Mechanism Study Of TRPM7-mediated Apoptosis And NGF-mediated Regulation Of TRPM7 In The Process Of Anoxia-induced Cell Damage

Part 1Mechanism study for implication of TRPM7 in oxygen-glucosedeprivation/reoxygenation-induced apoptosisSegment 1Establishment of stable HEK293 cell lines with tetracycline-inducible expression offull-length or kinase domain truncated TRPM7Transient receptor potential melastatin 7 (TRPM7),also named asChannel-Kinasel (ChaK1),is a unique bifunctional protein consisting of a proteinkinase domain fused to a non-selective cation channel.The aim of this study was toestablish stable HEK293 cell lines with tetracycline-inducible expression of full-lengthor kinase domain truncated TRPM7,so that we can investigate the functions of TRPM7and its kinase at cellular level.Eukaryotic expression plasmid pcDNA5/FRT/TO-HA-TRPM7/WT (expressingfull-length TRPM7 (TRPM7/WT)) or pcDNA5/FRT/TO-HA-TRPM7/â–³Kin(expressing kinase domain truncated TRPM7 (TRPM7/â–³Kin)) was cotransfected withthe Flp recombinase expression plasmid pOG44 into Flp-In^TM T-REx^TM 293 cells usingLipofectamine 2000^TM.After selection with hygromycin,cell clones were pooled anddefined as 293-TRPM7/WT (transfected with pcDNA5/FRT/TO-HA-TRPM7/WT) and293-TRPM7/â–³Kin (transfected with pcDNA5/FRT/TO-HA-TRPM7/â–³Kin),respectively.The tetracycline-inducible expression of TRPM7/WT in 293- TRPM7/WTcells or TRPM7/â–³Kin in 293-TRPM7/â–³Kin cells was confirmed by western blot andimmunofluorescence analysis.The results showed that TRPM7/WT or TRPM7/â–³Kinexpression was switched on by application of tetracycline in a concentration dependent manner.In conclusion,we successfully established tetracycline-inducible HEK293stable cell lines expressing full-length or kinase domain truncated TRPM7.Segment 2The role of TRPM7 kinase domain in oxygen-glucosedeprivation/reoxygenation-induced apoptosis and the relationship betweenTRPM7 and Annexin-1Neuronal death after cerebral ischemia is the main cause of neurologicimpairment.It is recently revealed that the activity of TRPM7 channel is closely relatedto anoxic neuronal death,however,the exact function of TRPM7 kinase in ischemicneuronal injury is unknown.In this study,by using stable HEK293 cell lines with tetracycline (Tet)-inducibleexpression of full-length and kinase domain truncated TRPM7 (293-TRPM7/WT cellsand 293-TRPM7/â–³Kin cells) and establishing in vitro model of oxygen-glucosedeprivation followed by reoxygenation (OGD/R) mimicking in vivo ischemicreperfusion process,we investigated the role of TRPM7 kinase domain inOGD/R-induced cell damage and cell apoptosis and the possible mechanisms.SinceAnnexin-1 is an important substrate for TRPM7 kinase,changes of Annexin-1 wasobserved in this study to explored the mechanism of action of TRPM7 kinase domain.Cell viability was determined by MTT assay.Apoptosis was quantitativelyanalyzed by Annexin V-FITC and PI double-staining followed by flow cytometry.Immunofluorescence and western blot were used to detect the localization andexpression of TRPM7 and Annexin-1,respectively.Interaction between TRPM7 andAnnexin-1 was measured by immunoprecipitation.The results showed that:1.TRPM7 kinase domain was implicated in OGD/R-induced,TRPM7-mediatedcell damage and apoptosis of HEK293 cells. 