Introduction

TNFα is a pleiotropic inflammatory cytokine, which was discovered decades ago (Carswell et al., 1975; Pennica et al., 1984). Since then, it has been shown to exert many functions in various physiological and pathological processes. In particular, its implication in the survival and death of normal and cancerous cells has been widely investigated. For most mammalian cells, TNFα is non-cytotoxic, but triggers, after ligation with TNF-receptor 1 at the membrane, a TRADD–TRAF2–RIP1–c-IAP1/2 complex (complex I)-dependent signaling pathway that ends up in the activation of the transcription factor NF-κB (Walczak, 2011; Micheau and Tschopp, 2003; Andrew, 2004; Wang et al., 2008). NF-κB in turn induces genes for cell survival (such as those encoding Bcl-x_L, c-IAPs, c-FLIP, etc.) and pro-inflammatory mediators (TNFα itself, COX-2, iNOS, etc.). Upon c-IAPs inhibition by so called Smac mimetics, low expression of the caspase-8 inhibitor c-FLIP, or protein synthesis inhibition by cycloheximide (CHX), complex I dissociates from the membrane and forms in the cytosol a second complex (complex II) which now includes caspase-8 and its activator FADD to cleave the BH3-only protein Bid to tBid (Micheau and Tschopp, 2003; Andrew, 2004; Wang et al., 2008). tBid activates mitochondrial outer membrane permeabilization (MOMP) via Bax/Bak (i.e. Bax and Bak) activation leading to the release of cytochrome c, the formation of the Apaf-1–caspase-9 apoptosome and the processing and activation of effector caspases-3/7 (i.e. caspase-3 and -7), the main executioners of apoptotic programmed cell death (Kroemer et al., 2007). Recently, another type of TNFα-induced cell death, called programmed necrosis or necroptosis has been discovered when caspases are inhibited by the pan-caspase inhibitor Z-VAD-fmk. This pathway includes RIP1 and other novel signaling components (RIP3, MLK, etc.) whose functions are only now being unveiled (Degterev and Yuan, 2008, He et al., 2009; Zhang et al., 2009).

A characteristic feature of TNFα-induced apoptotic and necroptotic cell death is the production of reactive oxygen species (ROS) such as superoxide anions (O₂⁻·) and hydrogen peroxide. The intracellular source of these ROS, their link to cell death signaling components and their exact role in the effector phase of apoptosis or necroptosis have been largely debated (Van Herreweghe et al., 2010; Taujimoto et al., 1986; Meier et al., 1989; Yazdanpanah et al., 2009; Kim et al., 2010; Woo et al., 2000). While in macrophages and neutrophils TNFα triggers ROS production as a quick oxidative burst required for an early innate immune response, in fibroblasts and other nonphagocytic cells this production occurs continuously over a time period of hours (Van Herreweghe et al., 2010; Taujimoto et al., 1986; Meier et al., 1989; Yazdanpanah et al., 2009). This suggests that phagocytic and nonphagocytic cells may exploit distinct molecular machineries for ROS generation. In innate immune cells TNFα is known to stimulate the activity of the plasma membrane-residing Nox2/gp91 phox subunit of NADPH oxidase which produces superoxides within seconds in order to kill invasive microorganisms (Yazdanpanah et al., 2009; Lambeth, 2004). Interestingly, after treating with TNFα, mouse fibroblasts can also produce ROS via NADPH oxidase. In this case, ROS generation depends on a signaling complex composed of TRADD, RIP1, Nox1 and the small GTPase Rac1 (Lin et al., 2004). Other reports further implicate calcium mobilization, activation of the Rac-cPLA2-LTB₄ cascade and activation of acid sphingomyelinases in this process (Taujimoto et al., 1986; Yazdanpanah et al., 2009; Woo et al., 2000; Shoji et al., 1995). Due to the role of RIP1, it was suggested that this pathway of ROS production is crucial for necrotic cell death. However, the other important component RIP3, which forms a pro-necrotic complex with RIP1 after phosphorylation (Cho et al., 2009), did not activate NADPH oxidase but enzymes controlling glycolytic flux and glutaminolysis, thereby causing ROS formation via mitochondrial complex I (Zhang et la., 2009; Vandenabeele et al., 2010). Hence for necroptosis, and probably also for apoptosis, ROS may be rather produced by mitochondria, preferentially at the ubisemiquinone site (Schulze et al., 1993). To trace mitochondrial ROS, the Murphy group succeeded to generate synthetic compounds, called MitoSOX™ and MitoQ, which after removal of the targeting sequence by mitochondrial esterases, are stably inserted into the inner mitochondrial membrane and act as ROS detection probe and antioxidant, respectively (James et al., 2005; Smith et al., 2011).

