High oxidative stress adversely affects NFκB mediated induction of inducible nitric oxide synthase in human neutrophils: Implications in chronic myeloid leukemia.
Increasing evidence support bimodal action of nitric oxide (NO) both as a promoter and as an impeder of oxygen free radicals in neutrophils (PMNs), however impact of high oxidative stress on NO generation is less explored. In the present study, we comprehensively investigated the effect of high oxidative stress on inducible nitric oxide synthase (iNOS) expression and NO generation in human PMNs. Our findings suggest that PMA or diamide induced oxidative stress in PMNs from healthy volunteers, and high endogenous ROS in PMNs of chronic myeloid leukemia (CML) patients attenuate basal as well as LPS/cytokines induced NO generation and iNOS expression in human PMNs. Mechanistically, we found that under high oxidative stress condition, S-glutathionylation of NFκB (p50 and p65 subunits) severely limits iNOS expression due to its reduced binding to iNOS promoter, which was reversed in presence of DTT. Furthermore, by using pharmacological inhibitors, scavengers and molecular approaches, we identified that enhanced ROS generation via NOX2 and mitochondria, reduced Grx1/2 expression and GSH level associated with NFκB S-glutathionylation in PMNs from CML patients. Altogether data obtained suggest that oxidative status act as an important regulator of NO generation/iNOS expression, and under enhanced oxidative stress condition, NOX2-mtROS-NFκB S-glutathionylation is a feed forward loop, which attenuate NO generation and iNOS expression in human PMNs.
Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, India.
Abhishek Kumar
AK
Singh
Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, India.
Deepika
D
Awasthi
Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, India.
Megha
M
Dubey
Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, India.
Sheela
S
Nagarkoti
Department of Pathology, King George's Medical University, Lucknow, India.
Ashutosh
A
Kumar
Department of Transfusion Medicine, King George's Medical University, Lucknow, India.
Tulika
T
Chandra
Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, India.
Manoj Kumar
MK
Barthwal
Department of Clinical Haematology & Medical Oncology, King George's Medical University, Lucknow, India.
Anil Kumar
AK
Tripathi
Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, India. Electronic address: madhu_dikshit@cdri.res.in.
Madhu
M
Dikshit
D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000503:physiopathology*; D000074662:NADPH Oxidase 2 / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D018384:Oxidative Stress / Q000502:physiology*; D011401:Promoter Regions, Genetic; D017382:Reactive Oxygen Species / Q000378:metabolism
D016328:NF-kappa B; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D005978:Glutathione; D005033:Ethylmaleimide; D004229:Dithiothreitol
Inducible nitric oxide synthase; NFκB; Nitric oxide; Reactive oxygen species; S-glutathionylation