Apelin-13 Attenuates ER Stress-mediated Neuronal Apoptosis By Activating G?_i/G?_q-CK2? Signaling In Ischemic Stroke

Ischemic stroke is mostly caused by thrombosis,which causes the interruption of oxygen and nutrients in the blood supply area of the obstructed blood vessels.Neurons in the ischemic core are unable to save because of acute oxygen-glucose deprivation-induced necrosis,while the neurons in the penumbra still sustain a certain level of metabolism which unexpectedly would bring in progressive apoptosis during reperfusion,making it possible to rescue those neurons.Therefore apoptosis induced from ischemic stroke is a focus of current research.It is of great practical significance to develop a treatment that can effectively protect neurons against apoptosis.Ischemia-reperfusion injury could activate endoplasmic reticulum stress(ERS)and further stimulate the unfolded protein response(UPR).If ERS does not exceed the cellular compensatory capacity,the cell can get rid of ERS through UPR,thus cellular apoptosis will not be induced;but,if the injury is so strong that ERS is too robust and prolonged,the proapoptotic factor CHOP(C/EBP-homologous protein CHOP/GADD153)will be activated which in turn induces apoptosis.Protein kinase CK2(Casein kinase II,CK2)is a multifunctional,highly conserved serine/threonine protein phosphotransferase.CK2holoenzyme consists of catalytic subunits and regulatory subunits.Its activity mainly comes from catalytic subunit CK2?.CK2 showed pro-survival effect and resistance to apoptosis in a variety of cell models.Upregulation of CK2 in tumor cells makes the cells insensitive to proapoptotic stimulus,while inhibition of CK2 induces ERS in tumor cells then finally apoptosis.But whether CK2 is involved in the modulation of ERS in the nervous system has not been reported.This study will focus on one of the ERS branches GRP78/e IF2?-ATF4-CHOP signaling pathway,investigate whether GRP78/eIF2?-ATF4-CHOP pathway is induced by the cerebral I/R injury,and explore whether CK2 is involved in modulating its activation in neurons.APJ(putative receptor protein related to the angiotensin receptor AT1)is a G-protein-coupled receptor,one of whose endogenous ligands is Apelin.Recently it has been reported that Apelin plays a neuroprotective role against excitotoxicity,oxidative stress,serum deprivation-induced apoptosis,ischemia/reperfusion-induced apoptosis,but its mechanism is not yet clear.This study will particularly stress on whether Apelin/APJ is involved in regulating the expression of CK2,modulating the activation of ERS and investigating the possible signal transduction mechanism.This study first detected the expression of all the molecules of GRP78/eIF2?-ATF4-CHOP pathway at different time points of MCAO/R(middle cerebral artery occlusion/reperfusion)by Western blot.We found that the important branch of ERS GRP78/eIF2?-ATF4-CHOP pathway was activated by MCAO/R.GRP78,as the upstream chaperone,got to the peak at MCAO-2h/R-12h;the phosphorylation level of eIF2?,expression of ATF4 and CHOP all increased significantly,and reached the maximum at MCAO-2h/R-24h.In addition,we detected the expression of catalytic subunit CK2?and regulatory subunit CK2?at different time points of MCAO/R by Western blot,and found that CK2?and CK2?decreased progressively with the development of MCAO/R.It was also confirmed that GRP78/eIF2?-ATF4-CHOP pathway was activated by oxygen-glucose deprivation/reoxygenation(OGD/R)model of primary cortical neurons,accompanied by down-regulation of CK2?and CK2?.Considering that CK2 can resist ERS and apoptosis in other cell lines,it is presumed that MCAO/R-induced or OGD/R-induced CK2 inhibition may be the incentive of ERS.Next,we studied the distribution of catalytic subunit CK2?in neurons by immunocytochemical staining,and found there was co-localization of CK2?positive and ER specific marker staining.In other words,CK2?partially localizes in endoplasmic reticulum,indicating a possibility that CK2?could interact with proteins on the surface of or in the lumen of endoplasmic reticulum.In primary cortical neurons exposed to the chemical CK2inhibitor CX-4945 or CK2?specific siRNA respectively,the detection of CK2 enzyme activity indicated that CX-4945 could effectively suppress CK2 activity,and CK2?siRNA also significantly inhibited CK2 enzyme activity by inhibiting CK2?expression.Western blot results showed that inhibition of CK2 activity and CK2?expression both activated each molecule of GRP78/eIF2?