Molecular Mechanisms Of Autophagy Research Articles

Identification of Key Genes and Potential Mechanisms Based on the Autophagy Regulatory Network in Osteoclasts Using a Murine Osteoarthritis Model.

BackgroundOsteoarthritis (OA) is a degenerative joint disease that acts as a major cause of early disability in the old population. However, the molecular mechanisms of autophagy in osteoclasts involved in OA remain unclear.MethodsThe gene expression profiles were downloaded from the Gene Expression Omnibus (GEO) repository. The NCBI GEO2R and ScanGEO analysis tool were used to identify differentially expressed genes (DEGs). The protein-protein interaction (PPI) network was predicted by the STRING website and visualized with Cytoscape software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were performed to enrich GO terms and signaling pathways using Metascape database. To predict LC3-interacting region (LIR) motif among these DEGs, the iLIR database was selected to assess specific short linear sequences. To obtain potential upstream miRNA targets of these DEGs, the mRNA-miRNA interaction networks were predicted by miRWalk database. The knee OA model was performed in mice, and autophagy related mRNAs of osteoclasts were identified. Experimental specimens were further verified with histopathological staining.ResultsBecn1, Atg3, Atg12, Pik3c3, and Gabarapl2 were obtained as coexpressed differential genes. PPI network was constructed and deduced the other 60 related genes. GO and KEGG enrichment networks indicated that autophagy-animal, selective autophagy, and mitophagy mainly participated in autophagy regulation in osteoclasts. The possible LIR sequences were collected to predict motifs. The mRNA–miRNA interaction networks suggested that many miRNAs could regulate autophagy-related genes individually and collectively. The RT–PCR results suggested that these five genes were upregulated in the OA group. Histopathological staining revealed that osteoclasts were increased in subchondral bone, and higher expression of these DEGs in the OA group was compared to the sham group.ConclusionOur results reveal that the role of autophagy in osteoclasts could be a regulatory mechanism in OA and that these autophagy-related genes might be targets for the intervention of OA disease.

First person – Gianna Fote

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Gianna Fote is first author on ‘ Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins’, published in JCS. Gianna conducted the research described in this article while a MD/PhD student in Joan S. Steffan and Leslie M. Thompson's lab at the University of California Irvine School of Medicine, USA, working on the influence of genetic variants on molecular mechanisms of autophagy, and therapeutic modulation of autophagic balance in neurological disease.

Establishment and validation of five autophagy-related signatures for predicting survival and immune microenvironment in glioma.

Gliomas, especially Glioblastoma multiforme, are the most frequent type of primary tumors in central nervous system. Increasing researches have revealed the relationship between autophagy and tumor, while the molecular mechanism of autophagy in glioma is still rarely reported. Our research aims to conform the autophagy-related genes (ARGs) implicated in the development and progression of glioma and improve our understanding of autophagy in glioma. 20 candidate ARGs were screened through the protein-protein interaction network. We also downloaded the publicly accessible glioma data for 665 individuals from TCGA and 970 individuals from CGGA with RNA sequences and clinicopathological information. Subsequently, univariate and multivariate Cox regression analysis identified 5 key ARGs among the 20 candidate genes as key prognostic genes for survival, GSEA and immune response analysis. ATG5, BCL2L1, CASP3, CASP8, GAPDH were identified as key ARGs in our research. Further studies showed that the high-risk population was linked to a dismal prognosis and suggested an immune-inhibitory microenvironment. GSEA results demonstrated that high risk population was closely related to DNA repair, hypoxia pathways, implicated in immunosuppression and carcinogenesis. Through CMap, we finally identified 14 candidate drugs for the ARG high risk population. This study established and verified an ARG risk model, which can serve as an independent predictor for prognosis, reflect on the strength of the immune response and predict the potential drugs in glioma. Our findings offer new understandings of ARG molecular mechanism and promising therapeutic targets for glioma treatment.

Meeting reports

Meeting reports

Selected Abstracts from the Meeting of \u201cMolecular Mechanisms of Autophagy in Diseases\u201d

Selected Abstracts from the Meeting of \u201cMolecular Mechanisms of Autophagy in Diseases\u201d

Autophagy induced by H. pylori VacA regulated the survival mechanism of the SGC7901 human gastric cancer cell line

