Molecular Biology, Department of Cancer Biology and Diagnosis, Bldg. 37, Rm. 4B19 .... produced by skin fibroblasts as expected from previous data. (Linker and .... the Finnish Medical Council, Finnish Cancer Foundation, Orion Corporation.
The EMBO Journal vol.3 no.3 pp.581-584, 1984
Integrity of the pericellular fibronectin matrix of fibroblasts is independent of sulfated glycosaminoglycans
Klaus Hedmanl*, Tapio Vartiol,2, Staffan Johansson3, Lena Kjellen3, Magnus Hook4, Alfred Linker5, EevaMarjatta Salonen1 and Antti Vaheri1 'Department of Virology, and 2Department of Pathology, University of Helsinki, Helsinki, Finland, 3Biomedical Center, Uppsala, Sweden, 4Diabetes Research and Training Center, University of Alabama, Birmingham, AL 35233, and 5Veterans Administration Hospital, Department of Biochemistry, University of Utah, Salt Lake City, UT 84112, USA *To whom reprint requests should be sent. Present address: Laboratory of Molecular Biology, Department of Cancer Biology and Diagnosis, Bldg. 37, Rm. 4B19, National Cancer Institute, National Institute of Health, Bethesda, MD 20205, USA Communicated by A. Vaheri
The pericellular matrix fibers of cultured human fibroblasts contain fibronectin, other glycoproteins, and heparan and chondroitin sulfate proteoglycans. In the present study, cellfree pericellular matrices were isolated from metabolically labeled fibroblast cultures. The isolated matrices were digested with heparinase from Flavobacterium heparinum, and then analyzed for sulfated glycosaminoglycans (GAGs). Nitrous acid degradation was used to distinguish the N-sulfated GAGs (heparan sulfate) from chondroitin sulfate. Fibronectin and the other major matrix polypeptides were studied using gel electrophoresis, enzyme immunoassay and immunofluorescence. Upon heparinase digestion, > 9507o of sulfated GAGs were degraded in the matrix without detectable release of fibronectin or other matrix polypeptides or alteration of the fibrillar matrix structure. We conclude that in fibroblast cultures the integrity of the fibrillar matrix is independent of sulfated GAGs. Together with earlier observations, this suggests that filamentous polymerization of fibronectin forms the backbone of early connective tissue matrix. Key words: extracellular matrix/fibroblast/fibronectin/ glycosaminoglycans/heparan sulfate Introduction The pericellular matrix of cultured human fibroblasts consists of fibronectin and of procollagen chains of types I and III as major polypeptides, and of hyaluronic acid, heparan sulfate and chondroitin sulfate proteoglycans (Hedman et al., 1979). Fibronectin and the procollagens are closely co-distributed in a fibrillar matrix network (Vaheri et al., 1978b; Hedman et al., 1979). We and others have shown that the sulfated proteoglycans are also integral components of the matrix fibers and occur in close association with fibronectin (Perkins et al., 1979; Hedman et al., 1982b; Hayman et al., 1982). The fibronectin-containing matrix has a number of effects on the cellular phenotype, including cell adhesion and spreading, locomotion and organization of cytoskeletal proteins (Vaheri et al., 1978a; see Yamada, 1983), cellular differentiation (Sieber-Blum et al., 1981; Couchman et al., 1982), and, in some instances, growth control (Gospodarowicz et © IRL Press Limited, Oxford, England.
