Recognition of collagen by fibroblasts through cell surface glycoproteins reactive with Phaseolus vulgaris agglutinin

Collagen is a major component of extracellular matrices (ECM) of multicellular animals, and its roles can be classified into: (1) supporting multicellular body organization, and (2) regulation of physiological functions of cells. Interactions of cells with ECM involve binding between cell surface receptors and ECM components as ligands. Recently, cell surface receptors for collagen were identified and characterized (Wayner and Carter, 1987; Kramer and Marks, 1989; Gullberg et al. 1989, 1990b).

Collagen gel culture of fibroblasts (Elsdale and Bard, 1972) has been a useful experimental system for the study of the cell to collagen interaction: fibroblasts recognize, bind and reorganize collagen fibrils during culture, resulting in the contraction of collagen gel (Bell et al. 1979; Steinberg et al. 1980; Harris et al. 1981; Stopak and Harris, 1982; Allen and Schor, 1983; Buttle and Ehrlich, 1983; Grinnell and Lamke, 1984; Yoshi-zato et al. 1985b; Guidry and Grinnell, 1985, 1986; Gillery et al. 1986; Guidry and Grinnell, 1987; Adams and Priestley, 1988; Montesano and Orci, 1988; Schafer et al. 1989; Kono et al. 1990; Gullberg et al. 1990a; Asaga et al. 1991). Recently, we proposed that there is an indirect interaction between fibroblasts and collagen fibrils via cellular fibronectin (cFN) but not plasma fibronectin (pFN), using monoclonal antibody A3A5 (mAb A3A5), which specifically inhibits the fibroblast-mediated gel contraction (Asaga et al. 1991).

Monoclonal antibody A3A5 is a potent inhibitor of collagen gel contraction. However, complete inhibition was not obtained even in the presence of relatively high amounts of the antibody, when the gel culture was extended to more than 3 days (Asaga et al. 1991). This suggests the involvement of other type of mechanism of binding between fibroblasts and collagen in the process of gel contraction.

There have been several studies showing that cell surface glycoproteins act as receptors for ECM. In the present study, we have tested the possibility of involvement of this type of receptor in the fibroblast-mediated collagen gel culture using several kinds of lectins as a probe. We showed that fibroblast-surface glycoproteins recognized by Phaseolus vulgaris agglutinin (PHA, especially PHA-E₄) act as a collagen receptor in the process of collagen gel contraction. The chemical nature of the PHA-E₄-reactive glycoproteins on cell membranes was analyzed and its function is discussed here in relation to the integrin family.

These were obtained as follows: Dulbecco’s modified Eagle’s medium (DMEM) from Kyokuto Pharmaceutical Industrial Co., Ltd. (Tokyo); fetal bovine serum (FBS) from Japan Biotest Laboratory (Tokyo); ethylenediaminetetraacetic acid (EDTA) and N-(2-hydroxyethyl)-piperazine-N’-2-ethanesul-fonic acid (Hepes) from Dojin Chemical Institute (Kumamoto, Japan); streptomycin and penicillin from Meiji Seika Co. (Tokyo); trypsin from Difco (Detroit); Falcon culture dishes from Becton Dickinson Labware (Oxnard, CA); Millipore filters and nitrocellulose papers from Millipore Japan (Yonezawa, Japan); Centricon-10 from W.R. Grace and Co. (Danvers, MA); human plasma fibronectin (pFN) from Hoechst Japan (Tokyo); concanavalin A (ConA), lentil seed (Lens culinaris) agglutinin (LCA), mushroom (Agaricus bisporus) agglutinin (ABA), pea (Pisum sativum) agglutinin (PSA), peanut agglutinin (PNA), Phaseolus vulgaris agglutinin (PHA), PHA-E4, PHA-L4, pokeweed mitogen (PWM), Ricinus communis agglutinin-60 (RCA), soybean agglutinin (SBA), wheat germ agglutinin (WGA), PHA-E₄-agarose, and peroxidase-PHA-E₄ from Seikagaku Kogyo Co. Ltd. (Tokyo); A-acetyl-D-galactosamine (GalNAc) and Nonidet P-40 (NP40) from Sigma Chemical Co. (St. Louis., MO); Pepstatin A from Peptide Institute Inc. (Osaka); tunicamycin, monensin (sodium salt) and Silver Stain Kit Wako from Wako Pure Chemical Industries (Osaka). Collagen (Type I) from Koken Co., Ltd. (Tokyo), which was prepared from calf dermis by digestion with pepsin in a dilute HC1 solution and characterized as described previously (Yoshizato et al. 1985a). All other chemicals were of reagent grade and purchased from Nacalai Tesque, Inc. (Kyoto) or Wako Pure Chemical Industries.

