MITOCHONDRIAL AUTOPHAGY: CYTOPROTECTIVE MECHANISM IN ISCHEMIA/REPERFUSION INJURY TO LIVER
소속 :
연사 : 김재숭 교수 (University of Florida College of Medicine Gainesville)
일시 : 2009-06-25 10:30 ~
장소 : 500동 목암홀
일시: 2009년 6월 25일 오전 10:30
장소: 500동 목암홀
-Abstract-
Autophagy selectively removes abnormal or damaged organelles such as dysfunctional mitochondria. The mitochondrial permeability transition (MPT) is a marker of impaired mitochondrial function that is evident in hepatic ischemia/reperfusion (I/R) injury. However, the relationship between mitochondrial dysfunction and autophagy in I/R injury is unknown. Cultured rat hepatocytes and mouse livers were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. Expression of Atg7, Beclin-1 and Atg12, autophagy regulatory proteins, was analyzed by Western blots. Some hepatocytes were incubated with calpain 2 inhibitors or infected with adenoviruses encoding GFP (control), Atg7 and Beclin-1 to augment autophagy. To induce nutrient depletion, a condition stimulating autophagy, hepatocytes were incubated in amino acid- and serum-free medium for 3 hours prior to onset of anoxia. For confocal imaging, hepatocytes were co-loaded with calcein and tetramethylrhodamine methylester to visualize onset of the MPT and mitochondrial depolarization, respectively. To further examine autophagy, hepatocytes were infected with adenovirus expressing GFP-LC3 and subjected to A/R. Calpain activity was fluorometrically determined with succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin. A/R markedly decreased Atg7 and Beclin-1, concomitant with a progressive increase in calpain activity. I/R of livers also decreased both proteins. However, inhibition of calpain isoform 2, adenoviral overexpression and nutrient depletion all substantially suppressed A/R-induced loss of autophagy proteins, prevented onset of the MPT and decreased cell death after reoxygenation. Confocal imaging of GFP-LC3 confirmed A/R-induced depletion of autophagosomes, which was reversed by nutrient depletion and adenoviral overexpression. Conclusions: Calpain 2-mediated degradation of Atg7 and Beclin-1 impairs mitochondrial autophagy that subsequently leads to MPT-dependent hepatocyte death after A/R.
장소: 500동 목암홀
-Abstract-
Autophagy selectively removes abnormal or damaged organelles such as dysfunctional mitochondria. The mitochondrial permeability transition (MPT) is a marker of impaired mitochondrial function that is evident in hepatic ischemia/reperfusion (I/R) injury. However, the relationship between mitochondrial dysfunction and autophagy in I/R injury is unknown. Cultured rat hepatocytes and mouse livers were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. Expression of Atg7, Beclin-1 and Atg12, autophagy regulatory proteins, was analyzed by Western blots. Some hepatocytes were incubated with calpain 2 inhibitors or infected with adenoviruses encoding GFP (control), Atg7 and Beclin-1 to augment autophagy. To induce nutrient depletion, a condition stimulating autophagy, hepatocytes were incubated in amino acid- and serum-free medium for 3 hours prior to onset of anoxia. For confocal imaging, hepatocytes were co-loaded with calcein and tetramethylrhodamine methylester to visualize onset of the MPT and mitochondrial depolarization, respectively. To further examine autophagy, hepatocytes were infected with adenovirus expressing GFP-LC3 and subjected to A/R. Calpain activity was fluorometrically determined with succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin. A/R markedly decreased Atg7 and Beclin-1, concomitant with a progressive increase in calpain activity. I/R of livers also decreased both proteins. However, inhibition of calpain isoform 2, adenoviral overexpression and nutrient depletion all substantially suppressed A/R-induced loss of autophagy proteins, prevented onset of the MPT and decreased cell death after reoxygenation. Confocal imaging of GFP-LC3 confirmed A/R-induced depletion of autophagosomes, which was reversed by nutrient depletion and adenoviral overexpression. Conclusions: Calpain 2-mediated degradation of Atg7 and Beclin-1 impairs mitochondrial autophagy that subsequently leads to MPT-dependent hepatocyte death after A/R.
