In the past several years, it has been demonstrated that the reactive oxygen species (ROS) may act as intracellular signalling molecules to activate or inhibit specific signalling pathways and regulate physiological cellular functions. It is now well-established that ROS regulate autophagy, an intracellular degradation process. However, the signalling mechanisms through which ROS modulate autophagy in a regulated manner have only been minimally clarified. NADPH oxidase (Nox) enzymes are membrane-bound enzymatic complexes responsible for the dedicated generation of ROS. Different isoforms of Nox exist with different functions. Recent studies demonstrated that Nox-derived ROS can promote autophagy, with Nox2 and Nox4 representing the isoforms of Nox implicated thus far. Nox2- and Nox4-dependent autophagy plays an important role in the elimination of pathogens by phagocytes and in the regulation of vascular- and cancer-cell survival. Interestingly, we recently found that Nox is also important for autophagy regulation in cardiomyocytes. We found that Nox4, but not Nox2, promotes the activation of autophagy and survival in cardiomyocytes in response to nutrient deprivation and ischaemia through activation of the PERK (protein kinase RNA-like endoplasmic reticulum kinase) signalling pathway. In the present paper, we discuss the importance of Nox family proteins and ROS in the regulation of autophagy, with a particular focus on the role of Nox4 in the regulation of autophagy in the heart.

INTRODUCTION

The NADPH oxidase (Nox) family proteins appear to be the only cellular enzymes that are devoted to the purposeful production of ROS (reactive oxygen species), including O2 (superoxide anion) and H2O2 (hydrogen peroxide) [13]. To date, seven members of the Nox family have been identified: Nox1, Nox2 (gp91phox), Nox3, Nox4, Nox5, Duox (dual oxidase) 1 and Duox2 [13]. These proteins have been found to be involved in the regulation of a myriad of cellular processes, such as differentiation during embryonal development, migration and proliferation, as well as cellular senescence and cell death [13]. Importantly, Nox isoforms were shown to be activated during different types of stress, either as a survival mechanism promoting the up-regulation of adaptive mechanisms or as a maladaptive response promoting cellular damage [13]. This dichotomous role of Nox during stress may depend on the specific Nox isoform activated, the sub-cellular compartment where Nox proteins are up-regulated and, most importantly, on the level of Nox isoform activation. In fact, it is now well established that at high levels, ROS contribute to oxidative damage, cellular abnormalities and death. In contrast, physiological and compartmentalized levels of ROS are critical for cell signalling [4].

ROS can oxidize downstream signalling targets, activating or inhibiting specific signalling pathways and regulating cellular functions [5]. Among these cellular functions, ROS are able to significantly modulate autophagy during cellular stress [68]. Autophagy is an evolutionarily conserved intracellular degradation process for elimination of damaged proteins and organelles [9]. Autophagy is usually activated during cellular stress where it reduces the accumulation of misfolded proteins and damaged organelles. In addition, during starvation, autophagy guarantees substrates for energy production and provides amino acids for the synthesis of important cellular proteins [911]. In general, oxidative stress promotes autophagosome formation and flux, although some evidence indicates that RNS (reactive nitrogen species) can inhibit mitophagy through inactivation of Parkin and can reduce autophagosome formation by facilitating the interaction between Beclin-1 and Bcl-2 [12,13]. The mechanisms through which ROS activate autophagy have only been minimally clarified. In most cases, autophagy is secondarily activated by accumulation of ROS as a compensatory/death-limiting mechanism, although it was demonstrated that ROS can directly oxidize components of the autophagic machinery, such as ATG4 to induce autophagy [6]. Interestingly, recent studies provided compelling evidence that Nox, in particular Nox4, can promote autophagy [1421]. Activation of Nox2 in phagocytic cells promotes the activation of autophagy and the recruitment of autophagosomes to phagosomes [14]. Autophagy promotes phagosome maturation and favours pathogen elimination [14]. Activation of Nox2 promotes endothelial function through the activation of AMPK (AMP-activated protein kinase) and autophagy [17]. Nox4 is activated in vascular cells in response to misfolded protein accumulation [15] and oxidized lipoproteins [16], where it promotes autophagy and cell survival through the activation of the Ras/ERK (extracellular-signal-regulated kinase) pathway and the oxidation of Atg4 respectively. Nox4 was also found to promote autophagy and survival in cancer cells [19,21]. Nox2 and Nox4 are the main isoforms of Nox in the cardiovascular system [2,3,22]. We found that Nox4, but not Nox2, promotes the activation of autophagy in cardiomyocytes in response to nutrient deprivation and ischaemia [20]. Nox4 is activated in the ER (endoplasmic reticulum) of energy-deprived cardiomyocytes where it produces ROS that in turn promote autophagy through the activation of the PERK (protein kinase RNA-like ER kinase)/ATF4 signalling pathway. This Nox4-dependent activation of the PERK pathway during energy deprivation promotes cardiomyocyte survival [20]. Of note, in all of these studies, the effects of Nox2 and Nox4 on autophagy were dependent on ROS production [15,16,1820]. This emerging evidence indicates the importance of Nox in the regulation of autophagy and cell survival in different cell types under a variety of cellular conditions. In addition, they highlight the biological relevance of ROS, which represent crucial signalling factors that can be devoted to the activation of autophagy during stress.

In the present review, we will discuss the importance of Nox and ROS in the regulation of autophagy, with a particular focus on the heart. Autophagy plays a critical role in the regulation of cardiomyocyte survival during stress and the Nox isoforms that are also activated during cardiac stress may promote either maladaptive or adaptive mechanisms. The mechanisms through which Nox-derived ROS control autophagic machinery will also be discussed in depth.

BIOLOGY OF NADPH OXIDASE

Nox enzymes are membrane-bound enzymatic complexes that generate O2 by transferring a single electron from NADPH to molecular oxygen [13,23], although some Nox isoforms were found to be able to use NADH as a substrate and directly produce H2O2 [2426]. Nox enzymes have been implicated in numerous physiological and pathological processes, including immune function, aging, embryonic development and physiological regulation in cells and tissues across essentially all organ systems [13,27]. Of the seven Nox family members identified to date, Nox2 and Nox4 are the two isoforms that have been demonstrated to play the most significant role in the regulation of cellular function and response to stress, particularly in the cardiovascular system [2,3,22].

Nox2, also known as gp91phox, is the prototypical Nox originally described in neutrophils and phagocytes [13]. Characterization of subsequent Nox isoforms was based on shared sequence homologies with gp91phox. Common to all members of the Nox protein family are binding sites for NADPH and FAD, six transmembrane domains and conserved histidine pairs that allow binding of haem [13]. Yet distinct features also exist among the various isoforms. In particular, signalling via Nox2 is very tightly regulated and activation occurs through initial phosphorylation of p47phox by kinases like PKC (protein kinase C) [28]. p47phox, which forms a cytosolic multiprotein complex with p40phox and p67phox in the inactive state, translocates to cellular membranes where it binds to the heterodimer Nox2/p22phox. Nox2 activation also involves recruitment of p67phox to the oxidase complex where it binds to Rac–GTP. The formation of a heterodimer with p22phox is a property conserved only among Nox1, Nox3 and Nox4 [13] (Figure 1). These mechanisms appear to occur at the plasma membrane, where Nox2 is mainly localized. Although Nox2 expression is seen in tissues throughout the body, its particular expression pattern in the human cardiovascular system is critical for its numerous roles in regulation of vessel lumen size, embryological development and inflammation and cardiac stress, including hypertrophy and cardiac remodelling [13,22]. In addition, Nox2 appears to be increased in response to cardiovascular bio-mechanical stress, which elicits maladaptive functions. Nox2 is activated in vascular cells and in the heart in response to growth factors and cytokines such as angiotensin II and TNF-α (tumour necrosis factor-α), where it promotes oxidative stress, endothelial dysfunction and cardiac remodelling and fibrosis [2,3,22,29,30]. Nox2 promotes cardiac hypertrophy in response to pressure overload and phenylephrine stimulation [3133]. In addition, increased expression of Nox2 in human cardiac tissue following acute myocardial infarction or coronary artery disease is well established and cardiac Nox2 deletion was shown to reduce cardiac remodelling in response to chronic myocardial infarction [3436].

