The Role of Nitric-oxide Synthase in the Regulation of UVB Light-induced Phosphorylation of the α Subunit of Eukaryotic Initiation Factor 2

Abstract

UV light induces phosphorylation of the ␣ subunit of the eukaryotic initiation factor 2 (eIF2␣) and inhibits global protein synthesis. Both eIF2 kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control of nonderepressible protein kinase 2 (GCN2), have been shown to phosphorylate eIF2␣ in response to UV irradiation. However, the roles of PERK and GCN2 in UV-induced eIF2␣ phosphorylation are controversial. The one or more upstream signaling pathways that lead to the activation of PERK or GCN2 remain unknown. In this report we provide data showing that both PERK and GCN2 contribute to UV-induced eIF2␣ phosphorylation in human keratinocyte (HaCaT) and mouse embryonic fibroblast cells. Reduction of expression of PERK or GCN2 by small interfering RNA decreases phosphorylation of eIF2␣ after UV irradiation. These data also show that nitric-oxide synthase (NOS)-mediated oxidative stress plays a role in regulation of eIF2␣ phosphorylation upon UV irradiation. Treating the cells with the broad NOS inhibitor N G -methyl-L-arginine, the free radical scavenger N-acetyl-L-cysteine, or the NOS substrate L-arginine partially inhibits UV-induced eIF2␣ phosphorylation. The results presented above led us to propose that NOS mediates UV-induced eIF2␣ phosphorylation by activation of both PERK and GCN2 via oxidative stress and L-arginine starvation signaling pathways.UV irradiation inhibits translation initiation through activation of kinases that phosphorylate the ␣-subunit of eukaryotic initiation factor 2 (eIF2␣).2 Two eIF2␣ kinases, double strand RNA-dependent protein kinase-like ER kinase (PERK) and general control of amino acid biosynthesis kinase (GCN2), are known to phosphorylate the serine 51 of eIF2␣ in response to UV irradiation (1-4). However, the one or more upstream pathways that activate eIF2␣ kinase(s) upon UV irradiation are not known. In this report, we provide evidence that UV-induced nitric-oxide synthase (NOS) activation and nitric oxide (NO ⅐ ) production regulate both PERK and GCN2 activation upon UVB irradiation.Expression of inducible nitric-oxide synthase in a mouse macrophage cell line leads to the phosphorylation of eIF2␣ and inhibition of translation (5). In cultured neuronal and pancreatic cell lines, production of NO ⅐ and peroxynitrite (ONOO Ϫ ) induces endoplasmic reticulum (ER) stress, which activates PERK and results in cell dysfunction and apoptosis (6 -9). Cytokine-stimulated inducible nitric-oxide synthase activation in astrocytes depletes L-arginine and activates GCN2, which phosphorylates eIF2␣ (10). UV irradiation also activates NOS and elevates cellular NO ⅐ (11-13). However, the UV-induced NOS activation and NO ⅐ production have never been shown to be related to the activation of eIF2␣ kinase(s). Now we demonstrate that UV-induced activation of NOS mediates the activation of both PERK and GCN2, which coordinately regulate the phosphorylation of eIF2␣. EXPERIMENTAL PROCEDURESCell Culture-HaCaT cells (kindly provided by Dr. Hongtao Yu, Jackson State University) wer...

The Role of Constitutive Nitric-oxide Synthase in Ultraviolet B Light-induced Nuclear Factor κB Activity

“…6, lanes 11-13 versus lanes 5-7). These results agreed with our previous study suggesting that cNOS-mediated IB reduction after UVB irradiation is independent of ubiquitin and proteasome pathway (15). IKK␣ phosphorylates IB and promotes its degradation via ubiquitin and proteasome pathway (23)(24)(25)35).…”

“…Our previous studies also showed that UVB radiation induced an immediate activation of cNOS, which mediated the activation of eIF2␣ kinases PERK and GCN2 (15,33). In this study, we demonstrated that cNOS activation contributed to the activation of NF-B post-UVB irradiation.…”

“…Immediately after UVB radiation, in the first pathway, the coupled cNOS-catalyzed NO⅐ production depletes L-Arg, which leads to the activation of GCN2, and in the second pathway, the uncoupled cNOS-catalyzed O 2 . production rapidly reacts with NO⅐ to form ONOO Ϫ and activates PERK (15). To further determine the mechanism of cNOS-mediated NF-B activation, we examined the effect of SNAP, a NO⅐ donor (19), on the protective effect of L-NAME on UVBinduced IB reduction.…”

Polypeptide from Chlamys farreri suppresses ultraviolet-B irradiation-induced apoptosis through restoring ER redox homeostasis, scavenging ROS generation, and suppressing the PERK-eIF2a-CHOP pathway in HaCaT cells

“…Farrukh et al have declared that UVB irradiation induces ER stress and PERK-eIF2a pathway only in Hs68 fibroblasts but not in HaCaT cells [8]. However, the studies from Lu et al was in contrast with these findings and showed that eIF2a, the downstream factor of PERK, could be phosphorylated by UVB irradiation in HaCaT cells [23,24]. Therefore, further in-depth studies are still needed to clarify the association between UVB irradiation and ER stress-induced PERK-eIF2a pathway activation in the HaCaT cells.…”

The roles of nitric oxide synthase and eIF2alpha kinases in regulation of cell cycle upon UVB-irradiation

“…In a recent paper published in the September issue of The Journal of Biological Chemistry , we provided evidence that UVB activates constitutive nitric oxide synthase (cNOS), which coordinately regulates the activation of both PERK/GCN2 and sequentially the phosphorylation of eIF2α in human keratinocytes HaCaT 29. We demonstrated that UVB-activated NOS rapidly generates NO • from L-Arg, which had a concentration of less than 2.5 µM in HaCaT cells.…”