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.

Introduction

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.

Immunoblotting

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.

Chondrocyte culture

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).

Northern blotting

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.

Proteoglycan synthesis

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).

DNA synthesis

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).

Results

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).

Fig. 1.

SDS-PAGE and immunoblotting of the chondrocyte membrane treated with various concentrations of retinoic acid, and the effect of retinoic acid pretreatment on MTf expression by chondrocyte cultures. (A) Chondrocytes were exposed or not to 10–6 M retinoic acid 4 days before the end of incubation. The protein in the crude membrane fraction (6 μg) was analysed by SDS-PAGE and stained with silver. (B) Chondrocytes were exposed to retinoic acid at 0 M, 10–8 M, 10–7 M and 10–6 M 4 days before the end of incubation. The protein in the ConA-bound membrane fraction (2 μg) was analysed by SDS-PAGE and stained with silver. (C) Chondrocytes were exposed to retinoic acid at 10–6 M 0 hours, 24 hours, 48 hours and 72 hours before the end of the incubation. The proteins in the ConA-bound fraction (2 μg) were resolved by SDS-PAGE and stained with silver. (D) Chondrocytes were exposed to retinoic acid at 10–6 M 0 hours, 24 hours, 48 hours and 72 hours before the end of the incubation. The MTf level in the chondrocyte cultures was analyzed by immunoblotting. (E) Chondrocytes were exposed, or not exposed, to retinoic acid at 10–6 M for 4 days (left) and then incubated in the absence of retinoic acid for 3 days (right). The MTf level in the chondrocyte cultures was analysed by immunoblotting. (F)Chondrocytes in confluent cultures were incubated with retinoic acid at 10–6 M for 4 days. The MTf and GAPDH mRNA levels in the chondrocytes were determined by northern blot analysis.

Fig. 1.

SDS-PAGE and immunoblotting of the chondrocyte membrane treated with various concentrations of retinoic acid, and the effect of retinoic acid pretreatment on MTf expression by chondrocyte cultures. (A) Chondrocytes were exposed or not to 10–6 M retinoic acid 4 days before the end of incubation. The protein in the crude membrane fraction (6 μg) was analysed by SDS-PAGE and stained with silver. (B) Chondrocytes were exposed to retinoic acid at 0 M, 10–8 M, 10–7 M and 10–6 M 4 days before the end of incubation. The protein in the ConA-bound membrane fraction (2 μg) was analysed by SDS-PAGE and stained with silver. (C) Chondrocytes were exposed to retinoic acid at 10–6 M 0 hours, 24 hours, 48 hours and 72 hours before the end of the incubation. The proteins in the ConA-bound fraction (2 μg) were resolved by SDS-PAGE and stained with silver. (D) Chondrocytes were exposed to retinoic acid at 10–6 M 0 hours, 24 hours, 48 hours and 72 hours before the end of the incubation. The MTf level in the chondrocyte cultures was analyzed by immunoblotting. (E) Chondrocytes were exposed, or not exposed, to retinoic acid at 10–6 M for 4 days (left) and then incubated in the absence of retinoic acid for 3 days (right). The MTf level in the chondrocyte cultures was analysed by immunoblotting. (F)Chondrocytes in confluent cultures were incubated with retinoic acid at 10–6 M for 4 days. The MTf and GAPDH mRNA levels in the chondrocytes were determined by northern blot analysis.

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.

Fig. 2.

Effects of retinoic acid pretreatment on the shape of cultured chondrocytes in the presence or absence of ConA. Poorly differentiated chondrocytes were not exposed to retinoic acid for 4 days and then incubated in the absence (A)or presence (B) of 5 μg ml–1 ConA for 24 hours. Poorly differentiated chondrocytes were exposed to retinoic acid at 10–6 M for 4 days and then incubated in the absence (C) or presence (D) of 5 μg ml–1 ConA for 24 hours. Poorly differentiated chondrocytes were exposed to retinoic acid at 10–6 for 4 days and then incubated in the absence (E) or presence (F) of 5 μg ml–1 ConA for 72 hours. Bar, 30μm.

Fig. 2.

