Autophagy activation reduces renal tubular injury induced by urinary proteins

Autophagy is shown to be beneficial for renal tubular injury caused by nephrotoxic drugs. To investigate whether autophagy could protect renal tubular epithelial cells (TECs) from injury induced by urinary proteins, we studied the activity and action of autophagy in TECs after urinary protein overload in vivo and in vitro. We found that autophagic vacuoles increased in TECs from patients with minimal change nephrotic syndrome (MCNS) and rat models with severe proteinuria induced by cationic BSA. In HK-2 cells, exposure to urinary proteins extracted from patients with MCNS led to a significant increase in autophagosome and autolysosome formation and decrease in SQSTM1/p62 protein level. Urinary protein addition also induced lysosomal turnover of LC3-II and perinuclear clustering of lysosomes. These changes were mediated by a reactive oxygen species (ROS)-dependent mechanism. Furthermore, pretreatment of HK-2 cells with rapamycin reduced the production of LCN2/NGAL and HAVCR1/KIM-1 and the level of apoptosis induced by urinary proteins. In contrast, blocking autophagy with chloroquine or BECN1 siRNAs exerted an opposite effect. Similar results were also observed in animal models with proteinuria after treatments with rapamycin and chloroquine. Taken together, our results indicated an increase in autophagic flux, which mounts an adaptive response in TECs after urinary protein overload.     

TPT1 (tumor protein,translationally-controlled 1) negatively regulates autophagy through the BECN1 interactome and an MTORC1-mediated pathway

TPT1/TCTP (tumor protein, translationally-controlled 1) is highly expressed in tumor cells, known to participate in various cellular activities including protein synthesis, growth and cell survival. In addition, TPT1 was identified as a direct target of the tumor suppressor TP53/p53 although little is known about the mechanism underlying the anti-survival function of TPT1. Here, we describe a role of TPT1 in the regulation of the MTORC1 pathway through modulating the molecular machinery of macroautophagy/autophagy. TPT1 inhibition induced cellular autophagy via the MTORC1 and AMPK pathways, which are inhibited and activated, respectively, during treatment with the MTOR inhibitor rapamycin. We also found that the depletion of TPT1 potentiated rapamycin-induced autophagy by synergizing with MTORC1 inhibition. We further demonstrated that TPT1 knockdown altered the BECN1 interactome, a representative MTOR-independent pathway, to stimulate autophagosome formation, via downregulating BCL2 expression through activating MAPK8/JNK1, and thereby enhancing BECN1-phosphatidylinositol 3-kinase (PtdIns3K)-UVRAG complex formation. Furthermore, reduced TPT1 promoted autophagic flux by modulating not only early steps of autophagy but also autophagosome maturation. Consistent with in vitro findings, in vivo organ analysis using Tpt1 heterozygote knockout mice showed that autophagy is enhanced because of haploinsufficient TPT1 expression. Overall, our study demonstrated the novel role of TPT1 as a negative regulator of autophagy that may have potential use in manipulating various diseases associated with autophagic dysfunction.     

Autophagy is a protective response to ethanol neurotoxicity

Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II) and upregulated LC3 puncta in SH-SY5Y neuroblastoma cells. It also enhanced the levels of LC3-II and BECN1 in the developing brain; meanwhile, ethanol reduced SQSTM1 (p62) levels. Bafilomycin A₁, an inhibitor of autophagosome and lysosome fusion, increased p62 levels in the presence of ethanol. Bafilomycin A₁ and rapamycin potentiated ethanol-increased LC3 lipidation, whereas wortmannin and a BECN1-specific shRNA inhibited ethanol-promoted LC3 lipidation. Ethanol increased mitophagy, which was also modulated by BECN1 shRNA and rapamycin. The evidence suggested that ethanol promoted autophagic flux. Activation of autophagy by rapamycin reduced ethanol-induced ROS generation and ameliorated ethanol-induced neuronal death in vitro and in the developing brain, whereas inhibition of autophagy by wortmannin and BECN1-specific shRNA potentiated ethanol-induced ROS production and exacerbated ethanol neurotoxicity. Furthermore, ethanol inhibited the MTOR pathway and downregulation of MTOR offered neuroprotection. Taken together, the results suggest that autophagy activation is a neuroprotective response to alleviate ethanol toxicity. Ethanol modulation of autophagic activity may be mediated by the MTOR pathway.     