Human skin fibroblasts were obtained from expiants of the normal dermis and cultured as described previously (Yoshizato et al. 1981; Asaga et al. 1991). Fibroblasts were grown and maintained in 75 cm² plastic dishes in DMEM containing 10% FBS, 10 mM NaHCO₃, 20 mM Hepes, 100 i.u./ml penicillin and 100 μg/ml streptomycin in a moist atmosphere of 5% CO₂/95% air at 37°C. The population doubling level of cells used in the present study was within 18. Cells were detached from dishes with calcium- and magnesium-free Hanks’ solution (CMF-Hanks’) containing 0.1% trypsin and 1 mM EDTA, collected by centrifugation at 500 g for 5 min and were used for experiments after washing twice with Hanks’ solution.

Fibroblasts were populated three-dimensionally in hydrated collagen gels by the method of Elsdale and Bard (1972) with slight modifications (Asaga et al. 1991). The following stock solutions were prepared and kept at 4°C: 0.5% (w/v) collagen solution; 4x concentrated DMEM, which contains 80 mM Hepes, 40 mM NaHCOj, 0.4 mg/ml streptomycin and 400 i.u./ml penicillin. Fibroblasts were harvested from monolayer cultures by treating with 0.1% trypsin and 1 mM EDTA in CMF-Hanks’, counted, adjusted to the desired cell number and collected by centrifugation in a plastic tube. The tube was placed on ice, and the cell pellet was resuspended in DMEM containing 0.1% collagen, 20 mM Hepes, 10 mM NaHCOs, 0.1 mg/ml streptomycin and 10% FBS, which had been prepared by quickly mixing the stock solutions and redistilled water. Then pH was adjusted to 7.4 by adding 1 M NaOH. One milliliter of medium containing 2 × 10⁵ fibroblasts was inoculated in 25 mm bacteriological dishes and cultured at 37°C. Collagen had gelled within 10 min and cells were embedded three-dimensionally in gels. When necessary, lectin, tunicamycin or monensin was introduced into gel culture. The extent of contraction was quantified during culture by measuring the diameter of collagen gels.

Collagen-fibril-coated (100 μg/cm²) plastic dishes were prepared according to Yoshizato et al. (1985a, 1988). Culture medium was added to collagen-coated dishes and the dishes were kept at 37°C to polymerize collagen molecules into fibrils prior to use.

Gelatin-coated (100 μg/cm²) plastic dishes were prepared as follows. Collagen solution (0.5%, w/v) was treated for 5 min at 100°C, poured into dishes, and dried at 60°C.

Fibroblasts harvested from monolayer cultures were suspended in DMEM containing 10% FBS at a concentration of 2 × 10⁴ cells/ml, inoculated on dishes to give a density of 10⁵ cells/cm², and cultured. Cells were allowed to attach for about 30 min, and then medium was replaced with DMEM containing 10% FBS and lectins. Morphology of fibroblasts was observed by phase-contrast microscopy (Nikon, TMD) and recorded by taking photomicrographs.

In order to examine whether PHA-E₄ binds collagen and FN or not, the dot blot analysis was performed as follows. One microliter of a solution of collagen (1 mg/ml) or human pFN (1 mg/ml) was dotted to nitrocellulose papers and dried at room temperature. The papers were soaked in 2% bovine serum albumin for 1 h to block any remaining protein binding sites, incubated for 1 or 2 h with PBS containing 10 μg/ml peroxidase-PHA-E₄, washed three times with PBS, and incubated in PBS containing 0.05% H₂O₂ and 0.5 mg/ml 4-chloro-l-naphthol.