Effects of retinoic acid pretreatment on the shape of cultured chondrocytes in the presence or absence of ConA. Poorly differentiated chondrocytes were not exposed to retinoic acid for 4 days and then incubated in the absence (A)or presence (B) of 5 μg ml–1 ConA for 24 hours. Poorly differentiated chondrocytes were exposed to retinoic acid at 10–6 M for 4 days and then incubated in the absence (C) or presence (D) of 5 μg ml–1 ConA for 24 hours. Poorly differentiated chondrocytes were exposed to retinoic acid at 10–6 for 4 days and then incubated in the absence (E) or presence (F) of 5 μg ml–1 ConA for 72 hours. Bar, 30μm.

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.

Fig. 3.

Specificity of anti-MTf antibodies and the appearance of chondrocytes not exposed (none) or exposed to ConA or ConA plus anti-MTf antibodies for 24 hours. (A) The proteins in the chondrocyte cultures were resolved by SDS-PAGE. MTf in the samples was analysed by immunoblotting with pAb1 (lane 1), pAb2(lane 2), pAb3 (lane 3), pAb4 (lane 4) or mAb2 (lane 5). (B) Poorly differentiated chondrocytes were incubated with 1% control serum in the absence (control) or presence of 5 μg ml–1 ConA for 24 hours. Alternatively, these cells were incubated with 1% pAb1-4 serum in the presence of 5 μg ml–1 ConA for 24 hours (ConA+pAb1-4). In the other studies, these cells were incubated with control mouse IgG (40 μg ml–1) or mAb2 (10-40 μg ml–1) in the presence of 5 μg ml–1 ConA for 24 hours. (C) Chondrocytes were incubated with the F(ab′)2 fragment of mAb2 (40 μg ml–1) in the presence of 5 μg ml–1 ConA for 24 hours. Bar, 30 μm.

Fig. 3.

Specificity of anti-MTf antibodies and the appearance of chondrocytes not exposed (none) or exposed to ConA or ConA plus anti-MTf antibodies for 24 hours. (A) The proteins in the chondrocyte cultures were resolved by SDS-PAGE. MTf in the samples was analysed by immunoblotting with pAb1 (lane 1), pAb2(lane 2), pAb3 (lane 3), pAb4 (lane 4) or mAb2 (lane 5). (B) Poorly differentiated chondrocytes were incubated with 1% control serum in the absence (control) or presence of 5 μg ml–1 ConA for 24 hours. Alternatively, these cells were incubated with 1% pAb1-4 serum in the presence of 5 μg ml–1 ConA for 24 hours (ConA+pAb1-4). In the other studies, these cells were incubated with control mouse IgG (40 μg ml–1) or mAb2 (10-40 μg ml–1) in the presence of 5 μg ml–1 ConA for 24 hours. (C) Chondrocytes were incubated with the F(ab′)2 fragment of mAb2 (40 μg ml–1) in the presence of 5 μg ml–1 ConA for 24 hours. Bar, 30 μm.

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).

Fig. 4.

Effects of mAb2 on proteoglycan synthesis and DNA synthesis in chondrocyte cultures, and on DNA synthesis in lymphocyte cultures in the presence or absence of ConA or sConA. (A-C) Poorly differentiated chondrocytes were incubated in the absence or presence of 5 μg ml–1 ConA or 10 μg ml–1 sConA with or without mAb2 or control mouse IgG for 24 hours. Alternatively, these cells were exposed to DBcAMP or insulin in the presence or absence of mAb2 at 40 μg ml–1. (D)Lymphocytes were incubated in the presence or absence of 3 μg ml–1 ConA with or without mAb2 or control IgG at 40 μg ml–1 for 72 hours. The values are averages ± s.d. for the four cultures. (A: ConA/sConA versus ConA/sConA + mAb2;*P<0.01; **P<0.005; ***P<0.0001. B:*P<0.01; **P<0.001.)

Fig. 4.

Effects of mAb2 on proteoglycan synthesis and DNA synthesis in chondrocyte cultures, and on DNA synthesis in lymphocyte cultures in the presence or absence of ConA or sConA. (A-C) Poorly differentiated chondrocytes were incubated in the absence or presence of 5 μg ml–1 ConA or 10 μg ml–1 sConA with or without mAb2 or control mouse IgG for 24 hours. Alternatively, these cells were exposed to DBcAMP or insulin in the presence or absence of mAb2 at 40 μg ml–1. (D)Lymphocytes were incubated in the presence or absence of 3 μg ml–1 ConA with or without mAb2 or control IgG at 40 μg ml–1 for 72 hours. The values are averages ± s.d. for the four cultures. (A: ConA/sConA versus ConA/sConA + mAb2;*P<0.01; **P<0.005; ***P<0.0001. B:*P<0.01; **P<0.001.)

