RESUMO
Ultrastructural alterations of podocytes are closely associated with loss of glomerular filtration function. In the present study, we explored changes at the proteome level that paralleled the disturbances of podocyte architecture in the early stages of puromycin aminonucleoside (PA) nephrosis in vivo. Using two-dimensional fluorescence difference gel electrophoresis and vacuum matrix-assisted laser desorption/ionization mass spectrometry combined with postsource decay fragment ion analysis and high-energy collision-induced dissociation tandem mass spectrometry, 23 differentially expressed protein spots, corresponding to 16 glomerular proteins that are involved in various cellular functions, were unambiguously identified, and a subset was corroborated by Western blot analysis. The majority of these proteins were primarily related to fatty acid metabolism and redox regulation. Key enzymes of the mitochondrial beta-oxidation pathway and antioxidant enzymes were consistently down-regulated in PA nephrosis. These changes were paralleled by increased expression levels of CD36. PA treatment of murine podocytes in culture resembled these specific protein changes in vitro. In this cell system, the modulatory effects of albumin-bound fatty acids on the expression levels of Mn-superoxide dismutase in response to PA were demonstrated as well. Taken together, these results indicate that a disrupted fatty acid metabolism in concert with an impaired antioxidant defense mechanism in podocytes may play a role in the early stages of PA-induced lesions in podocytes.
Assuntos
Antioxidantes/metabolismo , Ácidos Graxos/metabolismo , Nefrose/fisiopatologia , Podócitos/metabolismo , Podócitos/ultraestrutura , Animais , Antimetabólitos Antineoplásicos/toxicidade , Western Blotting , Antígenos CD36/biossíntese , Antígenos CD36/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Imunofluorescência , Masculino , Microscopia Eletrônica de Transmissão , Nefrose/induzido quimicamente , Proteoma , Puromicina Aminonucleosídeo/toxicidade , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Superóxido Dismutase/metabolismoRESUMO
In a recent screening for genes down regulated in replicatively senescent human umbilical vein endothelial cells (HUVECs), we have isolated the novel protein SNEV. Since then SNEV has proven as a multifaceted protein playing a role in pre-mRNA splicing, DNA repair, and the ubiquitin/proteosome system. Here, we report that SNEV mRNA decreases in various cell types during replicative senescence, and that it is increased in various immortalized cell lines, as well as in breast tumors, where SNEV transcript levels also correlate with the survival of breast cancer patients. Since these mRNA profiles suggested a role of SNEV in the regulation of cell proliferation, the effect of its overexpression was tested. Thereby, a significant extension of the cellular life span was observed, which was not caused by altered telomerase activity or telomere dynamics but rather by enhanced stress resistance. When SNEV overexpressing cells were treated with bleomycin or bleomycin combined with BSO, inducing DNA damage as well as reactive oxygen species, a significantly lower fraction of apoptotic cells was found in comparison to vector control cells. These data suggest that high levels of SNEV might extend the cellular life span by increasing the resistance to stress or by improving the DNA repair capacity of the cells.
Assuntos
Neoplasias da Mama/genética , Senescência Celular/genética , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Regulação da Expressão Gênica , Proteínas Nucleares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/diagnóstico , Proliferação de Células , Células Cultivadas , Senescência Celular/fisiologia , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Enzimas Reparadoras do DNA , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Fatores de Processamento de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The need for standardized experimental conditions to gain relevant and reproducible results has increased the demand for well characterized continuously growing cell lines that exhibit the characteristics of their normal counterparts. Immortalization of normal human cells by ectopic expression of the catalytic subunit of human telomerase (hTERT) has shown to result in highly differentiated cell lines. However, the influence of the increased telomerase activity on the protein expression profile was not investigated so far. Therefore, we have immortalized human umbilical vein endothelial cells (HUVECs) by hTERT overexpression and compared them to their normal early passage and senescent counterparts. This study, including a proteomic approach, shows that ectopic hTERT expression leads to a stable growing cell line. Although these cells are highly differentiated, the protein expression profile of the cell line is different to that of normal early passage and senescent cells.
