O silenciamento do proteoglicano sindecam-4 e a mutação na proteína Ras levam à tranformação de células endoteliais
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Data
2014
Autores
Cavalheiro, Renan Pelluzzi [UNIFESP]
Orientadores
Nader, Helena Bonciani [UNIFESP]
Tipo
Tese de doutorado
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Os sindecans constituem uma familia de quatro membros de proteoglicanos transmembranicos, que apresentam em geral cadeias de glicosaminoglicanos de heparam sulfato covalentemente ligadas ao esqueleto proteico. O sindecam-4 (Syn4) desempenha importante papel no comportamento celular, devido a sua interacao pelas cadeias de heparam sulfato (HS), com uma variedade de moleculas da regiao extracelular. O dominio citoplasmatico do sindecam-4 interage com proteinas citoplasmaticas, ativando proteinas de vias de sinalizacao, como por exemplo, a via Ras/Raf/MAP quinases. A transducao de sinal de Ras/Raf/MAP quinases tem papel chave na regulacao de eventos de crescimento celular, diferenciacao celular e sobrevivencia celular, e as mutacoes pontuais em Ras podem ocorrer, estando associada com o desenvolvimento de canceres humanos, inclusive na superexpressao do proteoglicano sindecam-4. Para elucidar o papel do sindecam-4 e da proteina Ras em eventos celulares de celulas endoteliais, realizamos o silenciamento para a porcao proteica deste proteoglicano pela tecnica de short hairpin RNA e a mutacao da proteina Ras pela transfeccao com oncogene EJ-ras. Assim, celulas endoteliais silenciadas para o sindecam-4 (shRNA-Syn4-EC) e celulas transfectadas com oncogene EJ-ras (EJ-ras-EC), foram analisadas em estudo comparativo com celulas endoteliais selvagens (EC). As celulas shRNA-Syn4-EC apresentam diminuicao na sintese de HS e CS (condroitim sulfato); ja EJ-ras-EC apresentam aumento da sintese de HS. Ambas as celulas mostram alteracoes na morfologia celular e desorganizacao do citoesqueleto, e nitida perda das formacoes de adesoes focais. A deficiente adesao celular aos substratos de fibronectina e laminina foi observada para as celulas shRNA-Syn4-EC, contrastando com EJ-ras-EC, que independem da concentracao desses substratos para adesao. A diminuicao da expressao de fibronectina por essas celulas e evidente, e a distribuicao e expressao da integrina 1 esta alterada apenas para as celulas shRNASyn4- EC. As celulas shRNA-Syn4-EC mostram diminuicao da expressao da proteina Ras, diferentemente de EJ-ras-EC, que nao so super expressam Ras, mas tambem secretam essa proteina via microvesiculas para o ambiente extracelular. O silenciamento de sindecam-4 e a mutacao em Ras levam a alteracao do ciclo celular das celulas endoteliais. As celulas que super expressam Ras apresentam nitido aumento da proliferacao celular quando comparadas com as celulas EC selvagens. Mudancas na ativacao/fosforilacao de proteinas intracelulares como Src, MEK e ERK, tambem foram observadas em ambas as celulas. O processo de angiogenese realizado pelas celulas endoteliais em membrana basal reconstituida e afetado pelo silenciamento de sindecam-4; ja a superexpressao de Ras nao afeta esse evento. Por outro lado, ambas as celulas aumentam o conteudo intracelular e a secrecao do fator de crescimento VEGF, diferentemente do fator TGF-β que esta presente apenas nos extratos celulares, e em baixa quantidade. Os resultados tambem mostram que as celulas shRNA-Syn4-EC e EJ-ras-EC apresentam crescimento independente de ancoragem, pelo ensaio de formacao de colonia em agar semi solido (osofto agar) e formam tumores quando injetadas no tecido subcutaneo de camundongos BALB / c-SCID e NUDE; alem disso, a mutacao em Ras torna essas celulas capazes de induzir metastase in vivo. Cabe ainda dizer, que o silenciamento de sindecam-4 e a mutacao de Ras em celulas endoteliais, nao induzem o processo de diferenciacao e transicao dessas celulas para o tipo mesenquimal. Em conclusao, as celulas endoteliais com mutacoes na proteina Ras mostram alteracoes fenotipicas e fisiologicas, incluindo superexpressao do sindecam-4, como observado para outros tipos celulares. Nossos resultados demonstram pela primeira vez na literatura que alteracoes na expressao do sindecam-4 promovem mudancas fenotipicas e na fisiologia normal das celulas endoteliais. O simples silenciamento do proteoglicanos de heparam sulfato levou a alteracoes na arquitetura da celula, no ciclo celular, na capacidade angiogenica, entre outras mudancas, incluindo a capacidade de induzir tumores in vivo. Podemos concluir que tanto o proteoglicano sindecam-4, como a proteina Ras, sao moleculas essenciais na homeostasia celular, e que, o silenciamento de sincecam-4 ou a mutacao em Ras, alteram uma serie de eventos celulares que afetam o correto comportamento celular de celulas endoteliais
