Membrane-bound transferrin-like protein (MTf), a glycosylphosphatidylinositol-anchored protein, is expressed at high levels in many tumors and in several fetal and adult tissues including cartilage and the intestine, as well as in the amyloid plaques of Alzheimer's disease, although its role remains unknown. MTf is one of the major concanavalin A-binding proteins of the cell surface. In this study, we examined the effects of anti-MTf antibodies and concanavalin A on cell shape and gene expression,using cultures of chondrocytes and MTf-overexpressing ATDC5 and C3H10T1/2 cells. In cultures expressing MTf at high levels, concanavalin A induced cell-shape changes from fibroblastic to spherical cells, whereas no cell-shape changes were observed with wild-type ATDC5 or C3H10T1/2 cells expressing MTf at very low levels. The cell-shape changes were associated with enhanced proteoglycan synthesis and expression of cartilage-characteristic genes,including aggrecan and type II collagen. Some anti-MTf antibodies mimicked this action of concanavalin A, whereas other antibodies blocked the lectin action. The findings suggest that the crosslinking of MTf changes the cell shape and induces chondrogenic differentiation. MTf represents the first identification of a plant lectin receptor involved in cell-shape changes and the differentiation of animal cells.
Because of their powerful biological actions in animal cells, plant lectins and their derivatives are promising for various aspects of biotechnology(Lis and Sharon, 1986). The elucidation of lectin-glycoprotein interactions on the cell surface will be useful for drug design and studies on the roles of glycoproteins, because the tertiary structure of several plant lectins and the structure of lectin-binding polysaccharides have previously been determined. There has been a vast accumulation of data on the actions of concanavalin A (ConA) and other plant lectins in animal cells, whereas the functional receptors for plant lectins have not been identified. Because many plant lectins, with different sugar-binding properties, induce cell-shape changes and proliferation and/or differentiation in lymphocyte cultures, we used chondrocytes or chondrogenic cells that responded to ConA but not to other lymphocyte-activating lectins(Yan et al., 1990) in order to identify a real ConA receptor. In chondrocyte cultures, ConA induced the conversion of fibroblastic cells to well-differentiated spherical chondrocytes and enhanced their synthesis of cartilage-matrix proteoglycan (aggrecan)within 24 hours (Yan et al.,1990; Yan et al.,1997). This effect of ConA was much greater than that of known growth factors (Yan et al.,1990; Yan et al.,1997). However, chondrocytes exposed to retinoic acid lost their responsiveness to ConA, suggesting that retinoic acid decreases the expression of ConA receptors on the cell surface (Yan et al., 1990). The ConA-induced chondrocyte differentiation can be observed in vivo: injection of ConA induced ectopic cartilage in the perichondrorium of mice (Wlodarski and Galus, 1992).
MTf was originally identified with the use of monoclonal antibodies as a 97 kDa human tumor-associated antigen(Woodbury et al., 1980; Dippold et al., 1980). Its amino acid sequence shows ∼40% identity with transferrin and lactoferrin(Rose et al., 1986). It binds to iron (Brown et al., 1982)and stimulates iron uptake in the absence of transferrin and transferrin receptor (Kennard et al.,1995). Interestingly, MTf can cross the blood-brain barrier(Demeule et al., 2002) and it accumulates in amyloid plaques of Alzheimer's disease, where iron is also concentrated (Jefferies et al.,1996). Furthermore, it has been suggested that MTf is involved in the proliferation and differentiation of melanoma cells and eosinophils(Estin et al., 1989; McNagny et al., 1996). However, the precise physiological roles of MTf remain unknown.
Previous studies have shown that MTf is a major ConA-binding protein on the chondrocyte surface (Kawamoto et al.,1998). Because cartilage contains MTf at a much higher level than other normal tissues, chondrogenic cells are a good model for studies on MTf. In the present study, we examined the effect of anti-MTf antibodies on cell shape and the expression of differentiation-related genes in cultures of chondrocytes or chondrogenic cells (ATDC5 and C3H10T1/2 cells) in the presence or absence of ConA. The anti-MTf antibodies markedly suppressed the effect of ConA on the cell shape and the phenotypic expression in these cultures, or they mimicked the action of ConA only when the cells synthesized MTf at high levels. These effects of ConA and the anti-MTf antibodies on MTf-expressing cells were observed within 24-48 hours. The findings on the ConA-MTf system obtained in this study will be useful in the understanding of the remarkable actions of plant lectins on animal cells.
