Stimulation by Concanavalin A of Cartilage-Matrix Proteoglycan Synthesis in Chondrocyte Cultures*

The effect of concanavalin A on proteoglycan syn- thesis by rabbit and articular chondrocytes

Faculty of Dentistry, Osaka University, I-8, Yamadaoka, Suita, Osaka 565, Japan The effect of concanavalin A on proteoglycan synthesis by rabbit costal and articular chondrocytes was examined.
Chondrocytes were seeded at low density and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. These results indicate that concanavalin A is a potent modulator of proteoglycan synthesis by chondrocytes.
A group of plant lectins induces blastoid transformation of lymphocytes and has been used extensively as a tool for studying the mechanism of cell proliferation (1). However, no information about the effect of lectins on chondrocyte proliferation or differentiation or both is currently available. Since certain lectin(s) may mimic chondrotrophic actions of growth factor or hormones, in the present study we tested various lectins with a broad spectrum of sugar-binding specificity for their ability to stimulate DNA or proteoglycan synthesis by rabbit chondrocytes.
Results show that none of lectins increase ["Hlthymidine incorporation into DNA in chondro- Relatl~~ Hydrodynamic Sizes of Proteoglycans-Chondrocytes were seeded at a density of 10" tells/35-mm plastic culture dish and grown in 2 ml of Medium A. They were then incubated for 24 h in the presence or absence of 3 pg/ml of ConA in 2 ml of a 1:l mixture of DME and Ham's F-12 medium with 0.3% fetal bovine serum. Twenty ~1 of DME supplemented with 100 PCi of ["'Slsulfate was added 6 h before the end of incubation.
Alternatively, 20 ~1 of DME supplemented with 60 FCi of [ 'Hlglucosamine was added 12 h before the end of incubation.
The medium was kept frozen at -70 "C until analyzed.
The cell layers were overlaid with 1.0 ml of 50 mM Tris-HCI (pH 8.0) buffer containing 4 M guanidine HCI, 0.1 mM iodoacetic acid, 1 mM phenylmethylsulfonyl fluoride, 0.1 M 6-amino-n-caproic acid, 20 mM EDTA, and 1 mg/ml benzamidine HCl. The culture dishes were then put on a shaker for 24 h at 4 "C. After the clarification by centrifugation (4000 x g for 15 min), the cell extracts were stored at -70 "C until analyzed.
Aliquots (1.0 ml) of the medium were mixed with 1 ml of 8 M guanidine HCl in water and 0. Determination of Cyclzc AMP-Confluent cultures in 35-mm culture dishes were preincubated for 24 h in 2 ml of DME with 0.3% fetal bovine serum. The medium was replaced by 2 ml of DME, and the cells were incubated for 3 h at 37 "C. They were then exposed to ConA (3 Kg/ml) or parathyroid hormone fragment(l-34) (lo-' M) for 2 min to 2 h. Intracellular cyclic AMP was determined using cyclic AMP assay kit (Yamasa Shoyu Co., Chiba, Japan) (10).
Determinations of DNA, Protein, and Hexuronic Acid-DNA for 6-36 h in DME supplemented with 0.3% fetal bovine serum alone (0) or DME supplemented with 0.3% fetal bovine serum and 3 pg/ml of ConA (0). The cells were exposed to ['l"S] sulfate for 6 h before the end of incubation. Values are averages + S.D. for four cultures.

ConA-induced
Morphologic Differentiation of Chondrocytes-Rabbit chondrocytes were seeded in 35-mm plastic culture dishes in the presence of DME supplemented with 10% fetal bovine serum, 50 pg/ml of ascorbic acid, and antibiotics. When cultures became confluent, the serum concentration was reduced to 0.3%. In the low serum concentration, cells adopted a fibroblastic configuration (Fig. 1A). Addition of 0.3 (Fig. lB), 2 ( Fig. lC), or 5 pg/ml ( Fig. 1D) of ConA induced a cell shape change from fibroblastic to polygonal or spherical. Within 6 h, the 5 pg/ml of ConA-exposed cells began to round, and after 24 h more than 90% of the cells assumed a spherical configuration (Fig. 1D). The effect of ConA on the chondrocyte morphology was maximal at 5 pg/ ml. the effect of ConA on proteoglycan synthesis by chondrocytes was examined. Proteoglycan synthesis was estimated by measuring incorporation of [3"S]sulfate into macromolecules (glycosaminoglycans) precipitated with cetylpyridinium chloride after protease digestion (5). In cartilage, the majority of sulfated glycosaminoglycans are associated with proteoglycans. When rabbit chondrocytes were incubated with ConA, the incorporation of [35S]sulfate into glycosaminoglycans increased in a dose-dependent manner (Fig. W). This effect of ConA was detected at a concentration of 0.5 pg/ml and maximal at 3 pg/ml. The level of [""Slsulfate incorporation into glycosaminoglycans in the presence of 3 pg/ml of ConA was 2.5 times that in its absence. At a high concentration (20 pg/ml), ConA slightly suppressed [""Slsulfate incorporation into glycosaminoglycans ( Fig. 2A).