Biology of Nox

Figure 1
Biology of Nox

Schematic representation of the basic mechanisms of action and activation of Nox.

Figure 1
Biology of Nox

Schematic representation of the basic mechanisms of action and activation of Nox.

Nox4, originally discovered in the kidney, is now known to be expressed in tissues throughout the body and is highly abundant in the cardiovascular system [13,22]. Nox4 is the predominant isoform in endothelial cells and VSMCs (vascular smooth muscle cells) [13,22,37]. Structurally, Nox4 and Nox2 share a similar hydrophobicity profile [13,22]. However, key differences in the first intracellular loop and in the C-terminal portion were shown to confer on Nox4 the unique ability to directly generate H2O2, unlike Nox1 and Nox2 [26], although whether or not Nox4 is able to directly produce H2O2 in all cell types requires further clarification. Like Nox2, ROS production by Nox4 is p22phox-dependent [13,22]. Unlike Nox2, Nox4 activity is mostly transcriptionally regulated and is not dependent on either Rac–GTPase or cytosolic oxidase components, although Nox4 can be regulated by Poldip2 (polymerase δ-interacting protein 2), as elegantly shown by Griendling's group [13,22,38]. Thus, Nox4 appears to be constitutively active [1,39] (Figure 1). The transcription of Nox4 has been shown to be regulated by different mechanisms in response to different cellular stimuli. TGF-β (transforming growth factor β) induces Nox4 expression and activity in fibroblasts, vascular cells and cancer cells [41,42] and E2F1 positively regulates Nox4 in VSMC under unstressed conditions [43]. NF-κB (nuclear factor κB) promotes Nox4 expression in response to hypoxia in vascular cells, whereas PPAR-γ (peroxisome-proliferator-activated receptor γ) is inhibitory under the same conditions [44,45].

Nox4 can be found in multiple sub-cellular compartments and its localization appears to be highly dynamic and dependent on the cell type and conditions [13,22,46]. This may be due to the presence of multiple localization signal sequences for different sub-cellular organelles in the primary structure of Nox4 [13,22,25,33,47]. In cardiomyocytes, Nox4 can be found in mitochondria, ER and the nucleus, with its localization varying during different conditions: Nox4 is rapidly activated in the ER during energy deprivation [20], in the nucleus during phenylephrine stimulation [33] and in the mitochondria during chronic stress [48,49]. This property of dynamic and rapidly varying localization is counter-intuitive, considering that Nox4 exists as a membrane-bound protein, and the mechanism by which this occurs is currently under investigation. Alternative splicing of either the Nox4 coding sequence or Nox4 mRNA UTR may be involved in the regulation of Nox4 localization [5054]. Post-translational modifications of nascent Nox4 protein, such as myristoylation and glycosylation [46,55], retro-translocation from the ER [56] or other yet unknown mechanisms may also be critical in the regulation of Nox4 cellular localization.

Nox4 is activated during stress in the cardiovascular system [3]. Nox4 is a major source of ROS in the failing heart and cardiomyocytes and Nox4 inhibition reduces cardiac dysfunction and hypertrophy in response to pressure overload [49]. Nox4 also mediates reperfusion injury after a brief period of ischaemia [48] and Nox4 up-regulation in cardiomyocytes and vascular cells due to hyperglycaemia contributes to diabetic cardiomyopathy and diabetes-induced vascular abnormalities [5759]. In addition, Nox4 is up-regulated in the heart during aging and cardiac-specific Nox4 overexpression promotes cardiac dysfunction in the late phase of life [25]. However, Nox4 activation also plays an important role in promoting the activation of adaptive signalling pathways and preserving cellular function in response to stress [60]. Nox4 stabilizes HIF-1α (hypoxia-inducible factor 1-α) levels and promotes vascular cellular proliferation and migration in response to hypoxia [44,6164]. In VSMC, Nox4 interacts with protein disulfide isomerase in the ER and may regulate the unfolded-protein response [46,65,66]. Nox4 promotes vascular cell survival in response to misfolded protein accumulation, through focal activation of the Ras/ERK pathway and autophagy [15] and following exposure to oxidized lipoproteins [16]. Nox4 activation protects endothelial cells against inflammatory stress through the activation of eNOS (endothelial nitric oxide synthase) [67]. Nox4 also promotes activation of the Nrf2 (nuclear factor-erythroid 2-related factor 2) signalling pathway in cardiomyocytes and vascular cells, through which it regulates the antioxidant response to stress [68,69]. Cardiac Nox4 promotes angiogenesis during pressure overload [70] and Nox4 activation in cardiomyocytes during energy deprivation promotes survival through the activation of autophagy [20]. There may be several possible explanations for these dichotomous outcomes of Nox4 activation in the cardiovascular system. First of all, exaggerated activation of Nox4 in response to certain stressors may be detrimental, whereas moderate activation may not be. Activation of Nox4 in specific organelles, such as mitochondria, may be detrimental [48,49,71], whereas activation in the ER may be adaptive [15,20]. Finally, activation of Nox4 under specific cellular conditions may be protective because Nox4 may have unique functions that are not shared by Nox2 in these contexts. On the other hand, in the conditions where Nox4 activation is maladaptive, it is very likely that Nox4 and Nox2 perform overlapping physiological functions. In these cases, in the absence of simultaneous Nox2 inhibition, inhibition of Nox4 may reduce only the detrimental effects of its activation without affecting much of its physiological functions. For example, we found that single deletion of Nox4 or Nox2 reduces ROS-induced reperfusion injury but that combined deletion of these isoforms actually aggravates reperfusion injury through the activation of PHD2 (prolyl hydroxylase domain 2) protein and inhibition of HIF-1α [48]. This is presumably due to the fact that both Nox4 and Nox2 can inhibit the activity of PHD2, such that deletion of only one of these isoforms is not sufficient to impair this mechanism.

AUTOPHAGY BIOLOGY WITH FOCUS ON AUTOPHAGY IN THE HEART

Autophagy (from the Greek, ‘auto’ self, ‘phagein’ to eat) is an evolutionarily conserved mechanism, originally characterized in yeast and now known to occur at a basal, homoeostatic level in virtually all eukaryotic cells [9,72]. At least three forms of autophagy have been described: microautophagy, chaperone-mediated autophagy and macroautophagy, which is the main autophagic pathway [9,72]. Macroautophagy, hereafter referred to as ‘autophagy’, is characterized by the sequestration and subsequent degradation of the long-lived proteins or organelles by double-membraned vesicles called the autophagosomes. Autophagy has two main functions. The first is to maintain homoeostasis in the cell at baseline and during stress through degradation of long-lived proteins and organelles. This mechanism is especially important in cardiomyocytes, since the heart is characterized by a very low cellular turnover rate [73], making an effective system for maintaining homoeostasis indispensable. The second function is to recycle amino acids derived from the degradation of proteins. These amino acids can be used to produce ATP when energy is scarce in the cell or to synthesize important proteins needed for the cellular response to stress. This becomes particularly relevant under stress conditions like glucose deprivation or ischaemia [9,72].

