RESUMO
During epithelial ovarian cancer (EOC) progression, intraperitoneally disseminating tumor cells and multicellular aggregates (MCAs) present in ascites fluid adhere to the peritoneum and induce retraction of the peritoneal mesothelial monolayer prior to invasion of the collagen-rich submesothelial matrix and proliferation into macro-metastases. Clinical studies have shown heterogeneity among EOC metastatic units with respect to cadherin expression profiles and invasive behavior; however, the impact of distinct cadherin profiles on peritoneal anchoring of metastatic lesions remains poorly understood. In the current study, we demonstrate that metastasis-associated behaviors of ovarian cancer cells and MCAs are influenced by cellular cadherin composition. Our results show that mesenchymal N-cadherin-expressing (Ncad+) cells and MCAs invade much more efficiently than E-cadherin-expressing (Ecad+) cells. Ncad+ MCAs exhibit rapid lateral dispersal prior to penetration of three-dimensional collagen matrices. When seeded as individual cells, lateral migration and cell-cell junction formation precede matrix invasion. Neutralizing the Ncad extracellular domain with the monoclonal antibody GC-4 suppresses lateral dispersal and cell penetration of collagen gels. In contrast, use of a broad-spectrum matrix metalloproteinase (MMP) inhibitor (GM6001) to block endogenous membrane type 1 matrix metalloproteinase (MT1-MMP) activity does not fully inhibit cell invasion. Using intact tissue explants, Ncad+ MCAs were also shown to efficiently rupture peritoneal mesothelial cells, exposing the submesothelial collagen matrix. Acquisition of Ncad by Ecad+ cells increased mesothelial clearance activity but was not sufficient to induce matrix invasion. Furthermore, co-culture of Ncad+ with Ecad+ cells did not promote a 'leader-follower' mode of collective cell invasion, demonstrating that matrix remodeling and creation of invasive micro-tracks are not sufficient for cell penetration of collagen matrices in the absence of Ncad. Collectively, our data emphasize the role of Ncad in intraperitoneal seeding of EOC and provide the rationale for future studies targeting Ncad in preclinical models of EOC metastasis.
Assuntos
Caderinas/metabolismo , Modelos Animais de Doenças , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/secundário , Animais , Caderinas/genética , Carcinoma Epitelial do Ovário , Adesão Celular , Agregação Celular , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 14 da Matriz/química , Metaloproteinase 14 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/metabolismo , Técnicas de Cultura de Órgãos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismoRESUMO
We have measured the optical properties of cancer and normal whole cells and lysates using light transmission spectroscopy (LTS). LTS provides both the optical extinction coefficient in the wavelength range from 220 to 1100nm and (by spectral inversion using a Mie model) the particle distribution density in the size range from 1 to 3000nm. Our current work involves whole cells and lysates of cultured human oral cells in liquid suspension. We found systematic differences in the optical extinction between cancer and normal whole cells and lysates, which translate to different particle size distributions (PSDs) for these materials. Specifically, we found that cancer cells have distinctly lower concentrations of nanoparticles with diameters less than 100nm and have higher concentrations of particles with diameters from 100 to 1000nm-results that hold for both whole cells and lysates. We also found a power-law dependence of particle density with diameter over several orders of magnitude.
Assuntos
Luz , Neoplasias Bucais/patologia , Nanopartículas , Fenômenos Ópticos , Análise Espectral , Linhagem Celular Tumoral , Humanos , Queratinócitos/citologia , Queratinócitos/patologia , Nanopartículas/química , Tamanho da Partícula , Espalhamento de RadiaçãoRESUMO
Epithelial ovarian cancer (OvCa) is associated with high mortality and, as the majority (>75%) of women with OvCa have metastatic disease at the time of diagnosis, rates of survival have not changed appreciably over 30 years. A mechanistic understanding of OvCa initiation and progression is hindered by the complexity of genetic and/or environmental initiating events and lack of clarity regarding the cell(s) or tissue(s) of origin. Metastasis of OvCa involves direct extension or exfoliation of cells and cellular aggregates into the peritoneal cavity, survival of matrix-detached cells in a complex ascites fluid phase and subsequent adhesion to the mesothelium lining covering abdominal organs to establish secondary lesions containing host stromal and inflammatory components. Development of experimental models to recapitulate this unique mechanism of metastasis presents a remarkable scientific challenge, and many approaches used to study other solid tumors (for example, lung, colon and breast) are not transferable to OvCa research given the distinct metastasis pattern and unique tumor microenvironment (TME). This review will discuss recent progress in the development and refinement of experimental models to study OvCa. Novel cellular, three-dimensional organotypic, and ex vivo models are considered and the current in vivo models summarized. The review critically evaluates currently available genetic mouse models of OvCa, the emergence of xenopatients and the utility of the hen model to study OvCa prevention, tumorigenesis, metastasis and chemoresistance. As these new approaches more accurately recapitulate the complex TME, it is predicted that new opportunities for enhanced understanding of disease progression, metastasis and therapeutic response will emerge.
