Loss of Pten,a tumor suppressor,causes the strong inhibition of autophagy without affecting LC3 lipidation

1Pten (phosphatase and tensin homolog deleted on chromosome ten), a tumor suppressor, is a phosphatase with a variety of substrate specificities. Its function as a negative regulator of the class I phosphatidyl-inositol 3-kinase/Akt pathway antagonizes insulin-dependent cell signaling. The targeted deletion of Pten in mouse liver leads to insulin hypersensitivity and the upregulation of the phosphatidyl-inositol 3-kinase/Akt signaling pathway. In this study, we investigated the effects of Pten deficiency on autophagy, a major cellular degradative system responsible for the turnover of cell constituents. The autophagic degradation of [14C]-leucine-labeled proteins of hepatocytes isolated from Pten-deficient livers was strongly inhibited, compared with that of control hepatocytes. However, no significant difference was found in the levels of the Atg12-Atg5 conjugate and LC3-II, the lipidated form of LC3, an intrinsic autophagosomal membrane marker, between control and Pten-deficient livers. Electron microsopic analyses showed that numerous autophagic vacuoles (autophagosomes plus autolysosomes) were present in the livers of control mice that had been starved for 48 hours, whereas they were markedly reduced in Pten-deficient livers under the same conditions. In vivo administration of leupeptin to control livers caused the inhibition of autophagic proteolysis, resulting in the accumulation of autolysosomes. These autolysosomes could be separated as a denser autolysosomal fraction from other cell membranes by Percoll density gradient centrifugation. In leupeptin-administered mutant livers, however, the accumulation of denser autolysosomes was reduced substantially. Collectively, we conclude that enhanced insulin signaling in Pten deficiency suppresses autophagy at the formation and maturation steps of autophagosomes, without inhibiting ATG conjugation reactions.     

Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy

During starvation-induced autophagy in mammals, autophagosomes form and fuse with lysosomes, leading to the degradation of the intra-autophagosomal contents by lysosomal proteases. During the formation of autophagosomes, LC3 is lipidated, and this LC3-phospholipid conjugate (LC3-II) is localized on autophagosomes and autolysosomes. While intra-autophagosomal LC3-II may be degraded by lysosomal hydrolases, recent studies have regarded LC3-II accumulation as marker of autophagy. The effect of lysosomal turnover of endogenous LC3-II in this process, however, has not been considered. We therefore investigated the lysosomal turnover of endogenous LC3-II during starvation-induced autophagy using E64d and pepstatin A, which inhibit lysosomal proteases, including cathepsins B, D and L. We found that endogenous LC3-II significantly accumulated in the presence of E64d and pepstatin A under starvation conditions, increasing about 3.5 fold in HEK293 cells and about 6.7 fold in HeLa cells compared with that in their absence, whereas the amount of LC3-II in their absence is cell-line dependent. Morphological analyses indicated that endogenous LC3-positive puncta and autolysosomes increased in HeLa cells under starvation conditions in the presence of these inhibitors. These results indicate that endogenous LC3-II is considerably degraded by lysosomal hydrolases after formation of autolysosomes, and suggest that lysosomal turnover, not a transient amount, of this protein reflects starvation-induced autophagic activity.     

Lysosomal Turnover,but Not a Cellular Level,of Endogenous LC3 is a Marker for Autophagy

During starvation-induced autophagy in mammals, autophagosomes form and fuse with lysosomes, leading to the degradation of the intra-autophagosomal contents by lysosomal proteases. During the formation of autophagosomes, LC3 is lipidated, and this LC3-phospholipid conjugate (LC3-II) is localized on autophagosomes and autolysosomes. While intra-autophagosomal LC3-II may be degraded by lysosomal hydrolases, recent studies have regarded LC3-II accumulation as marker of autophagy. The effect of lysosomal turnover of endogenous LC3-II in this process, however, has not been considered. We therefore investigated the lysosomal turnover of endogenous LC3-II during starvation-induced autophagy using E64d and pepstatin A, which inhibit lysosomal proteases, including cathepsins B, D, and L. We found that endogenous LC3-II significantly accumulated in the presence of E64d and pepstatin A under starvation conditions, increasing about 3.5 fold in HEK293 cells and about 6.7 fold in HeLa cells compared with that in their absence, whereas the amount of LC3-II in their absence is cell-line dependent. Morphological analyses indicated that endogenous LC3-positive puncta and autolysosomes increased in HeLa cells under starvation conditions in the presence of these inhibitors. These results indicate that endogenous LC3-II is considerably degraded by lysosomal hydrolases after formation of autolysosomes, and suggest that lysosomal turnover, not a transient amount, of this protein reflects starvation-induced autophagic activity.     

