The Role Of EIF2α Phosphorylation In UVA Irradiation-Induced The Pathway Of Nrf2-HO-1 Via Oxidative Damage In JB6 Cells

Background:It’s now clear that UV irradiation is a crucial environmental factor resulting in various skin diseases. primarily through the activation of different cell signaling pathways with altered gene expression and reprogrammed protein expression. Such an important translational control mechanism is executed by eukaryotic initiation factor 2α subunit(eIF2α). UV irradation as one of oxidative stress can induce phosphorylation of eIF2α. Phosphorylation of eIF2α could inhibit the synthesis of proteins, and renew the homeostasis of endoplasmic reticulum(ER) through altered production of preteins. Excessive and continuous ER stress can easily phosphorylated eIF2α and then activate the CHOP and Caspase series, lead to cell death finally. UVA generated oxidative stress that leads to the strong up-regulation of Nrf2 and heme oxygenase-1(HO-1). UVA generated ROS can activate Nrf2 and promote migrate to nucleus to take its position antioxidant response elements(ARE), which can initiate the downstream phase II detoxifying enzymes,such as:(HO-1, NQO1, and SOD etc.). The expression of HO-1 is controlled by Nrf2-Bach1 oxidative stress response pathway. HO-1 is the first and the rate limiting enzyme which catalyzes the degradation of heme to carbon monoxide(CO), bilirubin and Fe. HO-1 has an important influence an antioxidant and anti-inflammatory. Both Nrf2-HO-1 induction and eIF2α activation are cellular responses to oxidative stress. However, UVA irradiation induced effects of eIF2α phosphorylation on the Nrf2-HO-1 pathway are still not in knowledge. Thus, this led us to invest the role of eIF2α phosphorylation in UVA irradiation-induced pathway of Nrf2- HO-1 via oxidative damage in JB6 cells and its function in cells response to ER stress. Objects:We aimed to explore the oxidative stress damaging effects of different doses of UVA irradiation(50, 100, 150, 200, 250 KJ/m2) on JB6 cells. In addition, investigated whether UVA can induce Ser51 phosphorylation of eIF2α in a dose- and time-dependent cell response fashion. We also verify UVA can activate Nrf2-HO-1 oxidative stress response pathway. In order to know the relationship between phosphorylation of eIF2α and the Nrf2-HO-1 pathway. we choose Salubrinal(Sal). which is a potent inhibitor of eIF2α dephosphorylation to maintain the eIF2α phosphorylation in homeostasis, while GSK2606414 can inhibit eIF2α phosphorylation. Finally chosen the optimal concentrations of salubrinal(3μM) and GSK2606414(0.5μM). The cells were pretreated with 3.0 μM Salubrinal and 0.5 μM GSK2606414 and then exposed to 150 kJ/m2 UVA irradiation to collect the RNA and protein at different time points to explore the relationship between phosphorylation of eIF2α with Nrf2-HO-1 pathway. The oxidative stress damaging effects and cell cycle assays were done to accessed by flowcytometery on control, salubrinal group, GSK2606414 group, UVA irradiation group, and combined salubrinal/GSK2606414 with UVA irradiation groups in order to explore the effect of phosphorylation of eIF2α. Methods:MTS assay was used for detecting JB6 cell proliferation, cytomorphological changes were observed by immunofluorescent staining assay. QT-PCR for the detection of Nrf2,Bach1 and HO-1 gene expression. Western blot technique was applied to detect protein levels including Grp78, P-eIF2α, eIF2α, Nrf2, Bach1 and HO-1etc. Flow cytometry analyses were used for detecting cell cycle arrest. Results: 1. UVA irradiation caused oxidative damage in JB6 cells in a dose dependent manner.Compared with the control, there is no any significant difference in cell morphology and cell viability after less doses of 50 and 100 kJ/m2 of UVA irradiation(P>0.05). Obviously there is some increased oxidative damage with the increased dosage of UVA. 2. UVA induce Ser51 phosphorylation of eIF2α in a dose- and time-dependent cellresponse fashion.It was found that irradiation of JB6 cell with UVA, phosphorylation of eIF2α occurs as a response to stimuli. The phosphorylation of eIF2α in a dose-dependent manner with increasing dosages of UVA. However, 250 kJ/m2 UVA irradiation compared to 200 kJ/m2 group, the phosphorylation of eIF2α decreased. We detected the protein level of P-eIF2α after 150 kJ/m2 UVA irradiation at different time points, which speculated that irradiation of JB6 cell with UVA stimuli eIF2α phosphorylates in a time-dependent manner. 