The Involvement of Proteoglycans in the Human Plasma Prekallikrein Interaction with the Cell Surface

Introduction: the aim of this work was to evaluate the role of human plasma prekallikrein assembly and processing in cells and to determine whether proteoglycans, along with high molecular weight kininogen (H-kininogen), influence this interaction.Methods: We used the endothelial cell line ECV304 and the epithelial cell lines CHO-K1 (wild type) and CHO-745 (deficient in proteoglycans). Prekallikrein endocytosis was studied using confocal microscopy, and prekallikrein cleavage/activation was determined by immunoblotting using an antibody directed to the prekallikrein sequence C364TTKTSTR371 and an antibody directed to the entire H-kininogen molecule.Results: At 37 degrees C, prekallikrein endocytosis was assessed in the absence and presence of exogenously applied H-kininogen and found to be 1,418.4 +/- 0.010 and 1,070.3 +/- 0.001 pixels/cell, respectively, for ECV304 and 1,319.1 +/- 0.003 and 631.3 +/- 0.001 pixels/cell, respectively, for CHO-K1. No prekallikrein internalization was observed in CHO-745 in either condition. Prekallikrein colocalized with LysoTracker in the absence and presence of exogenous H-kininogen at levels of 76.0% and 88.5%, respectively, for ECV304 and at levels of 40.7% and 57.0%, respectively, for CHO-K1. After assembly on the cell surface, a plasma kallikrein fragment of 53 kDa was predominant in the incubation buffer of all the cell lines studied, indicating specific proteolysis; plasma kallikrein fragments of 48-44 kDa and 34-32 kDa were also detected in the incubation buffer, indicating non-specific cleavage. Bradykinin free H-kininogen internalization was not detected in CHO-K1 or CHO-745 cells at 37 degrees C.Conclusion: the prekallikrein interaction with the cell surface is temperature-dependent and independent of exogenously applied H-kininogen, which results in prekallikrein endocytosis promoted by proteoglycans. Prekallikrein proteolysis/activation is influenced by H-kininogen/glycosaminoglycans assembly and controls plasma kallikrein activity.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao de Apoio a Universidade Federal de São Paulo-FAP/UNIFESPUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, São Paulo, BrazilUniv Bandeirante São Paulo, Biomat & Biotechnol Res Grp, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, São Paulo, BrazilFAPESP: FAPESP 09/51319-1FAPESP: 09/13160-0FAPESP: FAPESP 13/05822-9FAPESP: FAPESP 2012/50219-6CNPq: CNPq 472403/2007-9Web of Scienc

Bradykinin Release Avoids High Molecular Weight Kininogen Endocytosis

Human H-kininogen (120 kDa) plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate H-kininogen endocytosis. in the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type) and CHO-745 (mutant deficient in proteoglycans biosynthesis) cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. in CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. the H-kininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. the anti-pain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular kininogenases. the present data also demonstrates that serine or cysteine proteases in lipid raft domains/caveolae on the CHO cell can hydrolyze H-kininogen, thus releasing kinins.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao de Apoio a Universidade Federal de São Paulo-FAP/UNIFESPUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Bioquim, São Paulo, SP, BrazilUniv Anhanguera São Paulo UNIAN SP, Programa Biomat, São Paulo, SP, BrazilUniv Anhanguera São Paulo UNIAN SP, Programa Biotecnol, São Paulo, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Biofis, São Paulo, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Bioquim, São Paulo, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Biofis, São Paulo, SP, BrazilCNPq: CNPq 472403/2007-9FAPESP: FAPESP 13/05822-9FAPESP: FAPESP 2012/50219-6Web of Scienc

Educomunicação e suas áreas de intervenção: Novos paradigmas para o diálogo intercultural

oai:omp.abpeducom.org.br:publicationFormat/1O material aqui divulgado representa, em essência, a contribuição do VII Encontro Brasileiro de Educomunicação ao V Global MIL Week, da UNESCO, ocorrido na ECA/USP, entre 3 e 5 de novembro de 2016. Estamos diante de um conjunto de 104 papers executivos, com uma média de entre 7 e 10 páginas, cada um. Com este rico e abundante material, chegamos ao sétimo e-book publicado pela ABPEducom, em seus seis primeiros anos de existência. A especificidade desta obra é a de trazer as “Áreas de Intervenção” do campo da Educomunicação, colocando-as a serviço de uma meta essencial ao agir educomunicativo: o diálogo intercultural, trabalhado na linha do tema geral do evento internacional: Media and Information Literacy: New Paradigms for Intercultural Dialogue

High molecular weight human kininogen interact with cell surface: Involvement endocytosis and proteolysis process

