Rac1 Mediates Type I Collagen-dependent MMP-2 Activation
2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês
10.1074/jbc.m010190200
ISSN1083-351X
Autores Tópico(s)Cell Adhesion Molecules Research
ResumoCell migration and proteolysis are two essential processes during tumor invasion and metastasis. Matrix metalloproteinase (MMP)-2 (type IV collagenase; gelatinase A), is implicated in tumor metastasis as well as in primary tumor growth. The Rho family of small GTPases regulates the dynamics of actin cytoskeleton associated with cell motility. In this report, we provide evidence that Rac1, one member of Rho-related small GTPases, is a mediator of MMP-2 activation in HT1080 fibrosarcoma cells cultured in three-dimensional collagen gel (3D-col) and that MMP-2 activation is required for Rac1-promoted cell invasion through collagen barrier. Stable expression of dominant negative (Rac1V12N17) and constitutively active Rac1 (Rac1V12), respectively, in HT1080 cells demonstrates that Rac1 promoted cell invasiveness across type I collagen and collagen-dependent MMP-2 activation. Active Rac1 is sufficient to induce MMP-2 activation in cells cultured in fibrin gel, an extracellular matrix component that does not support MMP-2 activation. The Rac1-dependent MMP-2 activation occurred in a cell-associated fashion and required MMP activities. Because the cell membrane-mediated MMP-2 activation requires MT1-MMP and low amount of issue inhibitor of matrix metalloproteinase-2 (TIMP-2), their expression was examined. Rac1 modulated MT1-MMP mRNA level and the accumulation of a 43-kDa form of MT1-MMP protein, in correlation with MMP-2 activation profile. However, TIMP-2 expression was independent of Rac1 activity. The coordinate modulation of MMP-2 activity and MT1-MMP expression/processing by Rac1 is consistent with cell collagenolytic activity. The C-terminal hemopexin-like domain of MMP-2, which interferes with the cell membrane activation of MMP-2, reduced Rac1-promoted cell invasiveness as monitored by collagen invasion assay. These results suggest that collagen-dependent MMP-2 activation and MT1-MMP expression/processing contribute to Rac-promoted tumor cell invasion through interstitial collagen barrier. Cell migration and proteolysis are two essential processes during tumor invasion and metastasis. Matrix metalloproteinase (MMP)-2 (type IV collagenase; gelatinase A), is implicated in tumor metastasis as well as in primary tumor growth. The Rho family of small GTPases regulates the dynamics of actin cytoskeleton associated with cell motility. In this report, we provide evidence that Rac1, one member of Rho-related small GTPases, is a mediator of MMP-2 activation in HT1080 fibrosarcoma cells cultured in three-dimensional collagen gel (3D-col) and that MMP-2 activation is required for Rac1-promoted cell invasion through collagen barrier. Stable expression of dominant negative (Rac1V12N17) and constitutively active Rac1 (Rac1V12), respectively, in HT1080 cells demonstrates that Rac1 promoted cell invasiveness across type I collagen and collagen-dependent MMP-2 activation. Active Rac1 is sufficient to induce MMP-2 activation in cells cultured in fibrin gel, an extracellular matrix component that does not support MMP-2 activation. The Rac1-dependent MMP-2 activation occurred in a cell-associated fashion and required MMP activities. Because the cell membrane-mediated MMP-2 activation requires MT1-MMP and low amount of issue inhibitor of matrix metalloproteinase-2 (TIMP-2), their expression was examined. Rac1 modulated MT1-MMP mRNA level and the accumulation of a 43-kDa form of MT1-MMP protein, in correlation with MMP-2 activation profile. However, TIMP-2 expression was independent of Rac1 activity. The