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.

Fig. 5.

Effects of anti-MTf-antibodies on cell shape and proteoglycan synthesis by chondrocytes in the absence of ConA. (A) Poorly differentiated chondrocytes were incubated with 1% control serum, 1% pAb2-4, 100 μg ml–1 control mouse IgG or 100 μg ml–1 IgG purified from the pAb4 serum for 24 hours. (B) Poorly differentiated chondrocytes were incubated with the control serum or pAb4 serum at concentrations of 0.05-1% for 24 hours. (C) Poorly differentiated chondrocytes were incubated with 1% control serum or 1% pAb4 for 7 days. Bar, 30 μm. The values are averages ± s.d. for the four cultures.*P<0.01.

Fig. 5.

Effects of anti-MTf-antibodies on cell shape and proteoglycan synthesis by chondrocytes in the absence of ConA. (A) Poorly differentiated chondrocytes were incubated with 1% control serum, 1% pAb2-4, 100 μg ml–1 control mouse IgG or 100 μg ml–1 IgG purified from the pAb4 serum for 24 hours. (B) Poorly differentiated chondrocytes were incubated with the control serum or pAb4 serum at concentrations of 0.05-1% for 24 hours. (C) Poorly differentiated chondrocytes were incubated with 1% control serum or 1% pAb4 for 7 days. Bar, 30 μm. The values are averages ± s.d. for the four cultures.*P<0.01.

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).

Fig. 6.

Effects of MTf overexpression on chondrogenic differentiation of ATDC5 cells in response to ConA. ConA (5-20 μg ml–1) or sConA(35 μg ml–1) was added to the cultures in the presence of 5% serum, transferrin and selenite with no other growth factors from day 10,every four days or every other day, respectively. (A) Rabbit MTf levels in the cell layer of Pc1 (lane 1), Pc2 (lane 2), MTf1 (lane 3) and MTf5 (lane4) were analysed by immunoblotting. The proportion of spherical/polygonal chondrocytes among total cells (B) and the uronic acid content (C) were determined on day 23. (D) The mRNA levels of aggrecan, collagen type IIA, collagen type IIB and GAPDH were determined on day 23 by RT-PCR and Southern-blot analysis. (E) The appearance of ATDC5-MTf5 cells exposed to 15 μg ml–1 ConA in the presence or absence of mAb2 (50 μg ml–1)(Experiment A) or pAb1 serum (1%) (Experiment B) on day 12. The lectin and/or antibodies were added to the cultures on day 10. Bar, 30 μm.

Fig. 6.

Effects of MTf overexpression on chondrogenic differentiation of ATDC5 cells in response to ConA. ConA (5-20 μg ml–1) or sConA(35 μg ml–1) was added to the cultures in the presence of 5% serum, transferrin and selenite with no other growth factors from day 10,every four days or every other day, respectively. (A) Rabbit MTf levels in the cell layer of Pc1 (lane 1), Pc2 (lane 2), MTf1 (lane 3) and MTf5 (lane4) were analysed by immunoblotting. The proportion of spherical/polygonal chondrocytes among total cells (B) and the uronic acid content (C) were determined on day 23. (D) The mRNA levels of aggrecan, collagen type IIA, collagen type IIB and GAPDH were determined on day 23 by RT-PCR and Southern-blot analysis. (E) The appearance of ATDC5-MTf5 cells exposed to 15 μg ml–1 ConA in the presence or absence of mAb2 (50 μg ml–1)(Experiment A) or pAb1 serum (1%) (Experiment B) on day 12. The lectin and/or antibodies were added to the cultures on day 10. Bar, 30 μm.

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).

Fig. 7.

