Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

The Involvement of the Fibronectin Type II-like Modules of Human Gelatinase A in Cell Surface Localization and Activation

1998; Elsevier BV; Volume: 273; Issue: 32 Linguagem: Inglês

10.1074/jbc.273.32.20622

ISSN

1083-351X

Autores

Bjorn Steffensen, Heather F. Bigg, Christopher M. Overall,

Tópico(s)

Cell Adhesion Molecules Research

Resumo

Recombinant collagen-binding domain (rCBD) comprising the three fibronectin type II-like modules of human gelatinase A was found to compete the zymogen form of this matrix metalloproteinase from the cell surface of normal human fibroblasts in culture. Upon concanavalin A treatment of cells, the induced cellular activation of gelatinase A was markedly elevated in the presence of the rCBD. Therefore, the mechanistic aspects of gelatinase A binding to cells by this domain were further studied using cell attachment assays. Fibroblasts attached to rCBD-coated microplate wells in a manner that was inhibited by soluble rCBD, blocking antibodies to the β1-integrin subunit but not the α2-integrin subunit, and bacterial collagenase treatment. Addition of soluble collagen rescued the attachment of collagenase-treated cells to the rCBD. As a probe on ligand blots of octyl-β-d-thioglucopyranoside-solubilized cell membrane extracts, the rCBD bound 140- and 160-kDa protein bands. Their identities were likely procollagen chains being both bacterial collagenase-sensitive and also converted upon pepsin digestion to 112- and 126-kDa bands that co-migrated with collagen α1(I) and α2(I) chains. A rCBD mutant protein (Lys263 → Ala) with reduced collagen affinity showed less cell attachment, whereas a heparin-binding deficient mutant (Lys357 → Ala), heparinase treatment, or heparin addition did not alter attachment. Thus, a cell-binding mechanism for gelatinase A is revealed that does not involve the hemopexin COOH domain. Instead, an attachment complex comprising gelatinase A-native type I collagen-β1-integrin forms as a result of interactions involving the collagen-binding domain of the enzyme. Moreover, this distinct pool of cell collagen-bound proenzyme appears recalcitrant to cellular activation. Recombinant collagen-binding domain (rCBD) comprising the three fibronectin type II-like modules of human gelatinase A was found to compete the zymogen form of this matrix metalloproteinase from the cell surface of normal human fibroblasts in culture. Upon concanavalin A treatment of cells, the induced cellular activation of gelatinase A was markedly elevated in the presence of the rCBD. Therefore, the mechanistic aspects of gelatinase A binding to cells by this domain were further studied using cell attachment assays. Fibroblasts attached to rCBD-coated microplate wells in a manner that was inhibited by soluble rCBD, blocking antibodies to the β1-integrin subunit but not the α2-integrin subunit, and bacterial collagenase treatment. Addition of soluble collagen rescued the attachment of collagenase-treated cells to the rCBD. As a probe on ligand blots of octyl-β-d-thioglucopyranoside-solubilized cell membrane extracts, the rCBD bound 140- and 160-kDa protein bands. Their identities were likely procollagen chains being both bacterial collagenase-sensitive and also converted upon pepsin digestion to 112- and 126-kDa bands that co-migrated with collagen α1(I) and α2(I) chains. A rCBD mutant protein (Lys263 → Ala) with reduced collagen affinity showed less cell attachment, whereas a heparin-binding deficient mutant (Lys357 → Ala), heparinase treatment, or heparin addition did not alter attachment. Thus, a cell-binding mechanism for gelatinase A is revealed that does not