Role In Regulation Of Autophagy Research Articles

Eukaryotic elongation factor-2 kinase (eEF2K): a potential therapeutic target in cancer.

Eukaryotic elongation factor-2 kinase (eEF2K), encoded by the EEF2K gene, is well-known to be a Ca(2+)/calmodulin (CaM)-dependent kinase which can negatively modulate protein synthesis. It is highly conserved among eukaryotes from mammals to invertebrates, of which human and mouse may have 99 % overall amino acid identity. This kinase can phosphorylate eukaryotic elongation factor-2 (eEF2) or undergo the process of autophosphorylation at multiple sites to inhibit its function in translation elongation. Due to the fact that regulation of eEF2 by eEF2K is an evolutionarily conserved mechanism, eEF2K activity may confer tumor cell adaption to metabolic stress under acute nutrient depletion, and the high expressed level of eEF2K has been found in several types of malignancies. eEF2K may modulate the expression of some apoptotic proteins such as XIAP, c-FLIPL, Bcl-XL, PI3KCI and p70(S6K) to inhibit apoptotic process in cancer. On the other hand, it plays a regulatory role in autophagy involved in mTORC1, AMPK and Atg8, thereby promoting cancer cell survival. Additionally, eEF2K may play a crucial role in the crosstalk between apoptosis and autophagy in cancer. Collectively, these findings have led to the conclusions that eEF2K may contribute to carcinogenesis, and thus being utilized as a potential target for future cancer therapy.

SIRT1 Positively Regulates Autophagy and Mitochondria Function in Embryonic Stem Cells Under Oxidative Stress

SIRT1, an NAD-dependent deacetylase, plays a role in regulation of autophagy. SIRT1 increases mitochondrial function and reduces oxidative stress, and has been linked to age-related reactive oxygen species (ROS) generation, which is highly dependent on mitochondrial metabolism. H2O2 induces oxidative stress and autophagic cell death through interference with Beclin 1 and the mTOR signaling pathways. We evaluated connections between SIRT1 activity and induction of autophagy in murine (m) and human (h) embryonic stem cells (ESCs) upon ROS challenge. Exogenous H2 O2 (1 mM) induced apoptosis and autophagy in wild-type (WT) and Sirt1-/- mESCs. High concentrations of H2O2 (1 mM) induced more apoptosis in Sirt1-/-, than in WT mESCs. However, addition of 3-methyladenine, a widely used autophagy inhibitor, in combination with H2O2 induced more cell death in WT than in Sirt1-/- mESCs. Decreased induction of autophagy in Sirt1-/- mESCs was demonstrated by decreased conversion of LC3-I to LC3-II, lowered expression of Beclin-1, and decreased LC3 punctae and LysoTracker staining. H2O2 induced autophagy with loss of mitochondrial membrane potential and disruption of mitochondrial dynamics in Sirt1-/- mESCs. Increased phosphorylation of P70/85-S6 kinase and ribosomal S6 was noted in Sirt1-/- mESCs, suggesting that SIRT1 regulates the mTOR pathway. Consistent with effects in mESCs, inhibition of SIRT1 using Lentivirus-mediated SIRT1 shRNA in hESCs demonstrated that knockdown of SIRT1 decreased H2O2-induced autophagy. This suggests a role for SIRT1 in regulating autophagy and mitochondria function in ESCs upon oxidative stress, effects mediated at least in part by the class III PI3K/Beclin 1 and mTOR pathways.

Understanding the non-canonical pathways involved in p53-mediated tumor suppression

In the last three decades since the discovery of p53, it has become increasingly apparent that p53 plays a very important role in tumor suppression. Previously, it was thought that the tumor suppressive functions lied solely in the canonical p53-mediated apoptosis, cell cycle arrest and senescence. However, more recent research has shown that anti-oncogenic activity of p53 can still occur in the absence of these downstream functions. These results suggest that more non-canonical roles of p53 may have a much larger impact on other p53-regulated programs then initially anticipated. Recently, the non-canonical activities of p53 such as cell metabolism, autophagy and necrosis have been the subject of intense study. p53 affects many aspects of cellular metabolism including catabolism, anabolism and reactive oxygen species levels. p53 has a dual role in autophagy regulation. Initiation of autophagy occurs through direct transcription of pro-autophagy genes and inhibition transpires through a transcription-independent mechanism. The role of p53 in these cellular processes is quite complex and evidence suggests that p53 can play both a pro- and anti-oncogenic role in these non-conical pathways. Despite of more than 60,000 publications on p53 in the literature, the mechanisms for p53-mediated tumor suppression apparently needs to be further elucidated.