Functionally, two different types of autophagy exist: non-selective autophagy and cargo-specific autophagy. The first type refers to an autophagic process that is not selective for specific proteins and organelles. On the other hand, cargo-specific autophagy refers to a type of autophagy that sequesters and degrades specific organelles, such as mitochondria (mitophagy) and ribosomes (ribophagy), through specific mechanisms of regulation. Mitophagy is the most studied type of cargo-specific autophagy and accumulating lines of evidence indicate that mitophagy plays a crucial role in the regulation of cardiac homoeostasis and mitochondrial function. In fact, mitophagy is required for appropriate mitochondrial turnover [9,72,74].

In the initial stage of autophagy, an isolation membrane, primarily derived from either the ER or the mitochondria, is formed. This double-membraned vesicle, also termed a phagophore, engulfs its target macromolecule to form an autophagosome, which then transports its cargo to the lysosomes [9,72]. The molecular machinery of autophagy is tightly regulated and can be conceptualized in four separate phases: induction, expansion, maturation and fusion [9,72] (Figure 2). The AMPK/Rheb (Ras homologue enriched in brain)/mTOR (mammalian target of rapamycin) pathway and the SIRT1/FOXO1 (forkhead box O1) pathway appear to be the master regulators of cellular stress-induced autophagy. Activated AMPK can induce autophagy directly by phosphorylating ULK1 (Unc-51-like kinase 1) (a mammalian homologue of yeast Atg1) or indirectly through the inhibition of mTOR complex 1 via phosphorylation of TSC2 (tuberous sclerosis 2) and regulatory-associated protein of mTOR (Raptor) and inhibition of Rheb. mTOR phosphorylates and inhibits the multiprotein complex consisting of ULK1 and ULK2, Atg13 and focal adhesion kinase FIP200 (family-interacting protein of 200 kDa) [9,72]. This, in turn, activates the autophagy-specific class III PI3K (phosphoinositide 3-kinase) complex [Vps (vacuolar protein sorting) 34, Vps150, Beclin-1 and mAtg14], which allows for the nucleation of the phagophore membrane from the phagophore assembly site. Once formed, membrane elongation and phagophore expansion are regulated by two additional ubiquitin-like complexes [Atg5–Atg12 and PE (phosphatidylethanolamine)–LC3-I that forms LC3-II], which are recruited to the phagophore by the PI3K complex [9,72]. Upon completion of the autophagosome, LC3-II, the only autophagy protein associated with the completed vesicle, is cleaved from PE by Atg4 and returned unchanged to the cytosol. Autophagosome–lysosome fusion in mammalian cells is regulated by LAMP-2 (lysosomal associated membrane protein-2) and the small GTPase Rab7 and it is activated by the SIRT1/FOXO1 pathway and by the transcription factor EB [911,72].

Role of autophagy in the heart

Figure 2
Role of autophagy in the heart

Schematic representation of the autophagic process and of the pathophysiological implications of autophagy during cardiac stress.

Figure 2
Role of autophagy in the heart

Schematic representation of the autophagic process and of the pathophysiological implications of autophagy during cardiac stress.

Autophagy plays a critical role in the preservation of cardiac structure and function. Indeed, mice with genetic disruption of autophagy through cardiac-specific deletion of Atg5 develop significant left ventricular dilatation and severe contractile dysfunction, which are accompanied by aberrant mitochondrial accumulation and disorganized sarcomeres [75]. Dysregulation of autophagic flux has significant consequences as well: LAMP-2 deficiency, as seen in Danon disease, is characterized by progressive myopathy leading to cardiac dysfunction and failure in the context of abnormally large autophagosomal clusters [911,72,76]. Autophagy is progressively inhibited during cardiac aging, contributing to the development of age-related cardiac abnormalities. Indeed, pro-aging substances like ROS, ubiquitinylated proteins and damaged mitochondria are normally eliminated by autophagy [911,72].

Autophagy is also up-regulated during cardiac stress, when it usually limits cardiomyocyte damage by limiting energy stress, relieving ER stress and mitochondrial dysfunction and reducing cell death [911,72,7779] (Figure 2). In fact, mice with cardiac-specific AMPK inhibition, Rheb overexpression or obesity/metabolic syndrome display significantly increased ischaemic injury due to autophagy inhibition [7779]. Moderate activation of autophagy is also protective during pressure overload and chronic myocardial infarction [75,80]. However, autophagy activation during cardiac stress can also be maladaptive and promote cell death. For example, exaggerated activation of autophagy during mechanical stress promotes cardiac remodelling [81]. Similarly, we observed that autophagy is extensively activated by ROS during myocardial reperfusion after ischaemia, contributing to myocardial injury [77,82] (Figure 2). It is likely that massive up-regulation of autophagy leads to degradation of important proteins and organelles, thereby causing cell death. In fact, a form of cell death distinct from apoptosis and necrosis has recently been described. This new type of cell death, named ‘autosis’ because it was found to be associated with autophagy, is characterized by cellular nuclear convolution at early stages and focal swelling of the perinuclear space at late stages. It is inhibited by autophagy inhibition and by digitalis, which blocks the Na+/K+–ATPase pump [83].

ROS AND AUTOPHAGY

Over the past decade, the role of ROS and RNS as second messengers in intracellular signalling has been increasingly recognized [5]. This process, called redox signalling, is made possible by the precisely-controlled balance of pro-oxidants and antioxidants under normal physiological conditions. A controlled, localized increase in ROS/RNS can modify a signalling target via post-translational oxidative modifications, thereby acting as an on-off switch for specific signalling pathways [5]. Redox signalling has emerged as a crucial mechanism of regulation of physiological cellular functions in the heart. For example, it has recently been demonstrated that progressive mitochondrial ROS accumulation in the mammalian heart, which occurs during the initial days of postnatal life and is secondary to the passage from the embryonic hypoxic environment to the postnatal normoxic environment, is responsible for the cell-cycle arrest of cardiomyocytes and for the switch from hyperplastic to hypertrophic growth in the postnatal mammalian heart [84].

Accumulating lines of evidence demonstrate that oxidative stress significantly modulates autophagy during cellular stress [68]. In general, oxidative stress was shown to be associated with increased autophagosome formation and flux, but some evidence indicates that RNS can inhibit mitophagy through inactivation of Parkin and can reduce autophagosome formation by facilitating the interaction between Beclin-1 and Bcl-2 [12,13]. Nitric oxide (NO)-based cell signalling occurs through S-nitrosylation, the covalent incorporation of nitric oxide into thiol groups. By S-nitrosylating JNK1 (c-Jun N-terminal kinase 1), NO impairs autophagosome formation by enhancing Bcl-2–Beclin-1 dimerization [12]. NO also independently inhibits the IKK-β (inhibitor of NF-κB kinase-β), which ultimately results in an mTOR-dependent down-regulation of autophagy [12].

