RESUMO
Mineralization of elastic fibers in pseudoxanthoma elasticum (PXE) has been associated with low levels of carboxylated matrix gla protein (MGP), most likely as a consequence of reduced vitamin K (vit K) availability. Unexpectedly, vit K supplementation does not exert beneficial effects on soft connective tissue mineralization in the PXE animal model. To understand the effects of vit K supplementation and in the attempt to interfere with pathways leading to the accumulation of calcium and phosphate within PXE-mineralized soft connective tissues, we have conducted in vitro studies on dermal fibroblasts isolated from control subjects and from PXE patients. Cells were cultured in standard conditions and in calcifying medium (CM) in the presence of vit K1 and K2, or levamisole, an alkaline phosphatase (ALP) inhibitor. Control and PXE fibroblasts were characterized by a similar dose-dependent uptake of both vit K1 and vit K2, thus promoting a significant increase of total protein carboxylation in all cell lines. Nevertheless, MGP carboxylation remained much less in PXE fibroblasts. Interestingly, PXE fibroblasts exhibited a significantly higher ALP activity. Consistently, the mineralization process induced in vitro by a long-term culture in CM appeared unaffected by vit K, whereas it was abolished by levamisole.
Assuntos
Fosfatase Alcalina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Pseudoxantoma Elástico/tratamento farmacológico , Pseudoxantoma Elástico/metabolismo , Vitamina K/farmacologia , Adjuvantes Imunológicos/farmacologia , Adulto , Fosfatase Alcalina/genética , Antifibrinolíticos/farmacocinética , Antifibrinolíticos/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Calcificação Fisiológica/fisiologia , Calcinose/tratamento farmacológico , Calcinose/metabolismo , Calcinose/patologia , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Derme/citologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Proteínas da Matriz Extracelular/genética , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Levamisol/farmacologia , Pessoa de Meia-Idade , Fosfatos/metabolismo , Pseudoxantoma Elástico/patologia , Vitamina K/farmacocinética , Proteína de Matriz GlaRESUMO
The ability of cells to respond to changes in oxygen availability is critical for many physiological and pathological processes (i.e. development, aging, wound healing, hypertension, cancer). Changes in the protein profile of normal human dermal fibroblasts were investigated in vitro after 96 h in 5% CO(2) and 21% O(2) (pO(2) = 140 mm Hg) or 2% O(2) (pO(2) = 14 mm Hg), these parameters representing a mild chronic hypoxic exposure which fibroblasts may undergo in vivo. The proliferation rate and the protein content were not significantly modified by hypoxia, whereas proteome analysis demonstrated changes in the expression of 56 proteins. Protein identification was performed by mass spectrometry. Data demonstrate that human fibroblasts respond to mild hypoxia increasing the expression of hypoxia inducible factor (HIF1a) and of the 150-kDa oxygen-regulated protein. Other differentially expressed proteins appeared to be related to stress response, transcriptional control, metabolism, cytoskeleton, matrix remodelling and angiogenesis. Furthermore, some of them, like galectin 1, 40S ribosomal protein SA, N-myc-downstream regulated gene-1 protein, that have been described in the literature as possible cancer markers, significantly changed their expression also in normal hypoxic fibroblasts. Interestingly, a bovine fetuin was also identified that appeared significantly less internalised by hypoxic fibroblasts. In conclusion, results indicate that human dermal fibroblasts respond to an in vitro mild chronic hypoxic exposure by modifying a number of multifunctional proteins. Furthermore, data highlight the importance of stromal cells in modulating the intercellular cross-talk occurring in physiological and in pathologic conditions.
