c-Jun N-terminal kinase 2 phosphorylates endothelial nitric oxide synthase at serine 116 and regulates nitric oxide production.
ABSTRACT The c-Jun N-terminal kinases (JNKs) belonging to the mitogen-activated protein kinase (MAPK) superfamily play important roles in foam-cell formation, hypercholesterolemia-mediated endothelial dysfunction, and the development of obesity. Although decreased nitric oxide (NO) production via decreased phosphorylation of endothelial NO synthase at serine 1179 (eNOS-Ser(1179)) was reported to be partly involved in JNK2-derived endothelial dysfunction, JNK2 seems likely to be indirectly involved in this signaling pathway. Here, using bovine aortic endothelial cells, we examined whether JNK2 directly phosphorylated eNOS-Ser(116), a putative substrate site for the MAPK superfamily, and this phosphorylation resulted in decreased NO release. JNK inhibitor SP60012 increased NO release in a time- and dose-dependent manner, which was accompanied by increased eNOS-Ser(116) phosphorylation. Purified JNK2 directly phosphorylated eNOS-Ser(116)in vitro. Ectopic expression of dominant negative JNK2 repressed eNOS-Ser(116) phosphorylation and increased NO production. Coimmunoprecipitation and confocal microscopy studies revealed a colocalization of eNOS and JNK2. However, all these observed effects were not manifested when JNK1 probes were used. Overall, this study indicates that JNK2 is a physiological kinase responsible for eNOS-Ser(116) phosphorylation and regulates NO production.
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ABSTRACT: Chronic (>24 h) exposure of arsenite, an environmental toxicant, has shown the decreased nitric oxide (NO) production in endothelial cells (EC) by decreasing endothelial NO synthase (eNOS) expression and/or its phosphorylation at serine 1179 (eNOS-Ser(1179) in bovine sequence), which is associated with increased risk of vascular diseases. Here, we investigated the acute (<24 h) effect of arsenite on NO production using bovine aortic EC (BAEC). Arsenite acutely increased the phosphorylation of eNOS-Thr(497), but not of eNOS-Ser(116) or eNOS-Ser(1179), which was accompanied by decreased NO production. The level of eNOS expression was unaltered under this condition. Treatment with arsenite also induced reactive oxygen species (ROS) production, and pretreatment with a ROS scavenger N-acetyl-L-cysteine (NAC) completely reversed the observed effect of arsenite on eNOS-Thr(497) phosphorylation. Although protein kinase C (PKC) and protein phosphatase 1 (PP1) were reported to be involved in eNOS-Thr(497) phosphorylation, treatment with PKC inhibitor, Ro318425, and overexpression of various PKC isoforms did not affect the arsenite-stimulated eNOS-Thr(497) phosphorylation. In contrast, treatment with PP1 inhibitor, calyculin A, mimicked the observed effect of arsenite on eNOS-Thr(497) phosphorylation. Lastly, we found decreased cellular PP1 activity in arsenite-treated cells, which was reversed by NAC. Overall, our study demonstrates firstly that arsenite acutely decreases NO production at least in part by increasing eNOS-Thr(497) phosphorylation via ROS-PP1 signaling pathway, which provide the molecular mechanism underlying arsenite-induced increase in vascular disease.Biomolecules and Therapeutics 11/2014; 22(6):510-518. · 0.84 Impact Factor
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ABSTRACT: Tumor necrosis factor-α (TNFα) is a pro-inflammatory cytokine that causes endothelial dysfunction. Endocytosis of TNFα receptors (TNFR) precedes endosomal reactive oxygen species (ROS) production, which is required for NF-κB activation in vascular smooth muscle cells. It is unknown how endocytosis of TNFRs impacts signaling in endothelial cells. We hypothesized that TNFα-induced endothelial dysfunction is induced by both endosomal and cell surface events, including NF-κB and mitogen activated protein kinases (MAPKs) activation, and endocytosis of the TNFR modifies signaling. Mesenteric artery segments from C57BL/6 mice were treated with TNFα (10ng/ml) for 22h in tissue culture, with or without signaling inhibitors (dynasore for endocytosis, SP600125 for JNK, SB203580 for p38, U0126 for ERK) and vascular function was assessed. Endothelium-dependent relaxation to acetylcholine (ACh) was impaired by TNFα and dynasore exacerbated this, while JNK or p38 inhibition prevented these effects. In cultured endothelial cells from murine mesenteric arteries, dynasore potentiated JNK and p38, but not ERK phosphorylation and promoted cell death. NF-κB activation by TNFα was decreased by dynasore. JNK inhibition dramatically increased both the magnitude and duration of TNFα-induced NF-κB activation and potentiated intercellular adhesion molecule-1 (ICAM-1) activation. Dynasore still inhibited NF-κB activation in the presence of SP600125. Thus, TNFα-induced endothelial dysfunction is both JNK and p38-dependent. Endocytosis modulates the balance of NF-κB and MAPK signaling and inhibition of NF-κB activation by JNK limits this pro-proliferative signal, which may contribute to endothelial cell death in response to TNFα.AJP Heart and Circulatory Physiology 02/2014; · 4.01 Impact Factor
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ABSTRACT: Valproic acid (VPA) with inhibitory activity of histone deacetylase has been used in the treatment of epilepsy and bipolar disorder that are associated with cerebrovascular dysfunction. Because nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays a role in maintenance of vascular function, NO is likely to mediate VPA's drug effect, but its effect on NO production remains controversial. We investigated whether and how VPA regulates NO production in bovine aortic endothelial cells (BAEC) and mice. VPA increased NO production in BAEC, which was accompanied by decrease in phosphorylations of eNOS at serine 116 (eNOS-Ser(116)) and cyclin-dependent kinase 5 at tyrosine 15 (CDK5-Tyr(15)). Ectopic expression of p25, a CDK5 activator, restored the VPA-inhibited eNOS-Ser(116) phosphorylation. In silico analysis revealed that the CDK5-Tyr(15) residue might be a substrate for SH2 domain-containing protein tyrosine phosphatase 1 (SH-PTP1), and CDK5 actually interacted with SH-PTP1. VPA increased SH-PTP1 expression and its activity. Stibogluconate, a specific SH-PTP1 inhibitor, reversed the VPA-inhibited phosphorylations of CDK5-Tyr(15) and eNOS-Ser(116). Knockdown of SH-PTP1 using small interfering RNA also reversed all the observed VPA's effects. Finally, both serum NO level and acetylcholine-induced aortic relaxation increased in the VPA-medicated male mice. These increases were accompanied by increased SH-PTP1 expression and decreased phosphorylations of CDK5-Tyr(15) and eNOS-Ser(116) in mouse aortas. In conclusion, VPA increases NO production by inhibiting CDK5-Tyr(15)-eNOS-Ser(116) phosphorylation axis; this process is mediated by SH-PTP1. VPA may be useful in the treatment of NO-related cerebrocardiovascular diseases.Free Radical Biology and Medicine 08/2014; · 5.71 Impact Factor