Assuntos
Modelos Biológicos , Neoplasias Epiteliais e Glandulares , Neoplasias Ovarianas , Animais , Animais Geneticamente Modificados , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Humanos , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Microambiente TumoralRESUMO
Compared to force-resisting elements of the mammalian feeding apparatus, data on jaw-muscle plasticity are less common. This hinders our understanding of the role of force-producing structures in craniofacial development and integration. Thus, we investigated fiber-type abundance and cross-sectional area in the masseter muscle of growing rabbits subjected to diet-induced variation in masticatory stresses. Three loading cohorts were obtained as weanlings and raised until adult on different diets. Immediately following euthanasia, left-sided masseters were dissected away, weighed, and then divided into anterior, intermediate and posterior sections for fiber-type immunohistochemistry. These data were compared to mandibular proportions and biomineralization from the same subjects. Results indicate that growing mammals fed a tougher, fracture-resistant diet develop: absolutely and relatively lower numbers of Type I jaw-muscle fibers; absolutely larger fiber cross-sectional areas; and relative increases in the amount of Type II fibers. These analyses indicate that an early postweaning dietary shift can induce significant variation in muscle fiber types. Such norms of reaction are comparable to those observed in bony elements. Functionally, the processing of fracture-resistant foods results in jaw adductors potentially characterized by faster contraction times and higher force production capabilities, which may influence the frequency and amplitude of forces experienced by oral tissues.
Assuntos
Força de Mordida , Mandíbula/crescimento & desenvolvimento , Músculo Masseter/crescimento & desenvolvimento , Fibras Musculares Esqueléticas/fisiologia , Desenvolvimento Musculoesquelético/fisiologia , Sistema Estomatognático/crescimento & desenvolvimento , Adaptação Fisiológica/fisiologia , Animais , Fenômenos Biomecânicos , Comportamento Alimentar/fisiologia , Imuno-Histoquímica , Masculino , Mandíbula/anatomia & histologia , Mandíbula/diagnóstico por imagem , Músculo Masseter/citologia , Mastigação/fisiologia , Contração Muscular/fisiologia , Fibras Musculares de Contração Rápida/citologia , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares Esqueléticas/citologia , Fibras Musculares de Contração Lenta/citologia , Fibras Musculares de Contração Lenta/fisiologia , Coelhos , Sistema Estomatognático/anatomia & histologia , Estresse Mecânico , Microtomografia por Raio-XRESUMO
A predominant characteristic of metastatic cells is the ability to invade host tissues and establish distant metastatic foci. Release of metastatic cells from a primary tumor results from disruption of tissue architecture and requires reversible modulation of cell-matrix and cell-cell contacts, cytoskeletal rearrangement, and acquisition of enhanced proteolytic potential. Malignant cells produce a spectrum of extracellular proteinases including matrix metalloproteinases (MMPs) that process extracellular matrix components, cell surface proteins, and immune modulators. Dysregulated proteolysis has been implicated in tumor invasion and metastasis in multiple model systems. This review will focus on data that highlight the influence of cell-matrix and cell-cell interactions and their associated signal transduction pathways on proteinase regulation. These data highlight cell adhesion signaling as a mechanism for a versatile cellular proteolytic response to changing microenvironmental cues.
