ISOLATION OF THE PERICELLULAR MATRIX OF H U M A N FIBROBLAST CULTURES
KLAUS H E D M A N , M A R K K U KURKINEN, K A R I ALITALO, A N T I ' I V A H E R I , STAFFAN JOHANSSON, and MAGNUS HOOK From the Department of Virology, University of Helsinki, SF-00290 Helsinki 29, Finland, and the Department of Medical and PhysiologicalChemistry, Swedish University of Agricultural Sciences Biomedical Center, S-751 Uppsala, Sweden
ABSTRACT T h e pericellular matrix o f h u m a n fibroblast cultures was isolated, using sequential extraction with sodium deoxycholate and hypotonic buffer in the presence of protease inhibitor. T h e matrix attached to the growth substratum had a "sackcloth-like" structure as seen by phase contrast, immunofluorescence, and scanning electron microscopy, and it had a vaguely filamentous ultrastructure similar to that seen in intact cell layers. The matrix consisted of hyaluronic acid and heparan sulfate as the m a j o r glycosaminoglycan c o m p o n e n t s and fibronectin and procollagen as m a j o r polypeptides as shown by metabolic labeling, gel electrophoresis, immunofluorescence, and collagenase digestion. This pericellular matrix can be regarded as an in vitro equivalent of the loose connective tissue matrix. KEY WORDS connective tissue extracellular matrix flbroblast procollagen proteoglycan
MATERIALS A N D METHODS fibronectin
The connective tissue matrix in vivo is composed of several types of proteoglycans, collagen, elastin, fibronectin and other glycoproteins (16, 23). When grown in vitro, connective tissue cells produce a pericellular matrix, which, in the case of fibroblasts, is known to contain fibronectin (8), procollagen types I and III (26), and both sulfated and nonsulfated glycosaminoglycans (10). Components of the pericellular matrix in vitro are clearly involved in cell adhesion (6). Loss of the pericellular matrix appears to be closely associated with malignant transformation of fibroblastic cells in culture (26). It is for these reasons that we wanted to study the matrix more directly. This paper describes a method for isolation of the pericellular matrix from human fibroblast cultures and reports on the biochemical and structural properties of the isolated matrix.
Cell Cultures Human adult (ES) and embryonic skin (HES-L) fibroblasts, -of locally established strains, were studied between the tenth and twenty-fifth passages. The cells were grown in Eagle's Basal Medium (BMED) supplemented with 10% fetal calf serum, as described previously (8), with or without glass or plastic coverslips, in plastic Petri dishes.
Isolation o f Pericellular Matrix 3 d after subculture, the growth medium was changed and, when indicated, reagents for metabolic labeling were added. On day six, when the cultures had become dense, the matrix was isolated as follows: The cell layers were briefly rinsed three times with phosphate-buffered saline (PBS) at room temperature. Then, they were treated at ~0~ on a slowly moving four-way shaker for three periods of 10 min, with 0.5% sodium deoxycholate (DOC) and 1 mM phenylmethyl sulfonylfluoride (PMSF, stock solution, 0.4 M in ethanol) in 10 mM Tris-Cl buffered saline, pH 8.0. Finally, the dishes were treated 3 x 5 min at -+0~ with
J. CELLBIOLOGY~) The Rockefeller University Press 9 0021-9525/79/04/0083/09 $1.00 Volume 81 April 1979 83-91
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a low ionic strength buffer: 2 mM Tris-C1, pH 8.0, containing 1 mM PMSF. In all these treatments, 5 ml of the solutions were applied per 20-cm 2 dish. The solutions were pipetted and sucked off gently to avoid detachment of the matrix from the dish/coverslip bottom.
Microscopic Studies For light and immunofluorescence microscopy, the matrices on coverslips were fixed for 30 min with 3% paraformaldehyde in PBS at room temperature and rinsed three times. Indirect immunofluorescence staining for fibronectin, procollagen I and procollagen IIl (26), for actin (12), and for the 10-nm filament protein (11) was performed as described. The antisera against procollagens were kindly provided by Dr. R. Timpl, Munich, and that against the 10-nm filament protein by Dr. I. Virtanen, Helsinki. The isolated matrices were fixed for scanning electron microscopy with 3 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, rinsed and dehydrated with ethanol, critical point dried, coated with carbon and gold according to conventional techniques, and observed and photographed in a JSM-U3 scanning electron microscope. Immunoferritin staining for fibronectin and transmission electron microscopy were carried out as described previously (8). The specificity controls for our immunostaining procedures were those originally documented for untreated cell cultures (8, 11, 12, 26). The isolated matrices were further tested by staining with corresponding nonimmune sera for immunofluorescence, and with antifibronectin blocked with fibronectin (8) for immunoelectron microscopy. As for whole cultures, these treatments resulted in minimal or no staining of the isolated matrices, indicating specificity.