The mechanisms through which ROS activate autophagy have only been partially clarified. In most cases, autophagy is secondarily activated by accumulation of ROS as a compensatory/death-limiting mechanism (Figure 3) [68]. Increased oxidative stress can promote secondary stimulation of the FOXO1 pathway that activates autophagy and the antioxidant defence [68]. We previously showed that cardiomyocyte starvation, a condition characterized by increased ROS production, stimulates autophagy and autophagic flux through the activation of the SIRT1/FOXO1 pathway and up-regulation of Rab7 [85]. Oxidative stress can also activate autophagy through activation of the HIF-1α/Bnip3 signalling pathway and up-regulation of autophagic proteins such as Beclin-1 and Atg5 [86,87]. In fact, it was shown that HIF-1α mediates the activation of autophagy in hypoxic cells through up-regulation of Bnip3 in multiple cell lines [86,87] and compelling evidence from Gustafsson's group showed that Bnip3 mediates ROS-induced autophagy during cardiomyocyte hypoxia/re-oxygenation and that Bnip3 overexpression induces mitophagy through the translocation of Drp-1 to mitochondria in cardiomyocytes [8890]. ROS can also stimulate autophagy through activation of the NF-κB signalling pathway in the heart [91]. Intriguingly, IKK-β, the canonical activator of NF-κB, which is known to be activated by oxidative stress [92], can induce autophagy independently of NF-κB [93]. Inhibition of IKK-β limits autophagy activation in response to energy deprivation in cancer cells, whereas activation of IKK-β induces autophagy that cannot be inhibited by concomitant inhibition of NF-κB. The antioxidant protein, Nrf2, is also an inducer of autophagy in response to ROS [93]. Oxidative stress promotes nuclear translocation of Nrf2, which induces autophagy through up-regulation of the autophagy adaptor protein NDP52 [94]. Nrf2 is also a strong inducer of autophagy in the heart, where it favours the clearance of misfolded proteins during pressure overload, preventing necrosis and cardiac dysfunction in response to proteotoxic stress [95]. ROS were also shown to induce autophagy through the pro-autophagic Beclin-1 binding protein, HMGB1 (high-mobility group box 1), in MEF (mouse embryonic fibroblast) cells. Upon oxidative stress, HMGB1 translocates from the nucleus to the cytosol, where it enhances autophagic flux by binding to Beclin-1 via its intramolecular disulfide bridge (C23/45) [96]. Finally, p53-regulated sestrin proteins also activate autophagy in response to oxidative stress through binding to AMPK and TSC1 and 2 [7,97,98].

Role of ROS in the regulation of autophagy and the ubiquitin/proteasome system

Figure 3
Role of ROS in the regulation of autophagy and the ubiquitin/proteasome system

Schematic representation of the most established signalling mechanisms through which ROS regulate autophagy and the ubiquitin/proteasome system.

Figure 3
Role of ROS in the regulation of autophagy and the ubiquitin/proteasome system

Schematic representation of the most established signalling mechanisms through which ROS regulate autophagy and the ubiquitin/proteasome system.

ROS can also induce autophagic cell death. For example, we found that ROS induce maladaptive autophagy during ischaemia/reperfusion, thereby enhancing myocardial injury through the up-regulation of Beclin-1 [77,82]. In this manner, ROS can induce caspase-independent autophagic cell death through a Beclin-1 dependent pathway [99]. Under normal circumstances, Beclin-1 is bound to Bcl-2 as an inactive complex. However, when the concentration of ROS far exceeds the antioxidant defences of the cell, the ubiquitin–proteasome system is activated, resulting in the degradation of Bcl-2. This allows Beclin-1 to ultimately signal for autophagic cell death [99]. Of note, we recently found that the serine/threonine kinase Mst1 inhibits autophagy through phosphorylation of Beclin-1 at Thr108, which contributes to cardiac remodelling after myocardial infarction [80]. Since Mst1 is known to be activated by ROS, it will be interesting to investigate in the future whether oxidative stress can promote cell death through Mst1-dependent inhibition of autophagy.

Finally, recent evidence suggests that ROS can also indirectly regulate autophagy through the control of the ubiquitin–proteasome system (Figure 3) [100,101]. The latter represents another important cellular mechanism for protein degradation. In response to oxidative stress, oxidized proteins accumulate and subsequently trigger the activation of the ubiquitin–proteasome system, despite the fact that severe oxidative stress can transiently inhibit proteasome activity through ROS-induced S-glutathionylation [100,101]. The transcription factor Nfr2, which translocates to the nucleus in response to ROS-mediated Keap1 (kelch-like ECH-associated protein 1) inactivation, was previously shown to promote the up-regulation of proteasomal genes [102]. TFC11/Nrf1 transcription factor also up-regulates proteasomal genes in response to the accumulation of oxidized proteins, through direct binding to antioxidant response elements located in the promoters of these genes [103]. TFC11/Nrf1 also promotes the up-regulation of the TXNL1 (thioredoxin-like protein 1), which associates with the 26S proteasome, probably regulating its substrate [100,101,103]. Interestingly, recent work demonstrated that the ubiquitin–proteasome system can also regulate autophagy and mitophagy [104]. Mitofusin-1, DRP-1 and Fis-1, key regulators of mitochondrial dynamics and, therefore, mitophagy, can be degraded by the ubiquitin–proteasome system [104]. Parkin, an E3 ubiquitin ligase associated with autosomal recessive Parkinsonism, activates mitophagy through the ubiquitylation of mitochondrial proteins [104]. Proteasome inhibition was shown to be sufficient to activate autophagy in cardio-myocytes [105]. Finally, the ubiquitin–proteasome system may indirectly control autophagy by regulating the unfolded protein response, which is another important signalling network that promotes autophagosome formation [104,106].

The evidence above indicates that ROS can activate autophagy indirectly through the regulation of specific signalling pathways. However, ROS were also shown to stimulate autophagy by directly oxidizing the components of the autophagic machinery. In the seminal study by Scherz-Shouval et al. [107], it was demonstrated that ROS promote autophagy in response to starvation in the ovarian cells by oxidizing Atg4. For autophagy to occur, Atg4 is initially required for the cleavage of specific residues at the C-terminus of LC3-I, allowing conjugation of LC3-I with PE [107]. However, following this initial step, Atg4 has to be inactivated to prevent reaction with the LC3–PE complex, which would result in deconjugation and inhibition of autophagy [107]. In the setting of starvation, H2O2 is produced, which oxidizes Cys81 of the C-terminal portion of Atg4, inactivating it. This inactivation of Atg4 by ROS prevents the premature dissociation of LC3–PE from the autophagosomal membrane, thereby inducing autophagy [6,107]. The mechanisms through which ROS regulate autophagy have been summarized in Table 1.

