Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor FLCN, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally occurring FLCN mutations predisposing to BHD are predicted to produce truncated proteins unable to bind AMPK, pointing to the critical role of this interaction in the tumor suppression mechanism. Here, we demonstrate that FLCN is an evolutionarily conserved negative regulator of AMPK. Using Caenorhabditis elegans and mammalian cells, we show that loss of FLCN results in constitutive activation of AMPK which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function.
The FLCN gene is responsible for the hereditary human tumor disease called Birt-Hogg-Dube syndrome (BHD). Patients that inherit an inactivating mutation in the FLCN gene develop lung collapse as well as tumors in the kidney, colon, and skin. It is not clear yet what the exact function of this protein is in the cell or an organism. In this study, we used a simple model organism (the round worm C. elegans) to study the function of FLCN. We found that it is involved in the regulation of energy metabolism in the cell. FLCN normally binds and blocks the action of another protein (AMPK), which is involved in the maintenance of energy levels. When energy levels fall, AMPK is activated and drives a recycling pathway called autophagy, where cellular components are recycled producing energy. In the absence of FLCN in worms and mammalian cells, like in tumors of BHD patients, AMPK and autophagy are chronically activated leading to an increased energy level, which makes the cells/organism very resistant to many stresses that would normally kill them, which in the end could lead to progression of tumorigenesis.
AMP-activated protein kinase (AMPK) is a known physiological cellular energy sensor and becomes phosphorylated at Thr-172 in response to changes in cellular ATP levels. Activated AMPK acts as either an inducer or suppressor of apoptosis depending on the severity of energy stress and the presence or absence of certain functional tumor suppressor genes.
Here we show that energy stress differentially affects AMPK phosphorylation and cell-death in brain tumor tissue and in tissue from contra-lateral normal brain. We compared TSC2 deficient CT-2A mouse astrocytoma cells with syngeneic normal astrocytes that were grown under identical condition in vitro. Energy stress induced by glucose withdrawal or addition of 2-deoxyglucose caused more ATP depletion, AMPK phosphorylation and apoptosis in CT-2A cells than in the normal astrocytes. Under normal energy conditions pharmacological stimulation of AMPK caused apoptosis in CT-2A cells but not in astrocytes. TSC2 siRNA treated astrocytes are hypersensitive to apoptosis induced by energy stress compared to control cells. AMPK phosphorylation and apoptosis were also greater in the CT-2A tumor tissue than in the normal brain tissue following implementation of dietary energy restriction. Inefficient mTOR and TSC2 signaling, downstream of AMPK, is responsible for CT-2A cell-death, while functional LKB1 may protect normal brain cells under energy stress.
Together these data demonstrates that AMPK phosphorylation induces apoptosis in mouse astrocytoma but may protect normal brain cells from apoptosis under similar energy stress condition. Therefore, using activator of AMPK along with glycolysis inhibitor could be a potential therapeutic approach for TSC2 deficient human malignant astrocytoma.
Autophagy is activated in response to a variety of cellular stresses including metabolic stress. While elegant genetic studies in yeast have identified the core autophagy machinery, the signaling pathways that regulate this process are less understood. AMPK is an energy sensing kinase and several studies have suggested that AMPK is required for autophagy. The biochemical connections between AMPK and autophagy, however, have not been elucidated. In this report, we identify a biochemical connection between a critical regulator of autophagy, ULK1, and the energy sensing kinase, AMPK. ULK1 forms a complex with AMPK, and AMPK activation results in ULK1 phosphorylation. Moreover, we demonstrate that the immediate effect of AMPK-dependent phosphorylation of ULK1 results in enhanced binding of the adaptor protein YWHAZ/14-3-3ζ; and this binding alters ULK1 phosphorylation in vitro. Finally, we provide evidence that both AMPK and ULK1 regulate localization of a critical component of the phagophore, ATG9, and that some of the AMPK phosphorylation sites on ULK1 are important for regulating ATG9 localization. Taken together these data identify an ULK1-AMPK signaling cassette involved in regulation of the autophagy machinery.
14-3-3 proteins; AMP-activated protein kinase; Atg9; autophagy; energy metabolism; metabolic stress; phosphorylation; Unc-51-like kinase 1
Human Cytomegalovirus (HCMV) infection induces several metabolic activities that have been found to be important for viral replication. The cellular AMP-activated protein kinase (AMPK) is a metabolic stress response kinase that regulates both energy-producing catabolic processes and energy-consuming anabolic processes. Here we explore the role AMPK plays in generating an environment conducive to HCMV replication. We find that HCMV infection induces AMPK activity, resulting in the phosphorylation and increased abundance of several targets downstream of activated AMPK. Pharmacological and RNA-based inhibition of AMPK blocked the glycolytic activation induced by HCMV-infection, but had little impact on the glycolytic pathway of uninfected cells. Furthermore, inhibition of AMPK severely attenuated HCMV replication suggesting that AMPK is an important cellular factor for HCMV replication. Inhibition of AMPK attenuated early and late gene expression as well as viral DNA synthesis, but had no detectable impact on immediate-early gene expression, suggesting that AMPK activity is important at the immediate early to early transition of viral gene expression. Lastly, we find that inhibition of the Ca2+-calmodulin-dependent kinase kinase (CaMKK), a kinase known to activate AMPK, blocks HCMV-mediated AMPK activation. The combined data suggest a model in which HCMV activates AMPK through CaMKK, and depends on their activation for high titer replication, likely through induction of a metabolic environment conducive to viral replication.