GAGs, the cultures were labeled with Naa~SO4 (10/xCi/ ml, carrier free, 3 d) followed by CPC precipitation. Protein was also determined according to Lowry et al. (15). Fibronectin was quantitated by a double antibody radioimmunoassay (17). For these two latter assays, the matrices or control cultures were solubilized in 250 ~1 of a mixture of 6 M urea, 1% Triton X-100 and 1 mM PMSF in Tris-buffered saline. Phospholipid was quantitated according to Bartlett (2). Polypeptide analysis by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE) was performed, as described (1), on 5% gels. The polypeptide bands were visualized by scintillation autoradiography (13). Fibronectin in the fixed SDS-PAGE slab gels was identified by a modification of published methods (3, 19). The "staining" reagents, antihuman fibronectin rabbit serum and l~ZI-labeled staphylococcal protein A, were applied in a longitudinal groove on the slot which permitted simultaneous visualization of the 3H and 1~I labels with minimal background. Hydroxyproline assays were done, as described (25), using polypeptide bands cut out from Coomassie blue-stained SDSPAGE gels.
Enzymic Digestion The matrix preparations were exposed either to 10 tzg/ ml of bovine pancreatic trypsin (Trypsin-TPCK, Worthington Biochemical Corp., Freehold, N. J.) for 10 rain or to 50/zg/ml of bacterial collagenase (form III, 500 U/ rag, Advance Biofacturers, Lynbrook, N. Y.) for 60 min at 37~ in serum-free medium and then processed either for SDS-PAGE or for immunofluorescence.
Analysis of Composition of the Matrix
Isolation and Identification of Matrix Glycosaminoglycans
The relative amount of proteins present in the isolated matrix was first measured by metabolic labeling with L[~S]methionine (10 p.Ci/ml; 380 Ci/mmol; this and the other radiochemicals were obtained from the Radiochemical Centre, Amersham, U. K.) or pH]glycine and [3H]proline (23 Ci/mmol, and 29 Ci/mmol and both 5 /zCi/ml) for the last 3 d in culture. The matrices were isolated as described above, while control cultures were briefly rinsed with PBS only. Then, the material remaining on the dish bottom was scraped into a mixture of 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% 2mercaptoethanol, and 0.002% bromophenol blue, 250 /xl per 20-cm ~ dish. To determine acid-insoluble radioactivity, aliquots of the samples were dried on Whatman 3 MM filters (Whatman, Inc., Clifton, N. J.), precipitated three times with cold trichloroacetic acid, dried, and counted in a scintillation counter. The relative amounts of total glycosaminoglycans (GAG) were quantitated by metabolic labeling of the cultures with [3H]glucosamine (10/xCi/ml, 10 Ci/mmol, 3 d) followed by precipitation of the samples with cetyl pyridinium chloride (CPC, see reference 27). To analyze sulfated
The pericellular matrix, metabolically labeled with [3H]glucosamine, was digested with papain overnight, essentially as described (18), After addition of 0.5 mg of hyaluronic acid, 1.0 mg of ehondroitin sulfate, and 2.0 mg of heparin, the digest was applied to a column (2 • 80 cm) of Sephadex G-50, equilibrated with 1 M NaCI. The labeled macromolecules eluting in the void volume were desalted by dialysis, freeze-dried, and subjected to ion-exchange chromatography on diethylaminoethyl (DEAE)-cellulose (9). The nature of glycosaminoglycans separated by ionexchange chromatography was further investigated by subjecting the material to various specific degradation procedures. Incubations with chondroitinase ABC (Miles-Seravac, Maidenhead, Buckinghamshire, U, K.) and testicular hyaluronidase (AB Leo, Helsinborg, Sweden) were performed as described (18). Digestion with leech hyaluronidase (Biotrics, Boston, Mass.) was carried out as follows: samples containing
KLAUS H E D M A N , M A R K K U KURKINEN, K A R I ALITALO, A N T I ' I V A H E R I , STAFFAN JOHANSSON, and MAGNUS HOOK From the Department of Virology, University of Helsinki, SF-00290 Helsinki 29, Finland, and the Department of Medical and PhysiologicalChemistry, Swedish University of Agricultural Sciences Biomedical Center, S-751 Uppsala, Sweden