Effects of MTf overexpression on chondrogenic differentiation of C3H10T1/2 cells in response to ConA. C3H10T1/2 cells were transfected with MTf-expression or empty vector. (A) MTf levels in the cells transfected with the empty vector (lane 1) or MTf-expression vector (lane 2) were analyzed by immunoblotting. (B) ConA (5 μg ml–1) was added to confluent cultures of these cells for 48 hours. (C) The proportion of spherical/polygonal/spindle-like cells among total cells was calculated. (D)The antibody mAb2 or control IgG (100 μg ml–1) was added to confluent cultures of MTf-overexpressing C3H10T1/2 cells for 4 days. ConA(5 μg ml–1) was added to these cultures for 48 hours. (E)The proportion of spherical/polygonal/spindle-like cells among total cells was calculated. Bar, 30 μm.

Fig. 7.

Effects of MTf overexpression on chondrogenic differentiation of C3H10T1/2 cells in response to ConA. C3H10T1/2 cells were transfected with MTf-expression or empty vector. (A) MTf levels in the cells transfected with the empty vector (lane 1) or MTf-expression vector (lane 2) were analyzed by immunoblotting. (B) ConA (5 μg ml–1) was added to confluent cultures of these cells for 48 hours. (C) The proportion of spherical/polygonal/spindle-like cells among total cells was calculated. (D)The antibody mAb2 or control IgG (100 μg ml–1) was added to confluent cultures of MTf-overexpressing C3H10T1/2 cells for 4 days. ConA(5 μg ml–1) was added to these cultures for 48 hours. (E)The proportion of spherical/polygonal/spindle-like cells among total cells was calculated. Bar, 30 μm.

Discussion

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.

Acknowledgements

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.

