p62/SQSTM1 upregulation constitutes a survival mechanism that occurs during granulocytic differentiation of acute myeloid leukemia cells

The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34⁺ progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA.p62 or sequestosome 1 (p62/SQSTM1) is a scaffold protein, implicated in a variety of biological processes including those that control cell death, inflammation, and metabolism.^{1, 2} Through its multi-domain structure, p62/SQSTM1 interacts specifically with key signaling proteins, including atypical PKC family members, NF-κB, and mTOR to control cellular responses.^{3, 4, 5, 6, 7} p62/SQSTM1 functions also as a key mediator of autophagy. Through its interaction with LC3, an essential protein involved in autophagy, p62/SQSTM1 selectively directs ubiquitinated substrates to autophagosomes leading to their subsequent degradation in lysosomes.^{8, 9} At the molecular level, p62/SQSTM1 acts as a pro-tumoral molecule by ensuring efficient and selective activation of cell signaling axes involved in cell survival, proliferation, and metabolism (i.e., NF-κB, mTOR, and Nrf-2 pathways).^{3, 5, 6, 7, 10, 11, 12, 13} p62/SQSTM1 can also signal anti-tumoral responses either by inactivating the pro-oncogenic signaling through BCR-ABL¹⁴ and Wnt pathways^{15, 16} or by inducing the activation of caspase 8, a pro-death protein.^{17, 18} Interestingly, in response to stress, autophagy promotes the degradation of p62, thus limits the activation of p62-regulatory pathways that control tumorigenesis.¹⁰ In addition, p62/SQSTM1 controls pathways that modulate differentiation of normal and cancerous cells. For example, p62/SQSTM1 has been shown to antagonize basal ERK activity and adipocyte differentiation.¹⁹ In contrast, p62/SQSTM1 favors differentiation of osteoclasts,²⁰ osteoblasts,²¹ neurons,²² megakaryocytes²³ and macrophages.²⁴ The role and regulation of p62/SQSTM1 during leukemia cell differentiation has been poorly documented.Acute myeloid leukemia (AML) is a hematological disease characterized by multiple deregulated pathways resulting in a blockade of myeloid precursors at different stages of maturation.^{25, 26} Acute promyelocyte leukemia (APL) is the M3 type of AML characterized by an arrest of the terminal differentiation of promyelocytes into granulocytes and frequently associated with the expression of the oncogenic PML-RAR alpha fusion gene.^{27, 28} All-trans retinoic acid (ATRA), a potent activator of cellular growth arrest, differentiation, and death of APL cells, has been shown to effectively promote complete clinical remission of APL when combined with chemotherapy.^{29, 30, 31} Despite the success of this treatment, some APL patients are refractory to ATRA treatment or relapse owing to the development of resistance to ATRA in leukemia cells.^{32, 33, 34}Our previous results revealed that autophagy flux is activated during granulocyte differentiation of myeloid leukemia cell lines induced by ATRA.³⁵ In the present study, we observed that p62/SQSTM1, an autophagic substrate, is markedly upregulated at both mRNA and protein levels during the granulocytic differentiation process. Here, we investigated the regulation and the function of p62/SQSTM1 during AML cells differentiation into neutrophils/granulocytes.     

Dysferlin regulates cell membrane repair by facilitating injury-triggered acid sphingomyelinase secretion