Fibroblasts (5 × 10⁷) were harvested from culture dishes with rubber policemen, washed three times with PBS, and homogenized with 5 ml of PBS containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM N-ethylmaleimide (NEM) and 1 μg/ml pepstatin A at 4°C. The homogenate was centrifuged at 1,000 g for 10 min, and the supernatant was centrifuged successively at 12,000 g for 20 min, and at 105,000 g for 1 h. The pellet was washed once with 5 ml of the homogenization medium described above and was homogenized in 0.5 ml of PBS containing 2% NP40, 1 mM PMSF, 2 mM NEM and 1 μg/ml pepstatin A, pH 8.0. The homogenate was diluted three-fold with the same buffer solution and was centrifuged at 12,000 g for 20 min. Affinity separation of PHA-E₄-reactive glycoproteins was performed according to Fleischmann et al. (1985). The supernatant (membrane fraction) was loaded onto a column (5 mm in diameter) of PHA-E₄-agarose (2 ml), which had been equilibrated with PBS (pH 8.0) containing 0.5% NP40, and was allowed to stand at 4°C for 12 h. The unbound fraction was collected. The column was washed with 20 ml of PBS (pH 8.0) containing 0.5% NP40 and was eluted with PBS (pH 8.0) containing 0.5% NP40, 0.2 mM K₂B₄O₇ and 200 mM GalNAc. Both unbound and bound fractions were concentrated by ultrafiltration using Centricon-10 and were subjected to electrophoretic analysis.

Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) was performed by the method of Laemmli (1970) with modifications described previously (Asaga et al. 1991). Samples to be analyzed were given SDS and 2-mercaptoethanol at a final concentration of 6% (w/v) and 5% (v/v), respectively, treated for 5min at 100°C, and were subjected to SDS-PAGE. SDS-PAGE was carried out with a stacking gel of 3% acrylamide and a separation gel of 7.5% acrylamide. Lower gel buffer (0.4% SDS in 1.5 M Tris-HC1, pH 8.8) was used instead of upper gel buffer (0.4% SDS in 0.5 M Tris-HCl, pH 6.8) for stacking gels. After electrophoresis, polypeptides in gels were stained with highly sensitive silver staining (Oakley et al. 1980; Morissey, 1981) using Silver Stain Kit Wako.

Fibroblasts were cultured in collagen gels in the presence of ConA, WGA, RCA, PHA, PSA, LCA, SBA, ABA, PNA or PWM at various concentrations (10, 20 or 50 μg/ml). Lectins of ConA, WGA, RCA, PHA, PSA and LCA inhibited fibroblast-mediated collagen gel contraction in a concentration-dependent manner, while other lectins did not affect the gel contraction (Fig. 1). Fibroblasts cultured in collagen gels spread three-dimensionally prior to gel contraction. However, when the gel contraction was inhibited by lectins, most fibroblasts remained round in shape (data not shown). The effect of lectins of ConA, WGA, PHA, PSA and LCA does not seem to be due to cytotoxicity, because viability of fibroblasts cultured on plain plastic was not affected by these lectins at a concentration of 100 μg/ml (data not shown). RCA showed a strong toxicity for fibroblasts.

Lectins did not need to be present throughout culture to show their effects on gel contraction. Fibroblasts were harvested by treating them with trypsin and EDTA, preincubated in Hanks’ solution containing 100 μg/ml of a lectin (ConA, PHA, WGA, LCA or PSA) at 4°C for 30 min, washed three times with Hanks’ solution to remove free lectin molecules and then’introduced into collagen gels that did not contain lectins. As shown in Fig. 2, all of the lectins tested significantly delayed gel contraction.

