Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase-Dependent Activation of Phosphoinositide 3-Kinase and p38 Mitogen-Activated Protein Kinase Signal Pathways Is Required for Lipopolysaccharide-Induced Microglial Phagocytosis

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

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The Farnesyltransferase Inhibitor LB42708 Suppresses Vascular Endothelial Growth Factor-Induced Angiogenesis by Inhibiting Ras-dependent Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase/Akt Signal Pathways

Molecular Pharmacology ◽

10.1124/mol.110.063586 ◽

2010 ◽

Vol 78 (1) ◽

pp. 142-150 ◽

Cited By ~ 22

Author(s):

Chun-Ki Kim ◽

Yoon Kyung Choi ◽

Hansoo Lee ◽

Kwon-Soo Ha ◽

Moo-Ho Won ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Protein Kinase ◽

Mitogen Activated Protein Kinase ◽

Signal Pathways ◽

Vascular Endothelial ◽

Phosphatidylinositol 3 Kinase ◽

Farnesyltransferase Inhibitor ◽

Mitogen Activated Protein ◽

Endothelial Growth Factor

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00206.2001 ◽

2002 ◽

Vol 282 (4) ◽

pp. L782-L795 ◽

Cited By ~ 72

Author(s):

Sukhdev S. Brar ◽

Thomas P. Kennedy ◽

Anne B. Sturrock ◽

Thomas P. Huecksteadt ◽

Mark T. Quinn ◽

...

Keyword(s):

Smooth Muscle ◽

Nadph Oxidase ◽

Airway Smooth Muscle ◽

Nicotinamide Adenine Dinucleotide ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Nuclear Factor Κb ◽

Nicotinamide Adenine ◽

Phagocytic Cells ◽

Diphenylene Iodonium ◽

Reduced Nicotinamide Adenine Dinucleotide

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[Formula: see text]) 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[Formula: see text] 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 p22phox. Transfection with p22phoxantisense oligonucleotides reduced human AWSM proliferation. Inhibition of NADPH oxidase activity with DPI prevented serum-induced activation of nuclear factor-κB (NF-κB), and overexpression of a superrepressor form of the NF-κB inhibitor IκBα significantly reduced human AWSM growth. These findings suggest that an NADPH oxidase containing p22phoxregulates growth-factor responsive human AWSM proliferation, and that the oxidase signals in part through activation of the prototypical redox-regulated transcription factor NF-κB.

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