Effects of Concanavalin A on Chondrocyte Hypertrophy and Matrix Calcification*

(Received for publication, June 11, 1996, and in revised form, October 11, 1996)

Weiqun Yan , Haiou Pan §, Hideyuki Ishida , Kazuhisa Nakashima par , Fujio Suzuki par , Masahiro Nishimura , Akitoshi Jikko **, Ryo Oda ‡‡ and Yukio Kato §§

From the Departments of  Biochemistry, ‡‡ Operative Dentistry, and  Prosthodontics, School of Dentistry, Hiroshima University, Hiroshima 734, the § Laboratory for Bone Research, Nippon Hoechst Marion Roussel, Kawagoe 350, and the Departments of par  Biochemistry and ** Radiology, Faculty of Dentistry, Osaka University, 1-6, Yamadaoka, Suita 565, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Resting chondrocytes do not usually undergo differentiation to the hypertrophic stage and calcification. However, incubating these cells with concanavalin A resulted in 10-100-fold increases in alkaline phosphatase activity, binding of 1,25(OH)2-vitamin D3, type X collagen synthesis, 45Ca incorporation into insoluble material, and calcium content. On the other hand, other lectins tested (including wheat germ agglutinin, lentil lectin, pea lectin, phytohemagglutinin-L, and phytohemagglutinin-E) marginally affected alkaline phosphatase activity, although they activate lymphocytes. Methylmannoside reversed the effect of concanavalin A on alkaline phosphatase within 48 h. Concanavalin A did not increase alkaline phosphatase activity in articular chondrocyte cultures. In resting chondrocyte cultures, succinyl concanavalin A was as potent as concanavalin A in increasing alkaline phosphatase activity, the incorporation of [35S]sulfate, D-[3H]glucosamine, and [3H]serine into proteoglycans, and the incorporation of [3H]serine into protein, although concanavalin A, but not succinyl concanavalin A, induced a rapid change in the shape of the cells from flat to spherical. These findings suggest that concanavalin A induces a switch from the resting, to the growth-plate stage, and that this action of concanavalin A is not secondary to changes in the cytoskeleton. Chondrocytes exposed to concanavalin A may be useful as a novel model of endochondral bone formation.


INTRODUCTION

Many plant lectins, including concanavalin A (ConA)1 and phytohemagglutinin (PHA), with various sugar-binding properties induce blastoid transformation of lymphocytes and have been extensively applied in studies of proliferation and differentiation (1). However, the mechanism by which plant lectins activate lymphocytes is unknown because of the lack of lectin specificity. ConA and wheat germ agglutinin (WGA) also have insulin-like activity in adipocytes (2-4). Our group has shown that in chondrocyte cultures, ConA, but not other lectins, stimulates the synthesis of cartilage-matrix proteoglycan (aggrecan) by chondrocytes, and that this effect is greater than that of growth factors and hormones (5). Furthermore, ConA induces cartilage in amphibian early gastrula ectoderm (6). These findings suggest that cell-surface glycoproteins with N-linked sugar chains specific for ConA play crucial roles in chondrocyte differentiation.

In growth plates and bone fracture callus, chondrocytes undergo a sequence of cell changes including proliferation, cessation of cell division, matrix synthesis, maturation (hypertrophy), and calcification. The hypertrophic chondrocytes produce alkaline phosphatase (ALPase) (7), type X collagen (8), and 1,25-dihydroxyvitamin D3 (1,25(OH)2-vitamin D) receptor (9), and they induce matrix calcification (10-13), which is essential for elongation and repair of the skeleton.

Chondrocytes in the resting zone provide a reserve chondrocyte pool in the growth plate, and articular chondrocytes contribute to resilience of the tissue. Neither of these chondrocyte subtypes usually undergoes endochondral ossification. Cells from the resting zone undergo hypertrophy at slower rates than growth-plate chondrocytes when maintained at high density in the presence of 10% serum (9). On the other hand, articular chondrocytes rarely undergo hypertrophy even at high density in the presence of 10% serum (14), although they become hypertrophic in arthritic joints (15). The mechanism by which hypertrophy is suppressed in permanent cartilages is not known. However, lectins may alter the differentiation program in permanent chondrocytes by cross-linking cell-surface glycoproteins, because some surface glycoproteins serve as growth factor receptors, extracellular matrix receptors, and modulators of signal transduction. To test this hypothesis, we examined the effect of 11 lectins with different sugar-binding properties on the expression of maturation-related phenotypes by resting and articular chondrocytes. The results showed that among the tested lectins, ConA alone induces ALPase activity, type X collagen synthesis, vitamin D receptor synthesis, and matrix calcification in resting, but not in articular chondrocyte cultures.


EXPERIMENTAL PROCEDURES

Materials

Lectins, alpha -methylmannoside, collagenase (Type IA), insulin, retinoic acid, and triiodothyronine (T3) were purchased from Sigma; Eagle's medium, alpha -modification was from Flow Laboratories (McLean, VA); fetal bovine serum was from Life Technologies, Inc.; and fibronectin and type II collagen were from Koken Co. (Osaka, Japan). Human bone morphogenetic protein-2 (BMP-2, recombinant) was a gift of Dr. J. M. Wozney (Genetics Institute, Cambridge, MA) and Dr. K. Takahashi (Yamanouchi Pharmaceutical Co., Tokyo). Human transforming growth factor beta -1 (TGF-beta 1, recombinant) and human insulin-like growth factor-I (IGF-I, recombinant) were purchased from Wako Pure Chemical (Osaka, Japan). Human calcitonin was purchased from Peninsula Laboratories Inc. (Belmont, CA). 1,25(OH)2-Vitamin D, 1,25-dihydroxy[26,27-methyl-3H]cholecalciferol (180 Ci/mmol), and parathyroid hormone (PTH-(1-34), human) were supplied by Dr. K. Sato (Chugai Pharmaceutical Co., Tokyo). 45CaCl2 (37 mCi/mg) and [35S]sulfate (carrier-free) were obtained from DuPont NEN and the Japan Atomic Energy Research Institute (Tokyo), respectively. D-[6-3H]Glucosamine (27 Ci/mmol), L-[3-3H]serine (28 Ci/mmol), and [6-3H]thymidine (2 Ci/mmol) were purchased from Amersham (UK).