Mitochondrial ROS production has been closely linked to a fall in the mitochondrial membrane potential. Although this process is seen in all apoptotic cells, including when cells die in response to TNFα, it is not an early event of cell death signaling but rather a consequence of Bax/Bak-mediated MOMP. Ricci et al. convincingly showed that caspase-3, which is activated after MOMP, cytochrome c release and apoptosome formation, feeds back on complex I of the respiratory chain by cleaving the p75 NDUFS1 subunit of this complex and thereby provoking a fall in the mitochondrial membrane potential and ROS production (Ricci et al., 2004). However, this feedback loop has not yet been investigated for TNFα. By contrast, a recent study using HEK 293, HeLa and cervix carcinoma cells reported that TNFα-induced mitochondrial ROS production was mediated by the recruitment of the survival factor Bcl-x_L to a mitochondrial protein called Romo1, which then resulted in a reduction of the mitochondrial membrane potential (Kim et al., 2010). Other studies claimed that MOMP itself triggered ROS production (Kirkland et al., 2002; Kirkland and Franklin, 2003; Kirkland and Franklin, 2007; Jiang et al., 2008). Finally, complex III of the respiratory chain was found to be involved in mitochondrial ROS formation induced by Bak in vascular endothelial cells (Childs et al., 2008). Thus, it is still unclear at which step in the apoptotic pathway ROS are produced and how they impact on the cell death machinery.

One widely discussed mode of action of ROS in the cell death pathway is their destabilizing effect on the lysosomal membrane leading to lysosomal membrane permeability (LMP) of various extent. Certain ROS species such as hydrogen peroxides can easily penetrate the organelle and be converted into highly reactive hydroxyl radicals due to the iron content of lysosomes (Fenton reaction). These radicals are known to effectively damage membranes by lipid peroxidation. While a full lysis of lysosomes has been associated with necrosis, a partial, more selective lysosomal membrane perforation is supposed to assist apoptosis by releasing cathepsins (Boya and Kroemer, 2008). An oxidative burst elicited by interferon-γ was also reported to induce LMP and cathepsin release (Kowanko and Ferrante, 1987). Cathepsins are cysteine, serine or aspartyl proteases, which, once in the cytosol can impinge on the apoptotic machinery by either directly processing and activating caspases or cleaving Bid to tBid (Boya and Kroemer, 2008; Blomgran et al., 2007; Guicciardi et al., 2000; Guicciardi et al., 2005; Droga-Mazovec et al., 2008). The problem with this pathway is that it was previously claimed to be an early event of apoptosis occurring before MOMP and effector caspase-3/7 activation and prior to ROS production (Werneburg et al., 2002; Werneburg et al., 2004; Gyrd-Hansen et al., 2006). We, however, recently published that for a variety of apoptotic stimuli, LMP is not an early requiring, but a late amplifying event of apoptosis induction because it strictly depends on Bax/Bak (i.e. MOMP), apoptosome formation and caspase-3/7 activation (Oberle et al., 2010). The link from caspase-3/7 to LMP has however not been sorted out yet. Moreover, our study did not include TNFα, which has so far been the golden standard stimulus for implying LMP as an early apoptotic trigger (Guicciardi et al., 2000; Guicciardi et al., 2005; Droga-Mazovec et al., 2008; Werneburg et al., 2002; Werneburg et al., 2004).

Here we show that also for TNFα, LMP is a late amplifying process downstream of Bax/Bak, the apoptosome and caspase-3/7. In addition, we uncovered the connection between caspase-3/7 activation and LMP. As previously shown by Ricci et al., 2004, caspase-3 cleaves the p75 subunit of complex I and thereby produces ROS, in particular O₂⁻·, which then damage the lysosomal membrane leading to LMP.

Results

TNFα/CHX-triggered ROS are O₂⁻· of mitochondria origin

TNFα is only able to trigger apoptosis of mammalian cells when the expression of NF-κB induced survival genes is blocked by transcriptional or translational inhibition (Walczak, 2011; Micheau and Tschopp, 2003; Andrew, 2004; Wang et al., 2008). We therefore used in our study a combination of TNFα and CHX (designated TNFα/CHX, T/C in figures) to treat mouse embryo fibroblasts (MEFs). We first monitored the origin and amount of ROS generated by this treatment. For that purpose, we used dihydroethidium (DHE) and 2′,7′-dichlorodihydrofluorescein (DCDHF) to estimate O₂⁻· and H₂O₂/nitric oxide (NO) by FACS analysis and fluorescence microscopy detection, respectively. After exposure to 10 ng/ml TNFα and 1 µg/ml CHX for 4 h MEFs were strongly stained with DHE (Fig. 1A,B), but not with DCDHF (data not shown) indicating that this treatment majorly induced the production of O₂⁻·. To detect the source of O₂⁻· production, we incubated the cells with the mitochondrial red fluorescent ROS sensor MitoSOX™. As shown in Fig. 1A,B, TNFα/CHX-induced O₂⁻· were effectively produced in mitochondria, and this effect was strongly inhibited by a pretreatment with the mitochondria-specific antioxidant MitoQ. This compound mimics the endogenous mitochondrial antioxidant coenzyme Q10 (James et al., 2005; Smith et al., 2011) thereby markedly scavenging the ROS produced at the ubisemiquinone site. As a positive control for ROS production at the same site, we used rotenone (Fig. 1A,C). Interestingly, the amount of ROS/O₂⁻· produced by the treatment of MEFs with 0.5 µM rotenone was approximately the same (46% by ScanR cytometry, Fig. 1C) as that generated by TNFα/CHX for the same time period (50%, FACS analysis, Fig. 1B). Again the rotenone-induced ROS production was completely ablated by MitoQ (Fig. 1A) confirming that the ROS induced by TNFα/CHX is of mitochondrial origin. It should be noted that DHE can be intracellularly oxidized to ethidium (ETH) by ROS and then stains DNA. Indeed, both DHE and MitoSOX™ (which is a DHE analog) show a clear DNA and other intracellular (mitochondrial) staining after cellular treatment with TNFα/CHX or rotenone (Fig. 1A).