MIR376A Is a Regulator of Starvation-Induced Autophagy

BackgroundAutophagy is a vesicular trafficking process responsible for the degradation of long-lived, misfolded or abnormal proteins, as well as damaged or surplus organelles. Abnormalities of the autophagic activity may result in the accumulation of protein aggregates, organelle dysfunction, and autophagy disorders were associated with various diseases. Hence, mechanisms of autophagy regulation are under exploration.MethodsOver-expression of hsa-miR-376a1 (shortly MIR376A) was performed to evaluate its effects on autophagy. Autophagy-related targets of the miRNA were predicted using Microcosm Targets and MIRanda bioinformatics tools and experimentally validated. Endogenous miRNA was blocked using antagomirs and the effects on target expression and autophagy were analyzed. Luciferase tests were performed to confirm that 3′ UTR sequences in target genes were functional. Differential expression of MIR376A and the related MIR376B was compared using TaqMan quantitative PCR.ResultsHere, we demonstrated that, a microRNA (miRNA) from the DLK1/GTL2 gene cluster, MIR376A, played an important role in autophagy regulation. We showed that, amino acid and serum starvation-induced autophagy was blocked by MIR376A overexpression in MCF-7 and Huh7 cells. MIR376A shared the same seed sequence and had overlapping targets with MIR376B, and similarly blocked the expression of key autophagy proteins ATG4C and BECN1 (Beclin 1). Indeed, 3′ UTR sequences in the mRNA of these autophagy proteins were responsive to MIR376A in luciferase assays. Antagomir tests showed that, endogenous MIR376A was participating to the control of ATG4C and BECN1 transcript and protein levels. Moreover, blockage of endogenous MIR376A accelerated starvation-induced autophagic activity. Interestingly, MIR376A and MIR376B levels were increased with different kinetics in response to starvation stress and tissue-specific level differences were also observed, pointing out to an overlapping but miRNA-specific biological role.ConclusionsOur findings underline the importance of miRNAs encoded by the DLK1/GTL2 gene cluster in stress-response control mechanisms, and introduce MIR376A as a new regulator of autophagy.

Oncolytic Adenovirus With Temozolomide Induces Autophagy and Antitumor Immune Responses in Cancer Patients

Oncolytic adenoviruses and certain chemotherapeutics can induce autophagy and immunogenic cancer cell death. We hypothesized that the combination of oncolytic adenovirus with low-dose temozolomide (TMZ) is safe, effective, and capable of inducing antitumor immune responses. Metronomic low-dose cyclophosphamide (CP) was added to selectively reduce regulatory T-cells. Preclinically, combination therapy inhibited tumor growth, increased autophagy, and triggered immunogenic cell death as indicated by elevated calreticulin, adenosine triphosphate (ATP) release, and nuclear protein high-mobility group box-1 (HMGB1) secretion. A total of 41 combination treatments given to 17 chemotherapy-refractory cancer patients were well tolerated. We observed anti- and proinflammatory cytokine release, evidence of virus replication, and induction of neutralizing antibodies. Tumor cells showed increased autophagy post-treatment. Release of HMGB1 into serum--a possible indicator of immune response--increased in 60% of treatments, and seemed to correlate with tumor-specific T-cell responses, observed in 10/15 cases overall (P = 0.0833). Evidence of antitumor efficacy was seen in 67% of evaluable treatments with a trend for increased survival over matched controls treated with virus only. In summary, the combination of oncolytic adenovirus with low-dose TMZ and metronomic CP increased tumor cell autophagy, elicited antitumor immune responses, and showed promising safety and efficacy.

Abstract SY08-01: AMPK and mTOR in nutrient signaling, metabolism, and autophagy regulation.

