Elsevier

Free Radical Biology and Medicine

Volume 65, December 2013, Pages 26-37
Free Radical Biology and Medicine

Original Contribution
Noxa couples lysosomal membrane permeabilization and apoptosis during oxidative stress

https://doi.org/10.1016/j.freeradbiomed.2013.05.051Get rights and content

Highlights

  • H2O2 induced lysosomal membrane permeabilization (LMP) independently of Noxa.

  • Noxa-dependent cytochrome c release occurred downstream of LMP.

  • Preventing LMP blocked oxidative DNA damage and p53-dependent Noxa induction.

  • Stabilizing lysosomal membranes inhibited Noxa-mediated apoptosis.

Abstract

The exact roles of lysosomal membrane permeabilization (LMP) in oxidative stress-triggered apoptosis are not completely understood. Here, we first studied the temporal relation between LMP and mitochondrial outer membrane permeabilization (MOMP) during the initial stage of apoptosis caused by the oxidative stress inducer H2O2. Despite its essential role in mediating apoptosis, the expression of the BH3-only Bcl-2 protein Noxa was dispensable for LMP. In contrast, MOMP was dependent on Noxa expression and occurred downstream of LMP. When lysosomal membranes were stabilized by the iron-chelating agent desferrioxamine, H2O2-induced increase in DNA damage, Noxa expression, and subsequent apoptosis were abolished by the inhibition of LMP. Importantly, LMP-induced Noxa expression increase was mediated by p53 and seems to be a unique feature of apoptosis caused by oxidative stress. Finally, exogenous iron loading recapitulated the effects of H2O2 on the expression of BH3-only Bcl-2 proteins. Overall, these data reveal a Noxa-mediated signaling pathway that couples LMP with MOMP and ultimate apoptosis during oxidative stress.

Introduction

In multicellular organisms, apoptosis is a cellular suicide process critical for the maintenance of normal tissue homeostasis, which preserves a proper balance between the rate of cell proliferation and the rate of cell death [1], [2]. Apoptosis can be induced by ligation of death receptors through the extrinsic pathways or by various death stimuli through the intrinsic pathways. Recent genetic and biochemical studies have revealed a conserved network that modulates the well-organized self-destruction of cells. In mammalian cells, intrinsic apoptotic signal leads to mitochondrial outer membrane permeabilization (MOMP) and the release of apoptogenic factors such as cytochrome c into the cytosol, where they activate a cascade of aspartate-directed cysteine proteases (caspases) subsequently leading to apoptosis [3]. MOMP and activation of caspases are usually considered as the molecular hallmarks of apoptosis [4]. Apoptosis signaling is regulated by the Bcl-2 family of proteins, which can be either proapoptotic or antiapoptotic [5], [6], [7]. Proapoptotic Bcl-2 proteins promote apoptosis by increasing MOMP, whereas antiapoptotic Bcl-2 proteins inhibit MOMP and prevent or delay apoptosis [8], [9], [10].

Although mitochondria play a central role in apoptosis regulation, other organelles including the endoplasmic reticulum (ER), the golgi aparatus, and lysosomes are also involved in apoptotic signaling [11], [12], [13], [14]. Lysosomes are major intracellular organelles responsible for degrading and recycling of cellular components. Lysosomes contain at least 50 hydrolytic enzymes including nucleases, proteases, phospholipases, lipases, phosphatases, sulfatases, and glycosidases which, on release, can degrade macromolecules in the cytosol [15]. The best characterized lysosomal enzymes belong to the cathepsin protease family [16]. A wide variety of stressors including osmotic stress, growth factor deprivation, death receptor activation, proteasome inhibitors, and oxidative stress inducers have been shown to target lysosomes and cause lysosomal membrane permeabilization (LMP) through which lysosomal hydrolytic enzymes are released into the cytosol [17]. The level of damage to the lysosome determines the fate of the cell. Massive lysosomal damage causes an excessive release of lysosomal contents into the cytosol resulting in indiscriminate degradation of cellular contents and cytoplasmic acidification, which in turn promotes cell death by necrosis. On the other hand, selective or partial lysosomal damage induces cell death by apoptosis [18], [19], [20]. For instance, in tumor necrotic factor alpha (TNFα)-treated cells, cathepsin B, D, and L released into the cytosol trigger apoptosis by converting the inactive proapoptotic BH3-only Bcl-2 protein Bid into its truncated active form (tBid), promoting subsequent MOMP and caspase activation [17], [21].