Human Cytomegalovirus (HCMV) is a ubiquitous human pathogen that is a major cause of birth defects. HCMV can also cause severe disease in immunocompromised individuals including transplant recipients, leukemia patients and those infected with HIV. It is clear that upon infection, HCMV takes control of numerous cellular processes that are important for the virus to generate the next round of infectious virions. We have previously found that upon infection, HCMV reprograms the metabolic activity of the host-cell. Here, we find that this metabolic reprogramming largely depends on the viral activation of a cellular protein called the AMP-activated protein kinase (AMPK). AMPK is a central regulator of cellular energy production that is typically only activated when cellular energy stores are very low. Our results indicate that HCMV-mediated activation of AMPK is necessary to flip the metabolic switch thereby driving host-cell metabolic activation and viral replication. As inhibition of AMPK blocked viral replication, and had little impact on uninfected host-cell metabolism, targeting AMPK could have therapeutic potential to treat HCMV-associated disease.
Long noncoding RNAs (lncRNAs) have emerged as critical regulators in various cellular processes. However, the potential involvement of lncRNAs in kinase signaling remains largely unknown. AMP-activated protein kinase (AMPK) acts as a critical sensor of cellular energy status. Here we show that lncRNA NBR2 (neighbor of BRCA1 gene 2) is induced by the LKB1-AMPK pathway under energy stress. Upon energy stress, NBR2 in turn interacts with AMPK and promotes AMPK kinase activity, thus forming a feed-forward loop to potentiate AMPK activation during energy stress. Depletion of NBR2 attenuates energy stress-induced AMPK activation, resulting in unchecked cell cycling, altered apoptosis/autophagy response, and increased tumor development in vivo. NBR2 is down-regulated and its low expression correlates with poor clinical outcomes in some human cancers. Together, our study uncovers a mechanism coupling lncRNAs with metabolic stress response, and provides a broad framework to further understand the regulation of kinase signaling by lncRNAs.
Proline dehydrogenase (oxidase, PRODH/POX), the first enzyme in the pathway of proline catabolism, has been identified as a mitochondrial, metabolic tumor suppressor, which is downregulated in a variety of human tumors. However, our recent findings show that PRODH/POX is upregulated by hypoxia in vitro and in vivo. The combination of low glucose and hypoxia produces additive effects on PRODH/POX expression. Both hypoxia and glucose depletion enhance PRODH/POX expression through AMP-activated protein kinase (AMPK) activation to promote tumor cell survival. Nevertheless, the mechanisms underlying PRODH/POX prosurvival functions are different for hypoxia and low-glucose conditions. Glucose depletion with or without hypoxia elevates PRODH/POX and proline utilization to supply ATP for cellular energy needs. Interestingly, under hypoxia PRODH/POX induces protective autophagy by generating reactive oxygen species (ROS). AMPK is the main initiator of stress-triggered autophagy. Thus, PRODH/POX acts as a downstream effector of AMPK in the activation of autophagy under hypoxia. This regulation was confirmed to be independent of the mechanistic target of rapamycin (MTOR) pathway, a major downstream target of AMPK signaling.
apoptosis; autophagy; hypoxia; metabolic stress; proline dehydrogenase/oxidase
Adenosine monophosphate-activated protein kinase (AMPK) is a key player in maintaining energy homeostasis in response to metabolic stress. Beyond diabetes and metabolic syndrome, there is a growing interest in the therapeutic exploitation of the AMPK pathway in cancer treatment in light of its unique ability to regulate cancer cell proliferation through the reprogramming of cell metabolism. Although many studies support the tumor-suppressive role of AMPK, emerging evidence suggests that the metabolic checkpoint function of AMPK might be overridden by stress or oncogenic signals so that tumor cells use AMPK activation as a survival strategy to gain growth advantage. These findings underscore the complexity in the cellular function of AMPK in maintaining energy homeostasis under physiological versus pathological conditions. Thus, this review aims to provide an overview of recent findings on the functional interplay of AMPK with different cell metabolic and signaling effectors, particularly histone deacetylases, in mediating downstream tumor suppressive or promoting mechanisms in different cell systems. Although AMPK activation inhibits tumor growth by targeting multiple signaling pathways relevant to tumorigenesis, under certain cellular contexts or certain stages of tumor development, AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation, low oxygen, and low pH, or as a downstream effectors of oncogenic proteins, including androgen receptor, hypoxia-inducible factor-1α, c-Src, and MYC. Thus, investigations to define at which stage(s) of tumorigenesis and cancer progression or for which genetic aberrations AMPK inhibition might represent a more relevant strategy than AMPK activation for cancer treatment are clearly warranted.
AMPK; metabolic homeostasis; cancer therapy; LKB1; mTORC1; HDAC; Foxo3a; HIF-1α
AMP-activated protein kinase (AMPK) is an evolutionarily conserved master regulator of metabolism and a therapeutic target in type 2 diabetes. As an energy sensor, AMPK activity is responsive to both metabolic inputs, for instance the ratio of AMP to ATP, and numerous hormonal cues. As in mammals, each of two genes, aak-1 and aak-2, encode for the catalytic subunit of AMPK in C. elegans. Here we show that in C. elegans loss of aak-2 mimics the effects of elevated serotonin signaling on fat reduction, slowed movement, and promoting exit from dauer arrest. Reconstitution of aak-2 in only the nervous system restored wild type fat levels and movement rate to aak-2 mutants and reconstitution in only the ASI neurons was sufficient to significantly restore dauer maintenance to the mutant animals. As in elevated serotonin signaling, inactivation of AAK-2 in the ASI neurons caused enhanced secretion of dense core vesicles from these neurons. The ASI neurons are the site of production of the DAF-7 TGF-β ligand and the DAF-28 insulin, both of which are secreted by dense core vesicles and play critical roles in whether animals stay in dauer or undergo reproductive development. These findings show that elevated levels of serotonin promote enhanced secretions of systemic regulators of pro-growth and differentiation pathways through inactivation of AAK-2. As such, AMPK is not only a recipient of hormonal signals but can also be an upstream regulator. Our data suggest that some of the physiological phenotypes previously attributed to peripheral AAK-2 activity on metabolic targets may instead be due to the role of this kinase in neural serotonin signaling.
