FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2045857371> ?p ?o ?g. }

• W2045857371 endingPage "23385" @default.
• W2045857371 startingPage "23375" @default.
• W2045857371 abstract "Matrix metalloprotease (MMP)-2 plays a key role in many biological and pathological processes related to cell migration, invasion, and mitogenesis. MMP-2 is synthesized as a zymogen that is activated through either a conformational change or proteolysis of the propeptide. Several activating enzymes for pro-MMP-2 have been proposed, including metalloproteases and serine proteases. The mechanism of pro-MMP-2 activation by metalloproteases is well established, and the most studied activation mechanism involves cleavage of the propeptide by membrane type 1-MMP (MT1-MMP). In contrast, serine protease activation has not been thoroughly studied, although studies suggest that MT1-MMP may be involved in activation by thrombin and plasmin. Here, we demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 in vascular smooth muscle cells and endothelial cells. Factor Xa and thrombin directly cleaved the propeptide on the carboxyl terminal sides of the Arg98 and Arg101 residues, whereas plasmin only cleaved the propeptide downstream of Arg101. Moreover, processed MMP-2 showed enzymatic activity that was enhanced by intermolecular autoproteolytic processing at the Asn109-Tyr peptide bond. In addition to its role in activation, factor Xa rapidly degraded MMP-2, thereby restricting excessive MMP-2 activity. Thrombin also degraded MMP-2, but the degradation was reduced greatly under cell-associated conditions, resulting in an increase in processed MMP-2. Overall, factor Xa and thrombin regulate MMP-2 enzymatic activity through its activation and degradation. Thus, the net enzymatic activity results from a balance between MMP-2 activation and degradation. Matrix metalloprotease (MMP)-2 plays a key role in many biological and pathological processes related to cell migration, invasion, and mitogenesis. MMP-2 is synthesized as a zymogen that is activated through either a conformational change or proteolysis of the propeptide. Several activating enzymes for pro-MMP-2 have been proposed, including metalloproteases and serine proteases. The mechanism of pro-MMP-2 activation by metalloproteases is well established, and the most studied activation mechanism involves cleavage of the propeptide by membrane type 1-MMP (MT1-MMP). In contrast, serine protease activation has not been thoroughly studied, although studies suggest that MT1-MMP may be involved in activation by thrombin and plasmin. Here, we demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 in vascular smooth muscle cells and endothelial cells. Factor Xa and thrombin directly cleaved the propeptide on the carboxyl terminal sides of the Arg98 and Arg101 residues, whereas plasmin only cleaved the propeptide downstream of Arg101. Moreover, processed MMP-2 showed enzymatic activity that was enhanced by intermolecular autoproteolytic processing at the Asn109-Tyr peptide bond. In addition to its role in activation, factor Xa rapidly degraded MMP-2, thereby restricting excessive MMP-2 activity. Thrombin also degraded MMP-2, but the degradation was reduced greatly under cell-associated conditions, resulting in an increase in processed MMP-2. Overall, factor Xa and thrombin regulate MMP-2 enzymatic activity through its activation and degradation. Thus, the net enzymatic activity results from a balance between MMP-2 activation and degradation. Matrix metalloprotease (MMP) 3The abbreviations used are: MMPmatrix metalloproteaseMT1-MMPmembrane type 1 matrix metalloproteaseSMCsmooth muscle cellrTAPrecombinant tick anticoagulant proteinHBMEChuman brain microvascular endothelial cell lineDMEMDulbecco's modified Eagle's mediumHUVEChuman umbilical vein endothelial cellTIMPtissue inhibitor of metalloprotease.3The abbreviations used are: MMPmatrix metalloproteaseMT1-MMPmembrane type 1 matrix metalloproteaseSMCsmooth muscle cellrTAPrecombinant tick anticoagulant proteinHBMEChuman brain microvascular endothelial cell lineDMEMDulbecco's modified Eagle's mediumHUVEChuman umbilical vein endothelial cellTIMPtissue inhibitor of metalloprotease.