Materials and Methods
Preparation of antibodies
MTf was purified from rabbit chondrocyte plasma membrane(Kawamoto et al., 1998). Three female BALB/c mice were immunized by a subcutaneous injection of Ribi adjuvant solution (0.2 ml per mouse) containing 20 μg pure MTf. The mice received two subcutaneous injections of the same solution (0.2 ml per mouse) 21 and 35 days after the first immunization. 3 days after the last injection, one mouse showed a high titer of anti-MTf antibodies in a serum sample. This serum(anti-MTf-pAb1) was taken and the spleen of the mouse was used for cell fusion with P3-X63-Ag8-U1 BALB/c myeloma cells. A hybridoma cell clone producing a monoclonal antibody to MTf (anti-MTf-mAb2) was obtained by limiting dilution. In other studies, three female BALB/c mice were immunized by four subcutaneous injections of the antigen (10 μg MTf in 0.15 ml of Ribi adjuvant solution per injection) on days 0, 14, 28 and 35. Sera (anti-MTf-pAb2, -pAb3, and-pAb4) were taken 7 days after the last injection.
The proteins extracted from the cultured chondrocytes (10 μg protein per lane) were resolved by 4-20% SDS-PAGE under nonreducing conditions. After blotting onto a PVDF membrane (Towbin et al., 1979) and blocking with 4% nonfat milk in PBS for 2 hours at room temperature, the membrane was incubated at 4°C with anti-MTf sera(1:500 dilution in PBS) or anti-MTf-mAb2 (5 μg ml–1) for 14 hours and then incubated with 125I-labeled sheep anti-mouse IgG(Fab′)2 fragment in PBS for 2 hours at room temperature.
The chondrocytes were isolated from the resting cartilage of the ribs of 4-week-old male Japanese White rabbits as described previously(Shimomura et al., 1975; Kato and Gospodarowicz, 1985). The cells were seeded at a density of 106 cells per 150-mm tissue culture dish and maintained in 30 ml α-modified Eagle's medium supplemented with 10% fetal bovine serum, 50 μg ml–1ascorbic acid, 50 U ml–1 penicillin, 60 μg ml–1 kanamycin and 250 ng ml–1 amphotericin B (medium A). When the cultures became subconfluent, the cells were harvested with trypsin and EDTA, and seeded at 5×104 cells per 16-mm well in 0.5 ml medium A. When the cultures again became subconfluent, the cells were preincubated in 0.5 ml of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium (Nissui Pharmaceutical, Tokyo, Japan)supplemented with 0.5% fetal bovine serum (medium B) for 24 hours. The medium was replaced by 0.5 ml of fresh medium B in the absence or presence of anti-MTf antibodies, ConA (5 μg ml–1) or both, and the incubation was continued for 24 hours.
ConA affinity chromatography of plasma membrane from retinoic-acid-exposed cultures
The cell membrane was isolated from retinoic-acid-exposed or -free chondrocyte cultures in three 150 mm dishes by the method of Mollenhauer et al. (Mollenhauer et al., 1984)with some modifications. The plasma membrane fraction was dissolved in 8 ml of buffer A [10mM Tris/HCl, pH 7.4, 10 μM (p-amidinophenyl)methanesulfonyl fluoride, 10 μM pepstatin A and 1% sodium deoxycholic acid]and applied to a ConA-Sepharose column (1 cm × 3 cm) (Amersham,Piscataway, NJ) equilibrated with buffer A. The ConA-bound glycoprotein was eluted with buffer A containing 0.5 M methyl-α-mannopyranoside as described previously (Kawamoto et al.,1998).
Total RNA was prepared from chondrocytes which had been treated with 10–6 M retinoic acid for 4 days using the guanidine-thiocyanate method (Smale and Sasse, 1992). RNA samples (10 μg) were electrophoresed on a 1%agarose gel containing 2.2 M formaldehyde and transferred to a Hybond-N membrane (Amersham). The membrane was hybridized with a 32P-labeled rabbit MTf cDNA probe or a 32P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe as described previously(Kawamoto et al., 1998). The membrane was exposed to BioMax X-ray film at –80°C with an intensifying screen.