coordinate modulation of MMP-2 activity and MT1-MMP expression/processing by Rac1 is consistent with cell collagenolytic activity. The C-terminal hemopexin-like domain of MMP-2, which interferes with the cell membrane activation of MMP-2, reduced Rac1-promoted cell invasiveness as monitored by collagen invasion assay. These results suggest that collagen-dependent MMP-2 activation and MT1-MMP expression/processing contribute to Rac-promoted tumor cell invasion through interstitial collagen barrier. p130Crk-associated substrate protein matrix metalloproteinase-2 membrane type 1-matrix metalloproteinase MP-1, collagenase-1 collagenase-3 three-dimensional collagenase extracellular matrix c-CrkII Dulbecco's modified Eagle's medium plasminogen activator tissue inhibitor of matrix metalloproteinase-2 C-terminal hemopexin-like domain fibronectin type-II-like modules concanavalin A During metastasis, invasive cells must traverse tissue barriers comprised largely of type I collagen. This process depends on the ability of tumor cells to degrade the surrounding collagen matrix and then migrate through the matrix defects (1MacDougall J.R. Matrisian L.M. Cancer Metastasis Rev. 1995; 14: 351-362Crossref PubMed Scopus (402) Google Scholar, 2Stetler-Stevenson W.G. Liotta L.A. Kleiner Jr., D.E. FASEB J. 1993; 7: 1434-1441Crossref PubMed Scopus (580) Google Scholar, 3Morton D.L. Essner E.R. Kirkwood J.M. Parker R.G. Holland J.F. Bast R.C. Morton D.L. Frei E. Kufe D.W. Weichselbaum R.R. Cancer Medicine. Williams & Wilkins, Baltimore, MD1997: 2467-2499Google Scholar). The actin dynamics regulated by Rho family of small GTPases play a critical role in cell migration (4Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3291) Google Scholar). The initiation of cell migration is characterized by actin polymerization at the leading edge and extension of a lamella in the direction of motion. 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Cell Biol. 2000; 148: 615-624Crossref PubMed Scopus (554) Google Scholar). Therefore MT1-MMP and MMP-2 activities at the cell surface provide a powerful combination for the localized ECM remodeling (33D'Ortho M.P. Will H. Atkinson S. Butler G. Messent A. Gavrilovic J. Smith B. Timpl R. Zardi L. Murphy G. Eur. J. Biochem. 1997; 250: 751-757Crossref PubMed Scopus (387) Google Scholar, 37Nakahara H. Howard L. Thompson E.W. Sato H. Seiki M. Yeh Y. Chen W.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7959-7964Crossref PubMed Scopus (367) Google Scholar). To evaluate the hypothesis that MT1-MMP/MMP-2 proteolytic cascade might play a functional role in Rac1-induced tumor cell invasion through type I collagen-rich tissue barrier, we examined an invasive HT1080 fibrosarcoma cell line that showed elevated level of active MMP-2 during cell-fibrillar collagen interaction (38Azzam H.S. Thompson E.W. Cancer Res. 1992; 52: 4540-4544PubMed Google Scholar). In this report, we provide evidence that Rac1 is a mediator of collagen-stimulated MMP-2 activation and MT1-MMP expression/processing, collagenolytic activity, and cell invasion through 3D-col. Furthermore, active MMP-2 contributes to Rac1-induced collagen invasive activity. Our findings suggest that Rac1 mediates MMP-2 activation and MT1-MMP expression/processing during the encounter between invading tumor cells and type I collagen-rich stroma, thereby facilitating collagenolysis and cell invasion. HT1080 fibrosarcoma and HEp3 epidermoid carcinoma cell lines were maintained in DMEM containing 10% heat-inactivated fetal bovine serum (Hyclone) supplemented with 2 mm glutamine, 1 mm sodium pyruvate, 100 units/ml penicillin and streptomycin (Life Technologies, Gaithersburg, MD). Stably transfected cell lines were maintained in