Biomedical subjects

M S Stack

Publications and source records attributed to M S Stack.

At least 37 records · Page 2Linked to original sources

PubMed1998

Processing of laminin-5 and its functional consequences: role of plasmin and tissue-type plasminogen activator.

The laminin-5 component of the extracellular matrices of certain cultured cells such as the rat epithelial cell line 804G and the human breast epithelial cell MCF-10A is capable of nucleating assembly of cell- matrix adhesive devices called hemidesmosomes when other cells are plated upon them. These matrices also impede cell motility. In contrast, cells plated onto the laminin-5-rich matrices of pp126 epithelial cells fail to assemble hemidesmosomes and are motile. To understand these contradictory phenomena, we have compared the forms of heterotrimeric laminin-5 secreted by 804G and MCF-10A cells with those secreted by pp126 cells, using a panel of laminin-5 subunit-specific antibodies. The alpha3 subunit of laminin-5 secreted by pp126 cells migrates at 190 kD, whereas that secreted by 804G and MCF-10A cells migrates at 160 kD. The pp126 cell 190-kD alpha3 chain of laminin-5 can be specifically proteolyzed by plasmin to a 160-kD species at enzyme concentrations that do not apparently effect the laminin-5 beta and gamma chains. After plasmin treatment, pp126 cell laminin-5 not only impedes cell motility but also becomes competent to nucleate assembly of hemidesmosomes. The possibility that plasmin may play an important role in processing laminin-5 subunits is supported by immunofluorescence analyses that demonstrate colocalization of laminin-5 and plasminogen in the extracellular matrix of MCF-10A and pp126 cells. Whereas tissue-type plasminogen activator (tPA), which converts plasminogen to plasmin, codistributes with laminin-5 in MCF-10A matrix, tPA is not present in pp126 extracellular matrix. Treatment of pp126 laminin-5-rich extracellular matrix with exogenous tPA results in proteolysis of the laminin-5 alpha3 chain from 190 to 160 kD. In addition, plasminogen and tPA bind laminin-5 in vitro. In summary, we provide evidence that laminin-5 is a multifunctional protein that can act under certain circumstances as a motility and at other times as an adhesive factor. In cells in culture, this functional conversion appears dependent upon and is regulated by tPA and plasminogen.

Animals↗

PubMed1998

Human mast cell tryptase fibrinogenolysis: kinetics, anticoagulation mechanism, and cell adhesion disruption.

Tryptase is a 31 kDa, glycosylated, trypsin-like enzyme stored in and released from mast cell granules. Human tryptase exists as a tetramer, binds heparin, and has a limited substrate specificity, yet it displays remarkable resistance to inhibition by blood plasma proteinase inhibitors. In this study we have examined the cleavage of human fibrinogen by tryptase. alpha chain cleavage was shown to occur in the carboxyl terminal region at Arg572 and beta chain cleavage was found to occur at Lys21. Kinetic analyses of these reactions yielded Km values of 0.2 microM for alpha chain cleavage and 0.26 microM for beta chain cleavage, as well as kcat/KM values of 7 x 10(5) and 4.6 x 10(5) M-1 s-1 for alpha and beta chain reactions, respectively. Proteolysis at Arg572 destroyed the Arg-Gly-Asp (RGD) sequence motif recognized by cell surface alphavbeta3 integrins, and endothelial cell binding to tryptase-modified fibrinogen was significantly reduced, consistent with loss of the RGD motif. Tryptase competed with thrombin in clotting assays using pure fibrinogen with heparin or blood plasma in the absence of heparin. Thrombin failed to initiate the clotting of fibrinogen following modification by tryptase, and fibrin clotting initiated with Ancrod was stopped and partially reversed by tryptase. These data provide insight concerning the mechanism by which tryptase renders fibrinogen unclottable by thrombin and suggests a novel role for tryptase in the modulation of cellular interactions with fibrin(ogen).

Amino Acid Sequence↗

PubMed1998

Intact vitronectin induces matrix metalloproteinase-2 and tissue inhibitor of metalloproteinases-2 expression and enhanced cellular invasion by melanoma cells.

