Lysophosphatidic acid receptor LPA3 prevents oxidative stress and cellular senescence in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare laminopathy that produces a mutant form of prelamin A, known as Progerin, resulting in premature aging. HGPS cells show morphological abnormalities of the nuclear membrane, reduced cell proliferation rates, accumulation of reactive oxygen species (ROS), and expression of senescence markers. Lysophosphatidic acid (LPA) is a growth factor-like lipid mediator that regulates various physiological functions via activating multiple LPA G protein-coupled receptors. Here, we study the roles of LPA and LPA receptors in premature aging. We report that the protein level of LPA3 was highly downregulated through internalization and the lysosomal degradation pathway in Progerin-transfected HEK293 cells. By treating Progerin HEK293 cells with an LPA3 agonist (OMPT, 1-Oleoyl-2-O-methyl-rac-glycerophosphothionate) and performing shRNA knockdown of the Lpa3r transcript in these cells, we showed that LPA3 activation increased expression levels of antioxidant enzymes, consequently inhibiting ROS accumulation and ameliorating cell senescence. LPA3 was shown to be downregulated in HGPS patient fibroblasts through the lysosomal pathway, and it was shown to be crucial for ameliorating ROS accumulation and cell senescence in fibroblasts. Moreover, in a zebrafish model, LPA3 deficiency was sufficient to cause premature aging phenotypes in multiple organs, as well as a shorter lifespan. Taken together, these findings identify the decline of LPA3 as a key contributor to the premature aging phenotypes of HGPS cells and zebrafish.

Human non­small cell lung cancer cells can be sensitized to camptothecin by modulating autophagy.

Lung cancer is a prevalent disease and is one of the leading causes of mortality worldwide. Despite the development of various anticancer drugs, the prognosis of lung cancer is relatively poor. Metastasis of lung cancer, as well as chemoresistance, is associated with a high mortality rate for patients with lung cancer. Camptothecin (CPT) is a well-known anticancer drug, which causes cancer cell apoptosis via the induction of DNA damage; however, the cytotoxicity of CPT easily reaches a plateau at a relatively high dose in lung cancer cells, thus limiting its efficacy. The present study demonstrated that CPT may induce autophagy in two human non­small cell lung cancer cell lines, H1299 and H460. In addition, the results of a viability assay and Annexin V staining revealed that CPT-induced autophagy could protect lung cancer cells from programmed cell death. Conversely, the cytotoxicity of CPT was increased when autophagy was blocked by 3-methyladenine treatment. Furthermore, inhibition of autophagy enhanced the levels of CPT-induced DNA damage in the lung cancer cell lines. Accordingly, these findings suggested that autophagy exerts a protective role in CPT-treated lung cancer cells, and the combination of CPT with a specific inhibitor of autophagy may be considered a promising strategy for the future treatment of lung cancer.

Grb7 Protein Stability Modulated by Pin1 in Association with Cell Cycle Progression.

Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein that is co-opted by numerous tyrosine kinases involved in various cellular signaling and functions. The molecular mechanisms underlying the regulation of Grb7 remain unclear. Here, we revealed a novel negative post-translational regulation of Grb7 by the peptidyl-prolyl cis/trans isomerase, Pin1. Our data show that phosphorylation of Grb7 protein on the Ser194-Pro motif by c-Jun N-terminal kinase facilitates its binding with the WW domain of Pin1. Subsequently, Grb7 is degraded by the ubiquitin- and proteasome-dependent proteolytic pathway. Indeed, we found that Pin1 exerts its peptidyl-prolyl cis/trans isomerase activity in the modulation of Grb7 protein stability in regulation of cell cycle progression at the G2-M phase. This study illustrates a novel regulatory mechanism in modulating Grb7-mediated signaling, which may take part in pathophysiological consequences.

Armillaridin induces autophagy-associated cell death in human chronic myelogenous leukemia K562 cells.

