The Role Of The NDRG2 In Endoplasmic Reticulum Stress-mediated Cell Apoptosis And Hepatic Lipid Metabolism

Endoplasmic reticulum(ER)is the site for protein synthesis,folding,modification,assembly and secretion in eukaryocytes.It’s also involved in many intracellular physiological processes including lipid metabolism,calcium storage and carbohydrate metabolism.When cells suffer from endogenous or exogenous stimulation such as energy or nutrition deficiency,hypoxia,calcium disorder,oxidative stress,glycosylation inhibition or virus infection,cellular homeostasis is disrupted,leading to blockage of protein processing and transportations,and accumulation of unfolded or misfolded protein in the ER,thus disrupting the morphology and physiological function of ER,which is termed endoplasmic reticulum stress(ERS).To cope with such stress,cells devolop an adaptive reponse to retore ER function.Unfolded protein response(UPR)is the most important ER stress response and has been well investigated as for the signaling pathway.UPR activation and signal transduction is induced by three transmembrane proteins of ER,IRE1,PERK and ATF6,which are also known as ER stress sensors.Under non-stress conditions,the three sensors remain inactive by binding to the molecular chaperon GRP78(glucose-regulated protein of 78 kDa)/BIP(immunoglobulin-binding protein)inside ER.Upon ER stress,BIP dissociated from the three UPR sensors because of the competitive interaction of BIP with unfolded or misfolded proteins,leading to activation of the three UPR signaling pathway.Appropriate ERS triggers UPR and the expression of a series targe genes,which exert their roles in combination to decrese protein synthesis,promoting protein folding and degradation,so as to restore ER homeostasis and function.However,continuous or excessive ERS will promote cell dysfunction or cell apoptosis.Extensive studies have shown that ERS/UPS dysregulation is related to the initiation and progression of many diseases,i.e.,metabolic diseases,cancers,etc.However,the molecuar mechanism by which ERS/UPS is regulated and the functional effects are not thoroughly understood.Human NDRG2(N-Myc Downstream Regulated Gene 2)is a newly identified tumor suppressor gene,firstly reported by our group.NDRG2 belongs to the NDRG family,which consists four members,NDRG1-4.In recent years,studies from our lab and others’ have demontrated that NDRG2 is downregulated in many tumor tissues;overexpression of NDRG2 could inhibit the biological behavior of cancer cells,such as proliferation,invasion and apoptosis-resistance,chemoradiotherapy resistance,metabolic reprogramming and EMT,etc.NDRG2 is also involved in cellular response to various stresses,including hypoxia,DNA damaging drugs,tissue ischemia reperfusion,temperature variation,lipotoxicity or inflammation.These studies suggested that NDRG2 is not only a tumor suppressor,but also a stress response gene.However,it has not been reported as to whether or how NDRG2 is involved in ERS/UPR and the related physiological and pathological processes.In this study,we therefore investigateed the role of NDRG2 in ERS-mediated cell apoptosis and hepatic lipid metabolism.Firstly,human hepatocellular carcinoma SK-Hep-1 cells were treated with the classic ERS inducer-thapsigargin(Tg)or tunicamycin(Tm)for different time.Changes in expression of NDRG2 and ERS/UPR markers were detected by Western blot and real-time PCR.We found that NDRG2 was upregulated,along with induction of ERS/UPR markers,demonstrating that NDRG2 could be induced by ERS.This findings implicated that NDRG2 might be involved in ERS/UPR.To determine how NDRG2 regulates ERS/UPR,we constructed stable NDRG2-overexpressing SK-Hep-1 cells and Cherry-expressing cells as control.Cells were treated with Tg/Tm and then subjected to Western blot and real-time PCR.Among the three UPR branches,NDRG2 was found to affect PERK pathway,enhancing the expression of PERK downstream genes,ATF4 and CHOP.Therefore,NDRG2 might be a new regulator of UPR and promote PERK signal pathway.To confirm the conclusion above,Ndrg2 KO and WT mice were treated with normal feeding,high fat feeding,or intraperitoneal injection of Tm to established in vivo ERS models.The expression markers and key player of ERS/UPR in mouse liver tissues were determined.The results of Western blot and real-time PCR demonstrated that Perk downstream genes Atf4 and Chop,were slightly decreased in Ndrg2 KO mice compared with that of WT mice under basal condition,but such reduction became more significant under the condition of high fat feeding and Tm-injection treatment.This implies that Ndrg2 deficiency leads to an attenuated UPR.PERK downstream signal ATF4/CHOP plays a vital role in ERS-induced cell apoptosis,we therefore explored whether the regulation of PERK signal by NDRG2 affects cell fate.Upon Tg or Tm treatment,flow cytometry analysis showed that NDRG2 overexpression enhances apoptosis rate in SK-Hep-1 cells;but such effect was significantly compromised when ATF4 or CHOP were knocked down in NDRG2-overexpressing SK-Hep-1 cells by siRNA.TUNEL staining of mice liver also indicated that number of apoptosis cells was reduced in Ndrg2 KO mice compared with those in WT mice after Tm injection.These findings indicated that NDRG2 promotes ERS-induced apoptosis via activating PERK-ATF4-CHOP signal.It has been reported that ERS/UPR is involved in hepatic lipid metabolism directly or indirectly.This promoted us to ask whether NDRG2 affects lipid metabolism.By examing the lipids in the liver of Ndrg2 KO and WT mice,we found an obvious lipid accumulation in KO mice,demonstrated by oil red staining and triglyceride content examination.Furthermore,the real-time PCR analysis indicated that Pgc1 a was downregulated in the liver of Ndrg2 KO mice.Thus,the role of NDRG2 in hepatic lipid metabolism still needs to be investigated.In conclusion,our study demonstrated that NDRG2,as a new ERS responsive gene,is involved in ERS,and plays a vital role in ERS-induced cell apoptosis.Thus,this study revealed a new function of NDRG2,and enriched the mechanism of UPR regulation.