The initial site of melanoma cell metastasis is frequently the regional lymph nodes, and the appearance of lymph node metastasis correlates with poor prognosis. Lymph node adhesion is mediated by an interaction between the tumor cell integrin alphavbeta3 and lymph node vitronectin. In this study, we explored the relationship between adhesion and proteolysis by examining the direct effect of vitronectin receptor ligation on matrix metalloproteinase-2 (MMP-2) production by B16F1 and B16F10 melanoma cells. We report a dose-dependent increase in secretion of both MMP-2 and tissue inhibitor of metalloproteinases-2 (TIMP-2) in response to vitronectin. Cellular invasiveness was also enhanced by vitronectin, as shown by the increased ability of vitronectin-treated cells to invade a synthetic basement membrane (Matrigel). Both the vitronectin-induced MMP-2 production and vitronectin-enhanced invasion were blocked by the peptide ligand Arg-Gly-Asp-Ser (RGDS). Furthermore, neither plasmin-degraded vitronectin nor the peptide ligand RGDS stimulated MMP-2 secretion or invasiveness, indicating that a multivalent ligand-receptor interaction rather than simple receptor occupancy was required for MMP-2 induction. MMP-2 and MMP-2/TIMP-2 interaction with the plasma membrane of melanoma cells resulted in enhanced catalytic activity against 14C-labeled gelatin, suggesting that membrane association may function in posttranslational regulation of MMP-2 activity. This is supported by data showing increased cellular invasion by cells containing membrane-bound MMP-2. Binding of proMMP-2 and proMMP-2/TIMP-2 to melanoma cells was not inhibited by RGDS, and melanoma cell adhesion to vitronectin was unaffected by pro- or active MMP-2, indicating that MMP-2 did not interact with the murine vitronectin receptor. Together, these data provide evidence for a functional link between adhesion and proteolysis and suggest a potential mechanism whereby adhesion of an invasive cell to the extracellular matrix regulates subsequent invasive behavior.

Animals↗

PubMed1998

Metastatic dissemination of human ovarian epithelial carcinoma is promoted by alpha2beta1-integrin-mediated interaction with type I collagen.

Metastatic dissemination of epithelial ovarian carcinoma is thought to be mediated via tumor cell exfoliation into the peritoneal cavity, followed by adhesion to and invasion through the mesothelium which overlies the contents of the peritoneal cavity. In this study, we have utilized short-term primary cultures to analyze the effect of specific extracellular matrix proteins on properties of human ovarian epithelial carcinoma cells which contribute to the invasive phenotype. Analysis of cell:matrix adhesive profiles indicated that ovarian carcinoma cells adhere preferentially to type I collagen. Immunoprecipitation analyses demonstrated the presence of the collagen-binding alpha2beta1 integrin in biotin-labeled ovarian carcinoma cell membranes, and cellular adhesion was inhibited by blocking antibodies directed against the alpha2 and beta1 integrin subunits. The alpha2beta1-binding peptide Asp-Gly-Glu-Ala (DGEA) was also moderately effective at blocking adhesion to collagen relative to the control peptide Ala-Gly-Glu-Ala (AGEA). Analysis of cell motility on protein-coated colloidal gold coverslips demonstrated that ovarian carcinoma cells migrate preferentially on type I collagen coated surfaces. Type I collagen promoted migration in a concentration-dependent, saturable manner, with maximal migration observed at a collagen-coating concentration of 50 microg/ml. Migration on collagen was inhibited by antibodies directed against the alpha2 and beta1 integrin subunits and by DGEA peptide, providing evidence for the role of the alpha2beta1 integrin in ovarian carcinoma cell motility. Culturing ovarian carcinoma cells on type I collagen gels led to a significant increase in conversion of the matrix metalloproteinase 2 zymogen to the 66-kD form, suggesting that adhesion to collagen also influences matrix-degrading proteinases. These data suggest that alpha2beta1-integrin-mediated interaction of ovarian carcinoma cells with type I collagen, a protein prevalent both in the mesothelial extracellular matrix and in the peritoneal cavity of ovarian carcinoma patients, may function on multiple levels to promote metastatic dissemination of ovarian carcinoma cells.

Cell Adhesion↗

PubMed1998

The role of proteolytic enzymes in the pathology of epithelial ovarian carcinoma.