Assuntos
Comunicação Celular , Metaloproteinases da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Metástase Neoplásica , Animais , Caderinas/metabolismo , Adesão Celular , Regulação Enzimológica da Expressão Gênica , Humanos , Integrinas/metabolismo , Metaloproteinases da Matriz Associadas à Membrana , Glicoproteínas de Membrana , Modelos Biológicos , Complexo Glicoproteico GPIb-IX de Plaquetas , Transdução de Sinais , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismoRESUMO
Via their integration of the intermediate filament cytoskeleton into the cell membrane, desmosomes facilitate the maintenance of cell shape and tissue integrity as well as intercellular communication. The transmembrane components of the desmosome, the desmogleins and desmocollins, are members of the cadherin family of cell-cell adhesion molecules. Each of these proteins exists as three distinct isoforms, which are the products of individual genes and expressed in a cell-type and differentiation-specific manner. Previous work has suggested that desmoglein 1 binds to its catenin partner, plakoglobin, in an approximately 6:1 stoichiometry. In this study, the molecular organization of complexes formed by plakoglobin and desmoglein 1, 2, or 3 are further examined through immunoprecipitation, size exclusion chromatography and sucrose density sedimentation analysis. It is shown that the complex formed between plakoglobin and desmoglein 1 has an overall molecular weight greater than that of plakoglobin/desmoglein 2 or plakoglobin/desmoglein 3; however, the stoichiometry of the plakoglobin/desmoglein 1 complex does not appear to exceed 2:1.
Assuntos
Caderinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Desmossomos/metabolismo , Animais , Fenômenos Químicos , Química , Desmocolinas , Desmogleína 1 , Desmogleína 3 , Desmogleínas , Desmoplaquinas , Matemática , Camundongos , Peso Molecular , Testes de Precipitina , gama CateninaAssuntos
Epitopos/química , Fibroblastos/enzimologia , Genes myc/imunologia , Proteínas Recombinantes/química , Animais , Caderinas/imunologia , Caderinas/metabolismo , Proteínas do Citoesqueleto/imunologia , Proteínas do Citoesqueleto/metabolismo , Desmogleína 1 , Desmoplaquinas , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Fibroblastos/metabolismo , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , CamundongosRESUMO
Type I collagen stimulation of pro-matrix metalloproteinase (pro-MMP)-2 activation by ovarian cancer cells involves beta(1) integrin receptor clustering; however, the specific cellular and biochemical events that accompany MMP processing are not well characterized. Collagenolysis is not required for stimulation of pro-MMP-2 activation, and denatured collagen does not elicit an MMP-2 activation response. Similarly, DOV13 cells bind to intact collagen utilizing both alpha(2)beta(1) and alpha(3)beta(1) integrins but interact poorly with collagenase-treated or thermally denatured collagen. Antibody-induced clustering of alpha(3)beta(1) strongly promotes activation of pro-MMP-2, whereas alpha(2)beta(1) integrin clustering has only marginal effects. Membrane-type 1 (MT1)-MMP is present on the DOV13 cell surface as both an active 55-kDa TIMP-2-binding species and a stable catalytically inactive 43-kDa form. Integrin clustering stimulates cell surface expression of MT1-MMP and co-localization of the proteinase to aggregated integrin complexes. Furthermore, cell surface proteolysis of the 55-kDa MT1-MMP species occurs in the absence of active MMP-2, suggesting MT1-MMP autolysis. Cellular invasion of type I collagen matrices requires collagenase activity, is blocked by tissue inhibitor of metalloproteinases-2 (TIMP-2) and collagenase-resistant collagen, is unaffected by TIMP-1, and is accompanied by pro-MMP-2 activation. Together, these data indicate that integrin stimulation of MT1-MMP activity is a rate-limiting step for type I collagen invasion and provide a mechanism by which this activity can be down-regulated following collagen clearance.