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Fig. 1.

TNFα/CHX-induced ROS production involves O₂⁻· and is blocked by the mitochondrially localized antioxidant MitoQ and general caspase inhibition. (A) MitoSOX™ or DHE fluorescence microscopy of MEFs (magnification 400 ×) treated with a combination of 10 ng/ml TNFα and 1 µg/ml CHX, or 0.5 µM rotenone in the presence or absence of the antioxidant MitoQ (5 µM) or the general caspase inhibitor Z-VAD.fmk (100 µM) for 4 h. Note that TNFα/CHX triggers O₂⁻· production (red fluorescence) specifically in mitochondria (detected by the mitochondrially targeted MitoSOX™ ROS sensor) and that this is blocked by the mitochondrial antioxidant MitoQ and by caspase inhibition (therefore implying caspases in ROS production). Rotenone, which inhibits the transfer of electrons from iron-sulfur centers in complex I to ubiquinone, serves as a positive control of mitochondrial ROS production detected by MitoSOX™ and blocked by MitoQ. Only a low level of ROS are produced in untreated cells. (B) FACS histogram of DHE fluorescence (FL-3) showing that 50% of the cells produce O₂⁻· after treatment with 10 ng/ml TNFα and 1 µg/ml CHX for 4 h. This increased ROS production is blocked by MitoQ and is not seen in untreated cells (control). (C) ScanR cytometry of MEFs (magnification 40 ×) treated with 0.5 µM rotenone for 4 h. Note that after this time, about the same percentage of cells (46%) are positive for mitochondrial O₂⁻· production/staining as with TNFα/CHX treatment (see B, 50%).

TNFα/CHX-triggered ROS production is dependent on the Bax/Bak–apoptosome–caspase gateway and the caspase-3-mediated cleavage of the p75 NDUFS1 subunit of complex I of the respiratory chain

To elucidate if ROS production induced by TNFα/CHX is an early event occurring before or simultaneously with MOMP or rather a late event downstream of MOMP, we determined the amount of O₂⁻· generation by DHE FACS analysis in WT, Bax/Bak^−/− [double knockout (KO) in both Bax and Bak], Apaf-1^−/−, caspase-9^−/− and caspase-3/7^−/− (double KO in both caspase-3 and -7) MEFs. As shown in Fig. 2A, the ROS production observed after 4 h of TNFα/CHX treatment (see Fig. 1B) was completely ablated in all the KO cell lines, although Apaf-1^−/− MEFs retained a slightly higher ROS level above background (which was however not significant). These data clearly show that ROS is generated downstream of MOMP and caspase-3/7 in response to TNFα/CHX. In order to exclude possible respiratory chain deficiencies in Bax/Bak^−/− MEFs, we measured oxygen consumption of intact cells. As shown in supplementary material Fig. S1A,C, Bax/Bak^−/− cells did not show any respiration deficit as they even consumed 55±17% more oxygen than WT cells. After treating with TNFα/CHX, WT cells expectedly respired less but the Bax/Bak^−/− counterparts still consumed oxygen at a higher rate (supplementary material Fig. S1A,C). This is consistent with the finding that the Bax/Bak-deficient cells were protected from TNFα/CHX-induced apoptosis (Fig. 6A) and cytochrome c release (MOMP) (Kroemer et al., 2007) and did not produce any ROS (Fig. 2A). In the presence of the complex I inhibitor rotenone, MEFs did hardly respire indicating that we indeed measured the respiration of mitochondria (supplementary material Fig. S1A).

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Fig. 2.

TNFα/CHX-induced ROS production occurs downstream of Bax/Bak and the apoptosome, and involves the caspase-3-dependent cleavage of the p75 NDUFS1 subunit of complex I. (A) Quantitative FACS analysis of DHE fluorescence measuring the amount of O₂⁻· produced in WT, Bax/Bak^−/−, Apaf-1^−/−, caspase-9^−/− or caspase-3/7^−/− MEFs exposed to 10 ng/ml TNFα and 1 µg/ml CHX (grey bars) or medium only (black bars) for 4 h. Note that O₂⁻· production depends on Bax/Bak and the Apaf-1–caspase-9 apoptosome, as well as caspase-3/7. The data are the means±s.d. of at least three independent experiments; P<0.001 between WT and KO cells. (B) FACS histogram of DHE fluorescence (FL-3) of WT HeLa cells expressing the WT p75 NDUFS1 subunit of complex I or its D255A mutant, which cannot be cleaved by caspase-3, treated either with 10 ng/ml TNFα and 1 µg/ml CHX (T/C) for 4 h or 1 µg/ml tunicamycin (Tuni) or 100 µM etoposide (Etop) for 24 h. Gray histograms represent untreated cells. Note that O₂⁻· production induced by T/C, but not induced by tunicamycin or etoposide is less in HeLa cells expressing the p75^D255A mutant, indicating that caspase-3-mediated cleavage of p75 is required for T/C-induced ROS production. (C) Western blot analysis showing the levels of mitochondrial full-length (FL) p75 and caspase-3 cleaved (CL) p28 NDUFS1 or full-length p17 NDUFB6 subunits of complex I or cytosolic cytochrome c (Cyto C) in p75^WT or mutant p75^D255A HeLa cells treated with 10 ng/ml TNFα and 1 µg/ml CHX or 1 µg/ml CHX alone for 0–4 h, or 1 µg/ml tunicamyin (Tuni) or 100 µM etoposide (Etop) for 0–24 h. Note that although p75 is cleaved into p28 after 2–4 h of T/C treatment, the levels of p75 in response to CHX, Tuni or Etop, as well as those of the p75^D255A mutant, stay the same. Cytochrome c appears in the cytosol and diminishes in the mitochondria after 2 h of T/C treatment or 14 h of Tuni or Etop treatment in both p75^WT and p75^D255A cells, although the kinetics were a bit slower for T/C-treated p75^D255A cells. The loss of cytochrome c in the cytosol after 4 h of T/C treatment is due to secondary necrosis, releasing cytochrome into the medium. Anti-actin and anti-COX-IV western blots serve a control for equal cytosolic and mitochondrial protein loading, respectively. Molecular masses are indicated in kDa on the right side of the blots.

