Detection of NADPH oxidase subunits NOX1 and NOX4 in lung adenocarcinoma A549 cells, and impact of NOX1 on Nrf2- and HIF-1-dependent gene regulation

摘要

NADPH oxidase 1 and 4 are ROS generating enzyme complexes that play an importantrole in cellular signaling. The core subunits of NADPH oxidase 1 and 4 are NOX1 andNOX4, respectively.The immuno-detection of NOX1 and NOX4 proteins is a commonly faced problem.Protein bands of different molecular weights suggested to represent NOX1 and NOX4have been described in previous studies. These bands could represent, for example,different NOX splice variants, posttranslational modifications, proteolytic products ornon-specifically immunoreacting proteins. Thus, well validated NOX1 and NOX4antibodies are critical for further functional and structural characterization of theseproteins. Based on this, the aims of the first part of the work were: 1. To test and to selectmost suitable antibodies for the detection of NOX1 and NOX4 in human adenocarcinomaA549, CaCo2 cells and HUVEC, 2. To determine the subcellular localization of NOX1and NOX4, 3. To identify putative NOX1 and NOX4 proteins by mass spectroscopic(MS) analysis.Employing several commercially available and custom made NOX1 and NOX4antibodies it was obvious that neither of the antibodies detected any common band.Further, by employing specific siRNAs, custom made antibodies termed NOX1wch andNOX4jh were identified as suitable for NOX1 and NOX4 detection. Using theseantibodies we also determined the presence of NOX1 and NOX4 in certain subcellularfractions. The results showed that both NOX1 and NOX4 were predominantly localizedin the cytoskeleton fraction of A549 cells. This observation was partially in contrast toprevious studies demonstrating the presence of endogenous NOX1 in the membranefraction of different cell types including those that we used.In addition to proteins of the predicted size, the NOX1wch and NOX4jh antibodies alsodetected other protein species of lower or higher molecular weight. To identify theseadditional bands and to confirm the identity of NOX1 and NOX4 full length proteins,purification and MALDI-TOF MS analysis was performed. However, neither of theanalysed proteins were identified as NOX-related which does not mean that this is not thecase. This might be explained, for example, by low abundance of NOX1 and NOX4proteins, or by technical limits of the protein purification and identification procedure.Furthermore, we determined that NOX1 predominates in ROS generation in comparisonto NOX4 in A549 cells which was in accordance with a higher expression level of NOX1mRNA than NOX4 mRNA in A549 cells.In conclusion, this part of the work identified NOX1 and NOX4 antibodies that aresuitable for the detection of NOX1 and NOX4 by Western blot. These antibodies wereemployed for the detection of NOX1 and NOX4 proteins in certain biochemical fractionsrepresenting different subcellular compartments. Furthermore, the open attempt for thepurification and identification of NOX1 and NOX4 proteins was performed, and theseresults may be considered as the basis for further proteomic experiments.NADPH oxidase 1 is a significant source of ROS in A549 cells. ROS are involved in theregulation of the transcription factors HIF-1 and Nrf2. Trx1 represents a target gene ofNrf2 that is known to induce HIF-1a. Intermittent hypoxia occurs in differentpathophysiological conditions and is characterized by fluctuations of oxygen and ROSlevels with impact on both HIF-1 and Nrf2.In this context, the aims of this part of the study were: 1. To analyse the expression ofNOX1, HIF-1a, Nrf2 and Trx1 under different conditions of oxygenation in A549 cells,2. To analyse possible cross-talk(s) between these components, particularly underconditions of intermittent hypoxia.Initial experiments revealed, that whereas HIF-1a was up-regulated both in continuousand intermittent hypoxias, the Nrf2, Trx1 and NOX1 as well as NOX1-derived ROS wereonly up-regulated in intermittent hypoxia. NOX1 was determined as crucial for enhancedROS production in intermittent hypoxia that in turn mediated induction of Nrf2 and Trx1.The regulation of Nrf2 and Trx1 by NOX1 was confirmed by both inhibition ofendogenous NOX1 and overexpression of recombinant NOX1 protein. Employing aproteosomal inhibitor, NOX1 was demonstrated to activate Nrf2 at the level of proteinstability. Subsequently, Nrf2-dependent Trx1 induction turned out to enhance HIF-1asignaling in intermittent hypoxia.In sum, we identified a signal transduction pathway that causes the enhancement of HIF-1a mediated by NOX1, Nrf2 and Trx1 in response to intermittent hypoxia.