Syndecans constitute a family of four transmembrane proteoglycans to which heparan sulphate glycosaminoglycan chains are generally covalently linked to the protein core. The syndecan-4 (Syn4) plays an important role in cell behavior due to its interaction, through heparan sulfate chains (HS), with a variety of molecules in the extracellular environment. The cytoplasmic domain of syndecan-4 interacts with proteins that activate signaling pathways such as the Ras/Raf/MAP kinases signaling pathway. Ras/Raf/MAP kinases signal transduction plays a key role in the regulation of cell growth, cell differentiation, and cell survival, and when mutations in Ras occur, they are associated with the development of human cancers as well as in the overexpression syndecan-4 proteoglycan. In order to elucidate the role of syndecan-4 and Ras protein in cellular events of endothelial cells, we performed silencing for the protein moiety of this proteoglycan using short hairpin RNA technique and mutation of the Ras protein by transfecting the wild types cells with EJ-ras oncogene. Consequently, endothelial cells silenced for syndecan4 (shRNA-Syn4-EC), and cells transfected with the oncogene EJ-ras (EJ-ras-CS), were examined in a comparative study with their originator wild type endothelial cells (EC). The shRNA-Syn4-EC cells displayed reduced synthesis of HS and CS (chondroitin sulfate ), whereas EJ-ras-EC showed increased synthesis of HS when compared to EC. Both cells showed changes in cell morphology and cytoskeletal disorganization as well as a net loss of focal adhesions formations compared to wild type cells. Weak cell adhesion to fibronectin and laminin substrates was observed for shRNASyn4-EC cells, contrasting with EJ-ras-EC, which are independent of the concentration of these substrates for adhesion. The decrease in fibronectin expression by both cells was evident, and the distribution as well as the expression of β1 integrin is changed only for shRNA-Syn4-EC cells. The shRNA-Syn4-EC cells showed decreased expression of Ras protein, unlike EJ-ras-EC, which not only super express Ras but also secretes such protein to the extracellular environment via microvesicles. Syndecan-4 silencing and Ras mutation lead to alteration in EC cell cycle. Cells expressing Ras have a noteworthy increase in cell proliferation when compared to the wild EC cells. Changes in the activation/phosphorylation of intracellular proteins such as Src, MEK and ERK, were also observed for both cells compared to wild type. The tube formation process - angiogenesis - conducted by endothelial cells when plated in reconstituted basement membrane is affected by silencing syndecan-4, whereas overexpression of Ras does not affect such event. Moreover, both cells increased intracellular content and secretion of the growth factor VEGF, in contrast to TGF-β, which is only present in cell extracts and in low quantity. The results also showed that the shRNA-Syn4 and EJ-ras-EC cells exhibited a anchorage independent growth as shown in the colony formation assay using semisolid agar ("soft" agar) and formed tumors when injected subcutaneously in BALB / c-SCID NUDE mice. Additionally, EJras-EC cells were capable to induce metastasis in vivo. Furthermore, it is important to stress that neither the silencing of syndecan-4 and Ras mutation in endothelial cells, did not induce the differentiation and transition process of such cells to a mesenchymal phenotype. In conclusion, the endothelial cells with mutations in the Ras protein showed phenotypic and physiological changes, including overexpression of syndecan-4, as observed for other cell types. Our results demonstrate for the first time in the literature that changes in syndecan-4 expression promotes phenotypic and physiological changes in normal endothelial cells. The simple silencing syndecan-4, a heparan sulfate proteoglycan, led to changes in cellular architecture, cell cycle, angiogenic capability, among others, including the ability to induce the formation of tumors in vivo. Finally, we can conclude that both the proteoglycan syndecan-4 and the Ras protein, are key molecules in cellular homeostasis, and that both syndecan-4 silencing or Ras mutation alter a number of cellular events that affect normal cell behavior of endothelial cells.
