Ultraviolet light (UV) induces phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2a) and inhibits global protein synthesis. Both eIF2 kinases PERK and GCN2 have been shown to phosphorylate eIF2a in response to UV-irradiation. However, the roles of PERK and GCN2 in UV-induced translation inhibition are controversial. The upstream signaling pathway(s) that leads to the activation of PERK or GCN2 remains unknown. In this research, evidences are provided to demonstrate that both PERK and GCN2 contribute to UVB-induced eIF2a phosphorylation in human keratinocyte (HaCaT) and mouse embryonic fibroblast (MEF) cells. Reducing expression of PERK or GCN2 by small interfering RNA (siRNA) decreases phosphorylation of eIF2a after UVB irradiation. The data also show that nitric oxide synthase (NOS)- mediated oxidative stress plays a role in regulation of eIF2a phosphorylation upon UVB irradiation. Treating the cells with the broad NOS inhibitor NG-Methyl-L-Arginine, the free radical scavenger N-Acetyl-L-Cysteine or the NOS substrate L-Arginine partially inhibits the UVB-induced eIF2a phosphorylation. The same treatments also partially rescue the translation inhibition induced by UVB irradiation. The results presented above led us to propose that NOS mediates UVB-induced eIF2a phosphorylation by activation of both PERK and GCN2 via oxidative-stress and L-Arginine starvation signaling pathways.
While NOS activation regulates translation initiation, it is not known if the NOS-mediated translation regulation affects NOS expression. In this study the impact of eIF2a phosphorylation on three NOS isoforms (nNOS, iNOS and eNOS) are investigated in both human keratinocyte (HaCaT) and mouse embryonic fibroblast (MEF) cells. Our results indicate that no apparent changes of nNOS and eNOS are observed. While iNOS is dynamically regulated in different time points post UVB irradiation. By comparing the results obtained from MEFS/S (wild type) and MEFA/A (contain a mutated site, 51 SàA, on eIF2a thus cannot be phosphorylated), it suggests that UVB-induced eIF2a phosphorylation negatively regulates the expressions of iNOS, on the translational level in a feedback manner.