The chondrocytes obtained from the rabbit primary cultures were seeded at 104 cells per 6-mm microwell and maintained in medium A. When the cultures became confluent, the cells were preincubated in 0.1 ml of medium B for 24 hours. The cells were incubated with ConA (5 μg ml–1), succinyl-ConA (sConA) (10 μg ml–1), dibutyryl cyclic AMP (DBcAMP) (1 mM) or insulin (10μg ml–1) in 0.1 ml of fresh medium B in the absence or presence of anti-MTf antibodies for 24 hours. [35S]sulfate (5μCi ml–1 final concentration) was added 6 hours before the end of the incubation (Kato et al.,1980). Uronic acid was determined by the method of Bitter and Muir(Bitter and Muir, 1962).
The chondrocytes obtained from the rabbit primary cultures were seeded at 104 cells per 6-mm microwell and maintained in medium A. When the cultures became confluent, the cells were preincubated in 0.1 ml ofα-modified Eagle's medium supplemented with 0.5% fetal bovine serum for 24 hours and then incubated with ConA (5 μg ml–1) or sConA(10 μg ml–1) in 0.1 ml of α-modified Eagle's medium supplemented with 0.5% fetal bovine serum in the absence or presence of anti-MTf-mAb2 or control mouse IgG for 24 hours. [3H]thymidine (10μCi ml–1) was added 3 hours before the end of the incubation (Kato et al.,1983). The lymphocytes were isolated from the thymus of 4-week-old rabbits. The cells were seeded at a density of 6×105 cells per 16-mm well and maintained in 0.2 ml medium A for 72 hours. The cells were exposed to anti-MTf-mAb2, control mouse IgG and/or ConA for 72 hours.[3H]Thymidine (3 μCi ml–1) was added 6 hours before the end of the incubation.
Plasmids, transfections and ATDC5 and C3H10T1/2 cells
Rabbit full-length MTf cDNA was inserted into the mammalian expression vector pDNA3.1/Zeo (Invitrogen, Carlsbad, CA) at the EcoRI-NotI site to construct pcDNA-MTf. pcDNA3.1/Zeo or pcDNA-MTf was transfected into ATDC5 and C3H10T1/2 cells (RIKEN, Tsukuba,Japan) using the SuperFect transfection reagent (Qiagen, Valencia, CA). The transfected cells were selected with 0.2 mg ml–1 Zeocin(Invitrogen). Individual colonies were isolated, and the expression levels of MTf were determined by immunoblotting with anti-MTf-mAb2.
ATDC5 cells were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 5% fetal bovine serum, 10 μg ml–1 human transferrin (Boehringer Mannheim, Mannheim,Germany) and 3×10–8 M sodium selenite (Sigma, St Louis,MO) in the presence or absence of 10 μg ml–1 bovine insulin (Sigma) as previously described(Atsumi et al., 1990). The inoculum density of the cells was 4×104 cells per 16-mm dish or 20×104 cells per 10 cm dish. The medium was replaced every other day.
C3H10T1/2 cells were maintained in medium A. The inoculum density of the cells was 8×104 cells per 35-mm dish or 20×104 cells per 10-cm dish. The medium was replaced every other day. On day 8, the culture medium was changed to Dulbecco's modified Eagle's medium and Ham's F-12 medium (1:1) containing 1% fetal bovine serum,insulin (6.25 μg ml–1), transferrin (6.25 μg ml–1), selenite (6.25 ng ml–1), ascorbic acid (50 μg ml–1), dexamethasone (10–10M) and transforming growth factor β1 (TGF-β1, 5 ng ml–1). The medium was replaced every other day.
Number of spherical/polygonal/spindle-like cells
Spherical/polygonal/spindle-like cells and spread cells were counted separately under a phase-contrast microscope. At least 300 cells were evaluated and the proportion of spherical/polygonal/spindle-like cells among total cells was calculated.