involve the hemopexin COOH domain. Instead, an attachment complex comprising gelatinase A-native type I collagen-β1-integrin forms as a result of interactions involving the collagen-binding domain of the enzyme. Moreover, this distinct pool of cell collagen-bound proenzyme appears recalcitrant to cellular activation. The plasma membrane of various human cancer cells contains high levels of collagenolytic and gelatinolytic proteinases (1Zucker S. Wieman J.M. Lysik R.M. Wilkie D.P. Ramamurthy N. Lane B. Biochim. Biophys. Acta. 1987; 924: 225-237Crossref PubMed Scopus (49) Google Scholar, 2Emonard H.P. Remacle A.G. Noel A.C. Grimaud J.-A. Stetler-Stevenson W.G. Foidart J.-M. Cancer Res. 1992; 52: 5845-5848PubMed Google Scholar) with a positive correlation shown between the expression of the matrix metalloproteinase (MMP) 1The abbreviations used are: MMPmatrix metalloproteinaseC domainMMP COOH-terminal hemopexin-like domainConAconcanavalin Aα-MEMα-minimal essential mediumMT-MMPmembrane type MMPTIMPtissue inhibitor of metalloproteinasesPAGEpolyacrylamide gel electrophoresisPBSphosphate-buffered salineCBDcollagen-binding domainrCBDrecombinant CBDBSAbovine serum albuminDTTdithiothreitol. gelatinase A and invasive potential (3Stetler-Stevenson W.G. Aznavoorian S. Liotta L.A. Annu. Rev. Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1522) Google Scholar). Moreover, certain tumor cell lines, which do not express gelatinase A, can bind the enzyme to their cell membranes by a membrane-associated receptor in trans (2Emonard H.P. Remacle A.G. Noel A.C. Grimaud J.-A. Stetler-Stevenson W.G. Foidart J.-M. Cancer Res. 1992; 52: 5845-5848PubMed Google Scholar, 4Tryggvason K. Hoyhtya M. Pyke C. Breast Cancer Res. Treat. 1993; 24: 209-218Crossref PubMed Scopus (236) Google Scholar). Activation of progelatinase A by cell membranes of concanavalin A (ConA)-stimulated (5Overall C.M. Sodek J. J. Biol. Chem. 1990; 265: 21141-21151Abstract Full Text PDF PubMed Google Scholar, 6Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J.P. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Crossref PubMed Scopus (193) Google Scholar) or 12-O-tetradecanoyl-phorbol-13-acetate-stimulated (7Brown P.D. Levy A.T. Margulies I.M.K. Liotta L.A. Stetler-Stevenson W.G. Cancer Res. 1990; 50: 6184-6191PubMed Google Scholar, 8Fridman R. Fuerst T.R. Bird R.E. Hoyhtya M. Oelkuct M. Kraus S. Komarek D. Liotta L.A. Berman M.L. Stetler-Stevenson W.G. J. Biol. Chem. 1992; 267: 15398-15405Abstract Full Text PDF PubMed Google Scholar) normal cells requires a specific mode of enzyme-cell interaction that utilizes the COOH-terminal domains of gelatinase A and the tissue inhibitor of MMPs, TIMP-2 (8Fridman R. Fuerst T.R. Bird R.E. Hoyhtya M. Oelkuct M. Kraus S. Komarek D. Liotta L.A. Berman M.L. Stetler-Stevenson W.G. J. Biol. Chem. 1992; 267: 15398-15405Abstract Full Text PDF PubMed Google Scholar, 9Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar, 10Murphy G. Willenbrock F. Ward R.V. Cockett M.I. Eaton D. Docherty A.J.P. Biochem. J. 1992; 283: 637-641Crossref PubMed Scopus (246) Google Scholar). Four membrane type (MT)-MMPs possessing a hydrophobic transmembrane domain have been shown to activate progelatinase A at the cell surface (11Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2371) Google Scholar, 12Kolkenbrock H. Hecker-Kia A. Orgel D. Ulbrich N. Will H. Biol. Chem. Hoppe-Seyler. 1997; 378: 71-76Crossref PubMed Scopus (56) Google Scholar) in an activation complex comprising progelatinase A, TIMP-2, and MT-MMP (12Kolkenbrock H. Hecker-Kia A. Orgel D. Ulbrich N. Will H. Biol. Chem. Hoppe-Seyler. 