Phosphatase and tensin homolog regulation of islet growth and glucose homeostasis

The Irs2 branch of the insulin/insulin-like growth factor signaling cascade activates the phosphatidylinositol 3-kinase --> Akt --> Foxo1 cascade in many tissues, including hepatocytes and pancreatic beta-cells. The 3'-lipid phosphatase Pten ordinarily attenuates this cascade; however, its influence on beta-cell growth or function is unknown. To determine whether decreased Pten expression could restore beta-cell function and prevent diabetes in Irs2(-/-) mice, we generated wild type or Irs2 knock-out mice that were haploinsufficient for Pten (Irs2(-/-)::Pten(+/-)). Irs2(-/-) mice develop diabetes by 3 months of age as beta-cell mass declined progressively until insulin production was lost. Pten insufficiency increased peripheral insulin sensitivity in wild type and Irs2(-/-) mice and increased Akt and Foxo1 phosphorylation in the islets. Glucose tolerance improved in the Pten(+/-) mice, although beta-cell mass and circulating insulin levels decreased. Compared with Irs2(-/-) mice, the Irs2(-/-)::Pten(+/-) mice displayed nearly normal glucose tolerance and survived without diabetes, because normal but small islets produced sufficient insulin until the mice died of lymphoproliferative disease at 12 months age. Thus, steps to enhance phosphatidylinositol 3-kinase signaling can promote beta-cell growth, function, and survival without the Irs2 branch of the insulin/insulin-like growth factor signaling cascade.     

Cadmium results in accumulation of autophagosomes-dependent apoptosis through activating Akt-impaired autophagic flux in neuronal cells

Environmental exposure to cadmium (Cd) links to neurodegenerative disorders. Autophagy plays an important role in controlling cell survival/death. However, how autophagy contributes to Cd's neurotoxicity remains enigmatic. Here, we show that Cd induced significant increases in autophagosomes with a concomitant elevation of LC3-II and p62 in PC12 cells and primary neurons. Using autophagy inhibitor 3-MA, we demonstrated that Cd-increased autophagosomes contributed to neuronal apoptosis. Impairment of Cd on autophagic flux was evidenced by co-localization of mCherry and GFP tandem-tagged LC3 puncta in the cells. This is further supported by the findings that administration of chloroquine (CQ) potentiated the basic and Cd-elevated LC3-II and p62 levels, autophagosome accumulation and cell apoptosis, whereas rapamycin relieved the effects in the cells in response to Cd. Subsequently, we noticed that Cd evoked the phosphorylation of Akt and BECN1. Silencing BECN1 and especially expression of mutant BECN1 (Ser295A) attenuated Cd-increased autophagosomes and cell death. Of note, inhibition of Akt with Akt inhibitor X, or ectopic expression of dominant negative Akt (dn-Akt), in the presence or absence of 3-MA, significantly alleviated Cd-triggered phosphorylation of Akt and BECN1, autophagosomes, and apoptosis. Importantly, we found that Cd activation of Akt functioned in impairing autophagic flux. Collectively, these results indicate that Cd results in accumulation of autophagosomes-dependent apoptosis through activating Akt-impaired autophagic flux in neuronal cells. Our findings underscore that inhibition of Akt to improve autophagic flux is a promising strategy against Cd-induced neurotoxicity and neurodegeneration.     