While it is well appreciated that food availability has profound effects on behavior, physiology, and metabolism, the molecular systems that link these complex processes together still remain poorly understood. An ancient cellular sensor of energy is AMP-activated protein kinase, AMPK. Here we show that in the genetically tractable C. elegans, loss of AMPK in the nervous system mimics many of the outcomes also seen upon elevated serotonin signaling, a neural indicator of food availability. We show that similar to elevated serotonin signaling, loss of neural AMPK causes reduced movement while enhancing fat metabolism and secretions of neuroendocrine hormones known to be systemic regulators of energy balance, development and aging. While AMPK is generally considered a mediator of hormonal signaling, our findings indicate that it also regulates their release. Our findings suggest that some previous results attributed to roles of AMPK in the regulation of peripheral metabolism may in fact be due to the roles of this kinase complex in the nervous system as a mediator of serotonin signaling.
The interaction between neurons, astrocytes and endothelial cells plays a central role coupling energy supply with changes in neuronal activity. For a long time it was believed that glucose was the only source of energy for neurons. However, a growing body of experimental evidence indicates that lactic acid, generated by aerobic glycolysis in perivascular astrocytes, is also a source of energy for neuronal activity, particularly when the supply of glucose from the intravascular space is interrupted. Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionary conserved kinase that couples cellular activity with energy consumption via induction of the uptake of glucose and activation of the glycolytic pathway. The uptake of glucose by the blood-brain barrier (BBB) is mediated by the transporter GLUT1, which is abundantly expressed in endothelial cells and astrocytic end-feet processes. Tissue-type plasminogen activator (tPA) is a serine proteinase that is found in endothelial cells, astrocytes and neurons. Genetic overexpression of neuronal tPA or treatment with recombinant tPA (rtPA) protects neurons from the deleterious effects of metabolic stress or excitotoxicity, via a mechanism independent of tPA’s ability to cleave plasminogen into plasmin. The work presented here shows that exposure to metabolic stress induces the rapid release of tPA from neurons but not from astrocytes. This tPA induces AMPK activation, membrane recruitment of GLUT1, and GLUT1-mediated glucose uptake in astrocytes and endothelial cells. Our data indicate that this is followed by the synthesis and release of lactic acid from astrocytes, and that the uptake of this lactic acid via the monocarboxylate transporter-2 (MCT-2) promotes survival in neurons exposed to metabolic stress.
Tissue-type plasminogen activator (tPA); Glucose metabolism; Neuroprotection; Adenosine monophosphate-activated protein kinase (AMPK); Monocarboxylate transporter-2 (MCT-2)
Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid involved in regulating pathways promoting cellular protection. We have previously shown that EETs trigger a protective response limiting mitochondrial dysfunction and reducing cellular death. Considering it is unknown how EETs regulate cell death processes, the major focus of the current study was to investigate their role in the autophagic response of HL-1 cells and neonatal cardiomyocytes (NCMs) during starvation. We employed a dual-acting synthetic analog UA-8 (13-(3-propylureido)tridec-8-enoic acid), possessing both EET-mimetic and soluble epoxide hydrolase (sEH) inhibitory properties, or 14,15-EET as model EET molecules. We demonstrated that EETs significantly improved viability and recovery of starved cardiac cells, whereas they lowered cellular stress responses such as caspase-3 and proteasome activities. Furthermore, treatment with EETs resulted in preservation of mitochondrial functional activity in starved cells. The protective effects of EETs were abolished by autophagy-related gene 7 (Atg7) short hairpin RNA (shRNA) or pharmacological inhibition of autophagy. Mechanistic evidence demonstrated that sarcolemmal ATP-sensitive potassium channels (pmKATP) and enhanced activation of AMP-activated protein kinase (AMPK) played a crucial role in the EET-mediated effect. Our data suggest that the protective effects of EETs involve regulating the autophagic response, which results in a healthier pool of mitochondria in the starved cardiac cells, thereby representing a novel mechanism of promoting survival of cardiac cells. Thus, we provide new evidence highlighting a central role of the autophagic response in linking EETs with promoting cell survival during deep metabolic stress such as starvation.
autophagy; epoxyeicosatrienoic acid; cardiac cells
Autophagy is a stress response protecting cells from unfavorable conditions, such as nutrient starvation. The class III phosphatidylinositol-3 kinase, Vps34, forms multiple complexes and regulates both intracellular vesicle trafficking and autophagy induction. Here, we show that AMPK plays a key role in regulating different Vps34 complexes. AMPK inhibits the non-autophagy Vps34 complex by phosphorylating T163/S165 in Vps34, therefore suppresses overall PI(3)P production and protects cells from starvation. In parallel, AMPK activates the pro-autophagy Vps34 complex by phosphorylating S91/S94 in Beclin1 to induce autophagy. Atg14L, an autophagy essential gene present only in pro-autophagy Vps 34 complex, inhibits Vps34 phosphorylation but increases Beclin1 phosphorylation by AMPK. As such, Atg14L dictates the differential regulation (either inhibition or activation) of different Vps34 complexes in response to glucose starvation. Our study reveals an intricate molecular regulation of Vps34 complexes by AMPK in nutrient stress response and autophagy.