Chondrocyte Cultures

Chondrocytes were isolated from the growth plate and resting cartilage of ribs of 4-week-old male Japanese White rabbits, as described (14, 16). Unlike growth plates of long bones, the rib growth plate does not contain resting chondrocytes. The resting cell population is not contaminated by growth-plate chondrocytes, although the growth-plate cell population may be contaminated by a few (<10%) resting chondrocytes (9). The majority of "growth-plate chondrocytes" start to proliferate after seeding and recapitulate the differentiation program after cell division stops, even though they originate from different (proliferating, matrix-forming, and hypertrophic) zones (10). Articular chondrocytes were isolated from the surface (0.2 mm) of the articular cartilage from the femur at knee joints of the same rabbits, since the deep zone contains maturing chondrocytes (14).

Cells were seeded at 104 or 3 × 103 cells/6-mm plastic microwell, and grown in 0.1 ml of Eagle's medium, alpha -modification, supplemented with 10% fetal bovine serum, 50 µg/ml ascorbic acid, 32 units/ml penicillin, and 40 µg/ml streptomycin (Medium A). Alternatively, cells were seeded at densities of 2 × 105 cells/35-mm dish and 106 cells/100-mm dish, and grown in 2 and 10 ml of Medium A, respectively. The cultures were supplied with fresh Medium A every other day. After reaching confluence in 6-, 35-, and 100-mm dishes (day 5 or 7), the cells were transferred to Eagle's medium, alpha -modification in the presence of various lectins and 0-2% fetal bovine serum for 6 or 24 h, after which the cell layers were washed three times with Medium A. Because lectins bind to serum glycoproteins, the serum concentration was reduced during the incubation with lectins for 6-24 h. Thereafter, the cells were maintained for 0-28 days in Medium A, unless otherwise specified.

Determination of ALPase Activity

Chondrocytes were seeded at 104 or 3 × 103 cells/6-mm well, and maintained in Medium A. On day 5 or 7, they were transferred to alpha -modified Eagle's medium supplemented with 2% fetal bovine serum in the presence or absence of lectins or growth factors for 24 h. Thereafter, the cells were maintained for 0-28 days in Medium A in the absence of lectins and in the presence or absence of growth factors. The media were replaced every other day. Unless otherwise specified, lectins were added to the cultures only once on day 5 or 7, whereas growth factors were added to the cultures every other day from day 5 or 7.

Cells were disrupted in a glass homogenizer in 1 ml of 0.9% NaCl, 0.2% Triton X-100 at 0-4 °C and centrifuged for 15 min at 12,000 × g. ALPase activity in the supernatant, which accounted for 95% of the total, was assayed in 0.5 M Tris-HCl buffer (pH 9.5) supplemented with 0.5 mM p-nitrophenyl phosphate and 0.5 mM MgCl2 (17).

Determination of DNA, Protein, and Hexuronic Acid

The DNA content was determined by means of a fluorometric procedure (18). Total protein was determined by dye binding (19). Hexuronic acid was determined as described by Bitter and Muir (20).

Collagen Analysis

After incubation with or without ConA for 24 h on day 5, resting chondrocytes in 6-mm wells were incubated for 10 days in 0.1 ml of Medium A in the presence of 100 µg/ml ascorbic acid. Cell layers were washed five times with saline and twice with distilled water at 4 °C. They were homogenized at 4 °C with a solution containing 0.9% NaCl, 0.2% Triton X, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 25 mM EDTA, and 10 mM N-ethylmaleimide). The suspension was centrifuged at 3,000 × g for 15 min at 4 °C, and the pellet was incubated with pepsin at 0.4 mg/ml in 0.5 M acetic acid for 48 h at 4 °C. The pepsin-resistant material was solubilized with Laemmli buffer, and proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, then stained with silver nitrate.

1,25(OH)2-Vitamin D Binding

Chondrocytes in 100-mm dishes were homogenized at 0 °C in 3 ml of buffer A (10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 5 mM dithiothreitol) containing 0.3 M KCl. The homogenate was centrifuged at 500 × g for 10 min at 4 °C, after which the supernatant was centrifuged at 4 °C at 226,000 × g for 60 min. Portions (0.5 mg of protein in 0.4 ml of buffer A) of cytosols were transferred to polypropylene tubes containing 1,25-dihydroxy[26,27-methyl-3H]cholecalciferol (20,000 dpm/26 pg/32 fmol). After an incubation at 20 °C for 1.5 h, 50 µl of buffer A containing 5% charcoal and 0.5% dextran was added to the solution, and the suspension was agitated every 5 min with a Vortex mixer. After 30 min at 4 °C, unbound vitamin D metabolites were removed by centrifugation at 500 × g for 20 min to precipitate the charcoal, and the radioactivity in the vitamin D receptor complex in the supernatant was determined. Nonspecific binding was determined by incubating the cells with a 200-fold excess of unlabeled 1,25(OH)2-vitamin D.

Determination of 45Ca Uptake and Calcium and Alizarin Red Staining

Chondrocytes in 35-mm dishes were exposed to 45CaCl2 (20 µCi/culture) in 2 ml of alpha -modified Eagle's medium 3 h before the end of the incubation. Cell-matrix layers were homogenized in 0.9% NaCl, 0.2% Triton X at 0 °C, then centrifuged for 15 min at 12,000 × g. The precipitate was washed with 0.1 M CaCl2 in 0.005 M Tris-HCl, pH 7.4, at 20 °C for 30 min to remove exchangeable 45Ca, then solubilized by incubation in 0.5 M HCl for 3 h at 20 °C (21). The levels of 45Ca were determined in aliquots.

To determine the calcium content, the cell layers were washed five times with saline, then heated at 800 °C for 10 h. The ash was dissolved in 4 M HCl containing 1% lanthanum chloride, and the calcium content was determined by atomic absorption spectrometry (model AA-640; Shimadzu, Kyoto, Japan). The cell-matrix layers were stained with alizarin red-S as described (22).