RT-PCR and Southern blot analysis
The first-strand cDNA was synthesized with 1 μg ml–1total RNA from ATDC5 cells. PCR was performed using an aliquot of the first-strand cDNA as a template under standard conditions with Klentaq Polymerase (Clonetech, Palo Alto, CA) for 18, 23 and 18 cycles for aggrecan,type II collagen and GAPDH, respectively. These cycles were optimal for comparison between the amplified products. The PCR products were separated on 1.5% agarose gels and transferred to NytranN membranes (Schleicher &Schuell, Dassel, Germany). The membranes were hybridized with 32P-labeled mouse aggrecan, 32P-labeled mouse-type II collagen or 32P-labeled mouse GAPDH cDNA(Nakamasu et al., 1999).
Close relationship between MTf levels and responsiveness of chondrocytes to ConA
To identify the retinoic-acid-sensitive membrane proteins that were potential ConA receptors (Yan et al.,1990), we incubated chondrocytes with retinoic acid at various concentrations for 1, 2 and 4 days, and examined retinoic-acid-induced changes in the membrane fraction by SDS-PAGE. The membrane fraction was resolved with over 40 bands on SDS-PAGE, but retinoic acid at 10–6 M for 4 days had little effect on the electrophoresis profile of the membrane proteins(Fig. 1A). The proteins were purified by ConA-affinity chromatography. About 5% of the membrane proteins were recovered in the ConA-bound fraction. When this fraction was analyzed by SDS-PAGE, the levels of 76 kDa (p76) and 140 kDa proteins (p140) were markedly affected (Fig. 1B,C): retinoic acid decreased the level of p76, whereas it increased the level of p140 dose-dependently. The p76 level decreased 24-48 hours after the retinoic acid was added (Fig. 1C and data not shown), and scarcely any of the p76 remained in cultures exposed to retinoic acid at 10–7 M or 10–6 M for 4 days(Fig. 1B). Because p76 had been identified as rabbit MTf by N-terminal sequencing(Kawamoto et al., 1998), the changes in p76 levels were examined by immunoblotting with anti-MTf antibody:Immunoblotting showed that incubation with 10–6 M retinoic acid for 24-72 hours markedly decreased the expression of p76/MTf(Fig. 1D). However, p76/MTf expression was recovered 3 days after the removal of retinoic acid(Fig. 1E). Northern blot analysis showed that the MTf mRNA level also decreased after the addition of retinoic acid (Fig. 1F).
In monolayer cultures in a low-serum medium, the chondrocytes took a fibroblastic configuration (Fig. 2A) and the addition of ConA induced a cell shape change from fibroblastic to spherical chondrocytes within 24 hours(Fig. 2B)(Yan et al., 1990). However,pretreatment with retinoic acid for 4 days prevented MTf expression, as indicated by immunoblotting (Fig. 1D), and the responsiveness of the chondrocytes to ConA(Fig. 2D), and removal of retinoic acid for 3 days restored the MTf level(Fig. 1E) and responsiveness to ConA (Fig. 2F). These findings indicate a close relationship between MTf level and responsiveness to ConA.
Inhibition of ConA-induced chondrocyte phenotypic expression by pAb1 and mAb2 and ConA-like actions of pAb4
If MTf is a receptor for ConA, antibodies to MTf should modulate the lectin action on chondrocytes. To test this hypothesis, we purified MTf from rabbit chondrocyte plasma membrane using HPLC and lectin-affinity chromatography(Kawamoto et al., 1998). Using the purified MTf, we prepared four polyclonal antisera (anti-MTf-pAb1, -pAb2,-pAb3 and -pAb4) and a monoclonal antibody (anti-MTf-mAb2) that had proven to be specific for MTf in immunoblot analysis with chondrocyte extracts(Fig. 3A). Rabbit chondrocytes were incubated with anti-MTf-pAb1 serum or the control serum in the absence or presence of ConA for 24 hours. The chondrocytes adopted a fibroblastic configuration in monolayer cultures at a low serum concentration(Fig. 3B, control)(Yan et al., 1990). The addition of ConA to the cultures altered the cell shape within 12 hours, with almost all of the lectin-exposed cells becoming spherical at 24 hours(Fig. 3B, ConA)(Yan et al., 1990). This effect of ConA was eradicated by the anti-MTf-pAb1 serum(Fig. 3B, ConA+pAb1). Anti-MTf-pAb2 and -pAb3 suppressed the action of ConA on the cell shape to a lesser extent, whereas anti-MTf-pAb4 had little effect on the cell-shape change (Fig. 3B, ConA+pAb2,ConA+pAb3 and ConA+pAb4). The anti-MTf-mAb2 suppressed the ConA-induced cell-shape change dose-dependently (Fig. 3B, ConA+mAb2), and the F(ab′)2 fragment of mAb2 also suppressed the cell-shape change (Fig. 3C, ConA+ F(ab′)2). The anti-MTf-mAb2 was prepared from the mouse that produced anti-MTf-pAb1.