1) Under normal condition,cell viability among Tet-induced,non-Tet-induced293-TRPM7/WT and 293-TRPM7/â–³Kin cells were no statistic difference.After 1h ofOGD and 0h,6h,12h or 24h of reoxygenation,the viability of Tet-induced293-TRPM7/WT or 293-TRPM7/â–³Kin cells was significantly lower than that ofnon-Tet-induced 293-TRPM7/WT or 293-TRPM7/â–³Kin cells at each time point(P＜0.01).At 12h after reoxygenation,the viability of Tet-induced 293-TRPM7/WTcells was significantly lower than that of Tet-induced 293-TRPM7/â–³Kin cells (P＜0.05).The difference of cell viability between Tet-induced 293-TRPM7/WT cells and293-TRPM7/â–³Kin cells was more significant at 24h after reoxygenation (P＜0.01).2) Under normal condition,the apoptotic rate of Tet-induced,non-Tet-induced293-TRPM7/WT and 293-TRPM7/â–³Kin cells were 1.4%±0.16%,1.5%±0.12%,1.4%±0.13%,1.4%±0.15%,respectively,and there was no statistic difference among them.However,after 1h of OGD and 24h of reoxygenation,the apoptotic rate of Tet-induced293-TRPM7/WT cells (34.1%±1.22%) was significantly higher than that ofnon-Tet-induced 293-TRPM7/WT cells (10.2%±1.05%,P＜0.05) and Tet-induced293-TRPM7/â–³Kin cells (13.5%±1.01%,P＜0.05).2.OGD/R promoted the association between TRPM7 and Annexin-1 in acalcium-independent and TRPM7 kinase domain-dependent manner.1) Double immunofluorescence staining assay showed colocalization ofTRPM7/WT protein with Annexin-1 in Tet-induced 293-TRPM7/WT cells.TRPM7/WT protein was mainly located at plasma membrane,while Annexin-1 wasevenly distributed throughout the cell.Colocalization of TRPM7/WT with Annexin-1was on plasma membrane.2) Immunoprecipitation assay showed that under normal condition,a fewassociation of Annexin-1 with TRPM7/WT was detected in Tet-induced293-TRPM7/WT cells.After 1h of OGD,the association between Annexin-1 andTRPM7/WT was gradually increased with the increasing duration of reoxygenation.After 1 h of OGD and 24h of reoxygenation,chelating intracellular or extracellular Ca²⁺, using BAPTA-AM or EDTA,did not abrogate the association of Annexin-1 withTRPM7/WT in Tet-induced 293-TRPM7/WT cells,and no association betweenAnnexin-1 and TRPM7/â–³Kin was detected in Tet-induced 293-TRPM7/â–³Kin cells.3.OGD/R induced TRPM7 kinase domain-dependent high expression ofAnnexin-1 and translocation of Annexin-1 to nucleus and cell membrane in HEK293cells.1) After lh of OGD,the expression of Annexin-1 in Tet-induced293-TRPM7/WT cells was gradually upregulated during reoxygenation,which startedat 6h and reached a peak at 24h.After lh of OGD and 24h of reoxygenation,theexpression level of Annexin-1 in Tet-induced 293-TRPM7/â–³Kin cells was no statisticdifference from that in non-Tet-induced 293-TRPM7/â–³Kin cells and non-Tet-induced293-TRPM7/WT cells,but it was significantly lower than that in Tet-induced293-TRPM7/WT cells (P＜0.05).In addition,Tet-induced 293-TRPM7/WT cellsexpressed higher level of Annexin-1 protein than non-Tet-induced 293-TRPM7/WTcells at the time points of 6h,12h and 24h after reoxygenation (P＜0.05).2) Immunofluorescence staining assay showed that Annexin-1 in Tet-induced293-TRPM7/WT cells was translocated to cell membrane and nucleus after 1h of OGDand 24h of reoxygenation,whereas Annexin-1 in Tet-induced 293-TRPM7/â–³Kin ornon-Tet-induced 293-TRPM7/WT cells did not.Expression of Annexin-1 in cellmembrane,cytoplasm and nucleus fractions was further measured by immunoblot.Itshowed that after 1h of OGD and 24h of reoxygenation,expression level of Annexin-1in cytosolic fraction in Tet-induced 293-TRPM7/WT cells was lower than normal,however,expression level of Annexin-1 in membrane,especially in nuclear fraction wasobviously higher than normal.On the other hand,Annexin-1 expression level in cellmembrane or nuclear fraction in non-Tet-induced 293-TRPM7/WT cells was nodifference from