Oxidative stress inducers, including hydrogen peroxide (H2O2), are capable of inducing cell death by both necrosis and apoptosis; mild oxidative stress causes apoptosis whereas severe oxidative stress triggers necrosis [22]. The extent of oxidative stress determines the level of lysosomal membrane damage. H2O2 interacts with intralysosomal iron to generate highly reactive hydroxyl radicals that initiate lipid peroxidation of lysosomal membranes and subsequent LMP. In support of this model, the iron-chelating agent desferrioxamine (DFO) has been shown to abolish oxidative stress-triggered LMP and apoptosis [23]. However, the involvement of LMP in oxidative stress-induced apoptosis signaling and how LMP is modulated by the complex Bcl-2 protein network are still unclear. Here, we investigated the temporal relation between LMP and MOMP during oxidative stress-induced apoptosis. In mouse embryonic fibroblasts (MEFs), H2O2 was able to induce LMP prior to MOMP during apoptosis. MOMP and subsequent apoptosis signaling, but not LMP, depended on Noxa expression. The iron-chelating agent DFO prevented H2O2-induced MOMP and apoptosis by inhibiting LMP, oxidative DNA damage, and subsequent p53-dependent Noxa expression increase. Therefore, LMP-induced Noxa expression is critical for MOMP and subsequent activation of the apoptosis cascade during oxidative stress.

Section snippets

Oxidative stress-induced LMP is independent of Noxa expression

In lysosomes, the oxidative stress inducer H2O2 causes lysosomal membrane permeabilization and subsequent apoptosis [24], [25], [26]. Our previous studies have shown that the elevated expression of the proapoptotic BH3-only Bcl-2 protein Noxa mediates apoptosis triggered by H2O2 [27]. To explore the involvement of Noxa in H2O2-induced LMP, we first examined lysosomal membrane stability in mouse embryonic fibroblasts deficient in Noxa expression (Noxa-KO) and their wild-type counterparts. The

Discussion

Oxidative stress-triggered LMP is well documented, but the detailed mechanisms through which LMP is coupled with MOMP and eventual apoptosis are still not completely understood. Here we investigated the role of lysosomal membrane rupture in regulating apoptosis caused by the oxidative stress inducer H2O2. On H2O2 exposure, the integrity of lysosomal membranes was disrupted independently of endogenous Noxa, despite its essential role in apoptosis signaling. Noxa-dependent MOMP appeared to occur

Reagents

Desferrioxamine (DFO), etoposide, doxorubicin, actinomycin D, hydrogen peroxide (H2O2), acridine orange (AO), and ferric ammonium citrate were purchased from Sigma (St. Louis, MO). Staurosporine, cisplatin, and carboplatin were purchased from Enzo (Farmingdale, NY). Propidium iodide (PI), MitoTracker Green, MitoTracker Red, and puromycin were obtained from Invitrogen (Carlsbad, CA). Unless otherwise stated, all reagents were dissolved in dimethyl sulfoxide (DMSO). Dulbecco's modified Eagle's

Acknowledgments

We are grateful to Dr. John Eaton for critical reading of the paper, Dr. Onno Kranenberg (University Medical Center Utrecht; Utrecht, Netherlands) for providing HCT116 cells; Professor Andreas Strasser (Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia) for providing Noxa-KO MEFs; and Sabine Waigel and Vennila Arumugam at the University of Louisville Microarray facility for helping with microarray experiments. This work was supported by NIH Grants CA106599, RR018733

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    1

    Current address: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105.

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