After knowing that ROS production was downstream of MOMP we wanted to know how it was caused. The most likely mediator was caspase-3 because it was previously shown to feed back on mitochondria by cleaving the p75 NDUFS1 subunit of complex I, thereby triggering a fall in the mitochondrial membrane potential and ROS production (Ricci et al., 2004). We first treated WT MEFs with the general caspase inhibitor Z-VAD-fmk and found that TNFα/CHX-induced mitochondrial O₂⁻· production measured by DHE and MitoSOX™ fluorescence was entirely inhibited (Fig. 1A). We then used HeLa cells expressing either WT p75 or a mutant version that cannot be cleaved by caspase-3 any more (p75^D255A). As shown in Fig. 2B, while p75^WT HeLa cells produced significant amounts of O₂⁻· in response to TNFα/CHX, this was not the case with the caspase non-cleavable p75 mutant cells. The p75^WT, but not the mutant protein, was indeed cleaved concomitantly with the release of cytochrome c from mitochondria into the cytosol whereas another subunit of complex I, p17 NDUFB6 remained intact (Fig. 2C, left panel). Importantly, the kinetics of cytochrome c release was only slightly delayed in p75^D255A as compared to p75^WT cells indicating that caspase-3-mediated cleavage of p75 did not majorly affect MOMP, but a process downstream of it (ROS production) (Fig. 2C, left panel). To determine whether this caspase-3-mediated feedback effect for ROS production was specific for TNFα/CHX, we performed the same DHE FACS analysis with p75^WT and p75^D255A HeLa cells treated with the genotoxic agent etoposide or the ER stress stimulus tunicamycin, which are also known to induce MOMP and ROS production (Kroemer et al., 2007). Indeed, both stimuli triggered ROS production but no difference was detected between p75^WT and p75^D255A mutant cells (Fig. 2B). Moreover, the p75 protein was not proteolyzed despite effective cytochrome c release (Fig. 2C, right panel). These data indicate that TNFα/CHX specifically provokes ROS generation via the caspase-3-mediated cleavage of p75 of complex I which supports the finding above that ROS production originates from a mitochondrial source.

p75 cleavage and mitochondrial ROS mediate TNFα/CHX-induced LMP downstream of MOMP, and lysosomes can be spontaneously permeabilized by oxidative damage

Like other apoptotic stimuli TNFα is able to induce LMP. However based on previous studies, it has been postulated that LMP is an early event occurring before MOMP, i.e. right after TNF-R1 stimulation (Boya and Kroemer, 2008; Blomgran et al., 2007; Guicciardi et al., 2000; Guicciardi et al., 2005; Droga-Mazovec et al., 2008; Werneburg et al., 2002; Werneburg et al., 2004; Gyrd-Hansen et al., 2006). Surprisingly, the acid test, namely the role of Bax/Bak and the apoptosome in TNFα-induced LMP has not been performed yet. We therefore looked at TNFα/CHX-triggered LMP in WT, Bax/Bak^−/−, Apaf-1^−/−, caspase-9^−/− and caspase-3/7^−/− MEFs by two means, the decrease of lysosomal red fluorescent acridine orange (AO) staining and the release of cathepsin B and -L (cathepsin B/L, CTB/L) activity and/or protein into the cytosol. As shown in Fig. 3A,B, the TNFα/CHX-induced diminishment of AO staining and the concomitant appearance of cathepsin B/L activity (Z-FRase) in the cytosol were both entirely prevented in all KO MEFs indicating that LMP triggered by TNFα/CHX clearly occurred downstream of MOMP. To identify if mitochondrial ROS production via caspase-3-mediated p75 cleavage was the mediator of this LMP, we quantified diminished AO staining and cathepsin B protein release in WT MEFs co-treated with TNFα/CHX and either MitoQ or the general antioxidant N-acetylcysteine (NAC) and in TNFα/CHX-treated p75^WT and p75^D255A HeLa cells. For comparison, we again exposed MEFs to etoposide and tunicamycin. While LMP was markedly (although not completely) inhibited by both MitoQ and NAC when MEFs were treated with TNFα/CHX, much less LMP inhibition was detected by MitoQ in the case of etoposide and tunicamycin (Fig. 3C,D). As discussed below, the partial dependence of LMP on ROS in the case of these two drugs could be due to ROS production independent of p75 cleavage. Moreover, the release of cathepsin B induced by TNFα/CHX was significantly diminished by MitoQ (Fig. 3E). In addition, while only 49% of p75^WT HeLa cells retained lysosomal AO staining (Fig. 3D), and cathepsin B largely appeared in the cytosol (Fig. 3F) after 4 h of TNFα/CHX treatment, 82.5% of the p75^D225A mutant cells exhibited AO staining (Fig. 3D) and no cytosolic cathepsin B release was observed in these cells (Fig. 3F). Consistent with these data, a cellular treatment with rotenone, which provokes mitochondrial ROS production (see Fig. 1C), also effectively triggered LMP (Fig. 5A). Thus, our results show for the first time that TNFα/CHX-induced LMP is mediated via caspase-3-triggered p75 cleavage and mitochondrial ROS production.