-2 is a member of the zinc-dependent endopeptidase family, which comprises 24 enzymes (1.Ra H.J. Parks W.C. Matrix Biol. 2007; 26: 587-596Crossref PubMed Scopus (437) Google Scholar). MMP-2 plays a key role in many biological and pathological processes, including organ growth, endometrial cycling, wound healing, bone remodeling, tumor invasion, and metastasis (2.Woessner Jr., J.F. Ann. N.Y. Acad. Sci. 1994; 732: 11-21Crossref PubMed Scopus (435) Google Scholar). This enzyme functions through proteolysis of non-structural extracellular molecules and components of the basement membrane, including type IV collagen, fibronectin, elastin, laminin, aggrecan, and fibrillin (3.Somerville R.P. Oblander S.A. Apte S.S. Genome Biol. 2003; 4: 216Crossref PubMed Scopus (244) Google Scholar). matrix metalloprotease membrane type 1 matrix metalloprotease smooth muscle cell recombinant tick anticoagulant protein human brain microvascular endothelial cell line Dulbecco's modified Eagle's medium human umbilical vein endothelial cell tissue inhibitor of metalloprotease. matrix metalloprotease membrane type 1 matrix metalloprotease smooth muscle cell recombinant tick anticoagulant protein human brain microvascular endothelial cell line Dulbecco's modified Eagle's medium human umbilical vein endothelial cell tissue inhibitor of metalloprotease. Like most MMPs, MMP-2 is synthesized as a zymogen that is activated by conformational change (4.Bescond A. Augier T. Chareyre C. Garçon D. Hornebeck W. Charpiot P. Biochem. Biophys. Res. Commun. 1999; 263: 498-503Crossref PubMed Scopus (97) Google Scholar) or proteolysis within the propeptide, which may involve membrane type MMPs (MT-MMPs) (5.Strongin 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 (1432) Google Scholar, 6.Morrison C.J. Butler G.S. Bigg H.F. Roberts C.R. Soloway P.D. Overall C.M. J. Biol. Chem. 2001; 276: 47402-47410Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 7.Nakada M. Yamada A. Takino T. Miyamori H. Takahashi T. Yamashita J. Sato H. Cancer Res. 2001; 61: 8896-8902PubMed Google Scholar, 8.Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 9.Nie J. Pei D. Cancer Res. 2003; 63: 6758-6762PubMed Google Scholar). The most studied activation mechanism for pro-MMP-2 is cleavage of the propeptide by MT1-MMP, which requires cooperative activity between MT1-MMP and tissue inhibitor of metalloprotease (TIMP)-2 (5.Strongin 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 (1432) Google Scholar, 10.Caterina J.J. Yamada S. Caterina N.C. Longenecker G. Holmbäck K. Shi J. Yermovsky A.E. Engler J.A. Birkedal-Hansen H. J. Biol. Chem. 2000; 275: 26416-26422Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 11.Hernandez-Barrantes S. Toth M. Bernardo M.M. Yurkova M. Gervasi D.C. Raz Y. Sang Q.A. Fridman R. J. Biol. Chem. 2000; 275: 12080-12089Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 12.Wang Z. Juttermann R. Soloway P.D. J. Biol. Chem. 2000; 275: 26411-26415Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Serine proteases, such as thrombin, factor Xa, activated protein C, and plasmin as well as the cysteine protease legumain are all known activators of pro-MMP-2 (13.Rauch B.H. Bretschneider E. Braun M. Schrör K. Circ. Res. 2002; 90: 1122-1127Crossref PubMed Scopus (52) Google Scholar, 14.Baramova E.N. Bajou K. Remacle A. L'Hoir C. Krell H.W. Weidle U.H. Noel A. Foidart J.M. FEBS Lett. 1997; 405: 157-162Crossref PubMed Scopus (238) Google Scholar, 15.Lafleur M.A. Hollenberg M.D. Atkinson S.J. Knäuper V. Murphy G. Edwards D.R. Biochem. J. 2001; 357: 107-115Crossref PubMed Scopus (113) Google Scholar, 16.Jackson M.T. Smith M.M. Smith S.M. Jackson C.J. Xue M. Little C.B. Arthritis Rheum. 2009; 60: 780-791Crossref PubMed Scopus (41) Google Scholar, 17.Chen J.M. Fortunato M. Stevens R.A. Barrett A.J. Biol. Chem. 2001; 382: 777-783Crossref PubMed Google Scholar). In addition to its role in coagulation, thrombin is involved in multiple cellular processes, including mitogenesis of fibroblasts (18.Kahan C. Seuwen K. Meloche S. Pouysségur J. J. Biol. Chem. 1992; 267: 13369-13375Abstract Full Text PDF PubMed Google Scholar), lymphocytes (19.Chen D. Carpenter A. Abrahams J. Chambers R.C. Lechler R.I. McVey J.H. Dorling A. J. Exp. Med. 2008; 205: 1739-1746Crossref PubMed Scopus (71) Google Scholar), mesenchymal cells (20.Ozaki Y. Nishimura M. Sekiya K. Suehiro F. Kanawa M. Nikawa H. Hamada T. Kato Y. Stem Cells Dev. 2007; 16: 119-129Crossref PubMed Scopus (163) Google Scholar), and smooth muscle cells (SMCs) (21.Wang Z. Kong L. Kang J. Morgan 3rd, J.H. Shillcutt S.D. Robinson Jr., J.S. Nakayama D.K. Neurosci. Lett. 2009; 451: 199-203Crossref PubMed Scopus (9) Google Scholar, 22.McNamara C.A. Sarembock I.J. Gimple L.W. Fenton 2nd, J.W. Coughlin S.R. Owens G.K. J. Clin. Invest. 