Armillaridin (AM) is an aromatic ester compound isolated from Armillaria mellea. Treatment with AM markedly reduced the viability of human chronic myelogenous leukemia K562, chronic erythroleukemia HEL 92.1.7, and acute monoblastic leukemia U937 cells, but not normal human monocytes, in a dose- and time-dependent manner. Treatment of K562 cells with AM caused changes characteristic of autophagy. Only a small amount of AM-treated K562 cells exhibited apoptosis. By contrast, AM treatment resulted in extensive apoptotic features in U937 and HEL 92.1.7 cells without evident autophagy. The autophagy of K562 cells induced by AM involved autophagic flux, including autophagosome induction, the processing of autophagosome-lysosome fusion and downregulation of BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3). By bcr-abl knockdown, the growth inhibition of K562 cells caused by AM was partially blocked, suggesting that AM-induced cell death might be a bcr-abl-dependent mode of autophagy-associated cell death. In conclusion, AM is capable of inhibiting growth and inducing autophagy-associated cell death in K562 cells, but not in normal monocytes. It may have potential to be developed as a novel therapeutic agent against leukemia.

Cardioprotective Actions of TGFßRI Inhibition Through Stimulating Autocrine/Paracrine of Survivin and Inhibiting Wnt in Cardiac Progenitors.

Heart failure due to myocardial infarction (MI) is a major cause of morbidity and mortality in the world. We found previously that A83-01, a TGFßRI inhibitor, could facilitate cardiac repair in post-MI mice and induce the expansion of a Nkx2.5 + cardiomyoblast population. This study aimed to investigate the key autocrine/paracrine factors regulated by A83-01 in the injured heart and the mechanism of cardioprotection by this molecule. Using a previously described transgenic Nkx2.5 enhancer-green fluorescent protein (GFP) reporter mice, we isolated cardiac progenitor cells (CPC) including Nkx2.5-GFP + (Nkx2.5+), sca1+, and Nkx2.5+/sca1 + cells. A83-01 was found to induce proliferation of these three subpopulations mainly through increasing Birc5 expression in the MEK/ERK-dependent pathway. Survivin, encoded by Birc5, could also directly proliferate Nkx2.5 + cells and enhance cultured cardiomyocytes viability. A83-01 could also reverse the downregulation of Birc5 in postinjured mice hearts (n = 6) to expand CPCs. Moreover, the increased Wnt3a in postinjured hearts could decrease CPCs, which could be reversed by A83-01 via inhibiting Fzd6 and Wnt1-induced signaling protein 1 expressions in CPCs. Next, we used inducible αMHC-cre/mTmG mice to label cardiomyocytes with GFP and nonmyocytes with RFP. We found A83-01 preserved more GFP + myocytes (68.6% ± 3.1% vs. 80.9% ± 3.0%; p < .05, n = 6) and fewer renewed RFP + myocytes (0.026% ± 0.005% vs. 0.062% ± 0.008%; p < .05, n = 6) in parallel with less cardiac fibrosis in isoprenaline-injected mice treated with A83-01. TGFßRI inhibition in an injured adult heart could both stimulate the autocrine/paracrine activity of survivin and inhibit Wnt in CPCs to mediate cardioprotection and improve cardiac function.

Increased Autophagy Markers Are Associated with Ductular Reaction during the Development of Cirrhosis.

Autophagy is a regulatory pathway in liver fibrosis. We investigated the roles of autophagy in human cirrhotic livers. Cirrhotic and noncirrhotic liver tissues were obtained from patients with hepatocellular carcinoma, and liver tissues from live donors served as control. Patients with cirrhotic livers had significantly increased levels of various essential autophagy-related genes compared with noncirrhotic livers. In addition, colocalization of autophagy marker microtubule-associated protein 1 light chain 3B (LC3B) with lysosome-associated membrane protein-1, increased levels of lysosome-associated membrane protein-2, and increased maturation of lysosomal cathepsin D were observed in cirrhotic livers. By using dual-immunofluorescence staining, we demonstrated that increased LC3B was located mainly in the cytokeratin 19-labeled ductular reaction (DR) in human cirrhotic livers and in an experimental cirrhosis induced by 2-acetylaminofluorene (AAF) with carbon tetrachloride (CCl4), indicating a conserved response to chronic liver damage. Furthermore, an AAF/CCl4-mediated increase in DR and fibrosis were attenuated after chloroquine treatment, suggesting that the autophagy-lysosome pathway was essential for AAF/CCl4-induced DR-fibrosis. In conclusion, we demonstrated that increased autophagy marker positively correlated with DR during the development of cirrhosis. Therefore, targeting autophagy may hold therapeutic value for liver cirrhosis.

LC3 overexpression reduces Aß neurotoxicity through increasing α7nAchR expression and autophagic activity in neurons and mice.