Assuntos
Colágeno/metabolismo , Metaloproteinases da Matriz/metabolismo , Adesão Celular , Linhagem Celular , Ativação Enzimática , Precursores Enzimáticos/metabolismo , Feminino , Gelatinases/metabolismo , Humanos , Imuno-Histoquímica , Integrinas/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloendopeptidases/metabolismo , Peso Molecular , Neoplasias Ovarianas/enzimologia , Relação Estrutura-Atividade , Propriedades de Superfície , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Células Tumorais CultivadasRESUMO
The activity of matrix metalloproteinase-2 (MMP-2) is regulated stringently on the posttranslational level. MMP-2 efficiently undergoes autolysis into inactive polypeptides in vitro, prompting the hypothesis that MMP-2 autolysis may function as an alternative mechanism for posttranslational control of MMP-2 in vivo. Moreover, MMP-2 binds to intact type I collagen fibrils; however, the functional consequences of this interaction have not been fully elucidated. To test the hypothesis that MMP-2 binding to type I collagen functions as a positive regulator of MMP-2 proteolytic potential, the effect of type I collagen on MMP-2 activity, inhibition by tissue inhibitor of metalloproteinase-2 (TIMP-2), and enzyme stability was examined. Here, we report that purified MMP-2 binds but does not cleave intact type I collagen. The presence of type I collagen affects neither enzymatic activity against a quenched fluorescent peptide substrate nor the kinetics of inhibition by TIMP-2. However, MMP-2 is stabilized from autolysis in the presence of type I collagen, but not by elastin, fibrinogen, or laminin. These data provide biochemical evidence that MMP-2 exosite interactions with type I collagen may function in the posttranslational control of MMP-2 activity by reducing the rate of autolytic inactivation.
Assuntos
Colágeno/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Autólise , Colágeno/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Humanos , Técnicas In Vitro , Cinética , Metaloproteinase 2 da Matriz/química , Inibidores de Metaloproteinases de Matriz , Ligação Proteica , Processamento de Proteína Pós-Traducional , Inibidor Tecidual de Metaloproteinase-2/farmacologiaRESUMO
Ovarian cancer is an highly metastatic disease characterized by ascites formation and diffuse i.p. adhesion, invasion, and metastasis. Levels of lysophosphatidic acid (LPA) are elevated in the plasma of patients with ovarian carcinoma, including 90% of patients with stage I disease, suggesting that LPA may promote early events in ovarian carcinoma dissemination. Expression of matrix metalloproteinases (MMPs) is also up-regulated in ovarian cancer tissues and ascites, and numerous studies have provided evidence for a direct role of MMPs in i.p. invasion and metastasis. Using three-dimensional type I collagen cultures or immobilized beta1 integrin subunit-specific antibodies, we previously demonstrated that beta1 integrin clustering promotes activation of proMMP-2 and processing of membrane type 1 MMP in ovarian cancer cells (S. M. Ellerbroek et al., Cancer Res., 59: 1635-1641, 1999). In the current study, the effect of LPA on MMP expression and invasive activity was investigated. Treatment of ovarian cancer cells with pathophysiological levels of LPA increased cellular adhesion to type I collagen and beta1 integrin expression. A significant up-regulation of MMP-dependent proMMP-2 activation was observed in LPA-treated cells, leading to enhanced pericellular MMP activity. As a result of increased MMP activity, haptotactic and chemotactic motility, in vitro wound closure, and invasion of a synthetic basement membrane were enhanced. These data indicate that LPA contributes to metastatic dissemination of ovarian cancer cells via up-regulation of MMP activity and subsequent downstream changes in MMP-dependent migratory and invasive behavior.