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Fig. 3.

TNFα/CHX-triggered LMP and cathepsin B release from lysosomes depend on the Bax/Bak–apoptosomal pathway, the caspase-3-mediated cleavage of the p75 NDUFS1 subunit of complex I and consequent ROS production. (A) Quantitative FACS measurement of lysosomal AO staining (red fluorescence) and (B) fluorometric determination of cytosolic cathepsin B/L activities (Z-FRase) in WT, Bax/Bak^−/−, Apaf-1^−/−, caspase-9^−/− or caspase-3/7^−/− MEFs treated (gray bars) or not (black bars) with T/C for 4 h. Note that WT cells exhibit a lack of retention of lysosomal AO staining and increased cytosolic cathepsin B/L activities in response to T/C, which reflects induction of LMP. Both changes do not occur at all in the KO cell lines, indicating that LMP and cathepsin B/L release is dependent on Bax/Bak, the caspase-9–Apaf-1 apoptosome and caspase-3/7. (C) The diminished AO staining in WT MEFs exposed to T/C (dark gray bars) is almost completely ablated by the co-treatment with 5 µM of the mitochondrial antioxidant MitoQ (light gray bars), indicating that T/C-induced LMP depends on mitochondrial ROS production. Treatment of these cells with 100 µM etoposide (Etop) for 4 h also induces LMP which is partially inhibited by MitoQ, whereas treatment with 1 µg/ml tunicamycin (Tuni) only slightly affects LMP. The data presented in A–C are the means±s.d. of at least three experiments. P-values were as follows: (A,B) P<0.001, n = 6 between WT and KO MEFs; (C) P<0.001 (T/C), 0.005 (Etop) and 0.01 (Tuni) between untreated and treated cells, P<0.001 (T/C), P<0.004 (Etop) and P<0.01 (Tuni) between ± Mito Q cells, n = 6. (D) FACS dot-plot analysis of AO staining of MEFs treated with T/C in the presence or absence of 100 µM of the general antioxidant N-acetylcysteine (NAC) or 5 µM of the mitochondrially targeted antioxidant MitoQ for 4 h (left panels) or of p75^WT and p75^D255A mutant HeLa cells treated with T/C for the same time period (right panels). T/C induces a loss of the red fluorescence of AO in lysosomes of MEFs (FL-3, red, y-axis) (59.4% less staining) concomitant with a slight gain of green fluorescence of AO in the cytosol (FL-1, green, x-axis) indicating that AO is released into the cytosol due to LMP. Both processes are reduced in cells treated with MitoQ and NAC (only 18.2% and 19.4% less FL-3 red staining, respectively), implicating ROS in the LMP process. Similarly, AO fluorescence decreases in the lysosomes of T/C-treated p75^WT HeLa cells (from 94.7 to 49%), but not in the p75^D255A mutant counterparts (red staining stays at 82.5%) showing that LMP depends on the caspase-3-mediated cleavage of p75 at D255. For unknown reasons, the green fluorescence of released AO in the cytosol does not increase but instead decreases. (E) Cathepsin B western blot of the cytosolic and postmitochondrial membrane (containing lysosomes) fractions of p75^WT HeLa cells treated with 10 ng/ml TNFα and 1 µg/ml CHX in the presence (+) or absence (−) of 5 µM MitoQ for 0–4 h. Note that CTB release into the cytosol is blocked by the MitoQ antioxidant, indicating that it required mitochondrially generated ROS. (F) The same western blot analysis as in E but showing p75^WT and p75^D255A cells treated with T/C for up to 4 h. Whereas, in WT cells, CTB levels increase in the cytosol and decrease in the lysosomal fraction, this does not occur in the p75^D255A cells, suggesting that caspase-3-mediated cleavage at D255A of p75 is required for cathepsin B release from lysosomes to the cytosol. Anti-actin and anti-Lamp-1 western blotting serves as a control for equal loading of cytosolic and lysosomal proteins. Molecular masses are indicated in kDa on the right side of the blots.

To validate this mechanism, we needed to show that ROS production was prior to LMP. For that purpose, we performed a kinetic FACS analysis of TNFα/CHX-treated HeLa cells, using AO (Fig. 4A) and DHE (Fig. 4B) stainings for LMP and ROS measurements, respectively. Since AO exhibits both green and red fluorescence and DHE stains red, the data could not be depicted on a single FACS dot-plot. But by comparing the extent of LMP and ROS at each hour post-treatment we could show that ROS production peaked at 2 h (Fig. 4B, black bars) when only 20% of the cells had undergone LMP (Fig. 4A). This confirms that mitochondrial ROS production in response to TNFα/CHX occurs before the induction of LMP.