Autophagy is an intracellular degradation pathway with dynamic interactions for eliminating damaged organelles and protein aggregates by lysosomal digestion. The EGFP-conjugated microtubule-associated protein 1 light chain 3 (EGFP-LC3) serves to monitor autophagic process. Extracellular ß-amyloid peptide accumulation is reported as a major cause in Alzheimer's disease (AD) pathogenesis; large numbers of autophagic vacuoles accumulate in patients' brains. We previously demonstrated that extracellular Aß (eAß) induces strong autophagic response and α7nAChR acts as a carrier to bind with eAß; which further inhibits Aß-induced neurotoxicity via autophagic degradation. In the present study, we overexpressed LC3 in both neuroblastoma cells (SH-SY5Y/pEGFP-LC3) and mice (TgEGFP-LC3) to assess the effect of LC3 overexpression on Aß neurotoxicity. SH-SY5Y/pEGFP-LC3 cells and primary cortical neuron cultures derived from E17 (embryonic day 17) TgEGFP-LC3 mice showed not only better resistance against Aß neurotoxicity but also higher α7nAChR expression and autophagic activity than control. Administration of α-bungarotoxin (α-BTX) to block α7nAChR antagonized the neuroprotective action of SH-SY5Y/pECGF-LC3 cells, suggesting that eAß binding with α7nAChR is an important step in Aß detoxification. LC3 overexpression thus exerts neuroprotection through increasing α7nAChR expression for eAß binding and further enhancing autophagic activity for Aß clearance in vitro and in vivo.

Assays for autophagy I: the Cvt pathway and nonselective autophagy.

Autophagy is a major intracellular degradation pathway. It is responsible for the bulk removal of obsolete or damaged cytoplasmic components, including both soluble proteins and membrane-bound organelles. As a fundamental function of eukaryotic cells, autophagy plays a key role in protecting the cells from stressful conditions. Budding yeast Saccharomyces cerevisiae has been the pioneering model system in autophagy-related research. In this chapter, we describe three basic assays of autophagy in S. cerevisiae: the Ape1 maturation assay, the GFP-Atg8 processing assay, and the Pho8Δ60 assay. These assays cover the selective cytoplasm to vacuole targeting (Cvt) pathway and starvation-induced nonselective autophagy.

Construction and application of a protein interaction map for white spot syndrome virus (WSSV).

White spot syndrome virus (WSSV) is currently the most serious global threat for cultured shrimp production. Although its large, double-stranded DNA genome has been completely characterized, most putative protein functions remain obscure. To provide more informative knowledge about this virus, a proteomic-scale network of WSSV-WSSV protein interactions was carried out using a comprehensive yeast two-hybrid analysis. An array of yeast transformants containing each WSSV open reading frame fused with GAL4 DNA binding domain and GAL4 activation domain was constructed yielding 187 bait and 182 prey constructs, respectively. On screening of ∼28,000 pairwise combinations, 710 interactions were obtained from 143 baits. An independent coimmunoprecipitation assay (co-IP) was performed to validate the selected protein interaction pairs identified from the yeast two-hybrid approach. The program Cytoscape was employed to create a WSSV protein-protein interaction (PPI) network. The topology of the WSSV PPI network was based on the Barabási-Albert model and consisted of a scale-free network that resembled other established viral protein interaction networks. Using the RNA interference approach, knocking down either of two candidate hub proteins gave shrimp more protection against WSSV than knocking down a nonhub gene. The WSSV protein interaction map established in this study provides novel guidance for further studies on shrimp viral pathogenesis, host-viral protein interaction and potential targets for therapeutic and preventative antiviral strategies in shrimp aquaculture.

Presenilin-1 regulates the expression of p62 to govern p62-dependent tau degradation.

Mutations in presenilin-1 (PS1) are tightly associated with early-onset familial Alzheimer's disease (FAD), which is characterized by extracellular amyloid plaques and the accumulation of intracellular Tau. In addition to being the catalytic subunit of γ-secretase, PS1 has been shown to regulate diverse cellular functions independent of its proteolytic activity. We found that cells deficient in PS1 exhibit reduced levels of p62 protein, a cargo-receptor shuttling Tau for degradation. The downregulation of PS1 led to a significant decrease in both the protein and mRNA transcript of p62, concomitant with attenuated p62 promoter activity. This PS1-dependent regulation of p62 expression was mediated through an Akt/AP-1 pathway independent of the proteolytic activity of PS1/γ-secretase. This p62-mediated Tau degradation was significantly impaired in PS1-deficient cells, which can be rescued by ectopic expression of either p62 or wild-type PS1 but not mutant PS1 containing FAD-linked mutations. Our study suggests a novel function for PS1 in modulating p62 expression to control the proteostasis of Tau.