We performed a spreading assay of fibroblasts on plain or collagen-fibril-coated (100 μg/cm³) plastics. Lectins of ConA, WGA, LCA and PSA inhibited cell spreading on plastic (Fig. 3). These lectins also inhibited spreading on collagen fibrils and on gelatin-coated dishes (data not shown). The effect of PHA was noteworthy, because this lectin inhibited spreading only on collagen-fibrils, not on plastic (Figs 3, 4). In the experiment shown in Fig. 4, cells were cultured on plastic, collagen-fibrils or within a collagen gel in the presence of PHA at a higher concentration (100 μg/ml). PHA did not show any effect on fibroblast spreading on plain plastic, but inhibited the spreading on collagen fibrils or within a collagen gel. PHA appears to recognize cell surface glycochains that are specifically involved in the interaction with collagen fibrils. We examined also the effect of PHA on fibroblast spreading on gelatin-coated plastic. PHA showed no effect on the spreading on gelatin as in the case of plastic (data not shown), indicating that the mode of cell binding to gelatin is different from that to native collagen.

We introduced into collagen gel cultures of fibroblasts tunicamycin, which inhibits glycochain synthesis of glycoprotein (Alonso-Caplen and Compans, 1983), or monensin, which inhibits glycoprotein secretion (Tar-takoff, 1983) (Fig. 5). These antibiotics dose-depen-dently inhibited gel contraction. Gels containing each inhibitor were rinsed three times with inhibitor-free medium to remove unbound inhibitors and were cultured again in an inhibitor-free condition. The gels started to contract, indicating that the effect of these antibiotics is reversible and, therefore, that the drugs are not cytotoxic. These antibiotics could produce their inhibitory effects at any time during contraction (data not shown). This contrasts with the effect of lectins; lectins added after gels had started to contract did not inhibit the contraction.

The PHA molecule is a tetramer composed of two types of subunits, called E and L, both of which have the same molecular weights but differ in ability of glycochain recognition (Leavitt et al. 1977). PHA is a mixture of five forms of molecules, which are named E₄, E₃L_1; E₂L₂, E₂L₃ and L₄, respectively. In order to know which kind of glycochain plays a key role in cell to collagen binding we introduced PHA-E₄ and PHA-L₄ into the collagen gel culture of fibroblasts and compared their effects on gel contraction. Both types of lectins dose-dependently inhibited gel contraction, the effect of PHA-E₄ being more intense (Fig. 6).

It has been shown that cFN is involved in fibroblast-mediated collagen gel contraction (Asaga et al. 1991). PHA-E₄ did not bind glycochains of either collagen or fibronectin, which was shown by dot blot analysis using peroxidase-PHA-E₄ (data not shown).

To understand the chemical nature of glycoproteins recognized by PHA-E₄, the membrane fraction of fibroblasts was obtained as an extract with NP40, and subjected to affinity chromatography with a PHA-E₄-agarose column (Fig. 7). Five bands were obtained in the eluate (Fig. 7, lane 4). When PHA-E₄ was mixed with membrane fraction, precipitates were formed. The same banding pattern of proteins was obtained when the precipitates were subjected to SDS-PAGE (data not shown). The M_r values of three of five bands were 130 × 10³, 150 × 10³ and 180 × 10³, respectively. M_r values of the other two were larger than 230 × 10. The 130 kDa protein could be β₁-integrin, because this protein was reported to be involved in the fibroblast-mediated collagen gel contraction (Gullberg et al. 1990a).

The present study clearly demonstrates that PHA specifically inhibits cell to collagen interactions shown by inhibition of the spreading of fibroblasts on collagen fibrils or in collagen gels and fibroblast-mediated collagen gel contraction. PHA-E₄, an isolectin of PHA, shows a relatively strong inhibition of collagen gel contraction.

The interaction of cells with ECM, or artificial substrata, such as plastic, has been extensively studied. Kundsen et al. (1981) reported that a 140 kDa membrane glycoprotein is involved in cell to substratum adhesion. Lehto and his associates showed that a 140 kDa glycoprotein of plasma membranes is involved in cell spreading (Lehto, 1983; Virtanen et al. 1982). Oppenheimer-Marks and Grinnell (1981, 1982, 1984) showed that WGA inhibits FN receptor (FNR) funetion, and that the antibodies against the membrane glycoproteins recognized by WGA also inhibit the spreading of cells on pFN-coated substrata. WGA was used to identify or purify the glycoprotein in all of these studies, but PHA has not been a target of study.