MTOR overactivation and interrupted autophagy flux in obese hearts

As a central controller of cell growth, mechanistic target of rapamycin (MTOR) affects an array of biological processes, in particular protein synthesis, autophagy and cardiac homeostasis. Conflicting findings have been seen with regard to the role of MTOR signaling and autophagy in cardiac and adipocyte function under metabolic syndrome. AKT, an essential insulin-signaling molecule upstream of MTOR, participates in the regulation of glucose homeostasis and cardiac metabolism. Akt2 knockout may rescue against high-fat diet-disrupted autophagy flux, en route to cardioprotection. Thus, inhibition of MTOR may serve as a possible avenue to retard pathological cardiac hypertrophy via rescuing interrupted autophagic flux.     

Reactivation of autophagy ameliorates LMNA cardiomyopathy

Mutations in the LMNA gene, which encodes lamin A and C (lamin A/C), cause a diverse spectrum of tissue-selective diseases termed laminopathies. The most prevalent form affects striated muscles as dilated cardiomyopathy with variable skeletal muscle involvement, which includes autosomal Emery-Dreifuss muscular dystrophy. Mechanisms underlying the disease pathogenesis are beginning to be understood and they point toward defects in cell signaling. We therefore assessed putative signaling defects in a mouse model carrying a point mutation in Lmna (Lmna^H222P/H222P) that faithfully recapitulates human Emery-Dreifuss muscular dystrophy. We found that AKT-mechanistic target of rapamycin (MTOR) signaling was hyperactivated in hearts of Lmna^H222P/H222P mice and that reducing MTOR activity by pharmacological intervention ameliorated cardiomyopathy. Given the central role of MTOR in regulating autophagy, we assessed fasting-induced autophagic responses and found that they were impaired in hearts of these mice. Moreover, the improved heart function associated with pharmacological blockade of MTOR was correlated with enhanced autophagy. These findings demonstrated that signaling defects that impair autophagy underlie pathogenesis of dilated cardiomyopathy arising from LMNA mutation.     

The ciliary protein RPGRIP1L governs autophagy independently of its proteasome-regulating function at the ciliary base in mouse embryonic fibroblasts

Previously, macroautophagy/autophagy was demonstrated to be regulated inter alia by the primary cilium. Mutations in RPGRIP1L cause ciliary dysfunctions resulting in severe human diseases summarized as ciliopathies. Recently, we showed that RPGRIP1L deficiency leads to a decreased proteasomal activity at the ciliary base in mice. Importantly, the drug-induced restoration of proteasomal activity does not rescue ciliary length alterations in the absence of RPGRIP1L indicating that RPGRIP1L affects ciliary function also via other mechanisms. Based on this knowledge, we analyzed autophagy in Rpgrip1l-negative mouse embryos. In these embryos, autophagic activity was decreased due to an increased activation of the MTOR complex 1 (MTORC1). Application of the MTORC1 inhibitor rapamycin rescued dysregulated MTORC1, autophagic activity and cilia length but not proteasomal activity in Rpgrip1l-deficient mouse embryonic fibroblasts demonstrating that RPGRIP1L seems to regulate autophagic and proteasomal activity independently from each other.     

Autophagy is a protective response to ethanol neurotoxicity

Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II) and upregulated LC3 puncta in SH-SY5Y neuroblastoma cells. It also enhanced the levels of LC3-II and BECN1 in the developing brain; meanwhile, ethanol reduced SQSTM1 (p62) levels. Bafilomycin A₁, an inhibitor of autophagosome and lysosome fusion, increased p62 levels in the presence of ethanol. Bafilomycin A₁ and rapamycin potentiated ethanol-increased LC3 lipidation, whereas wortmannin and a BECN1-specific shRNA inhibited ethanol-promoted LC3 lipidation. Ethanol increased mitophagy, which was also modulated by BECN1 shRNA and rapamycin. The evidence suggested that ethanol promoted autophagic flux. Activation of autophagy by rapamycin reduced ethanol-induced ROS generation and ameliorated ethanol-induced neuronal death in vitro and in the developing brain, whereas inhibition of autophagy by wortmannin and BECN1-specific shRNA potentiated ethanol-induced ROS production and exacerbated ethanol neurotoxicity. Furthermore, ethanol inhibited the MTOR pathway and downregulation of MTOR offered neuroprotection. Taken together, the results suggest that autophagy activation is a neuroprotective response to alleviate ethanol toxicity. Ethanol modulation of autophagic activity may be mediated by the MTOR pathway.     