Table 1
Summary of the most known signalling mechanisms through which ROS may regulate autophagy
Signalling pathway Role in autophagy Effects of ROS References 
Parkin Promotes mitophagy through mitochondrial protein ubiquitinylation RNS inhibit mitophagy by inactivation of Parkin [12,13
Beclin1 Promotes autophagosome formation Beclin forms an inactive complex with Bcl2. ROS can degrade Bcl-2 allowing Beclin-1 to promote autophagy [12,99
JNK1 Phosphorylates Bcl-2 to activate autophagy S-nitrosylation of JNK1 promotes Bcl2–Beclin1 interaction and thus, inhibits autophagosome formation [12
SIRT1/FOXO1 Stimulates autophagy and autophagic flux through Rab7 up-regulation Starvation and ROS promote stimulation of FOXO1 pathway, thus autophagy and autophagic flux [68,85
HIF-1α/Bnip3 Up-regulates autophagy. Bnip3-induced mitophagy through mitochondrial translocation of Drp1 ROS activate both HIF-1α and Bnip3 [8690
IKK-β and NF-κB Promote autophagy Reperfusion injury activates NF-κB pathway. ROS also stimulate autophagy through the activation of IKK-β, independent of NF-κB [9193
Nrf2 Promotes autophagy through up-regulation of NDP52 ROS promote nuclear translocation of Nrf2 though the inactivation of Keap1 [94,95
HMGB1 Stimulates autophagic flux by binding to Beclin-1 Oxidative stress promotes cytosolic translocation of HMGB1 [96
Sestrin proteins Bind to AMPK, TSC1 and TSC2 to stimulate autophagy Oxidative stress activates sestrin proteins [7,97,98
Mst1 Inhibits autophagy through the phosphorylation of Beclin-1 ROS activate Mst1 [80
Atg4 Atg4 mediates LC3-I conjugation to PE (lipidation) and then LC3–PE deconjugation (delipidation) ROS inactivate Atg4, thereby inducing LC3 lipidation and autophagy [6,100
Signalling pathway Role in autophagy Effects of ROS References 
Parkin Promotes mitophagy through mitochondrial protein ubiquitinylation RNS inhibit mitophagy by inactivation of Parkin [12,13
Beclin1 Promotes autophagosome formation Beclin forms an inactive complex with Bcl2. ROS can degrade Bcl-2 allowing Beclin-1 to promote autophagy [12,99
JNK1 Phosphorylates Bcl-2 to activate autophagy S-nitrosylation of JNK1 promotes Bcl2–Beclin1 interaction and thus, inhibits autophagosome formation [12
SIRT1/FOXO1 Stimulates autophagy and autophagic flux through Rab7 up-regulation Starvation and ROS promote stimulation of FOXO1 pathway, thus autophagy and autophagic flux [68,85
HIF-1α/Bnip3 Up-regulates autophagy. Bnip3-induced mitophagy through mitochondrial translocation of Drp1 ROS activate both HIF-1α and Bnip3 [8690
IKK-β and NF-κB Promote autophagy Reperfusion injury activates NF-κB pathway. ROS also stimulate autophagy through the activation of IKK-β, independent of NF-κB [9193
Nrf2 Promotes autophagy through up-regulation of NDP52 ROS promote nuclear translocation of Nrf2 though the inactivation of Keap1 [94,95
HMGB1 Stimulates autophagic flux by binding to Beclin-1 Oxidative stress promotes cytosolic translocation of HMGB1 [96
Sestrin proteins Bind to AMPK, TSC1 and TSC2 to stimulate autophagy Oxidative stress activates sestrin proteins [7,97,98
Mst1 Inhibits autophagy through the phosphorylation of Beclin-1 ROS activate Mst1 [80
Atg4 Atg4 mediates LC3-I conjugation to PE (lipidation) and then LC3–PE deconjugation (delipidation) ROS inactivate Atg4, thereby inducing LC3 lipidation and autophagy [6,100

NADPH OXIDASE AND AUTOPHAGY IN NON-CARDIOMYOCYTE CELLS

In the past several years, emerging data have indicated that Nox is involved in the positive regulation of autophagy in several different cell types [1421] (Table 2). This is not surprising given that Nox proteins are dedicated generators of ROS and that a close relationship exists between ROS and autophagy [68]. However, we believe that the fact that Nox regulates autophagy is biologically important because it proves that ROS do not only activate autophagy by stimulating a secondary autophagic response to oxidative stress, but also that they act as molecular signalling molecules, purposefully generated by Nox proteins in a regulated manner, to modulate specific autophagy signalling pathways. Both Nox2 and Nox4 have been implicated in the regulation of autophagy [1421]. However, the majority of existing evidence indicates that Nox4 plays a major role in control of the autophagy machinery. This is reasonable if we consider that Nox4 is intracellularly localized and possibly capable of directly generating H2O2, which is a stable signalling molecule [13,1422].

Table 2
Summary of the studies providing evidence of the role of Nox in the regulation of autophagy
Study Nox isoform: localization Cell type Effect on autophagy Outcome of Nox- induced autophagy Reference 
Immune cells      
 Huang et al. [14Nox2 Phagocytes Nox2 stimulates autophagy during pathogen phagocytosis; other Nox isoforms activate autophagy in non-phagocytic cells lacking Nox2 in response to bacterial invasion Elimination of phagocytized pathogens [14
 Romao et al. [108Nox2 Phagocytes Nox2-derived ROS activate autophagy, leading to phagocytosis regulation Efficient processing of extracellular antigen for MHC class II presentation [108
Vascular cells      
 Shafique et al. [17Nox2 Endothelial cells Nox2 induces autophagy through activation of the AMPK pathway Improves endothelial function [17
 Teng et al. [18Nox2 Pulmonary artery endothelial cells Persistent pulmonary hypertension promotes autophagy through Nox2 activation Inhibits angiogenesis; maladaptive [18
 Wu et al. [15Nox4:ER Endothelial cells Nox4 activates autophagy via the Ras/ERK signalling pathway in response to misfolded protein accumulation Increases endothelial cell survival [15
 He et al. [16Nox4:ER VSMC Nox4 promotes autophagy by inhibiting Atg4 in response to 7-KC Promotes ER stress relief and, thus, cell survival [16
Cancer cells      
 Sobhakumari et al. [19Nox4 Cancer cells EGFR inhibitor erlotinib promotes autophagy through Nox4 activation Increases cell survival during chemotherapy [19
 Yoon et al. [21Unspecified Nox Cancer cells Starvation stimulates Nox and autophagy and, thus, activation of the JAK2/STAT3 pathway and IL-6 Promotes cells survival [21
Cardiomyocytes      
 Sciarretta et al. [20Nox4:ER Cardiomyocytes Glucose deprivation promotes autophagy through the PERK/ATF4 signalling pathway Promotes cardiomyocyte survival, limits infarct size during prolonged ischaemia [20
Study Nox isoform: localization Cell type Effect on autophagy Outcome of Nox- induced autophagy Reference 
Immune cells      
 Huang et al. [14Nox2 Phagocytes Nox2 stimulates autophagy during pathogen phagocytosis; other Nox isoforms activate autophagy in non-phagocytic cells lacking Nox2 in response to bacterial invasion Elimination of phagocytized pathogens [14
 Romao et al. [108Nox2 Phagocytes Nox2-derived ROS activate autophagy, leading to phagocytosis regulation Efficient processing of extracellular antigen for MHC class II presentation [108
Vascular cells      
 Shafique et al. [17Nox2 Endothelial cells Nox2 induces autophagy through activation of the AMPK pathway Improves endothelial function [17
 Teng et al. [18Nox2 Pulmonary artery endothelial cells Persistent pulmonary hypertension promotes autophagy through Nox2 activation Inhibits angiogenesis; maladaptive [18
 Wu et al. [15Nox4:ER Endothelial cells Nox4 activates autophagy via the Ras/ERK signalling pathway in response to misfolded protein accumulation Increases endothelial cell survival [15
 He et al. [16Nox4:ER VSMC Nox4 promotes autophagy by inhibiting Atg4 in response to 7-KC Promotes ER stress relief and, thus, cell survival [16
Cancer cells      
 Sobhakumari et al. [19Nox4 Cancer cells EGFR inhibitor erlotinib promotes autophagy through Nox4 activation Increases cell survival during chemotherapy [19
 Yoon et al. [21Unspecified Nox Cancer cells Starvation stimulates Nox and autophagy and, thus, activation of the JAK2/STAT3 pathway and IL-6 Promotes cells survival [21
Cardiomyocytes      
 Sciarretta et al. [20Nox4:ER Cardiomyocytes Glucose deprivation promotes autophagy through the PERK/ATF4 signalling pathway Promotes cardiomyocyte survival, limits infarct size during prolonged ischaemia [20