The cell surface receptors for various ECM components were identified and characterized using affinity gels conjugated with ECM or monoclonal antibodies against cell surface proteins (Wayner and Carter, 1987; Pytela et al. 1985a, 1985b; Horwitz et al. 1985; Akiyama et al. 1986; Wayner et al. 1988; Kramer and Marks, 1989; Gullberg et al. 1989,1990b). The majority of these receptors have structural relativity with one another. Therefore, these receptors are generally called “integrin(s)” (Buck et al. 1986; Hynes, 1987). All integrins are plasma membrane-bound complexes with α and β subunits noncovalently associated in a heterodimeric structure. These subunits showed apparent values of about 110–180 (×10³) by SDS-PAGE.

The significance of glycosylation of FNR was recently studied by Akiyama et al. (1989), who showed that the processing of oligosaccharide to a mature form of FNR is not important for subunits assembly and insertion into the plasma membrane, but is important for its FN-binding function. However, to our knowledge, the functional significance of the giycochain of receptors in binding has not been reported for other ECM components including collagen.

The results of the present study suggest the possibility that PHA-E4 recognizes the glycochain of collagen receptor, and that the glycochain plays important roles for binding collagen fibril. Other lectins, ConA, WGA, LCA and PSA, may also recognize specific glycochains involved in binding to collagen. However, these lectins also recognize other glycochains that are not involved in binding to collagen, because their inhibition of fibroblast-spreading seems nonspecific. There is a possibility that WGA inhibited FNR function in the experiments described in the present paper, because it was demonstrated that WGA inhibits FNR function (Oppenheimer-Marks and Grinnell, 1981) and fibroblast-mediated collagen gel contraction requires cFN (Asaga et al. 1991).

The inhibition of cell spreading or gel contraction by ConA, LCA and PSA may be an indirect effect, since it was suggested that ConA indirectly inhibits cell spreading by modulating the cytoskeleton or preventing receptor redistribution (Oppenheimer-Marks and Grinnell, 1981).

The glycochain structure recognized by PHA-E₄ is characterized in detail. High-affinity binding to PHA-E₄-agarose occurs only with biantennary glycopeptides containing two outer galactose residues and a residue of N-acetylglucosamine-linked β (l,4)-linked mannose residue in the core (Cummings and Komfeid, 1982). Only Asn-linked oligosaccharides interact strongly with PHA-E₄ (Kornfeld and Kornfeld, 1970). In the present study, it was also demonstrated that tunicamycin, an inhibitor of synthesis of Asn-linked glycochain (Alonso-Caplen and Compans, 1983), suppresses fibroblast-mediated collagen gel contraction. Therefore, it may be reasonably suggested that Asn-linked glycochains play a critical role in fibroblast-mediated collagen gel contraction.

The SDS-PAGE pattern of membrane glycoproteins recognized by PHA-E₄ revealed five bands. Three of them have apparent M_r values of 130, 150 and 180 (×10³), respectively. The chemical nature of these proteins has not been described further. However, the 130 kDa band appears to be β₁-integrin, because Gullberg et al. (1990a) suggested the involvement of 130 kDa β ₁-integrin in fibroblast-mediated collagen gel contraction. Therefore, there is a possibility that PHA-E₄ recognizes and blocks glycochains of collagen receptors of integrin family. M_r values of the other two bands were larger than 230 × 10³, suggesting that PHA-E₄ might block glycochains of unknown collagen receptors.

We proposed that collagen gel contraction by fibroblasts requires cFN but not pFN, in the previous study (Asaga et al. 1991). It, therefore, appears that there are at least two binding mechanism between fibroblasts and collagen in the process of collagen gel contraction, indirect binding via cFN and direct binding via the glycoprotein recognized by PHA-E₄. Detailed characterization of the glycoprotein described in the present paper will be the subject of further study.

This work was supported in part by a grant-in-aid for special project research from the Ministry of Education, Science and Culture of Japan (01870034) to K.Y. and H.A. is the recipient of postdoctoral fellowship from the Japan Society for Promotion of Science.