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EXPRESSION OF FIBRONECTIN AND TYPE I COLLAGEN BY HUMAN DENTAL PULP CELLS AND GINGIVA FIBROBLASTS GROWN ON FIBRONECTIN SUBSTRATE M.-H. VERON,’ M.-L. COUBLE,’ G. CAILLOT,*D.-J. HARTMANN’ and H. MAGLOIRE’ ’ Laboratoire d’Histophysiologie et de Pathologie des Tissus Dentaires, UA CNRS 244, FacultC d’Odontologie, 69372 Lyon, Cedex 08 and 2Centre de Radioanalyse, Institut Pasteur, Lyon, France (Received

6 October

1989; accepted 2 February

1990)

Summary-Specific antibodies and indirect immunoperoxidase labelling were used to study the intracellular production of collagen and fibronectin by cells grown on fibronectin-coated glass; the same cell populations seeded on uncoated glass were used as controls. Strong intracellular staining for type I collagen was seen in all cases, but immunostaining for fibronectin was very faint or negative in both gingival and pulp cells grown on the fibronectin substrate, in contrast to control cells. Thus, fibronectin substrate inhibited fibronectin synthesis by the cultured cells, but did not seem to influence type I collagen synthesis. Key words: fibronectin, collagen, dental pulp, gingiva, fibroblasts.

Fibronectins are a class of high molecular-weight glycoprotein with functional domains that specifically interact with numerous extracellular molecules such

as collagens and heparin. Fibronectin has been implicated in a variety of cell functions including adhesion, migration, growth and differentiation (Yamada er al., 1984; Dufour, Duband and Thiery, 1986) and can influence cytoskeletal organization during the differentiation of many cell lines (Courtois et al., 1981; Zanetti and Solursh, 1984; Spiegelman and Ginty, 1983). In turn, extracellular matrix components can regulate their own protein synthesis through the same cytoskeletal components (Sugrue and Hay, 1986). Hedin et al. (1988) established that plasma fibronectin can phenotypically modulate smooth muscle cells, by showing that the cells were able to sense the macromolecular composition of the pericellular matrix and to modify their secretory activity accordingly. During tooth formation, odontoblast differentiation is controlled by epithelial-mesenchymal interactions and is thought to be matrix-mediated (Thesleff, Lehtonen and Saxen, 1978; Karcher-Djuritic et al., 1978; Thesleff and Pratt, 1980; Thesleff and Hurmerinta, 1981; Ruth et al., 1983; Ruth, 1985). Fibronectin has been demonstrated in association with the dental basement membrane and may play a crucial role during the various steps of differentiation (Andujar, Magloire and Grimaud, 1984; Lesot, Osman and Ruth, 1981; Linde et al., 1982; Thesleff

Abbreviation:

PBS, phosphate-buffered

saline.

et al., 1981). Indeed, it is generally well-accepted that dental papilla cells are derived from the cephalic neural crest (Lumsden, 1988) and can use fibronectin substrate as a suitable matrix for migration (Duband and Thiery, 1982; Duband et al., 1986). They do not, however, contain significant amounts of fibronectin mRNA at this stage (Ffrench-Constant and Hynes, 1988). At a later stage, polarized odontoblasts elaborate collagen and non-collagenous proteins constituting the predentine matrix but do not express fibronectin (Takita et al., 1987). We have now examined the synthesis of fibronectin and type I collagen by dental pulp cells grown on a fibronectin substrate to assess the role of extracellular fibronectin in controlling its own synthesis. Pulp cells were compared with gingival fibroblasts, in which fibronectin corresponds to the major glycoprotein elaborated in association with type I and III collagen (Baum and Wright, 1980). Pulp cells were cultured from explants obtained from permanent premolar teeth removed for orthodontic reasons from 10 children (aged 12-13 yr). The whole pulp, except the apical part, was dissected out. Gingival fibroblasts were cultured from 5 biopsies of clinically healthy human gingiva from volunteers who had given their consent. The biopsies were of buccal attached gingiva from upper or lower teeth. Explants were grown on glass coverslips in Leighton tubes, as described by Magloire et al. (1984). Cells were dissociated with a trypsin-EDTA solution (trypsin 0.05%, Biomerieux, France), counted on a Coulter Counter (Coultronics, France) and seeded (30,00O/ml) on fibronectin-coated or uncoated glass 565

M.-H. VERONet al.