Abstract Autophagy is a stress response protecting cells from unfavorable conditions, such as nutrient starvation. The cellular energy sensor AMPK and key growth controller mTOR, both are regulated by nutrient status, play major roles in autophagy regulation. The class III phosphatidylinositol-3 kinase, Vps34, forms multiple complexes and regulates both intracellular vesicle trafficking and autophagy induction. We show that AMPK plays a key role in regulating different Vps34 complexes. AMPK inhibits the non-autophagy Vps34 complex by phosphorylating Vps34 and suppresses overall PI(3)P production and protects cells from starvation. In parallel, AMPK activates the pro-autophagy Vps34 complex by phosphorylating Beclin1 to induce autophagy. Atg14L, an autophagy essential gene present only in the pro-autophagy Vps34 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. In addition, the Vps34 complex is regulated by mTOR, which is inhibited by nutrient starvation. mTOR phosphorylates Beclin to inhibit the autophagy specific Vps34 complex. Our study reveals an intricate molecular regulation of Vps34 complexes by AMPK and mTOR in nutrient stress response and autophagy. Citation Format: Joungmok Kim, Ryan Russell, Haxin Yuan, Kun-Liang Guan. AMPK and mTOR in nutrient signaling, metabolism, and autophagy regulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY08-01. doi:10.1158/1538-7445.AM2013-SY08-01

UCP2 inhibition triggers ROS-dependent nuclear translocation of GAPDH and autophagic cell death in pancreatic adenocarcinoma cells

Mitochondrial uncoupling protein 2 (UCP2) can moderate oxidative stress by favoring the influx of protons into the mitochondrial matrix, thus reducing electron leakage from respiratory chain and mitochondrial superoxide production. Here, we demonstrate that UCP2 inhibition by genipin or UCP2 siRNA strongly increases reactive oxygen species (ROS) production inhibiting pancreatic adenocarcinoma cell growth. We also show that UCP2 inhibition triggers ROS-dependent nuclear translocation of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), formation of autophagosomes, and the expression of the autophagy marker LC3-II. Consistently, UCP2 over-expression significantly reduces basal autophagy confirming the anti-autophagic role of UCP2. Furthermore, we demonstrate that autophagy induced by UCP2 inhibition determines a ROS-dependent cell death, as indicated by the apoptosis decrease in the presence of the autophagy inhibitors chloroquine (CQ) or 3-methyladenine (3-MA), or the radical scavenger NAC. Intriguingly, the autophagy induced by genipin is able to potentiate the autophagic cell death triggered by gemcitabine, the standard chemotherapeutic drug for pancreatic adenocarcinoma, supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to standard chemotherapy. Our results demonstrate for the first time that UCP2 plays a role in autophagy regulation bringing new insights into mitochondrial uncoupling protein field.

Abstract 40: Induced Basal Autophagy in Atg7 Mice Ameliorates Cardiac Proteinopathy

Backgrounds: Basal autophagy is a cellular homeostatic mechanism that can eliminate damaged and potentially toxic proteins and organelles. We hypothesized that cardiac proteinopathies, such as desmin-related cardiomyopathy (DRC) that lead to accumulations of misfolded proteins, can trigger alterations in cardiomyocyte autophagy as an adaptive process. However, a mouse model of cardiac specific-induced basal autophagy is lacking and the effects of acute or chronic increased basal autophagy remains unclear. Hypothesis: To determine whether induction of basal autophagy in the induced Atg7 mice can ameliorate cardiac proteinopathy in a model of DRC. Methods and Results: The protein Atg7 is thought to be essential for autophagy and, in an attempt to induce high levels of basal autophagy we generated an inducible-transgenic (Tg) mouse. This mouse is able to undergo cardiac specific overexpression of the Atg7 gene, where the transgene can be switched on or off by doxycycline. The Atg7-Tg mouse showed 10 fold overexpression of Atg7 protein with normal heart morphology and function being maintained even after 10 months. Overexpression of Atg7 had no detectable effect on other proteins involved in the autophagy-lysosomal pathways. However, the hearts show increased basal autophagy, as measured by increased autophagic flux activity and cardiomyocyte ultrastructural examination. We showed earlier that autophagy can be down-regulated in cardiac proteinopathy as observed in a mutant αB-crystallin (CryAB R120G ) mouse model of DRC. Therefore, we crossed the CryAB R120G mice to transgenic mice with cardiac specific overexpression of Atg7. Sustained Atg7-induced autophagy in CryAB R120G hearts prolonged CryAB R120G survival by 40% and is associated with the maintenance of cardiac function and decreased cardiac hypertrophy. Conclusions: Cardiac-specific Atg7 inducible-Tg mice with increased basal autophagy may serve as a novel model to study the regulatory role of autophagy in the heart. Induced basal autophagy in Atg7-Tg mice can extend the life-span in a model of cardiac proteinopathy. Modulating the overall autophagic state may be useful therapeutically in heart disease.