1993; 91: 94-98Crossref PubMed Scopus (462) Google Scholar). Factor Xa acts as a potent mitogen for endothelial cells (23.Bono F. Herault J.P. Avril C. Schaeffer P. Lormeau J.C. Herbert J.M. J. Cell. Physiol. 1997; 172: 36-43Crossref PubMed Scopus (35) Google Scholar), fibroblasts (24.Blanc-Brude O.P. Chambers R.C. Leoni P. Dik W.A. Laurent G.J. Am. J. Physiol. Cell Physiol. 2001; 281: C681-C689Crossref PubMed Google Scholar), and vascular SMCs (25.Koo B.H. Kim D.S. J. Biol. Chem. 2003; 278: 52578-52586Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 26.Herbert J. Bono F. Herault J. Avril C. Dol F. Mares A. Schaeffer P. J. Clin. Invest. 1998; 101: 993-1000Crossref PubMed Scopus (70) Google Scholar). Both proteases can also elicit endothelial cell and SMC migration through pro-MMP-2 activation and subsequent extracellular matrix degradation (13.Rauch B.H. Bretschneider E. Braun M. Schrör K. Circ. Res. 2002; 90: 1122-1127Crossref PubMed Scopus (52) Google Scholar, 27.Henderson N. Markwick L.J. Elshaw S.R. Freyer A.M. Knox A.J. Johnson S.R. Am. J. Physiol. Lung Cell Mol. Physiol. 2007; 292: L1030-L1038Crossref PubMed Scopus (43) Google Scholar, 28.Galis Z.S. Kranzhöfer R. Fenton 2nd, J.W. Libby P. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 483-489Crossref PubMed Scopus (121) Google Scholar). However, despite studies suggesting that MT1-MMP is involved in thrombin-mediated activation of pro-MMP-2, a detailed mechanism for MMP-2 activation has yet to be elucidated (15.Lafleur M.A. Hollenberg M.D. Atkinson S.J. Knäuper V. Murphy G. Edwards D.R. Biochem. J. 2001; 357: 107-115Crossref PubMed Scopus (113) Google Scholar, 27.Henderson N. Markwick L.J. Elshaw S.R. Freyer A.M. Knox A.J. Johnson S.R. Am. J. Physiol. Lung Cell Mol. Physiol. 2007; 292: L1030-L1038Crossref PubMed Scopus (43) Google Scholar). In this study, we investigated the roles of factor Xa and thrombin in MMP-2 regulation. Data are presented to demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 by cleavage of specific sites within the propeptide. Furthermore, factor Xa-processed MMP-2 showed enzymatic activity that was enhanced following intermolecular autoproteolytic cleavage. Thrombin also activated pro-MMP-2 through the same cleavage reaction. Interestingly, factor Xa and thrombin were also found to be involved in MMP-2 degradation. However, this activity was reduced greatly in thrombin-treated MMP-2 by the cell surface, which resulted in an increase in processed MMP-2. Human factor Xa, thrombin, and plasmin were purchased from Hematologic Technologies (Essex Junction, VT). Monoclonal antibodies specific to MMP-2 (sc-13595), TIMP-2 (sc-21735), and rabbit polyclonal MT1-MMP antibody (sc-30074) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal anti-integrin αvβ3 antibody, polyclonal anti-integrin αv, monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase, GM6001, and human TIMP-2 were purchased from Chemicon (Temecula, CA), and anti-Myc antibody (clone 9E10) was from Invitrogen. Type IV collagen, Pefabloc TH, and concanavalin A were purchased from Sigma, and the EnzChek gelatinase/collagenase assay kit was obtained from Molecular Probes, Inc. (Eugene, OR). siGENOME SMARTpool small interfering RNA against integrin αv was purchased from Dharmacon (Lafayette, CO). Recombinant tick anticoagulant protein (rTAP) was kindly provided by Dr. Yangsoo Jang. BLAST programs from the National Center for Biotechnology Information were used to search for expressed sequence tags. Human testis cDNA (Marathon cDNA; Clontech, Palo Alto, CA) was used as a template to amplify the full-length cDNA for MMP-2 (GenBankTM accession number NM_004530). Oligonucleotide primers 5′-GCTACGATGGAGGCGCTAATGGCC-3′ (start codon underlined) and 5′-TCAGCAGCCTAGCCAGTCGGATTTG-3′ (stop codon underlined) were