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Fig. 4.

TNFα/CHX triggers ROS production prior to LMP. (A) FACS dot-plot analysis of AO staining of HeLa cells treated with 10 ng/ml TNFα and 1 µg/ml CHX (T/C) for 0–6 h. T/C induces a loss of the red fluorescence of AO in lysosomes (FL-3, Red, y-axis) indicating that AO is released into the cytosol owing to LMP. For unknown reasons, the green fluorescence of released AO does not increase but instead decreases in these cells. (B) The same cells as in A were analyzed in parallel for ROS production by DHE FACS analysis. Note that O₂⁻· production is already maximal after 2 h of T/C treatment and stays at the same level up to 5 h (black bars). LMP (i.e. a drop in red fluorescence), however, only starts at 2 h and further increases until 6 h, confirming that ROS production occurs before LMP. The data represent the means±s.d. of at least three experiments, P<0.05 between untreated and 1 h T/C-treated cells, P<0.01 between 1 h with 2 h T/C-treated cells, n = 6.

To get an idea if it is principally possible that ROS can provoke LMP in vitro, we incubated a post-mitochondrial fraction containing lysosomes with a buffer containing no antioxidants (oxidizing) or reducing agents for up to 60 min and then measured the amount of cathepsin B/L activity (Z-FRase) remaining in the lysosomes. As shown in Fig. 5B, left panel, the lysosomal Z-FRase activity gradually decreased with time of incubation, and this was markedly inhibited by the addition of NAC or glutathione (GSH) to the buffer system (Fig. 5B, right panel). Simultaneously, the AO staining, which was diminished in these lysosomes after 60 min buffer incubation also recovered by the presence of the two antioxidants (Fig. 5B, right panel). We could therefore demonstrate that ROS is able to effectively mediate LMP in vitro.

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Fig. 5.

Rotenone triggers a similar extent of LMP to that induced by TNFα/CHX, and isolated lysosomes can be permeabilized by a process that is prevented by the antioxidants NAC and GSH. (A) WT MEFs were treated with 0.5 µM rotenone for 4 h followed by staining with AO, as described in the Materials and Methods. AO fluorescence in lysosomes was then quantified by ScanR cytometry (magnification 40 ×). Although 96% of the untreated cells showed lysosomal AO staining, only 33% of the cells retained this staining after rotenone treatment, indicating that most of the AO was not kept in lysosomes due to LMP to a similar extent as observed upon TNFα/CHX treatment (see Fig. 3A, WT). (B) The post-mitochondrial fraction (containing lysosomes) was extracted from cells loaded with 5 µM AO, washed and resuspended in 1× AT buffer, as described in the Materials and Methods. The fractions were then incubated at 37°C with or without 1 mM N-acetylcysteine (NAC) or 1 mM glutathione (GSH) for up to 60 min, followed by centrifugation and measurement of cathepsin B/L activity (Z-FRase activity, measured as relative fluorescence units, RFU) (left panel) or AO fluorescence (right panel) in the membrane (lysosomal) fraction. Note that cathepsin activity decreased in the lysosomes by simply incubating them at 37°C. This decrease, as well as the reduction in AO staining, were reversed by NAC or GSH, suggesting that LMP is mediated by ROS. In the right panel, the remaining Z-FRase activity and AO staining after 60 min of incubation (without antioxidant addition) was taken as 100% (blue column, boxed). The data in B are the means±s.d. of at least three experiments. P<0.001 comparing 30 and 60 min to 0 min (left panel), and comparing NAC and GSH to no addition (right panel), n = 4.

TNFα/CHX-induced apoptosis is partially prevented by the mitochondria-specific antioxidant MitoQ and in cells expressing the p75^D255A mutant

We finally needed to show that the caspase-3-mediated p75 cleavage and mitochondrial O₂⁻· production had any physiological significance for TNFα/CHX-induced apoptosis, at least as an amplification mechanism. As reported before, this apoptosis was entirely prevented in Bax/Bak^−/−, Apaf-1^−/−, caspase-9^−/− and caspase-3/7^−/− MEFs (Fig. 6A) confirming that in these cells TNFα/CHX used the intrinsic mitochondrial pathway for apoptosis induction. The mitochondria-specific antioxidant MitoQ (Fig. 6B) or the presence of the caspase-3 non-cleavable p75^D225A mutant (Fig. 6C) also partially protected the cells from TNFα/CHX-induced apoptosis. By contrast NAC was unable to interfere with this type of apoptosis (Fig. 6B) despite the fact that it blocked LMP (Fig. 3D), most likely because this antioxidant was slightly cytotoxic on its own (data not shown). For unknown reasons untreated p75^D225A cells respired less effectively than their WT counterparts (supplementary material Fig. S1B,C). Like WT MEFs, p75^WT cells consumed less oxygen upon TNFα/CHX treatment. p75^D225A cells showed a slightly improved respiration under these conditions confirming that their respiratory chain was less impaired and they produced less ROS than p75^WT cells (Fig. 2B).

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Fig. 6.

