RESUMEN
The plasma membrane Ca(2+)-ATPase (PMCA) located in the hepatocyte is a controversial molecule in itself since it displays different features to those regarded as canonical for P-type Ca(2+)-ATPases, and from which transcript expression as well as catalytic activity continues to be under active investigation. Our aim in this study was to explore at a first glance, pmca isoform distribution using isolated parenchymal and non-parenchymal cells from rat liver tissue. Expression of pmca transcripts was analyzed in fresh or cell-enriched culture preparations, confirming pmca1 and pmca4 as the housekeeping isoforms in all cell types studied (hepatocytes, Kupffer cells, and stellate cells). However, for the first time we show expression of pmca3 transcripts edited at two different sites in both hepatocytes and non-parenchymal cells. Interestingly, employing non-parenchymal cells we demonstrate the specific expression of pmca3e transcripts previously considered nearly exclusive of excitable tissues. Real-time PCR quantification shows a significant decrease of pmca3 transcripts in cultured Kupffer and hepatic stellate cells in comparison with fresh cells. The presence of pmca2 along with pmca3 in all liver cell types studied suggests that high affinity isoforms are relevant to the adequate management of calcium in liver tissue, particularly when hepatic cells become activated by diverse stimuli.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Isoenzimas/metabolismo , Hígado/enzimología , Animales , Secuencia de Bases , ATPasas Transportadoras de Calcio/genética , Membrana Celular/enzimología , Cartilla de ADN , Masculino , ARN Mensajero/genética , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
AII (angiotensin II) is a vasoactive peptide that plays an important role in the development of liver fibrosis mainly by regulating profibrotic cytokine expression such as TGF-beta (transforming growth factor-beta). Activated HSCs (hepatic stellate cells) are the major cell type responsible for ECM (extracellular matrix) deposition during liver fibrosis and are also a target for AII and TGF-beta actions. Here, we studied the effect of AII on the mRNA levels of TGF-beta isoforms in primary cultures of rat HSCs. Both quiescent and activated HSCs were stimulated with AII for different time periods, and mRNA levels of TGF-beta1, TGF-beta2 and TGF-beta3 isoforms were evaluated using RNaseI protection assay. The mRNA levels of all TGF-beta isoforms, particularly TGF-beta2and TGF-beta3, were increased after AII treatment in activated HSCs. In addition, activated HSCs were able to produce active TGF-beta protein after AII treatment. The mRNA expression of TGF-beta isoforms induced by AII required both ERK1/2 and Nox (NADPH oxidase) activation but not PKC (protein kinase C) participation. ERK1/2 activation induced by AII occurs via AT1 receptors, but independently of either PKC and Nox activation or EGFR (epidermal growth factor receptor) transactivation. Interestingly, AII has a similar effect on TGF-beta expression in quiescent HSCs, although it has a smaller but significant effect on ERK1/2 activation in these cells.
Asunto(s)
Angiotensina II/metabolismo , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , ARN Mensajero/análisis , Receptor de Angiotensina Tipo 1/genética , Factor de Crecimiento Transformador beta/genética , Animales , Western Blotting , Células Cultivadas , Matriz Extracelular/metabolismo , Luciferasas , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , ARN Mensajero/genética , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia ArribaRESUMEN
SnoN and Ski oncoproteins are co-repressors for Smad proteins and repress TGF-beta-responsive gene expression. The smad7 gene is a TGF-beta target induced by Smad signaling, and its promoter contains the Smad-binding element (SBE) required for a positive regulation by the TGF-beta/Smad pathway. SnoN and Ski co-repressors also bind SBE but regulate negatively smad7 gene. Ski along with Smad4 binds and represses the smad7 promoter, whereas the repression mechanism by SnoN is not clear. Ski and SnoN overexpression inhibits smad7 reporter expression induced through TGF-beta signaling. Using chromatin immunoprecipitation assays, we found that SnoN binds smad7 promoter at the basal condition, whereas after a short TGF-beta treatment for 15-30 min SnoN is downregulated and no longer bound smad7 promoter. Interestingly, after a prolonged TGF-beta treatment SnoN is upregulated and returns to its position on the smad7 promoter, functioning probably as a negative feedback control. Thus, SnoN also seems to regulate negatively the TGF-beta-responsive smad7 gene by binding and repressing its promoter in a similar way to Ski.
Asunto(s)
Regulación hacia Abajo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Proteína smad7/genética , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Unión Proteica , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Transducción de Señal , Factores de Tiempo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Proteasome pathway regulates TGF-beta signaling; degradation of activated Smad2/3 and receptors turns TGF-beta signal off, while degradation of negative modulators such as Ski and SnoN maintains the signal. We have found that anisomycin is able to downregulate Ski and SnoN via proteasome as TGF-beta does, but through a mechanism independent of Smad activation. The mechanism used by anisomycin to downregulate Ski and SnoN is also independent of MAPK activation and protein synthesis inhibition. TGF-beta signal was the only pathway described causing Ski and SnoN degradation, thus this new effect of anisomycin on endogenous Ski and SnoN proteins suggests alternative processes to downregulate these negative modulators of TGF-beta signaling.