Epithelial ovarian cancer is the leading cause of death from gynecologic malignancy among North American women. The vast majority of women are diagnosed after the cancer has metastasized into the peritoneum, resulting in a low 5-year survival. Because of difficulties associated with early detection of ovarian carcinoma and the invasive potential of these malignancies, a more detailed understanding of the mechanism(s) by which ovarian carcinomas metastasize may suggest novel therapeutic approaches which could impact favorably on long-term survival. Connective tissue degrading proteinases are necessary for tumor cell invasion and enzymes in the plasminogen activator (PA) and matrix metalloproteinase (MMP) families have been implicated in ovarian cancer metastasis. The goal of this review is to summarize current data regarding the role of these proteinases in ovarian carcinoma invasion.

Carcinoma↗

PubMed1997

Production of extracellular matrix-degrading proteinases by primary cultures of human epithelial ovarian carcinoma cells.

BACKGROUND: The authors analyzed the secretion of extracellular matrix-degrading proteinases, including urinary-type plasminogen activator (u-PA), matrix metalloproteinase-2 (MMP-2, gelatinase A), and MMP-9 (gelatinase B), by short term primary cultures of epithelial ovarian carcinoma cells derived from primary ovarian tumors, intraperitoneal metastases, or ascites. The presence of these enzymatic activities in samples of ascites was also evaluated. The effect of adhesive substratum on proteinase production was determined. METHODS: A coupled spectrophotometric assay was utilized to evaluate the initial rate of plasminogen activation by u-PA in conditioned medium; this involved monitoring the activity of generated plasmin with a colorimetric substrate. MMP activity was evaluated by gelatin zymography. RESULTS: Ascitic fluids from 18 patients contained u-PA, MMP-2, and MMP-9. However, short term primary cultures of cells derived from primary ovarian tumors (OVET), metastatic lesions (OVEM), or ascites (OVEA) produced very low levels of u-PA. Production of u-PA by OVET and OVEM cells was regulated by adhesive substratum. Conditioned media from OVET, OVEM, and OVEA cells contained high levels of both MMP-2 and MMP-9. MMP-9 levels decreased with increasing passage in culture, whereas MMP-2 activity was maintained. Production of neither MMP-2 nor MMP-9 was regulated by adhesive substratum. CONCLUSIONS: These results demonstrate that primary cultures of epithelial ovarian carcinoma cells derived from three distinct anatomic locations produce MMP-2 and MMP-9, with low level secretion of u-PA. These data suggest that MMPs, particularly MMP-2, may play a significant role in the intraperitoneal invasion of ovarian carcinoma cells.

Adult↗

PubMed1997

The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin.

Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin. We have now identified two components released by PC-3 cells, urokinase (uPA) and free sulfhydryl donors (FSDs), that are sufficient for angiostatin generation. Furthermore, in a defined cell-free system, plasminogen activators [uPA, tissue-type plasminogen activator (tPA), or streptokinase], in combination with one of a series of FSDs (N-acetyl-L-cysteine, D-penicillamine, captopril, L-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant plasminogen was converted to angiostatin in the setting of uPA/FSD; however, a plasminogen activation site mutant and a catalytically inactive mutant failed to generate angiostatin. Cell-free derived angiostatin inhibited angiogenesis in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases. These findings define a direct mechanism for cancer-cell-mediated angiostatin generation and permit large-scale production of bioactive angiostatin for investigation and potential therapeutic application.

Angiostatins↗

PubMed1997

Biochemical characterization of primary peritoneal carcinoma cell adhesion, migration, and proteinase activity.

Primary papillary serous carcinoma of the peritoneum (PPC) is clinically and histologically similar to advanced stage epithelial ovarian carcinoma. PPC classically presents with widespread intraperitoneal dissemination, superficial invasion, and minimal ovarian involvement. Surgical cytoreduction and combination chemotherapy utilized for patients with epithelial ovarian carcinoma have produced varying results for patients with PPC. These differences in response may be secondary to the stage of disease or due to biological differences in metastatic behavior between these carcinomas. In this study, short-term primary cultures of PPC and epithelial ovarian carcinoma (OVCA) were compared to enable biochemical comparison with respect to components of the metastatic cascade including adhesion, migration, and proteinase activity. These data demonstrated similar properties in adhesive profiles of PPC and OVCA, with preferential adhesion to type I collagen and vitronectin. Matrix-degrading proteinases including matrix metalloproteinases (MMP)-2, MMP-9, and urinary-type plasminogen activator were produced by both cell types. PPC migration was stimulated by multiple extracellular matrix proteins, whereas OVCA cells demonstrated maximal migration on type I collagen coated surfaces. Together our data suggest biochemical similarities between PPC and OVCA with respect to individual components of the metastatic cascade.

