iNOS expression requires NADPH oxidase-dependent redox signaling in microvascular endothelial cells

Redox regulation of inducible nitric oxide synthase (iNOS) expression was investigated in lipopolysaccharide and interferon-γ (LPS + IFNγ)-stimulated microvascular endothelial cells from mouse skeletal muscle. Unstimulated endothelial cells produced reactive oxygen species (ROS) sensitive to inhibition of NADPH oxidase (apocynin and DPI), mitochondrial respiration (rotenone) and NOS (L-NAME). LPS + IFNγ caused a marked increase in ROS production; this increase was abolished by inhibition of NADPH oxidase (apocynin, DPI and p47phox deficiency). LPS + IFNγ induced substantial expression of iNOS protein. iNOS expression was prevented by the rintioxidant ascorbate and by NADPH oxidase inhibition (apocynin, DPI and p47phox deficiency), but not by inhibition of mitochondrial respiration (rotenone) and xanthine oxidase (allopurinol). iNOS expression also was prevented by selective antagonists of ERK. JNK, Jak2, and NFκB activation. LPS + IFNγ stimulated activation/phosphorylation of ERK, JNK, and jak2 and activation/degradation of IκB, but only the activation of JNK and Jak2 was sensitive to ascorbate, apocynin and p47phox deficiency. Ascorbate, apocynin and p47phox deficiency also inhibited the LPS + IFNγ-induced DNA binding activity of transcription factors IRFI and API but not NFκB. In conclusion, LPS+ IFNγ-induced NFκB activation is necessary for iNOS induction but is not dependent on ROS signaling. LPS -IFNγ-stimulated NADPH oxidase activity produces ROS that activate the JNK-API and Jak2-IRF1 signaling pathways required for iNOS induction. Since blocking either NFκB activation or NADPH oxidase activity is sufficient to prevent iNOS expression, they are separate targets for therapeutic interventions that aim to modulate iNOS expression in sepsis.