IRE1α/RACK1 Axis Orchestrates ER Stress-elicited Cytoprotection From Ischemia Reperfusion Injury In Liver

Disturbance in the folding capacity of the endoplasmic reticulum(ER), caused by a variety of physiologic conditions changing, prompts a cellular stress condition known as ER stress(ERS). An intricate homeostatic adaptive response to tovercome the accumulation of unfolded protein which has been termed the unfolded protein response(UPR). The UPR alleviates ER stress by decreasing protein synthesis and increasing the expression of molecular chaperones that promote proper folding and cell recovery. However, prolonged ER stress and increasing protein loading on the ER, ultimately leading to cell death. The UPR is distinguished by the action of three signaling proteins named IRE1α, PERK, and ATF6. Under physiological conditions, these signaling proteins were activated and induce signal transduction events that alleviate the ERS or lead the cell death.ERS is involved in ischemic preconditioning(IPC), which protects various organs from ischemia/reperfusion(I/R) injury. However, the underlying molecular mechanisms have remained elusive. Here we show that IRE1α /RACK1 orchestrates ER stress-elicited cytoprotection from ischemia reperfusion injury in liver during IPC.In this study,we established an ER stress preconditioning model that protects rat liver from I/R injury using tunicamycin(TM) prior to partial hepatectomies. Serum aminotransferase levels and the ultra-structure of livers were scored. Levels of GRP78 and phosphorylated IRE1α were used to quantify ER stress. Interestingly, ischemic injury was alleviated by administration of TM, which activated IRE1α and up-regulated GRP78. The present study provides evidence that UPR is involved in ischemic preconditioning(IPC).Our previous study have showed that the interaction and cooperation between IRE1α and RACK1. To elusive the role of IRE1α /RACK1 in IPC, an ER stress preconditioning model was established with HL-7702(L-02) human liver cells by addition of TM to the culture medium prior to H/R injury. The results showed that TM at a concentration of 0.20 μg/ml in the culture medium for 48 hours before hypoxia reduced the apoptosis caused by the subsequent H/R injury. This protective effect was abolished by knock-down of RACK1 expression by use of si RNA. To probe the role of IRE1α and its signaling network in ER stress response, we carried out immunoprecipitation of Hep G2 cells treated with 0.20 μg/ml tunicamycin for 12 hours. Mass spectrometric identification revealed the co-precipitation of RACK1 and AMPK in the IRE1α fraction. To evaluate if AMPK and RACK1 interact with IRE1α in a stress-dependent manner, we performed comparative western blotting analyses using tunicamycin-treated and control Hep G2 cells. Quantitative analyses revealed that levels of RACK1 and phospho-AMPK are significantly elevated in IRE1 precipitates from tunicamycin-treated cells.We next examine the subcellular localization of IRE1α relative to RACK1 and AMPK in control and tunicamycin-treated cells.The results suggested that stress signaling induced dynamic changes of IRE1α and RACK1 distribution. Moreover, tunicamycin treatment elicits stress granule-like distribution of p T172-AMPK. Thus, we conclude that ER stress elicits a spatial reorganization of IRE1α complex containing active AMPK.ER stress preconditioning was applied to HL-7702 cells to establish the role of RACK1 in the protective mechanism against I/R injury. The results show that ER stress preconditioning provides protection against hepatic I/R injury and this protective effect was abolished by knock-down of RACK1 expression by use of si RNA. Moreover, the spliced form of XBP-1 was down-regulated in cells exposed to RACK1 si RNA.To further examine if ER stress preconditioning modulates cell survival activity, we carried out western blotting analyses to evaluate the status of BCL2, an anti-apoptotic modulator. To this end, Hep G2 cells were transiently transfected to suppress IRE1α with a si RNA followed by tunicamycin treatment. The si RNA transfection resulted in 82±5% of IRE1 reduction. This reduction of IRE1 induced a comparable suppression of phosphorylated BCL2(p T70) but not BCL2 protein level. The level of AMPK phosphorylation and activity is not changed in the absence of suppressed IRE1, which suggest that IRE1α activation does not act on the upstream of AMPK. Thus, we conclude that ER stress preconditioning prevents apoptotic process by phosphorylation of BCL2 via IRE1.In summary, we demonstrate that ER stress preconditioning induces an interaction of IRE1α interacts with RACK1 which may serve as an interacting hub for signaling molecules such as AMPK. Active AMPK1 kinase further phosphorylates IRE1α in a spatially regulated pattern. Active IRE1α phosphorylates Bcl-2 to exert anti-apoptotic and cytoprotective effects, providing mechanistic insight into a better understanding of spatiotemporal dynamics underlying ER stress preconditioning. Our findings provide novel insights for better understanding the molecular pathway underlying ER stress preconditioning-elicited cytoprotective effect in liver transplantation.