The Protective Effect Of MLKL On Autophagy Flux In Pulmonary Microvascular Endothelial Cell In Acute Lung Injury Under Short-term Starvation

Background:P PMVEC(pulmonary microvascular endothelial cells),the main target cell attacked during the occurrence and development of ALI(acute lung injury),is an important component of pulmonary vascular endothelial barrier.In LPS induction of ALI,the necroptosis of PMVEC often occurs,which is characterized by the destruction of cell membrane integrity accompanied by obvious inhibition of autophagy.MLKL(mixed series protein kinase-like domain)serves as a core component of necroptosis.As an executor of necroptosis,phosphorylated MLKL is regulated by RIP3(receptor-interacting serine/threonine kinase 3).However,recent studies have found that the phosphorylation of MLKL is involved in the endosomal trafficking,which is crucial for cell survival,independent of the kinase activity of RIP3.The pro-cell survival function of activated MLKL usually initiates by Ca2+influx.Ca2+ and its downstream effector CaMKII(calmodulindependent kinase kinase)are well-considered as essential pathways for autophagy enhancement in a variety of cell types and experimental models.Obviously,Ca2+and CaMKII play an important role in autophagy,cell survival and functional repair during MLKL activation.Objective:The purpose of the study is to clarify the protective effect of MLKL on autophagy flux and cell function of PMVEC in ALI.We aim to elucidate the specific mechanism that MLKL phosphorylation regulated by CaMKII activity participates in the fusion of autophagosome and lysosome,which helps promote cell survival and maintain the normal autophagy flow in PMVEC,thus providing a new theoretical basis for the protection of PMVEC in ALI.Methods:PMVEC(primary mouse pulmonary microvascular endothelial cells)and Ehy926(human umbilical vein endothelial cells)were divided into Ctrl group(control group),LPS group(10 mg/L,12-24 h),ECS group(serum and amino acid deprived extracellular solution was pretreated for 1 h and restored to normal medium without LPS.12-24 h)and ECS+LPS group(serum and amino acid deprived extracellular solution was pretreated for 1 h and restored to normal medium containing 10 mg/L LPS,12-24 h).Cell death was determined by PI staining and LDH activity assay.EdU infiltration cell flow cytometry and CCK-8 OD value were used to reflect cell proliferation.Cell migration ability was detected by the migration distance of cell scratches.LC3II and p62(markers of autophagy degradation)were detected by Western Blot to reflect autophagy levels.The autophagy flow was detected by confocal microscopy after transfection of dual fluorescence PK-hLC3 plasmid.Then,siCaMKII PMVEC,shMLKL PMVEC,N2a(mouse neuroblastoma),L929(mouse fibroblast),L929 RIP 1 KO,L929 RIP3 KO,L929 MLKL KO,HEK293(human embryonic kidney cell 293)and HT29(human colon cancer cell),classical necroptosis inducers(TSZ)and various drug inhibitors(RIP1 inhibitor NEC-1,RIP3 inhibitor GSK’872,MLKL inhibitor GW,CaMKII inhibitor KN93 and KN62,intracellular Ca2+chelating agent)were used.The regulation of CaMKII on MLKL phosphorylation under starvation was detected by Western Blot,immunoprecipitation and in vitro kinase activity assay.LC3II and p62 were detected by Western Blot to reflect the opposite effect of MLKL phosphorylation induced by CaMKII and RIP3 on autophagy under starvation and necroptosis conditions respectively.The effects of MLKL phosphorylation induced by CaMKII and RIP3 on autophagy flux were determined by confocal microscopy after transfection of dual fluorescence PK-hLC3 plasmids.Results:In the 1st and 2nd part of the study,we found that the phosphorylation level of MLKL in PMVEC increased after LPS stimulation,resulting in significant necroptosis,accompanied by inhibition of autophagy flus,characterized by the increasing expression levels of LC3-Ⅱ/LC3-Ⅰ and SQSTM1/p62(a marker of autophagy degradation)and the accumulation of LC3 fluorescent puncta.Meanwhile,the proliferation and migration of PMVEC decreased significantly.Necroptosis and autophagic flux inhibition in this process were blocked by MLKL or RIP3 inhibitors.However,short-term starvation(the deprivation of cell serum and amino acids within 1 hour)induced the phosphorylation of MLKL in PMVEC.Interestingly,the MLKL phosphorylation induced by short-term starvation followed by LPS-stimulation was accompanied by decreasing LC3-Ⅱ/LC3-Ⅰ and SQSTM1/p62 levels and accelerating autophagic lysosome degradation,suggesting that short-term starvation promoted autophagy flux even under the LPS stimulation.In this process,PMVEC mortality was significantly reducing and cell proliferation and migration were improved,which surprisingly could not be reversed by RIP3 suppression.In the 3rd part of the study,we found that the phosphorylation of MLKL induced by short-term starvation was dependent on CaMKII activity rather than the well-established RIP3.Similar to the inhibition of MLKL,by using genetic manipulation and pharmacological methods,the suppression of CaMKII prevented the promoting autophagy flux and accelerating autophagic lysosomal degradation induced by starvation,independent of RIP3.Additionally,mouse N2a and L929 cells,as well as human HEK293 and HT29 cells were involved,which are commonly used in necroptosis research,to support the above results that CaMKII-MLKL pathway promotes autophagy flus during short-term starvation,while RIP3-MLKL pathway inhibits autophagy flow under necroptosis.Contrary to the necroptosis pathway,starvation-induced the phosphorylation of MLKL,mediated by CaMKII,protected cells from cell death.In addition,immunoprecipitation and in vitro kinase activity assays showed that CaMKII bound to MLKL to form a protein complex under starvation,and the starvation-induced oligomer formed by MLKL could be blocked by CaMKII inhibitors.Conclusion:Therefore,we propose that CaMKⅡ activates MLKL in the presence of nutrient deficiency,thereby promoting the maturation of autophagosomes and the proper fusion of autophagosomes and lysosomes,thus accelerating subsequent material degradation.This new function is independent of RIP3 and does not participate in necroptosis.Unlike the classical necroptosis executor,MLKL may play a dual role in cell survival.Our study reveals a new role of MLKL in cell survival,signaling and metabolism,which may provide a new theoretical basis and therapeutic target for maintaining normal function of PMVEC in ALI.