3. UVA irradiation activated Nrf2-HO-1 pathway in a dose- and time-dependent cellresponse fashion.Compare to control group, UVA irradiation activated Nrf2-HO-1 oxidative stress response pathway with the increase in UVA dose. the expression of Nrf2 mRNA and protein levels were significantly increased. But at 250 kJ/m2 UVA irradiation there has a little decline in expression levels compared to 200 kJ/m2 UVA dose. Similar to Nrf2, UVA irradiation induce HO-1 expression in a dose-dependent manner both for mRNA and in protein expression level. Furthermore, we detected the protein and mRNA level of Nrf2 and HO-1 expressed following 150 kJ/m2 UVA irradiation at different time points. which further increased both Nrf2 and HO-1 expression in a time-dependent. The peak highest protein expression of both Nrf2 and HO-1 was observed at 12 h post UVA irradiation. 4. The effect of salubrinal or GSK2606414 treatment to UVA irradiated cells about phosphorylation of eIF2αWe selected the optimum concentration of Salubrinal(3μM) and GSK2606414(0.5μM) by measuring cell viability and western blot. There is no cytotoxicity no matter(0-10μM) salubrinal or(0-8μM) GSK2606414 was used. Salubrinal, a selective inhibitor of eIF2α dephosphorylation, may increased the eIF2α phosphorylation in a dose dependent manner. But 5μM concentration of salubrinal, the phosphorylation of eIF2α was decreased compared to 3μM salubrinal. On the other hand GSK2606414, a selective inhibitor of P-PERK, inhibited the eIF2α phosphorylation at the optimum concentration of 0.5 μM without any dose dependence.5. The change of eIF2α phosphorylation influence Nrf2-HO-1 pathway.Compare to 150 kJ/m2 UVA group, salubrinal(3μM) pretreated exposed to 150 kJ/m2 UVA irradiation cells could increased the phosphorylation of eIF2α and the expression of Grp78. GSK2606414(0.5μM) pretreatment following 150 kJ/m2 UVA irradiation, have decreased the phosphorylation of eIF2α and the expression Grp78. Salubrinal pretreatment combined with UVA group may delayed the expression of Nrf2, Bach1 and HO-1. GSK2606414 pretreated cells combined with UVA exposure may further advanced the expression of Nrf2, Bach1 and HO-1. 6. The change of eIF2α phosphorylation influence cell cycle.Comparing with control, there is not any significant difference in cell morphology and cell viability with Salubrinal group pretreated and GSK2606414 pretreated groups. Compared to 150 kJ/m2 UVA group, 3μM Salubrinal pretreated following 150kJ/m2 UVA irradiation, may increased cell viability and protect cell morphology. GSK2606414(0.5μM) pretreatment with 150 kJ/m2 UVA irradiation decreased cell viability and suppressed cell morphology. Moreover, compare to control, 3μM SAL+150 kJ/m2 UVA irradiation increased the S phase in cell cycle, while 0.5μM GSK+150 kJ/m2 UVA irradiation suppressed the S phase in cell cycle, but pretreatment of GSK2606414 alone has no any significant influence. Conclusion:UVA irradiation creats a stimuli, which can induce eIF2α phosphorylation in a doseand time-dependent manner. UVA irradiation can also induce the expression of important transcription factor Nrf2 and HO-1 in a dose and time dependent manner. Salubrinal, as a selective inhibitor of eIF2α dephosphorylation can increase this process in a dose dependent way. Anbother drug GSK2606414 a selective inhibitor of P-PERK, inhibited the eIF2α phosphorylation at the optimum concentration of 0.5 μM is not dose dependent. Alteration in the expression of P-eIF2α will influence the pathway of Nrf2- HO-1. By controlled the phosphorylation of eIF2α, may protect the cells against oxidative damage and promote cell proliferation. Inhibition of eIF2α phosphorylation can increase the oxidative damage and suppressed cell proliferation, which is not a desirable significant influence.