Alkaline stress-induced autophagy is mediated by mTORC1 inactivation

The activation of autophagic pathway by alkaline stress was investigated. Various types of mammalian cells were subjected to alkaline stress by incubation in bicarbonate buffered media in humidified air containing atmospheric 0.04% CO(2) . The induction of autophagy following alkaline stress was evaluated by assessing the conversion of cytosolic LC3-I into lipidated LC3-II, the accumulation of autophagosomes, and the formation of autolysosomes. Colocalization of GFP-LC3 with endolysosomal marker in HeLa GFP-LC3 cells undergoing autophagic process by alkaline stress further demonstrates that autophagosomes triggered by alkaline stress matures into autolysosomes for the lysosome dependent degradation. We found that the inactivation of mTORC1 is important for the pathway leading to the induction of autophagy by alkaline stress since the expression of RhebQ64L, a constitutive activator of mTORC1, downregulates the induction of autophagy after alkaline stress in transfected human 293T cells. These results imply that activation of autophagic pathway following the inactivation of mTORC1 is important cellular events governing alkaline stress-induced cytotoxicity and clinical symptoms associated with alkalosis.     

Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells

Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP⁺RFP⁺ puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 μM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.     

Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.     

Muscle-specific Pten deletion protects against insulin resistance and diabetes

Pten (phosphatase with tensin homology), a dual-specificity phosphatase, is a negative regulator of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Pten regulates a vast array of biological functions including growth, metabolism, and longevity. Although the PI3K/Akt pathway is a key determinant of the insulin-dependent increase in glucose uptake into muscle and adipose cells, the contribution of this pathway in muscle to whole-body glucose homeostasis is unclear. Here we show that muscle-specific deletion of Pten protected mice from insulin resistance and diabetes caused by high-fat feeding. Deletion of muscle Pten resulted in enhanced insulin-stimulated 2-deoxyglucose uptake and Akt phosphorylation in soleus but, surprisingly, not in extensor digitorum longus muscle compared to littermate controls upon high-fat feeding, and these mice were spared from developing hyperinsulinemia and islet hyperplasia. Muscle Pten may be a potential target for treatment or prevention of insulin resistance and diabetes.     

The reciprocal interaction between autophagic dysfunction and ER stress in adipose insulin resistance

Autophagy, a predominantly cytoprotective process, is an important regulator in diabetic metabolism and endoplasmic reticulum (ER) stress responses. However, the interaction and biological significance between autophagic imbalance and ER stress involved in insulin resistance remain not fully elucidated. In the present study, when compared with normal glucose tolerance (NGT) subjects, enhanced ER stress and pronounced protein and mRNA levels of the autophagic genes such as Atg7, LC3A, and LC3B were evident in adipose tissue of patients with type 2 diabetes. An increased number of autophagosomes and elevated autophagy flux in adipose explants incubated with lysomoal inhibitor were also observed in type 2 diabetes. In addition, adipocytes differentiation was significantly repressed by exogenous ER stress and defective autophagy in vitro. Tunicamycin-induced ER stress in adipocytes can trigger autophagic response and insulin insensitivity that was partially attributed to the upregulation of IRE1-JNK pathway, whereas autophagy deficiency resulted in ER stress and impaired insulin signaling, further supporting the crucial roles of autophagy in ER stress and insulin resistance. Moreover, disturbance of autophagy and insulin sensitivity induced by tunicamycin can be effectively corrected by the addition of osteocalcin in an NFκB-dependent manner in vitro. In conclusion, our results demonstrated a reciprocal functional interaction among autophagy, ER stress, and insulin signaling in adipose tissue of type 2 diabetes and adipocytes, supporting an adaptive role of autophagy-dependent mechanism in response to ER stress-induced insulin resistance in type 2 diabetes.     

Autophagic degradation of nuclear components in mammalian cells

Autophagy is an evolutionally conserved intracellular mechanism for the degradation of organelles and proteins. Here we demonstrate the presence of perinuclear autophagosomes/autolysosomes containing nuclear components in nuclear envelopathies caused by mutations in the genes encoding A-type lamins (LMNA) and emerin (EMD). These autophagosomes/autolysosomes were sometimes bigger than nucleus. The autophagic nature is further supported by up-regulation of LC3-II in Lmna^H222P/H222P fibroblasts. In addition, inhibition of autophagy led to the accumulation of nuclear abnormalities and reduced cell viability, highly suggesting a beneficial role of autophagy, at least in these cells. Similar giant autophagosomes/autolysosomes were seen even in wild-type cells, albeit rarely, implying that this “nucleophagy” is not confined to the diseased condition, but may be seen even in physiologic conditions to clean up nuclear wastes produced by nuclear damage.     