Extrinsic sphingosine 1-phosphate activates S1P5 and induces autophagy through generating endoplasmic reticulum stress in human prostate cancer PC-3 cells.

Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid that binds to a family of G protein-coupled receptors (GPCRs), termed S1P1-S1P5. Our previous study has reported that S1P induces autophagy in human prostate cancer PC-3 cell. In addition, S1P-induced autophagy plays a prosurvival role in PC-3 cells. Accumulating evidence has shown that the autophagy responses triggered by ER stress signaling have cytoprotective effects. Thus, we attempted to investigate whether S1P-induced autophagy is a result of triggering ER stress in PC-3 cells. By monitoring XBP-1 mRNA splicing, a characteristic of ER stress, we demonstrate that S1P triggers ER stress in a concentration-dependent and time-dependent manner. Moreover, DiH S1P, a membrane-nonpermeable S1P analog without intracellular effects also enhances ER stress. Meanwhile, we also show that S1P5 is required for S1P-induced ER stress by using RNA interference experiments. Furthermore, signaling analyses revealed that PI3K, PLC, and ROS production were involved in S1P's effects on ER stress induction. On the other hand, knockdown of XBP-1 abolished S1P-induced autophagy. In summary, our results demonstrate for the first time that the extracellular S1P-triggered ER stress is responsible for autophagy induction in PC-3 cells.

Dopamine- and zinc-induced autophagosome formation facilitates PC12 cell survival.

Dopamine oxidation and divalent cations have been reported to induce neuronal cell death. Although autophagy is involved in neuronal cell death, it has also been suggested to facilitate cell survival. We sought to investigate the role of autophagy in PC12 cells and cultured neurons treated with dopamine and Zn2+. Cells expressing EGFP-LC3 were treated with high concentrations of dopamine and Zn2+, and the formation of EGFP-LC3 fluorescence aggregates was monitored. Our results showed a significant increase in the number of fluorescent puncta in the cytosol of PC12 cells treated with these chemicals. These treatments enhanced LC3 lipidation levels in PC12 cells. Decreasing the ATG7 protein level using specific small interference RNA (siRNA) and pretreating with phosphatidylinositol 3-phosphate kinase blockers, wortmannin and LY294002, inhibited puncta formation. Dopamine or Zn2+ treatment significantly elevated the intracellular Zn2+ concentration ([Zn2+] i ); however, inhibiting the [Zn2+] i elevation in dopamine-treated cells suppressed the puncta formation. LY294002 or siRNA-directed members of the autophagy pathway increased the fraction of phosphatidylserine present on the outer membrane leaflet in PC12 cells treated with dopamine or Zn2+, suggesting an increase in apoptosis. Primary embryonic midbrain neurons expressing EGFP-LC3 also displayed a significant increase in the number of fluorescent aggregates in cells upon treatment with dopamine or Zn2+. Dopamine or Zn2+ treatment significantly elevated the [Zn2+] i in neurons and caused neuronal death. Our results indicate that treating cells with dopamine and Zn2+ results in the activation of the autophagy pathway in an effort to enhance cell survival.

3C protein of feline coronavirus inhibits viral replication independently of the autophagy pathway.

Feline coronavirus (FCoV) can cause either asymptomatic enteric infection or fatal peritonitis in cats. Although the mutation of FCoV accessory gene 3c has been suggested to be related to the occurrence of feline infectious peritonitis (FIP), how the 3C protein is involved in this phenomenon remains unknown. To investigate the role of the 3C protein, a full-length 3c gene was transiently expressed and the cytoplasmic distribution of the protein was found to be primarily in the perinuclear region. Using 3c-stable expression cells, the replication of a 3c-defective FCoV strain was titrated and a significant decrease in replication (p<0.05) was observed. The mechanism underlying the decreased FIPV replication caused by the 3C protein was further investigated; neither the induction nor inhibition of autophagy rescued the viral replication. Taken together, our data suggest that the 3C protein might have a virulence-suppressing effect in FCoV-infected cats. Deletion of the 3c gene could therefore cause more efficient viral replication, which leads to a fatal infection.

Sorafenib induces autophagy in human myeloid dendritic cells and prolongs survival of skin allografts.