Analysis of Secreted Proteins and Amino Acid Sequence

Resting chondrocytes were seeded at a density of 106 cells/100-mm dish, and maintained in Medium A. On days 7, 12, and 17, the cells were exposed or not to 10 µg/ml ConA in the absence of serum for 6 h. They were incubated in 10 ml of Medium A with 10% serum from days 7 to 12 and from days 15 to 17, and in 10 ml of alpha -modified Eagle's medium without serum from days 12 to 15 and 17 to 20. Proteins in the conditioned media on days 12-15 and 17-20 were separated by SDS-PAGE, then stained with silver nitrate.

Alternatively, proteins in the media were transferred electrophoretically to a polyvinylidene difluoride membrane (23), and stained with Coomassie Brilliant Blue. The N-terminal amino acids of a 15-kDa protein were determined using an automatic protein sequencer (Applied Biosystems model 476A).

Determination of Proteoglycan, Protein, and DNA Synthesis

When resting chondrocyte cultures in 6-mm wells became confluent, they were incubated in 0.1 ml of Dulbecco's modified Eagle's medium containing 0.3% fetal bovine serum, 32 units/ml penicillin, and 40 µg/ml streptomycin (Medium B). They were then incubated in 0.1 ml of fresh Medium B in the presence of lectins and/or methyl-alpha -D-mannopyranoside for 24 h. The cells were exposed to [35S]sulfate (0.5 µCi/culture) for 17 h, D-[3H]glucosamine (3 µCi/culture) for 6 h, L-[3H]serine (3 µCi/culture) for 6 h, or [3H]thymidine (1 µCi/culture) for 3 h before the end of the incubation. We estimated the level of proteoglycan synthesis by measuring the incorporation of [35S]sulfate, D-[3H]glucosamine, and [3H]serine into material precipitated with cetylpyridinium chloride after digestion with Pronase E (24). Total protein and DNA synthesis were determined by measuring the incorporation of [3H]serine and [3H]thymidine into 5% trichloroacetic acid-insoluble cell precipitates, respectively (24).

Coating the Culture Dishes

Plastic tissue culture dishes (diameter, 35 mm) were incubated with 1 ml of 0.1 M NaHCO3 containing type II collagen (10 µg/ml) or fibronectin (10 µg/ml) at 37 °C for 3 h, then washed three times with saline.

Cell Adhesion

Chondrocytes in primary cultures were harvested with phosphate-buffered saline containing 0.01% trypsin and 0.01% EDTA, then seeded at a density of 2 × 105 cells/35-mm plastic, fibronectin- or collagen-coated well. They were incubated with lectins at 37 °C in 2 ml of Dulbecco's modified Eagle's medium for 3 h before being photographed.


RESULTS

Effect of Lectins, Hormones, and Growth Factors on ALPase Activity in Chondrocytes

Resting chondrocytes in high or low density cultures were exposed for 24 h to ConA on day 5 or 7, respectively, after the cultures reached confluence. Thereafter, they were maintained in the presence of 10% serum for 10 days. ConA (10 µg/ml) was added to the cultures only once on day 5 or 7, because ConA bound to the cells and extracellular matrix macromolecules within 24 h.2 The incubation with ConA caused a >12-fold increase in ALPase activity in high and low density cultures on days 15 and 17, respectively (Fig. 1).


Fig. 1. Comparison of the effect of ConA on ALPase with that of hormones and growth factors in resting chondrocyte cultures. Rabbit resting chondrocytes were seeded at 104 (A) or 3 × 103 cells (B) per 6-mm well, and maintained in Medium A. On day 5 (A) or 7 (B), the cells were exposed or not to ConA, T3, BMP-2, IGF-I, PTH, or TGF-beta 1 at the indicated concentrations in the presence of 2% fetal bovine serum for 24 h. They were then incubated in Medium A in the presence or absence of growth factors or hormones. Hormones and growth factors were added to the cultures every other day until the end of the incubation, whereas ConA was added only once on day 5 or 7. ALPase activity was determined on day 15 (A) or 17 (B). The values are averages ± S.D. of four cultures.
[View Larger Version of this Image (39K GIF file)]


Among growth factors and hormones, BMP, thyroid hormone, calcitonin, and retinoic acid increase ALPase and/or type X collagen synthesis by chondrocytes (25-30). These compounds were added to ConA-free cultures every other day, and their effect on ALPase was compared with that of ConA. Incubating resting chondrocytes with BMP-2 from day 5 or 7 for 10 days consistently increased ALPase activity in high, but not low density cultures (Fig. 1; data not shown). T3 also increased ALPase activity in high and low density cultures, whereas IGF-I induced a low level of ALPase in low, but not high density cultures. In all studies, the effect of ConA on ALPase was greater than that of BMP-2, IGF-I, or T3 (Fig. 1; data not shown). The effect of retinoic acid and calcitonin on ALPase was much lower than that of ConA.2

PTH and TGF-beta 1 decreased ALPase activity in ConA-free resting chondrocyte cultures (Fig. 1). These peptides also suppressed the ConA stimulation of ALPase synthesis by resting chondrocytes,2 as predicted from published studies (10, 27, 31, 32). Basic fibroblast growth factor, a potent inhibitor of hypertrophy (14), also decreased ALPase activity in the absence and presence of ConA.2 Thus, ConA did not down-regulate receptors for these inhibitors of hypertrophy.

In articular chondrocyte cultures, ConA (10 µg/ml) had little effect on ALPase activity, although BMP and T3 markedly increased ALPase activity (Fig. 2). In MC3T3-E1 osteoblast cultures, ConA (10 µg/ml) decreased ALPase activity at 48-72 h, whereas BMP-2 increased this activity 2-20-fold at 24-72 h.2 These findings suggested that ConA and BMP-2 modulate ALPase synthesis by different mechanisms. The effect of ConA on ALPase in resting chondrocyte cultures was detectable at 1 µg/ml and maximal at 5-10 µg/ml (Fig. 3A). ConA at 10 µg/ml increased ALPase activity in resting chondrocytes 50-fold (Fig. 3A), whereas it increased ALPase activity in growth-plate chondrocytes only 1.3-fold because of a high basal level (Fig. 3B).


