Macroautophagy and chaperone-mediated autophagy are required for hepatocyte resistance to oxidant stress.

Excessive production of reactive oxygen species (ROS) can overwhelm cellular antioxidant defenses and lead to oxidative stress that mediates cell injury and death. Oxidative stress has been implicated as a central mechanism of a variety of forms of hepatic injury including that caused by toxins,1 ischemia/reperfusion,2 viral infection3 and steatohepatitis.4,5 ROS may promote cellular injury through the direct oxidative modification of cellular macromolecules including proteins, lipids and DNA. Alternatively, recent investigations have emphasized that oxidant injury results from the direct activation of cell signaling pathways that regulate death. In the model of hepatocyte oxidative stress mediated by the superoxide generator menadione, apoptosis results from activation of the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) and its downstream effector c-Jun.6,7 Overactivation of the JNK/c-Jun pathway is central to many forms of hepatocyte death including chronic as well as acute oxidant stress.8–11 Although it is known that ROS can increase JNK signaling by activating upstream kinases or inactivating phosphatases, other as yet unknown mechanisms likely contribute to ROS-induced increases in JNK/c-Jun signaling. Studies performed in nonhepatic cell types have demonstrated a relationship between the pathways of cell death and autophagy. Autophagy is a process in which cellular components are targeted to the lysosome for degradation.12 There are three types of mammalian autophagy which differ in their substrates and regulation.13 Macroautophagy sequesters whole organelles or portions of cytoplasm into autophagic vacuoles or autophagosomes that fuse with lysosomes and undergo degradation. In microautophagy the lysosomal membrane itself surrounds and sequesters portions of cytosol for degradation. In chaperone-mediated autophagy (CMA), proteins with a specific pentapeptide motif are selectively targeted for degradation by heat shock cognate chaperone of 70 kD and translocate to the lysosome for uptake by lysosome-associated membrane protein type 2A (LAMP-2A) and degradation. Autophagy has two principal functions: to remove unwanted or damaged cellular constituents and to supply the cell with substrates for energy generation in times of nutrient deprivation.14 The central role of autophagy in the elimination of damaged cellular components has suggested that it may be an important protective pathway in cellular injury, and oxidized proteins are one autophagic substrate.15 However, the relationship of autophagy to cell death is controversial. In nonhepatic cells macroautophagy has been implicated as a mechanism of cell death from a variety of stimuli including radiation and viruses.16,17 In contrast, other investigations have shown that macroautophagy mediates survival from injury induced by nutrient deprivation, ischemia/reperfusion injury and endoplasmic reticulum stress.18–20 Little is known about the function of autophagy specifically in hepatocyte injury and cell death. Autophagic function is known to be decreased in the liver under certain conditions such as steatosis and aging.21,22 Thus, if autophagy serves a protective function in hepatocytes, then these conditions that impair autophagic function may promote liver injury and resultant cell death. To determine the function of autophagy in hepatocyte injury, the effects of a knockdown of the critical macroautophagy gene atg5 on menadione toxicity were examined in the nontransformed rat hepatocyte line RALA255-10G. Inhibition of macroautophagy sensitized these cells to apoptotic and necrotic death from normally nontoxic concentrations of menadione. Death was mediated by JNK/c-Jun overactivation that triggered the mitochondrial death pathway. Compensatory up regulation of other forms of autophagy failed to protect against cell death despite the ability of CMA to also mediate resistance to death from menadione. These findings demonstrate a critical and mutually exclusive function of both macroautophagy and CMA in hepatocyte resistance to death from oxidant stress.