al., 1980; Hsieh and Chen, 1983). The surfaces of malignantly transformed mesenchymal cells often lack the ability to assemble pericellular matrix fibers in vitro, although the cells have partially retained the capacity to bind to, and to phenotypically respond to, exogenous fibronectin matrices (Vaheri et al., 1978a; Alitalo and Vaheri, 1982; Hsieh and Chen, 1983). Despite extensive study, the molecular mechanism of pericellular matrix fibrillogenesis is largely unknown. The formation of matrix fibers seems to correlate with cell spreading and the formation of cell-cell contacts (Couchman et al., 1982; see Furcht, 1983; Yamada, 1983). Fibrillar assembly of fibronectin at the cell surface follows soon after cell adhesion to fibronectin (Avnur and Geiger, 1981), or after the addition of soluble fibronectin to cell culture media (McKeown-Longo and Mosher, 1983). Thus, the cell surface seems to actively induce matrix fibrillogenesis; in these events attention has been paid recently to the potential role of sulfated cell surface proteoglycans. There are indications that heparan sulfate of high charge density induces filamentous precipitation of fibronectin (Stathakis and Mosesson, 1977; Jilek and Hormann, 1979) and, unlike chondroitin sulfate, stabilizes the fibronectincollagen interaction (Ruoslahti et al., 1979; Jilek and Hormann, 1979; Johansson and Hook, 1980). The attachment or spreading of some cell types seems to involve cell surface heparan sulfate, in addition to fibronectin (Laterra et al., 1983; Stamatoglou and Keller, 1983). Chondroitin sulfate proteoglycans, in turn, inhibited fibronectin-mediated attachment of fibroblasts to collagen (Rich et al., 1981), and chondroitin sulfate was, indeed, recently found at the plasma membranes of cultured fibroblastic cells (Oldberg et al., 1981; Hedman et al., 1983). These complex findings have prompted us to study glycosaminoglycans (GAGs) in relation to matrix fibrillogenesis. Here we provide evidence that removal of sulfated GAGs from the matrix fibers has no detectable effect on the fibronectin-containing polypeptide core of the pericellular matrix. Results Effect of heparinase on matrix GAGs Metabolically labeled cell-free pericellular matrices of human fibroblasts were treated with crude heparinase, which degrades all types of GAG produced by fibroblasts. Table I shows the amounts of [35S]sulfate solubilized by the enzyme or the buffer. Heparinase at 1 Ag/ml released 60% of the total 35S, and 100 Ag/ml released 86% of the total 35S from the matrix. Both concentrations of the enzyme released < 10% of the [3H]glycine of the matrix (not shown). To document the extent of degradation of both heparan and chondroitin sulfates in the matrix, radiosulfate-labeled matrices treated with heparinase were degraded extensively with Pronase and analysed for the amount of nitrous acidsensitive versus resistant non-dialyzable GAGs. Figure 1 shows the profile of sulfated GAGs in the enzyme-treated or 581
K. Hedman et al.
Table I. [35S]Sulfate radioactivity solubilized by heparinase treatment of isolated pericellular matrices Enzyme concentration (ug/ml)
35S Released
0 1
100
Matrix bound
Total
64420 (19.7) 299 120 (59.6) 300 680
263 200 (80.3) 155 300
327 620 (100) 384420
(40.4)
(100)
(85.9)
(14.1)
349 980
49 300
(100)
FN14094-
The figures represent the mean values of two culture dishes. Percentage of total in a dish is indicated within parentheses.
68-
200
Fig. 2. Effect of heparinase on polypeptides of the matrix. Metabolically labeled ([35H]glycine) isolated matrices were treated with 100 Ag/ml (lane 1) or 1 yg/ml (lane 2) of crude heparinase or with digestion buffer (lane 3). The rinsed insoluble matrices were analyzed by SDS- polyacrylamide gel electrophoresis followed by fluorography. Mol. wt. standards in kd. are on the left. FN = migration position of human plasma fibronectin.
Cs
10
seen in Figure 2, the [3H]glycine-labeled matrices contained fibronectin as the major polypeptide. The 140 000 dalton polypeptide (Hedman et al., 1979; Carter and Hakomori, 1981; Lehto et al., 1983), and some other matrix polypeptides (Hedman et al., 1979), were found in the matrices. Heparinase treatment had no detectable effect on the matrix poly-
HS
40 20 30 FRACTION NUMBER
50
60
Fig. 1. Effect of heparinase on sulfated GAGs of the matrix. Metabolically labeled (35SO0 isolated matrices were treated with the digestion buffer (top graph), or with 100 Ag/ml of crude heparinase in the digestion buffer (bottom graph). The rinsed matrices were extensively degraded with Pronase, dialyzed and the glycosaminoglycan chains were treated with nitrous acid followed by gel filtration on Sephadex G-75. The buffer control sample contains both macromolecular, nitrous acid-resistant material (chondroitin sulfate,CS) and degraded, nitrous acid-sensitive material (heparan sulfate, HS). The heparinase-treated sample contains no nitrous acid-resistant and only trace amounts of degraded material.
control matrices after nitrous acid degradation followed by gel filtration. The upper graph shows the proportion of chondroitin sulfate to heparan sulfate in a buffer-treated control matrix; the lower graph shows the proportion in a matrix treated with heparinase. In the control, the two classes of GAG were present, in roughly equal amounts. From the digested matrix, 99% of the nitrous acid-resistant sulfated macromolecules, and >90/o of the nitrous acid-sensitive, were removed. This suggested that >9507o of the matrix GAGs were degraded by heparinase.
