Dataset Information

Direct Activation of NADPH Oxidase 2 by 2-Deoxyribose-1-Phosphate Triggers Nuclear Factor Kappa B-Dependent Angiogenesis.

ABSTRACT:

Aims

Deoxyribose-1-phosphate (dRP) is a proangiogenic paracrine stimulus released by cancer cells, platelets, and macrophages and acting on endothelial cells. The objective of this study was to clarify how dRP stimulates angiogenic responses in human endothelial cells.

Results

Live cell imaging, electron paramagnetic resonance, pull-down of dRP-interacting proteins, followed by immunoblotting, gene silencing of different NADPH oxidases (NOXs), and their regulatory cosubunits by small interfering RNA (siRNA) transfection, and experiments with inhibitors of the sugar transporter glucose transporter 1 (GLUT1) were utilized to demonstrate that dRP acts intracellularly by directly activating the endothelial NOX2 complex, but not NOX4. Increased reactive oxygen species generation in response to NOX2 activity leads to redox-dependent activation of the transcription factor nuclear factor kappa B (NF-?B), which, in turn, induces vascular endothelial growth factor receptor 2 (VEGFR2) upregulation. Using endothelial tube formation assays, gene silencing by siRNA, and antibody-based receptor inhibition, we demonstrate that the activation of NF-?B and VEGFR2 is necessary for the angiogenic responses elicited by dRP. The upregulation of VEGFR2 and NOX2-dependent stimulation of angiogenesis by dRP were confirmed in excisional wound and Matrigel plug vascularization assays in vivo using NOX2^-/- mice.

Innovation

For the first time, we demonstrate that dRP acts intracellularly and stimulates superoxide anion generation by direct binding and activation of the NOX2 enzymatic complex.

Conclusions

This study describes a novel molecular mechanism underlying the proangiogenic activity of dRP, which involves the sequential activation of NOX2 and NF-?B and upregulation of VEGFR2. Antioxid. Redox Signal. 28, 110-130.

SUBMITTER: Vara D

PROVIDER: S-EPMC5725637 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Direct Activation of NADPH Oxidase 2 by 2-Deoxyribose-1-Phosphate Triggers Nuclear Factor Kappa B-Dependent Angiogenesis.

Vara Dina D Watt Joanna M JM Fortunato Tiago M TM Mellor Harry H Burgess Matthew M Wicks Kate K Mace Kimberly K Reeksting Shaun S Lubben Anneke A Wheeler-Jones Caroline P D CPD Pula Giordano G

Antioxidants & redox signaling 20170918 2

<h4>Aims</h4>Deoxyribose-1-phosphate (dRP) is a proangiogenic paracrine stimulus released by cancer cells, platelets, and macrophages and acting on endothelial cells. The objective of this study was to clarify how dRP stimulates angiogenic responses in human endothelial cells.<h4>Results</h4>Live cell imaging, electron paramagnetic resonance, pull-down of dRP-interacting proteins, followed by immunoblotting, gene silencing of different NADPH oxidases (NOXs), and their regulatory cosubunits by sm ...[more]

PMID: 28793782

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Project description:In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40(phox), p47(phox), and p67(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40(phox) remains less well defined than those of p47(phox) and p67(phox). We investigated the biological role of p40(phox) in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40(phox) using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67(phox) levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40(phox) reduces both the maximal rate and total amount of ROS produced without altering the K(M) value of the oxidase for NADPH. Using a series of mutants, p47PX-p40(phox) chimeras, and deletion constructs, we found that the p40(phox) PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67(phox) requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40(phox), however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40(phox) can increase oxidase activity approximately 2.5-fold above that of wild-type p40(phox) but maintain the requirement for PX and SH3 domain function. We present a model where p40(phox) translocates p67(phox) to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.

Dataset Information

Direct Activation of NADPH Oxidase 2 by 2-Deoxyribose-1-Phosphate Triggers Nuclear Factor Kappa B-Dependent Angiogenesis.

Aims

Results

Innovation

Conclusions

Publications

Direct Activation of NADPH Oxidase 2 by 2-Deoxyribose-1-Phosphate Triggers Nuclear Factor Kappa B-Dependent Angiogenesis.

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