Assuntos
Lisofosfolipídeos/farmacologia , Metaloproteinases da Matriz/metabolismo , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Colágeno/metabolismo , Ativação Enzimática/efeitos dos fármacos , Precursores Enzimáticos/metabolismo , Feminino , Gelatinases/metabolismo , Humanos , Integrina beta1/biossíntese , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/metabolismo , Metaloendopeptidases/metabolismo , Invasividade NeoplásicaRESUMO
Activation of the epidermal growth factor (EGF) receptor regulates many processes associated with metastasis, including modulation of cell:cell and cell:substrate interactions, production of matrix-degrading proteinases, and cellular migration. We have demonstrated previously that EGF stimulates migration and matrix metalloproteinase (MMP)-9-dependent invasion of ovarian cancer cells. In this study, we compare the roles of EGF-induced phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) activities in regulation of cellular responses associated with ovarian tumor cell metastasis. Inhibition of PI3K and MAPK activity impairs EGF-stimulated cell migration, in vitro invasion, and MMP-9 production. PI3K activity is not required for growth factor disruption of cell:cell junctions, whereas inhibitors of extracellular signal-regulated kinase (ERK)1/ERK2 activation and p38 MAPK activity block EGF-dependent junction dissolution. EGF promotes pro-MMP-9 binding to the cell surface through a mechanism that is independent of extracellular enzyme concentration. Interestingly, inhibition of PI3K activity abolishes EGF-induced cell surface association of pro-MMP-9, whereas inhibitors of MAPKs only partially block the response. These data suggest that EGF receptor activation promotes a PI3K-dependent induction of a cell surface pro-MMP-9 binding component that may facilitate gelatinase-mediated cellular invasion and supports an expanded role for elevated PI3K activity in cellular responses associated with ovarian tumor metastasis. In addition, our findings support the hypothesis that divergent kinase activities regulate distinct cellular events associated with growth factor-induced invasion of ovarian cancer cells.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Metaloproteinase 9 da Matriz/biossíntese , Fosfatidilinositol 3-Quinases/metabolismo , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/fisiologia , Membrana Celular/enzimologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Desmossomos/efeitos dos fármacos , Desmossomos/fisiologia , Receptores ErbB/fisiologia , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Metaloproteinase 9 da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Invasividade Neoplásica , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Células Tumorais CultivadasRESUMO
OBJECTIVE: The metastatic process in epithelial ovarian cancer is thought to involve surface shedding and subsequent dissemination of ovarian cancer cells, facilitated by localized proteolysis at the interface between ovarian cancer cells and peritoneal surfaces. The factors regulating the metastatic process, however, are not well understood. Transforming growth factor-beta (TGF-beta) is a multifunctional peptide that elicits numerous cellular effects pertinent to the metastatic process. The purpose of this study was to evaluate the regulatory role of TGF-beta on metastasis in ovarian cancer. METHOD: We evaluated the effect of TGF-beta on the metastatic characteristics (adhesion, invasion, motility, proteolysis) of five ovarian cancer cell lines (DOV-13 and OVCA 420, 429, 432, and 433), two short-term primary ovarian cancer cell cultures (OVCA 10 and OVCA 208), and five normal ovarian surface epithelial (NOSE) cell cultures (OSE 133, 185, 186, 188, and 189). The effect of TGF-beta on invasion and proteolysis was quantified using a modified Boyden chamber invasion assay, zymography, a coupled colorimetric activity assay, and an HPLC-based quantitation of synthetic substrate cleavage. RESULTS: TGF-beta significantly increased invasion in five of seven ovarian cancer cell lines in amounts ranging from 2- to 20-fold. In contrast, TGF-beta significantly decreased invasion in two of five NOSE isolates by 50 to 80% and had no significant effect on invasion in three. TGF-beta treatment increased matrix metalloproteinase (MMP) expression in OVCA 420 and 433 and DOV-13, resulting in MMP-dependent collagen cleavage and invasive activity. Addition of the MMP inhibitor GI12947 neutralized the enhancing effect of TGF-beta on invasion. TGF-beta had no effect on ovarian cancer cell motility and only increased adhesion in DOV-13. CONCLUSIONS: These data suggest that TGF-beta may enhance the invasiveness of ovarian cancers through induction of MMP activity.
Assuntos
Neoplasias Ovarianas/patologia , Fator de Crescimento Transformador beta/fisiologia , Membrana Basal/citologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Epiteliais/patologia , Feminino , Humanos , Metaloproteinases da Matriz/biossíntese , Invasividade Neoplásica , Neoplasias Ovarianas/enzimologia , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/enzimologia , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The laminin family contains a number of complex, multi-domain proteins that participate in a large variety of biologic processes. Limited proteolysis has been utilized extensively as a tool with which to determine laminin structure/function relationships. In addition, proteolytic modification of laminins may occur as a component of heterotrimer assembly and secretion, or may follow incorporation of mature laminin into the extracellular matrix. Conversely, laminin binding to cellular receptors may also influence proteinase expression. This review will highlight specific examples to demonstrate the functional interplay between laminins and proteinases in the regulation of laminin structure and function as well as in the subsequent control of proteinase expression.