Ksp1 Kinase Regulates Autophagy via the Target of Rapamycin Complex 1 (TORC1) Pathway

Macroautophagy (hereafter autophagy) is a bulk degradation system conserved in all eukaryotes, which engulfs cytoplasmic components within double-membrane vesicles to allow their delivery to, and subsequent degradation within, the vacuole/lysosome. Autophagy activity is tightly regulated in response to the nutritional state of the cell and also to maintain organelle homeostasis. In nutrient-rich conditions, Tor kinase complex 1 (TORC1) is activated to inhibit autophagy, whereas inactivation of this complex in response to stress leads to autophagy induction; however, it is unclear how the activity of TORC1 is controlled to allow precise adjustments in autophagy activity. In this study, we performed genetic analyses in Saccharomyces cerevisiae to identify factors that regulate TORC1 activity. We determined that the Ksp1 kinase functions in part as a negative regulator of autophagy; deletion of KSP1 facilitated dephosphorylation of Atg13, a TORC1 substrate, which correlates with enhanced autophagy. These results suggest that Ksp1 down-regulates autophagy activity via the TORC1 pathway. The suppressive function of Ksp1 is partially activated by the Ras/cAMP-dependent protein kinase A (PKA), which is another negative regulator of autophagy. Our study therefore identifies Ksp1 as a new component that functions as part of the PKA and TORC1 signaling network to control the magnitude of autophagy.

Exogenous Calcium Induces Autophagy via Classical Autophagic Machinery and Recruits Endoplasmic Reticulum Membranes to Form Autophagosomes

Calcium plays a role in regulation of autophagy. We have reported that exogenously added calcium as the calcium phosphate precipitate (CPP) induces autophagy. Here we further show that CPP‐induced autophagy applies classical autophagic machinery including LC3 conjugation molecules Atg4, Atg5 and Atg3, and autophagosome nucleation molecules FIP200 and Beclin1; however, Atg9 is not involved. CPP does not induce mTOR inhibition, neither over‐production of radical oxygen species, and only slightly increases endoplasmic reticulum (ER) stress. CPP activates DFCP1 in a PI3k and calcium dependent manner, and increases the association of DFCP1 with autophagosomes. ER membrane, but not Golgi and mitochondria membranes, comprises part of the LC3 positive autophagsomes. These data suggests CPP works through non‐mTOR dependent mechanisms to activate downstream classical autophagosome nucleation and expansion molecules, and the growth of autophagosomes induced by CPP is closely related to ER.

A Role for Rac3 GTPase in the Regulation of Autophagy

The process of autophagy is situated at the intersection of multiple cell signaling pathways, including cell metabolism, growth, and death, and hence is subject to multiple forms of regulation. We previously reported that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational prenylation of so-called CAAX proteins, results in the induction of autophagy which enhances cell death in some cancer cells. In this study, using siRNA-mediated knockdown of a group of small GTPases that are predicted Icmt substrates, we identify Rac3 GTPase as a negative regulator of the process of autophagy. Knockdown of Rac3, but not the closely related isoforms Rac1 and Rac2, results in induction of autophagy. Ectopic expression of Rac3, significantly rescues cells from autophagy and cell death induced by Icmt inhibition, strengthening the notion of an isoform-specific autophagy regulatory function of Rac3. This role of Rac3 was observed in multiple cell lines with varying Rac subtype expression profiles, suggesting its broad involvement in the process. The identification of this less-studied Rac member as a novel regulator provides new insight into autophagy and opens opportunities in identifying additional regulatory inputs of the process.