Autophagy controls everolimus (RAD001) activity in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive neoplasm, which lacks effective therapy. The mechanistic target of rapamycin (MTOR) kinase inhibitor everolimus (RAD001) has shown activity in preclinical and clinical models of MCL, despite the fact that its mechanism of action has not been fully elucidated. We found that everolimus activity in MCL cells is closely linked to AKT phosphorylation status, and that the prevention of AKT rephosphorylation upon everolimus treatment by means of a selective AKT inhibitor, greatly enhances everolimus activity. Furthermore, our data show that an accumulation of autophagic vacuoles correlates with a lack of efficacy of dual AKT-MTOR targeting and that the complete therapeutic potential of this strategy can be restored by ATG gene selective knockdown or secondary inhibition of autolysosome formation by hydroxychloroquine. We thus demonstrated for the first time that the use of an autophagy inhibitor can overcome resistance to the combination of MTOR and AKT inhibitors in MCL cell lines and primary samples, demonstrating the prosurvival role of autophagy in AKT-MTOR compromised cells, and pointing out some potential opportunities using this triple combinational strategy in hematological malignancies.     

Role for DUSP1 (dual-specificity protein phosphatase 1) in the regulation of autophagy

Accumulating evidence suggests that mitogen-activated protein kinases (MAPKs) regulate macroautophagy/autophagy. However, the involvement of dual-specificity protein phosphatases (DUSPs), endogenous inhibitors for MAPKs, in autophagy remains to be determined. Here we report that DUSP1/MKP-1, the founding member of the DUSP family, plays a critical role in regulating autophagy. Specifically, we demonstrate that DUSP1 knockdown by shRNA in human ovarian cancer CAOV3 cells and knockout in murine embryonic fibroblasts, increases both basal and rapamycin-increased autophagic flux. Overexpression of DUSP1 had the opposite effect. Importantly, knockout of Dusp1 promoted phosphorylation of ULK1 at Ser555, and BECN1/Beclin 1 at Ser15, and the association of PIK3C3/VPS34, ATG14, BECN1 and MAPK, leading to the activation of the autophagosome-initiating class III phosphatidylinositol 3-kinase (PtdIns3K) complex. Furthermore, knockdown and pharmacological inhibitor studies indicated that DUSP1-mediated suppression of autophagy reflected inactivation of the MAPK1-MAPK3 members of the MAPK family. Knockdown of DUSP1 sensitized CAOV3 cells to rapamycin-induced antigrowth activity. Moreover, CAOV3-CR cells, a line that had acquired cisplatin resistance, exhibited an elevated DUSP1 level and were refractory to rapamycin-induced autophagy and cytostatic effects. Knockdown of DUSP1 in CAOV3-CR cells restored sensitivity to rapamycin. Collectively, this work identifies a previously unrecognized role for DUSP1 in regulating autophagy and suggests that suppression of DUSP1 may enhance the therapeutic activity of rapamycin.     