1997; 378: 71-76Crossref PubMed Scopus (56) Google Scholar, 13Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar). Here, the active site of MT-MMP functions as a receptor for the inhibitory NH2 domain of TIMP-2, leaving the TIMP-2 COOH domain free to interact with progelatinase A. Recent site-directed mutagenesis studies have mapped the TIMP-2-binding site on gelatinase A to the junction of the outer rim of β-blades III and IV of the hemopexin-like COOH-terminal domain (C domain) 2C. M. Overall, A. King, D. Sam, A. Ong, T. T. Y. Lau, U. M. Wallon, Y. A. DeClerck, and J. J. Atherstone, submitted for publication. . However, alternative interactions of the gelatinase A C domain with TIMP-4 (14Bigg H.F. Shi Y.E. Liu Y.E. Steffensen B. Overall C.M. J. Biol. Chem. 1997; 272: 15496-15500Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) and cell surface components such as the αvβ3integrin receptor (15Brooks P.C. Stromblad S. Sanders L.C. von Schalscha T.L. Aimes R.T. Stetler-Stevenson W.G. Cherish D. Cell. 1996; 85: 683-693Abstract Full Text Full Text PDF PubMed Scopus (1431) Google Scholar), fibronectin (16Wallon U.M. Overall C.M. J. Biol. Chem. 1997; 272: 7473-7481Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), and heparin (16Wallon U.M. Overall C.M. J. Biol. Chem. 1997; 272: 7473-7481Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 17Overall C.M. Wallon U.M. Steffensen B. De Clerck Y. Tschesche H. Abbey R.S. Edwards D. Hawkes S. Khokha R. Inhibitors of Metalloproteinases in Development and Disease. Gordon and Breach, Amsterdam, Holland1998Google Scholar, 18Crabbe T. Joannou C. Docherty A.J.P. Eur. J. Biochem. 1993; 218: 431-438Crossref PubMed Scopus (89) Google Scholar) have also been identified. matrix metalloproteinase MMP COOH-terminal hemopexin-like domain concanavalin A α-minimal essential medium membrane type MMP tissue inhibitor of metalloproteinases polyacrylamide gel electrophoresis phosphate-buffered saline collagen-binding domain recombinant CBD bovine serum albumin dithiothreitol. The C domain of MMPs is involved in several important protein-protein interactions. In gelatinase B the C domain binds TIMP-1, whereas interstitial and neutrophil collagenases utilize the C domain for binding and cleavage of native type I collagen (19Windsor L.J. Birkedal-Hansen H. Birkedal-Hansen B. Engler J.A. Biochemistry. 1991; 30: 641-647Crossref PubMed Scopus (72) Google Scholar). However, the gelatinase A C domain does not bind collagen (16Wallon U.M. Overall C.M. J. Biol. Chem. 1997; 272: 7473-7481Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 20Murphy G. Nguyen Q. Cockett M.I. Atkinson S.J. Allan J.A. Knight C.G. Willenbrock F. Docherty A.J.P. J. Biol. Chem. 1994; 269: 6632-6636Abstract Full Text PDF PubMed Google Scholar). Instead, a different collagen-binding domain (CBD) is found in gelatinases A and B consisting of three fibronectin type II-like modules inserted in the catalytic domain (21Collier I.E. Wilhelm S.M. Eisen A.Z. Marmer B.L. Grant G.A. Seltzer J.L. Kronberger A. He C. Bauer E.A. Goldberg G.I. J. Biol. Chem. 1988; 263: 6579-6587Abstract Full Text PDF PubMed Google Scholar, 22Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar). In addition to binding denatured type I collagen (23Banyai L. Patthy L. FEBS Lett. 1991; 282: 23-25Crossref PubMed Scopus (63) Google Scholar, 24Collier I.E. Krasnov P.A. Strongin A.Y. Birkedal-Hansen H. Goldberg G.I. J. Biol. Chem. 1992; 267: 6776-6781Abstract Full Text PDF PubMed Google Scholar, 25Steffensen B. Wallon U.M. Overall C.M. J. Biol. Chem. 1995; 270: 11555-11566Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), our characterization of recombinant human gelatinase A CBD (rCBD) showed that this domain accounts for all of the binding properties of the enzyme to native and denatured collagen types I, V, and X and elastin and also contains a heparin-binding site (17Overall C.M. Wallon U.M. Steffensen B. De Clerck Y. Tschesche H. Abbey R.S. Edwards D. Hawkes S. Khokha R. Inhibitors of Metalloproteinases in Development and Disease. Gordon and Breach, Amsterdam, Holland1998Google Scholar,25Steffensen B. Wallon U.M. Overall C.M. J. Biol. Chem. 1995; 270: 11555-11566Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). 