medium that included 200 μg/ml G418 in addition to the above mentioned supplements. Plasmids RSV-neo-%-galactosidase, c-Myc-tagged Rac1V12N17, and Rac1V12 were kindly provided by Dr. Lorne Taichman, SUNY at Stony Brook, and Dr. Alan Hall, University College London, London, United Kingdom. Plasmids containing cDNAs for MT1-MMP, MMP-2, TIMP-2, and 36B4 were purchased from ATCC. Subconfluent cell culture was co-transfected with one of testing plasmids (Rac1V12N17, Rac1V12, and vector) and a plasmid construct containing the neomycin-resistant gene by the calcium phosphate precipitation technique as previously described (39Xu J. Zutter M.M. Santoro S.A. Clark R.A. J. Cell Biol. 1996; 134: 1301-1311Crossref PubMed Scopus (43) Google Scholar). After transfection, individual colonies from Rac1V12N17- and Rac1V12-transfected cells were isolated after 2–3 weeks of 500 μg/ml G418 selection. As control, vector-transfected clones were pooled. All stably transfected cell lines were maintained in growth medium containing 200 μg/ml G418. Cells grown on tissue culture plates in growth medium (10% heat-inactivated fetal bovine serum/DMEM) were washed twice with serum-free medium before the addition of serum-free medium supplemented with 0.2% heat-inactivated lactoalbumin and 50 μg/ml concanavalin A (Sigma). Medium was then collected after 16–24 h for zymography. Type I collagen or fibrin gel cultures were prepared according to a procedure previously described with some modification (40Xu J. Clark R.A. J. Cell Biol. 1996; 132: 239-249Crossref PubMed Scopus (186) Google Scholar). Pepsin-solubilized bovine dermal collagen dissolved in 0.012 m HCl was 99.9% pure containing 95–98% type I collagen and 2–5% type III collagen (Vitrogen 100, Collagen Corp). Briefly, cells detached from tissue culture plates in growth medium were washed with warm DMEM twice before being seeded into a serum-free solution that contained 2 mg/ml vitrogen or 3 mg/ml fibrinogen (Calbiochem-Novabiochem). Subsequently, cell-collagen or cell-fibrinogen suspension (5 × 105cells/ml) was plated onto 24-well plates at 250 μl/well or 35-mm plastic dishes at 1.5 ml/dish. Cell-collagen cultures were incubated at 37 °C to form gel. The gelling of cell-fibrin cultures occurred in less than 5 min at room temperature after the addition of thrombin to a final concentration of 0.2 unit/ml. After both collagen and fibrin cell cultures formed gel, serum-free DMEM supplemented with 0.2% heat-inactivated lactoalbumin was added. Gels remained attached to the plastic dish for the duration of incubation. Conditioned medium was collected from cells cultured for 18–24 h in serum-free medium. To analyze the activity of cell-associated MMP-2, cells (7.5 × 105) in 35-mm plates were released from collagen or fibrin gel after digestion with bacterial collagenase D (Roche Molecular Biochemicals, Indianapolis, IN) for 10 min or dispase (Becton Dickinson, Bedford, MA) for 5 min, respectively. Cells were washed gently with ice-cold phosphate-buffered saline 10–12 times and suspended in 150 μl of 1 × SDS sample buffer. Aliquots (total cell lysates) were immediately processed for enzymatic assay using zymography. Substrate zymography was performed as described previously with some modifications (41Herron G.S. Banda M.J. Clark E.J. Gavrilovic J. Werb Z. J. Biol. Chem. 1986; 261: 2814-2818Abstract Full Text PDF PubMed Google Scholar, 42Gogly B. Groult N. Hornebeck W. Godeau G. Pellat B. Anal. Biochem. 