Effect of ConA on ["S]Sulfate Incorporation into Proteoglycans-Because
ConA increased the number of spherical chondrocytes that were surrounded by a refractile matrix (Fig. l), into glycosaminoglycans in the cell layer and medium fractions in 0.3-3% serum, but not in lo-15% serum (Fig. 3). The lack of the ConA effect on proteoglycan synthesis in the presence of high concentrations of serum may be due to its binding to serum glycoproteins.    (Fig. 4B) (6, 18).
The 3H-labeled macromolecules from cultures treated or not with ConA were digested with chondroitinase AC, and the resulting disaccharides were analyzed by thin-layer chromatography (Table II). ConA decreased by 40% the proportion of 3H radioactivity incorporated into hyaluronic acid, but had little effect on the proportion of 3H radioactivity incorporated into chondroitin 6-sulfate, chondroitin 4-sulfate, or unsulfated chondroitin.
Because ConA increased the incorporation of 3H radioactivity with glucosamine into total glycosaminoglycans 2-to 4-fold (Table II)

Lack of Effects of ConA on Proteoglycan Synthesis in Dedifferentiated
Chondrocytes-Rabbit chondrocytes exposed to retinoic acid became fibroblastic and lost their ability to synthesize the large proteoglycan (Fig. 5), as expected from previous studies (19). ConA did not increase [Yllsulfate incorporation into proteoglycans synthesized by the dedifferentiated cartilage cells (Fig. 5) Samples were applied onto a Sepharose CL-2B column as described in Fig. 4. ' Transforming growth factor-B-l. sulfate incorporation into glycosaminoglycans (data not shown). Chondrocytes exposed to WGA or garden pea lectin became spherical or polygonal within 2 days, although they had a marginal effect on proteoglycan synthesis (Table IV). Abrin lectin (Abrus precatorius), at 1 Fg/ml, caused cell death (not shown).

Effects of Various Lectins on pH]Thymidine Incorporation-Although
ConA is a potent mitogen for lymphocytes (l), in chondrocyte cultures it decreased [3H]thymidine incorporation into DNA in a dose-dependent manner with an EDso of 0.4-l pg/ml in the presence of various concentrations of serum ranging from 0.3 to 20% (Fig. 6). Note that ConA stimulated proteoglycan synthesis by chondrocytes in 0.3-3% serum, but not in lo-15% serum (see Fig. 3). This discrepancy suggests that ConA affects the syntheses of DNA and proteoglycans in chondrocytes by different mechanisms. ConA, at l-3 pg/ml, also suppressed the proliferation of chondrocytes, dedifferentiated cartilage cells, and fibroblasts in monolayer cultures (data not shown). The inhibition of cell division was not explained by binding of ConA to serum mitogens, because ConA, at l-5 rg/ml, suppressed DNA synthesis in chondrocytes in the absence of serum or in the presence of serum fraction which eluted from a ConA-Sepharose column.'