used for PCR with Advantage 2 polymerase (Clontech). The 2-kb PCR product was cloned into TOPO cloning vectors (Invitrogen) and sequenced completely. For the full-length MMP-2 expression plasmid, its open reading frame was digested with EcoRI and then recloned into the pcDNA3.1/Myc-His(−) B vector (Invitrogen) digested with EcoRI. All mutants used for the present study were generated by site-directed mutagenesis (Intron Biotechnology). The human MT1-MMP expression plasmid was kindly provided by Dr. Suneel Apte. Rat aortic SMCs (Bio-Bud), HEK293F, COS-1, MCF-7 (human breast adenocarcinoma) (ATCC number HTB-22), and human brain microvascular endothelial cell line (HBMEC) (29.Callahan M.K. Williams K.A. Kivisäkk P. Pearce D. Stins M.F. Ransohoff R.M. J. Neuroimmunol. 2004; 153: 150-157Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. Human umbilical vein endothelial cells (HUVECs) were maintained on gelatin-coated plastic dishes in M199 culture medium containing 20% fetal bovine serum, 10 units/ml heparin, and 3 ng/ml basic fibroblast growth factor. The endothelial cells used in all experiments were from passages 3–5. Transfection with plasmids and small interfering RNA was performed using Lipofectamine 2000 according to the manufacturer's recommendations (Invitrogen). For secreted MMP-2, transfected COS-1 and HEK293F cells were cultured and then transferred to 293 SFM-II medium (Invitrogen). Cell invasion assays were performed using Transwell inserts with 6.5-mm diameter polycarbonate 8-μm microporous membranes (Costar, Cambridge, MA). The filters were coated with Matrigel (BD Biosciences) at 7 μg/well. Trypsinized cells were pelleted by centrifugation at 1,500 rpm for 5 min and resuspended in DMEM containing 1% fetal bovine serum. A 100-μl cell suspension containing 1 × 104 cells in the presence and absence of 1 μm rTAP or 10 μm GM6001 was placed in the upper chamber, and factor Xa was subsequently added. Then 600 μl of DMEM containing 10% fetal bovine serum was added to the lower chamber. After 16 h, any cells remaining on the upper surface of the membrane were removed with a cotton swab. The lower side of the membrane was fixed in 4% formaldehyde and stained with 10% Giemsa. The cell number was counted using a light microscope. Cells were detached from 6-well plates using phosphate-buffered saline containing 5 mm EDTA and incubated with a monoclonal antibody against integrin αvβ3 at 4 °C for 2 h. The cells were washed and further incubated with fluorescein isothiocyanate-conjugated goat anti-mouse antibody (Chemicon) for 1 h. Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences), and data were analyzed using WinMDI software version 2.8 (Scripps Research Institute, La Jolla, CA). MMP-2 present in the conditioned medium of transfected cells was quantitated using an MMP-2 enzyme-linked immunosorbent assay kit according to the manufacturer's recommendations (Calbiochem). The conditioned medium was analyzed for proteins with gelatinolytic activity by substrate lysis using 8% SDS-polyacrylamide gels containing 2 mg/ml gelatin. The gels were washed with 1% Triton X-100 for 1 h and incubated for 14–20 h at 37 °C in 50 mm Tris-HCl, pH 7.5, containing 20 mm CaCl2. Then the gels were stained with 0.2% Coomassie Brilliant Blue R-250 in 40% methanol and 10% acetic acid. Cells were lysed in buffer containing 50 mm Tris-HCl, pH 7.5, 5 mm EDTA, 150 mm NaCl, 1% Nonidet P-40, and protease inhibitor mixture (Roche Applied Science) for 1 h at 4 °C and then centrifuged. The soluble portion of the lysate was used for Western blotting, which was performed by separation of reduced or nonreduced samples on SDS-PAGE, followed by electroblotting to polyvinylidine difluoride membrane and detection of bound antibody by enhanced chemiluminescence (Amersham Biosciences). MMP-2 was purified from the conditioned medium using gelatin-Sepharose according to the manufacturer's recommendations (Amersham Biosciences). The signal intensity of relevant bands from the zymogram and Western blot was quantitated using ImageJ software (National Institutes of Health, Bethesda, MD). To determine the N-terminal sequence of cleaved MMP-2, treated protein was blotted onto polyvinylidine difluoride membrane prior to Edman degradation (Tufts University Protein Chemistry Facility, Boston, MA). Substrates were incubated with gelatin-Sepharose-purified rat MMP-2 or conditioned medium containing MMP-2 (20 μg/ml per reaction) in 50 mm Tris-HCl, pH 7.5, plus 5 mm CaCl2 and 10 μg/ml leupeptin at 37 °C for 6 h with collagen IV as substrate (5 μg/reaction) or 1 h with fluorescein-conjugated gelatin, according to the manufacturer's recommendations. The digested substrates were analyzed by SDS-PAGE or a fluorescence microplate reader set for excitation at 495 nm and emission detection at 515 nm. Data are represented as the mean and S.D. of n experiments. Statistical analysis was performed using an unpaired Student's t test or analysis of variance. A p value less than 0.05 was considered statistically significant. Factor Xa stimulates mitogenesis and extracellular matrix invasion of human vascular SMCs through activation of pro-MMP-2 (13.Rauch B.H. Bretschneider E. Braun M. Schrör K. Circ. Res. 2002; 90: 1122-1127Crossref PubMed Scopus (52) Google Scholar). However, the exact mechanism of pro-MMP-2 activation in factor Xa-treated vascular SMC remains to be elucidated. Therefore, we investigated the molecular role of factor Xa-mediated activation of pro-MMP-2 in SMCs and endothelial cells. Zymography of conditioned medium isolated from factor Xa-treated rat aortic SMCs demonstrated that MMP-2 propeptide processing was induced by factor Xa over time (Fig. 1A). To investigate whether factor Xa catalytic activity or generation of active thrombin is involved, pro-MMP-2 processing was assessed in factor Xa-treated cells incubated with rTAP, a specific factor Xa inhibitor, or Pefablock TH, a thrombin inhibitor. Zymographic and Western blot analysis of the conditioned medium showed that pro-MMP-2 processing was inhibited significantly only in the presence of rTAP (Fig. 1B). These results indicate that enzymatic activity of factor Xa is essential for pro-MMP-2 processing, but the processing is thrombin-independent. We then examined factor Xa-induced extracellular matrix invasion of rat aortic SMCs. In these invasion assays, cells were incubated with factor Xa in the presence and absence of rTAP or GM6001, an MMP inhibitor, for 16 h. As shown in Fig. 1C, factor Xa increased rat aortic SMC invasion significantly across the matrix gel relative to untreated cells. However, treatment with rTAP or GM6001 suppressed this invasion, suggesting that factor Xa-stimulated cell invasion is dependent on the proteolytic activation of pro-MMP-2. Similar to SMCs, pro-MMP-2 was also found to be processed in factor Xa-treated HUVECs, and factor Xa catalytic activity was essential for this processing independent of active thrombin (data not shown). Because MT1-MMP has been suggested to be a major activator of pro-MMP-2 (30.Hotary K. Allen E. Punturieri A. Yana I. Weiss S.J. J. Cell Biol. 2000; 149: 1309-1323Crossref PubMed Scopus (505) Google Scholar, 31.Gilles C. Polette M. Piette J. Munaut C. Thompson E.W. Birembaut P. Foidart J.M. Int. J. Cancer. 1996; 65: 209-213Crossref PubMed Scopus (157) Google Scholar, 32.Ellerbroek S.M. Fishman D.A. Kearns A.S. Bafetti L.M. Stack M.S. Cancer Res. 1999; 59: 1635-1641PubMed Google Scholar), we hypothesized that this metalloprotease, as well as TIMP-2 and integrin αvβ3 (10.Caterina J.J. Yamada S. Caterina N.C. Longenecker G. Holmbäck K. Shi J. Yermovsky A.E. Engler J.A. Birkedal-Hansen H. J. Biol. Chem. 2000; 275: 26416-26422Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 11.Hernandez-Barrantes S. Toth M. Bernardo M.M. Yurkova M. Gervasi D.C. Raz Y. Sang Q.A. Fridman R. J. Biol. Chem. 2000; 275: 12080-12089Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 12.Wang Z. Juttermann R. Soloway P.D. J. Biol. Chem. 2000; 275: 26411-26415Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 33.Deryugina E.I. Ratnikov B. Monosov E. Postnova T.I. DiScipio R. Smith J.W. Strongin A.Y. Exp. Cell Res. 2001; 263: 209-223Crossref PubMed Scopus (329) Google Scholar), mediated the activation of pro-MMP-2 in factor Xa-treated cells. Therefore, we examined the expression levels of MT1-MMP, TIMP-2, and integrin αvβ3 in these cells. Western blot analysis of cell lysates showed no difference in MT1-MMP expression between untreated and factor Xa-treated