TNFα/CHX-induced apoptosis depends on the Bax/Bak-apoptosomal pathway and is amplified by the caspase-3-mediated cleavage of the p75 NDUFS1 subunit of complex I, mitochondrial ROS production and cathepsin B/L activities. (A) Quantitative annexin-V/PI FACS analysis of WT, Bax/Bak^−/−, caspase-9^−/−, Apaf-1^−/− or caspase-3/7^−/− MEFs treated (gray bars) or not (black bars) with T/C for 4 h showing that the T/C-induced reduction of the number of annexin-V/PI-negative (surviving) cells is dependent on the Bax/Bak–apoptosomal pathway. (B) Dot-plot analysis of annexin-V (FL-1-green)/PI (FL-3-red) stained WT MEFs treated or not (control) with T/C in the presence or absence of 100 µM NAC or 5 µM MitoQ for 4 h. Note that the number of apoptotic (lower right quadrant, 20.6%) and secondary necrotic (upper right quadrant, 18.3%) in response to T/C is largely not affected by NAC (25.8% and 13.6%, respectively), but markedly decreased by MitoQ (9.0% and 7.2%, respectively), indicating that this cell death is partially mediated via mitochondrial, not general ROS production. (C) Quantitative annexin-V FACS analysis of p75^WT and p75^D255A HeLa cells, as well as WT and cathepsin-B/L-deficient MEFs treated or not with TNFα/CHX (or E64d) for 4 h. The number of dying cells (apoptotic and necrotic) upon T/C treatment is significantly reduced in the presence of the caspase non-cleavable p75^D255A mutant, indicating that cleavage of p75 at its D255 site is not only crucial for ROS production (see Fig. 2B) and LMP (Fig. 3D,E) but also amplifies T/C-induced cell death. Cathepsin B and L appear to amplify T/C-induced cell death in a similar way. The data in A and C represent the means±s.d. of at least three experiments. For A, P<0.001 when comparing WT with mutant cells; for C, P = 0.0004 between p75^WT and p75^D255A cells, P = 0.0008 between WT and CTB/L^−/− MEFs and P = 0.001 between untreated and E64d-treated WT MEFs, n = 5.

However, the p75^D225A cells were not entirely protected against TNFα/CHX-induced apoptosis since the p75/ROS pathway is not expected to be an initiating, requiring event leading to MOMP but a downstream amplifying process to further enhance apoptosis through LMP, cathepsin release and additional caspase activation. Indeed, the released cathespins seemed to contribute to this amplification loop as both cathepsin B/L^−/− MEFs as well as MEFs pre-treated with the cysteine protease inhibitor E64d showed a similar diminishment of TNFα/CHX-induced apoptosis as p75^D225A HeLa cells (Fig. 6C).

Discussion

TNFα, the founding member of the TNF-superfamily, mediates both necroptotic and apoptotic cell death (Walczak, 2011; Micheau and Tschopp, 2003; Andrew, 2004; Wang et al., 2008; Kroemer et al., 2007; Degterev and Yuan, 2008, He et al., 2009; Zhang et al., 2009). Although many investigations have been conducted over the last few years the detailed mechanisms of these death signaling pathways are still not completely known. This particularly concerns our understanding about where ROS are produced intracellularly, how ROS and LMP are mechanistically linked and how both processes contribute to TNFα-induced apoptosis (Boya and Kroemer, 2008; Shen and Pervaiz, 2006; Zdolsek et al., 1993; Brunk et al., 1995). Our study here bridges these missing gaps. We identified that TNFα/CHX-induced LMP is not an apoptosis initiating, but amplifying process downstream of MOMP, and it is triggered by mitochondrial ROS generated as the result of a caspase-3-mediated cleavage of the p75 NDUFS1 subunit of complex I. A similar finding was obtained in two different cellular systems, MEFs and HeLa cells, although molecular tools for manipulating p75 levels were not available for MEFs, and HeLa cells were less sensitive to TNFα/CHX-induced apoptosis due to the expression of HPV E6 and E7 genes (Garnett et al., 2006; Filippova et al., 2002).

The mitochondrial production of ROS by TNFα has been shown in previous reports, and it was also suggested that these ROS somehow contribute to the apoptotic or necrotic process (Kim et al., 2010; Lin et al., 2004; Shoji et la., 1995; Schulze et la., 1993; Shen and Pervaiz, 2006). However, since other intracellular sources of ROS exist, such as the plasma membrane residing NADPH oxidase complex (Yazdanpanah et al., 2009; Woo et al., 2000; Lambeth, 2004), we had to make sure that the majority of ROS implicated in TNFα-induced death signaling is of mitochondrial origin. Indeed, the mitochondria specific antioxidant MitoQ, but not the general antioxidant NAC, partially protected fibroblasts from TNFα/CHX-induced apoptosis. Moreover, DHE and MitoSOX™ stainings were completely abrogated by MitoQ and in Bax/Bak^−/− MEFs indicating that the major source of ROS production induced by TNFα/CHX is indeed the mitochondria. We further showed that rotenone which interferes with complex I, triggered similar ROS production as TNFα/CHX, and this was also blocked by MitoQ. Finally, the fact that HeLa cells expressing the caspase non-cleavable p75^D255A mutant did not produce any ROS in response to TNFα/CHX confirms that complex I is one major source of this ROS generation. It has been reported that ROS can be a direct consequence of Bax/Bak-mediated MOMP (Kirkland et al., 2002; Kirkland and Franklin, 2003; Kirkland and Franklin, 2007; Jiang et al., 2008, Childs et al., 2008), because of the loss of cytochrome c from the respiratory chain complex III/IV and the fall in the mitochondrial membrane potential. This might be the case for apoptotic stimuli such as etoposide or tunicamycin, which did not provoke p75 cleavage despite effective cytochrome c release, and did not depend on p75 for ROS production and apoptosis. As with TNFα/CHX, the kinetics of cytochrome c release induced by these two drugs were also similar between p75^WT and p75^D255A cells. However, the situation is clearly different for TNFα/CHX. Although it similarly triggers cytochrome c release and ROS production as other apoptotic stimuli, the latter event is entirely blocked in Apaf-1^−/−, caspase-9^−/− or caspase-3/7^−/− MEFs. This reinforces the notion that in response to TNFα/CHX ROS is generated downstream of MOMP, e.g. by a caspase-3-mediated cleavage of p75, as has previously been shown by Ricci et al., 2004. In their report they also clearly revealed that the fall in the mitochondrial membrane potential occurs after cytochrome c release in a caspase-3/p75 cleavage dependent manner (Ricci et al., 2004). More studies are required to determine if other apoptotic stimuli (for example other members of the TNF-superfamily such as FasL or TRAIL) use the same mechanism to amplify apoptosis via LMP.