Syndecans constitute a family of four transmembrane proteoglycans to which heparan sulphate glycosaminoglycan chains are generally covalently linked to the protein core. The syndecan-4 (Syn4) plays an important role in cell behavior due to its interaction, through heparan sulfate chains (HS), with a variety of molecules in the extracellular environment. The cytoplasmic domain of syndecan-4 interacts with proteins that activate signaling pathways such as the Ras/Raf/MAP kinases signaling pathway. Ras/Raf/MAP kinases signal transduction plays a key role in the regulation of cell growth, cell differentiation, and cell survival, and when mutations in Ras occur, they are associated with the development of human cancers as well as in the overexpression syndecan-4 proteoglycan. In order to elucidate the role of syndecan-4 and Ras protein in cellular events of endothelial cells, we performed silencing for the protein moiety of this proteoglycan using short hairpin RNA technique and mutation of the Ras protein by transfecting the wild types cells with EJ-ras oncogene. Consequently, endothelial cells silenced for syndecan4 (shRNA-Syn4-EC), and cells transfected with the oncogene EJ-ras (EJ-ras-CS), were examined in a comparative study with their originator wild type endothelial cells (EC). The shRNA-Syn4-EC cells displayed reduced synthesis of HS and CS (chondroitin sulfate ), whereas EJ-ras-EC showed increased synthesis of HS when compared to EC. Both cells showed changes in cell morphology and cytoskeletal disorganization as well as a net loss of focal adhesions formations compared to wild type cells. Weak cell adhesion to fibronectin and laminin substrates was observed for shRNASyn4-EC cells, contrasting with EJ-ras-EC, which are independent of the concentration of these substrates for adhesion. The decrease in fibronectin expression by both cells was evident, and the distribution as well as the expression of β1 integrin is changed only for shRNA-Syn4-EC cells. The shRNA-Syn4-EC cells showed decreased expression of Ras protein, unlike EJ-ras-EC, which not only super express Ras but also secretes such protein to the extracellular environment via microvesicles. Syndecan-4 silencing and Ras mutation lead to alteration in EC cell cycle. Cells expressing Ras have a noteworthy increase in cell proliferation when compared to the wild EC cells. Changes in the activation/phosphorylation of intracellular proteins such as Src, MEK and ERK, were also observed for both cells compared to wild type. The tube formation process - angiogenesis - conducted by endothelial cells when plated in reconstituted basement membrane is affected by silencing syndecan-4, whereas overexpression of Ras does not affect such event. Moreover, both cells increased intracellular content and secretion of the growth factor VEGF, in contrast to TGF-β, which is only present in cell extracts and in low quantity. The results also showed that the shRNA-Syn4 and EJ-ras-EC cells exhibited a anchorage independent growth as shown in the colony formation assay using semisolid agar ("soft" agar) and formed tumors when injected subcutaneously in BALB / c-SCID NUDE mice. Additionally, EJras-EC cells were capable to induce metastasis in vivo. Furthermore, it is important to stress that neither the silencing of syndecan-4 and Ras mutation in endothelial cells, did not induce the differentiation and transition process of such cells to a mesenchymal phenotype. In conclusion, the endothelial cells with mutations in the Ras protein showed phenotypic and physiological changes, including overexpression of syndecan-4, as observed for other cell types. Our results demonstrate for the first time in the literature that changes in syndecan-4 expression promotes phenotypic and physiological changes in normal endothelial cells. The simple silencing syndecan-4, a heparan sulfate proteoglycan, led to changes in cellular architecture, cell cycle, angiogenic capability, among others, including the ability to induce the formation of tumors in vivo. Finally, we can conclude that both the proteoglycan syndecan-4 and the Ras protein, are key molecules in cellular homeostasis, and that both syndecan-4 silencing or Ras mutation alter a number of cellular events that affect normal cell behavior of endothelial cells.
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Citação
CAVALHEIRO, Renan Pelluzzi. O silenciamento do proteoglicano sindecam-4 e a mutação na proteína Ras levam à tranformação de células endoteliais. 2014. 153 f. Tese (Doutorado em Ciências) – Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2014.