1998; 255: 211-216Crossref PubMed Scopus (105) Google Scholar). SDS-polyacrylamide (12% unless indicated in the figure legend) gels were co-polymerized with 1 mg/ml gelatin or 0.5 mg/ml type I collagen (Sigma). Samples (conditioned medium or total cell lysates) were resolved under nonreducing conditions. Gels were washed twice in 2.5% Triton X-100 for 30 min and incubated overnight in a buffer containing 50 mm Tris-HCl, pH 7.5, 5 mm CaCl2, and 0.02% NaN3 (gelatin substrate) or 100 mm Tris-HCl, pH 8.0, 5 mmCaCl2, 0.005% Brij-35, and 0.02% NaN3 (type I collagen substrate). At the end of the incubation, gels were stained with Coomassie Blue and destained. Actin organization was visualized by staining with Texas Red-conjugated phalloidin (Molecular Probes, Eugene, OR). Cells in serum-free vitrogen solution were plated in 8-well chamber slides to form gel. After 24 h, cells in collagen gel were fixed in 4% paraformaldehyde, permeabilized with acetone at −20 °C, and stained with Texas Red-conjugated phalloidin for 30 min at room temperature. The images were captured using an epifluorescence microscope by the University Microscopy Imaging Center, SUNY at Stony Brook. Total RNA was isolated from cells cultured in collagen or fibrin gel for 10 h and Northern analysis was performed as previously described (40Xu J. Clark R.A. J. Cell Biol. 1996; 132: 239-249Crossref PubMed Scopus (186) Google Scholar). RNA was detected with α-32P-labeled cDNA probes for MT1-MMP and TIMP-2. Control probe was 36B4 cDNA. Cell extracts were prepared using a detergent extraction method described by Lee et al. (30Lee A.Y. Akers K.T. Collier M. Li L. Eisen A.Z. Seltzer J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4424-4429Crossref PubMed Scopus (59) Google Scholar) with a few modifications. Briefly, cells cultured in collagen or fibrin gel were washed gently with cold phosphate-buffered saline three times, transferred into a 1.5-ml microcentrifuge tube, and subject to centrifugation to remove as much solution as possible. The pellet was suspended in a Triton X-100 lysis buffer (2% Triton X-100 in 100 mm Tris buffer, pH 7.5, and 150 mm NaCl in the presence of protein inhibitors: 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) and forced through a syringe with a 26-gauge needle several times. The supernatant after centrifugation was designated as cell extracts for Western analysis. Western blotting analysis was performed as previously described (43Xu J. Zutter M.M. Santoro S.A. Clark R.A. J. Cell Biol. 1998; 140: 709-719Crossref PubMed Scopus (99) Google Scholar). After detection by monoclonal antibodies against Rac1 (Upstate Biotechnology, Lake Placid, NY), MMP-2 (Chemicon, Temecula, CA), TIMP-2, and MT1-MMP (Calbiochem-Novabiochem), the blots were visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). The capacity of cells to degrade type I collagen fibrils was assessed based on the modification of a procedure as described (35Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1112) Google Scholar). Briefly, 300 μl of vitrogen solution at 1 mg/ml was added to each well of a 24-well plate and allowed to air-dry overnight. The collagen fibril film was washed with several changes of distilled water and serum-free DMEM before a pellet of 4 × 104 cells in 25 μl of 10% fetal bovine serum/DMEM was dotted onto the center of each well. Cells were allowed to attach for 5 h, washed twice with serum-free DMEM, and then incubated for 3 days in serum-free DMEM supplemented with 0.2% lactoalbumin. Following the incubation, cells were removed with trypsin/EDTA and collagen remaining in the wells was visualized by staining with Coomassie Blue. For quantification of fibrillar type I collagen degradation, type I rat tail collagen (Becton Dickinson) was labeled with [3H]acetic anhydride (Amersham Pharmacia Biotech) according to the protocols described (44Birkedal-Hansen H. Methods Enzymol. 1987; 144: 140-171Crossref PubMed Scopus (130) Google Scholar, 45Mookhtiar K.A. Mallya S.K. Van Wart H.E. Anal. Biochem. 