Avaliação de glicosaminoglicanos e proteínas de matriz extracelular em queratinócitos submetidos ao estresse oxidativo e ao choque hiperosmótico.
Data
2013
Autores
Leme Júnior, Luiz Eugênio Garcez 
Orientadores
Justo, Giselle Zenker
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A pele e o maior orgao do corpo, atuando como uma barreira contra a radiacao e patogenos, mantendo a temperatura corporal, prevenindo contra desidratacao e funcionando como orgao sensorial. Os glicosaminoglicanos (GAGs) tem importantes funcoes nas celulas da pele, tais como a participacao no controle da proliferacao e divisao celular, comunicacao e adesao celular e modulacao das respostas celulares aos estimulos externos. O estresse oxidativo e o choque hiperosmotico sao os tipos de estresses que mais afetam os queratinocitos da pele. Este trabalho avaliou os efeitos do estresse oxidativo, induzido pelo peroxido de hidrogenio (H2O2) e do choque hiperosmotico, induzido pelo sorbitol, sobre o perfil de GAGs e sobre a expressao genica e/ou proteica de sindecans e enzimas associadas ao metabolismo dessas moleculas em queratinocitos humanos da linhagem HaCaT. Proteoglicanos (PGs) foram metabolicamente marcados com [35S]-sulfato apos exposicao das celulas a 1,2 (IC25) e 2,0 mM (IC50) de H2O2 por 4 h (estresse oxidativo) e 0,4 (IC25) e 1,0 M (IC50) de sorbitol por 2 h (choque hiperosmotico). A seguir, os GAGs foram identificados pela diGestão com enzimas especificas. Os resultados demonstraram que celulas HaCaT sintetizam e secretam heparam sulfato (HS), acido hialuronico (AH) e um hibrido de condroitim sulfato e dermatam sulfato (CS/DS). O estresse oxidativo induzido por 2,0 mM de H2O2 (IC50) causou um aumento significativo na sintese de GAGs sulfatados ao mesmo tempo que provocou uma diminuicao na sintese e secrecao de AH. O tratamento com 1,2 mM de H2O2 (IC25) diminuiu a sintese e secrecao de AH, sem causar alteracoes no perfil de GAGs sulfatados. O choque hiperosmotico induzido pelo tratamento com sorbitol nas concentracoes de 0,4 M (IC25) e 1,0 M (IC50) provocou diminuicao na sintese e secrecao de GAGs sulfatados e aumento na concentracao de AH. Os niveis de expressao de genes relacionados ao metabolismo de proteoglicanos de HS (HPSE1, SDC1 e SDC4), genes relacionados ao metabolismo do AH (HAS2, HAS3, HYAL1 e HYAL2) e genes codificantes de gelatinases e seus inibidores (MMP2, MMP9, TIMP1, TIMP2 e RECK), analisados por qPCR, diminuiram significativamente tanto apos o estresse oxidativo como apos o choque hiperosmotico nas duas concentracoes estudadas. Uma diminuicao na expressao de HYAL3 foi observada em celulas expostas ao estresse oxidativo causado pela concentracao de H2O2 referente ao IC50. O gene HAS1 nao e expresso em celulas HaCaT. As concentracoes das proteinas HYAL1 e HYAL2, avaliadas por western blotting, aumentaram significativamente apos exposicao ao H2O2 e ao sorbitol nas concentracoes equivalentes aos valores de IC50. Esse aumento nas concentracoes de HYAL1 e HYAL2, juntamente com a degradacao quimica causada pelo H2O2, pode explicar a diminuicao observada nos niveis de AH apos o estresse oxidativo. A atividade da proteina HPSE1 nao variou nos tratamentos estudados, sugerindo que outros fatores contribuem para os niveis reduzidos de HS induzidos pelo choque hiperosmotico
The skin is the largest organ in the body, acting as a barrier against radiation and pathogens, maintaining the body temperature, preventing dehydration and acting as a sensory organ. Glycosaminoglycans (GAG) play important roles in skin cells, such as regulating cell proliferation and division, cell communication and adhesion, as well as modulating cellular responses to external stimuli. Oxidative stress and hyperosmotic shock are the most important stress conditions that affect skin keratinocytes. In this study, the effects of hydrogen peroxide (H2O2)induced oxidative stress and sorbitolinduced hyperosmotic shock in human keratinocyte (HaCaT cell lineage) GAG profile were evaluated. In addition, alterations in gene and/or protein expression of syndecans and enzymes involved in GAG metabolism were studied by qPCR and immunoblotting, respectively. Proteoglycans (PG) were labeled with [35S]sulfate after cell exposure to 1.2 (IC25) or 2.0 mM (IC50) of H2O2 during 4 h (oxidative stress) and to 0.4 M (IC25) or 1.0 M (IC50) of sorbitol during 2 h (hyperosmotic shock), and GAG were then identified by specific enzyme digestion. The results demonstrated that HaCaT