Fig. 2. Effects of ConA, T3, and BMP-2 on ALPase activity in high density cultures of articular chondrocytes. Rabbit articular chondrocytes were seeded at 104 cells/6-mm well, and maintained in Medium A. On day 5, the cells were exposed to ConA at 10 µg/ml, T3 at 10-7 M, or BMP-2 at 100 ng/ml in the presence of 2% fetal bovine serum for 24 h. Thereafter, they were incubated in ConA-free medium containing 10% fetal bovine serum and the growth factors. T3 and BMP-2 were added to the cultures every other day until the end of the incubation, whereas ConA was added only once on day 5. ALPase activity was determined on days 14 and 18. The values are averages ± S.D. of four cultures.
[View Larger Version of this Image (39K GIF file)]



Fig. 3. Effect of increasing concentrations of various lectins on ALPase activity in cultures of resting chondrocytes (RC, A) and growth-plate chondrocytes (GC, B). Cells were seeded at 104 cells/6-mm well and maintained in Medium A. On day 5, they were exposed to ConA (closed circles), succinyl ConA (open squares), PHA-E, lentil lectin, lima bean lectin, garden pea lectin, Scotch broom lectin, M. promifera (MPA), lotus lectin, WGA, PHA-L, or U. europeaus agglutinin (I + II) at the indicated concentrations in the presence of 2% fetal bovine serum for 24 h, then incubated in medium containing 10% fetal bovine serum. ALPase activity was determined on day 15. The values are averages ± S.D. of triplicate cultures.
[View Larger Version of this Image (35K GIF file)]


The lectin specificity of the ConA effect on ALPase induction was examined by exposing resting chondrocytes to the various lectins that activate lymphocytes. A divalent derivative of ConA (succinyl ConA) was as potent as the native tetravalent ConA in stimulating ALPase activity (Fig. 3, A and B). Lentil lectin, garden pea lectin (pea), and Phaseolus vulgaris agglutinin-E (PHA-E) induced very low levels of ALPase activity (Fig. 3A). Maclura promifera, lima bean lectin, WGA, Scotch broom lectin, Ulex europeaus agglutinin (I + II), lotus lectin, and P. vulgaris agglutinin-L (PHA-L) did not affect ALPase activity (Fig. 3A).

Effects of ConA on the Levels of DNA, Uronic Acid, and ALPase in Chondrocyte Cultures

Fig. 4 shows the time courses of the increases in DNA, uronic acid, and ALPase contents in cultures exposed or not to ConA. Resting chondrocytes seeded at 104 cells/6-mm well proliferated for two generations, yielding a confluent monolayer on day 5, then underwent one or two rounds of cell division to form a multi-cell layer by day 10. The DNA content reached a plateau on day 16 (Fig. 4A). Exposing chondrocytes to ConA for 24 h on day 5 suppressed DNA synthesis on days 6, 8, and 10 (Fig. 4A). The cells exposed to ConA started to proliferate from day 14, then the DNA content reached the level in cultures not exposed to ConA by day 24 (Fig. 4A).


Fig. 4. Effects of ConA on the levels of DNA and uronic acid, and ALPase activity in resting chondrocyte cultures. Resting chondrocytes were seeded at 104 cells/6-mm well, and maintained in Medium A. On day 5, they were exposed (closed circles) or not to 10 µg/ml ConA (open circles) in the presence of 2% fetal bovine serum for 24 h. They were then incubated in the medium containing 10% fetal bovine serum. The DNA (A) and uronic acid levels (B) and ALPase activity (C) were determined on the indicated days. The values are averages ± S.D. of four cultures.
[View Larger Version of this Image (22K GIF file)]


Most of the macromolecules containing uronic acid synthesized by chondrocytes exposed or not to ConA are large, chondroitin-sulfate proteoglycans (aggrecan) (5). ConA selectively stimulates aggrecan synthesis without increasing the synthesis of hyaluronic acid and small proteoglycans (5). In ConA-free cultures, the uronic acid level increased from day 10, and reached a plateau on day 18 (Fig. 4B). In cultures exposed to ConA, the level promptly increased from day 5-6, and reached a maximum on day 8. Thereafter, it decreased gradually, and returned to the level of ConA-free cultures on day 20. On day 8, the uronic acid level in the exposed cultures was 4-fold higher than that in ConA-free cultures.

In ConA-free chondrocyte cultures, the level of ALPase activity was very low throughout a 24-day culture period (Fig. 4C). However, upon exposure to ConA, it markedly increased from day 8, and reached a maximum on day 14 (Fig. 4C). The high ALPase level was sustained at least until day 24. Some cultures were exposed twice to ConA on days 5 and 14. The second exposure on day 14 increased ALPase activity 1.4-fold within 48 h,2 even though the uronic acid content reached a plateau on day 8. These findings suggested that the ConA induction of ALPase is not secondary to the accumulation of proteoglycan.

Incubation of chondrocytes with ConA for 6 h had the same effects on uronic acid and ALPase levels as that for 24 h,2 perhaps because the lectin binds to the cell-matrix layer within 6 h.

Effects of Methyl-alpha -mannopyranoside (MeMan) on ALPase Activity

The addition of MeMan on day 5 for 24 h suppressed the effect of ConA on ALPase activity dose-dependently with an ED50 of 10 mM (Fig. 5A). MeMan alone had little effect on ALPase activity. Furthermore, the addition of MeMan on day 12 reversed the effect of ConA on ALPase activity within 48 h, suggesting that prolonged action of the lectin is required for ALPase synthesis after the accumulation of proteoglycan. In contrast, the addition of MeMan on day 16 had little effect on ALPase activity (Fig. 5B), perhaps because the ConA-activated cells had already differentiated to the irreversible hypertrophic stage.