Angiogenesis and hemostasis are among the most consistent host responses associated with cancer, and these two pathways interrelate with blood coagulation and fibrinolysis influencing tumor angiogenesis directly, thereby contributing to tumor growth. Since the tumor survival depends on blood supplement it has inspired many researchers to search for anti-angiogenic factors and to draw anti-angiogenic strategies for cancer treatment. The plasma kallikrein-kinin system is related to vascular biology and interacts with many physiologic processes such as coagulation, fibrinolysis, complement and renin-angiotensin, it regulates local blood pressure by bradykinin release (BK) and has both anti-thrombotic and pro-fibrinolitic activities. High molecular weight kininogen (HK) plays two roles in angiogenesis process, because BK promotes angiogenesis and kinin free HK (HKa) inhibits angiogenesis. Heparan sulfate proteoglycans (HSPG) may act as structural constituents of extracellular matrix, and those HSPG on cell surface may participate on processes such as endocytosis and vesicular uptake, regulating molecule traffic inside and outside cellular compartments. Different vesicles contribute on endocytosis depending on cell type and environment conditions of growth. The signaling pathways regulate all cell processes activities. The effectors in the pathways include transcription factors that control gene expression, the regulation of secretion apparatus, the metabolic enzymes, the structural and motor elements on cytoskeleton, the cell surface receptors, the cell cycle regulating proteins and membrane ionic channels. Some signaling pathways converge on all these effectors systems. The integration of different signals determine cell behavior, for instance, if cell secretes, moves, growths, proliferates and differentiates. The aim of the present study was analyze the HK interaction with surface of epithelial cells from human breast and CHO. The specific objectives in our work were to study the possibility of HK endocytosis, its processing and activity, in non-metastatic comparing to metastatic cells. In both CHO-K1 and CHO-745 cell lines we confirmed that HSPG plays role in ATP dependent and caveolae mediated endocytosis driven to endosomes the HK assembled on cell surface; we verified kinin release activities of both serine and cysteine proteases on cell surface in pH 7,35 of both cells lines CHO-K1 and CHO-745; both BK and HSPG are important in HK endocytosis; in both cells lines, and absence of Zn2+ and presence of 2% or 10% serum in medium, HKa has proliferative action, and glycosaminoglycans influence in HK activity on CHO-K1 proliferation. Both HK and HKa interactions were evaluated in mammary epithelium cells with different degrees of metastasis. The MCF-10A (non metastatic) interact more with both HK and HKa comparing to other two tumor cells, probably because their higher chondroitin sulfate content in tumor cells surface; the ratio between BK release in medium/BK internalized is higher in MDA-MB-231 cells (high degree of metastasis). In the absence of Zn2+ and presence of serum 2% or 10% in medium, within 24 h, HKa increased the proliferation of both MCF-10A and MCF-7 (middle metastatic) cells, and within 48 h the proliferation decreased in MCF-7 cells; in MDA-MB-231 both HK and HKa decreased cell proliferation and the presence of the serum changes the effect of both in 24 h, in 48 h only HK decreased cell proliferation in the presence of serum 2%. In MCF-7 Akt expression increased compared to non treated cells (control), except in cells treated with HKa in 48 h, ERK1/2 was detected in all conditions tested and c-Myc expression increased compared to non treated cells (control); in MDA-MB-231, HK or its fragments, and HKa increased Akt expression in 24 h; although p-38 is present in these cells in all conditions, HK or its fragments, and HKa promoted changes in p-38 expression, in similar manner in the presence of serum 2% or 10% in medium. The MDA-MD-231 cells migrated after in incubation with either HK or HKa in medium containing 2% or 10% serum and absence of zinc. The present study confirms the HSPG participation in cellular uptake of HK/HKa assembled on cell surface and fused with endosomes; the enzyme action of both serine and cysteine proteases classes in kinin release in incubation buffer in pH 7,35; the importance of both BK and HSPG in HK endocytosis; the involvement of caveolae in this process, and suggests the importance of zinc ions in proliferation processes related to uPAR or suPAR participation. Future studies on cell signaling may help in evaluation of cell cycle under HK/HKa treatment.A angiogênese e a hemostasia estão entre as mais consistentes respostas do hospedeiro associadas ao câncer, e essas duas vias interrelacionam-se, com a coagulação sanguínea e a fibrinólise