Participation of autophagy in renal ischemia/reperfusion injury

Renal ischemia-reperfusion (I/R) injury is inevitable in transplantation, and it results in renal tubular epithelial cells undergoing cell death. We observed an increase in autophagosomes in the tubular epithelial cells of I/R-injured mouse models, and in biopsy specimens from human transplanted kidney. However, it remains unclear whether autophagy functions as a protective pathway, or contributes to I/R-induced cell death. Here, we employed the human renal proximal tubular epithelial cell line HK-2 in order to explore the role of autophagy under hypoxia (1% O₂) or activation of reactive oxygen species (500 μM H₂O₂). When compared to normoxic conditions, 48 h of hypoxia slightly increased LC3-labeled autophagic vacuoles and markedly increased LAMP2-labeled lysosomes. We observed similar changes in the mouse IR-injury model. We then assessed autophagic generation and degradation by inhibiting the downstream lysosomal degradation of autophagic vacuoles using lysosomal protease inhibitor. We found that autophagosomes increased markedly under hypoxia in the presence of lysosomal protease inhibitors, thus suggesting that hypoxia induces high turnover of autophagic generation and degradation. Furthermore, inhibition of autophagy significantly inhibited H₂O₂-induced cell death. In conclusion, high turnover of autophagy may lead to autophagic cell death during I/R injury.     

TARDBP/TDP-43 regulates autophagy in both MTORC1-dependent and MTORC1-independent manners

In a recent paper we addressed the mechanism by which defective autophagy contributes to TARDBP/TDP-43-mediated neurodegenerative disorders. We demonstrated that TARDBP regulates MTORC1-TFEB signaling by targeting RPTOR/raptor, a key component and an adaptor protein of MTORC1. Loss of TARDBP decreased the mRNA stability of RPTOR and this regulation in turn enhanced autophagosomal and lysosomal biogenesis in an MTORC1-dependent manner. Meanwhile, loss of TARDBP could also impair autophagosome-lysosome fusion in an MTORC1-independent manner. Importantly, we found that modulation of MTOR activity by treatment with rapamycin and phosphatidic acid had strong effects on the neurodegenerative phenotypes of TBPH (Drosophila TARDBP)-depleted flies. Taken together, our data reveal that multiple dysfunctions in the autophagic process contribute to TARDBP-linked neurodegeneration and may help to identify potential therapeutic targets in the future.     

Hypoxia-induced MIR155 is a potent autophagy inducer by targeting multiple players in the MTOR pathway

Hypoxia activates autophagy, an evolutionarily conserved cellular catabolic process. Dysfunction in the autophagy pathway has been implicated in an increasing number of human diseases, including cancer. Hypoxia induces upregulation of a specific set of microRNAs (miRNAs) in a variety of cell types. Here, we describe hypoxia-induced MIR155 as a potent inducer of autophagy. Enforced expression of MIR155 increases autophagic activity in human nasopharyngeal cancer and cervical cancer cells. Knocking down endogenous MIR155 inhibits hypoxia-induced autophagy. We demonstrated that MIR155 targets multiple players in MTOR signaling, including RHEB, RICTOR, and RPS6KB2. MIR155 suppresses target-gene expression by directly interacting with their 3′ untranslated regions (UTRs), mutations of the binding sites abolish their MIR155 responsiveness. Furthermore, by downregulating MTOR signaling, MIR155 also attenuates cell proliferation and induces G₁/S cell cycle arrest. Collectively, these data present a new role for MIR155 as a key regulator of autophagy via dysregulation of MTOR pathway.     

Rapamycin Induces Apoptosis When Autophagy is Inhibited in T-47D Mammary Cells and Both Processes are Regulated by Phlda1

Autophagy is an evolutionarily conserved lysosomal degradation pathway and plays a critical role in the homeostatic process of recycling proteins and organelles. Functional relationships have been described between apoptosis and autophagy. Perturbations in the apoptotic machinery have been reported to induce autophagic cell deaths. Inhibition of autophagy in cancer cells has resulted in cell deaths that manifested hallmarks of apoptosis. However, the molecular relationships and the circumstances of which molecular pathways dictate the choice between apoptosis and autophagy are currently unknown. This study aims to identify specific gene expression of rapamycin-induced autophagy and the effects of rapamycin when the autophagy process is inhibited. In this study, we have demonstrated that rapamycin is capable of inducing autophagy in T-47D breast carcinoma cells. However, when the autophagy process was inhibited by 3-MA, the effects of rapamycin became apoptotic. The Phlda1 gene was found to be up-regulated in both autophagy and apoptosis and silencing this gene was found to reduce both activities, strongly suggests that Phlda1 mediates and positively regulates both autophagy and apoptosis pathways.     