3B. Steffensen, R. Maurus, E. Rydberg, and C. M. Overall, submitted for publication. The importance of these functions is shown by CBD deletion, which reduces gelatinase A cleavage of denatured type I collagen by 90% (20Murphy G. Nguyen Q. Cockett M.I. Atkinson S.J. Allan J.A. Knight C.G. Willenbrock F. Docherty A.J.P. J. Biol. Chem. 1994; 269: 6632-6636Abstract Full Text PDF PubMed Google Scholar) and abolishes elastin binding and cleavage (26Shipley J.M. Doyle G.A.R. Fliszar C.J. Ye Q.-Z. Johnson L.L. Shapiro S.D. Welgus H.G. Senior R.M. J. Biol. Chem. 1996; 271: 4335-4341Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The gelatinase A CBD may also serve to localize the enzyme to matrix components in tissues (17Overall C.M. Wallon U.M. Steffensen B. De Clerck Y. Tschesche H. Abbey R.S. Edwards D. Hawkes S. Khokha R. Inhibitors of Metalloproteinases in Development and Disease. Gordon and Breach, Amsterdam, Holland1998Google Scholar, 20Murphy G. Nguyen Q. Cockett M.I. Atkinson S.J. Allan J.A. Knight C.G. Willenbrock F. Docherty A.J.P. J. Biol. Chem. 1994; 269: 6632-6636Abstract Full Text PDF PubMed Google Scholar, 25Steffensen B. Wallon U.M. Overall C.M. J. Biol. Chem. 1995; 270: 11555-11566Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). These properties may similarly provide another mode of cell binding to membrane-associated matrix proteins, including collagen and heparan sulfate proteoglycans, and thus may play a role in gelatinase A activation (18Crabbe T. Joannou C. Docherty A.J.P. Eur. J. Biochem. 1993; 218: 431-438Crossref PubMed Scopus (89) Google Scholar) and its physiological function on the cell surface. Here we report experiments that establish that the fibronectin-like CBD localizes gelatinase A to fibroblast cell surfaces by the formation of a gelatinase A-type I collagen-β1-integrin complex. Notably, this distinct pool of cell-bound enzyme shows a lowered cellular activation potential compared with soluble progelatinase A. This finding has important implications for the role of cell membrane-bound stromal gelatinase A on tumor cells. rCBD (Val191–Gln364) and the rC domain (Gly417–Cys631) of human gelatinase A were expressed in Escherichia coli and purified by Zn2+-chelate and gelatin-Sepharose chromatography as appropriate (14Bigg H.F. Shi Y.E. Liu Y.E. Steffensen B. Overall C.M. J. Biol. Chem. 1997; 272: 15496-15500Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 25Steffensen B. Wallon U.M. Overall C.M. J. Biol. Chem. 1995; 270: 11555-11566Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Electrospray mass spectrometry of the recombinant proteins was performed on a SCIEX API 300 (Perkin-Elmer) mass spectrometer. The convention used in this paper to distinguish between the recombinant protein comprised of the gelatinase A triple fibronectin type II-like repeat and the domain present in the natural enzyme will be to refer to the recombinant collagen-binding domain as the rCBD and to the domain in the enzyme as the CBD (no r). Rabbit polyclonal antibody (αCBD) was raised against rCBD injected with sarcosyl-extracted rCBD inclusion bodies and was then affinity purified over rCBD-AffiGel 10 (Bio-Rad) columns. Anti-peptide