Mitochondria breathe for autophagy

Proper mitochondrial structure and function are established by continuous cycles of fusion and fission, and a unidirectional pathway of autophagy‐mediated degradation. In this issue of The EMBO Journal , Graef and Nunnari (2011) demonstrate that autophagy requires healthy mitochondria in cells under amino‐acid starvation. Defects in mitochondrial respiration cause activation of cAMP‐dependent protein kinase A (PKA), an evolutionarily conserved regulator that inhibits autophagic flux. This study reveals a crucial interplay between mitochondrial function and autophagy, and raises the possibility that deregulation in one of these processes simultaneously compromises the other to potentiate risks for various human disorders.

Abstract 3787: Serotonin signaling plays a role in regulation of autophagy in breast cancer cells

Abstract Breast cancer is the second most common cause of cancer death in women in the United States, 40,000 dying of this disease each year. One out of eight women will be diagnosed with breast cancer in their lifetime. There are several treatments for breast cancer, such as surgery, radiation therapy, chemotherapy, and hormone therapy, but still 5 year survival is less than 40% in advance cases. Due to the high incidence and mortality associated with this cancer better understanding of the biology of the disease and development of new treatments are urgently needed. The actions of local physiological mediators in the context of breast cancer are poorly understood. Serotonin (5HT, 5-hydroxytryptamine) is a critical local regulator of epithelial homeostasis in the breast and other organs. Serotonin is more commonly known as a monoamine neurotransmitter and a mediator of depression if the levels drop in the brain. Serotonin has been implicated in epithelial homeostasis of the breast, lung, pancreas, liver, and prostate. Deregulation of serotonin signaling in these epithelia may be associated with various pathologies, including cancer. Previous and our studies suggested serotonin can induce proliferation of some cancer cell lines. On the other hand serotonin can induce apoptosis or induce cell proliferation depending on the cancer type. Serotonin is believed to function as a growth factor working as an autocrin manner. Also tryptophan hydroxylase 1 (TPH1), rate-limiting step in 5-HT synthesis, has been shown to be overexpressed in breast cancer cell lines and biopsy samples from patients. The effect of serotonin signaling and its role in survival of breast cancer cells has not been well understood. We tried to address the questions that what is the role of serotonin signaling in breast cancer survival and if serotonin inhibits apoptosis or autophagy? We found that serotonin slightly induced cell proliferation in estrogen receptor positive MCF-7 breast cancer cells. Because autophagy is implicated as a survival pathway in some cancers we also examined if serotonin induces autophagy in MCF7 ER+ breast cancer cells. We found that serotonin induces autophagy that is detected Acridine orange staining and FACS analysis and LC-3 II induction, a hallmark of autophagy, by western blot analysis in MCF7 cells after serotonin treatment for 4 hours. We found that serotonin (10 uM) induces expression of cyclinD1, NF-kB and mTOR and leads induction of ERK and AMPK pathway as indicated by increased phosphorylation of ERK1/2 and AMPK (Thr 172), both of which are known to mediate autophagy. We are currently testing if induction ERK and AMPk signaling is involved in induction autophagy in these cells using specific inhibitors of MEK. In conclusion our studies showed for the first that induction of serotonin signaling induces autophagy in ER+ breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3787. doi:10.1158/1538-7445.AM2011-3787

Excess Leucine Intake Enhances Muscle Anabolic Signaling but Not Net Protein Anabolism in Young Men and Women1–3

Essential amino acids (EAA) stimulate skeletal muscle protein synthesis (MPS) in humans. Leucine may have a greater stimulatory effect on MPS than other EAA and/or decrease muscle protein breakdown (MPB). To determine the effect of 2 different leucine concentrations on muscle protein turnover and associated signaling, young men (n = 6) and women (n = 8) ingested 10 g EAA in 1 of 2 groups: composition typical of high quality proteins (CTRL; 1.8 g leucine) or increased leucine concentration (LEU; 3.5 g leucine). Participants were studied for 180 min postingestion. Fractional synthetic rate and leg phenylalanine and leucine kinetics were assessed on muscle biopsies using stable isotopic techniques. Signaling was determined by immunoblotting. Arterial leucine concentration and delivery to the leg increased in both groups and was significantly higher in LEU than in CTRL; however, transport into the muscle and intracellular availability did not differ between groups. MPS increased similarly in both groups 60 min postingestion. MPB decreased at 60 min only in LEU, but net muscle protein balance improved similarly. Components of mammalian target of rapamycin (mTOR) signaling were improved in LEU, but no changes were observed in ubiquitin-proteasome system signaling. Changes in light chain 3 and mTOR association with Unc-51-like kinase 1 indicate autophagy decreased more in LEU. We conclude that in 10 g of EAA, the leucine content typical of high quality proteins (~1.8 g) is sufficient to induce a maximal skeletal muscle protein anabolic response in young adults, but leucine may play a role in autophagy regulation.