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coverslips. First-third-passage cells were used and the term of culture was about 8-10 days. Human fibronectin was extracted and purified from human serum according to Caillot et al. (1986). The fibronectin coating was prepared using a solution of 30pg/ml fibronectin in PBS. This was either spread over the whole coverslip or only on one half, the uncoated side being used as control, and dried at 37°C. Immunohistochemical procedures were performed with anti-fibronectin and anti-type I collagen antibodies raised in rabbits against human plasma fibronectin and skin type I collagen. Anti-fibronectin antibodies were purified according to Linck et al. (1983) and the specificity and cross-reactivity of the anti-collagen antibodies were determined by immunoblotting and by radioimmunoassay as described by Magloire et al. (1984, 1986). Cell cultures were briefly rinsed, fixed in 4% paraformaldehydti. 1 M sodium cacodylate (pH 7.2t 0.025% saponin for 30 min at 4°C then rinsed again in PBSO.025% saponin-2 mg/ml bovine serum albumina. 1 M lysine HCl at 4°C. Intracellular detection of proteins is promoted by the permeabilizing effect of saponin. Cultures were then reacted with anti-fibronectin or anti-type I collagen antibodies overnight at 4°C. After washing, cells for light microscopy were incubated with a fluorescein-conjugated goat anti-rabbit gamma globulin (Ref. 74561, Institut Pasteur, Paris, France), mounted with buffered glycerin under a coverslip and examined with a Reichert Polyvar Microscope equipped for immunofluorescence. Electron microscope immunohistochemistry was carried out on pulp cells cultured on coverslides half-coated with fibronectin in order to demonstrate

the intracellular distribution of the glycoprotein and to confirm the immunofluorescence observations. Immunoperoxidase staining was performed with IgG peroxidase conjugate (Ref. 75011, Institut Pasteur, Paris, France), diluted in PBS and routinely visualized by diaminobenzidine according to Hedman (1980). Cells were post-fixed in a 2% OsOh PBS solution, dehydrated in a graded series of ethanol concentrations and embedded in Epon. Ultra-thin sections of coated and uncoated sides were observed under the electron microscope without further contrast. Immunohistochemical controls were carried out with the same steps but with the incubation done with the conjugate alone. Similar findings were obtained with pulp and gingival cells. Immunofluorescence showed that both cell types synthesized type I collagen on uncoated glass and on the fibronectin substrate (Figs 14) but whereas both cell populations synthesized fibronectin on uncoated glass, they did not express this protein when grown on the fibronection substrate (Figs 5-8). At the ultrastructural level (Figs 9 and lo), strong staining was clearly seen in the rough endoplasmic reticulum of control cells, whereas no labelling was seen in pulp cells grown on fibronectin substrate. Our findings suggest that fibronectin as substrate can reduce or inhibit its own synthesis by pulp or gingival cells without affecting type I collagen production. Immunoperoxidase staining was carried out on half-coated coverslides such that all cells were subjected to the same procedures. As cell cultures were processed at the same density and after the same culture period, the presence or absence of fibronectin was discriminant in this experiment.

Plate 1 Fig.

1.Immunofluorescent fluorescence

staining for type I collagen in pulp cells grown on uncoated glass. Very marked (arrowheads) is seen in the perinuclear area (N = nucleus). x 160

Fig. 2. Immunofluorescent staining for type I collagen in pulp cells grown on fibronectin cells show the same strong perinuclear staining (arrowhead). x 160 Fig. 3. Immunofluorescent Very marked

staining for type I collagen in gingival fibroblasts fluorescence (arrowhead) is seen in the perinuclear

grown area.