Fig. 5. Effect of MeMan on ALPase activity in the presence or absence of ConA. Resting chondrocytes were seeded at 104 cells/6-mm well and maintained in Medium A. A, on day 5, the cells were not exposed (open circles) or exposed to 10 µg/ml ConA (closed circles) in the presence of various concentrations of MeMan for 24 h. They were then incubated in the medium supplemented with 10% fetal bovine serum. ALPase activity was determined on day 15. B, On day 5, the cells were not exposed (open circles) or exposed to ConA at 10 µg/ml for 24 h (closed circles). MeMan at 40 mM was added to the cultures on the indicated days (open triangles). The values are averages ± S.D. of four cultures.
[View Larger Version of this Image (21K GIF file)]


Effect of ConA on the Chondrocyte Size

Because ConA was removed gradually from the cell-matrix layer (Fig. 4), chondrocytes were incubated three times with ConA on days 7, 12, and 17. Resting chondrocytes exposed to ConA (Fig. 6B), but not the untreated cells (Fig. 6A), became hypertrophic by day 20. On the other hand, articular chondrocytes became fibroblastic in the absence of ConA by day 20 (Fig. 6C), because of the low seeding cell density and the low serum concentration used in this study. Nevertheless, the articular chondrocytes exposed to ConA maintained a spherical phenotype, although they did not become hypertrophic (Fig. 6D). When articular chondrocytes were seeded at high density (104 cells/6-mm plastic microwell) and maintained in the presence of 10% serum, they assumed a spherical configuration in the absence of ConA. However, they did not become hypertrophic, even in the presence of 10 µg/ml ConA.2 The resting chondrocytes exposed to ConA (B, 1279 ± 591 µm2) were 2.5- and 3-fold larger than those that were not (A, 506 ± 176 µm2) and articular chondrocytes (D, 385 ± 85 µm2), respectively. The diameter of resting chondrocytes exposed to ConA (B, 39.0 ± 10.2 µm) was similar to that (30-40 µm) of hypertrophic chondrocytes in the growth plate in vivo.2


Fig. 6. Morphology of resting (A and B) and articular (C and D) chondrocytes maintained with (B and D) or without ConA (A and C). Resting chondrocytes were seeded at 2 × 105 cells/35-mm dish and maintained in Medium A. On day 7, they were exposed or not to 10 µg/ml ConA in the absence of serum for 24 h, then incubated in alpha -modified Eagle's medium containing 5% fetal bovine serum, 50 µg/ml ascorbic acid, and antibiotics. They were further exposed to 10 µg/ml ConA in the absence of serum for 24 h on days 12 and 17, and photographed on day 20. The size and diameter of 50 cells/population were measured using a computer-assisted video camera and the image-processing software, Image 1 (Universal Imaging Co., West Chester, PA). Bar, 37 µm.
[View Larger Version of this Image (163K GIF file)]


Induction of Type X Collagen in ConA-exposed Chondrocytes

Resting chondrocytes maintained without ConA synthesized type II collagen but not type X collagen, a marker of hypertrophic chondrocytes. However, ConA-exposed chondrocytes synthesized both type II and X collagens on day 15 (Fig. 7).


Fig. 7. Effects of ConA on type II and X collagen contents of chondrocyte cultures. Resting chondrocytes were seeded at 104 cells/6-mm well and maintained in Medium A. ConA (10 g/ml) was added or not on day 5 for 24 h. Collagen contents in day 15 cultures were determined as described under "Experimental Procedures."
[View Larger Version of this Image (41K GIF file)]


Induction of 1,25(OH)2-Vitamin D Receptor in ConA-exposed Chondrocytes

Next we examined whether ConA stimulated the expression of the other maturation-related phenotypes in resting chondrocyte cultures. Resting chondrocytes maintained without ConA showed only a low level of the 1,25(OH)2-vitamin D binding capacity on days 14 and 20 (Fig. 8). However, exposure to ConA on day 7 resulted in 30- and 5-fold increases in the binding of 1,25(OH)2-vitamin D to chondrocytes on days 14 and 20, respectively.


Fig. 8. Effects of ConA on the binding of 1,25(OH)2-vitamin D to chondrocytes. Resting chondrocytes were seeded at 2 × 105 cells/35-mm dish, and maintained in Medium A. On day 7, they were exposed or not to 10 µg/ml ConA in the absence of serum for 24 h, then incubated in Medium A. On days 14 and 20, the cells were homogenized and the cytosols were incubated with 3H-labeled 1,25(OH)2-vitamin D in the presence or absence of excess nonradioactive 1,25(OH)2-vitamin D at 20 °C for 1.5 h. The values are averages ± S.D. of four cultures.
[View Larger Version of this Image (17K GIF file)]


Calcification

No calcification was evident in ConA-free cultures of resting chondrocytes. However, exposure to ConA for 24 h on day 7 resulted in 10-20-fold increases in 45Ca incorporation into insoluble material and the calcium content on day 35 (Fig. 9, A and B). The cell-matrix layer of chondrocytes exposed to ConA was intensely stained with alizarin red (Fig. 9C).


Fig. 9. Effects of ConA on the incorporation of 45Ca into insoluble material and the calcium content in the cell layer of resting chondrocytes. Resting chondrocytes were seeded, maintained, and exposed (closed bars) or not to 10 µg/ml ConA (open bars) as described in the legend to Fig. 6. On day 35, the incorporation of 45Ca into insoluble material (A) and the calcium level (B) were determined, and the cell layers in the ConA-free (a) and ConA-exposed (b) cultures were stained with alizarin red (C). The values are averages ± S.D. of four cultures.
[View Larger Version of this Image (60K GIF file)]


Analysis of Secreted Proteins

ConA had little effect on the SDS-PAGE profiles of secreted proteins in the media conditioned by resting chondrocytes, except that it markedly increased the level of 15-kDa protein (Fig. 10). N-terminal analysis of the 15-kDa protein revealed a 28-amino acid sequence of LLGGLEDVDAQEKDVQRALGFAESSYNK, which was homologous to that of rat, bovine, mouse, and human cystatin C (61, 57, 57, and 50% identity, respectively) and human cystatins D, SA, S, and SN (54, 54, 58, and 54% identity, respectively). The molecular mass of the 15-kDa protein was also similar to that of cystatin. It is unlikely however, that the cystatin-like protein mediates the action of ConA on chondrocytes, because this protein partially purified from the conditioned medium had little effect on ALPase activity.3 The media conditioned by ConA-treated chondrocytes did not increase ALPase activity in resting chondrocyte cultures,3 suggesting that the ConA stimulation of ALPase is not mediated by secreted factors.