The first demonstration of the link between Nox and autophagy was reported in a study by Huang et al. [14], in which the authors found that Nox2-mediated autophagy in phagocytes is required for pathogen elimination after phagocytosis. Nox2 is activated during the process of pathogen phagocytosis in immune cells and Nox2-derived ROS may promote autophagosome formation and subsequent recruitment to phagosomes. This process is independent of mitochondrial ROS. Importantly, Nox2-generated ROS are important for phagosome maturation and, later, for elimination of phagocytized pathogens [14]. Nox2 is the main Nox isoform in phagocytes [13,22]. Interestingly, the authors of this paper also found that Nox is also important for autophagy activation in response to bacterial invasion in non-phagocytic cells lacking Nox2, suggesting that other Nox isoforms are involved in the autophagic response to stress in non-phagocytic cells [14]. The importance of Nox2-dependent autophagy in the immune response was further corroborated by a recent study demonstrating that the activation of autophagy by Nox2-derived ROS in LC3-positive phagosomes is crucial for efficient processing of extracellular antigens for MHC class II presentation [108].

Nox is important for the autophagic response to a variety of stresses in vascular cells. In their seminal study, Wu et al. [15] demonstrated that Nox4 is activated in the ER of endothelial cells with misfolded protein accumulation, where it promotes H2O2 accumulation. Nox4 activation in the ER promotes autophagy activation and cell survival through activation of the Ras/ERK signalling pathway. Interestingly, in this study, Nox2 was found to be dispensable for autophagy activation in response to ER stress [15]. However, recent evidence indicates that moderate overexpression of Nox2 is sufficient to induce autophagy in endothelial cells through activation of the AMPK pathway and Nox2 overexpression improves endothelial function [17]. Nox4 was found to promote autophagy in VSMC in response to 7-ketocholesterol (7-KC) [16]. 7-KC, a major component of oxidized lipoproteins, has garnered much attention due to its role in the pathogenesis of human atherosclerosis [16,109]. 7-KC was found to enhance Nox4 levels but not the levels of the other Nox isoforms in VSMC. Nox4-derived H2O2 promotes autophagy in response to 7-KC through the inhibition of Atg4, thereby promoting survival through relief of ER stress [16]. The study suggests that Nox4 activation and autophagy may limit the development of atherosclerosis in response to oxidized lipoproteins. However, Nox-dependent autophagy in vascular cells can also be maladaptive. It was recently demonstrated in pulmonary-artery endothelial cells from foetal lambs with persistent pulmonary hypertension that Nox2-derived ROS promote autophagy and inhibit angiogenesis [18]. Autophagy inhibition decreases ROS production, reduces apoptosis and enhances angiogenesis in these cells [18].

Recent studies also suggest that Nox4 is important in autophagy regulation in cancer cells. Autophagy is often activated in aggressive cancer cells, promoting cell survival, particularly in response to chemotherapy. Recent work demonstrated that erlotinib, an epidermal growth factor receptor (EGFR) inhibitor used to treat head and neck squamous cell carcinomas, may up-regulate autophagy in head and neck cancer cells through activation of Nox4 [19]. Nox4-dependent autophagy activation promotes cell survival and may explain the poor therapeutic response to erlotinib sometimes observed in head and neck squamous cell carcinomas [19]. In addition, it has recently been demonstrated that Nox is activated in cancer cells in response to starvation and Nox activation is paralleled by a significant activation of autophagy, which, in turn, promotes activation of the JAK2 (Janus kinase 2)/STAT3 (signal transducer and activator of transcription 3) pathway, up-regulation of IL (interleukin)-6 and ultimately cell survival [21,110].

Overall these studies highlight the importance of Nox in the regulation of autophagy. Of note, in the majority of these studies, reduction in ROS levels through expression of catalase or by antioxidants abrogated the Nox-dependent activation of autophagy, indicating that Nox proteins regulate autophagy through the production of ROS [15,16,1820].

NADPH OXIDASE AND AUTOPHAGY IN THE HEART

We recently clarified the role of Nox in the regulation of autophagy in the heart [20]. In cardiomyocytes, autophagy is activated during energy stress, limiting cell death [911,72]. This is highly clinically relevant since myocardial ischaemia, the most common and life-threatening acute cardiac pathological event, is characterized by severe energy stress. Acute energy stress is characterized by rapid ROS accumulation [107,111]. As discussed previously, Nox2 and Nox4 are the main Nox isoforms in the cardiovascular system [2,3,22]. We found that Nox4 expression levels are rapidly up-regulated during glucose deprivation, thereby promoting ROS accumulation [20]. This result is consistent with previous evidence that Nox4 is rapidly activated during glucose deprivation in cancer cells, where it is responsible for ROS production [112]. Nox4 protein levels are increased during starvation in vascular cells as well, where Nox4 activation inhibits apoptosis [113,114]. In addition, it is well established that Nox4 activity is enhanced during hypoxia, during which Nox4-derived ROS may be involved in stabilization of HIF-1α levels [44,6164]. Although the activation of Nox4 in the absence of oxygen and energy might seem paradoxical, it may be explained by the significant up-regulation of Nox4 protein levels under these conditions, which appears to happen in specific cellular compartments, as we will discuss later. Nox activity is maintained even at low oxygen levels [13]. In the early phase of nutrient deprivation, cellular energy content is still preserved and the NADPH pool can be maintained through multiple metabolic pathways and enzymes, such as NADH kinase, aldehyde dehydrogenase and transhydrogenase [13,115118]. Of note, other enzymes utilizing NADPH have been previously shown to be activated during hypoxia and nutrient deprivation. Shao et al. [119] recently found that thioredoxin-1, an enzyme that requires NADPH for its activity, is also activated during the early phase of ischaemia and nutrient deprivation, limiting redox modification of AMPK at Cys130/Cys174 and thereby promoting AMPK activation and cell survival. These data thus provide fascinating evidence indicating that, during the early phase of energy deprivation, cells appear to utilize some of the remaining energetic substrates to activate multiple crucial adaptive mechanisms, such as autophagy or antioxidant defence, which are critical for preserving cellular energy content or limiting oxidative damage in later phases of stress.

Of note, studies have indicated that NF-κB is rapidly activated and promotes up-regulation of Nox4 during cellular energy deprivation [44,45,120122]. We previously found that NF-κB regulates Nox4 expression levels [33]. We found that inhibition of NF-κB activity prevents the up-regulation of Nox4 during glucose deprivation, suggesting that NF-κB plays an important role in this mechanism. Intriguingly, since NF-κB is known to be rapidly activated in response to cellular stress, these results can explain how Nox4 levels and activity are rapidly up-regulated in response to glucose deprivation and hypoxia [120]. This is consistent with the rapid up-regulation of Nox4 protein levels (within minutes) in response to growth factors [33,123]. Nevertheless, it remains important to investigate whether translation and post-translational mechanisms of regulation are also involved in the rapid up-regulation of Nox4 during cellular energy stress. In particular, it will be important to understand the involvement of p22–Nox4 interaction in these mechanisms.

