Extracellular Acidification Impairs Macrophage Lipophagy Through ASIC1/RIP1 Pathway
Objective Our recent study has demonstrated that extracellular acidification promotes lipid accumulation in macrophages via the activation of acid sensing ion channel 1(ASIC1),but the underlying mechanism remains unclear.This study aims to explore the effect of extracellular acidification on macrophage lipophagy and the underlying mechanism.Methods RAW264.7 macrophages were incubated with 25 mg/L ox-LDL in a pH 6.5 culture medium for 24 h to build macrophage-derived foam cell models induced by extracellular acidification.Then,RAW264.7 macrophages were cultured in the acidic medium of pH 6.5 with or without PcTx-1(ASIC1 specific blocker,10 μg/L)or Nec-1(RIP1 specific inhibitor,20 μmol/L)for 24 h,intracellular lipid accumulation was observed by oil red O staining.The expressions of total ASIC1,plasma membrane ASIC1,RIP1,p-RIP1 Ser166,TFEB,p-TFEB Ser142,LC3 and p62 were measured by Western blot.The co-localization of lipids(indicated by Bodipy)with LC3Ⅱ(autophagosomes)and LAMP1(lysosomes)was analyzed by a confocal laser scanning microscopy,respectively.Morphological changes of lipophagy in the cells were observed by using transmission electron microscopy.ABCA1-mediated cholesterol efflux was determined by cholesterol fluorescence kits.Results Compared with pH 7.4+ox-LDL group,the intracellular lipid accumulation in the pH 6.5+ox-LDL group was significantly increased.Meanwhile,the expressions of plasma membrane ASIC1,p-RIP1 Ser166,p-TFEB Ser142,and p62 proteins were elevated significantly,while LC3Ⅱprotein level and LC3Ⅱ/LC3Ⅰ ratio were decreased.Accordingly,compared with pH 7.4+ox-LDL group,the macrophage lipophagy of the pH 6.5+ox-LDL group was inhibited as indicated by the decreased localization of lipid droplets with LC3 and LAMP1,a decrease in the number of lipophagosomes as well as an increase in lipid droplets.Furthermore,ATP binding cassette transporter A1(ABCA1)-dependent cholesterol efflux from the macrophages of pH 6.5+ox-LDL group reduced dramatically.However,these above effects of extracellular acidification on RAW264.7 macrophages were abolished by PcTx-1 and Nec-1,respectively.Conclusion These findings suggest extracellular acidification promotes the phosphorylation of TFEB at Ser142 via activating ASIC 1/RIP1 pathway,thereby impeding lipophagy in RAW 264.7 macrophages,and that ASIC1 may be a new potential target for preventing aberrant lipid accumulation diseases including atherosclerosis.
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