Assuntos
Derme/metabolismo , Fibroblastos/metabolismo , Oxigênio/metabolismo , Proteômica , Adulto , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular , Proliferação de Células , Derme/citologia , Eletroforese em Gel Bidimensional , Feminino , Fibroblastos/citologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Epidemiological and clinical studies suggest the possibility that estrogens might have a cytoprotective effect on the liver. The aim of the present study was to test the hypothesis that 17beta-estradiol (E2) prevents hepatocellular damage induced by deoxycholic acid (DCA), a hydrophobic bile acid. METHODS: HepG2 cells were exposed for 24 h to DCA (350 micromol/L). Cell viability, aspartate aminotransferase and lactate dehydrogenase activity and apoptosis were measured as indices of cell toxicity. The effect of DCA was compared to that observed using either a hydrophilic bile acid, ursodeoxycholic acid (UDCA; 100 micromol/L), or E2 at different concentrations (1 nmol/L, 10 nmol/L, 50 nmol/L and 50 micromol/L) or mixtures of E2/DCA or UDCA/DCA. The same experiments were performed using WRL-68 cells that, at variance with HepG2, express a higher level of nuclear estrogen receptor. RESULTS: High concentrations of E2 and UDCA prevented DCA-induced decrease in cell viability, increase in enzyme activity and apoptosis evaluated both by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and TdT-mediated dUTP nick-end labeling (TUNEL) assays. In addition, DCA-related apoptosis, assessed by caspase activity, was also prevented by E2 (P < 0.01) in physiological (1-10 nmol/L) doses. The cytoprotective effects of E2 and UDCA was also observed in the WRL-68 cell line. CONCLUSIONS: 17Beta-Estradiol prevents DCA-induced cell damage in HepG2 and WRL-68 cell lines to an extent comparable to UDCA. The hypothesis that the protective effect of E2 may be mediated by a mechanism that is nuclear estrogen receptor independent, deserves further verification.
Assuntos
Ácido Desoxicólico/antagonistas & inibidores , Ácido Desoxicólico/toxicidade , Detergentes , Estradiol/farmacologia , Hepatócitos/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Detergentes/toxicidade , Hepatócitos/patologia , Humanos , Interações Hidrofóbicas e HidrofílicasRESUMO
Pseudoxanthoma elasticum (PXE) is a genetic disease characterized by calcification and fragmentation of elastic fibres of the skin, cardiovascular system and eye, caused by mutations of the ABCC6 gene, which encodes the membrane transporter MRP6. The pathogenesis of the lesions is unknown. Based on studies of similar clinical and histopathological damage present in haemolytic disorders, our working hypothesis is that PXE lesions may result from chronic oxidative stress occurring in PXE cells as a consequence of MRP6 deficiency. Our results show that PXE fibroblasts suffer from mild chronic oxidative stress due to the imbalance between production and degradation of oxidant species. The findings also show that this imbalance results, at least in part, from the loss of mitochondrial membrane potential (DeltaPsi(m)) with overproduction of H2O2. Whether mitochondrial dysfunction is the main factor responsible for the oxidative stress in PXE cells remains to be elucidated. However, mild chronic generalized oxidative stress could explain the great majority of structural and biochemical alterations already reported in PXE.
Assuntos
Fibroblastos/fisiologia , Estresse Oxidativo/fisiologia , Pseudoxantoma Elástico/fisiopatologia , Nucleotídeos de Adenina/análise , Adulto , Antioxidantes/metabolismo , Biomarcadores/análise , Células Cultivadas , Doença Crônica , Feminino , Glutationa/análise , Dissulfeto de Glutationa/análise , Humanos , Peróxido de Hidrogênio/metabolismo , Malondialdeído/análise , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Mitocôndrias/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/deficiência , Pseudoxantoma Elástico/patologia , Pele/patologia , Superóxido Dismutase/metabolismoRESUMO
Cultured fibroblasts from the dermis of normal subjects and of Pseudoxanthoma elasticum (PXE) patients were analysed for enzyme activity, protein and mRNA expression of metalloproteases (MMP-2, MMP-3, MMP-9, MT1-MMP) and of their specific inhibitors (TIMP-1, TIMP-2 and TIMP-3). MMP-3, MMP-9 and TIMP-3 mRNAs and proteins failed to be detected in both the medium and the cell layer of both controls and PXE patients. MMP-2 mRNA was significantly more expressed in PXE than in control cell lines, whereas MT1-MMP, TIMP-1 and TIMP-2 mRNAs appeared unchanged. MMP-2 was significantly higher in the cell extracts from PXE fibroblasts than in control cells, whereas differences were negligible in the cell medium. Data suggest that PXE fibroblasts have an increased proteolytic potential, and that MMP-2 may actively contribute to connective tissue alterations in this genetic disorder.