antibody (αHis6) to the NH2-terminal His6fusion tag on the recombinant proteins was affinity purified as before (16Wallon U.M. Overall C.M. J. Biol. Chem. 1997; 272: 7473-7481Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Human gingival fibroblasts, kindly provided by Drs D. Brunette and H. Larjava (University of British Columbia), were maintained in α-minimal essential medium (α-MEM) (Life Technologies, Inc.) containing 10% newborn calf serum (Life Technologies, Inc.) and antibiotics at 37 °C. To minimize proteolysis of membrane proteins during cell harvesting for cell attachment assays, 0.2 mm EDTA with a low concentration of trypsin (0.05%) in phosphate-buffered saline (PBS) (140 mmNaCl, 2.7 mm KCl, 4.3 mmNa2HPO4·7H2O, 1.5 mmKH2PO4, pH 7.4) was used for 30–60 s only. Fibroblasts in 96-microwell tissue culture plates were treated with soluble rCBD (1.0 × 10−4 to 1.0 × 10−8m) or rC domain (5.6 × 10−6 to 1.0 × 10−8m) for 24–28 h during and/or after ConA treatment (20 μg/ml) (5Overall C.M. Sodek J. J. Biol. Chem. 1990; 265: 21141-21151Abstract Full Text PDF PubMed Google Scholar) of quiescent cells in serum-free conditions. Conditioned medium and cell extracts were analyzed by zymography on 10% polyacrylamide/40 μg/ml gelatin SDS-PAGE gels (27Overall C.M. Limeback H. Biochem. J. 1988; 256: 965-972Crossref PubMed Scopus (96) Google Scholar). To determine whether progelatinase A could bind unstimulated cells by the CBD, quiescent cells were thoroughly rinsed with PBS to remove unbound secreted enzyme. Gelatinase A was then competed from cell surfaces by incubation of the cell layers with 1.2 or 12 × 10−6m rCBD in serum-free α-MEM at 22 °C for 5 min only. This short time was selected to minimize contributions from newly synthesized enzyme to the medium during the incubation. After medium harvesting, the remaining cell-associated enzyme was assessed after lysis of the cell layer with SDS-PAGE sample buffer. Tissue culture surface treated 96-microwell plates were coated with 2-fold serially diluted rCBD (50–0.25 μg/ml) in 100 μl PBS/well for 18 h at 4 °C. After blocking with 10 mg/ml heat-denatured bovine serum albumin (BSA) for 30 min, 4 × 104 fibroblasts were added per well in serum-free α-MEM (to avoid cell attachment from serum proteins) and incubated for 90 min at 37 °C. Cells were then thoroughly rinsed with PBS and fixed with 4% formaldehyde in PBS. The attached cells were stained with 0.1% crystal violet in 200 mm boric acid, pH 6.0 (28Keung W. Silber E. Eppenberger U. Anal. Biochem. 1989; 182: 16-19Crossref PubMed Scopus (595) Google Scholar). After extensive rinses, cellular stain was dissolved in 10% acetic acid, and cell numbers were quantitated by measurement of the optical density at 590 nm in a microplate reader. Positive control wells were coated with fibronectin (Chemicon) or acid soluble type I collagen prepared from rat tail collagen (25Steffensen B. Wallon U.M. Overall C.M. J. Biol. Chem. 1995; 270: 11555-11566Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) or were nonblocked wells. Any cell attachment to BSA-blocked wells served to adjust for nonspecific attachment. Experiments were performed in duplicate or triplicate and repeated several times, but results were only compared for experiments on the same plate. For scanning electron microscopy, cells were seeded and grown in serum-free α-MEM on rCBD-coated glass coverslips (1 cm2) blocked with BSA. After 1 or 2 h, cells were rinsed and fixed with 2.5% glutaraldehyde in PBS. Slides were stained with 1% osmium in PBS, treated with 2% tannic acid, dried by critical point drying, and sputter-coated with gold for analysis on a Stereoscan 260 (Cambridge Instruments) scanning