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[CHOP promotes hepatocyte pyroptosis by downregulating endoplasmic reticulophagy and enhancing endoplasmic
Xiaoyu Guo1, Jiayu Wu1, Bing Han2
1Department of Pathophysiology, School of Basic Medical Sciences, Guizhou Medical University.
Objectives:
To investigate the role of C/EBP homologous protein (CHOP) in dithiothreitol (DTT)‑induced endoplasmic reticulophagy in murine hepatocytes cells and its mechanism for regulating hepatocytes pyroptosis through endoplasmic reticulum (ER)‑mitochondria interactions.
Methods:
The changes in CHOP, endoplasmic reticulophagy, mitochondrial-ER contact sites (MERCs)-associated protein, and pyroptosis-associated proteins in DTT-treated BRL-3A cells were examined using Western blotting, and CHOP enrichment in the promoter regions of ATG5, ATG12 and LC3 genes was detected using ChIP assay. The effect of bafilomycin A1 (an autophagy inhibitor) pretreatment on DTT-induced BRL-3A cell death was assessed using Hoechst 33342 and propidium iodide staining. The effects of lentivirus-mediated CHOP overexpression in the hepatocytes on ER-phagy, MERCs-associated proteins, pyroptosis-associated proteins, and colocalizations of ER with the autophagosomes, lysosomes and mitochondria were examined using Western blotting and immunofluorescence double staining; the changes in intramitochondrial Ca2+, mitochondrial membrane potential and intramitochondrial GSDMD-N levels were also analyzed.
Results:
DTT treatment for 12 h did not significantly affect CHOP protein expression but significantly upregulated ER-phagy, MERCs-associated proteins, and pyroptosis-associated proteins in BRL-3A cells. DTT treatment for 36 h markedly increased CHOP protein expression and further increased MERCs-associated proteins and pyroptosis-associated protein levels, but moderately decreased ER-phagy levels. DDT treatment significantly downregulated CHOP enrichment in the promoter regions of ATG5, ATG12 and LC3 genes in BRL-3A cells. Pretreatment with bafilomycin A1 significantly increased DTT-induced BRL-3A cell death. CHOP overexpression in BRL-3A cells downregulated the levels of endoplasmic reticulophagy-related proteins, upregulated MERCs- and pyroptosis-related proteins, and reduced the colocalization of ER with autophagosomes and lysosomes, and increased its colocalization with the mitochondria, resulting in also elevated intramitochondrial Ca²⁺ levels and GSDMD-N protein and decreased mitochondrial transmembrane potential.
Conclusions:
CHOP promotes hepatocytes pyroptosis by suppressing ER-phagy and enhancing ER-mitochondrial interactions, which leads to increased Ca²⁺ shuttling from the ER to the mitochondria.
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