-ATF4-CHOP branch.TUNEL staining showed inhibition of CK2activity or inhibition of CK2?expression could both induce neuronal apoptosis,confirming that CK2 may be a negative regulator of ERS,and CK2 inhibition may be the inducement for ERS and apoptosis.In the present study,Wistar rats exposed to intracerebroventricular injection of Apelin-13(0.5?g/g)pretreatment 30min before MCAO/R surgery.TTC staining showed MCAO/R led to obvious infarction,while Apelin-13 could significantly reduce infarct volume.Western blot results revealed that Apelin-13 effectively inhibited GRP78/eIF2?-ATF4-CHOP activation,with increase of CK2?and CK2?expression.Apelin-13(10nmol/L)pretreatment to the primary cortical neurons followed by OGD/R,significantly alleviated the release of lactate dehydrogenase(LDH),inhibited the expression of all the molecules in the GRP78/e IF2?-ATF4-CHOP pathway,and up-regulated CK2?and CK2?expression,and most importantly inhibited neuronal apoptosis.As confirmed in vivo and in vitro models,Apelin-13 could inhibit ERS and promote CK2 expression.Moreover CK2 may be the negative regulator of ERS.Consequently we presumed that Apelin-13 inhibited MCAO/R-induced or OGD/R-induced ERS and thus apoptosis possibly via upregulating CK2.In further experiments,CK2?siRNA transfection was performed,and it abolished the effect of Apelin-13 including CK2?upregulation,inhibiting GRP78/e IF2?-ATF4-CHOP activation and blocking apoptosis.But Apelin-13 still promoted the expression of CK2?,which was even close to the basal.The above confirmed 1)In neurons CK2 enzyme activity mainly comes from CK2?;2)CK2?is not involved in the modulation of ERS,while CK2?is the negative modulator of ERS;3)Apelin-13 could up-regulate both of CK2?and CK2?,but only CK2?participates in the battle against ERS and ERS-induced apoptosis.As a kind of GPCR,APJ combining with Apelin,first activates its coupled-G protein,further activates the second messenger and downstream signaling molecules,thereby actualizing the transduction of the biological information carried by Apelin/APJ from extracellular to intracellular.APJ is known to mainly couple with G?_i or G?_q.In order to verify the signal transduction by which Apelin/APJ modulated CK2,further experiment was performed using G?_i inhibitor PTX or G?_q inhibitor UBO-QIC and Apelin-13 in OGD/R-treated neurons.Western blot results showed that both of G?_i inhibitor and G?_q inhibitor could neutralize the effect of Apelin-13.Accordingly,the CK2 modulation of Apelin-13 relies on both G?_i and G?_q signaling.In other words,Apelin/APJ participates in the control of GRP78/eIF2?-ATF4-CHOP branch via G?_i/G?_q-CK2 signaling.In summary,this study showed that:1)One important branch of ERS,GRP78/eIF2?-ATF4-CHOP signaling pathway,can be stimulated by ischemic stroke.2)Ischemic stroke could also down-regulate catalytic subunit CK2?and regulatory subunit CK2?,that is to say,CK2 inhibition.3)CK2 is a negative modulator of ERS.Inhibition of CK2 enzyme activity or CK2?expression both could bring in ERS and ERS-mediated apoptosis.4)The CK2inhibition induced by ischemic stroke results in the activation of GRP78/e IF2?-ATF4-CHOP signaling pathway and then apoptosis,which is one of the main proapoptotic mechanisms of ischemic stroke.5)Apelin-13 inhibits ERS and ERS-related apoptosis through up-regulation of CK2?,while CK2?is not involved in the modulation of ERS,even though Apelin-13 does up-regulate CK2?.6)Apelin/APJ takes part in the control of GRP78/e IF2?-ATF4-CHOP branch and apoptosis via G?_i/G?_q-CK2?signaling,so as to protects neurons against ERS and ERS-induced apoptosis.The present study first proved that in neurons protein kinase CK2,mainly its catalytic subunit CK2?,is the negative modulator of ERS,and CK2 inhibition induced by ischemic stroke results in the activation of ERS and then apoptosis.Apelin-13 inhibits ERS and ERS-related apoptosis through G?_i/G?_q-CK2?signaling.The above results provide a theoretical basis for further study of the neuroprotective effects of Apelin/APJ and CK2.This study also confirmed that APJ,ERS pathways and CK2,especially CK2?,have the potential to be the new targets for the treatment of ischemic stroke.It is of high practical value in theory to develop inhibitors against ERS and agonists for APJ and CK2,but it still needs further study.