electron microscope. Phase contrast microscopy was used to quantitate cell spreading at different time points after seeding 5 × 103 cells on rCBD- or fibronectin-coated wells. Cells were fixed in 4% formaldehyde for 30 or 60 min at 22 °C, and cell spreading, as judged by the appearance of lamellar cytoplasm, was then quantitated. Harvested cells were treated with 0.075–7.5 units/100 μl highly pure bacterial collagenase (clostridiopeptidase A, Type III, fraction A (EC 3.4.24.3), Sigma) or 0.01 and 0.1 units/ml highly pure heparinase (Flavobacterium heparinum heparinase, Seikagaku Corporation) in α-MEM with 10 mm Ca2+ acetate and 0.1% BSA for 15–30 min at 37 °C. Enzymes were then removed by repeated cell sedimentation (120 × g, 5 min) and washes in serum-free α-MEM prior to seeding in rCBD (25 μg/ml)-coated wells. Attachment of bacterial collagenase-treated cells to native type I collagen bound to rCBD-coated wells was also quantitated. In addition, cells were seeded in the presence of blocking monoclonal antibody mAb13 (0.6–20 μg/ml) to the β1-integrin subunit (kindly provided by Dr. K. Yamada, NIDR, National Institutes of Health) or ascites fluid antibody (P1E6, Life Technologies, Inc.) to the α2-integrin subunit diluted 1:10 to 1:100. Affinity purified αCBD and αHis6 antibodies served as controls in the 90-min incubations. The effect of 1 or 10 μg of heparin (Sigma) in 100 μl of PBS added to rCBD-coated wells for 1 h prior to seeding was also assessed. Confluent fibroblast cultures were rinsed thoroughly with PBS and then treated with 50 mmoctyl-β-d-thioglucopyranoside (Sigma) in PBS for 30 min at 15 °C (29Pytela R. Pierschbacher M.D. Argraves S. Suzuki S. Ruoslahti E. Methods Enzymol. 1987; 144: 475-489Crossref PubMed Scopus (211) Google Scholar). After clarification at 10,000 × gfor 15 min at 22 °C, detergent-solubilized cell membrane protein was precipitated at 0 °C and then collected by centrifugation at 10,000 × g for 10 min at 0 °C. The protein pellet was dissolved in PBS, separated under nonreducing or reducing (65 mm DTT) conditions by 7.5% SDS-PAGE, and transferred to Immobilon-P polyvinylidene difluoride membrane (Millipore). The blots were BSA-blocked and then incubated with 20 μg/ml rCBD in 150 mm NaCl, 10 mm Tris, pH 7.2, with 0.2% BSA for 1 h at 22 °C. After washes, rCBD bound to the blotted proteins was detected using αCBD antibody and enhanced chemiluminescence reagents (Amersham Pharmacia Biotech). The rCBD-binding proteins were characterized by digestion with pepsin (0.1 mg/ml (Sigma) for 3 h at 15 °C, pH 2.0) or highly pure bacterial collagenase (4 units/100 μl for 18 h at 37 °C, pH 7.0). An aliquot of the pepsin-treated sample was adjusted to pH 7.0 and incubated with bacterial collagenase for 18 h at 37 °C. The efficiency and specificity of the enzyme digestions was verified using BSA, type I collagen and rCBD as control substrates. The rCBD mass was measured by electrospray mass spectrometry to be 21,218 Da, confirming NH2-terminal methionine processing of the recombinant protein (predicted mass 21, 212 Da), fidelity of expression, and homogeneity of the protein preparation. The typical yield of purified rCBD from 3.6 liters of culture was 120 mg. When rCBD was incubated with human fibroblasts for 24 h during and after ConA treatment (Fig.1 A) or for 24 h after ConA treatment only (not shown), an increase in gelatinase A activation was apparent in six separate experiments. At high rCBD concentrations, essentially all the soluble gelatinase A was converted to the 59-kDa (−DTT) activated form (5Overall C.M. Sodek J. J. Biol. Chem. 1990; 265: 21141-21151Abstract Full Text PDF PubMed Google Scholar). Although