Activated pancreatic stellate cells (PaSC) are key participants in the stroma of pancreatic cancer, secreting extracellular matrix proteins and inflammatory mediators. Tumors are poorly vascularized, creating metabolic stress conditions in cancer and stromal cells that necessitate adaptive homeostatic cellular programs. Activation of autophagy and the endoplasmic reticulum unfolded protein response (UPR) have been described in hepatic stellate cells, but the role of these processes in PaSC responses to metabolic stress is unknown. We reported that the PI3K/mTOR pathway, which AMPK can regulate through multiple inputs, modulates PaSC activation and fibrogenic potential. Here, using primary and immortalized mouse PaSC, we assess the relative contributions of AMPK/mTOR signaling, autophagy and the UPR to cell fate responses during metabolic stress induced by mitochondrial dysfunction. The mitochondrial uncoupler rottlerin at low doses (0.5–2.5 μM) was added to cells cultured in 10% FBS complete media. Mitochondria rapidly depolarized, followed by altered mitochondrial dynamics and decreased cellular ATP levels. This mitochondrial dysfunction elicited rapid, sustained AMPK activation, mTOR pathway inhibition, and blockade of autophagic flux. Rottlerin treatment also induced rapid, sustained PERK/CHOP UPR signaling. Subsequently, high doses (>5 μM) induced loss of cell viability and cell death. Interestingly, AMPK knock-down using siRNA did not prevent rottlerin-induced mTOR inhibition, autophagy, or CHOP upregulation, suggesting that AMPK is dispensable for these responses. Moreover, CHOP genetic deletion, but not AMPK knock-down, prevented rottlerin-induced apoptosis and supported cell survival, suggesting that UPR signaling is a major modulator of cell fate in PaSC during metabolic stress. Further, short-term rottlerin treatment reduced both PaSC fibrogenic potential and IL-6 mRNA expression. In contrast, expression levels of the angiogenic factors HGF and VEGFα were unaffected, and the immune modulator IL-4 was markedly upregulated. These data imply that metabolic stress-induced PaSC reprogramming differentially modulates neighboring cells in the tumor microenvironment.
Kidney ischemia-reperfusion is a form of acute kidney injury
resulting in a cascade of cellular events prompting rapid cellular damage
and suppression of kidney function. A cellular response to ischemic stress
is the activation of AMP-activated protein kinase (AMPK) where AMPK induces
a number of homeostatic and renoprotective mechanisms, including autophagy.
However whether autophagy is beneficial or detrimental in
ischemia-reperfusion remains controversial. We investigated the effects of
agonist induction of AMPK activity on autophagy and cell stress proteins in
the model of kidney ischemia-reperfusion.
AMPK agonists, AICAR (0.1g/kg) and Metformin (0.3g/kg), were
administered 24 hours prior to ischemia, with kidneys harvested at 24 hours
We observed a paradoxical decrease in AMPK activity accompanied by
increases in mTORC1 activity and p62/SQSTM1 expression. These results lead
us to propose that AMPK and autophagy was insufficient to properly counter
the cellular insults in ischemia-reperfusion. Agonist induction of AMPK
activity with AICAR or metformin increased macroautophagy protein LC3 and
normalizes p62/SQSTM1 expression and mTOR activity. Ischemia-reperfusion
increases in Beclin-1 and PINK1 expressions, consistent with increased
mitophagy, were also mitigated with AMPK agonists. Stress responsive and
apoptotic marker expressions increase in ischemia-reperfusion and are
significantly attenuated with agonist administration, as are early
indicators of fibrosis.
Our data suggest that levels of renoprotective AMPK activity and
canonical autophagy are insufficient to maintain cellular homeostasis
contributing to the progression of ischemia-reperfusion injury. We further
demonstrate that induction of AMPK activity can provide beneficial cellular
effects in containing injury in ischemia-reperfusion.
mitophagy; mTOR; p62/SQSTM1; PINK1
The adenosine monophosphate (AMP)–activated protein kinase (AMPK) has a crucial role in maintaining cellular energy homeostasis. This study shows that human and mouse T lymphocytes express AMPKα1 and that this is rapidly activated in response to triggering of the T cell antigen receptor (TCR). TCR stimulation of AMPK was dependent on the adaptors LAT and SLP76 and could be mimicked by the elevation of intracellular Ca2+ with Ca2+ ionophores or thapsigargin. AMPK activation was also induced by energy stress and depletion of cellular adenosine triphosphate (ATP). However, TCR and Ca2+ stimulation of AMPK required the activity of Ca2+–calmodulin-dependent protein kinase kinases (CaMKKs), whereas AMPK activation induced by increased AMP/ATP ratios did not. These experiments reveal two distinct pathways for the regulation of AMPK in T lymphocytes. The role of AMPK is to promote ATP conservation and production. The rapid activation of AMPK in response to Ca2+ signaling in T lymphocytes thus reveals that TCR triggering is linked to an evolutionally conserved serine kinase that regulates energy metabolism. Moreover, AMPK does not just react to cellular energy depletion but also anticipates it.