Nox4 is localized mainly in the ER and mitochondria in the cardiovascular system [2,3,22]. Surprisingly, we found that Nox4 levels and activity are selectively increased in the ER but not in mitochondria during the early response to cardiomyocyte glucose deprivation [20]. This provides additional evidence that Nox4 expression and cellular localization is dynamic and varies depending on cell type and cellular conditions. As we discussed above and as comprehensively elaborated upon in the recent review article by Laurindo et al. [46], it is possible that Nox4 is always initially expressed in the ER and that it then translocates to other organelles. Alternatively, Nox4 localization in the ER in cardiomyocytes may be energy deprivation-dependent. The varying cellular localizations of Nox4 may be dependent upon post-translational modifications and protein–protein interactions [46], as is the case with N-myristoylation of NADH-cytochrome b5 reductase, which determines its localization to mitochondria rather than the ER [55]. Alternative splicing of either the coding or the UTR that is differentially modulated under different cellular conditions may also drive the localization of Nox4 to different sub-cellular compartments [5054]. An interesting study from Shah and group [29] provided compelling evidence that a splice variant of the coding sequence of Nox4 can generate a Nox4 isoform that localizes to the nucleus. Intriguingly, it has been demonstrated that asymmetric localization of mRNAs is an alternative mechanism for regulating protein sub-cellular localization [51]. Alternative splicing of the 3′-UTR region of specific transcripts of genes, for example, like in the case of inositol monophosphatase-1 in neurons, can affect the fate of the mRNAs and of the nascent proteins, resulting in localization to different sub-cellular compartments [52]. We believe that transcriptomic and proteomic analyses of Nox4 in different cell types and under different cellular conditions would represent the only scientific approach to understanding the mechanisms behind variable and dynamic Nox4 cellular localization. This would potentially have a tremendous affect on the clarification of the biology of Nox isoforms.

We found that Nox4-dependent activation of ROS in the ER is responsible for autophagy up-regulation during energy deprivation [20]. In fact, ROS inhibition by catalase overexpression in the ER was able to inhibit autophagy, confirming that Nox regulates autophagy through ROS production. Why is ROS production in the ER so important for autophagy up-regulation during glucose deprivation? ER-specific signalling mechanisms were clearly involved in these effects. Nox4 activation was previously shown to regulate the unfolded protein response [15]. We found that Nox4-derived ROS promotes activation of autophagy through activation of the PERK/ATF4 signalling pathway in the early response to glucose deprivation [20]. This observation extends previous evidence suggesting that ROS accumulation during energy deprivation promotes the unfolded protein response, which represents a known inducer of autophagy [111,124,125]. However, our results suggest that Nox4-dependent PERK and autophagy activation during the early response to glucose deprivation occurs in the context of an integrated stress response, since we did not observe any modulation of the other branches of the unfolded protein response by either Nox4 or the same cellular conditions [20,126].

Interestingly, PERK activity is not regulated by direct oxidation of the cysteine residues in its regulatory domain [127], which excludes the possibility that Nox4 directly oxidizes and activates PERK. On the other hand, PERK has the potential to be hydroxylated [128]. We found that Nox4 can promote PERK signalling and autophagy activation indirectly through inhibition of the ER-resident PHD4 [129]. This result significantly expands upon the biological property of Nox4 of being a negative regulator of the PHD proteins, through which it up-regulates HIF-1α in response to hypoxia, for example [48,62,70]. In addition, our results provide a biological explanation for previous findings indicating that inhibition of PHD proteins activates the PERK pathway and inhibits ischaemic injury [128,130]. Of note, PHD proteins negatively regulate HIF-1α. Therefore, it is possible that Nox4-dependent PHD4 inhibition contributes to autophagy stimulation by activating HIF-1α [48,62,70,129]. However, autophagy was recently found to be activated during amino acid deprivation by inhibition of PHDs, independently of HIF-1α levels, in cancer cells [131]. In addition, we previously found that Nox4 inhibition alone does not affect HIF-1α levels in the heart, with only combined Nox4 and Nox2 deletion resulting in down-regulation of HIF-1α levels. This result would make unlikely the possibility that Nox4 regulates autophagy in the heart through HIF-1α activation during energy deprivation.

Previous works conducted on cancer cells showed that GCN2 (general control non-derepressible 2) promotes autophagy activation in response to amino acid starvation through the eIF-2α (eukaryotic initiation factor 2α)/ATF4 pathway. It will be interesting, in the future, to investigate whether Nox4 is involved in the regulation of GCN2 activity in cancer cells during stress [132].

It will also be important to investigate the mechanisms through which PERK and ATF4 regulate autophagy in response to Nox4 activation during energy deprivation. It is known that ATF4 has a tremendous positive affect on the transcriptional activation of autophagic proteins involved in both autophagosome formation and flux [124,132]. This suggests that the activation of the Nox4/PERK/ATF4 pathway is important because it provides support to the autophagic machinery by continuously promoting the transcription of autophagic genes. Of note, although in our study Nox4 appeared to regulate autophagy in cardiomyocytes mainly through the modulation of PERK and ATF4, we cannot exclude the possibility that Nox4 may also directly regulate autophagosome formation by oxidizing autophagy proteins involved in phagophore formation. In fact, accumulating evidence indicates that the ER is a major site of autophagosome formation and several autophagic proteins important for phagophore assembly, such as ULK1, Atg14L, Vps34 and Beclin-1, localize to the ER during the initiation of the autophagic process [9,72,80,133,134]. A recent work also suggested that autophagosome formation may start in the sites where ER and mitochondria are interconnected [135], thus raising the possibility that Nox4 activation in the ER might also signal to mitochondria to regulate mitochondrial autophagy. Additional studies are strongly warranted to directly test all these intriguing hypotheses.

We found that Nox4-dependent autophagy activation during energy stress promotes cardiomyocyte survival [20]. In addition, Nox4-dependent autophagy limited ischaemic injury in an animal model of prolonged ischaemia without reperfusion [20]. This model allowed us to selectively test the importance of Nox4 activation in the cardiac response to severe energy stress, whereas, with the usual models of brief ischaemia followed by reperfusion, it would have been impossible to distinguish the role of Nox4 during ischaemia from its pathophysiological involvement during reperfusion injury.

We observed that Nox4 and autophagy activation during energy stress promote the preservation of cellular ATP content [20]. This result has been recently confirmed by the elegant study by Tian's group in which it was demonstrated that transgenic mice overexpressing dominant-negative Nox4 protein in the heart (Tg–DN–Nox4) display increased cardiac ischaemic injury due to increased reductive stress in a Langendorff model of ischaemia/reperfusion [136]. Intriguingly, after low-flow global ischaemia, Tg–DN–Nox4 mice display a dramatic drop in ATP levels, which suggests a potential involvement of autophagy inhibition in these pathological events.

Of note, we previously found that combined deletion of both Nox2 and Nox4 isoforms in the heart is required to increase reperfusion injury after a brief period of in vivo regional ischaemia (30 min) followed by reperfusion (24 h). This occurs through down-regulation of HIF-1α levels and an increase in PPARα levels [48].