quantitation of enzyme levels from zymograms is only semiquantitative, less than ∼3% of the total soluble gelatinase A remained as the 66-kDa (−DTT) zymogen form in the presence of 100 μm rCBD (lane 100 +) compared with ∼28–34% in those cells not treated with rCBD (lanes 0 +). This trend was also apparent at 50 μm rCBD. In contrast, recombinant gelatinase A C domain reduced cellular activation of the enzyme as before (17Overall C.M. Wallon U.M. Steffensen B. De Clerck Y. Tschesche H. Abbey R.S. Edwards D. Hawkes S. Khokha R. Inhibitors of Metalloproteinases in Development and Disease. Gordon and Breach, Amsterdam, Holland1998Google Scholar) (not shown). Cell lysates containing gelatinase A that was bound to cells via the C domain of the enzyme or was intracellular in the cell secretory pathway were also prepared after rCBD treatment. Unlike the effect of rCBD on gelatinase A levels in the medium (Fig. 1 A), addition of rCBD to cells during and/or after ConA treatment did not alter the ratios of latent (66 kDa) to active (59 kDa) gelatinase A in the lysates (Fig. 1 B). As estimated from enzyme levels per microliter, the total enzyme recovered in the lysates of ConA-activated cells was ∼10-fold less than that in the medium. In other experiments, zymography also demonstrated that cell-bound progelatinase A (the 66-kDa zymogen form) was competitively displaced from unstimulated cells that had not been ConA-treated. This was found even after a short 5-min pulse of the rCBD intended to minimize accumulations of newly secreted progelatinase A during the experiment (Fig. 1 C). Extraction of the cell layer with SDS-PAGE sample buffer revealed that additional gelatinase A remained associated with the cells that was either not fully released by the short exposure to the rCBD or was bound by the C domain or was intracellular. That the increased gelatinase A activation upon ConA addition combined with rCBD treatment was not because of a direct cellular response to binding rCBD was shown in cultures incubated in the absence of ConA where rCBD addition for 24 h did not induce gelatinase A activation (not shown). Moreover, neither gelatinase A expression nor activation was altered in cells that were attached to rCBD-coated plates (see "The Collagen-binding Domain of Gelatinase A Mediates Cell Attachment") without ConA treatment (Fig. 1 D). Thus, these data show that in addition to interactions involving the C domain, progelatinase A can bind to cells via another domain of the enzyme, the CBD. Because only latent and not active gelatinase A was displaced in unstimulated cultures by the rCBD, these competition experiments also show that cell binding via the CBD of progelatinase A is not sufficient for enzyme activation. Indeed, because gelatinase A activation upon ConA treatment increases in the presence of excess rCBD, we conclude that cellular progelatinase A bound by the CBD has a lower cellular activation potential than the soluble enzyme in the medium. Hence, displacement of CBD-bound progelatinase A by the rCBD in ConA-treated cells may facilitate entry of the latent enzyme into the cellular activation pathway. The mechanistic aspects of gelatinase A cell binding via the CBD were further investigated by adaptation of cell attachment assays. Fibroblasts attached to rCBD-coated microwells in a concentration-dependent manner (Fig.2 A), but this was less efficient than cell attachment to fibronectin (Fig. 2 B). Incubation of fibroblasts with soluble rCBD prior to seeding inhibited attachment to rCBD-coated wells in a concentration-dependent manner, confirming binding specificity (Fig. 2 C). Attachment was not observed in wells coated with 10 mg/ml BSA, whereas