1986; 158: 322-333Crossref PubMed Scopus (27) Google Scholar). 150 μl of3H-labeled type I collagen (0.5–1 × 106cpm/mg protein) was allowed to polymerize in individual wells of a 48-well plate. Cells at 1.5 × 104/300 μl were added to each well and incubated at 37 °C. To follow the progressive degradation of collagen fibrils, aliquots (50 μl) of the medium were collected after 24 and 48 h. The soluble radioactivity was quantified in a liquid scintillation counter. A cDNA clone K-121 that contains the coding sequence for MMP-2 with a partially truncated propeptide domain (46Huhtala P. Eddy R.L. Fan Y.S. Byers M.G. Shows T.B. Tryggvason K. Genomics. 1990; 6: 554-559Crossref PubMed Scopus (62) Google Scholar) was used as a template for amplification with polymerase chain reaction. The regions corresponding to residues Gly417-Cys631 (C-terminal hemopexin-like domain; CTD) or Val191-Gln364(fibronectin type-II-like modules; CBD) of MMP-2 proenzyme, respectively, were amplified. The resulting fragments were inserted into a pGEX-3X plasmid, respectively (Amersham Pharmacia Biotech, Uppsala, Sweden). The glutathione S-transferase fusion proteins were purified on Sepharose 4B-coupled glutathione beads (Amersham Pharmacia Biotech) based on the manufacturer's instructions. The assay was performed using the modification of procedures previously described (47Albini A. Iwamoto Y. Kleinman H.K. Martin G.R. Aaronson S.A. Kozlowski J.M. McEwan R.N. Cancer Res. 1987; 47: 3239-3245PubMed Google Scholar). 50 μl of vitrogen solution at 1 mg/ml was applied to the upper compartment of each well in a 24-well Transwell plate (8-μm pore size; Costar) and allowed to gel at 37 °C. Cells at 5 × 104 in 200 μl of serum-free DMEM were added to the upper chamber. Culture medium was added to the lower compartment. In indicated experiments, inhibitors or controls were added to both upper and lower chambers at the following final concentrations: 25 μm SC68180 (formerly SC44463,N-[3-(N′-hydroxycarboxamido)-2-(2-methylpropyl)propanoyl]-O-methyl-l-tyrosine-N-methylamide; a generous gift of Dr. W. C. Parks, Washington University, St. Louis), 100 μg/ml aprotinin (Sigma), 500 ng/ml recombinant TIMP-1 (Chemicon), 500 ng/ml recombinant TIMP-2 (Chemicon), 100 μm furin inhibitor Dec-Arg-Val-Lys-Arg-CH2Cl (Bachem Biochemicals), 500 ng/ml CTD (Gly417-Cys631), and 500 ng/ml CBD (Val191-Gln364). The invasion proceeded for 24 h at 37 °C. After incubation, the filters were fixed and stained with Diff-Quick staining kit (Fisher Scientific). The cells that reached the underside of the filter were counted. For each filter, the number of cells in 10 randomly chosen microscope fields was determined and averaged. Three invasion chambers were used per condition. The final values were the average of triplicates. A human fibrosarcoma cell line, HT1080, was transfected with dominant negative Rac1V12N17, constitutively active Rac1V12, or a vector control. Ectopic Rac1 expression was examined by Western analysis in G418-resistant clones. In a representative experiment, a monoclonal antibody against Rac1 detected both ectopic Rac1 tagged with c-Myc (upper band) and endogenous Rac1 (lower band) in lysates of cells expressing Rac1V12N17 (HN) and Rac1V12 (HV), but only endogenous Rac1 in vector-transfected cell extracts (HW) (Fig.1 A). Clones that express comparable levels of Rac1 were selected for further study. Cells were embedded in 3D-col in serum-free medium for 24 h and subject to morphological examination. To view single cells in type I collagen matrix, the entire cell-containing 3D-col was stained with rhodamine phalloidin. As shown in Fig. 1 B, HW and HV cells developed long protrusions extending from cell body, whereas