Reactive oxygen species (ROS) initiate activation of the transcription factor NF-KB in a variety of cell systems. Perhaps the most potent biological source of ROS is the NADPH respiratory burst oxidase of phagocytic cells -neutrophils, eosinophils, monocytes, and macrophages. This multi- component enzyme catalyzes the formation of superoxide anion, which undergoes further reactions to generate a spectrum of ROS. Although phagocytes use this oxidase to kill ingested microorganisms, the products also mediate a broad range of biological oxidation reactions and some evidence exists for activation of NF-KB through this mechanism. It appears that the components of the phagocyte NADPH oxidase are also expressed in certain non-phagocytic cells. Moreover, we have recently discovered and cloned a widely expressed homologue (NOH-1) of the major catalytic component of the system. The basis of this proposal is a hypothesis that the products of NADPH oxidases of both the phagocyte and homologous types cause the activation of NF-KB, which in turn contributes to the changes in gene expression associated with aging.
Our specific aims are: 1) To explore using model systems and stimulated phagocytes, the ability of ROS generated by NADPH oxidase to activate NF-KB in various target cells, as assessed by gel mobility shift, nuclear translocation of GFP-tagged NF-KB, changes in transcription of specific genes, and expression of a heterologous reporter construct. The mechanisms responsible for major differences in susceptibility to ROS-dependent NF-KB activation among cell types will be explored. 2) To characterize the NAD(P)H oxidase activity of the recently- discovered homologue of the phagocyte catalytic subunit. NOH-1 will be expressed in the membranes of K562 human leukemia cells and other cell lines. Membrane fractions will be tested in a broken-cell oxidase system, along with cytosols from cells likely to express oxidase components/co- factors. This approach should permit a definition of the enzymatic activity of NOH-1 and its co-factor requirements. 3) To determine the role of the NOH-1 nonphagocyte oxidase system in cellular generation of ROS and activation of NF-KB. Cells that exhibit stable expression of functionally active NOH-1 will be developed and studied in parallel with cells that naturally express this protein. Mechanisms of oxidase activation, rates of ROS formation, and ability to activate NF-KB and modify gene expression will be determined. Collectively, these studies will establish a scientific basis for the potential role in aging of the activation of NF-KB by the products of NADPH oxidases of phagocytic and non-phagocytic cells.
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