The anti-MTf-mAb2 suppressed the ConA-stimulation of the incorporation of[35S]sulfate into glycosaminoglycans synthesized by chondrocytes(Fig. 4A, left panel). Under these conditions, the majority of 35S-labeled glycosaminoglycans were associated with cartilage-characteristic proteoglycan (aggrecan)(Yan et al., 1990).
Previous studies have shown that a divalent succinylated derivative of ConA(sConA) enhances chondrocyte phenotypic expression without inducing rapid changes in cell shape, although native tetravalent ConA enhances both rapid cell-shape change and chondrocyte phenotypic expression (proteoglycan synthesis) (Yan et al., 1997). ConA-but not sConA-induces extensive crosslinking of the cell surface glycoproteins and patch/cap formation (clustering of lectin-binding membrane proteins) (Gunther et al.,1973). In our study, the anti-MTf-mAb2 also suppressed the sConA-stimulation of proteoglycan synthesis by the chondrocytes(Fig. 4A, right panel).
Anti-MTf-mAb2 had little effect on the stimulation of proteoglycan synthesis by either a permeable analogue of cyclic AMP or insulin at the pharmacological level (Fig. 4B). The anti-MTf-mAb2 did not interfere with the effect of ConA on DNA synthesis in the chondrocytes (Fig. 4C). In addition, the anti-MTf-mAb2 had little effect on the ConA stimulation of lymphocyte proliferation(Fig. 4D), because MTf is barely expressed in lymphocytes (Kawamoto et al., 1998; Nakamasu et al.,1999).
Interestingly enough, the addition of the anti-MTf-pAb4 serum, as in the case of ConA, induced the expression of the spherical phenotype(Fig. 5A, pAb4). The anti-MTf-pAb2 and -pAb3 sera also induced cell-shape change, although their effect was far less than that of the anti-MTf-pAb4(Fig. 5A, pAb2 and pAb3). IgG purified from the anti-MTf-pAb4 serum using a Protein-A-affinity gel column also elicited the ConA-like action at 24 hours(Fig. 5A, pAb4 IgG), with the ConA-like action of the anti-MTf-pAb4 being indicated by the increase in incorporation of [35S]sulfate into glycosaminoglycans(Fig. 5B). In cultures exposed to the anti-MTf-pAb4 for 7 days, all of the cells were eventually surrounded by an abundant refractile matrix (Fig. 5C). All of the mice that were immunized with MTf – but none of the control mice – produced neutralizing and/or mimicking antibodies for the ConA action on chondrocytes.
Effects of ConA on chondrocyte phenotypic expression in ATDC5 cultures overexpressing MTf
MTf is upregulated in the mouse embryonic carcinoma-derived ATDC5 cells during chondrogenic differentiation(Nakamasu et al., 1999). These cells initiate chondrogenic differentiation only after the addition of some growth factor, such as insulin/insulin-like growth factor-I, TGF-β or bone morphogenic proteins (Shukunami et al., 1996; Atsumi et al.,1990; Fujii et al.,1999). Using this model, we examined the role of MTf in ConA-induced chondrogenic differentiation. Rabbit MTf cDNA was expressed under the control of the CMV promoter in stably transfected ATDC5 cells, and two MTf-expressing clones were isolated (ATDC5-MTf1 and ATDC5-MTf5);immunoblotting confirmed the expression of rabbit MTf in these clones(Fig. 6A). In the absence of added growth factors, parental ATDC5 cells and empty vector-integrated cells(Pc1 and Pc2) did not differentiate chondrogenic cells, regardless of the presence or absence of ConA (Fig. 6B), because of an absence of MTf expression(Nakamasu et al., 1999). By contrast, ∼30% of the MTf-overexpressing cells became spherical chondrocytes even in the absence of ConA, and ConA further increased the number of spherical chondrocytes dose-dependently(Fig. 6B), with the cell-shape change being accompanied by an increase in the uronic-acid-containing macromolecule (proteoglycan) (Fig. 6C). Even in the absence of ConA, the mRNA levels of aggrecan and collagen type IIB (chondrocyte specific), as well as the collagen type IIA(prechondrogenic stage characteristic) mRNA level, were much higher in the MTf-overexpressing cells than in the control cells(Fig. 6D), indicating the involvement of MTf in chondrogenic differentiation. Furthermore, sConA increased these mRNA levels in the MTf-overexpressing cells but not in the control cells. And, within 48 hours, the anti-MTf-mAb2 or anti-MTf-pAb1 suppressed expression of the spherical phenotype induced by the MTf-overexpressing ATDC5 cells in response to ConA(Fig. 6E).