Dysferlin deficiency compromises the repair of injured muscle, but the underlying cellular mechanism remains elusive. To study this phenomenon, we have developed mouse and human myoblast models for dysferlinopathy. These dysferlinopathic myoblasts undergo normal differentiation but have a deficit in their ability to repair focal injury to their cell membrane. Imaging cells undergoing repair showed that dysferlin-deficit decreased the number of lysosomes present at the cell membrane, resulting in a delay and reduction in injury-triggered lysosomal exocytosis. We find repair of injured cells does not involve formation of intracellular membrane patch through lysosome–lysosome fusion; instead, individual lysosomes fuse with the injured cell membrane, releasing acid sphingomyelinase (ASM). ASM secretion was reduced in injured dysferlinopathic cells, and acute treatment with sphingomyelinase restored the repair ability of dysferlinopathic myoblasts and myofibers. Our results provide the mechanism for dysferlin-mediated repair of skeletal muscle sarcolemma and identify ASM as a potential therapy for dysferlinopathy.Dysferlinopathy is a progressive muscle wasting disease, which is classified as limb-girdle muscular dystrophy type 2B (LGMD2B) or Miyoshi muscular dystrophy 1, based on its muscle involvement.^{1, 2} Dysferlin deficit leads to altered vesicle formation and trafficking,^{3, 4} poor repair of injured cell membranes,^{5, 6} and increased muscle inflammation.^{7, 8} Dysferlin contains C2 domains that are found in Ca²⁺-dependent membrane fusion proteins such as synaptotagmins.⁹ Thus, dysferlin is thought to regulate muscle function by regulating vesicle trafficking and fusion.^{10, 11, 12, 13} Dysferlin deficiency has also been implicated in conflicting reports regarding the fusion ability of dysferlinopathic myoblasts.^{4, 14, 15, 16} With such diverse roles for dysferlin, the mechanism through which dysferlin deficiency results in muscle pathology is unresolved. As skeletal muscle-specific re-expression of dysferlin rescues all dysferlinopathic pathologies,^{17, 18} myofiber repair has been suggested to be the unifying deficit underlying muscle pathology in dysferlinopathy.¹⁹ Repair of injured cell membranes requires subcellular compartments, which in mammalian cells include lysosomes,¹¹ enlargeosomes,²⁰ caveolae,²¹ dysferlin-containing vesicles,⁵ and mitochondria.²²Cells from muscular dystrophy patients that have normal dysferlin expression exhibit normal lysosome and enlargeosome exocytosis.²³ However, dysferlinopathic muscle cells exhibit enlarged LAMP2-positive lysosomes, reduced fusion of early endosomes, altered expression of proteins regulating late endosome/lysosome fusion, and reduced injury-triggered cell-surface levels of LAMP1.^{4, 11, 12} In non-muscle cells, lack of dysferlin reduces lysosomal exocytosis.²⁴ These findings implicate lysosomes in dysferlin-mediated muscle cell membrane repair. In one model for lysosome-mediated cell membrane repair, Ca²⁺ triggers vesicle–vesicle fusion near the site of injury, forming ‘membrane patch'', which fuses to repair the wounded cell membrane.^{25, 26, 27, 28} In another model, lysosome exocytosis following cell membrane injury by pore-forming toxins leads to secretion of the lysosomal enzyme acid sphingomyelinase (ASM), which causes endocytosis of pores in the damaged cell membranes.^{21, 29, 30} Both these models have been suggested to be involved in the repair of injured muscle cells.^{21, 28}To examine the muscle cell pathology in dysferlinopathy, we have developed dysferlinopathic mouse and human models. Use of these models shows that a lack of dysferlin does not alter myogenic differentiation but causes poor repair of even undifferentiated muscle cells. We show that dysferlin is required for tethering lysosomes to the cell membrane. Fewer lysosomes at the cell membrane in dysferlinopathic cells results in slow and reduced lysosome exocytosis following injury. This reduction in exocytosis reduces injury-triggered ASM secretion, which is responsible for the poor repair of dysferlinopathic muscle cells. Extracellular sphingomyelinase (SM) fully rescues the repair deficit in dysferlinopathic cells and mouse myofibers, offering a potential drug-based therapy for dysferlinopathy.     