HN cells possessed a compact morphology (Fig. 1 B, panels a-c). Interestingly, HV cells displayed multiple membrane protrusions (Fig.1 B, panel c). Cell organization in 3D-col was visualized by phase-contrast microscopy (Fig. 1 B, panels d-f). Consistent with the single cell morphology, HV cells were assembled into complex branching tubular networks (Fig. 1 B, panel f). In contrast, HN cells failed to demonstrate organized structure (Fig. 1 B, panel e). Because the branching phenotype of carcinoma cells has been associated with their metastatic capacity (47Albini A. Iwamoto Y. Kleinman H.K. Martin G.R. Aaronson S.A. Kozlowski J.M. McEwan R.N. Cancer Res. 1987; 47: 3239-3245PubMed Google Scholar), whether Rac1 mediates HT1080 cell invasion was assessed. Rac1V12 substantially increased, whereas Rac1V12N17 reduced, HT1080 cell invasion across 3D-col (Fig.1 C). To assess whether MMPs or plasminogen activator (PA)/plasmin system is responsible for this process, we monitored cell invasion in the presence of inhibitors for MMPs and PA/plasmin system, SC68180 and aprotinin, respectively. SC68180, formally known as SC44463, is a hydroxymate compound that has been shown to inhibit human keratinocyte migration on native type I collagen as a general MMP inhibitor (48Pilcher B.K. Dumin J.A. Sudbeck B.D. Krane S.M. Welgus H.G. Parks W.C. J. Cell Biol. 1997; 137: 1445-1457Crossref PubMed Scopus (498) Google Scholar). As shown in Fig. 1 D, invasion of HW and HV cells through 3D-col was inhibited by SC68180. Interestingly, the active Rac1-induced cell invasion was reduced ∼90% by the MMP inhibitor. In contrast, aprotinin did not impact on the collagen invasiveness. These data indicate that invasion of HT1080 cells across collagen depends on both Rac1 and MMP activities and that Rac1 requires MMP activities to promote cell invasion. To investigate the Rac1-mediated MMP activity in HT1080 cells surrounded by collagen matrix, we monitored MMP production by gelatin zymography in these cell lines when cultured in 3D-col for 24 h (Fig.2 A). The conditioned medium of HEp3, an epidermoid carcinoma cell line that secretes MMP-1, MMP-2, and MMP-9 as detected by gelatin zymography (49Kim J., Yu, W. Kovalski K. Ossowski L. Cell. 1998; 94: 353-362Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar), was used as a control (Fig. 2 A, lane 1). Four major gelatinolytic bands with different intensity were detected in the serum-free medium of HW cell culture (Fig. 2 A, lane 2). These bands corresponded to the inactive MMP-9 proenzyme (92 kDa) and three MMP-2 species, latent (L), intermediate (i), and active (A). Interstitial collagenase MMP-1 was undetectable in HT1080-derived cell lines in gelatin (Fig.2 A) as well as type I collagen zymography (Fig.2 B), a method that more sensitively detects MMP-1 than does MMP-2 (42Gogly B. Groult N. Hornebeck W. Godeau G. Pellat B. Anal. Biochem. 1998; 255: 211-216Crossref PubMed Scopus (105) Google Scholar). The distribution of three MMP-2 species was distinctly altered by the stable expression of Rac1V12N17 (HN) and Rac1V12 (HV). Active and latent MMP-2 became the major species in HV and HN cells, respectively (Fig. 2 A, lanes 2–4), suggesting Rac1 specifically mediates MMP-2 proenzyme processing cells cultured in 3D-col. Active MMP-9 was not detected in this system; instead, latent MMP-9 at 92 kDa was detected at low level in both HW and HV cells, but nearly undetectable in HN cells (Fig. 2 A). Cells cultured in fibrin gel were used as control. Unlike 3D-col that induced MMP-2 processing, fibrin gel did not promote MMP-2 activation (Fig. 2 A, compare lanes 2 and 5). Predominantly latent MMP-2, as well as low level of the i
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