Cell Adhesion↗

PubMed1997

Lumen formation and other angiogenic activities of cultured capillary endothelial cells are inhibited by thrombospondin-1.

The large secreted glycoprotein thrombospondin-1 is a potent inhibitor of neovascularization in vivo. In order to better understand its mechanism of action, we have determined the full range of deficits thrombospondin can impose on cultured capillary endothelial cells. Exogenously added thrombospondin-1 blocked the ability of these cells to organize into cords. It blocked the migration of endothelial cells and vascular smooth muscle cells, but not that of fibroblasts, neutrophils, or keratinocytes, demonstrating specificity. Conversely, when the endogenous thrombospondin-1 produced by the endothelial cells was inactivated using antibodies that can neutralize its inhibition of neovascularization in vivo, migration toward basic fibroblast growth factor and cord formation were stimulated, and sparsely plated cells developed cylindrical cavities. These cavities formed by vesicle fusion, extended the depth of the cell, and appeared to be incipient lumens, staining positively for the luminal marker angiotensin converting enzyme. Antiangiogenic levels of thrombospondin-1 had no measurable effect on the overall level of activity of soluble gelatinases or on urokinase plasminogen activator produced by activated endothelial cells. Coupled with previously published data, these results demonstrate thrombospondin-1 is a multifaceted inhibitor able to block the entire program of dedifferentiation and redifferentiation essential to the formation of new vessels. They also support the contention that the endogenously produced protein contributes to the quiescence of the normal vasculature.

Animals↗

PubMed1996

Human prostate carcinoma cells express enzymatic activity that converts human plasminogen to the angiogenesis inhibitor, angiostatin.

Angiostatin is an inhibitor of angiogenesis and metastatic growth that is found in tumor-bearing animals and can be generated in vitro by the proteolytic cleavage of plasminogen. The mechanism by which angiostatin is produced in vivo has not been defined. We now demonstrate that human prostate carcinoma cell lines (PC-3, DU-145, and LN-CaP) express enzymatic activity that can generate bioactive angiostatin from purified human plasminogen or plasmin. Affinity purified PC-3-derived angiostatin inhibited human endothelial cell proliferation, basic fibroblast growth factor-induced migration, endothelial cell tube formation, and basic fibroblast growth factor-induced corneal angiogenesis. Studies with proteinase inhibitors demonstrated that a serine proteinase is necessary for angiostatin generation. These data indicate that bioactive angiostatin can be generated directly by human prostate cancer cells and that serine proteinase activity is necessary for angiostatin generation.

Angiostatins↗

PubMed1996

Evidence for preferential adhesion of ovarian epithelial carcinoma cells to type I collagen mediated by the alpha2beta1 integrin.

Epithelial ovarian carcinoma, the leading cause of gynecologic cancer death, is characterized by widespread intra-abdominal metastases mediated primarily by surface shedding of tumor cells and peritoneal implantation. Whereas hematogenous metastasis is known to involve cellular adhesion, extracellular matrix proteolysis and cell migration, the role of these processes in the intraperitoneal dissemination of ovarian cancer remains unclear. To analyze further the role of adhesion and proteolysis in ovarian carcinoma dissemination, we have characterized the adhesive profiles of 4 primary cultures of ovarian carcinoma cells and 5 ovarian carcinoma cell lines. Our data demonstrate preferential adhesion of ovarian carcinoma cells to interstitial type I collagen. Analysis of adhesion molecule expression demonstrated the presence of the alpha2 and beta1 integrin subunits by cell surface ELISA, immunoprecipitation and immunohistochemistry. Furthermore, antibodies directed against the alpha2 and beta1 subunits inhibited adhesion of ovarian carcinoma cells to type I collagen by 56% and 95%, respectively. Plasminogen activator and matrix metalloproteinase production by adherent cells was not altered as a consequence of adhesion to individual extracellular matrix proteins; however, adhesion to an extracellular matrix comprised primarily of interstitial collagen increased plasminogen activator activity in 5 of 5 cell lines. Since the ovarian carcinoma micro-environment is rich in type I collagen, our data suggest that preferential adhesion to type I collagen followed by secretion of serine and metalloproteinases may represent a biochemical mechanism by which the intraperitoneal dissemination of ovarian carcinoma is mediated.

