Hahn Nynke E, Meischl Christof, Wijnker Paul J M, Musters Rene J P, Fornerod Maarten, Janssen Hans W R M, Paulus Walter J, van Rossum Albert C, Niessen Hans W M, Krijnen Paul A J
Department of Pathology, VU University Medical Centre, De Boelelaan 1117, Amsterdam, The Netherlands.
Cell Physiol Biochem. 2011;27(5):471-8. doi: 10.1159/000329968. Epub 2011 Jun 15.
NADPH oxidases play an essential role in reactive oxygen species (ROS)-based signaling in the heart. Previously, we have demonstrated that (peri)nuclear expression of the catalytic NADPH oxidase subunit NOX2 in stressed cardiomyocytes, e.g. under ischemia or high concentrations of homocysteine, is an important step in the induction of apoptosis in these cells. Here this ischemia-induced nuclear targeting and activation of NOX2 was specified in cardiomyocytes.
The effect of ischemia, mimicked by metabolic inhibition, on nuclear localization of NOX2 and the NADPH oxidase subunits p22(phox) and p47(phox), was analyzed in rat neonatal cardiomyoblasts (H9c2 cells) using Western blot, immuno-electron microscopy and digital-imaging microscopy.
NOX2 expression significantly increased in nuclear fractions of ischemic H9c2 cells. In addition, in these cells NOX2 was found to colocalize in the nuclear envelope with nuclear pore complexes, p22(phox), p47(phox) and nitrotyrosine residues, a marker for the generation of ROS. Inhibition of NADPH oxidase activity, with apocynin and DPI, significantly reduced (peri)nuclear expression of nitrotyrosine.
We for the first time show that NOX2, p22(phox) and p47(phox) are targeted to and produce ROS at the nuclear pore complex in ischemic cardiomyocytes.
NADPH氧化酶在心脏基于活性氧（ROS）的信号传导中起重要作用。此前，我们已经证明，应激心肌细胞中催化性NADPH氧化酶亚基NOX2的（核周）核表达，例如在缺血或高浓度同型半胱氨酸情况下，是这些细胞凋亡诱导过程中的重要一步。在此，对缺血诱导的心肌细胞中NOX2的核靶向和激活进行了明确研究。
在大鼠新生心肌母细胞（H9c2细胞）中，采用蛋白质免疫印迹法、免疫电子显微镜和数字成像显微镜，分析由代谢抑制模拟的缺血对NOX2以及NADPH氧化酶亚基p22（phox）和p47（phox）核定位的影响。
缺血H9c2细胞的核组分中NOX2表达显著增加。此外，在这些细胞中发现NOX2与核孔复合体、p22（phox）、p47（phox）以及硝基酪氨酸残基（ROS生成的标志物）共定位于核膜。用夹竹桃麻素和二苯基碘鎓抑制NADPH氧化酶活性，可显著降低硝基酪氨酸的（核周）核表达。
我们首次表明，在缺血心肌细胞中，NOX2、p22（phox）和p47（phox）靶向核孔复合体并在该处产生活性氧。
