In recent years, evidence has been accumulating that ROS are involved in the regulation of autophagy signaling [1
]. The obligatory role of ROS in apoptosis and the intertwining of apoptosis and autophagy pathways [8
], however, obscured the essentiality of ROS as messengers in autophagy. In this study, we dissected the apoptosis and autophagy signaling pathways in HeLa cells by knocking down cyt c
to assess the engagement of mitochondrial ROS in autophagy without interference by concomitant apoptosis. Counter to the prevailing view, our data suggest that mitochondrial ROS are not necessary to induce autophagy. It is tempting to speculate that collapse of CL asymmetry and appearance of non-oxidized CL on the surface of mitochondria are driving the recognition of damaged organelles for the mitophageal clearance. If the elimination of damaged or unnecessary mitochondria is delayed or inefficient, the inevitable production of ROS leads to the accumulation of oxidized CL – capable of inducing mitochondrial membrane permeabilization. This model of sequential events in damaged cells suggests that ROS production and CL peroxidation act as a switch from mitophagy to apoptosis [12
] or necroptosis [21
], but these oxidative stress events are not essential for the execution of autophagy.
According to current views - implying the involvement of ROS in autophagy - Atg4, a recently discovered family of cysteine proteases with at least four homologues in mammalian cells [22
], is one of the likely redox-sensitive components of autophageal machinery [7
]. It has been well documented that activation of Atg4s is required for the process of LC3 to LC3-I conversion [23
], which is conjugated to PE by Atg7, and E1-like enzyme, and Atg3, and E2-like enzyme [24
]. PE-lipidated LC3 is further deconjugated by Atg4s after completion of autophagosome. As one of the arguments in favor of ROS as essential for the execution of autophagy, Scherz-Shouval et al
indicated that a highly conserved cysteine residue, in proximity of the catalytic center, may be a target for the redox regulation of HsAtg4A and HsAtg4B [25
]. The authors suggested that formation of sulfenic acid or disulfide bond shield the catalytic center. No experimental data, however, were presented either in vitro
or in vivo
to explain how ROS were temporally and spatially controlled to ensure that Atg4 remains active [25
]. Another pressing issue is how mitochondria avoid oxidative damage, which could trigger apoptosis signaling by releasing pro-apoptotic factors, while generate sufficient amount of H2
to diffuse through the mitochondrial membrane into reducing cytosolic environment and oxidize Atg4. It is recognized that autophagy/mitophagy exert a protective role in limiting cell death by removing the dysfunctional/injured mitochondria before they release the deleterious contents. In fact, Scherz-Shouval et al
reported that starvation induced autophagy in the absence of programed cell death [25
]. Therefore, it would be more logical to speculate that ROS are the initial insults that cause mitochondrial injury, rather than serve as essential messengers in the execution of autophagic pathway. The demonstrated protective effects of antioxidants on autophagy could be attributed to their efficiency in preventing or eliminating the cause of the initial damage.
In the present study, we alleged that STS-induced autophagy in HeLa1.2 cells was not accompanied by superoxide or H2
generation. Moreover, STS induced autophagy in mt
DNA deficient ρ° HeLa1.2 cells, is consistent with a previous report [26
], in which the ability of mitochondrial ROS generation was completely abolished. The present data indicated that yet to be identified alternative mechanisms – other than mitochondrial ROS - are involved in the Atg4s regulation in STS-treated HeLa1.2 cells. Recently, several potential mechanisms of Atg4 regulation have been reviewed by Chen et al
]. Nair et al
] have demonstrated a role for Atg18 and Atg21 in the protection of Atg8-PE from cleavage by Atg4. In addition, it has been suggested that the substrate of human Atg4B (pro-LC3 or LC3-PE) cannot access the catalytic cysteine residue; prior conformational changes of Atg4B upon binding to LC3 are required for allowing LC3 access to the catalytic site [29
]. Assuming that ROS are not required for the execution of autophagy, they can be involved in the induction of oxidative damage to critical biomolecules and organelles thus acting as a cause and indirect initiators of autophagic responses. ROS have also been implicated in the autophagy regulation through the activation of Snf1 [31
] and of AMPK [32
In summary, by using cyt c deficient HeLa cells, in which the apoptosis and autophagy signaling pathways are separated, we were able to demonstrate that mitochondrial ROS are not necessary for autophagy induction.
- Dissection of autophagy from apoptosis in cytochrome c deficient HeLa1.2 cells;
- staurosporine-induced autophagy in HeLa1.2 cells was not accompanied by either superoxide or hydroperoxide generation;
- Autophagy was detectable in mtDNA deficient ρ0 HeLa1.2 cells in which reactive oxygen species generation was completely disrupted;
- Counter to the widespread view mitochondrial ROS are not required for the staurosporine-dependent induction of autophagy.