Amino Acid Sequence↗

PubMed1996

Fluorescence quenching studies of matrix metalloproteinases (MMPs): evidence for structural rearrangement of the proMMP-2/TIMP-2 complex upon mercurial activation.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases which are secreted from cells as zymogens and can be activated by treatment with organomercurial reagents or limited proteolysis. The proenzyme forms of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are found in complex with tissue inhibitor of metalloproteinases (designated proMMP-2/ TIMP-2 and proMMP-9/TIMP-1, respectively). The proposed mechanism of activation by mercurial compounds involves the induction of a conformational change in the zymogen which leads to propeptide autoprocessing. To investigate the possibility of conformational differences in MMPs, solute quenching of MMP intrinsic fluorescence was used to probe the relative exposure of tryptophan residues in latent and mercurial-activated MMPs. Our data demonstrate that fluorescence quenching of the proMMP-2/TIMP-2 complex by either acrylamide or iodide is significantly increased following mercurial activation. In contrast, no significant change in tryptophan accessibility accompanies mercurial treatment of either proMMP-2 or TIMP-2 alone, or mercurial-activated MMP-2 mixed with TIMP-2. To determine whether the enhanced fluorescence quenching was unique to the activated proMMP-2/TIMP-2 complex, similar experiments were performed using MMP-1, MMP-3, and MMP-9/TIMP-1 complex. In all cases, both latent and mercurialtreated MMPs exhibited similar fluorescence quenching profiles, suggesting that there are no significant conformational differences between the zymogen and activated forms of MMP-1, -2, -3, or -9/TIMP-1. The enhanced fluorescence quenching observed with mercurial-treated proMMP-2/TIMP-2 is indicative of increased exposure of a previously buried tryptophan residue(s), providing evidence for a structural rearrangement of the activated complex. These data, together with our previous biochemical observation that mercurial treatment of proMMP-2/TIMP-2 exposes the MMP-2 active site without propeptide processing (Y. Itoh et al. (1995) Biochem. J. 308, 645-651), suggest that the activated proMMP-2 in the complex may represent a transitional conformational intermediate in MMP activation.

Acrylamide↗

PubMed1996

Characterization of gelatinases linked to extracellular matrix invasion in ovarian adenocarcinoma: purification of matrix metalloproteinase 2.

Substantial evidence indicates that proteolytic degradation of the extracellular matrix is necessary for invasion and metastasis by cancer cells. Our previous work has demonstrated elevated secretion by cultured ovarian adenocarcinoma cells of two gelatinolytic metalloproteinases, a 72-kDa enzyme resembling matrix metalloproteinase 2 (MMP-2) and a 92-kDa enzyme resembling MMP-9 (Moser et al, Int. J. Cancer 56, 552-559, 1994). To assess the potential in vivo relevance of these enzymes, we have examined ovarian carcinoma ascites using gelatin substrate zymography. MMP species identical to those secreted from several well-characterized ovarian adenocarcinoma cell lines were found in the majority of ascites: MMP-2-like gelatinase (23 of 23 cases) and MMP-9-like gelatinase (18 of 23 cases), suggesting a prevalence of these species in the ovarian carcinoma microenvironment and their availability for tumor-associated proteolysis. The contribution of these proteinases to ovarian cancer invasion was further demonstrated by experiments measuring tumor cell-mediated proteolysis of native endothelial cell extracellular matrix (ECM) and tumor cell invasion of reconstituted basement membrane (Matrigel). These data showed that secretion of type IV collagenase activity by a series of independently isolated ovarian adenocarcinoma cell lines correlated well with the ability of these cells to proteolyze the ECM and invade the basement membrane. Furthermore, we have identified and characterized an ovarian carcinoma-associated gelatinase, the 72-kDa MMP found in conditioned media of the DOV 13 cell line, as MMP-2. This enzyme was identical to the previously described MMP-2 from other sources by Western blot, amino terminal sequence, and substrate specificity. Additionally, a large portion of the MMP-2 activity found in DOV 13 conditioned media is active without organomercurial treatment, suggesting that ovarian cancer cells have an endogenous activator of the zymogen. Together, these data suggest that ECM proteolysis mediated by tumor-associated proteinases plays an important role in the invasion and/or metastasis of ovarian carcinoma.