Targeting the autophagy pathway using ectopic expression of Beclin 1 in combination with rapamycin in drug-resistant v-Ha-<Emphasis Type="Italic">ras</Emphasis>-transformed NIH 3T3 cells

The effectiveness of an apoptosis-targeting therapy may be limited in tumor cells with defects in apoptosis. Recently, considerable attention in the field of cancer therapy has been focused on the mammalian rapamycin target (mTOR), inhibition of which results in autophagic cell death. In our study using multidrug-resistant v-Ha-rastransformed NIH3T3 (Ras-NIH 3T3/Mdr) cells, we demonstrated that rapamycin-induced cell death may result from 2 different mechanisms. At high rapamycin concentrations (≥ 100 nM), cell death may occur via an autophagy-dependent pathway, whereas at lower concentrations (≤ 10 nM), cell death may occur after G1-phase cell cycle arrest. This effect was accompanied by upregulation of p21^Cip1 and p27^Kip1 expression via an autophagy-independent pathway. We also tested whether inhibition of mTOR with low concentrations of rapamycin and ectopic Beclin-1 expression would further sensitize multidrug resistance (MDR)-positive cancer cells by upregulating autophagy. Rapamycin at low concentrations might be insufficient to initiate autophagosome formation in autophagy but Beclin-1 overexpression triggered additional processes downstream of mTOR during G₁ cell cycle arrest by rapamycin. Our findings suggest that these combination strategies targeting autophagic cell death may yield significant benefits for cancer patients, because lowering rapamycin concentration for cancer treatment minimizes its side effects in patients undergoing chemotherapy.     

Reciprocal regulation of cilia and autophagy via the MTOR and proteasome pathways

Primary cilium is an organelle that plays significant roles in a number of cellular functions ranging from cell mechanosensation, proliferation, and differentiation to apoptosis. Autophagy is an evolutionarily conserved cellular function in biology and indispensable for cellular homeostasis. Both cilia and autophagy have been linked to different types of genetic and acquired human diseases. Their interaction has been suggested very recently, but the underlying mechanisms are still not fully understood. We examined autophagy in cells with suppressed cilia and measured cilium length in autophagy-activated or -suppressed cells. It was found that autophagy was repressed in cells with short cilia. Further investigation showed that MTOR activation was enhanced in cilia-suppressed cells and the MTOR inhibitor rapamycin could largely reverse autophagy suppression. In human kidney proximal tubular cells (HK2), autophagy induction was associated with cilium elongation. Conversely, autophagy inhibition by 3-methyladenine (3-MA) and chloroquine (CQ) as well as bafilomycin A₁ (Baf) led to short cilia. Cilia were also shorter in cultured atg5-knockout (KO) cells and in atg7-KO kidney proximal tubular cells in mice. MG132, an inhibitor of the proteasome, could significantly restore cilium length in atg5-KO cells, being concomitant with the proteasome activity. Together, the results suggest that cilia and autophagy regulate reciprocally through the MTOR signaling pathway and ubiquitin-proteasome system.     

Epigenetic regulation of autophagy by the methyltransferase EZH2 through an MTOR-dependent pathway

Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the autophagy regulation machinery has been widely studied, the key epigenetic control of autophagy process still remains unknown. Here we report that the methyltransferase EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) epigenetically represses several negative regulators of the MTOR (mechanistic target of rapamycin [serine/threonine kinase]) pathway, such as TSC2, RHOA, DEPTOR, FKBP11, RGS16 and GPI. EZH2 was recruited to these genes promoters via MTA2 (metastasis associated 1 family, member 2), a component of the nucleosome remodeling and histone deacetylase (NuRD) complex. MTA2 was identified as a new chromatin binding protein whose association with chromatin facilitated the subsequent recruitment of EZH2 to silenced targeted genes, especially TSC2. Downregulation of TSC2 (tuberous sclerosis 2) by EZH2 elicited MTOR activation, which in turn modulated subsequent MTOR pathway-related events, including inhibition of autophagy. In human colorectal carcinoma (CRC) tissues, the expression of MTA2 and EZH2 correlated negatively with expression of TSC2, which reveals a novel link among epigenetic regulation, the MTOR pathway, autophagy induction, and tumorigenesis.