Saturated fatty acids (SFAs) induce hepatocyte cell death, wherein oxidative stress is mechanistically involved. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master transcriptional regulator of cellular antioxidant defense enzymes. Therefore, Nrf2 activation is regarded as an effective strategy against oxidative stress-triggered cellular damage. In this study, tert-butylhydroquinone (tBHQ), a widely used Nrf2 activator, was initially employed to investigate the potential protective role of Nrf2 activation in SFAs-induced hepatoxicity. As expected, SFAs-induced hepatocyte cell death was prevented by tBHQ in both AML-12 mouse hepatocytes and HepG2 human hepatoma cells. However, the protective effect of tBHQ is Nrf2-independent, because the siRNA-mediated Nrf2 silencing did not abrogate tBHQ-conferred protection. Alternatively, our results revealed that autophagy activation was critically involved in the protective effect of tBHQ on lipotoxicity. tBHQ induced autophagy activation and autophagy inhibitors abolished tBHQ’s protection. The induction of autophagy by tBHQ exposure was demonstrated by the increased accumulation of LC3 puncta, LC3-II conversion, and autophagic flux (LC3-II conversion in the presence of proteolysis inhibitors). Subsequent mechanistic investigation discovered that tBHQ exposure activated AMP-activated protein kinase (AMPK) and siRNA-mediated AMPK gene silencing abolished tBHQ-induced autophagy activation, indicating that AMPK is critically involved in tBHQ-triggered autophagy induction. Furthermore, our study provided evidence that tBHQ-induced autophagy activation is required for its Nrf2-activating property. Collectively, our data uncover a novel mechanism for tBHQ in protecting hepatocytes against SFAs-induced lipotoxicity. tBHQ-triggered autophagy induction contributes not only to its hepatoprotective effect, but also to its Nrf2-activating property.
Tert-Butylhydroquinone; autophagy; SFAs; lipotoxicity; AMPK; Nrf2
Elevated proteasome activity extends lifespan in model organisms such as yeast, worms and flies. This pro-longevity effect might be mediated by improved protein homeostasis, as this protease is an integral module of the protein homeostasis network. Proteasomes also regulate cellular processes through temporal and spatial degradation of signaling pathway components. Here we demonstrate that the regulatory function of the proteasome plays an essential role in aging cells and that the beneficial impact of elevated proteasome capacity on lifespan partially originates from deregulation of the AMPK signaling pathway. Proteasome-mediated lifespan extension activity was carbon-source dependent and cells with enhancement proteasome function exhibited increased respiratory activity and oxidative stress response. These findings suggested that the pro-aging impact of proteasome upregulation might be related to changes in the metabolic state through a premature induction of respiration. Deletion of yeast AMPK, SNF1, or its activator SNF4 abrogated proteasome-mediated lifespan extension, supporting this hypothesis as the AMPK pathway regulates metabolism. We found that the premature induction of respiration in cells with increased proteasome activity originates from enhanced turnover of Mig1, an AMPK/Snf1 regulated transcriptional repressor that prevents the induction of genes required for respiration. Increasing proteasome activity also resulted in partial relocation of Mig1 from the nucleus to the mitochondria. Collectively, the results argue for a model in which elevated proteasome activity leads to the uncoupling of Snf1-mediated Mig1 regulation, resulting in a premature activation of respiration and thus the induction of a mitohormetic response, beneficial to lifespan. In addition, we observed incorrect Mig1 localization in two other long-lived yeast aging models: cells that overexpress SIR2 or deleted for the Mig1-regulator HXK2. Finally, compromised proteasome function blocks lifespan extension in both strains. Thus, our findings suggest that proteasomes, Sir2, Snf1 and Hxk2 form an interconnected aging network that controls metabolism through coordinated regulation of Mig1.
Advanced cellular age is associated with decreased efficiency of the proteostasis network. The proteasome, a protease in the cytoplasm and nuclei of eukaryotic cells, is an important component of this network. Recent studies demonstrate that increased proteasome capacity has a positive impact on longevity. The underlying mechanisms, however, have not been fully identified. Here we report that proteasomes are involved in regulating the AMP-activated kinase (AMPK) pathway and thus participate in correct metabolic adaptation. We find that Mig1, a transcriptional repressor downstream of yeast AMPK, Snf1, is a proteasome target and a negative regulator of lifespan. Increased proteasome activity results in enhanced turnover and incorrect localization of Mig1. The reduced Mig1 levels result in the induction of respiration and upregulation of the oxidative stress response. Premature Mig1 inactivation is also observed in two additional long-lived strains that overexpress SIR2 or are deleted for HXK2 and lifespan extension in both strains requires correct proteasome function. Our results uncover an interconnected network comprised of the proteasome, Sir2 and AMPK/Hxk2 signaling that impacts longevity through regulation of Mig1 and modulates respiratory metabolism. Mechanistic information on the cross-communication between these pathways is expected to facilitate the identification of novel pro-aging interventions.