cells synthesize and secrete heparan sulfate (HS), hyaluronic acid (AH) and a hybrid of chondroitin/dermatan sulfate (CS/DS). Treatment of cells with 2.0 mM of H2O2 induced an increase in the synthesis of sulfated GAG, while it simultaneously decreased AH synthesis and secretion. Although the effects of 1.2 mM of H2O2 on AH synthesis and secretion was the same as those observed for 2.0 mM, no changes in sulfated GAG were induced by this stress condition. Hyperosmotic shock induced by sorbitol, at both concentrations, decreased GAG synthesis and secretion, whilst AH was increased. The expression of genes associated with HSPG metabolism (HPSE1, SDC1 and SDC4), AH metabolism (HAS2, HAS3, HYAL1 and HYAL2) and genes coding for gelatinases and theirs inhibitors (MMP2, MMP9, TIMP1, TIMP2 and RECK), were significantly reduced after oxidative stress and hyperosmotic shock at both concentrations. The levels of HYAL3 expression decreased in cells exposed to oxidative stress caused by 2.0 mM of H2O2. HAS1 expression was not detected in HaCaT cells. HYAL1 and HYAL2 protein expression significantly increased after cell exposure to 2.0 mM H2O2 and 1.0 M sorbitol. This increase in HYAL1 and HYAL2 concentration, as well as the chemical degradation caused by H2O2, can explain the decrease observed in AH concentration after oxidative stress. HPSE1 activity did not change after the stress conditions, suggesting that other factors contribute to the decreased levels of HS induced by hyperosmotic shock.
The skin is the largest organ in the body, acting as a barrier against radiation and pathogens, maintaining the body temperature, preventing dehydration and acting as a sensory organ. Glycosaminoglycans (GAG) play important roles in skin cells, such as regulating cell proliferation and division, cell communication and adhesion, as well as modulating cellular responses to external stimuli. Oxidative stress and hyperosmotic shock are the most important stress conditions that affect skin keratinocytes. In this study, the effects of hydrogen peroxide (H2O2)induced oxidative stress and sorbitolinduced hyperosmotic shock in human keratinocyte (HaCaT cell lineage) GAG profile were evaluated. In addition, alterations in gene and/or protein expression of syndecans and enzymes involved in GAG metabolism were studied by qPCR and immunoblotting, respectively. Proteoglycans (PG) were labeled with [35S]sulfate after cell exposure to 1.2 (IC25) or 2.0 mM (IC50) of H2O2 during 4 h (oxidative stress) and to 0.4 M (IC25) or 1.0 M (IC50) of sorbitol during 2 h (hyperosmotic shock), and GAG were then identified by specific enzyme digestion. The results demonstrated that HaCaT cells synthesize and secrete heparan sulfate (HS), hyaluronic acid (AH) and a hybrid of chondroitin/dermatan sulfate (CS/DS). Treatment of cells with 2.0 mM of H2O2 induced an increase in the synthesis of sulfated GAG, while it simultaneously decreased AH synthesis and secretion. Although the effects of 1.2 mM of H2O2 on AH synthesis and secretion was the same as those observed for 2.0 mM, no changes in sulfated GAG were induced by this stress condition. Hyperosmotic shock induced by sorbitol, at both concentrations, decreased GAG synthesis and secretion, whilst AH was increased. The expression of genes associated with HSPG metabolism (HPSE1, SDC1 and SDC4), AH metabolism (HAS2, HAS3, HYAL1 and HYAL2) and genes coding for gelatinases and theirs inhibitors (MMP2, MMP9, TIMP1, TIMP2 and RECK), were significantly reduced after oxidative stress and hyperosmotic shock at both concentrations. The levels of HYAL3 expression decreased in cells exposed to oxidative stress caused by 2.0 mM of H2O2. HAS1 expression was not detected in HaCaT cells. HYAL1 and HYAL2 protein expression significantly increased after cell exposure to 2.0 mM H2O2 and 1.0 M sorbitol. This increase in HYAL1 and HYAL2 concentration, as well as the chemical degradation caused by H2O2, can explain the decrease observed in AH concentration after oxidative stress. HPSE1 activity did not change after the stress conditions, suggesting that other factors contribute to the decreased levels of HS induced by hyperosmotic shock.
Descrição
Citação
LEME JR, Luiz Eugênio Garcez. Avaliação de glicosamiglicanos e proteínas de matriz extracelular em queratinócitos submetidos ao estresse oxidativo e ao choque hiperosmótico. 2013. 83f. Dissertação (Mestrado em Biologia Molecular) – Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2013.