Adenocarcinoma↗

PubMed1996

Membrane-type matrix metalloproteinase expression and matrix metalloproteinase-2 activation in primary human ovarian epithelial carcinoma cells.

Metastatic dissemination of epithelial ovarian carcinoma occurs primarily through exfoliation of cells from the primary tumor, with subsequent implantation, invasion, and growth throughout the organs within the peritoneal cavity. Previous studies have suggested a role for matrix metalloproteinases (MMPs), particularly MMP-2, in ovarian cancer invasion and metastasis. To characterize further the role of MMPs and their inhibitors in ovarian carcinoma, in this study the production and activation of MMPs by short-term primary cultures of human ovarian epithelial carcinoma cells were analyzed. We report that MMP-2 is the predominant gelatinolytic MMP secreted by primary ovarian cancer cells derived from both ovarian tumors and ascites fluid. Furthermore, zymographic analysis demonstrated that MMP-2 is present in conditioned media in both the latent and activated forms, indicating that primary ovarian cancer cells catalyze proMMP-2 activation. Presence of a proMMP-2 activator was confirmed by immunohistochemistry and immunoprecipitation studies which found membrane-type 1 MMP (MT1-MMP) in the membranes of unstimulated cells and levels of both MT1-MMP and tissue inhibitor of metalloproteinases-2 (TIMP-2) were enhanced by culturing cells in the presence of concanavalin A. In addition, interaction of MMP-2 with the ovarian carcinoma cell surface resulted in a 2.5- to 5-fold increase in invasiveness. These data suggest that MT1-MMP-catalyzed activation of proMMP-2 may play a physiologic role in intraperitoneal invasion of ovarian carcinoma cells.

Carcinoma↗

PubMed1995

Specific binding of urinary-type plasminogen activator (u-PA) to vitronectin and its role in mediating u-PA-dependent adhesion of U937 cells.

The present paper described interactions of urinary-type plasminogen activator (u-PA) with isolated protein components of the extracellular matrix (ECM) using kinetic and ligand-blotting analyses, as well as adhesion studies with u-PA-saturated U937 monocytic cells. Kinetic analyses showed that fibronectin and laminin were moderately effective at decreasing activation of plasminogen by u-PA (3-4-fold decrease in kcat/Km), while activation was stimulated slightly by collagen types I and IV (2-4-fold increase in kcat/Km). Ligand-blotting experiments using intact immobilized ECM proteins demonstrated that u-PA binds predominantly to vitronectin. This was supported by ELISA studies, which showed concentration dependent, saturable, reversible binding of u-PA to vitronectin (Kd,app. of 97 nM). Limited proteolysis of vitronectin followed by ligand-blotting analysis demonstrated u-PA binding to a specific vitronectin fragment (M(r) 49,000), and binding was shown to occur through the N-terminal fragment of u-PA. N-terminal sequence analysis indicated that this binding fragment of vitronectin originates with Thr-122 and comprises the hemopexin domain, including the heparin-binding region of the vitronectin molecule. Plasminogen activator inhibitor type I did not compete with u-PA for binding to vitronectin, suggesting both molecules may co-localize on vitronectin. In contrast, binding of u-PA to vitronectin was significantly inhibited by plasminogen, suggesting these molecules share a common binding site on vitronectin. In addition to in vitro studies, experiments were performed to assess the contribution of direct binding of u-PA to vitronectin on the adhesive behaviour of U937 cells. Binding of u-PA-saturated U937 cells to vitronectin was inhibited 66% by excess vitronectin, suggesting that direct binding of u-PA to vitronectin is the mechanism by which u-PA-dependent adhesion of U937 cells to vitronectin is mediated.