Effect of ConA on cell shape in cultures of C3H10T1/2 cells overexpressing MTf
We isolated the C3H10T1/2 cells (T4) overexpressing rabbit MTf to examine whether ConA would induce cell shape change in the mouse pluripotent mesenchymal cell line expressing MTf at high levels. Wild-type C3H10T1/2 and T4 cells showed MTf mRNA expression at low and high levels, respectively (data not shown), and immunoblotting confirmed the expression of rabbit MTf at a high level in T4 cells, and its absence in the wild-type C3H10T1/2 cells(Fig. 7A). ConA induced cell shape change from fibroblastic to spherical or spindle-like cells within 48 hours in T4 cells, but not in the wild-type or empty vector-integrated cells(Fig. 7B,C). Furthermore, the cell-shape change was suppressed in the presence of the anti-MTf-mAb2(Fig. 7D,E).
ConA enhances phenotypic expression by chondrocytes(Yan et al., 1990). Because this effect of ConA is greater than that of growth factors, identification of ConA receptors might give us insight into the process of chondrocyte differentiation. MTf is a major ConA-binding protein on the chondrocyte surface, has a potential N-glycosylation site and binds to ConA-Sepharose (Kawamoto et al.,1998). Digestion of MTf of 76 kDa with N-glycosidase F for 5-15 hours yielded a product of 66 kDa (R.O., E. Usui and Y.K.,unpublished). Accordingly, MTf elutes from a ConA-Sepharose column with methyl-α-mannopyranoside (Kawamoto et al., 1998). ConA actions on chondrocytes can be abolished by anti-MTf antibodies (Fig. 3B,C)or methyl-α-mannopyranoside (Yan et al., 1990; Yan et al.,1997). These findings suggest that sugar is needed for the interaction between ConA and MTf. When retinoic acid decreased the MTf level,the chondrocytes lost responsiveness to ConA. The responsiveness of the chondrocytes, ATDC5 cells and C3H10T1/2 cells to the lectin depended upon the expression of MTf. Furthermore, some anti-MTf antibodies mimicked the ConA actions. These findings strongly suggest that MTf is a ConA receptor in chondrocytes, prechondrogenic cells and mesenchymal cells.
ConA alters the shape, proliferation and/or differentiation of various animal cells. MTf is not always expressed in these cells, suggesting that other ConA receptors are present in non-chondrogenic cells including lymphocytes. However, it is noteworthy that MTf is expressed at high or moderate levels in many tumors and fetal tissues(Brown et al., 1981; Danielsen and van Deurs, 1995),as well as several adult tissues, including the capillaries(Rothenberger et al., 1996),salivary glands (Richardson,2000) and eosinophil precursors(McNagny et al., 1996). In these cells, MTf might work, at least partially, as a ConA receptor.
ConA modulated both cell shape and the expression of aggrecan and type II collagen genes in chondrocytes and ATDC5 cells, via crosslinking of MTf and/or simple binding to MTf. Crosslinking of MTf by ConA appears to be essential for rapid cell-shape change but not for the expression of the differentiation-related genes, because simple binding of sConA induced the gene expression in chondrocytes (Fig. 4A) but not the rapid cell-shape change(Yan et al., 1997). Unlike ConA, sConA cannot induce clustering of cell-surface proteins. The lack of effect of sConA on cell shape suggests that the ConA-induced cell-shape change from fibroblastic cells to spherical cells is not directly linked with the ConA-induced, aggrecan and type II collagen expressions. This conclusion was unexpected because the cell shape has a great effect on the differentiation of prechondrogenic cells and dedifferentiated cartilage cells in some experimental systems (Zanetti and Solursh, 1984; Brown and Benya, 1988). However, in cultures of MTf-overexpressing C3H10T1/2 cells, ConA altered cell shape but did not enhance the expression of aggrecan or type II collagen (data not shown), which also suggests that MTf has two distinct roles: modulating cell shape and stimulating chondrocyte phenotypic expression.