Fig. 4. Immunofluorescent staining for type I collagen in gingival fibroblasts substrate shows the same strong perinuclear staining (arrowhead). Fig. 5. Immunofluorescent area (arrowhead)

substrate.

on uncoated x 200

The glass.

grown on fibronectin x 200

staining for fibronectin in pulp cells grown on uncoated glass. The perinuclear is positively stained as is extracellular fibronectin (white arrow). x 160

Fig. 6. Immunofluorescent staining for fibronectin in pulp cells grown on fibronectin substrate. The intracellular staining is very faint; only extracellular fibronectin fluorescence (white arrow) is marked. x 160 Fig. 7. Immunofluorescent staining for fibronectin in gingival fibroblasts grown on uncoated glass. The perinuclear staining is positive (arrowhead) and extracellular fibronectin can also be seen (white arrow). x 160 Fig.

8. Immunofluorescent staining for fibronectin Intracellular staining is negative. Arrows

in gingival cells grown on fibronectin substrate. indicate extracellular fibronectin. x 160

Fig. 9. Electron micrograph showing indirect immunoperoxidase fibronectin staining of pulp cells grown on uncoated glass. The rough endoplasmic reticulum (rER) is strongly labelled by the peroxidase deposits. The mitochondria (M) appear racket-shaped because of the saponin permeation treatment. Note the presence of numerous microfilaments (m; N = nucleus). x 10,000 Fig. 10. Electron micrograph showing indirect immunoperoxidase fibronectin staining of pulp cells grown on fibronectin substrate. The rough endoplasmic reticulum (rER) is devoid of peroxidase deposits. Outside of the cell, the peroxidase deposits (arrows) correspond to extracellular fibronectin (M = mithochodria, N = nucleus). x 7500

Expression

of fibronectin

Plate

1

by pulp cells

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M.-H. VERON et al.

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It is generally well-accepted that matrix components are able to inform cell shape, mobility and differentiation (Hay, 1981). Thus, Yoshizato, Taira and Yamamoto (1985) showed that collagen fibrils strongly inhibit DNA synthesis and suppress growth of cultured fibroblasts. Similarly, cornea1 endothelial cells seeded on their own preformed extracellular matrix exhibit a dramatic decrease of collagen synthesis (Tseng et al., 1983). This typical feed-back mechanism of cell control has also been demonstrated for fibronectin (Holderbaum and Ehrhart, 1986) and type IV collagen (Sudhakaran, Stomatoglou and Hughes, 1986) with other cell types, in agreement with our findings. The extracellular matrix can therefore modulate the production of its own components, the mechanisms involved being signals generated by cellmatrix interactions. Roman et al. (1989) demonstrated that the fibronectin receptor is organized on the cell surface in response to extracellular fibronectin. It has been suggested that this receptor may serve as a signal transducer between the extracellular fibronectin matrix and the cell regulative-adhesive functions (cytodifferentiation, growth promotion) and also the regulation of gene expression. Thus, the strong concentration of fibronectin associated with the dental basement membrane may inhibit fibronectin synthesis by polarizing odontoblasts, and this feed-back regulation could be one step in odontoblast differentiation. Similarly, the substrate composition seems to affect human gingival cells in culture, thus suggesting a specific role of the extracellular matrix with significant changes in collagen synthesis as periodontitis develops (Narayanan and Page, 1983).

