CiGRP78 Resists Heavy Metal Ions-Induced ER Stress And Molecular Mechanism Of IKK?-Mediated UPR Pathway

The unfolded protein response?UPR?,which is activated in response to an accumulation of unfolded or misfolded proteins in endoplasmic reticulum lumen caused by stress,such as oxidative stress,bacterial infection,and virus invasion,is a regulatory system to restore cellular homeostasis in the endoplasmic reticulum?ER?.As an important member of Hsp70?heat shock protein 70?family,the 78-kDa glucose-regulated protein?GRP78?can enhance cell survival rates remarkably under thermal stress.Recent studies also demonstrated that the expression of GRP78enhances the cell survival under heavy metal stress.Heavy metal ion is one of the critical environmental pollutants accumulated in living organisms and causes toxic or carcinogenic effects once passed threshold levels.In this study,three most representative heavy metal ions,Pb²⁺,Hg²⁺and Cd²⁺,were used to stimulate Ctenopharyngodon idella kidney?CIK?cells.The results showed that cell viability under Pb²⁺,Hg²⁺and Cd²⁺stress decreased significantly.The longer and the greater the concentrations of stimulation from heavy metal ions,the higher the rate of cell death was observed.Among them,Hg²⁺is the most hazardous to cells.Under the same stress condition,Hg²⁺resulted in 50%of cell death,Cd²⁺(or Pb²⁺)led to 45%?or 35%?of cell death,respectively.Western immunoblotting indicated that C.idella GRP78?CiGRP78?protein expression level was enhanced obviously in CIK cells under Pb²⁺,Hg²⁺and Cd²⁺stress,meaning CiGRP78 is involved in heavy metal cytotoxicity.To further study the role of CiGRP78 in cytoprotection,we designed the siRNA against CiGRP78 and transfected it into CIK cells to silence endogenous CiGRP78.The viability rate of CIK cells transfected with or without siRNA incubated with HgCl₂ for 12 h showed a significant decrease from 50% to 21%.Our results showed that CiGRP78 protects cells against heavy metal stimuli to some extent.Heavy metal exposure impacts basic cellular processes and results in serious toxicological effects.Pb²⁺can activate the response to endoplasmic reticulum?ER?stress by protein denaturation,changing intracellular calcium homeostasis,and inducing cell death.As an ER retention protein,78-kDa glucoseregulated protein?GRP78?can relieve the Pb²⁺-induced ER stress and enhance cell viability.Here,we investigated the mechanism in which CiGRP78 exerted its protective function.Using metal ions affinity elution method and fluorescent spectral analysis,we showed that CiGRP78 could respectively form a complex with Calcium,Lead and Cadmium ions,especially with Lead ion in vitro.However,another ER retention protein CiGRP94could not bind to Pb²⁺,highlighting the functional differentiation might exist in CiGRP78 and CiGRP94 in regulating heavy metal cytotoxicity.Our results suggested that CiGRP78 might increase cellular tolerance to Pb²⁺via the direct interaction with it.The UPR pathways have been also reported to be involved in immune responses in mammals,including the classical NF-?B pathway.Recently,studies have shown that IKK?,a critical kinase in the NF-?B pathway,participates in inflammatory responses associated with UPR and plays an important role in ER stress-induced cell death.However,the molecular mechanism of their crosstalk remains to be elucidated.Previously,we demonstrated that IKK?also has some conserved functions between fish and human,as Ctenopharyngodon idella IKK??Ci IKK??can activate NF-?B pathway.In this study,we found that Ci IKK?level in nuclear was elevated under ER stress and CiIKK?can interact with grass carp XBP1S?CiXBP1S?,a key transcription factor in UPR.Consistently,fluorescent histochemical analysis of CIK cells indicated that Ci IKK?and CiXBP1S colocalized under ER stress.Furthermore,overexpression of CiIKK?in CIK cells enhanced ER stress tolerance by regulating UPR signaling and resulted in the significant increase of cell viability.These results provide a novel insight into molecular interaction between immune response and ER stress.