Assuntos
Laminina/fisiologia , Animais , Moléculas de Adesão Celular/metabolismo , Endopeptidases/metabolismo , Humanos , Laminina/química , Laminina/metabolismo , Relação Estrutura-Atividade , CalininaRESUMO
Cells in complex tissues contact extracellular matrix that interacts with integrin receptors to influence gene expression, proliferation, apoptosis, adhesion, and motility. During development, tissue remodeling, and tumorigenesis, matrix components are modified by enzymatic digestion with subsequent effects on integrin binding and signaling. We are interested in understanding the mechanisms by which broad spectrum proteinases such as plasmin are targeted to their extracellular matrix protein substrates. We have utilized plasmin-mediated cleavage of the epithelial basement membrane glycoprotein laminin-5 as a model to evaluate molecular events that direct plasmin activity to specific structural domains. We report that plasminogen and tissue plasminogen activator (tPA) exhibit high affinity, specific binding to the G(1) subdomain of the N terminus of the laminin-5 alpha(3) subunit, with equilibrium dissociation constants of 50 nm for plasminogen and 80 nm for tPA. No high affinity binding to the G(2), G(3), and G(4) subdomains was observed. As a result of binding to the G(1) subdomain, the catalytic efficiency of tPA-catalyzed plasminogen activation is enhanced 32-fold, leading to increased matrix-associated plasmin that is positioned favorably for cleavage within the G(4) subdomain as we have reported previously (Goldfinger, L. E., Stack, M. S., and Jones, J. C. R. (1998) J. Cell Biol. 141, 255-265). Thus, physical constraints dictated by interaction of proteinase and matrix macromolecule control not only enzymatic activity but may regulate substrate targeting of proteinases.
Assuntos
Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Fibrinolisina/química , Fibrinolisina/metabolismo , Membrana Basal/metabolismo , Sítios de Ligação , Western Blotting , Moléculas de Adesão Celular/genética , Clonagem Molecular , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Matriz Extracelular/metabolismo , Fibrinolisina/genética , Humanos , Cinética , Ligantes , Lisina/química , Plasminogênio/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Fatores de Tempo , Ativador de Plasminogênio Tecidual/metabolismo , CalininaRESUMO
Expression of urinary-type plasminogen activator (uPA) and its receptor (uPAR) is correlated with matrix proteolysis, cell adhesion, motility, and invasion. To evaluate the functional link between adhesion and proteolysis in gingival keratinocytes (pp126), cells were treated with immobilized integrin antibodies to induce integrin clustering. Clustering of alpha(3) and beta(1) integrin subunits, but not alpha(2), alpha(5), alpha(6), or beta(4), enhanced uPA secretion. Bead-immobilized laminin-5 and collagen I, two major alpha(3)beta(1) ligands, also induced uPA expression. Coordinate regulation of the serpin plasminogen activator inhibitor 1 was also apparent; however, a net increase in uPA activity was predominant. alpha(3)beta(1) integrin clustering induced extracellular signal-regulated kinase 1/2 phosphorylation, and both uPA induction and extracellular signal-regulated kinase activation were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059. Integrin aggregation also promoted a dramatic redistribution of uPAR on the cell surface to sites of clustered alpha(3)beta(1) integrins. Co-immunoprecipitation of beta(1) integrin with uPAR provided further evidence that protein-protein interactions between uPAR and beta(1) integrin control uPAR distribution. As a functional consequence of uPA up-regulation and uPA-mediated plasminogen activation, the globular domain of the laminin-5 alpha(3) subunit, a major pp126 matrix protein, was proteolytically processed from a 190-kDa form to a 160-kDa species. Laminin-5 containing the 160-kDa alpha(3) subunit efficiently nucleates hemidesmosome formation and reduces cell motility. Together, these data suggest that multivalent aggregation of the alpha(3)beta(1) integrin regulates proteinase expression, matrix proteolysis, and subsequent cellular behavior.