normal,while Annexin-1 expression level in cytosolic fraction washigher than normal.4.Annexin-1 was involved in OGD/R-induced,TRPM7-mediated cell damage and apoptosis of HEK293 cells.1) After 1h of OGD and 24h of reoxygenation,Annexin-1 antisenseoligodeoxynucleotides (ASODN) significantly downregulated OGD/R-induced highexpression of Annexin-1 in Tet-induced 293-TRPM7/WT cells to a level similar tonormal (P＜0.05),and markedly increased cell viability (P＜0.01).However,Annexin-1sense oligodeoxynucleotides (SODN) affected neither Annexin-1 expression inTet-induced 293-TRPM7/WT cells nor cell viability.2) After 1h of OGD and 24h of reoxygenation,Annexin-1 ASODN significantlyreduced OGD/R-induced high apoptotic rate of Tet-induced 293-TRPM7/WT cells by19.6%±1.01% (P＜0.05).However,Annexin-1 SODN did not affect the apoptotic rateof Tet-induced 293-TRPM7/WT cells,the percentage of apoptotic cells was 33.7%±1.56%.In conclusion,in this study,we firstly verify that TRPM7,through its kinasedomain,is implicated in upregulating Annexin-1 expression and promoting Annexin-1translocation from cytoplasm to nucleus and cell membrane in response to OGD/Rtreatment,and serves to aggravate OGD/R-induced cell damage and apoptosis inHEK293 cells.These findings provide theoretical evidence for further investigating therole of TRPM7 kinase in ischemic reperfusion-induced neuronal cell apoptosis. Part 2NGF inhibits Gd³⁺-sensitive calcium influx and reduces chemical anoxic neuronal deathThe aim of this study was to investigate whether glutamate receptor channels andvoltage-gated calcium channels independent calcium influx arises in the process ofacute anoxic neuronal damage and its possible relationship to neuronal protectivefunction of NGF.In in vitro model of acute anoxia,hippocampal cultures from newborn rats wereexposed to KCN.Changes of intracellular Ca²⁺ concentration ([Ca²⁺]_i) were monitoredby laser scanning confocal microscope and cell viability was assayed by PI and cFDAstaining.The results showed that:1.After treatment with 3mM KCN for 15min,[Ca²⁺]_i in hippocampal neuronswas significantly increased 6.27±0.05-fold compared with pre-anoxia level(P＜0.05) and 73.3%±12.1% of the cells died (P＜0.05).2.When combination of 20μM MK-801 (glutamate receptor antagonist),40μMCNQX (AMPA receptor antagonist) and 5μM nimodipine (voltage-gated calciumchannel antagonist) (hereafter denoted as MCN) were administrated to hippocampalneurons,KCN-induced increase of [Ca²⁺]_i and cell death were significantlyreduced (P＜0.05).Levels of [Ca²⁺]_i was 4.02±0.04-fold of pre-anoxia and cell deathrate was 38.5%±4.4%,both of which were still markedly higher than normal (P＜0.05).3.Gd³⁺ (10μM) completely blocked KCN-induced elevation of [Ca²⁺]_i and furtherreduced cell death in the presence of MCN.Levels of [Ca²⁺]_i was 1.2±0.03-fold ofpre-anoxia,which was no statistic difference from normal.Cell death rate was only8.2%±1.5%.4.Similar to Gd³⁺,NGF (100ng/ml),used in combination with MCN,significantly inhibited KCN-induced increase of [Ca²⁺]_i and cell death.Levels of [Ca²⁺]_iwas 1.42±0.06-fold of pre-anoxia,which was also no statistic difference from normal. Only 9.1%±2.3% of the cells died.The effects of NGF were abolished by PLCinhibitor U73122 (10μM).It is concluded that Gd³⁺-sensitive calcium influx and cell death,which areglutamate receptor channels and voltage-gated calcium channels independent,arise inthe process of acute anoxic neuronal damage.NGF can inhibit Gd³⁺-sensitive calciuminflux and reduce anoxic neuronal death through activating PLC pathway.