Effect on matrix polypeptides The heparinase-treated and control matrices were then studied by gel electrophoresis for matrix polypeptides. As 582
peptides; neither were detectable polypeptide fragments generated. This result suggested that the anchorage of fibronectin, and of the other proteins in the pericellular matrix fibers, might be independent of sulfated GAGs. This was studied using a more direct approach. Immunochemistry of fibronectin The enzyme and buffer supernatants were assayed for intact human fibronectin, or for possible proteolytic fragments containing the cell-binding domain. For this purpose, a sensitive species-specific monoclonal enzyme immunoassay for fibronectin was used. Data were obtained in four parallel samples of digestion supernatants containing crude heparinase at 100 jig/ml or 0 ,tg/ml. The resulting concentrations of fibronectin, as solubilized from the matrix, were 291 (i 12) ng/ml, and 281 (± 72) ng/ml, respectively. As calculated from this, the respective mean amounts of matrix fibronectin solubilized from each plastic dish were 873 ng and 843 ng, out of 160 itg total protein (Hedman et al., 1979) in the matrix. These figures indicated that the crude heparinase did not solubilize fibronectin from the matrix above background levels. To further exclude any effect of heparinase on the supramolecular organization of fibronectin in the pericellular matrix, the treated matrix samples were fixed with paraformaldehyde and stained for fibronectin using immuno-
fluorescence microscopy. As seen in Figure 3, the heparinasedigested matrix contained a dense network of fibronectin, indistinguishable from the control.
Glycosaminoglyeans and pericellular matrix
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Fig. 3. Immunofluorescence staining for fibronectin of heparinase (a) or buffer-treated (b) pericellular matrices (x 700).
Discussion Sulfated proteoglycans are components of the fibronectinprocollagen-containing pericellular matrix fibers in fibroblast cultures and also appear to exist as plasma membrane components of various types of cultured cells (Kjellen et al., 1981; Hedman et al., 1983). The functions and interrelations of these forms of ubiquitous molecules are not fully understood. There is evidence linking heparan sulfate, in particular, with cell adhesion or spreading (see Introduction); fibronectin is the major adhesion molecule of mesenchymal cells, and the pericellular matrix fibers appear to form in close association with the event of cell adhesion (Hynes and Destree, 1978; Rennard et al., 1981; Avnur and Geiger, 1981; Grinnell and Feld, 1982). Furthermore, there is in vitro evidence that the multiple interactions between the pericellular matrix components (Hedman et al., 1982b; Laterra and Culp, 1982; Yamada, 1983) might be stabilized by GAGs (see Introduction). Indeed, the extractability of fibronectin from tissues was reported to be enhanced by exogenous heparin (Bray et al., 1981). Heparan sulfate has been found to be enriched in early cell-substrate adhesion sites (Laterra et al., 1983). In the present work we have examined the proposed role of sulfated GAGs in the integrity of the fibronectin-procollagencontaining pericellular matrix. Isolated pericellular matrices of human fibroblast cultures were digested with a preparation of heparinase, which degraded all types of sulfated GAGs produced by skin fibroblasts as expected from previous data (Linker and Hovingh, 1975). The crude heparinase appeared to be relatively free from proteinase contamination, as fibronectin, which is very sensitive to most proteinases (Vartio et al., 1981), was not degraded. The proteinase-free nature of the heparinase has been previously observed (Gill et al., 1981). Using 100 ttg/ml of the enzyme we were able to degrade > 95%0 of all sulfated GAGs from the isolated cell-free matrix without effect on biochemically or immunochemically detectable fibronectin, or on the other major pericellular matrix glycoproteins. The structure of the fibronectin matrix also remained unaltered, as revealed by immunofluorescence