influenciando a angiogênese tumoral diretamente e contribuindo com o crescimento do tumor. O fato dos tumores serem dependentes do suprimento sanguíneo tem inspirado muitos pesquisadores a procurarem por moléculas antiangiogênicas e desenharem estratégias antiangiogênicas ao tratamento do câncer. O sistema calicreína-cinina plasmático está relacionado à biologia vascular e interage com os sistemas da coagulação, fibrinólise, complemento e renina-angiotensina, regula a pressão sanguínea local pela liberação de bradicinina (BK) e apresenta atividades antitrombóticas e pró-fibrinolíticas. O cininogênio de alta massa molecular (HK) apresenta duas faces no processo de angiogênese, pois a BK é pró-angiogênica e o HK de duas cadeias e livre de cinina (HKa) é antiangiogênico Os proteoglicanos de heparam sulfato (PGHS) podem atuar como constituintes estruturais de matriz, e aqueles presentes na superfície celular podem participar de processos como a endocitose e a captação vesicular, regulando o movimento de moléculas entre os compartimentos intra e extracelulares. Muitas classes de vesículas endocíticas contribuem de modo variável na atividade endocítica total, dependendo do tipo celular e das condições de crescimento do meio. As vias de sinalização regulam a atividade de todos os processos celulares. Os sistemas efetores incluem fatores de transcrição que controlam a expressão gênica, o aparato de secreção regulada, as enzimas metabólicas, os elementos estruturais e motores do citoesqueleto, os receptores de superfície celular, as proteínas reguladoras do ciclo celular e os canais iônicos de membrana. Várias vias de sinalização convergem sobre todos esses sistemas efetores. A integração de diversos sinais determina o comportamento celular, ou seja, se a célula secreta, move, cresce, divide ou diferencia. O objetivo geral proposto no projeto foi estudar a interação do HK com a superfície de células epiteliais derivadas de epitélio de mama humana e CHO. Os objetivos específicos foram estudar a possibilidade de endocitose do HK, seu processamento e atividade, em células não metastáticas comparando a células metastáticas. No presente estudo utilizando as linhagens CHO-K1 e CHO-745 confirmamos o papel desenvolvido pelo PGHS na endocitose dependente de ATP, e mediada por cavéola, do HK ligado à superfície celular e direcionado aos endossomos; verificamos a ação de enzimas das classes serino e cisteíno-proteases na liberação de cininas na superfície celular em pH 7,35 de ambas as linhagens CHO-K1 e CHO-745; ambos BK e PGHS são importantes na endocitose do HK; em ambas as linhagens, na ausência de Zn2+ e na presença de soro 2% ou 10%, o HKa exerce ação proliferativa, e a presença de glicosaminoglicanos influencia na ação do HK sobre a proliferação das CHO-K1. A interação do HK ou HKa foi avaliada em linhagens celulares de epitélio mamário com diferentes graus de metástase. As MCF-10A (não metastáticas) interagem mais com ambos HK e HKa comparadas a ambas as linhagens tumorais, possivelmente pelo maior conteúdo de condroitim sulfato presente na superfície das células tumorais; a relação BK liberada no sobrenadante comparada à BK internalizada é maior em células MDA-MB-231 (alto grau de metástase). Na ausência de Zn2+, e na presença soro 2% ou 10%, em 24 h, o HKa aumenta a proliferação em MCF-10A e MCF-7 (pouco metastáticas), e em 48 h a proliferação diminui em MCF-7; em MDA-MB-231 ambos HK e HKa diminuem a proliferação e a presença de soro reverte os efeitos de ambos em 24 h, e em 48 h apenas o HK diminui a proliferação na presença de soro 2%. Em MCF-7 ocorre o aumento na expressão de Akt em relação às células não tratadas (controle), exceto em células tratadas com HKa em 48 h, ocorre a presença constante de ERK1/2, e o aumento na expressão de c-Myc em relação às células não tratadas (controle); em MDA-MB-231 há o aumento na expressão de Akt por HK, ou seus fragmentos, e HKa em 24 h; apesar de p-38 estar presente nestas células, o HK, ou seus fragmentos, e HKa promovem alterações na expressão de p-38 de maneira semelhante na presença de soro 2% ou 10%. As MDA-MD-231 migram sob ação tanto de HK quanto HKa, na presença de soro 2% ou 10% e na ausência de zinco. O presente estudo confirma a participação do PGHS na internalização de HK/HKa associados à superfície celular e fusão com endossomos; a ação de enzimas das classes serino e cisteíno-proteases na liberação de cininas nos sobrenadantes celulares em pH 7,35; a importância de ambos BK e PGHS na endocitose do HK; o envolvimento de cavéolas presentes em microdomínios de membrana na endocitose e sugere a importância dos íons zinco nos processos de proliferação relacionados com a participação de uPAR ou suPAR. Os estudos de sinalização celular abrem perspectivas à avaliação do ciclo celular sob a ação de HK/HKa.TEDEBV UNIFESP: Teses e dissertaçõe