ABSTRACT T h e pericellular matrix o f h u m a n fibroblast cultures was isolated, using sequential extraction with sodium deoxycholate and hypotonic buffer in the presence of protease inhibitor. T h e matrix attached to the growth substratum had a "sackcloth-like" structure as seen by phase contrast, immunofluorescence, and scanning electron microscopy, and it had a vaguely filamentous ultrastructure similar to that seen in intact cell layers. The matrix consisted of hyaluronic acid and heparan sulfate as the m a j o r glycosaminoglycan c o m p o n e n t s and fibronectin and procollagen as m a j o r polypeptides as shown by metabolic labeling, gel electrophoresis, immunofluorescence, and collagenase digestion. This pericellular matrix can be regarded as an in vitro equivalent of the loose connective tissue matrix. KEY WORDS connective tissue extracellular matrix flbroblast procollagen proteoglycan
MATERIALS A N D METHODS fibronectin
The connective tissue matrix in vivo is composed of several types of proteoglycans, collagen, elastin, fibronectin and other glycoproteins (16, 23). When grown in vitro, connective tissue cells produce a pericellular matrix, which, in the case of fibroblasts, is known to contain fibronectin (8), procollagen types I and III (26), and both sulfated and nonsulfated glycosaminoglycans (10). Components of the pericellular matrix in vitro are clearly involved in cell adhesion (6). Loss of the pericellular matrix appears to be closely associated with malignant transformation of fibroblastic cells in culture (26). It is for these reasons that we wanted to study the matrix more directly. This paper describes a method for isolation of the pericellular matrix from human fibroblast cultures and reports on the biochemical and structural properties of the isolated matrix.
Cell Cultures Human adult (ES) and embryonic skin (HES-L) fibroblasts, -of locally established strains, were studied between the tenth and twenty-fifth passages. The cells were grown in Eagle's Basal Medium (BMED) supplemented with 10% fetal calf serum, as described previously (8), with or without glass or plastic coverslips, in plastic Petri dishes.
Isolation o f Pericellular Matrix 3 d after subculture, the growth medium was changed and, when indicated, reagents for metabolic labeling were added. On day six, when the cultures had become dense, the matrix was isolated as follows: The cell layers were briefly rinsed three times with phosphate-buffered saline (PBS) at room temperature. Then, they were treated at ~0~ on a slowly moving four-way shaker for three periods of 10 min, with 0.5% sodium deoxycholate (DOC) and 1 mM phenylmethyl sulfonylfluoride (PMSF, stock solution, 0.4 M in ethanol) in 10 mM Tris-Cl buffered saline, pH 8.0. Finally, the dishes were treated 3 x 5 min at -+0~ with
J. CELLBIOLOGY~) The Rockefeller University Press 9 0021-9525/79/04/0083/09 $1.00 Volume 81 April 1979 83-91
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a low ionic strength buffer: 2 mM Tris-C1, pH 8.0, containing 1 mM PMSF. In all these treatments, 5 ml of the solutions were applied per 20-cm 2 dish. The solutions were pipetted and sucked off gently to avoid detachment of the matrix from the dish/coverslip bottom.
Microscopic Studies For light and immunofluorescence microscopy, the matrices on coverslips were fixed for 30 min with 3% paraformaldehyde in PBS at room temperature and rinsed three times. Indirect immunofluorescence staining for fibronectin, procollagen I and procollagen IIl (26), for actin (12), and for the 10-nm filament protein (11) was performed as described. The antisera against procollagens were kindly provided by Dr. R. Timpl, Munich, and that against the 10-nm filament protein by Dr. I. Virtanen, Helsinki. The isolated matrices were fixed for scanning electron microscopy with 3 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, rinsed and dehydrated with ethanol, critical point dried, coated with carbon and gold according to conventional techniques, and observed and photographed in a JSM-U3 scanning electron microscope. Immunoferritin staining for fibronectin and transmission electron microscopy were carried out as described previously (8). The specificity controls for our immunostaining procedures were those originally documented for untreated cell cultures (8, 11, 12, 26). The isolated matrices were further tested by staining with corresponding nonimmune sera for immunofluorescence, and with antifibronectin blocked with fibronectin (8) for immunoelectron microscopy. As for whole cultures, these treatments resulted in minimal or no staining of the isolated matrices, indicating specificity.