Amino Acid Sequence↗

PubMed1995

A plasma membrane-associated component of ovarian adenocarcinoma cells enhances the catalytic efficiency of matrix metalloproteinase-2.

Several recent investigations have demonstrated that matrix metalloproteinase-2 (MMP-2) binds to the cell surface and undergoes zymogen activation via a plasma membrane-associated activity. The purpose of this study was to determine if association of MMP-2 with the plasma membrane also modulates the catalytic efficiency of the active enzyme. Using density gradient centrifugation, we isolated the plasma membrane fractions of two ovarian adenocarcinoma cell lines, DOV 13 and OVCA' 432, previously described either to express MMP-2 or to express no gelatinolytic metalloproteinases, respectively. While DOV 13 cells contained plasma membrane-associated MMP-2 and OVCA 432 did not, both cell types were able to bind exogenous MMP-2. Furthermore, plasma membrane fractions from these cells significantly enhanced the rate of cleavage of [14C]gelatin I substrate by both MMP-2 tissue inhibitor of metalloproteinases-2 (TIMP-2) complex (2.5-8-fold) and TIMP-2-free MMP-2 (5.9-fold). This stimulatory activity was dose-dependent, soluble in Triton X-100, and abolished by trypsin treatment of the membranes, but was stable to heat treatment. Plasma membrane stimulation of MMP-2 resulted in a 3.8-4.6-fold increase in the catalytic efficiency of gelatinolysis. These data suggest that, in addition to promoting zymogen activation, cell surface binding of MMP-2 may regulate enzyme activity by increasing the rate of substrate cleavage. Via this mechanism, tumor cell types that do not express MMPs (such as OVCA 432) nevertheless may be able to utilize exogenous MMP-2 to mediate proteolysis associated with invasion and metastasis.

Adenocarcinoma↗

PubMed1994

Comparison of plasminogen binding and activation on extracellular matrices produced by vascular smooth muscle and endothelial cells.

Plasminogen is the zymogen form of the serine proteinase plasmin. Although plasmin functions primarily as a fibrinolytic enzyme, recent evidence from numerous laboratories indicates that plasmin is also active in extracellular-matrix (ECM) proteolysis. The role of plasmin in ECM degradation suggests that activation of plasminogen may be regulated by interaction with components of the ECM. In the current study, we have investigated binding and kinetic interactions between plasminogen, plasminogen activators and ECM synthesized by either vascular smooth muscle cells (SMCECM) or endothelial cells (ECECM). We report binding of plasminogen, tissue-type plasminogen activator (t-PA) and urinary-type plasminogen activator (u-PA) to intact SMCECM with concentrations of ligand yielding half-maximal binding (B50) of 34, 5 and 15 nM, respectively. ECECM bound only plasminogen and t-PA, with B50 values of 32 nM and 10 nM, respectively. The initial rate of t-PA-catalyzed plasminogen activation was enhanced 41-fold in the presence of SMCECM and 27-fold on ECECM. In contrast, u-PA-catalyzed activation on SMCECM and ECECM was increased only 1.5-fold or 3-fold, respectively. These data suggest that the ECM may provide an alternative surface for assembly and regulation of plasminogen activation.

Amino Acid Sequence↗

PubMed1994

Human mast cell tryptase activates single-chain urinary-type plasminogen activator (pro-urokinase).

Human lung mast cell tryptase is a trypsin-like serine proteinase that is stored in mast cell granules and released by activated mast cells. Here we report that mast cell tryptase is a potent activator of single-chain urinary-type plasminogen activator (scu-PA, or prourokinase), the zymogen form of urinary-type plasminogen activator (u-PA). Activation was complete within 75 min using an enzyme:substrate molar ratio of 1:50 and was accompanied by cleavage of scu-PA at Lys158-Ile159, generating active two-chain u-PA. The reaction was dependent on enzyme concentration and obeyed Michaelis-Menten kinetics. Kinetic constants calculated for scu-PA activation by mast cell tryptase are Km = 34 microM, Vmax = 3.6 pmol of u-PA/min, and kcat = 0.08 s-1. These data suggest that tryptase from tumor-associated mast cells may participate in the activation of scu-PA.

Amino Acid Sequence↗