Cell shape is determined by extracellular proteins (e.g. adhesion factors,matrix proteins and proteases) and membrane-bound proteins (e.g. integrins,cytoskeleton-associated proteins, small G proteins and tyrosine kinases), and the crosslinking of MTf might affect the activity of these proteins. Signals for the cell-shape change induced by ConA remain unknown. However, in subconfluent cultures of rabbit chondrocytes, SB203580 [an inhibitor of p38 mitogen-activated protein (MAP) kinase] at 10 μM suppressed the ConA-induced cell-shape changes, whereas U0126 [an inhibitor of MAP kinase kinase (MEK)] or SP600125 [an inhibitor of c-Jun N-terminal kinase (JNK)] had little effect at the same concentrations (data not shown). These findings suggest that p38 MAP kinase is involved in the cell shape change induced by the ConA-MTf system.
MTf is a glycosylphosphatidylinositol (GPI)-anchored protein(Food et al., 1994) and,because MTf does not have a cytoplasmic domain, the effect of ConA might be mediated by the binding of a ConA-MTf complex to a transmembrane receptor. One GPI-anchored protein [CD14, which binds to lipopolysaccharide (LPS)] forms a complex with a transmembrane Toll-like receptor-4 to induce inflammatory responses (Chow et al., 1999). The ciliary neurotrophic factor receptor is also a GPI-anchored protein that binds to a transmembrane gp130 protein, the signaling component of the IL-6 receptor (Davis et al., 1993). Similarly, the GPI-anchored glial-cell-derived neurotrophic factor receptorα associates, after ligand binding, with a transmembrane tyrosine kinase receptor Ret (Jing et al.,1996). A more likely mechanism for ConA actions is activation of signaling molecules in lipid rafts via crosslinking of MTf or simple binding to MTf. GPI-anchored proteins are found on the outer surface of lipid rafts,and signalling molecules, such as the Src kinase family and G proteins, are located in the inner surface of lipid rafts(Rodgers et al., 1994; Mumby, 1997; Simons and Ikonen, 1997). Binding of specific antibodies to some GPI-anchored proteins (e.g. CD14,Thy-1, Ly-6 and Qa-2) elicits striking biological reactions, including tyrosine phosphorylation, increase of cytoplasmic calcium, cell aggregation,phagocytosis, IL-2 production and/or DNA synthesis in various cells(Horejsi et al., 1998).
It is clear that MTf is not a ConA receptor for lymphocyte activations. We speculate, however, that some GPI-anchored proteins might work as a ConA receptor in lymphocytes, because ConA and antibodies to some GPI-anchored proteins have similar effects on lymphocytes, including increases in cytoplasmic calcium, cell-shape change, DNA synthesis and IL-2 production(Robinson, 1991; Horejsi et al., 1998).
Some GPI-anchored proteins, including Thy-1, have been shown physiologically to be involved in signaling via immunoreceptors(Hueber et al., 1997; Romagnoli and Bron, 1997). We showed here that even in the absence of ConA, the overexpression of MTf moderately altered the shape of ATDC5 cells and enhanced the expression of cartilage-characteristic genes, which suggests that GPI-anchored MTf plays a specific physiological role in chondrocyte differentiation.
We are now using ConA to enhance chondrogenic differentiation of human bone marrow mesenchymal cells and to enhance the phenotypic expression of cultured chondrocytes in vitro. The cartilage-like tissue formed in the presence of ConA in vitro might be useful for cell therapy. The results obtained in this study should be useful in promoting the application of ConA to tissue engineering.
We thank T. Kawamoto and M. Noshiro for their contributions. We also thank the Research Center for Molecular Medicine, Hiroshima University School of Medicine, for the use of their facilities.