BackgroundVacuolating cytotoxin (VacA) is an important virulence factor of Helicobacter pylori (H. pylori). It was previously believed that VacA can trigger the cascade of apoptosis on mitochondria to lead to cell apoptosis. Recently, it was found that VacA can induce autophagy. However, the molecular mechanism by which VacA induces autophagy is largely unknown.ObjectiveWe aimed to explore the molecular mechanism of autophagy induced by H. pylori in gastric cancer cells and the effect of autophagy on the survival of gastric cancer cells.MethodsThe autophagy of human gastric cancer cell line SGC7901 was detected by Western blot and RT-PCR in the treatment of VacA protein of H. pylori. The relationship between autophagy and reactive oxygen species (ROS) in the proliferation of gastric cancer cells were studied by gene expression silences (siRNA) and CM-H2DCFDA (DCF) staining.ResultsThe results showed that VacA protein secreted by H. pylori in the supernatant stimulated autophagy in SGC7901 cells. After VacA protein treatment, the mRNA expressions of BECN1, ATG7 and PIK3C3, were up-regulated. ATG7 silencing by siRNA inhibited VacA-induced autophagy. Furthermore, our data demonstrated that VacA protein increased ROS levels. Addition of the antioxidant N-acetyl-l-cysteine (NAC) suppressed the levels of ROS, leading to inhibition of autophagy.ConclusionsH. pylori VacA is a key toxin that induces autophagy by increased ROS levels. And our findings demonstrated that VacA significantly inhibited proliferation in SGC7901 cells.

YAP promotes autophagy and progression of gliomas via upregulating HMGB1

BackgroundDue to the hypoxia and nutrient deficiency microenvironment, glioblastoma (GBM) exhibits high autophagy activity and autophagy plays an important role in the progression of GBM. However, the molecular mechanism of autophagy in GBM progression remains unclear. The aim of this study is to delve out the role and mechanism of yes-associated protein (YAP) in GBM autophagy and progression.MethodsThe level of autophagy or autophagy flux were assessed by using western blotting, GFP-LC3 puncta (Live) imaging, transmission electron microscopy and GFP-RFP-LC3 assay. The GBM progression was detected by using CCK8, EdU, nude mouse xenograft and Ki67 staining. Isobaric tags for relative and absolute quantification (iTraq) quantitative proteomics was used to find out the mediator of YAP in autophagy. Expression levels of YAP and HMGB1 in tissue samples from GBM patients were examined by Western blotting, tissue microarray and immunohistochemistry.ResultsYAP over-expression enhanced glioma cell autophagy under basal and induced conditions. In addition, blocking autophagy by chloroquine abolished the promoting effect of YAP on glioma growth. Mechanistically, YAP over-expression promoted the transcription and translocation of high mobility group box 1(HMGB1), a well-known regulator of autophagy, from nucleus to cytoplasm. Down-regulation of HMGB1 abolished the promoting effect of YAP on autophagy and glioma growth. Furthermore, the expression of YAP and HMGB1 were positively associated with each other and suggested poor prognosis for clinical GBM.ConclusionYAP promoted glioma progression by enhancing HMGB1-mediated autophagy, indicating that YAP-HMGB1 axis was a feasible therapeutic target for GBM. Our study revealed a clinical opportunity involving the combination of chemo-radiotherapy with pharmacological autophagy inhibition for treating GBM patients with YAP high expression.

Molecular Mechanism of Autophagy and Its Regulation by Cannabinoids in Cancer.

Simple SummaryThis review examines the complex function of autophagy in malignancy and explores its regulation by cannabinoids in different cancers. Autophagy is an important process in the maintenance of cellular homeostasis, through the degradation and recycling of cytoplasmic constituents. The action of autophagy is highly dependent on tumour stage and type and the receptors with which ligands interact. Cannabinoids are growingly being acknowledged for their anticancer activities and are known to stimulate several mechanisms such as apoptosis and autophagy. Better understanding the mechanism of action behind autophagy and its regulation by cannabinoids will allow the development of novel cancer therapeutics.Autophagy is a “self-degradation” process whereby malfunctioned cytoplasmic constituents and protein aggregates are engulfed by a vesicle called the autophagosome, and subsequently degraded by the lysosome. Autophagy plays a crucial role in sustaining protein homeostasis and can be an alternative source of energy under detrimental circumstances. Studies have demonstrated a paradoxical function for autophagy in cancer, displaying both tumour suppressive and tumour promotive roles. In early phases of tumour development autophagy promotes cancer cell death. In later phases, autophagy enables cancer cells to survive and withstand therapy. Cannabinoids, which are derivatives of the Cannabis sativa L. plant, have shown to be associated with autophagy induction in cells. There is an emerging interest in studying the signalling pathways involved in cannabinoid-induced autophagy and their potential application in anticancer therapies. In this review, the molecular mechanisms involved in the autophagy degradation process will be discussed. This review also highlights a role for autophagy in cancer progression, with cannabinoid-induced autophagy presenting a novel strategy for anticancer therapy.

