N-acetyl cysteine reverts the proinflammatory state induced by cigarette smoke extract in lung Calu-3 cells

Chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) are lethal pulmonary diseases. Cigarette consumption is the main cause for development of COPD, while CF is produced by mutations in the CFTR gene. Although these diseases have a different etiology, both share a CFTR activity impairment and proinflammatory state even under sterile conditions. The aim of this work was to study the extent of the protective effect of the antioxidant N-acetylcysteine (NAC) over the proinflammatory state (IL-6 and IL-8), oxidative stress (reactive oxygen species, ROS), and CFTR levels, caused by Cigarette Smoke Extract (CSE) in Calu-3 airway epithelial cells. CSE treatment (100 µg/ml during 24 h) decreased CFTR mRNA expression and activity, and increased the release of IL-6 and IL-8. The effect on these cytokines was inhibited by N-acetyl cysteine (NAC, 5 mM) or the NF-kB inhibitor, IKK-2 (10 µM). CSE treatment also increased cellular and mitochondrial ROS levels. The cellular ROS levels were normalized to control values by NAC treatment, although significant effects on mitochondrial ROS levels were observed only at short times (5´) and effects on CFTR levels were not observed. In addition, CSE reduced the mitochondrial NADH-cytochrome c oxidoreductase (mCx I-III) activity, an effect that was not reverted by NAC. The reduced CFTR expression and the mitochondrial damage induced by CSE could not be normalized by NAC treatment, evidencing the need for a more specific reagent. In conclusion, CSE causes a sterile proinflammatory state and mitochondrial damage in Calu-3 cells that was partially recovered by NAC treatment. Keywords: Cigarette smoke extract, Mitochondria, CFTR, ROS, COPD, Cystic fibrosi

APC senses cell-cell contacts and moves to the nucleus upon their disruption

Fil: Brocardo, Mariana G. Instituto de Investigaciones Bioquímicas. Fundación Campomar; Argentina.Fil: Bianchini, Michele. Instituto de Investigaciones Bioquímicas. Fundación Campomar; Argentina.Fil: Radrizzani, Martín. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Reyes, Gloria B. Instituto de Investigaciones Bioquímicas. Fundación Campomar; Argentina.Fil: Dugour, Andrea V. Instituto de Investigaciones Bioquímicas. Fundación Campomar; Argentina.Fil: Taminelli, Guillermo L. Instituto de Investigaciones Bioquímicas. Fundación Campomar; Argentina.Fil: Gonzalez Solveyra, César. Instituto de Investigaciones Bioquímicas. Fundación Campomar; Argentina.Fil: Santa-Coloma, Tomás A. Instituto de Investigaciones Bioquímicas. Fundación Campomar; Argentina.The adenomatous polyposis coli (APC) tumor suppressor protein is involved in the Wnt/wingless pathway, modulating beta-catenin activity. We report the development of a highly specific, chemically synthesized oligobody (oligonucleotide-based synthetic antibody), directed against the N-terminal region of APC. Using this reagent, we found that within 16 h of disrupting HT-29 cell-cell contacts by harvesting cells with trypsin/EDTA treatment and replating, APC was translocated from the cytoplasm to the nucleus. Five days after plating the cells, when the cells had returned to their normal confluent phenotype and cell-cell contacts were reestablished, APC returned to the cytoplasm. These results suggest that APC functions as part of a "sensor" system, and responds to the loss of cell-cell contacts by moving to the nucleus, and returning to the cytoplasm when the contacts are fully restored

NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl– concentration

Abstract: The impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) activity induces intracellular chloride (Cl– ) accumulation. The anion Cl– , acting as a second messenger, stimulates the secretion of interleukin-1β (IL-1β), which starts an autocrine positive feedback loop. Here, we show that NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) are indirectly modulated by the intracellular Cl– concentration, showing maximal expression and activity at 75 mM Cl– , in the presence of the ionophores nigericin and tributyltin. The expression of PYD and CARD domain containing (PYCARD/ASC) remained constant from 0 to 125 mM Cl– . The CASP1 inhibitor VX-765 and the NLRP3 inflammasome inhibitor MCC950 completely blocked the Cl– - stimulated IL-1β mRNA expression and partially the IL-1β secretion. DCF fluorescence (cellular reactive oxygen species, cROS) and MitoSOX fluorescence (mitochondrial ROS, mtROS) also showed maximal ROS levels at 75 mM Cl– , a response strongly inhibited by the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase (NOX) inhibitor GKT137831. These inhibitors also affected CASP1 and NLRP3 mRNA and protein expression. More importantly, the serum/glucocorticoid regulated kinase 1 (SGK1) inhibitor GSK650394, or its shRNAs, completely abrogated the IL-1β mRNA response to Cl– and the IL-1β secretion, interrupting the autocrine IL-1β loop. The results suggest that Cl– effects are mediated by SGK1, in which under Cl– modulation stimulates the secretion of mature IL-1β, in turn, responsible for the upregulation of ROS, CASP1, NLRP3 and IL-1β itself, through autocrine signalling