Role of Inositol Trisphosphate Receptors in Autophagy in DT40 Cells

Previous studies have shown that small interfering RNA knockdown and pharmacological inhibition of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) stimulate autophagy. We have investigated autophagy in chicken DT40 cell lines containing targeted deletions of all three IP(3)R isoforms (triple knock-out (TKO) cells). Using gel shifts of microtubule-associated protein 1 light chain 3 as a marker of autophagy, we find that TKO cells have enhanced basal autophagic flux even under nutrient-replete conditions. Stable DT40 cell lines derived from TKO cells containing the functionally inactive D2550A IP(3)R mutant did not suppress autophagy in the same manner as wild-type receptors. This suggests that the channel function of the receptor is important in its regulatory role in autophagy. There were no marked differences in the phosphorylation state of AMP-activated protein kinase, Akt, or mammalian target of rapamycin between wild-type and TKO cells. The amount of immunoprecipitated complexes of Bcl-2-Beclin-1 and Beclin-1-Vps34 were also not different between the two cell lines. The major difference noted was a substantially decreased mTORC1 kinase activity in TKO cells based on decreased phosphorylation of S6 kinase and 4E-BP1. The discharge of intracellular stores with thapsigargin stimulated mTORC1 activity (measured as S6 kinase phosphorylation) to a greater extent in wild-type than in TKO cells. We suggest that basal autophagic flux may be negatively regulated by IP(3)R-dependent Ca(2+) signals acting to maintain an elevated mTORC1 activity in wild-type cells and that Ca(2+) regulation of this enzyme is defective in TKO cells. The protective effect of a higher autophagic flux in cells lacking IP(3)Rs may play a role in the delayed apoptotic response observed in these cells.

Positive or Negative Roles of Different Cyclin-Dependent Kinase Pho85-Cyclin Complexes Orchestrate Induction of Autophagy in Saccharomyces cerevisiae

As a major intracellular degradation pathway, autophagy is tightly regulated to prevent cellular dysfunction in all eukaryotic cells. The rapamycin-sensitive Tor kinase complex 1 is a major regulator of autophagy. Several other nutrient-sensory kinases also play critical roles to precisely modulate autophagy; however, the network of regulatory mechanisms remains largely elusive. We used genetic analyses to elucidate the mechanism by which the stress-responsive, cyclin-dependent kinase Pho85 and its corresponding cyclin complexes antagonistically modulate autophagy in Saccharomyces cerevisiae. When complexed with cyclins Pho80 and Pcl5, Pho85 negatively regulates autophagy through downregulating the protein kinase Rim15 and the transcription factors Pho4 and Gcn4. The cyclins Clg1, Pcl1, and Pho80, in concert with Pho85, positively regulate autophagy through promoting the degradation of Sic1, a negative regulator of autophagy that targets Rim15. Our results suggest a model in which Pho85 and its cyclin complexes have opposing roles in autophagy regulation.

Control of autophagy initiation by phosphoinositide 3-phosphatase jumpy

The majority of studies on autophagy, a cytoplasmic homeostasis pathway of broad biological and medical significance, have been hitherto focused on the phosphatidylinositol 3-kinases as the regulators of autophagy. Here, we addressed the reverse process driven by phosphoinositide phosphatases and uncovered a key negative regulatory role in autophagy of a phosphatidylinositol 3-phosphate (PI3P) phosphatase Jumpy (MTMR14). Jumpy associated with autophagic isolation membranes and early autophagosomes, defined by the key factor Atg16 necessary for proper localization and development of autophagic organelles. Jumpy orchestrated orderly succession of Atg factors by controlling recruitment to autophagic membranes of the sole mammalian Atg factor that interacts with PI3P, WIPI-1 (Atg18), and by affecting the distribution of Atg9 and LC3, the two Atg factors controlling organization and growth of autophagic membranes. A catalytically inactive Jumpy mutant, R336Q, found in congenital disease centronuclear myopathy, lost the ability to negatively regulate autophagy. This work reports for the first time that initiation of autophagy is controlled not only by the forward reaction of generating PI3P through a lipid kinase but that its levels are controlled by a specific PI3P phosphatase, which when defective can lead to human disease.