References

Atsumi, T., Miwa, Y., Kimata, K. and Ikawa, Y.(
1990
). A chondrogenic cell line derived from a differentiating culture of AT805 teratocartinoma cells.
Cell Differ Dev.
30
,
109
-116.
Bitter, T. and Muir, H. M. (
1962
). Modified uronic acid carbazole reaction.
Anal. Biochem.
4
,
330
-334.
Brown, P. D. and Benya, P. D. (
1988
). Alterations in chondrocyte cytoskeletal architecture during phenotypic modulation by retinoic acid and dihydrocytochalasin B-induced reexpression.
J. Cell Biol.
106
,
171
-179.
Brown, J. P., Woodbury, R. G., Hart, C. E., Hellstrom, I. and Hellstrom, K. E. (
1981
). Quantitative analysis of melanoma-associated antigen p97 in normal and neoplastic tissues.
Proc. Natl Acad. Sci. USA
78
,
539
-543.
Brown, J. P., Hewick, R. M., Hellstrom, I., Hellstrom, K. E.,Doolittle, R. F. and Dreyer, W. J. (
1982
). Human melanoma-associated antigen p97 is structurally and functionally related to transferrin.
Nature
296
,
171
-173.
Chow, J. C., Young, D. W., Golenbock, D. T., Christ, W. J. and Gusovsky, F. (
1999
). Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction.
J. Biol. Chem.
274
,
10689
-10692.
Danielsen, E. M. and van Deurs, B. (
1995
). A transferrin-like GPI-linked iron-binding protein in detergent-insoluble noncaveolar microdomains at the apical surface of fetal intestinal epithelial cells.
J. Cell Biol.
131
,
939
-950.
Davis, S., Aldrich, T. H., Stahl, N., Pan, L., Taga, T.,Kishimoto, T., Ip, N. Y. and Yancopoulos, G. D. (
1993
). LIFR beta and gp130 as heterodimerizing signal transducers of the tripartite CNTF receptor.
Science
260
,
1805
-1808.
Demeule, M., Poirier, J., Jodoin, J., Bertrand, Y., Desrosiers,R. R., Dagenais, C., Nguyen, T., Lanthier, J., Gabathuler, R., Kennard, M. et al. (
2002
). High transcytosis of melanotransferrin (P97)across the blood-brain barrier.
J. Neurochem.
83
,
924
-933.
Dippold, W. G., Lloyd, K. O., Li, L. T., Ikeda, H., Oettgen, H. F. and Old, L. J. (
1980
). Cell surface antigens of human malignant melanoma: definition of six antigenic systems with mouse monoclonal antibodies.
Proc. Natl. Acad. Sci. USA
77
,
6114
-6118.
Estin, C. D., Stevenson, U., Kahn, M., Hellstrom, I. and Hellstrom, K. E. (
1989
). Transfected mouse melanoma lines that express various levels of human melanoma-associated antigen p97.
J. Natl. Cancer Inst.
81
,
445
-448.
Food, M. R., Rothenberger, S., Gabathuler, R., Haidl, I. D.,Reid, G. and Jefferies, W. A. (
1994
). Transport and expression in human melanomas of a transferrin-like glycosylphosphatidylinositol anchored protein.
J. Biol. Chem.
269
,
3034
-3040.
Fujii, M., Takeda, K., Imamura, T., Aoki, H., Sampath, T. K.,Enomoto, S., Kawabata, M., Kato, M., Ichijo, H. and Miyazono, K.(
1999
). Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation.
Mol. Biol. Cell
10
,
3801
-3813.
Gunther, G. R., Wang, J. L., Yahara, I., Cunningham, B. A. and Edelman, G. M. (
1973
). Concanavalin A derivatives with altered biological activities.
Proc. Natl. Acad. Sci. USA
70
,
1012
-1016.
Horejsi, V., Cebecauer, M., Cerny, J., Brdicka, T., Angelisova,P. and Drbal, K. (
1998
). Signal transduction in leucocytes via GPI-anchored proteins: an experimental artefact or an aspect of immunoreceptor function?
Immunol. Lett.
63
,
63
-73.
Hueber, A. O., Bernard, A. M., Battari, C. L., Marguet, D.,Massol, P., Foa, C., Brun, N., Garcia, S., Stewart, C., Pierres, M. and He, H. T. (
1997
). Thymocytes in Thy-1–/– mice show augmented TCR signaling and impaired differentiation.
Curr. Biol.
7
,
705
-708.
Jefferies, W. A., Food, M. R., Gabathuler, R., Rothenberger, S.,Yamada, T., Yasuhara, O. and McGeer, P. L. (
1996
). Reactive microglia specifically associated with amyloid plaques in Alzheimer's disease brain tissue express melanotransferrin.
Brain Res.
712
,
122
-126.
Jing, S., Wen, D., Yu, Y., Holst, P. L., Luo, Y., Fang, M.,Tamir, R., Antonio, L., Hu, Z., Cupples, R. et al. (
1996
). GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF.
Cell
85
,
1113
-1124.
Kato, Y. and Gospodarowicz, D. (
1985
). Effect of exogenous extracellular matrices on proteoglycan synthesis by cultured rabbit costal chondrocytes.
J. Cell Biol.
100
,
486
-495.
Kato, Y., Nomura, Y., Daikuhara, Y., Nasu, N., Tsuji, M., Asada,A. and Suzuki, F. (
1980
). Cartilage-derived factor (CDF) I. Stimulation of proteoglycan synthesis in rat and rabbit costal chondrocytes in culture.
Exp. Cell Res.
130
,
73
-81.
Kato, Y., Hiraki, Y., Inoue, H., Kinoshita, M., Yutani, Y. and Suzuki, F. (
1983