Excessive ethanol exposure is detrimental to the brain. The developing brain is particularly vulnerable to ethanol such that prenatal ethanol exposure causes fetal alcohol spectrum disorders (FASD). Neuronal loss in the brain is the most devastating consequence and is associated with mental retardation and other behavioral deficits observed in FASD. Since alcohol consumption during pregnancy has not declined, it is imperative to elucidate the underlying mechanisms and develop effective therapeutic strategies. One cellular mechanism that acts as a protective response for the central nervous system (CNS) is autophagy. Autophagy regulates lysosomal turnover of organelles and proteins within cells, and is involved in cell differentiation, survival, metabolism, and immunity. We have recently shown that ethanol activates autophagy in the developing brain. The autophagic preconditioning alleviates ethanol-induced neuron apoptosis, whereas inhibition of autophagy potentiates ethanol-stimulated reactive oxygen species (ROS) and exacerbates ethanol-induced neuroapoptosis. The expression of genes encoding proteins required for autophagy in the CNS is developmentally regulated; their levels are much lower during an ethanol-sensitive period than during an ethanol-resistant period. Ethanol may stimulate autophagy through multiple mechanisms; these include induction of oxidative stress and endoplasmic reticulum stress, modulation of MTOR and AMPK signaling, alterations in BCL2 family proteins, and disruption of intracellular calcium (Ca2+) homeostasis. This review discusses the most recent evidence regarding the involvement of autophagy in ethanol-mediated neurotoxicity as well as the potential therapeutic approach of targeting autophagic pathways.
alcohol; alcoholism; development; fetal alcohol spectrum disorders; neurodegeneration; oxidative stress; protein degradation; AD, Alzheimer disease; ALS, autophagy-lysosome system; AMPK, adenosine 5′-monophosphate-activated protein kinase;; ATG, autophagy-related; CNS, central nervous system; ER, endoplasmic reticulum; FASD, fetal alcohol spectrum disorders; FOXO3, forkhead box O3; GSK3B, glycogen synthase kinase 3 β; HD, Huntington disease, HNSCs, hippocampal neural stem cells; LC3, microtubule-associated protein 1 light chain 3; MTOR, mechanistic target of rapamycin (serine/threonine kinase); MTORC1, MTOR complex 1; NOX, NADPH oxidase; NFE2L2, nuclear factor, erythroid 2-like 2; PD, Parkinson disease; PI3K, class I phosphoinositide 3-kinase; ROS, reactive oxygen species; SQSTM1/p62, sequestosome 1; TSC1/2, tuberous sclerosis 1/ 2; UPR, unfolded protein response
The degradation of proteins by the ubiquitin proteasome system (UPS) is required for the maintenance of cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). AMPK activation inhibits protein synthesis and activates autophagy, but whether AMPK plays a role in regulating protein breakdown through the UPS in the heart is not known.
To determine whether AMPK enhances UPS-mediated protein degradation by directly regulating the ubiquitin ligases Atrogin-1 and MuRF1 in the heart.
Methods and Results
Nutrient deprivation, pharmacologic or genetic activation of AMPK increased mRNA expression and protein levels of Atrogin-1 and MuRF1, and consequently enhanced protein degradation in neonatal cardiomyocytes. Inhibition of AMPK abrogated these effects. Using gene reporter and chromatin immunoprecipitation assays we found that AMPK regulates MuRF1 expression by acting through the transcription factor MEF2. We further validated these findings in vivo using MEF2-LacZ reporter mice. Furthermore, we demonstrated in adult cardiomoycytes that MuRF1 is necessary for AMPK-mediated proteolysis through the UPS in the heart. Consequently, MuRF1 knockout mice were protected from severe cardiac dysfunction during fasting.
AMPK regulates the transcription of Atrogin-1 and MuRF1 and enhances UPS-mediated protein degradation in heart. Specifically, AMPK regulates MuRF1 through the transcription factor MEF2. The absence of MuRF1 in the heart preserves cardiac function during fasting. The results strengthen the hypothesis that AMPK serves as a modulator of intracellular protein degradation in the heart.
AMPK; protein degradation; ubiquitin ligases; transcriptional regulation
Autophagy is a tightly regulated process activated in response to metabolic stress and other microenvironmental changes. Astrocyte elevated gene 1 (AEG-1) reportedly induces protective autophagy. Our results indicate that AEG-1 also enhances the susceptibility of malignant glioma cells to TGF-β1-triggered epithelial-mesenchymal transition (EMT) through induction of autophagy. TGF-β1 induced autophagy and activated AEG-1 via Smad2/3 phosphorylation in malignant glioma cells. Also increased was oncogene cyclin D1 and EMT markers, which promoted tumor progression. Inhibition of autophagy using siRNA-BECN1 and siRNA-AEG-1 suppressed EMT. In tumor samples from patients with malignant glioma, immunohistochemical assays showed that expression levels of TGF-β1, AEG-1, and markers of autophagy and EMT, all gradually increase with glioblastoma progression. In vivo siRNA-AEG-1 administration to rats implanted with C6 glioma cells inhibited tumor growth and increased the incidence of apoptosis among tumor cells. These findings shed light on the mechanisms underlying the invasiveness and progression of malignant gliomas.
transforming growth factor-β1; astrocyte elevated gene-1; protective autophagy; epithelial mesenchymal transition; malignant glioma invasion
Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse directly throughout the peritoneum to seed secondary lesions. There is growing evidence that EOC spheroids are key mediators of metastasis, and they use specific intracellular signalling pathways to control cancer cell growth and metabolism for increased survival. Our laboratory discovered that AKT signalling is reduced during spheroid formation leading to cellular quiescence and autophagy, and these may be defining features of tumour cell dormancy. To further define the phenotype of EOC spheroids, we have initiated studies of the Liver kinase B1 (LKB1)-5′-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response. We demonstrate that activity of AMPK and its upstream kinase LKB1 are increased in quiescent EOC spheroids as compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Functional studies reveal that treatment with the AMP mimetic AICAR or allosteric AMPK activator A-769662 led to a cytostatic response in proliferative adherent ovarian cancer cells, but they fail to elicit an effect in spheroids. Targeted knockdown of STK11 by RNAi to reduce LKB1 expression led to reduced viability and increased sensitivity to carboplatin treatment in spheroids only, a phenomenon which was AMPK-independent. Thus, our results demonstrate a direct impact of altered LKB1-AMPK signalling function in EOC. In addition, this is the first evidence in cancer cells demonstrating a pro-survival function for LKB1, a kinase traditionally thought to act as a tumour suppressor.