GAGs, the cultures were labeled with Naa~SO4 (10/xCi/ ml, carrier free, 3 d) followed by CPC precipitation. Protein was also determined according to Lowry et al. (15). Fibronectin was quantitated by a double antibody radioimmunoassay (17). For these two latter assays, the matrices or control cultures were solubilized in 250 ~1 of a mixture of 6 M urea, 1% Triton X-100 and 1 mM PMSF in Tris-buffered saline. Phospholipid was quantitated according to Bartlett (2). Polypeptide analysis by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE) was performed, as described (1), on 5% gels. The polypeptide bands were visualized by scintillation autoradiography (13). Fibronectin in the fixed SDS-PAGE slab gels was identified by a modification of published methods (3, 19). The "staining" reagents, antihuman fibronectin rabbit serum and l~ZI-labeled staphylococcal protein A, were applied in a longitudinal groove on the slot which permitted simultaneous visualization of the 3H and 1~I labels with minimal background. Hydroxyproline assays were done, as described (25), using polypeptide bands cut out from Coomassie blue-stained SDSPAGE gels.
Enzymic Digestion The matrix preparations were exposed either to 10 tzg/ ml of bovine pancreatic trypsin (Trypsin-TPCK, Worthington Biochemical Corp., Freehold, N. J.) for 10 rain or to 50/zg/ml of bacterial collagenase (form III, 500 U/ rag, Advance Biofacturers, Lynbrook, N. Y.) for 60 min at 37~ in serum-free medium and then processed either for SDS-PAGE or for immunofluorescence.
Analysis of Composition of the Matrix
Isolation and Identification of Matrix Glycosaminoglycans
The relative amount of proteins present in the isolated matrix was first measured by metabolic labeling with L[~S]methionine (10 p.Ci/ml; 380 Ci/mmol; this and the other radiochemicals were obtained from the Radiochemical Centre, Amersham, U. K.) or pH]glycine and [3H]proline (23 Ci/mmol, and 29 Ci/mmol and both 5 /zCi/ml) for the last 3 d in culture. The matrices were isolated as described above, while control cultures were briefly rinsed with PBS only. Then, the material remaining on the dish bottom was scraped into a mixture of 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% 2mercaptoethanol, and 0.002% bromophenol blue, 250 /xl per 20-cm ~ dish. To determine acid-insoluble radioactivity, aliquots of the samples were dried on Whatman 3 MM filters (Whatman, Inc., Clifton, N. J.), precipitated three times with cold trichloroacetic acid, dried, and counted in a scintillation counter. The relative amounts of total glycosaminoglycans (GAG) were quantitated by metabolic labeling of the cultures with [3H]glucosamine (10/xCi/ml, 10 Ci/mmol, 3 d) followed by precipitation of the samples with cetyl pyridinium chloride (CPC, see reference 27). To analyze sulfated
The pericellular matrix, metabolically labeled with [3H]glucosamine, was digested with papain overnight, essentially as described (18), After addition of 0.5 mg of hyaluronic acid, 1.0 mg of ehondroitin sulfate, and 2.0 mg of heparin, the digest was applied to a column (2 • 80 cm) of Sephadex G-50, equilibrated with 1 M NaCI. The labeled macromolecules eluting in the void volume were desalted by dialysis, freeze-dried, and subjected to ion-exchange chromatography on diethylaminoethyl (DEAE)-cellulose (9). The nature of glycosaminoglycans separated by ionexchange chromatography was further investigated by subjecting the material to various specific degradation procedures. Incubations with chondroitinase ABC (Miles-Seravac, Maidenhead, Buckinghamshire, U, K.) and testicular hyaluronidase (AB Leo, Helsinborg, Sweden) were performed as described (18). Digestion with leech hyaluronidase (Biotrics, Boston, Mass.) was carried out as follows: samples containing