Fig. 10. SDS-PAGE profiles of secreted proteins in the media conditioned by chondrocytes treated with or without ConA. Resting chondrocytes were seeded at 106 cells/100-mm dish and maintained in Medium A. On days 7, 12, and 17, the cells were exposed (B and D) or not to 10 µg/ml ConA (A and C) in the absence of serum for 6 h. They were incubated in Medium A from days 7 to 12 and from days 15 to 17. These cells were incubated in alpha -modified Eagle's medium in the absence of serum from days 12 to 15 and from days 17 to 20. Proteins in the conditioned media on days 12-15 (A and B) and days 17-20 (C and D) were concentrated 20-fold using a Centriprep (Amicon, Mr 10,000 cut-off). The proteins in 10 µl of the solution were separated by SDS-PAGE and then stained with silver nitrate.
[View Larger Version of this Image (52K GIF file)]


Comparison between the Effects of ConA and Succinyl ConA on Protein Synthesis, Proteoglycan Synthesis, DNA Synthesis, and Cell Spreading

Native, tetravalent ConA stimulates lymphocyte proliferation at lower concentrations than the divalent ConA derivative, succinyl ConA (33). However, in chondrocyte cultures, succinyl ConA was as potent as ConA in stimulating the incorporation of [35S]sulfate (Fig. 11A), [3H]glucosamine (Fig. 11B), and [3H]serine (Fig. 11C) into proteoglycans and the incorporation of [3H]serine into total protein (Fig. 11D). On the other hand, succinyl ConA suppressed DNA synthesis to a lesser extent than ConA (Fig. 11E). The inhibition of DNA synthesis by these lectins was dose-dependently reversed by MeMan (Fig. 11E). The effect of ConA on proteoglycan synthesis was also reversed by MeMan (5).


Fig. 11. Effects of ConA and succinyl ConA on proteoglycan synthesis, total protein synthesis, and DNA synthesis. When resting chondrocyte cultures in 6-mm wells reached confluence, the cells were incubated in 0.1 ml of medium containing 0.3% fetal bovine serum (Medium B). They were then incubated in 0.1 ml of fresh Medium B containing various concentrations of lectins for 24 h (A-D). Alternatively, they were incubated in 0.1 ml of fresh Medium B containing various concentrations of MeMan, 10 µg/ml lectin, or both for 24 h (E). These cells were exposed to [35S]sulfate (A) for 17 h, D-[3H]glucosamine (B) for 6 h, L-[3H]serine (C and D) for 6 h, or [3H]thymidine (E) for 3 h before the end of incubation. Proteoglycan synthesis was estimated by measuring the incorporation of [35S]sulfate (A), D-[3H]glucosamine (B), or [3H]serine (C) into material precipitated with cetylpyridinium chloride after digestion with Pronase E. Total protein (D) and DNA syntheses (E) were determined by measuring the incorporation of [3H]serine and [3H]thymidine into 5% trichloroacetic acid-insoluble cell precipitate, respectively. The values are averages ± S.D. of four cultures.
[View Larger Version of this Image (36K GIF file)]


Succinyl ConA and ConA differed with respect to their actions on chondrocyte morphology. Resting chondrocytes assumed a fibroblastic configuration in monolayer cultures at a low serum concentration (0.3%). ConA at 10 µg/ml remarkably changed the cells from fibroblastic to spherical within 24 h (5), whereas succinyl ConA induced a moderate change from fibroblastic to polygonal at 48-96 h,2 perhaps because of the increase in the proteoglycan content. ConA, but not succinyl ConA, may directly alter the architecture of the cytoskeleton.

To test this notion, chondrocytes were seeded on fibronectin-coated dishes and incubated for 3 h in the absence or presence of ConA. ConA-free cells fully spread on the substrate within 3 h (Fig. 12A). ConA inhibited the formation of long and/or thin cellular projections at 1-3 µg/ml (Fig. 12, B and C), and abolished cell spreading at 10 µg/ml (Fig. 12D). In contrast, succinyl ConA had little effect on cell spreading at 30 µg/ml (Fig. 12E). Similar results were obtained with chondrocytes seeded on plastic tissue culture dishes and type II collagen-coated dishes.2


Fig. 12. Effects of ConA and succinyl ConA on chondrocyte spreading. Resting chondrocytes were seeded onto fibronectin-coated dishes and incubated in the presence of 0 (A), 1 (B), 3 (C), and 10 µg/ml (D) ConA or 30 µg/ml succinyl ConA (E) for 3 h.
[View Larger Version of this Image (127K GIF file)]



DISCUSSION

Exposure to ConA markedly increased ALPase activity and the size of resting chondrocytes. ALPase synthesis was stimulated more by ConA than by hormones and growth factors that supposedly enhance ALPase synthesis by chondrocytes. The specificity of the ConA action on ALPase was indicated by the following observations. (i) ConA did not stimulate ALPase synthesis and hypertrophy in articular chondrocyte cultures. The marginal effect of ConA on ALPase activity in growth-plate chondrocyte cultures could have been due to contamination of the growth-plate cell population by resting chondrocytes. (ii) Lentil and garden pea lectins had little effect on ALPase activity, although their monosaccharide-binding properties (mannose/glucose) are similar to those of ConA. The crystal structure of pea lectin is also similar to that of ConA (34). However, the oligosaccharide-binding properties of lentil and garden pea lectins differ from those of ConA (1). (iii) Of the lectins examined, only ConA stimulated chondrocyte hypertrophy and calcification. This is important, because many lectins with different sugar-binding properties activate lymphocytes. (iv) ALPase induction by ConA was suppressed by methylmannoside.