ovarian cancer; spheroid; LKB1; AMPK; tumour cell dormancy
Calcineurin signalling plays a critical role in the pathogenesis of many cardiovascular diseases. Calcineurin has been proven to affect a series of signalling pathways and to exert a proapoptotic effect in cardiomyocytes. However, whether it is able to regulate autophagy remains largely unknown. Here, we report that prolonged oxidative stress-induced activation of calcineurin contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signalling and inhibits autophagy in cardiomyocytes. Primary cardiomyocytes exhibited rapid formation of autophagosomes, microtubule-associated protein 1 light chain 3 (LC3) expression and phosphorylation of AMPK in response to hydrogen peroxide (H2O2) treatment. However, prolonged (12 h) H2O2 treatment attenuated these effects and was accompanied by a significant increase in calcineurin activity and apoptosis. Inhibition of calcineurin by FK506 restored AMPK function and LC3 expression, and decreased the extent of apoptosis caused by prolonged oxidative stress. In contrast, overexpression of the constitutively active form of calcineurin markedly attenuated the increase in LC3 induced by short-term (3 h) H2O2 treatment and sensitised cells to apoptosis. In addition, FK506 failed to induce autophagy and alleviate apoptosis in cardiomyocytes expressing a kinase-dead K45R AMPK mutant. Furthermore, inhibition of autophagy by 3-methylanine (3-MA) or by knockdown of the essential autophagy-related gene ATG7 abrogated the protective effect of FK506. These findings suggest a novel role of calcineurin in suppressing adaptive autophagy during oxidative stress by downregulating the AMPK signalling pathway. The results also provide insight into how altered calcineurin and autophagic signalling is integrated to control cell survival during oxidative stress and may guide strategies to prevent cardiac oxidative damage.
oxidative stress; cardiomyocyte; autophagy; calcineurin; AMPK
Autophagy is a survival mechanism activated in response to metabolic stress. In normal tissues autophagy plays a major role in energy homeostasis through catabolic self-digestion of damaged proteins and organelles. Contrary to its survival function, autophagy defects are implicated in tumorigenesis suggesting that autophagy is a tumor suppression mechanism. Although the exact mechanism of this tumor suppressor function is not known, it likely involves mitigation of cellular damage leading to chromosomal instability. The complex role of functional autophagy in tumors calls for model systems that allow the assessment of autophagy status, stress management and the impact on oncogenesis both in vitro as well as in vivo. We developed model systems that involve generation of genetically defined, isogenic and immortal epithelial cells from different tissue types that are applicable to both wild-type and mutant mice. This permits the study of tissue- as well as gene-specific tumor promoting functions. We successfully employed this strategy to generate isogenic, immortal epithelial cell lines from wild-type and mutant mice deficient in essential autophagy genes such as beclin 1 (beclin 1+/-) and atg5 (atg 5-/-). As these cell lines are amenable to further genetic manipulation, they allowed us to generate cell lines with apoptosis defects and stable expression of the autophagy marker EGFP-LC3 that facilitate in vitro and in vivo assessment of stress-mediated autophagy induction. We applied this model system to directly monitor autophagy in cells and 3D-morphogenesis in vitro as well as in tumor allografts in vivo. Using this model system we demonstrated that autophagy is a survival response in solid tumors that co-localizes with hypoxic regions, allowing tolerance to metabolic stress. Furthermore, our studies have established that autophagy also protects tumor cells from genome damage and limits cell death and inflammation as possible means to tumor suppression. Additionally these cell lines provide an efficient way to perform biochemical analyses, and high throughput screening for modulators of autophagy for potential use in cancer therapy and prevention.
Hematopoietic cells normally require cell extrinsic signals to maintain metabolism and survival. In contrast, cancer cells can express constitutively active oncogenic kinases such as BCR-Abl that promote these processes independent of extrinsic growth factors. When cells receive insufficient growth signals or when oncogenic kinases are inhibited, glucose metabolism decreases and the self-digestive process of autophagy is elevated to degrade bulk cytoplasm and organelles. While autophagy has been proposed to provide a cell-intrinsic nutrient supply for mitochondrial oxidative metabolism and to maintain cellular homeostasis through degradation of damaged organelles or protein aggregates, its acute role in growth factor deprivation or inhibition of oncogenic kinases remains poorly understood. We therefore developed a growth factor-dependent hematopoietic cell culture model in which autophagy can be acutely disrupted through conditional Cre-mediated excision of the autophagy-essential gene Atg3. Treated cells rapidly lost their ability to perform autophagy and underwent cell cycle arrest and apoptosis. While Atg3 was essential for optimal upregulation of mitochondrial oxidative pathways in growth factor withdrawal, this metabolic contribution of autophagy did not appear critical for cell survival, as provision of exogenous pyruvate or lipids could not completely rescue Atg3-deficiency. Instead, autophagy suppressed a stress response that otherwise led to p53 phosphorylation and upregulation of p21 and the pro-apoptotic Bcl-2 family protein Puma. Importantly, BCR-Abl-expressing cells had low basal levels of autophagy but were highly dependent on this process, and rapidly underwent apoptosis upon disruption of autophagy through Atg3 deletion or treatment with chemical autophagy inhibitors. This dependence on autophagy extended in vivo, as Atg3 deletion also prevented BCR-Abl-mediated leukemogenesis in a cell transfer model. Together these data demonstrate a critical role for autophagy to mitigate cell stress, and that cells expressing the oncogenic kinase BCR-Abl appear particularly dependent on autophagy for cell survival and leukemogenesis.