Enhanced insulin sensitivity, energy expenditure and thermogenesis in adipose-specific Pten suppression in mice

Pten is an important phosphatase, suppressing the phosphatidylinositol-3 kinase/Akt pathway. Here, we generated adipose-specific Pten-deficient (AdipoPten-KO) mice, using newly generated Acdc promoter-driven Cre transgenic mice. AdipoPten-KO mice showed lower body and adipose tissue weights despite hyperphagia and enhanced insulin sensitivity with induced phosphorylation of Akt in adipose tissue. AdipoPten-KO mice also showed marked hyperthermia and increased energy expenditure with induced mitochondriagenesis in adipose tissue, associated with marked reduction of p53, inactivation of Rb, phosphorylation of cyclic AMP response element binding protein (CREB) and increased expression of Ppargc1a, the gene that encodes peroxisome proliferative activated receptor gamma coactivator 1 alpha. Physiologically, adipose Pten mRNA decreased with exposure to cold and increased with obesity, which were linked to the mRNA alterations of mitochondriagenesis. Our results suggest that altered expression of adipose Pten could regulate insulin sensitivity and energy expenditure. Suppression of adipose Pten may become a beneficial strategy to treat type 2 diabetes and obesity.     

Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases

Pten (Phosphatase and tensin homolog deleted on chromosome 10) is a recently identified tumor suppressor gene which is deleted or mutated in a variety of primary human cancers and in three cancer predisposition syndromes [1]. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P(3)), a product of PI 3-kinase [2-5]. Pten has also been implicated in controlling cell migration [6], but the exact mechanism is not very clear. Using the isogenic Pten(+/+) and Pten(-/-) mouse fibroblast lines, here we show that Pten deficiency led to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduced the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125(FAK) was not changed in Pten(-/-) cells. Instead, significant increases in the endogenous activities of Rac1 and Cdc42, two small GTPases involved in regulating the actin cytoskeleton [7], were observed in Pten(-/-) cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reversed the cell migration phenotype of Pten(-/-) cells. Thus, our studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42.     

Autophagy inhibition enhances ursolic acid-induced apoptosis in PC3 cells

The phosphoinositol 3-kinase/Akt pathway plays a critical role in oncogenesis and the dysregulation of this pathway through loss of PTEN is a particularly common phenomenon in aggressive prostate cancers. Several recent studies have indicated that ursolic acid (UA), a pentacyclic triterpenoid, and its derivatives inhibit the growth of cancer cells by cell cycle arrest and the stimulation of apoptosis. In the present study, we report a novel autophagic response of UA in PTEN-deficient PC3 prostate cancer cells. As one of the major types of programmed cell death, autophagy has been observed in response to several anticancer drugs and demonstrated to be responsible for cell death. UA-induced autophagy in PC3 cells is associated with the reduced cell viability and the enhanced expression of LC3-II, an autophagosome marker in mammals, and monodansylcadaverine incorporation into autolysosomes. Furthermore, we found that UA exhibited anti-proliferative effects characterized by G1 phase arrest and autophagy at an early stage that precedes apoptosis. We also show that UA-induced autophagy in PC3 cells are mediated through the Beclin-1 and Akt/mTOR pathways. Inhibition of autophagy by either 3-methyladenine or Beclin-1/Atg5 small interfering RNA enhanced UA-induced apoptosis. Taken together, our data suggest that autophagy functions as a survival mechanism in PC3 cells against UA-induced apoptosis and a rational for the use of autophagy inhibitors in combination with UA as a novel modality of cancer therapy.     

3-methyladenine inhibits autophagy in tobacco culture cells under sucrose starvation conditions

