NADPH oxidase promotes NF-κB activation and proliferation in human airway smooth muscle

Abstract

Evidence is rapidly accumulating that low-activity-reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases homologous to that in phagocytic cells generate reactive oxygen species as signaling intermediates in both endothelium and vascular smooth muscle. We therefore explored the possibility of such an oxidase regulating growth of airway smooth muscle (AWSM). Proliferation of human AWSM cells in culture was inhibited by the antioxidants catalase and N-acetylcysteine, and by the flavoprotein inhibitor diphenylene iodonium (DPI). Membranes prepared from human AWSM cells generated superoxide anion (O 2 Ϫ ) measured by superoxide dismutase-inhibitable lucigenin chemiluminescence, with a distinct preference for NADPH instead of reduced nicotinamide adenine dinucleotide as substrate. Chemiluminescence was also inhibited by DPI, suggesting the presence of a flavoprotein containing oxidase generating O 2 Ϫ as a signaling molecule for cell growth. Examination of human AWSM cells by reverse transcriptase-polymerase chain reaction consistently demonstrated transcripts with sequences identical to those reported for p22 phox . Transfection with p22 phox antisense oligonucleotides reduced human AWSM proliferation. Inhibition of NADPH oxidase activity with DPI prevented serum-induced activation of nuclear factor-B (NF-B), and overexpression of a superrepressor form of the NF-B inhibitor IB␣ significantly reduced human AWSM growth. These findings suggest that an NADPH oxidase containing p22 phox regulates growth-factor responsive human AWSM proliferation, and that the oxidase signals in part through activation of the prototypical redox-regulated transcription factor NF-B. superoxide anion; diphenylene iodonium; p22 phox EVIDENCE IS RAPIDLY ACCUMULATING that low-activity-reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases homologous to that in phagocytic cells generate reactive oxygen species (ROS) as signaling intermediates in both endothelium and vascular smooth muscle (23). In the arterial circulation, ROS from these oxidases play important roles in regulation of blood pressure and in arterial wall inflammation and vascular smooth muscle proliferation characteristic of atherosclerosis (23). Recently, a C 242 to T transition of the p22 phox component of the oxidase, resulting in a histidine (H) 72 to tyrosine (Y) mutation, has even been associated with accelerated progression of coronary disease (15).In phagocytes, the NADPH oxidase consists of two membrane proteins, gp91 phox and p22 phox, that bind a flavin adenine nucleotide and form a unique cytochrome with a redox midpoint potential of Ϫ245 mV (4). At least two and possibly three cytosolic peptides (p47 phox , p67 phox , and p40 phox ) are also essential, and several other cytosolic components participate, including the small GTPases Rac1 or Rac2. The cytochrome appears to contain all the factors necessary for transporting electrons from the donor substrate NADPH via flavin adenine nucleotide and then heme to generate superoxide anion (O 2 Ϫ...

Regulation of Airway Mucin Gene Expression

“…The generation of ROS by NE appears to be mediated by different types of NADPH oxidases (77,78) and may also require PKC activation (74). This may mean that ROS act upstream of NF-κB in MUC5AC expression because ROS activation of NF-κB in airway epithelial and smooth muscle cells has been observed (79)(80)(81).…”

Airway Smooth Muscle Growth in Asthma: Proliferation, Hypertrophy, and Migration

“…Rac1 constitutes part of the NADPH oxidase complex that generates superoxide and H 2 O 2 (74,75). Intracellular H 2 O 2 is increased after mitogen treatment of rat tracheal myocytes (76), bovine tracheal myocytes (73), and human bronchial smooth muscle cells (77). Accordingly, treatment with antioxidants attenuates both mitogen-activated cyclin D1 expression and DNA synthesis in these cells (73,76,77).…”

“…Intracellular H 2 O 2 is increased after mitogen treatment of rat tracheal myocytes (76), bovine tracheal myocytes (73), and human bronchial smooth muscle cells (77). Accordingly, treatment with antioxidants attenuates both mitogen-activated cyclin D1 expression and DNA synthesis in these cells (73,76,77). Finally, in bovine cells, Rac1 induces transactivation of the cyclin D1 promoter CREB/ATF2 binding site, which is attenuated by antioxidants (71).…”

NADPH oxidase p22phox gene variants are associated with systemic oxidative stress biomarker responses to exercise training

“…As an adaptation to acute exercise-induced oxidative stress, the increased level of ROS induces antioxidant enzyme gene expression via an intracellular signaling pathway, which involves the redox regulated transcription factor, NF-B (17). Furthermore, using p22phox knockout mice, it has been demonstrated that the NADPH oxidase system plays a critical role in NF-B activation (4). Therefore, the NADPH oxidase system is a potential mechanism through which exercise training may modulate systemic oxidative stress levels.…”