Autophagy is involved in influenza A virus replication

Autophagy is involved in the replication of viruses, especially those that perform RNA assembly on the surface of cytoplasmic membrane in host cells. However, little is known about the regulatory role of autophagy in influenza A virus replication. Using fluorescence and electron microscopy, we observed that autophagosomes can be induced and identified upon influenza A virus infection. The virus increased the amount of the autophagosome marker protein microtubule-associated protein light chain 3-II (LC3-II) and enhanced autophagic flux. When autophagy was pharmacologically inhibited by either 3-methylademine or wortmannin, the titers of influenza A virus were remarkably decreased. Viral reduction via autophagy inhibition was further confirmed by RNA interference, through which two different proteins required for autophagy were depleted. Noticeably, the compounds utilized had no marked effect on virus entry or cell viability, either of which might limit viral replication. Furthermore, alteration of cellular autophagy via pharmacological reagents or RNA interference impaired viral protein accumulation. Taken together, these findings indicate that autophagy is actively involved in influenza A virus replication.

Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis

A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death–related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes—death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53—as well as Ras–Raf–mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in D. melanogaster cultured cells. During D. melanogaster oogenesis, we found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in Atg1 and Atg7 resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. Our study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo.

All-Trans-Retinoic Acid (ATRA) Induces Autophagy in Acute Promyelocytic Leukemia Cells.

All-trans-retinoic acid (ATRA), a naturally occurring derivative of vitamin A (retinol), is a potent inducer of cellular differentiation, growth arrest, and apoptosis in various tumor cell lines. ATRA induces terminal differentiation of immature leukemic promyelocytes into normal mature granulocytes in vitro and in vivo, leading to a complete remission in majority of acute promyelocytic leukemia (APL) patients. Thus, this system provides an excellent in vitro model for studying the molecular events taking place during the terminal differentiation of myeloid cells. Apoptosis (type I) and autophagy (type II) are both highly regulated forms of programmed cell death and play crucial roles in such physiological processes as the development, homeostasis and elimination of unwanted cells. In contrast to apoptosis, autophagic cell death is caspase-independent and does not involve classic DNA laddering. Recent studies suggest that cancer cells in general tend to undergo less autophagy than their normal counterparts, supporting the contention that defective autophagic cell death plays a role in the process of carcinogenesis. The present study provides the first evidence that ATRA induces autophagy in acute promyelocytic leukemia cells. We demonstrated that treatment of APL (NB4) cells with ATRA resulted in a marked increase in formation of the acidic vesicular organelles demonstrated by acridine orange staining detected by flow cytometry. ATRA treatment caused about 12-fold increase in number of acridine orange positive cells compared to untreated group. Furthermore, ATRA induced marked upregulation of microtubule-associated protein light chain 3 (LC3-II), an important hallmark of autophagy, by western blot analysis. To determine the mechanim by which ATRA induces autophagy we examined expression of Beclin-1, an autophagy-inducing protein, and Bcl-2, an inhibitor of Beclin-1 as well as levels of mTOR, a suppressor of autophagy, upon ATRA treatment. ATRA inhibited Bcl-2, upregulated Beclin-1 expression and reduced induction of activation/phosphorylation of mTOR in NB4 cells. However, rapamycin, a specific inhibitor of mTOR failed to induce autophagy, suggesting that ATRA-mediated autophagy was independent of the mTOR pathway. We are currently testing whether inhibition of Beclin-1 can prevent ATRA-induced autophagy in APL cells. In conclusion, our results revealed that ATRA plays a role in regulation of autophagy by reducing Bcl-2 and inducing protein Beclin-1, which in turn induces autophagy, suggesting a novel action and mechanism of ATRA in regulation of autophagy in APL cells.