Estudo do envolvimento da metalo-peptidase PHEX em processos tumorais
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Data
2013-03-28
Autores
Neves, Raquel Leão [UNIFESP]
Orientadores
Barros, Nilana Meza Tenorio de [UNIFESP]
Tipo
Dissertação de mestrado
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Resumo
A PHEX é uma metalo-peptidase codificada pelo gene PHEX (phosphate-regulating gene with homologies to endopeptidase on the X chromosome), identificado em 1995 como sendo o gene mutado em pacientes com uma forma prevalente (1:20.000) de raquitismo humano hereditário denominada Hipofosfatemia Ligada ao Cromossomo X (XLH). A XLH é caracterizada por retardo no crescimento, osteomalácia, hipofosfatemia, defeitos renais de reabsorção de fosfato e no metabolismo da vitamina D. Desde a identificação de PHEX (1995), a função da peptidase tem sido majoritariamente estudada e relacionada a ossos e dentes. Recentemente, a identificação pelo nosso grupo da osteopontina como substrato desta protease, permitiu que ampliássemos o estudo da PHEX para outros tumores, e não apenas ósseos. Os resultados obtidos no presente trabalho mostram o aumento na expressão de PHEX no carcinoma espinocelular (SCC) e ainda, um fragmento prevalente de OPN endógeno de SCC é degradado após a adição de PHEX exógena, indicando que esta peptidase pode participar da regulação da OPN tumoral, assim como ocorre no osso. No estudo de localização da PHEX, as análises de microscopia confocal mostraram que no período de 48h de cultivo, a PHEX não está localizada na membrana. No entanto, a curva de tempo realizada por citometria de fluxo mostrou que a peptidase é encontrada na membrana nos tempos iniciais de 1h e 4h de cultivo celular, porém nos períodos de 12h e 48h, a PHEX não é mais detectada na membrana, e apenas é localizada intracelularmente. A curva de tempo mostrou ainda que a ausência da PHEX na membrana não está relacionada à retenção da proteína dentro da célula. Nossos resultados também mostram que a ausência da PHEX na membrana pode ser resultado do processamento proteolítico de cisteíno proteases, visto que a PHEX exógena é hidrolisada pelas catepsinas B, L e S, e esta hidrólise resulta na inativação enzimática da PHEX, Os resultados aqui obtidos poderão esclarecer a possível participação da PHEX na regulação/modulação tumoral e poderão contribuir para melhor entendimento do papel desta protease nos diferentes tecidos.
The metallopeptidase PHEX is encoded by PHEX gene (Phosphate-regulating gene with homologies to endopeptidase on the X chromosome), identified in 1995 as the defective gene in patients with a prevalent form (1:20.000) of inherited rickets in humans, called XLinked Hypophosphatemia (XLH). The XLH is characterized by growth retardation, osteomalacia, hypophosphatemia, defective renal phosphate reabsorption and metabolism of vitamin D. Since the identification of PHEX (1995), the function of this peptidase has been strictly studied and related to bone and teeth. Recently, the identification of osteopontin (by our group) as a substrate of this protease, has allowed the study of PHEX for other tumors, not only osteomalacia. The results obtained in this study show the increased expression of PHEX in squamous cell carcinoma (SCC), indicating that this peptidase may also participate in tumor regulation, as well as OPN. The viability of tumor cells was not affected when the expression of PHEX is reduced. However, the cellular localization PHEX is not in the membrane. The cellular localization was examined by immunocytochemistry investigated in confocal microscopy and flow cytometry (FACS). To investigate the role of PHEX in SCC cells, it was performed the inhibition of PHEX expression in this cel line using shRNA. In the current phase of the project, the ability of adhesion and cell proliferation (in vitro) is being investigated. The results obtained here can contribute to the understanding of the possible role of PHEX in the regulation / modulation of the tumor.
The metallopeptidase PHEX is encoded by PHEX gene (Phosphate-regulating gene with homologies to endopeptidase on the X chromosome), identified in 1995 as the defective gene in patients with a prevalent form (1:20.000) of inherited rickets in humans, called XLinked Hypophosphatemia (XLH). The XLH is characterized by growth retardation, osteomalacia, hypophosphatemia, defective renal phosphate reabsorption and metabolism of vitamin D. Since the identification of PHEX (1995), the function of this peptidase has been strictly studied and related to bone and teeth. Recently, the identification of osteopontin (by our group) as a substrate of this protease, has allowed the study of PHEX for other tumors, not only osteomalacia. The results obtained in this study show the increased expression of PHEX in squamous cell carcinoma (SCC), indicating that this peptidase may also participate in tumor regulation, as well as OPN. The viability of tumor cells was not affected when the expression of PHEX is reduced. However, the cellular localization PHEX is not in the membrane. The cellular localization was examined by immunocytochemistry investigated in confocal microscopy and flow cytometry (FACS). To investigate the role of PHEX in SCC cells, it was performed the inhibition of PHEX expression in this cel line using shRNA. In the current phase of the project, the ability of adhesion and cell proliferation (in vitro) is being investigated. The results obtained here can contribute to the understanding of the possible role of PHEX in the regulation / modulation of the tumor.
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Citação
NEVES, Raquel Leao. Estudo do envolvimento da metalo-peptidase PHEX em processos tumorais. 2013. 71 f. Dissertação (Mestrado) - Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo (UNIFESP), Diadema, 2013.