rat aortic SMCs and HUVECs (data not shown). Likewise, TIMP-2 expression was not changed by factor Xa treatment (data not shown). Flow cytometric analysis of integrin αvβ3 cell surface expression in HUVECs was also unaffected by factor Xa treatment (data not shown). Expression of integrin αvβ3 could not be examined in rat aortic SMCs, because an antibody to rat integrin αvβ3 was not commercially available. These data suggest that factor Xa processing of pro-MMP-2 is most likely MT1-MMP-independent, since expression of components of MT1-MMP-mediated pro-MMP-2 activation was not altered in factor Xa-treated cells. Examination of pro-MMP-2 processing in conditioned medium isolated from factor Xa-treated cells incubated with GM6001 and TIMP-2, metalloprotease inhibitors that abrogate MTI-MMP catalytic activity, further confirmed these results. Zymographic data revealed that pro-MMP-2 processing in rat aortic SMCs and HUVECs was unaffected by these inhibitors (Fig. 2, A and B). The conditioned medium of concanavalin A-treated HT-1080 cells was assessed as a control to demonstrate that MT1-MMP-mediated processing of pro-MMP-2 could be completely inhibited by these metalloprotease inhibitors (Fig. 2C). Activation of pro-MMP-2 by MT1-MMP involves cleavage of pro-MMP-2 at the Asn66-Leu peptide bond (5.Strongin 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 (1432) Google Scholar). Thus, to further investigate the role of MT1-MMP in factor Xa-mediated activation of pro-MMP-2, we generated pro-MMP-2 mutants incapable of cleavage by MT1-MMP and/or autolysis, namely N66I/L67V, N109I/Y110F, and N66I/L67V/N109I/Y110F. Transient transfection of these mutant plasmids into COS-1 cells did not affect cleavage, since processed MMP-2 was still detected in the conditioned medium of factor Xa-treated cells (Fig. 2D). To demonstrate the resistance of these mutants to cleavage, COS-1 cells were co-transfected with mutant pro-MMP-2 and MT1-MMP. In particular, N66I/L67V and N66I/L67V/N109I/Y110F were not cleaved by MT1-MMP, and N109I/Y110F did not undergo autoproteolytic processing following MT1-MMP-cleavage (Fig. 2E). Interestingly, fully activated MMP-2 was observed weakly in the conditioned medium of cells co-expressing wild type pro-MMP-2 with MT1-MMP (Fig. 2E). Taken together, these data suggest that the processing of pro-MMP-2 by factor Xa occurs in an MT1-MMP-independent manner. Based on the results described above, we postulated that factor Xa-mediated pro-MMP-2 processing may involve direct cleavage of the propeptide by factor Xa. To test this hypothesis, we incubated purified pro-MMP-2 with factor Xa under cell-free conditions. Zymographic analysis showed that factor Xa converted the purified pro-MMP-2 to its processed form (Fig. 3A), demonstrating direct cleavage of the propeptide by factor Xa. Next, the factor Xa cleavage site within pro-MMP-2 was identified by N-terminal sequencing of the cleaved product. The N-terminal sequence of cleaved MMP-2, as determined by Edman degradation, was Cys102-Gly-Asn-Pro-Asp-Val, which lies within the conserved cysteine switch motif (1.Ra H.J. Parks W.C. Matrix Biol. 2007; 26: 587-596Crossref PubMed Scopus (437) Google Scholar). Mutation of Arg101 to Ala reduced factor Xa-mediated processing of pro-MMP-2 dramatically but did not completely abolish this activity (Fig. 3B). Therefore, further site-directed mutagenesis was performed to substitute Arg98 and Arg98/Arg101 with Ala, generating the pro-MMP-2 R98A and pro-MMP-2 R98A/R101A mutants. Analysis of conditioned medium from COS-1 cell cultures expressing these mutants showed that the pro-MMP-2 R98A/R101A mutant was completely resistant to factor Xa-induced cleavage (Fig. 3B), whereas the pro-MMP-2 R98A mutant was normally processed. These results demonstrate that factor Xa cleaves pro-MMP-2 immediately downstream of Arg98 and Arg101 with a preference for Arg101 (Fig. 3, B and C). Since thrombin has been shown to activate pro-MMP-2 in endothelial and smooth muscle cells through MT1-MMP-dependent and -independent pathways (15.Lafleur M.A. Hollenberg M.D. Atkinson S.J. Knäuper V. Murphy G. Edwards D.R. Biochem. J. 2001; 357: 107-115Crossref PubMed Scopus (113) Google Scholar, 27.Henderson N. Markwick L.J. Elshaw S.R. Freyer A.M. Knox A.J. Johnson S.R. Am. J. Physiol. Lung Cell Mol. Physiol. 