Assuntos
Gengiva/metabolismo , Integrinas/metabolismo , Queratinócitos/metabolismo , Receptores de Superfície Celular/isolamento & purificação , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Moléculas de Adesão Celular/metabolismo , Compartimento Celular , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica , Gengiva/citologia , Humanos , Integrina alfa3beta1 , Junções Intercelulares/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio , Lesões Pré-Cancerosas , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Células Tumorais Cultivadas , CalininaRESUMO
Hematogenous metastasis is postulated to involve tumor cell-initiated degradation of basement membrane barriers and underlying connective tissue matrices. Matrix metalloproteinases (MMP) are zinc-dependent endopeptidases that have been implicated in the proteolytic events of tumor cell invasion. Research has revealed a class of membrane-anchored metalloproteinases (MT-MMPs) and has provided convincing evidence that these enzymes activate latent MMP-2 (72 kDa gelatinase A) on the cell surface. The activation of plasma membrane associated MMP is a potential mechanism for facilitation of cellular metastasis and requires consideration when addressing potential roles of MMPs in tumor progression. This review focuses on potential in vivo regulatory mechanisms of membrane-associated MMP activity in the context of tumor cell interaction with matrix macromolecules. BioEssays 1999;21:940-949.
Assuntos
Membrana Celular/enzimologia , Metaloendopeptidases/química , Metástase Neoplásica , Membrana Basal/metabolismo , Tecido Conjuntivo/metabolismo , Ativação Enzimática , Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 2 da Matriz/metabolismoRESUMO
Ovarian carcinomas secrete single-chain urinary-type plasminogen activator (scuPA) and expression of uPA is up-regulated relative to normal ovarian epithelium, leading to an enhanced proteolytic capacity which may facilitate invasion. Furthermore, the uPA receptor (uPAR) is present on ovarian carcinoma cells and is occupied in tumour tissues. In the present study, incubation of scuPA with serum-free conditioned medium from ovarian carcinoma cells resulted in release of a 14 kDa polypeptide. N-terminal sequence analysis identified this fragment as the uPA N-terminal fragment (NTF), which contains a growth-factor and a kringle domain. NTF generation was abolished by serine-proteinase inhibitors, but not inhibitors of matrix metalloproteinases, and was not enhanced by the addition of plasminogen or plasmin. To determine whether ovarian carcinoma-cell growth is altered by uPA, the effect of exogenous scuPA or NTF on proliferation was analysed. Both NTF and scuPA induced a dose-dependent increase in proliferation, with maximal stimulation obtained at 10-20 nM. Furthermore, blocking the interaction of endogenous uPA with uPAR using anti-NTF antibodies significantly inhibited proliferation. Together these data indicate that, in addition to enhancing the invasive activity of ovarian carcinoma cells via increased pericellular proteolysis, uPA also acts as a mitogen for ovarian carcinoma cells, suggesting a biochemical mechanism whereby uPA may contribute to ovarian carcinoma progression by modulating both cell invasion and proliferation.
Assuntos
Divisão Celular/efeitos dos fármacos , Neoplasias Ovarianas/patologia , Fragmentos de Peptídeos/metabolismo , Serina Endopeptidases/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/química , Feminino , Humanos , Neoplasias Ovarianas/metabolismoRESUMO
Angiostatin, a kringle-containing fragment of plasminogen, is a potent inhibitor of angiogenesis. The mechanism(s) responsible for the anti-angiogenic properties of angiostatin are unknown. We now report that human angiostatin blocks plasmin(ogen)-enhanced in vitro invasion of tissue plasminogen activator (t-PA)-producing endothelial and melanoma cells. Kinetic analyses demonstrated that angiostatin functions as a non-competitive inhibitor of extracellular-matrix (ECM)-enhanced, t-PA-catalysed plasminogen activation, with a Ki of 0.9+/-0.03 microM. This mechanism suggests that t-PA has a binding site for the inhibitor angiostatin, as well as for its substrate plasminogen that, when occupied, prevents ternary complex formation between t-PA, plasminogen and matrix protein. Direct binding experiments confirmed that angiostatin bound to t-PA with an apparent Kd [Kd(app)] of 6.7+/-0.7 nM, but did not bind with high affinity to ECM proteins. Together, these data suggest that angiostatin in the cellular micro-environment can inhibit matrix-enhanced plasminogen activation, resulting in reduced invasive activity, and suggest a biochemical mechanism whereby angiostatin-mediated regulation of plasmin formation could influence cellular migration and invasion.