BACKGROUND: Sorafenib, a multikinase inhibitor approved for the treatment of advanced renal cell carcinoma and hepatocellular carcinoma, has been reported inhibitory on the function of dendritic cells. This study was aimed to determine the effects of sorafenib on inducing autophagy and immunomodulatory activity and its implication on graft rejection. METHODS: Cell viability and surface antigens were examined by 7-amino-actinomycin D and flow cytometric analysis. Autophagy was characterized using light microscopy and transmission electron microscopy for morphology, Western blotting for LC3B-I lipidation and mammalian target of rapamycin signaling molecules, and immunofluorescence staining for endogenous LC3B, GFP-LC3 transfection, and acidic component vacuoles. Skin allograft in mice was used as an experimental transplantation rejection model. Soluble factors contained in culture medium and serum were measured by enzyme-linked immunosorbent assay. RESULTS: We found that sorafenib inhibited the viability of dendritic cells accompanied by morphologic changes characteristic of autophagy and immature differentiation. This autophagic effect induced by sorafenib was validated by LC3B-I lipidation and autophagosome accumulation. Sorafenib treatment was associated with the down-regulation of phosphorylated mammalian target of rapamycin and its downstream substrate p70S6K. We next performed skin graft model to testify the role of sorafenib-induced immature and autophagic dendritic cells. Intriguingly, sorafenib prolonged the survival of skin allograft without major toxicity. Blockade of autophagic flux by chloroquine partially diminished the protective effect of sorafenib, indicating an autophagy-related mechanism in vivo. CONCLUSION: This study suggests that sorafenib, in addition to being an anticancer agent, may have potential to be developed as a new category of immunosuppressant drugs acting via autophagy induction of dendritic cells.

Receptor protein complexes are in control of autophagy.

In autophagic processes a variety of cargos is delivered to the degradative compartment of cells. Recent progress in autophagy research has provided support for the notion that when autophagic processes are operating in selective mode, a receptor protein complex will process the cargo. Here we present a concept of receptor protein complexes as comprising a functional tetrad of components: a ligand, a receptor, a scaffold and an Atg8 family protein. Our current understanding of each of the four components and their interaction in the context of cargo selection are considered in turn.

Autophagy: a double-edged sword in Alzheimer's disease.

Autophagy is a major protein degradation pathway that is essential for stress-induced and constitutive protein turnover. Accumulated evidence has demonstrated that amyloid-beta (A beta) protein can be generated in autophagic vacuoles, promoting its extracellular deposition in neuritic plaques as the pathological hallmark of Alzheimer's disease (AD). The molecular machinery for A beta generation, including APP, APP-C99 and beta-/gamma-secretases, are all enriched in autophagic vacuoles. The induction of autophagy can be vividly observed in the brain at early stages of sporadic AD and in an AD transgenic mouse model. Accumulated evidence has also demonstrated a neuroprotective role of autophagy in mediating the degradation of aggregated proteins that are causative of various neurodegenerative diseases. Autophagy is thus widely regarded as an intracellular hub for the removal of the detrimental A beta peptides and Tau aggregates. Nonetheless, compelling data also reveal an unfavorable function of autophagy in facilitating the production of intracellular A beta. The two faces of autophagy on the homeostasis of A beta place it in a very unique and intriguing position in AD pathogenesis. This article briefly summarizes seminal discoveries that are shedding new light on the critical and unique roles of autophagy in AD and potential therapeutic approaches against autophagy-elicited AD.

Co-interactive DNA-binding between a novel, immunophilin-like shrimp protein and VP15 nucleocapsid protein of white spot syndrome virus.

White spot syndrome virus (WSSV) is one of the most serious pathogens of penaeid shrimp. Although its genome has been completely characterized, the functions of most of its putative proteins are not yet known. It has been suggested that the major nucleocapsid protein VP15 is involved in packaging of the WSSV genome during virion formation. However, little is known in its relationship with shrimp host cells. Using the yeast two-hybrid approach to screen a shrimp lymphoid organ (LO) cDNA library for proteins that might interact with VP15, a protein named PmFKBP46 was identified. It had high sequence similarity to a 46 kDa-immunophilin called FKBP46 from the lepidopteran Spodoptera frugiperda (the fall armyworm). The full length PmFKBP46 consisted of a 1,257-nucleotide open reading frame with a deduced amino acid sequence of 418 residues containing a putative FKBP-PPIase domain in the C-terminal region. Results from a GST pull-down assay and histological co-localization revealed that VP15 physically interacted with PmFKBP46 and that both proteins shared the same subcellular location in the nucleus. An electrophoretic mobility shift assay indicated that PmFKBP46 possessed DNA-binding activity and functionally co-interacted with VP15 in DNA binding. The overall results suggested that host PmFKBP46 might be involved in genome packaging by viral VP15 during virion assembly.

