the 'T(;F-/l-inJuccd increiihe in the accumulation of some of the protcoglycans synthesized by libroblasts. In the cell-lines studied by Bassols and Mussaguk (19x8) there was a general increase in the production of small chondroitin sulphate/dermatan sulphate protcoglycans and they also found an increased GAG chain length i n these protcoglpcans. We have not observed any alterittions i n GAG chain length as a result of TGF-fi treatment. I n general. thcrc i h ;I lower .'H, .'