IL10 inhibits starvation-induced autophagy in hypertrophic scar fibroblasts via cross talk between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways

Hypertrophic scar (HS) is a serious skin fibrotic disease characterized by excessive hypercellularity and extracellular matrix (ECM) component deposition. Autophagy is a tightly regulated physiological process essential for cellular maintenance, differentiation, development, and homeostasis. Previous studies show that IL10 has potential therapeutic benefits in terms of preventing and reducing HS formation. However, no studies have examined IL10-mediated autophagy during the pathological process of HS formation. Here, we examined the effect of IL10 on starvation-induced autophagy and investigated the molecular mechanism underlying IL10-mediated inhibition of autophagy in HS-derived fibroblasts (HSFs) under starvation conditions. Immunostaining and PCR analysis revealed that a specific component of the IL10 receptor, IL10 alpha-chain (IL10Rα), is expressed in HSFs. Transmission electron microscopy and western blot analysis revealed that IL10 inhibited starvation-induced autophagy and induced the expression of p-AKT and p-STAT3 in HSFs in a dose-dependent manner. Blocking IL10R, p-AKT, p-mTOR, and p-STAT3 using specific inhibitors (IL10RB, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, rapamycin, and cryptotanshinone, respectively) showed that IL10 inhibited autophagy via IL10Rα-mediated activation of STAT3 (the IL10R-STAT3 pathway) and by directly activating the AKT-mTOR pathway. Notably, these results suggest that IL10-mediated inhibition of autophagy is facilitated by the cross talk between STAT3, AKT, and mTOR; in other words, the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways. Finally, the results also indicate that mTOR-p70S6K is the molecule upon which these two pathways converge to induce IL10-mediated inhibition of autophagy in starved HSFs. In summary, the findings reported herein shed light on the molecular mechanism underlying IL10-mediated inhibition of autophagy and suggest that IL10 is a potential therapeutic agent for the treatment of HS.Autophagy is a degradative process in eukaryotic cells that removes or turns over bulk cytoplasmic constituents through the endosomal and lysosomal fusion system (i.e., autophagosomes).^{1, 2} Autophagy is induced by stressful conditions such as starvation and pathogenic invasion.²Hypertrophic scar (HS) is a major skin fibrotic disorder caused by hypercellularity and extracellular matrix (ECM) component deposition.^{3, 4, 5} HS formation is usually recognized as the consequence of disturbed tissue repair processes and/or disrupted homeostasis in the skin after traumatic injury: HS negatively impacts on patient appearance, skeletal muscle function, and quality of life in general.^{6, 7, 8, 9} About 40–70% of surgeries and over 91% of burn injuries result in HS.¹⁰ A key feature of HS is a metabolic disorder of collagen-based ECM proteins.^{11, 12, 13} Autophagy has an important role in homeostasis of tissue structure and function.^{2, 14, 15} Skin autophagic capability is associated with HS and with the pathogenesis of many human diseases.^{16, 17, 18, 19, 20, 21, 22, 23}Existing studies suggest that cytokines are important regulators of the autophagic process in both immune and non-immune cells.^{24, 25, 26} Interleukin-10 (IL10), expressed by a variety of mammalian cell types, was first described as a cytokine-synthesis-inhibitory factor with immunosuppressive and anti-inflammatory functions.^{27, 28} IL10 has a pivotal role in wound healing^{29, 30} and is a promising therapeutic agent for scar improvement in both animal models and human cutaneous wounds.^{9, 31, 32}Fibroblasts are one of the most important effector cells responsible for HS formation.^{12, 33, 34} Thus, we were prompted to elucidate the mechanisms underlying the interactions among IL10, autophagy, and HS formation, with the aim of providing a molecular foundation for the therapeutic efficacy IL10. We used HS tissue, HS-derived fibroblasts (HSFs), and starvation-induced autophagy in HSFs as our research platform.Here, we report that IL10 inhibited autophagy by interfering with IL10R-mediated activation of IL10R-STAT3, as well as by activating the AKT-mTOR pathway. In addition, cross talk among STAT3, AKT, and mTOR and between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways collaboratively regulated starvation-induced autophagy in HSFs.     