Although the major ROS species, which we measured in our system were O₂⁻· (as evidenced by DHE or MitoSOX staining), we cannot exclude that they were further converted into hydrogen peroxide (H₂O₂) by mitochondrial superoxide dismutases. In fact, in contrast to O₂⁻·, H₂O₂ is more cell permeable and may be the ROS species that leave mitochondria and enter lysosomes where they react with iron to produce the highly reactive hydroxyl radicals (^•OH) (Fenton reaction) which then induce LMP via lipid peroxidation. Indeed the role of intralysosomal iron in oxidant-induced cell death has been previously reported (Yu et al., 2003). Moreover, we could show here that incubating a post-mitochondrial cellular fraction containing lysosomes with an oxidizing buffer triggered LMP in vitro over time. The reason why we did not detect major H₂O₂ production by DCDHF staining might have been due to the use of CHX, which was required for TNFα to effectively induce apoptosis (Walczak, 2011; Micheau and Tschopp, 2003; Andrew, 2004; Wang et al., 2008), but which also is known to downregulate the mitochondrial superoxide dismutase and ferritin heavy chain (Sasazuki et al., 2004; Pham et al., 2004). This may explain why ROS levels reached such high levels within a short time period (2 h) of the TNFα/CHX treatment (Fig. 4B). These levels were already at the maximum before a significant LMP was observed (Fig. 4A) reinforcing that notion that LMP is caused by ROS.

The surprising finding of our study was that TNFα-induced LMP turned out to be a process downstream of MOMP. Many previous studies proposed an early induction of LMP by this cytokine, for example via the activation of acid or neutral (FAN) sphingomyelinases, cathepsin B or caspase-8 (Boya and Kroemer, 2008; Blomgran et al., 2007; Guicciardi et al., 2000; Guicciardi et al., 2005; Droga-Mazovec et al., 2008; Werneburg et al., 2002; Werneburg et al., 2004; Gyrd-Hansen et al., 2006; Ullio et al., 2012). How the lysosomal membrane is damaged by these molecules has however not been unveiled so far, although a direct perforation by Bax or Bak (Werneburg et al., 2007, Castino et al., 2009; Kagedal et al., 2005; Feldstein et al., 2006) or the implication of ROS (without showing from where they in fact originated) were proposed (Zdolsek et al., 1993; Brunk et al., 1995; Roberg and Öllinger, 1998; Brunk and Svensson, 1999; Terman et al., 2006). LMP results in the release of cathepsins, which then either cleave Bid to tBid, process caspases or degrade Bcl-2-like survival factors to induce MOMP (Boya and Kroemer, 2008; Blomgran et al., 2007; Guicciardi et al., 2000; Guicciardi et al., 2005; Droga-Mazovec et al., 2008). These signaling pathways, and therefore LMP, were supposed to be initiating, requiring events for TNFα-induced apoptosis. Astonishingly, none of the previous studies verified if LMP was dependent on MOMP, i.e. if it was blocked in Bax/Bak^−/− cells. Our study now reveals for the first time that TNFα/CHX-induced LMP, as determined by a diminished red fluorescent staining of the lysosomotropic dye AO and the release of cathepsin B/L activity and cathepsin B protein into the cytosol, is entirely dependent on Bax/Bak, the apoptosome and caspase-3/7. Since ROS production is also dependent on this pathway, and that LMP is inhibited by MitoQ treatment or in p75^D225A HeLa cells, the mechanistic link between TNFα and LMP is mitochondrially generated ROS. Thus, rather than being an initiating process, LMP occurs as an amplification loop downstream of MOMP. Such a mechanism explains why inhibiting ROS generation or performing the experiments in p75^D225A HeLa cells did not entirely eliminate TNFα/CHX-induced apoptosis, because cell death could still proceed at a low level without the amplification loop. This adds TNFα to the growing list of apoptotic stimuli, which we have previously shown to trigger LMP downstream of MOMP (Oberle et al., 2010). We therefore propose that in most, if not all cases, LMP and the release of cathepsins are not required for apoptosis induction but rather amplify the process to make sure that cell death occurs effectively, rapidly and irreversibly.