Assuntos
Fibroblastos/enzimologia , Metaloproteinase 2 da Matriz/metabolismo , Pseudoxantoma Elástico/patologia , Pele/patologia , Adulto , Biópsia , Extratos Celulares/análise , Células Cultivadas , Meios de Cultura/química , Matriz Extracelular/enzimologia , Matriz Extracelular/metabolismo , Humanos , Pessoa de Meia-Idade , Pseudoxantoma Elástico/metabolismo , RNA Mensageiro/metabolismoRESUMO
Proteins present within the cell layer and those released in the cell medium from in vitro cultured normal human dermal fibroblasts were separated and characterized in terms of their isoelectric point and molecular weight, by two-dimensional (2-D) gel electrophoresis. All spots in the synthetic gel were firstly analyzed by the Melanie 3 software and compared with those of breast cancer cells, colorectal epithelial cells, HL60, lymphoma cells, and platelets, already available on-line. From the identification of 144 spots from both the cell layer and the medium, we were able to recognize 89 different proteins, since a certain number of spots represented different isoforms of the same molecule. Identifications were performed by matching with on-line 2-D databases, and by matrix assisted laser-desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), in order to confirm the identification by matching, or to identify new proteins. The procedure we used allows (i) to design a highly reproducible reference map of the proteome of adult human normal fibroblasts in culture, (ii) to evaluate protein species produced in the cell layer as well as those released in the culture medium, and (iii) to compare data from gel matching with those obtained by MS. This work represents an essential step for a better knowledge of mesenchymal cells, given the widespread use of this cell type in both clinical and experimental investigations.
Assuntos
Fibroblastos/química , Proteínas/isolamento & purificação , Proteômica/métodos , Pele/citologia , Adulto , Técnicas de Cultura de Células , Bases de Dados Factuais , Eletroforese em Gel Bidimensional , Humanos , Proteínas/análise , Proteínas/metabolismo , Proteômica/instrumentação , Software , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Human sterol 27-hydroxylase catalyses the first step in the alternative pathway of bile acids biosynthesis in hepatocytes. However the gene encoding this enzyme (CYP27 gene) is expressed in every tissue and some evidence suggests that this enzyme plays a role in cholesterol homeostasis. Although modulation of CYP27 expression has been reported, the mechanisms underlying the regulation of this gene in human tissues is still poorly understood. To elucidate the mechanism governing CYP27 expression we cloned a 4.3 kb fragment of the 5' flanking region of the human CYP27 gene and constructed deletion mutants which were transfected into HepG2 cells. Functional assays showed that the -217/-10 nucleotide region from the translation start site (minimal promoter), devoid of TATA and CAAT boxes, contains all the elements for basal transcription. Foot-printing analysis of minimal promoter showed four protected regions (A-D). Regions A, B and D each contain one Sp1 binding site, and region C contains a HNF4 site. Electrophoretic mobility shift assays demonstrated that Sp1, Sp3 and HNF4 transcription factors bind these sites. Mutagenesis of any of these sites resulted in the loss of promoter activity. Co-transfection of the minimal promoter with Sp1 and Sp3 expression vectors transactivated CYP27 gene promoter in Drosophila SL2 cells, which lack endogenous Sp proteins. Transactivation of the minimal promoter was also observed in HeLa cells co-transfected with HNF4 expression vector. Therefore, Sp1, Sp3 and HNF4 co-operate in the expression of the human CYP27 gene in HepG2 cells.