cell attachment to tissue culture-treated plastic alone or to type I collagen-coated wells was similar to that on fibronectin under saturating conditions. As assessed by phase contrast microscopy significantly fewer fibroblasts displayed cytoplasmic spreading on rCBD coated at 10 μg/ml (23%) compared with fibronectin (50%) after 30 min. Greater differences in cell spreading were apparent between rCBD and fibronectin using 25 μg/ml coated protein with 23 and 90%, respectively, of the cells spreading after 30 min. Although the kinetics of cell attachment and spreading differed at these early time points, spreading of cells on both substrates plateaued at 80–90% of the attached cells by 60 min. Scanning electron microscopy confirmed both cell attachment to rCBD protein and these differences. After 1 and 2 h on fibronectin (Fig.3, A and C, respectively), cells demonstrated typical cytoplasmic spreading (arrows) with a diameter of ∼100 μm. In contrast, cells on rCBD were smaller (diameter of ∼50 μm) and more rounded after 1 h (Fig. 3 B) with limited spreading and extension of only delicate filopodia (arrowheads) after 2 h (Fig.3 D). Thus, this novel use of cell attachment assays confirmed the potential for gelatinase A binding to cells via the CBD of the enzyme.Figure 3Morphological differences between cells cultured on rCBD and fibronectin. 1 × 103 human fibroblasts were seeded onto glass coverslips coated with 25 μg/ml rCBD or fibronectin and blocked with BSA. After 1 and 2 h at 37 °C, the cells were fixed with glutaraldehyde and processed for scanning electron microscopy. Bars, 25 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A role for β1-integrins in CBD-mediated gelatinase A cell binding was demonstrated using mAb13, an anti-β1-integrin blocking monoclonal antibody. At 2.5 μg/ml antibody, more than 50% of the cell attachment to rCBD-coated wells was inhibited (Fig. 4). This inhibition increased to 90% at antibody concentrations >5 μg/ml. In comparison, α2-integrin blocking antibody and affinity purified αCBD and αHis6 control antibodies showed no significant blocking effects at these concentrations. Ligand blotting was performed to identify cell proteins that may interact with the rCBD. On polyvinylidene difluoride blots of octyl-β-d-thioglucopyranoside solubilized cell membrane proteins, rCBD bound two distinct protein bands having apparent masses of 140 and 160 kDa under reducing conditions (Fig.5) in the approximate positions of α- and β-integrin subunits or procollagen chains. However, both bands were degraded by bacterial collagenase. The 140- and 160-kDa bands were also partially pepsin-sensitive, being degraded to pepsin-resistant, but collagenase-sensitive, 112- and 126-kDa proteins. These co-migrated with collagen α1(I) and α2(I) chains that were also bound by the rCBD (Fig. 5). Thus, these data exclude the identity of the 140- and 160-kDa protein bands as integrin chains. Rather, the data provide strong evidence that the rCBD can interact with procollagen chains in cell membrane protein extracts. Nonetheless, other proteins, including those that do not renature on these blots or that require subunit interactions, might also be involved in the CBD interaction. In addition to any direct interaction with other cell membrane proteins, the ligand blots indicated that binding of gelatinase A CBD to native cellular collagen might represent one mode of gelatinase A cell binding. To test this, rCBD-coated wells were incubated with 10 μg of soluble type I collagen in 100 μl of PBS/well to saturate rCBD collagen-binding sites prior to cell seeding. On the rCBD-collagen complexes, cell attachment levels approached

Referência(s)