Spermidine induces autophagy by inhibiting the acetyltransferase EP300

Several natural compounds found in health-related food items can inhibit acetyltransferases as they induce autophagy. Here we show that this applies to anacardic acid, curcumin, garcinol and spermidine, all of which reduce the acetylation level of cultured human cells as they induce signs of increased autophagic flux (such as the formation of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta and the depletion of sequestosome-1, p62/SQSTM1) coupled to the inhibition of the mammalian target of rapamycin complex 1 (mTORC1). We performed a screen to identify the acetyltransferases whose depletion would activate autophagy and simultaneously inhibit mTORC1. The knockdown of only two acetyltransferases (among 43 candidates) had such effects: EP300 (E1A-binding protein p300), which is a lysine acetyltranferase, and NAA20 (N(α)-acetyltransferase 20, also known as NAT5), which catalyzes the N-terminal acetylation of methionine residues. Subsequent studies validated the capacity of a pharmacological EP300 inhibitor, C646, to induce autophagy in both normal and enucleated cells (cytoplasts), underscoring the capacity of EP300 to repress autophagy by cytoplasmic (non-nuclear) effects. Notably, anacardic acid, curcumin, garcinol and spermidine all inhibited the acetyltransferase activity of recombinant EP300 protein in vitro. Altogether, these results support the idea that EP300 acts as an endogenous repressor of autophagy and that potent autophagy inducers including spermidine de facto act as EP300 inhibitors.Macroautophagy (herein referred to as ‘autophagy'') consist in the sequestration of cytoplasmic material in autophagosomes, followed by their fusion with lysosomes for the bulk degradation of autophagic cargo by lysosomal hydrolases.¹ This phenomenon can be measured by following the redistribution of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) fusion proteins from a diffuse location to autophagosomes (that results in the formation of the so-called GFP-LC3 ‘puncta''), the diminution of the overall abundance of autophagic substrates (such as sequestosome-1, p62/SQSTM1), and the stereotyped activation of proautophagic signals (such as the inhibition of the mammalian target of rapamycin complex 1, mTORC1).²There is growing consensus that the induction of autophagy by nutritional, pharmacological or genetic interventions can reduce age-related pathologies (such as neurodegenerative diseases or type 2 diabetes) and/or extend longevity.^{3, 4, 5, 6} This applies to caloric restriction or intermediate fasting,⁷ continuous or intermittent medication of rapamycin,^{8, 9, 10} administration of the sirtuin 1-activator resveratrol,^{11, 12} external supply of the polyamine spermidine,¹³ or genetic ablation of p53.¹⁴ In all these cases, inhibition of autophagy by deleting or silencing relevant genes abolishes the extension of health span and/or lifespan.^{13, 14, 15, 16, 17} Moreover, direct induction of autophagy by transgenic expression of autophagy-relevant genes such as ATG5 in mice is sufficient to increase lifespan.¹⁸Recently, acetyltransferases have emerged as a potential target for the pharmaceutical induction of autophagy. Thus, depletion of the sole donor of acetyl groups, acetyl-coenzyme A (acetyl-CoA), is sufficient to reduce the acetylation of cytoplasmic and nuclear proteins coupled to the induction of autophagy.^{19, 20, 21, 22} Culture of mammalian cells in nutrient-free (NF) conditions or starvation of mice for 24 h reduced the intracellular nucleocytosolic concentrations of acetyl-CoA at the same time as autophagy was induced, and replenishment of acetyl-CoA by external sources (for instance, by providing a membrane-permeant precursor of α-ketoglutarate for anaplerotic reactions or by microinjection of acetyl-CoA) was sufficient to inhibit starvation-induced autophagy.^{19, 20, 21, 22} Beyond the inhibition of acetyltransferases by acetyl-CoA depletion, direct pharmacological inhibition of acetyltransferases might also contribute to the induction of autophagy. A close correlation between autophagy induction and deacetylation of cytoplasmic proteins was observed in a screen conceived to identify autophagy-stimulating polyphenols²³ as well as in in vivo experiments designed to explore the health-improving effects of coffee.²⁴ Spermidine turned out to be an efficient inhibitor of histone acetyltransferases in vitro¹³ and reduced the global protein acetylation levels in cultured cells.^{25, 26}Driven by these premises, we investigated the hypothesis that several health-related compounds including anacardic acid, curcumin, garcinol and spermidine might induce autophagy by inhibition of acetyltranferases. Here we report results supporting this hypothesis. Moreover, we demonstrate that one particular acetyltransferase, EP300 (E1A-binding protein p300), negatively controls autophagy and that anacardic acid, curcumin, garcinol and spermidine may induce autophagy by directly inhibiting EP300.     