Our findings indicated that ConA does not randomly activate gene expression by chondrocytes, but that it initiates the maturation program in resting chondrocytes. Exposing resting chondrocytes to ConA for 6-24 h suppressed cell division, then increased the proteoglycan content, the cell size, ALPase activity, the vitamin D receptor level, the incorporation of 45Ca into insoluble material, and the calcium content. These cell-matrix changes proceeded in the same order as that seen in growth plates and bone fracture callus in vivo. The cell size as well as the ALPase and the vitamin D receptor levels in the ConA-exposed chondrocyte cultures were comparable to those in the growth plate in vivo (14).

Chondrocytes in rib resting cartilage start to mature only near the growth plate in vivo, although cells far from the growth plate can also become hypertrophic when cultured at high density in the absence of a tissue culture surface (9). Changes in cell-matrix interactions and/or growth factor levels may stimulate the maturation of resting chondrocytes in vivo. We postulated that specific lectins induce the maturation of "permanent chondrocytes" in vitro by cross-linking or activating growth factor receptors, extracellular matrix receptors, and/or modulators of signal transduction, because some of the cell-surface proteins are uniquely glycosylated. The finding that ConA, but not other lectins, induces maturation of resting chondrocytes suggests that ConA-binding surface glycoprotein(s) plays a crucial role in switching from the immature, to the mature stage.

Why only resting, but not articular, chondrocytes respond to ConA is unknown. Resting cartilage provides the reserved chondrocyte pool for the growth plate while remaining flexible, whereas articular cartilage supports high compressive loads in the body while remaining flexible. A fraction of resting chondrocytes may undergo hypertrophy even in normal cartilage, whereas articular chondrocytes do not undergo hypertrophy unless they become arthritic. The functional differences between these cartilages suggest that the factors regulating maturation in resting and articular cartilage should differ. In fact, PTH-related protein is involved in inhibiting hypertrophy in resting, but not articular cartilage in vivo (32), although BMP-2 and thyroid hormone induce hypertrophy in both resting and articular chondrocyte cultures.

Although ConA and WGA activate insulin receptors in adipocytes (2-4), insulin induced only a very low level of ALPase activity in chondrocytes.2 Thus, the stimulation of ALPase is not mediated by the binding of ConA to insulin receptors. In addition, ConA did not down-regulate PTH, TGF-beta 1, and fibroblast growth factor receptors in chondrocytes.

ConA enhanced the synthesis of 15-kDa cystatin-like protein by resting chondrocytes. Since ConA also enhanced this synthesis by articular chondrocytes,3 the cystatin-like protease inhibitor is unlikely to mediate the action of ConA on chondrocyte hypertrophy. Previous studies have shown that ConA stimulates and inhibits the synthesis of matrix-metalloproteinases and TIMP inhibitor, respectively, by fibroblasts (35). These findings suggest that some ConA-binding surface glycoproteins are involved in the control of matrix breakdown in connective tissues.

Interactions of chondrocytes with artificial substrate impair their phenotypic expression. Thus ConA may enhance proteoglycan and ALPase synthesis via inhibition of their spreading on tissue culture surface. We tested this notion by comparing the action of ConA with that of succinyl ConA. Tetravalent ConA induced extensive cross-linking of membrane glycoproteins, whereas the divalent ConA derivative did not. This accounted for the differences between the actions of ConA and succinyl ConA on lymphocytes (36). In this study, ConA, but not succinyl ConA, inhibited the spreading of chondrocytes, although these lectins exerted the same effects on ALPase activity, proteoglycan synthesis, and total protein synthesis. Thus, extensive cross-linking of cell-surface glycoproteins is required for the rapid cell-shape change, but not for chondrocyte differentiation.

The tertiary structures of ConA and the ConA-sugar complex, as well as its sugar-binding site, have been characterized (37). In addition, the oligosaccharide structure required for ConA binding has been determined (38), and a family of peptides that mimic the binding of MeMan to ConA have been identified from screening a hexapeptide epitope library (39). This information will be useful for future studies on the action of ConA on animal cells.

In conclusion, the present study showed that ConA selectively acts on resting chondrocytes to initiate the maturation program, and that the ConA stimulation of chondrocyte differentiation is not directly linked with lectin-induced changes in the cytoskeleton. Resting chondrocytes exposed to ConA may be useful as a novel model for studies on endochondral bone formation.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§§   To whom correspondence should be addressed: Dept. of Biochemistry, School of Dentistry, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima-city 734, Japan. Tel./Fax: 82-257-5629.
1   The abbreviations used are: ConA, concanavalin A; PHA, phytohemagglutinin; WGA, wheat germ agglutinin; T3, triiodothyronine; PTH, parathyroid hormone; PAGE, polyacrylamide gel electrophoresis; MeMan, methylmannoside; TGF-beta 1, transforming growth factor beta -1; IGF-I, insulin-like growth factor I; ALPase, alkaline phosphatase.
3   M. Nishimura, unpublished data.
2   W. Yan, unpublished data.

Acknowledgments

We gratefully acknowledge Dr. J. M. Wozney (Genetics Institute, Cambridge, MA) and Dr. K. Takahashi (Yamanouchi Pharmaceutical Co., Tokyo) for gifts of BMP-2. We thank the Research Center for Molecular Medicine, Hiroshima University School of Medicine, for the use of their facilities.