The inhibition
of chondrocyte proliferation does not seem to be specific to ConA. WGA, lentil lectin, PHA-P, garden pea, and UEA (I + II) also decreased, dose-dependently, (3H] thymidine incorporation into DNA in chondrocytes (Fig. 7). Other lectins had little effect on [3H]thymidine incorporation at 0.01-20 pg/ml (Fig. 7).  activity. Therefore, chemical analyses were carried out to confirm the ConA stimulation of proteoglycan synthesis (Fig.  8). In subconfluent cultures, chondrocytes were incubated in the presence of 2% serum with ConA (0.05-10 &g/ml) for 48 h. The addition of ConA, at 3-10 fig/ml, resulted in a 4-fold increase in the accumulation of macromolecules containing changes in pool sizes rather than real increase in the synthetic hexuronic acid (proteoglycans) (Fig. 8A), whereas it sup-pressed the increase of DNA during a 48-h period (Fig. BC). Accordingly, the uranic acid content1p.g DNA of the ConAexposed cultures was 10 times that of ConA-free cultures. ConA also increased the protein content to a lesser degree (Fig. 8s). In this situation, ConA promoted the conversion of proliferating chondrocytes into maturing chondrocytes that produced matrical proteoglycans.
Note that ConA did not increase the uranic acid content of chondrocyte cultures at 1 pg/ml (Fig. 8A) that abolished DNA synthesis (Fig. 8C). Cytosine arabinoside also had little effect on the uranic acid content (Fig. 8A), when it abolished DNA synthesis (Fig. 8C). Thus, the inhibition of DNA synthesis is not sufficient for the induction of proteoglycan synthesis. Effect of ConA on Proteoglycan Synthesis by Chondrocytes in Suspension Culture-Freshly isolated chondrocytes were exposed to ConA in suspension cultures. The addition of ConA resulted in 1.3-to 1.5-fold increase in [YS]sulfate incorporation into glycosaminoglycans in two independent series of experiments (Table VI). Effect of ConA on the Cyclic AMP Level-Previous studies have shown that parathyroid hormone increases the intracellular cyclic AMP concentration when it stimulates proteoglycan synthesis by chondrocytes (24,25). However, the addition of ConA (3 pg/ml) resulted in only 1.5-to 2.5-fold increase in the cyclic AMP level after lo-120 min (data not shown), whereas parathyroid hormone increased the cyclic AMP level 40-fold in 2-5 min (24,25). Thus, cyclic AMP does not seem to play a second messenger role in ConA-stimulated chondrocytes.

DISCUSSION
The present study showed that ConA has little effect on the monomer size of cartilage-matrix proteoglycan and the degree and position of sulfation of the glycosaminoglycan side   is insufficient to account for the stimulation of proteoglycan synthesis by ConA. It may not be surprising that plant lectins affect proliferation or differentiation of chondrocytes, because they can bind to many cell surface glycoproteins, some of which must be involved in receiving microenvironmental information. Interestingly enough, the ConA action on proteoglycan synthesis by chondrocytes is, however, specific (Table VII). The specificity of the ConA effect was demonstrated by the five observations. (a) While various plant lectins with distinct sugarbinding properties, as listed in Table VII, have potent mitogenie actions in lymphocytes (l), none of them, besides ConA, enhanced proteoglycan synthesis by chondrocytes. (b) Several lectins including ConA suppressed DNA synthesis in chondrocytes. (c) WGA, ConA, and garden pea lectin altered the morphology of chondrocytes, but only ConA enhanced their proteoglycan synthesis. (d) ConA increased proteoglycan synthesis by chondrocytes at low concentrations, whereas lentil lectin, whose sugar-binding specificity is similar to that of ConA (1) (Table VII), did not increase proteoglycan synthesis even at high concentrations. This could be explained by the fact that lentil lectin has lower affinity for mannose and glucose (1). Furthermore, lentil is a dimer, whereas ConA is a tetramer (1). (e) The stimulation of proteoglycan synthesis by ConA was greater than the stimulation by hormones or growth factors that were shown previously to increase proteoglycan synthesis. Furthermore, the ConA stimulation of proteoglycan synthesis was abolished by 10 mM methyl-a-Dmannopyranoside. These observations suggest that cell surface glycoprotein(s) that has N-linked sugar chains specific for ConA plays a special role in the control of proteoglycan synthesis by chondrocytes.
Recent studies showed that the COOH-terminal domain of cartilage proteoglycan core protein elicits lectin-like activity (31). However, it remains unknown whether animal lectins are involved in the control of proteoglycan synthesis in cartilage in vivo.
In conclusion, the present study showed that ConA is a specific modulator of proteoglycan synthesis by chondrocytes. Because the molecular structure of ConA and its mode of actions have been extensively characterized (1)) chondrocytes exposed to this lectin will be useful as a novel model in studying the role of cell surface glycoproteins in the control of cellular differentiation.