microscopy. Our conclusion is that the sulfated GAGs in the matrix are not essential for the structural stability of the fibronectin filaments. However, the sulfated GAGs at the cell surface may have inductive or modulatory functions in the very early stages of fibronectin fibrillogenesis, as pointed out in the Introduction. That the early fibrillar connective tissue matrix is critically dependent on fibronectin is suggested by the present findings, in combination with earlier observations: (i) the integrity of the pericellular matrix is independent of collagen (Vaheri et al., 1978b; Hedman et al., 1979); (ii) when examined by immunofluorescence, deposition of fibronectin precedes that of procollagen I or III in a matrix formed in vitro underneath subcultured fibroblasts (Vaheri et al., 1978b) or in experimental models of connective tissue formation in vivo (Kurkinen et al., 1980; Weiss and Reddi, 1981); (iii) isolated fibronectincontaining pericellular matrices from chick fibroblast cultures contain little procollagen (Chen et al., 1978); (iv) isolated soluble fibronectin can spontaneously polymerize into filamerntous structures that ultrastructurally resemble those observed in cell cultures (Vuento et al., 1980); (v) treatment of fibroblast cultures with a defined anti-fibronectin Fab' fragment prevents the fibrillogenesis of fibronectin-procollagen matrix (McDonald et al., 1982). Collagen, however, may have a role in cell adhesion and proliferation (Liotta et al., 1978). Later, upon maturation of the filamentous matrix, fibronectin is replaced by collagen as the principal structural element (Hedman et al., 1982a; see Furcht, 1983). Materials and methods Cell cultures and isolated matrices Human embryonic skin fibroblasts were grown on 5 cm plastic dishes with or without glass coverslips, as described (Hedman et al., 1982b). For the biochemical analyses of pericellular matrix GAGs or protein, the cultures were metabolically labeled during days 3-6 from subculture with Na235SO4 (carrier-free, 100 yCi/ml, Amersham,UK), or with 2-[3H]glycine (20 ACi/ml, 20 Ci/mM) or 5-[3H]proline (22 Ci/mM). The pericellular matrices were isolated in cell-free form, 6 days after subculture, by solubilizing the cells with 0.5% sodium deoxycholate in the presence of proteinase inhibitor, in low
583
K. Hedman et al.
ionic strength conditions (Hedman et al., 1982b). The matrices attached to the growth substratum were treated with crude heparinase from Flavobacterium heparinum (Linker and Hovingh, 1975) in situ. The enzyme, which is known to degrade all types of GAG produced by cultured skin fibroblasts, was used at 1 - 100 pg/ml in 2 ml of 0.1 M Na-acetate buffer, pH 6.5, with 5 mM Caacetate and 1 mM phenylmethylsulfonyl fluoride, for 20 h at 30°C. After digestion the dishes were rinsed with 1 ml of 0.14 M sodium chloride in 0.01 M sodium phosphate buffer, pH 7.4, which was combined with the (enzyme) buffer, to form the digestion supernatant. The digestion supernatants were scintillation counted for solubilized 3H or
35S.
Analyses of polypeptides and GA Gs After treatment of the matrix samples with heparinase or with enzyme-free buffer, the radiolabeled insoluble matrices were dissolved in Laemmli sample buffer containing 20%7o 2-mercaptoethanol SDS polyacrylamide gel electrophoresis (Laemmli, 1970) using vertical slab gels. The acrylamide concentration was 3.3Wno in the spacer gel and 6%m in the separating gel. After electrophoresis, the gels were fluorographed (Bonner and Laskey, 1974). To analyze the quantity and species of GAG, the insoluble matrices or dialyzed digestion supernatants were digested with Pronase (Calbiochem, La Jolla, CA) by three additions of 500 pg in I ml of 0.15 M Tris-HCI buffer, pH 8.0, containing 0.lWo SDS and 1 mM CaC12 for 16 h at 60°C. The samples were mixed with 5 mg of carrier heparin (Medica, Helsinki, Finland) and chondroitin sulfate (Jacobsson et al., 1979), dialyzed against H20 and freeze dried and assayed
for the relative content of N-sulfated GAG (heparan sulfate) by its sensitivity degradation by nitrous acid deamination (Shively and Conrad, 1976), followed by gel filtration on Sephadex G-75 (Pharmacia, Uppsala, Sweden). Enzyme immunoassay and immunofluorescence staining The amount of immunoreactive fibronectin solubilized by the heparinase or buffer treatments was measured by a solid-phase enzyme immunoassay (Salonen et al., 1984) based on the use of species- (human) specific monoclonal antibody directed against the cell-binding domain of fibronectin and polyclonal rabbit anti-human fibronectin as detector antibody. Fibronectin standards were also in the digestion buffer. The distribution of insoluble matrix fibronectin was visualized by indirect immunofluorescence microscopy of paraformaldehyde-fixed digestion or control matrix samples to
on
glass coverslips (Hedman et al., 1982b).
Acknowledgements We thank Ms. Anja Virtanen for expert technical assistance. The study was financially supported by the U.S. Veterans Administration and grants from the Finnish Medical Council, Finnish Cancer Foundation, Orion Corporation Research Foundation. Thanks to Scandinavia Foundation, and AM 13412 from the U.S. Public Health Service.
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