Navegando por Palavras-chave "PHEX"

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    Estudo da ação da osteopontina na inibição da expressão genica do cotransportador de sódio-fosfato NPT2A
    (Universidade Federal de São Paulo, 2014-03-26) Chiarantin, Gabrielly Maria Denadai [UNIFESP]; Barros, Nilana Meza Tenório de [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    The X-linked hypophosphatemia (XLH) is the most prevalent form of inherited rickets in humans, occurring as a consequence of inactivating mutations in the PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). XLH is characterized by growth retardation, rickets and osteomalacia leading to hypomineralized bones that deform, and soft tooth dentin prone to infection and abscesses. Associated with XLH and causing hypophosphatemia are defects in renal phosphate reabsorption and vitamin D metabolism. In vivo mouse studies have shown that the absence of PHEX leads to the release of a circulating factor(s) that decreases mineralization by inhibiting the sodium/phosphate co-transporter NPT2A, which mediates reabsorption of phosphate in the kidneys. Recently we demonstrated that osteopontin (OPN) is a physiologically relevant substrate for PHEX, whose inactivation by PHEX normally promotes bone mineralization. However, in PHEX-deficient Hyp mouse bone, OPN and its fragments accumulate to inhibit mineralization locally in the extracellular matrix. In the present study, we have extended these observations to show that OPN and a derived OPN fragment (ASARM peptide; acidic serine- and aspartate-rich motif) affect NPT2A expression. Using the supernatant from cultures of a constructed PHEX-deficient human osteosarcoma cell line (MG-63 shRNA-PHEX) we show that, in the absence of PHEX, a secreted factor found in the MG-63 osteosarcoma supernatant decreased NPT2A mRNA and protein expression in human kidney (HK-2) proximal tubule epithelial cell cultures. Based on this observation and on the fact that OPN is completely degraded by PHEX, we investigated the effects of exogenous OPN and its ASARM peptide on HK-2 cell NPT2A expression. RT-PCR revealed that OPN and its ASARM peptide significantly decreased NPT2A expression. In conclusion, these results provide new insight into circulating humoral factors that might influence phosphate handling in the kidney whose alterations contribute to the XLH phenotype, and they suggest that OPN and its ASARM peptide may be involved in this process.
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    Estudo do envolvimento da metalo-peptidase PHEX em processos tumorais
    (Universidade Federal de São Paulo, 2013-03-28) Neves, Raquel Leão [UNIFESP]; Barros, Nilana Meza Tenório de [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    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.
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    Expression and inactivation of osteopontin-degrading PHEX enzyme in squamous cell carcinoma
    (Pergamon-Elsevier Science Ltd, 2016) Neves, Raquel L. [UNIFESP]; Chiarantin, Gabrielly M. D. [UNIFESP]; Nascimento, Fabio D.; Pesquero, Joao B. [UNIFESP]; Nader, Helena B. [UNIFESP]; Tersariol, Ivarne L. S. [UNIFESP]; McKee, Marc D.; Carmona, Adriana K. [UNIFESP]; Barros, Nilana M. T. [UNIFESP]
    Proteolytic enzymes mediate the activation or inactivation of many physiologic and pathologic processes. The PHEX gene (Phosphate-regulating gene with homologies to endopeptidase on the X chromosome) encodes a metallopeptidase, which is mutated in patients with a prevalent form (1:20,000) of inherited rickets-X-linked hypophosphatemia (XLH). XLH shows growth retardation, hypophosphatemia, osteo-malacia, and defective renal phosphate reabsorption and metabolism of vitamin D. Most PHEX studies have focused on bone, and recently we identified osteopontin (OPN) as the first protein substrate for PHEX, demonstrating in the murine model of XLH (Hyp mice) an increase in OPN that contributes to the osteomalacia. Besides its role in bone mineralization, OPN is expressed in many tissues, and therein has different functions. In tumor biology, OPN is known to be associated with metastasis. Here, we extend our PHEX-OPN studies to investigate PHEX expression in a squamous cell carcinoma (SCC) cell line and its possible involvement in modulating OPN function. Real-time PCR showed PHEX-OPN co-expression in SCC cells, with sequencing of the 22 exons showing no mutation of the PHEX gene. Although recombinant PHEX hydrolyze SCC-OPN fragments, unlike in bone cells, SCC-PHEX protein was not predominantly at the plasma membrane. Enzymatic activity assays, FACs and immunoblotting analyses demonstrated that membrane PHEX is degraded by cysteine proteases and the decreased PHEX activity could contribute to inappropriate OPN regulation. These results highlight for the first time PHEX in tumor biology. (C) 2016 Elsevier Ltd. All rights reserved.
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    Proteolytic processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone, the murine model of X-linked hypophosphatemia
    (Wiley-Blackwell, 2013-03-01) Barros, Nilana Meza Tenório de [UNIFESP]; Hoac, Betty; Neves, Raquel Leão [UNIFESP]; Addison, William N.; Assis, Diego Magno [UNIFESP]; Murshed, Monzur; Carmona, Adriana Karaoglanovic [UNIFESP]; McKee, Marc D.; Universidade Federal de São Paulo (UNIFESP); McGill Univ; Harvard Univ
    X-linked hypophosphatemia (XLH/HYP)with renal phosphate wasting, hypophosphatemia, osteomalacia, and tooth abscessesis caused by mutations in the zinc-metallopeptidase PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome). PHEX is highly expressed by mineralized tissue cells. Inactivating mutations in PHEX lead to distal renal effects (implying accumulation of a secreted, circulating phosphaturic factor) and accumulation in bone and teeth of mineralization-inhibiting, acidic serine- and aspartate-rich motif (ASARM)-containing peptides, which are proteolytically derived from the mineral-binding matrix proteins of the SIBLING family (small, integrin-binding ligand N-linked glycoproteins). Although the latter observation suggests a local, direct matrix effect for PHEX, its physiologically relevant substrate protein(s) have not been identified. Here, we investigated two SIBLING proteins containing the ASARM motifosteopontin (OPN) and bone sialoprotein (BSP)as potential substrates for PHEX. Using cleavage assays, gel electrophoresis, and mass spectrometry, we report that OPN is a full-length protein substrate for PHEX. Degradation of OPN was essentially complete, including hydrolysis of the ASARM motif, resulting in only very small residual fragments. Western blotting of Hyp (the murine homolog of human XLH) mouse bone extracts having no PHEX activity clearly showed accumulation of an approximate to 35kDa OPN fragment that was not present in wild-type mouse bone. Immunohistochemistry and immunogold labeling (electron microscopy) for OPN in Hyp bone likewise showed an accumulation of OPN and/or its fragments compared with normal wild-type bone. Incubation of Hyp mouse bone extracts with PHEX resulted in the complete degradation of these fragments. in conclusion, these results identify full-length OPN and its fragments as novel, physiologically relevant substrates for PHEX, suggesting that accumulation of mineralization-inhibiting OPN fragments may contribute to the mineralization defect seen in the osteomalacic bone characteristic of XLH/HYP. (c) 2013 American Society for Bone and Mineral Research.