Nuclear ULK1 promotes cell death in response to oxidative stress through PARP1

Reactive oxygen species (ROS) may cause cellular damage and oxidative stress-induced cell death. Autophagy, an evolutionarily conserved intracellular catabolic process, is executed by autophagy (ATG) proteins, including the autophagy initiation kinase Unc-51-like kinase (ULK1)/ATG1. Although autophagy has been implicated to have both cytoprotective and cytotoxic roles in the response to ROS, the role of individual ATG proteins, including ULK1, remains poorly characterized. In this study, we demonstrate that ULK1 sensitizes cells to necrotic cell death induced by hydrogen peroxide (H₂O₂). Moreover, we demonstrate that ULK1 localizes to the nucleus and regulates the activity of the DNA damage repair protein poly (ADP-ribose) polymerase 1 (PARP1) in a kinase-dependent manner. By enhancing PARP1 activity, ULK1 contributes to ATP depletion and death of H₂O₂-treated cells. Our study provides the first evidence of an autophagy-independent prodeath role for nuclear ULK1 in response to ROS-induced damage. On the basis of our data, we propose that the subcellular distribution of ULK1 has an important role in deciding whether a cell lives or dies on exposure to adverse environmental or intracellular conditions.Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide (H₂O₂), are formed by the incomplete reduction of oxygen during oxidative phosphorylation and other enzymatic processes. ROS are signaling molecules that regulate cell proliferation, differentiation, and survival.^{1, 2, 3} Accumulation of ROS (i.e., oxidative stress) on exposure to xenobiotic agents or environmental toxins can cause cellular damage and death via apoptotic or nonapoptotic pathways.^{4, 5, 6} Oxidative stress-induced cellular damage and death have been implicated in aging, ischemia-reperfusion injury, inflammation, and the pathogenesis of diseases (e.g., neurodegeneration and cancer).⁷ Oxidative stress also contributes to the antitumor effects of many chemotherapeutic drugs, including camptothecin^{8, 9} and selenite.^{10, 11}Autophagy, an evolutionarily conserved intracellular catabolic process, involves lysosome-dependent degradation of superfluous and damaged cytosolic organelles and proteins.¹² It is typically upregulated under conditions of perceived stress and in response to cellular damage. The consequence of autophagy activation – whether cytoprotective or cytotoxic – appears to depend on the cell type and the nature and extent of stress. Although most studies indicate a cytoprotective role for autophagy, some evidence suggests that it contributes to cell death in response to oxidative stress.^{13, 14, 15, 16, 17} Studies have also indicated that autophagy may be suppressed in response to oxidative stress, thereby sensitizing certain cells to apoptosis.^{18, 19} Unc-51-like kinase/autophagy 1 (ULK1/ATG1) is a mammalian serine–threonine kinase that regulates flux through the autophagy pathway by activating the VPS34 PI(3) kinase complex and facilitating ATG9-dependent membrane recycling.²⁰ Results from two studies suggest that ULK1 expression is altered in response to oxidative stress, and that the corresponding effects on autophagy contribute to cell death.^{18, 21}For example, p53-mediated upregulation of ULK1 and increase in autophagy promote cell death in osteosarcoma cells exposed to sublethal doses of camptothecin,²¹ yet mutant p53-mediated suppression of ULK1 impairs autophagic flux and promotes apoptosis in selenite-treated NB4 cells.¹⁸ Here we investigated the role of ULK1 in cells exposed to H₂O₂.