Assuntos
Movimento Celular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Matriz Extracelular/fisiologia , Melanoma/patologia , Fragmentos de Peptídeos/farmacologia , Plasminogênio/metabolismo , Plasminogênio/farmacologia , Angiostatinas , Animais , Anticorpos/farmacologia , Bovinos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibrinolisina/antagonistas & inibidores , Fibrinolisina/metabolismo , Humanos , Cinética , Melanoma/enzimologia , Melanoma/metabolismo , Camundongos , Neovascularização Patológica , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tecidual/imunologia , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tecidual/farmacologia , Células Tumorais Cultivadas , alfa 2-Antiplasmina/metabolismo , alfa 2-Antiplasmina/farmacologiaRESUMO
Culturing DOV 13 ovarian carcinoma cells on three-dimensional collagen lattice but not on thin-layer collagen induces processing of promatrix metalloproteinase (MMP)-2 to a M(r) 62,000 form, suggesting that multivalent integrin aggregation may participate in proteinase regulation. To address the role of collagen-binding integrins in this event, we treated DOV 13 cells with soluble beta1 integrin antibodies (clones P4C10 or 21C8) or beta1 integrin antibodies immobilized on latex beads to promote integrin aggregation. Divalent ligation of beta1 integrins with soluble P4C10 antibodies stimulated expression of pro-MMP-2 and its inhibitor, tissue inhibitor of metalloproteinase-2, whereas soluble 21C8 antibodies had no effect. Aggregation of beta1 integrins with immobilized 21C8 or P4C10 antibodies stimulated MMP-dependent pro-MMP-2 activation and accumulation of a M(r) 43,000 form of membrane type 1 MMP (MT1-MMP), a cell surface activator of pro-MMP-2, in cell extracts. beta1 integrin-mediated MMP-2 activation required protein synthesis and tyrosine kinase signaling and was reduced by an inhibitor of gene transcription. Treatment of control cells with concanavalin A stimulated MMP-dependent pro-MMP-2 activation and accumulation of M(r) 55,000 and 43,000 forms of MT1-MMP in cell extracts. Addition of either the MMP inhibitor GM-6001-X or exogenous tissue inhibitor of metalloproteinase-2 to concanavalin A-treated cells resulted in loss of the M(r) 43,000 form of MT1-MMP and accumulation of the M(r) 55,000 form of the enzyme in cell extracts, suggesting that the M(r) 43,000 form is a product of MMP-dependent M(r) 55,000 MT1-MMP proteolysis. Together, these data suggest that beta1 integrin stimulation of pro-MMP-2 activation involves MT1-MMP posttranslational processing and requires multivalent integrin aggregation.
Assuntos
Gelatinases/biossíntese , Integrina beta1/fisiologia , Metaloendopeptidases/biossíntese , Neoplasias Ovarianas/metabolismo , Colágeno/metabolismo , Precursores Enzimáticos/metabolismo , Feminino , Humanos , Metaloproteinase 2 da Matriz , Metaloproteinases da Matriz Associadas à Membrana , Peso Molecular , Inibidores de Proteases/farmacologia , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Células Tumorais CultivadasRESUMO
Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and/or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins for plasminogen and angiostatin from human umbilical vein endothelial cells. Binding studies demonstrated that plasminogen and angiostatin bound in a concentration-dependent, saturable manner. Plasminogen binding was unaffected by a 100-fold molar excess of angiostatin, indicating the presence of a distinct angiostatin binding site. This finding was confirmed by ligand blot analysis of isolated human umbilical vein endothelial cell plasma membrane fractions, which demonstrated that plasminogen bound to a 44-kDa protein, whereas angiostatin bound to a 55-kDa species. Amino-terminal sequencing coupled with peptide mass fingerprinting and immunologic analyses identified the plasminogen binding protein as annexin II and the angiostatin binding protein as the alpha/beta-subunits of ATP synthase. The presence of this protein on the cell surface was confirmed by flow cytometry and immunofluorescence analysis. Angiostatin also bound to the recombinant alpha-subunit of human ATP synthase, and this binding was not inhibited by a 2,500-fold molar excess of plasminogen. Angiostatin's antiproliferative effect on endothelial cells was inhibited by as much as 90% in the presence of anti-alpha-subunit ATP synthase antibody. Binding of angiostatin to the alpha/beta-subunits of ATP synthase on the cell surface may mediate its antiangiogenic effects and the down-regulation of endothelial cell proliferation and migration.