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Hay E. D. (1981) Extracellular matrix. J. ce//. Biol. 91, 205s-223s. Hedin U., Bottger B.-A., Forsberg E., Johansson S. and Thyberg J. (1988) Diverse Effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells. J. cell. Biol. 107, 307-3 19. Hedman K. (1980) Intracellular localization of fibronectin using immunoperoxidase cytochemistry in light and electron microscopy. J. Hisrochem. Cyrochem. 28, 1233-1241. Holderbaum D. and Ehrhart L. A. (1986) Substratum influence on collagen and fibronectin biosynthesis by arterial smooth muscle cells in vitro. J. ceN. Physiol. 126, 216-224. Karcher-Djuricic V., Osman M., Meyer J. M., Staubli A. and Ruth J. V. (1978) Basement membrane reconstitution and cytodifferentiation of odontoblasts in isochronal and heterochronal reassociations of enamel organs and pulps. J. Biol. buccale 6, 257-265. Lesot H., Osman M. and Ruth J.-V. (1981) Immunofluorescent localisation of collagen, fibronectin, and laminin during terminal differentiation of odontoblasts. Devl Biol. 82, 371-381. Linck G., Stocker S., Grimaud J.-A. and Porte A. (1983) Distribution of immunoreactive fibronectin and collagen (type I, III, IV) in mouse joints. Fibronectin, an essential component of the synovial cavity border. Hisrochemistry 77, 323-328. Linde A., Johansson S., Jonsson R. and Jontell M. (1982) Localization of fibronectin during dentinogenesis in rat incisor. Arch.7 oral Biol. 27, 1069-1073. Lumsden A. G. S. (1988) Spatial organization of the epithelium and the role of neural crest cells in the initiation of the mammalian tooth germ. Developmenf 103, 155-169. Magloire H., Hartmann D. J., Couble M. L., Joffre A., Grimaud J. A., Herbage D. and Ville G. (1984) Cytodifferentiation of human pulp cells in explant culture. In: Tooth Morphogenesis and Dljf&-mtiation (Edited by Balcourt A. B. and Ruth J. V.) Vol. 125, pp. 119-126. INSERM, Paris. Magloire H., Call& A., Hartmann D. J., Joffre A., Serre B.. Grimaud J. A. and SchuC F. (1986) Type I collagen production by human odontoblast-like cells in explants cultured on cyanocrylate films. Electron immunolocalization of fibronectin at cell/film interface. Cc/l Tiss. Res. 244, 1233%1240. Narayanan A. S. and Page R. C. (1983) Connective tissues of the periodontium: a summary of current work. Coil. Rel. Res. 3, 3344. Roman J., Lachance R. M., Broekelmann T. J., Kennedy C. J. R., Wayner E. A., Carter W. G. and McDonald J. A. (1989) The fibronectin receptor is organized by extracellular matrix fibronectin: Implications for oncogenie transformation and for cell recognition of fibronectin matrices. J. cell. Biol. 108, 2529-2543. Ruth J.-V. (1985) Odontoblast differentiation and the formation of the odontoblast layer. J. den/. Res. 64,489-498. Ruth J.-V., Karcher-Djuricic V.. Meyer J.-M. and Mark M. (1983) Epitheliai~mesenchymal interactions in tooth germs: mechanisms of differentiation. J. Biol. huccale 11, 1744193. Spiegelman B. M. and Ginty C. A. (1983) Fibronectin modulation of cell shape and lipogenic gene expression in 3T3 adinocvtes. Cell 35. 657466. Sudhakaran 6. R., Stomatoglou S. C. and Hughes R. C. (1986) Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes. Expl Cell Res. 167, 505-516. Sugrue S. P. and Hay E. D. (1986) The identification of extracellular matrix (ECM) binding sites on the basal surface of embryonic cornea1 epithelium and the effect of ECM binding on epithelial collagen production. J. ceN. Bio/. 102, 190771916.

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Thesleff I. and Hurmerinta K. (1981) Tissue interactions in tooth development. Differentiation 18, 75-88. Thesleff I., Lehtonen E. and Saxen L. (1978) Basement membrane formation in transfiher tooth culture and its relation to odontoblast differentiation. Differentiation 10, 71-79.

Thesleff I., Barrach H. J., Foidart J. M., Vaheri A., Pratt R. M. and Martin G. R. (1981) Changes in the distribution of type IV collagen, laminin, proteoglycans and

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Yamada K. M., Hayashi M., Hirano H. and Akiyama S. K. (1984) Fibronectin and cell surface interactions. In: The Role of Extracellular Matrix in Development, pp. 89-121. Liss, New York. Yoshizato K., Taira T. and Yamamoto N. (1985) Growth inhibition of human fibroblasts by reconstituted collagen fibrils. Biomed. Res. 6, 61-71. Zanetti N. and Solursh M. (1984) Induction of chondrogenesis in limb mesenchymal cultures by disruption of the actin cytoskeleton. J. cell. Biol. 99, 115-123.

Expression of fibronectin and type I collagen by human dental pulp cells and gingiva fibroblasts grown on fibronectin substrate.

Specific antibodies and indirect immunoperoxidase labelling were used to study the intracellular production of collagen and fibronectin by cells grown...
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