Regulation of NADPH Oxidase 5 by Protein Kinase C Isoforms

Abstract

NADPH oxidase5 (Nox5) is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKCα inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKCα and PKCε, enhanced by loss of PKCδ and the silencing of PKCθ expression was without effect. A constitutively active form of PKCα robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKCε potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKCα directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKCα activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKCα inhibitor, Go 6976. In summary, our study reveals that PKCα is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular complications of diabetes and other diseases with increased ROS production.

The Novel Anticancer Drug Hydroxytriolein Inhibits Lung Cancer Cell Proliferation via a Protein Kinase Cα– and Extracellular Signal-Regulated Kinase 1/2–Dependent Mechanism

“…Gelsolin protein levels increase when lung cancer cells receive differentiation stimuli (Jarrard et al, 1998), and we previously detected cancer (glioma) cell differentiation after membrane lipid remodeling as an intermediate step before programmed cell death (Terés et al, 2012). Fatty acids can induce ROS production through mitochondrial events (Schonfeld and Wojtczak, 2008) and by enhanced activity of the NAD(P)H-oxidase stimulated by PKC (Chen et al, 2014). ROS are known to produce apoptosis and to act as mediators in signal transduction pathways, such as the mitogen-activated protein kinase pathway (Cagnol and Chambard, 2010).…”

PKCα promotes generation of reactive oxygen species via DUOX2 in hepatocellular carcinoma

“…A previous study demonstrated that PKC inhibitor Ro318220 specifically inhibited PMA-stimulated DUOX2 phosphorylation and H 2 O 2 production in COS-7 cells, suggesting PKC is a critical regulator of DUOX2 phosphorylation [28]. Moreover, it also has been reported that PKCa can phosphorylate NOX5 and enhance its activity [29]. Therefore, it is possible that PKCa may promote DUOX2 expression and activity by stimulating its phosphorylation.…”

Estradiol augments while progesterone inhibits arginine transport in human endothelial cells through modulation of cationic amino acid transporter-1

“…The level of nitrite (NO2)/nitrate (NO 3) concentrations in the conditioned medium was measured using a nitric oxide detection kit (ENZO Life Sciences) according to the manufacturer's instructions. In brief, following filtration in a 10-kDa spin column (Biovision) 50 l of the culture medium were diluted with 50 l of reaction buffer containing 50 M L-N 6 -(1-iminoethyl)lysine hydrochloride [L-NIL, a selective inducible NOS (iNOS) inhibitor] and mixed with 25 l NADH and 25 l nitrate reductase.…”