Autophagy; BCR-Abl; Puma; apoptosis; metabolism; p53
Autophagy has been shown to be protective against drug and alcohol-induced liver injury. CYP2E1 plays a role in the toxicity of ethanol, carcinogens and certain drugs. Inhibition of autophagy increased ethanol-toxicity and accumulation of fat in wild type and CYP2E1 knockin mice but not in CYP2E1 knockout mice as well as in HepG2 cells expressing CYP2E1 (E47 cells) but not HepG2 cells lacking CYP2E1 (C34 cells). The goal of the current study was to evaluate whether modulation of autophagy can affect CYP2E1-dependent cytotoxicity in the E47 cells. The agents used to promote CYP2E1 –dependent toxicity were a polyunsaturated fatty acid, arachidonic acid (AA), buthionine sulfoximine (BSO), which depletes GSH, and CCl4, which is metabolized to the CCl3 radical. These three agents produced a decrease in E47 cell viability which was enhanced upon inhibition of autophagy by 3-methyladenine (3-MA) or Atg 7 siRNA. Toxicity was lowered by rapamycin which increased autophagy and was much lower to the C34 cells which do not express CYP2E1. Toxicity was mainly necrotic and was associated with an increase in reactive oxygen production and oxidative stress; 3-MA increased while rapamycin blunted the oxidative stress. The enhanced toxicity and ROS formation produced when autophagy was inhibited was prevented by the antioxidant N-Acetyl cysteine. AA, BSO and CCl4 produced mitochondrial dysfunction, lowered cellular ATP levels and elevated mitochondrial production of ROS. This mitochondrial dysfunction was enhanced by inhibition of autophagy with 3-MA but decreased when autophagy was increased by rapamycin. The mitogen activated protein kinases p38 MAPK and JNK were activated by AA especially when autophagy was inhibited and chemical inhibitors of p38 MAPK and JNK lowered the elevated toxicity of AA produced by 3-MA. These results show that autophagy was protective against the toxicity produced by several agents known to be activated by CYP2E1. Since CYP2E1 plays an important role in the toxicity of ethanol, drugs and carcinogens and is activated under various pathophysiological conditions such as diabetes, NASH and obesity, attempts to stimulate autophagy may be beneficial in preventing/lowering CYP2E1/ethanol liver injury.
•Inhibition of autophagy in HepG2 E47 cells which express CYP2E1 promotes AA, BSO and CCl4 toxicity.•Decreased autophagy in E47 cells increased reactive oxygen production and oxidative stress and potentiated mitochondrial dysfunction.•The inhibition of autophagy enhanced activation of p38 MAPK and JNK by AA and this activation contributed to the toxicity.•Autophagy is protective against the toxicity produced by agents known to be activated by CYP2E1.
CYP2E1, cytochrome P4502E1, E47 cells, HepG2 cells which express CYP2E1; C34 cells, HepG2 cells which do not express CYP2E1; AA, arachidonic acid; BSO, L-buthionine sulfoximine; CCl4, carbon tetrachloride; 3-MA, 3-methyadenine; Rap, rapamycin; NAC, N-acetyl-cysteine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide; ROS, reactive oxygen species; DCFDA, 2′-7′-dichlorofluorescin-diacetate; SOD, superoxide dismutase; GSH, reduced glutathione; TBARs, thiobarbituric acid-reactive substances; Cox IV, cytochrome oxidase subunit 4; CYP2E1; Autophagy; P38 MAPK; JNK; Mitochondria dysfunction; ROS; Cytotoxicity
The overall goal of the investigation was to examine autophagy in the growth plate and to ascertain how this process was regulated. Herein, we show that while proliferating and terminally differentiated chondrocytes display low levels of LC-3 fluorescence, cells in the post-mitotic maturing zone strongly express particulate LC3 fluorescence. This robust immunohistochemical response of the maturing chondrocyte provides directl evidence that autophagy is a new and transient stage in the chondrocyte maturation pathway. We found that induction of autophagy was regulated by mTOR, a second sensor of cellular metabolism. When mTOR was inhibited, there was extensive reorganization of LC3, indicating that the kinase regulated development of the autophagic state. To determine if AMP kinase was required for chondrocyte autophagy, we suppressed AMPK expression in N1511 cells using siRNA technology. When these cells were serum-starved, a condition that triggers autophagy, we failed to observe a loss of organization of LC3. In other words, chondrocytes failed to induce autophagy. This result confirmed that AMPK was required for the induction of the autophagic response. Based on the two studies described above, and the observation that HIF-1 is required for the induction of autophagy, we put forward the hypothesis that the activity of AMPK and mTOR is regulated by HIF-1. HIF-1-dependent glycolytic activity would elevate AMP levels resulting in AMP kinase activation and suppression of mTOR, activities that lead to increased autophagy. Once autophagy is activated, the post-mitotic chondrocytes would be expected to remain viable in their unique microenvironment, and complete their life cycle. Thus, these two proteins serve to directly regulate the induction of the autophagic response. Once autophagy is activated, the post-mitotic chondrocytes would be expected to remain viable in their unique microenvironment, and complete their life cycle.
growth plate; chondrocytes; autophagy; AMP kinase; mTOR