REFERENCES

  1. Lis, H., and Sharon, N. (1986) Annu. Rev. Biochem. 55, 35-67 [CrossRef][Medline] [Order article via Infotrieve]
  2. Cuatrecasas, P., and Tell, G. P. E. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 485-489 [Abstract]
  3. Czech, M. P., Lawrence, J. C., Jr., and Lynn, W. S. (1974) J. Biol. Chem. 249, 7499-7505 [Abstract/Free Full Text]
  4. Katzen, H. M., Vicario, P. P., Mumford, R. A., and Green, B. G. (1981) Biochemistry 20, 5800-5809 [Medline] [Order article via Infotrieve]
  5. Yan, W., Nakashima, K., Iwamoto, M., and Kato, Y. (1990) J. Biol. Chem. 265, 10125-10131 [Abstract/Free Full Text]
  6. Makhailov, A. T., and Gorgolyuk, N. A. (1988) Cell Diff. 22, 145-154 [CrossRef][Medline] [Order article via Infotrieve]
  7. Robison, R. (1923) Biochem. J. 17, 286-293
  8. Schmid, T. M., and Conrad, H. E. (1982) J. Biol. Chem. 257, 12444-12450 [Free Full Text]
  9. Iwamoto, M., Sato, K., Nakashima, K., Shimazu, A., and Kato, Y. (1989) Dev. Biol. 136, 500-507 [Medline] [Order article via Infotrieve]
  10. Kato, Y., Iwamoto, M., Koike, T., Suzuki, F., and Takano, Y. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9552-9556 [Abstract]
  11. Leboy, P. S., Vaias, L., Uschmann, B., Golub, E., Adams, S. L., and Pacifici, M. (1989) J. Biol. Chem. 264, 17281-17286 [Abstract/Free Full Text]
  12. Wu, L. N. Y., Sauer, G. R., Genge, B. R., and Wuthier, R. E. (1989) J. Biol. Chem. 264, 21346-21355 [Abstract/Free Full Text]
  13. Tacchetti, C., Quarto, R., Campanile, G., and Cancedda, R. (1989) Dev. Biol. 132, 442-447 [Medline] [Order article via Infotrieve]
  14. Iwamoto, M., Shimazu, A., Nakashima, K., Suzuki, F., and Kato, Y. (1991) J. Biol. Chem. 266, 461-467 [Abstract/Free Full Text]
  15. von der Mark, K., Kirsch, T., Medici, A., Kumiss, A., Weseloh, G., Gluckert, K., and Stoss, H. (1992) Arthritis Rheum. 35, 806-811 [Medline] [Order article via Infotrieve]
  16. Shimomura, Y., Yoneda, T., and Suzuki, F. (1975) Calcif. Tissue Res. 19, 179-187 [Medline] [Order article via Infotrieve]
  17. Bessey, O. A., Lowry, O. H., and Brock, M. J. (1946) J. Biol. Chem. 154, 321-329
  18. Johnson-Wint, B., and Hollils, S. (1982) Anal. Biochem. 122, 338-344 [Medline] [Order article via Infotrieve]
  19. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  20. Bitter, T., and Muir, H. M. (1962) Anal. Biochem. 4, 330-334
  21. Maor, G., von der Mark, K., Reddi, H., Heinegard, D., Franzen, A., and Silberman, M. (1987) Collagen Relat. Res. 7, 351-370
  22. Dahl, L. K., and Dole, V. P. (1952) J. Exp. Med. 95, 341-346
  23. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  24. Kato, Y., Nomura, Y., Tsuji, M., Ohmae, H., Nakazawa, T., and Suzuki, F. (1981) J. Biochem. (Tokyo) 90, 1377-1386 [Abstract]
  25. Wozney, J. M., Rosen, V., Celesta, A. J., Mattock, L. M., Whitters, R. W., Kriz, R. W., Hewick, R. M., and Bang, E. A. (1988) Science 242, 1528-1534 [Medline] [Order article via Infotrieve]
  26. Vukicevic, S., Luyten, F. P., and Reddi, A. H. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8793-8797 [Abstract]
  27. Kato, Y., Shimazu, A., Nakashima, K., Suzuki, F., Jikko, A., and Iwamoto, M. (1990) Endocrinology 127, 114-118 [Abstract]
  28. Pacific, M., Golden, E. B., Iwamoto, M., and Adams, S. L. (1991) Exp. Cell Res. 195, 38-46 [Medline] [Order article via Infotrieve]
  29. Bohme, K., Conscience, E. M., Tschan, T., Winterhalter, K. H., and Bruckner, P. (1992) J. Cell Biol. 116, 1035-1042 [Abstract]
  30. Quarto, R., Campanile, G., Cancedda, R., and Dozin, B. (1992) J. Cell Biol. 119, 989-995 [Abstract]
  31. Iwamoto, M., Jikko, A., Murakami, H., Shimazu, A., Nakashima, K., Iwamoto, M., Takigawa, M., Baba, H., Suzuki, F., and Kato, Y. (1994) J. Biol. Chem. 269, 17245-17251 [Abstract/Free Full Text]
  32. Karaplis, A. C., Luz, A., Glowacki, J., Bronson, R. T., Tybulewocz, V., Kronenberg, H. M., and Mulligan, R. C. (1994) Genes Dev. 8, 277-289 [Abstract]
  33. Saito, M., Takaku, F., Hayashi, M., Tanaka, I., Abe, Y., Nagai, Y., and Ishii, S.-I. (1983) J. Biol. Chem. 258, 7499-7505 [Abstract/Free Full Text]
  34. Einspahr, H., Parks, E. H., Suguna, K., Subramanian, E., and Suddath, F. L. (1986) J. Biol. Chem. 261, 16518-16527 [Abstract/Free Full Text]
  35. Overall, C. M., and Sodek, J. (1990) J. Biol. Chem. 265, 21141-21151 [Abstract/Free Full Text]
  36. Gunther, G. R., Wang, J. L., Yahara, I., Cunnungham, B. A., and Edelman, G. M. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1012-1016 [Abstract]
  37. Derewenda, Z., Carib, J., Helliwell, J. R., Halo (Gibbon), A. J., Godson, E. J., Rapid, M. Z., Wan, T., and Campbell, J. (1989) EMBO J. 8, 2189-2193 [Abstract]
  38. Baenziger, J. U., and Fiete, D. (1979) J. Biol. Chem. 254, 2400-2407 [Medline] [Order article via Infotrieve]
  39. Scott, J. K., Loganathan, D., Bailey, R. B., Gong, X., and Goldstein, I. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5398-5402 [Abstract]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.