2007; 292: L1030-L1038Crossref PubMed Scopus (43) Google Scholar), we examined whether processing of pro-MMP-2 by thrombin in rat aortic SMCs and HUVECs involved MT1-MMP activity. GM6001 and TIMP-2 did not inhibit processing of the propeptide by thrombin (data not shown), suggesting that thrombin processing can occur independently of MT1-MMP activity. Furthermore, thrombin processing of pro-MMP-2 mutants incapable of MT1-MMP cleavage and/or autolysis was observed in COS-1 cells (Fig. 4A). Direct cleavage of purified pro-MMP-2 by thrombin occurred under cell-free conditions, albeit at a lower efficiency (data not shown). The MMP-2 propeptide contains one potential thrombin cleavage site (i.e. Xaa-Pro-Arg-Xaa-Xaa-Xaa, where Xaa is a non-acidic residue) (34.Jenny R.J. Mann K.G. Lundblad R.L. Protein Expr. Purif. 2003; 31: 1-11Crossref PubMed Scopus (199) Google Scholar), which corresponds to the factor Xa cleavage site (Arg98-Lys-Pro-Arg101-Cys). To investigate whether thrombin could cleave pro-MMP-2 within this motif, COS-1 cells expressing the pro-MMP-2 R101A mutant were treated with thrombin. Partial inhibition of thrombin-mediated pro-MMP-2 processing was observed (Fig. 4B). However, mutation of both Arg98 and Arg101 to Ala resulted in a complete loss of processing (Fig. 4B), suggesting that thrombin and factor Xa share MMP-2 cleavage sites. To further verify that pro-MMP-2 is cleaved immediately downstream of Arg98-Lys-Pro-Arg101 by factor Xa and thrombin, a pro-MMP-2 mutant in which a FLAG epitope was inserted on the C-terminal side of Arg98-Lys-Pro-Arg101 was generated (Fig. 4C). In these experiments, anti-FLAG M1 antibody was used to detect processed MMP-2 by Western blotting, since this clone only recognizes proteins with a free N-terminal FLAG tag. The results show that processed MMP-2 was recognized specifically by the M1 antibody (Fig. 4C, bottom), whereas pro-MMP-2 and processed MMP-2 were detected by the anti-MMP-2 antibody (Fig. 4C, top). Since plasmin has also been shown to activate pro-MMP-2 (14.Baramova E.N. Bajou K. Remacle A. L'Hoir C. Krell H.W. Weidle U.H. Noel A. Foidart J.M. FEBS Lett. 1997; 405: 157-162Crossref PubMed Scopus (238) Google Scholar, 35.Mazzieri R. Masiero L. Zanetta L. Monea S. Onisto M. Garbisa S. Mignatti P. EMBO J. 1997; 16: 2319-2332Crossref PubMed Scopus (370) Google Scholar), we examined whether this enzyme could cleave the same bonds within the propeptide. As shown in Fig. 4D, plasmin processing of the propeptide did not occur in COS-1 cells expressing the pro-MMP-2 R101A mutant, indicating cleavage on the C-terminal side of Arg101. The proteolytic activity of MMP-2 purified from conditioned medium of rat aortic SMCs treated with or without factor Xa using collagen IV as a substrate was assessed. SDS-PAGE analysis showed that factor Xa-processed MMP-2 had greater proteolytic activity than MMP-2 from untreated rat aortic SMC, which exhibited low levels of enzymatic activity (Fig. 5A). Because factor Xa cleaves pro-MMP-2 on the N-terminal side of the Cys102 residue, this amino acid was retained after processing and may be capable of interacting with the catalytic domain zinc ion, keeping MMP-2 in a latent state. To test this, we investigated whether factor Xa-cleaved MMP-2 possessed any proteolytic activity or whether further autoproteolytic processing at the Asn109-Tyr peptide bond (36.Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar) was required. We compared proteolysis of collagen IV and fluorescein-conjugated gelatin in the conditioned medium of HEK293F cells expressing pro-MMP-2, the pro-MMP-2 E404A mutant, or the pro-MMP-2 N109I/Y110F mutant treated with or without" @default.
• W2045857371 created "2016-06-24" @default.
• W2045857371 creator A5004968504 @default.
• W2045857371 creator A5041000287 @default.
• W2045857371 creator A5052623517 @default.
• W2045857371 creator A5064558238 @default.
• W2045857371 date "2009-08-01" @default.
• W2045857371 modified "2023-09-27" @default.
• W2045857371 title "Regulatory Mechanism of Matrix Metalloprotease-2 Enzymatic Activity by Factor Xa and Thrombin" @default.
• W2045857371 cites W1501988955 @default.
• W2045857371 cites W1921915279 @default.