Tobacco (Nicotiana tabacum) culture cells perform autophagy and degrade cellular proteins in response to sucrose starvation. When protein degradation is blocked by the cysteine protease inhibitor E-64c, lysosomes containing particles of cytoplasm (autolysosomes) accumulate in the cells. Therefore, using light microscopy, we can determine whether cells have performed autophagy. In this study, we investigated whether or not 3-methyladenine (3-MA), which is a known inhibitor of autophagy in mammalian cells, blocks autophagy in tobacco culture cells. The accumulation of autolysosomes was blocked by the addition to the culture media of 5 mM 3-MA together with E-64c. We did not detect autolysosomes or structures thought to be involved with autophagy, such as autophagosomes, accumulating in these cells, as observed by electron microscopy. 3-MA blocked cellular protein degradation without any effect on cellular protease activity. In mammalian cells, phosphatidylinositol 3-kinase (PtdIns 3-kinase) is a putative target of 3-MA. The PtdIns 3-kinase inhibitors wortmannin and LY294002 also inhibited the accumulation of autolysosomes in tobacco culture cells. These results suggest that (1) 3-MA inhibits autophagy by blocking the formation of autophagosomes in tobacco culture cells, and (2) PtdIns 3-kinase is essential for autophagy in tobacco cells.     

Elevated autophagic sequestration of mitochondria and lipid droplets in steatotic hepatocytes of chronic ethanol-treated rats: an immunohistochemical and electron microscopic study

Ethanol-induced hepatic steatosis may induce the progression of alcoholic liver disease. The involvement of autophagic clearance of damaged mitochondria (mitophagy) and lipid droplets (LDs) (lipophagy) in chronic ethanol-induced hepatic steatosis is not clearly understood. Adult Wistar rats were fed either 5 % ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 10 weeks. Light microscopy showed marked steatosis in hepatocytes of ethanol-treated rats (ETRs), which was further revealed by transmission electron microscopy (TEM), where significant numbers of large LDs and damaged mitochondria were detected in steatotic hepatocytes. Moreover, TEM demonstrated that hepatocyte steatosis was associated with greatly enhanced autophagic vacuole (AV) formation compared to control hepatocytes. Mitochondria and LDs were the predominant contents of AVs in steatotic hepatocytes. Immunohistochemistry of LC3, a specific marker of early AVs (autophagosomes), demonstrated an extensive punctate pattern in hepatocytes of ETRs, while LC3 puncta were much less frequent in control hepatocytes. This was confirmed by immunoelectron microscopy (IEM), which showed localization of LC3 to autophagosomes sequestering damaged mitochondria and LDs. In addition, IEM revealed that PINK1 (a sensor of mitochondrial damage and marker of mitophagy) was overexpressed in mitochondria of ETRs. Enhanced autophagic lysosomal activity was evidenced by increased immunolabeling of LAMP-2, a marker of late AVs (autolysosomes) in hepatocytes of ETRs and colocalization of LC3 and lysosomal cathepsins using double immunofluorescence labeling. Increased AVs in hepatocytes of ETRs reflect ethanol toxicity and could represent a possible protective mechanism via stimulation of mitophagy and lipophagy.     

An autophagic mechanism is involved in apoptotic death of rat striatal neurons induced by the non-N-methyl-D-aspartate receptor agonist kainic acid

Previous studies found that kainic acid (KA)-induced apoptosis involved the lysosomal enzyme cathepsin B, suggesting a possible mechanism of autophagy in excitotoxicity. The present study was sought to investigate activation and contribution of autophagy to excitotoxic neuronal injury mediated by KA receptors. The formation of autophagosomes was observed with transmission electron microscope after excitotoxin exposure. The contribution of autophagic mechanisms to KA-induced upregulation of microtubule-associated protein 1A/1B light chain 3 (LC3), lysosome- associated membrane protein 2 (LAMP2) and cathepsin B, release of cytochrome c, activation of caspase-3, down-regulation of Bcl-2, upregulation of Bax, p53, puma and apoptotic death of striatal neurons were assessed with co-administration of the autophagy inhibitor 3-methyladenine (3-MA). These studies showed that KA brought about an increase in the formation of autophagosomes and autolysosomes in the cytoplasm of striatal cells. KA-induced increases in the ratio of LC3-II/LC3-I, LAMP2, cathepsin B, release of cytochrome c and activation of caspase-3 were blocked by pre-treatment with 3-MA. 3-MA also reversed KA-induced down-regulation of Bcl-2 and upregulation of Bax protein levels, LC3, p53 and puma mRNA levels in the striatum. KA-induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by 3-MA. These results suggest that over-stimulation of KA receptors can activate autophagy. The autophagic mechanism participates in programmed cell death through regulating the mitochondria-mediated apoptotic pathway.