Roles of sphingosine 1-phosphate on tumorigenesis.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid with a variety of biological activities. It is generated from the conversion of ceramide to sphingosine by ceramidase and the subsequent conversion of sphingosine to S1P, which is catalyzed by sphingosine kinases. Through increasing its intracellular levels by sphingolipid metabolism and binding to its cell surface receptors, S1P regulates several physiological and pathological processes, including cell proliferation, migration, angiogenesis and autophagy. These processes are responsible for tumor growth, metastasis and invasion and promote tumor survival. Since ceramide and S1P have distinct functions in regulating in cell fate decision, the balance between the ceramide/sphingosine/S1P rheostat becomes a potent therapeutic target for cancer cells. Herein, we summarize our current understanding of S1P signaling on tumorigenesis and its potential as a target for cancer therapy.

Nuclear translocation of epidermal growth factor receptor by Akt-dependent phosphorylation enhances breast cancer-resistant protein expression in gefitinib-resistant cells.

Epidermal growth factor receptor (EGFR), an aberrantly overexpressed or activated receptor-tyrosine kinase in many cancers, plays a pivotal role in cancer progression and has been an attractive target for cancer therapy. Gefitinib and erlotinib, two EGFR-tyrosine kinase inhibitors, have been approved for non-small cell lung cancer. However, durable clinical efficacy of these EGFR inhibitors is severely limited by the emergence of acquired resistance. For example, the expression of breast cancer-resistant protein (BCRP/ABCG2) has been shown to confer acquired resistance of wild-type EGFR (wtEGFR)-expressing cancer cells to gefitinib. However, the underlying molecular mechanisms still remain unclear. Here, we show that wtEGFR expression is elevated in the nucleus of acquired gefitinib-resistant cancer cells. Moreover, nuclear translocation of EGFR requires phosphorylation at Ser-229 by Akt. In the nucleus, EGFR then targets the proximal promoter of BCRP/ABCG2 and thereby enhances its gene transcription. The nuclear EGFR-mediated BCRP/ABCG2 expression may contribute at least in part to the acquired resistance of wtEGFR-expressing cancer cells to gefitinib. Our findings shed light on the role of nuclear EGFR in the sensitivity of wtEGFR-expressing cancer cells to EGFR tyrosine kinase inhibitors and also deciphered a putative molecular mechanism contributing to gefitinib resistance through BCRP/ABCG2 expression.

Inhibition of the mammalian target of rapamycin promotes cyclic AMP-induced differentiation of NG108-15 cells.

To clarify the involvement of autophagy in neuronal differentiation, the effect of rapamycin, an mTOR complex inhibitor, on the dibutyryl cAMP (dbcAMP)-induced differentiation of NG108-15 cells was examined. Treatment of NG108-15 cells with 1 mM dbcAMP resulted in induction of differentiation, including neurite outgrowth and varicosity formation, enhanced voltage-sensitive Ca2+ channel activity and expression of microtubule-associated protein 2, and these effects involved phosphorylation of cAMP-response element binding protein (CREB) and extracellular signal regulated kinase (ERK). Simultaneous application of dbcAMP and rapamycin synergistically increased and accelerated differentiation. mTOR or raptor silencing with siRNA had a similar effect to rapamycin. Rapamycin and silencing of mTOR or raptor evoked autophagy, while blockade of autophagy by addition of 3-methyladenine or beclin 1 or Atg5 silencing prevented the potentiation of differentiation. Silencing of rictor also evokes autophagy, at a level 55% of that induced by raptor silencing and enhancement of differentiation is proportional. Rapamycin also caused increased ATP generation and cell cycle arrest in G0/G1 phase, but had no effect on CREB and ERK phosphorylation. dbcAMP also induced ATP generation, but not autophagy or cell cycle arrest. These results suggest that the increased autophagy, ATP generation and cell cycle arrest caused by mTOR inhibition promotes the dbcAMP-induced differentiation of NG108-15 cells.