). Differential and synergistic actions of somatomedin-like growth factors, fibroblast growth factor and epidermal growth factor in rabbit costal chondrocytes.
Eur. J. Biochem.
129
,
685
-690.
Kawamoto, T., Pan, H., Yan, W., Ishida, H., Usui, E., Oda, R.,Nakamasu, K., Noshiro, M., Kawashima-Ohya, Y., Fujii, M. et al.(
1998
). Expression of membrane-bound transferrin-like protein p97 on the cell surface of chondrocytes.
Eur. J. Biochem.
256
,
503
-509.
Kennard, M. L., Richardson, D. R., Gabathuler, R., Ponka, P. and Jefferies, W. A. (
1995
). A novel iron uptake mechanism mediated by GPI-anchored human p97.
EMBO J.
14
,
4178
-4186.
Lis, H. and Sharon, N. (
1986
). Biological properties of lectins. In
The Lectins
(ed. I. E. Liener, N. Sharon and I. J. Goldstein), pp.
265
-291. Orlando, FL: Academic Press
McNagny, K. M., Rossi, F., Smith, G. and Graf, T.(
1996
). The eosinophil-specific cell surface antigen, EOS47, is a chicken homologue of the oncofetal antigen melanotransferrin.
Blood
87
,
1343
-1352.
Mollenhauer, J., Bee, J. A., Lizarbe, M. A. and von der Mark,K. (
1984
). Role of anchorin CII, a 31,000-mol-wt membrane protein, in the interaction of chondrocytes with type II collagen.
J. Cell Biol.
98
,
1572
-1579.
Mumby, S. M. (
1997
). Reversible palmitoylation of signaling proteins.
Curr. Opin. Cell Biol.
9
,
148
-154.
Nakamasu, K., Kawamoto, T., Shen, M., Gotoh, O., Teramoto, M.,Noshiro, M. and Kato, Y. (
1999
). Membrane-bound transferrin-like protein (MTf): structure, evolution and selective expression during chondrogenic differentiation of mouse embryonic cells.
Biochim. Biophys. Acta
1447
,
258
-264.
Richardson, D. R. (
2000
). The role of the membrane-bound tumour antigen, melanotransferrin (p97), in iron uptake by the human malignant melanoma cell.
Eur. J. Biochem.
267
,
1290
-1298.
Robinson, P. J. (
1991
). Phosphatidylinositol membrane anchors and T-cell activation.
Immunol. Today
12
,
35
-41.
Rodgers, W., Crise, B. and Rose, J. K. (
1994
). Signals determining protein tyrosine kinase and glycosyl-phosphatidylinositol-anchored protein targeting to a glycolipid-enriched membrane fraction.
Mol. Cell. Biol.
14
,
5384
-5391.
Romagnoli, P. and Bron, C. (
1997
). Phosphatidylinositol-based glycolipid-anchored proteins enhance proximal TCR signaling events.
J. Immunol.
158
,
5757
-5764.
Rose, T. M., Plowman, G. D., Teplow, D. B., Dreyer, W. J.,Hellstrom, K. E. and Brown, J. P. (
1986
). Primary structure of the human melanoma-associated antigen p97 (melanotransferrin) deduced from the mRNA sequence.
Proc. Natl. Acad. Sci. USA
83
,
1261
-1265.
Rothenberger, S., Food, M. R., Gabathuler, R., Kennard, M. L.,Yamada, T., Yasuhara, O., McGeer, P. L. and Jefferies, W. A.(
1996
). Coincident expression and distribution of melanotransferrin and transferrin receptor in human brain capillary endothelium.
Brain Res.
712
,
117
-121.
Shimomura, Y., Yoneda, T. and Suzuki, F.(
1975
). Osteogenesis by chondrocytes from growth cartilage of rat rib.
Calcif. Tissue Res.
19
,
179
-187.
Shukunami, C., Shigeno, C., Atsumi, T., Ishizeki, K., Suzuki, F. and Hiraki, Y. (
1996
). Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor.
J. Cell Biol.
133
,
457
-468.
Simons, K. and Ikonen, E. (
1997
). Functional rafts in cell membranes.
Nature
387
,
569
-572.
Smale, G. and Sasse, J. (
1992
). RNA isolation from cartilage using density gradient centrifugation in cesium trifluoroacetate: an RNA preparation technique effective in the presence of high proteoglycan content.
Anal. Biochem.
203
,
352
-356.
Towbin, H., Staehelin, T. and Gordon, J.(
1979
). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Natl. Acad. Sci. USA
76
,
4350
-4354.
Wlodarski, K. H. and Galus, K. (
1992
). Osteoblastic and chondroblastic response to a variety of locally administered immunomodulators in mice.
Folia Biol. (Praha)
38
,
284
-292.
Woodbury, R. G., Brown, J. P., Yeh, M. Y., Hellstrom, I. and Hellstrom, K. E. (
1980
). Identification of a cell surface protein, p97, in human melanomas and certain other neoplasms.
Proc. Natl. Acad. Sci. USA
77
,
2183
-2187.
Yan, W., Nakashima, K., Iwamoto, M. and Kato, Y.(
1990
). Stimulation by concanavalin A of cartilage-matrix proteoglycan synthesis in chondrocyte cultures.
J. Biol. Chem.
265
,
10125
-10131.
Yan, W., Pan, H., Ishida, H., Nakashima, K., Suzuki, F.,Nishimura, M., Jikko, A., Oda, R. and Kato, Y. (
1997
). Effects of concanavalin A on chondrocyte hypertrophy and matrix calcification.
J. Biol. Chem.
272
,
7833
-7840.
Zanetti, N. C. and Solursh, M. (
1986
). Epithelial effects on limb chondrogenesis involve extracellular matrix and cell shape.
Dev. Biol.
113
,
110
-118.