• W2045857371 cites W1964550340 @default.
• W2045857371 cites W1970490335 @default.
• W2045857371 cites W1975052707 @default.
• W2045857371 cites W1975578801 @default.
• W2045857371 cites W1977238595 @default.
• W2045857371 cites W1979248947 @default.
• W2045857371 cites W1990771793 @default.
• W2045857371 cites W1991408607 @default.
• W2045857371 cites W1994591574 @default.
• W2045857371 cites W1998705080 @default.
• W2045857371 cites W1999639028 @default.
• W2045857371 cites W2003384516 @default.
• W2045857371 cites W2003699309 @default.
• W2045857371 cites W2010027735 @default.
• W2045857371 cites W2010264164 @default.
• W2045857371 cites W2015117848 @default.
• W2045857371 cites W2022512688 @default.
• W2045857371 cites W2029527760 @default.
• W2045857371 cites W2035143124 @default.
• W2045857371 cites W2040166170 @default.
• W2045857371 cites W2045096405 @default.
• W2045857371 cites W2049220062 @default.
• W2045857371 cites W2049831929 @default.
• W2045857371 cites W2057143876 @default.
• W2045857371 cites W2064143875 @default.
• W2045857371 cites W2065599741 @default.
• W2045857371 cites W2072103796 @default.
• W2045857371 cites W2076718353 @default.
• W2045857371 cites W2079152631 @default.
• W2045857371 cites W2091009027 @default.
• W2045857371 cites W2093226982 @default.
• W2045857371 cites W2093979559 @default.
• W2045857371 cites W2101564903 @default.
• W2045857371 cites W2101844493 @default.
• W2045857371 cites W2110404024 @default.
• W2045857371 cites W2117690071 @default.
• W2045857371 cites W2118523009 @default.
• W2045857371 cites W2121164372 @default.
• W2045857371 cites W2129115464 @default.
• W2045857371 cites W2153055943 @default.
• W2045857371 cites W2161961353 @default.
• W2045857371 cites W2166170833 @default.
• W2045857371 cites W2166494171 @default.
• W2045857371 cites W2166588664 @default.
• W2045857371 cites W2266449379 @default.
• W2045857371 cites W4238705525 @default.
• W2045857371 cites W4245415564 @default.
• W2045857371 doi "https://doi.org/10.1074/jbc.m109.036848" @default.
• W2045857371 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2749111" @default.
• W2045857371 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19581306" @default.
• W2045857371 hasPublicationYear "2009" @default.
• W2045857371 type Work @default.
• W2045857371 sameAs 2045857371 @default.
• W2045857371 citedByCount "22" @default.
• W2045857371 countsByYear W20458573712012 @default.
• W2045857371 countsByYear W20458573712013 @default.
• W2045857371 countsByYear W20458573712014 @default.
• W2045857371 countsByYear W20458573712015 @default.
• W2045857371 countsByYear W20458573712016 @default.
• W2045857371 countsByYear W20458573712018 @default.
• W2045857371 countsByYear W20458573712021 @default.
• W2045857371 crossrefType "journal-article" @default.
• W2045857371 hasAuthorship W2045857371A5004968504 @default.
• W2045857371 hasAuthorship W2045857371A5041000287 @default.
• W2045857371 hasAuthorship W2045857371A5052623517 @default.
• W2045857371 hasAuthorship W2045857371A5064558238 @default.
• W2045857371 hasBestOaLocation W20458573712 @default.
• W2045857371 hasConcept C106487976 @default.
• W2045857371 hasConcept C109523444 @default.
• W2045857371 hasConcept C111472728 @default.
• W2045857371 hasConcept C138885662 @default.
• W2045857371 hasConcept C181199279 @default.
• W2045857371 hasConcept C185592680 @default.
• W2045857371 hasConcept C203014093 @default.
• W2045857371 hasConcept C2777292125 @default.
• W2045857371 hasConcept C43617362 @default.
• W2045857371 hasConcept C55493867 @default.
• W2045857371 hasConcept C86803240 @default.
• W2045857371 hasConcept C89560881 @default.
• W2045857371 hasConcept C89611455 @default.
• W2045857371 hasConceptScore W2045857371C106487976 @default.
• W2045857371 hasConceptScore W2045857371C109523444 @default.
• W2045857371 hasConceptScore W2045857371C111472728 @default.
• W2045857371 hasConceptScore W2045857371C138885662 @default.
• W2045857371 hasConceptScore W2045857371C181199279 @default.
• W2045857371 hasConceptScore W2045857371C185592680 @default.
• W2045857371 hasConceptScore W2045857371C203014093 @default.

• next