On the other hand, we found that Nox2 is not activated and is not necessary for autophagy activation during cardiomyocyte glucose deprivation [20]. This result is consistent with the evidence that only Nox4, and not other Nox isoforms, is selectively up-regulated in response to misfolded protein accumulation and oxidized lipoproteins in vascular cells, where it promotes autophagy activation. Other Nox isoforms are not necessary for autophagy activation under the same conditions [15,16]. The lack of involvement of Nox2 in autophagy regulation during energy stress may be explained by the fact that it is not up-regulated in this condition, whereas Nox4 is activated. In addition, Nox2 may not be localized in the ER during cardiomyocyte glucose deprivation and, if so, it cannot regulate PHD4 and PERK. As such, we previously demonstrated that Nox2 can regulate PHD2, which is not localized in the ER but rather mainly in the cytosol [48,137].

PERSPECTIVES

Future studies are strongly needed to elucidate the biological relevance of Nox-dependent autophagy in different cell types and conditions. Different NADPH isoforms may regulate autophagy through different molecular mechanisms depending on cell type and condition, as well as on their sub-cellular localization. For example, activation of Nox4 activates autophagy through the PERK/ATF4 pathway during energy deprivation in cardiomyocytes [20], whereas it can regulate autophagy in response to vascular proteotoxic stress through Ras signalling or Atg4 inhibition [15,16]. The role of Nox4-interacting proteins, such as p22phox, in the regulation of autophagy in response to different cellular stresses also deserves clarification. The cellular outcome of Nox-dependent autophagy may also be different depending on the mechanisms involved and on the role of autophagy in different cell types and conditions. For example, in endothelial cells, Nox4-dependent autophagy may be protective [15], whereas Nox2-dependent autophagy may be maladaptive [18] during stress. In this regard, exaggerated accumulation of Nox2-mediated ROS may even impair autophagy and lysosome biogenesis in some conditions, for example, in a mouse model of muscular dystrophy [138]. These divergent functions of Nox isoforms depending on cell type and conditions could be particularly relevant to the heart, where autophagy and Nox4 have dichotomous effects depending on the cardiac conditions, sub-cellular localization and level of activation. Nox4 activation is physiological during cardiomyocyte energy stress. On the other hand, cardiomyocyte Nox4 activation is detrimental during reperfusion injury and pressure overload. This may be partially explained by the fact that autophagy is protective in the former condition whereas it is maladaptive in the latter conditions [911,72]. Whether autophagy activation is detrimental or protective may also depend on the cellular signalling promoting it. Nox4 is activated in the ER during acute energy stress, whereas it is strongly up-regulated in mitochondria during chronic stress. It is possible that Nox4 activation in the ER promotes adaptive autophagy signalling, such as the PERK/ATF4 pathway or Ras/ERK signalling, whereas its up-regulation in mitochondria promotes autophagic cell death, which, for example, may be mediated by Bnip3 activation or activation of the Na+/K+ ATPase pump, promoting autosis.

The clarification of all these aspects appears to be highly relevant clinically. In fact, it is well established that activation of Noxs is detrimental in clinical conditions like diabetes and hypertension, where they contribute to vascular damage, endothelial dysfunction and atherosclerosis development [58,139]. Nox activation also appears to be detrimental during cerebrovascular ischaemia [140]. For these reasons, pharmacological Nox inhibitors have been recently developed with the potential to be protective in these conditions [22]. However, we speculate that the use of these compounds under conditions in which Noxs play physiological functions or in which autophagy is required for cell survival in response to stress may not be beneficial, but actually detrimental. Future studies are needed to better elucidate this issue. Of note, small molecules activating autophagy have also been recently developed and they also represent potentially-efficacious agents to reduce myocardial damage during cardiac stress [10,141].

Nox4-dependent autophagy may be highly involved in cancer progression. Indeed, Nox4 is activated in cancer cells in response to energy stress or chemotherapy, where it promotes survival and cellular adaptation [142]. Autophagy activation also promotes survival and invasiveness of cancer cells under the same conditions and studies demonstrated that PERK activation is involved in autophagy activation and survival of cancer cells [143]. Interestingly, PHD proteins appear to be down-regulated in cancer cells [144]. These data suggest that Nox4 may be responsible for autophagy activation and survival in cancer and introduce the possibility that inhibition of Nox4 and autophagy could be beneficial in cancer treatment.

Nox4-dependent autophagy may also be relevant during aging. Indeed, autophagy has the potential to exert multiple anti-aging effects [9,74]. It has been shown that, despite ROS accumulation during the aging process, reduction in cellular ROS by systemic over-expression of antioxidant genes does not increase life span [145]. The only ROS-scavenging intervention that appeared to be consistently successful in extending life span was over-expression of catalase in mitochondria [146]. This evidence suggests that mitochondrial H2O2 accumulation contributes to the aging process, whereas ROS production in other cellular compartments may not or may even perform critical physiological functions during aging. For this reason, it will be interesting to evaluate the role of Nox4 activity in the ER in the regulation of autophagy during aging.

Abbreviations

     
  • 7-KC

    7-ketocholesterol

  •  
  • AMPK

    AMP-activated protein kinase

  •  
  • Duox

    dual oxidase

  •  
  • EGFR

    epidermal growth factor receptor

  •  
  • ER

    endoplasmic reticulum

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • FOXO

    forkhead box O

  •  
  • GCN2

    general control non-derepressible 2

  •  
  • HIF-1α

    hypoxia-inducible factor 1-α

  •  
  • HMGB1

    high-mobility group box 1

  •  
  • IKK-β

    inhibitor of κB kinase

  •  
  • IL

    interleukin

  •  
  • JAK

    Janus kinase

  •  
  • JNK1

    c-Jun N-terminal kinase-1

  •  
  • Keap1

    kelch-like ECH-associated protein

  •  
  • LAMP

    lysosomal associated membrane protein

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • NF-κB

    nuclear factor κB

  •  
  • Nox

    NADPH oxidase

  •  
  • Nrf2

    nuclear factor-erythroid 2-related factor 2

  •  
  • PE

    phosphatidylethanolamine

  •  
  • PERK

    protein kinase RNA-like endoplasmic reticulum kinase

  •  
  • PHD

    prolyl hydroxylase domain

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • PPAR

    peroxisome-proliferator-activated receptor

  •  
  • Rheb

    Ras homologue enriched in brain

  •  
  • RNS

    reactive nitrogen species

  •  
  • ROS

    reactive oxygen species

  •  
  • STAT

    signal transducer and activator of transcription

  •  
  • TSC

    tuberous sclerosis

  •  
  • ULK

    Unc-51-like kinase

  •  
  • Vps

    vacuolar protein sorting

  •  
  • VSMC

    vascular smooth muscle cell

We thank Daniela Zablocki and Christopher D. Brady for critical reading of the manuscript and suggestions.

FUNDING

This work was supported by the U.S. Public Health Service [grant numbers HL102738, HL67724, HL69020, HL91469, AG23039 and AG27211]; the Fondation Leducq Transatlantic Networks of Excellence; Postdoctoral Fellowship from the Founders Affiliate, American Heart Association [grant number 10POST4260019 (to S.S.)]; the Italian Society of Cardiology and Italian Society of Hypertension; and the New Jersey Commission on Cancer Research.

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Author notes

1

These authors contributed equally to the article.