Exploring the Regulation and Molecular Mechanism of Autophagy Using Saccharomyces cerevisiae as the Model System

Exploring the Regulation and Molecular Mechanism of Autophagy Using Saccharomyces cerevisiae as the Model System

Identification of autophagy-related protein 3 in the ancient protist Trichomonas vaginalis

Background: Autophagy has been suggested to be involved in the pathogenesis of protists. While the molecular mechanisms of autophagy are mainly studied in model organisms, functional characterization of autophagy-related (Atg) proteins is poorly understood in deep-branching protists. Trichomoniasis is the most common nonviral sexually transmitted infection caused by Trichomonas vaginalis. Bioinformatics analysis of the T. vaginalis genome reveals that the parasite possesses the genes encoding proteins of the Atg8 conjugation system. Herein, we sought to characterize whether the T. vaginalis Atg3 ortholog (TVAG_447140), a putative component of the TvAtg8 conjugation system, regulates autophagy in this parasite. Methods: The recombinant protein of T. vaginalis Atg3 ortholog (TvAtg3) (rTvAtg3) and the polyclonal antibody against rTvAtg3 were generated. The expression and localization was monitored upon autophagy induction by glucose restriction (GR) compared with glucose-rich cultivation. The role of TvAtg3 in autophagy was clarified using small interfering RNA targeting TvAtg3 gene. Results: Phylogenic analysis of Atg3 proteins from different organisms showed that T. vaginalis was not in a close evolutionary relationship with any other protozoan. The expression of TvAtg3 was upregulated in the late-stationary phase of GR culture, implying its involvement in autophagy. Immunofluorescence analysis revealed a much higher TvAtg3 fluorescent intensity located on the round and/or linear structures close to the nucleus. Silencing Tvatg3 expression suppressed GR-induced TvAtg8 expression and autophagic vacuoles formation. Conclusions: These findings suggest the potential role of TvAtg3 in T. vaginalis autophagy and enhance our understanding of the autophagy regulatory network in the deep-branching eukaryotes.

Differential mechanisms of autophagy in cancer stem cells: Emphasizing gastrointestinal cancers

Gastrointestinal (GI) cancers are one of the most common forms of malignancies and still are the most important cause of cancer-related mortality worldwide. Autophagy is a conserved catabolic pathway involving lysosomal degradation and recycling of whole cellular components, which is essential for cellular homeostasis. For instance, it acts as a pivotal intracellular quality control and repair mechanism but also implicated in cell reformation during cell differentiation and development. Indeed, GI cancer stem cells (CSCs) are thought to be responsible for tumour initiation, traditional therapies resistance, metastasis and tumour recurrence. Molecular mechanisms of autophagy in normal vs CSCs gain great interest worldwide. Here, we shed light on the role of autophagy in normal stem cells differentiation for embryonic progression and its role in maintaining the activity and self-renewal capacity of CSCs which offer novel viewpoints on promising cancer therapeutic strategies based on the differential roles of autophagy in CSCs.

Cloning, Expression Analysis, 20-Hydroxyecdysone Induction, and RNA Interference Study of Autophagy-Related Gene 8 from Heortia vitessoides Moore.

Autophagy is a highly conserved and regulated process in eukaryotic cells and remodels cytoplasm, recovers essential nutrients, and disposes of unwanted cytoplasmic components. Autophagy-related gene (ATG) 8, identified in Heortia vitessoides Moore, which is an oligophagous pest of Aquilaria sinensis (Lour.), was characterized (HvATG8). Multiple sequence alignment showed that HvATG8 possesses highly conserved domain structures. Stage- and tissue-specific expressions indicated that HvATG8 is highly expressed in prepupal, pupal, and adult stages and in the midgut of larvae and abdomen of adults. Lack of function of HvATG8 by RNA interference resulted in a significant decrease in survival rate and an increase in abnormal or nonviable phenotypes in H. vitessoides. Transition rate from larval to pupal stages was 33.0% and from pupal to adult stages was 15.0% after injection. Reduction of ATG8 expression reduced survival of H. vitessoides. Therefore, HvATG8 possibly plays a key role in normal growth stage of H. vitessoides. HvATG8 suppression downregulates HvATG3 expression, suggesting that the two genes are interconnected. Further, HvATG8 expression increased by 20-hydroxyecdysone treatment, starvation, and extreme temperature exposure. Starvation also altered expression of other ATGs in H. vitessoide. This study may be used to guide research on molecular mechanisms of autophagy in insects.

Autophagy and the potential linkage with the human oral diseases

Autophagy is a cell survival mechanism that has a key role in both physiologic and pathologic conditions. In this cellular ‘clearance’ process, damaged organelles and macromolecules are transported to the lysosomes for degradation. With autophagy, a protection mechanism occurs in the cell under difficult conditions including oxidative stress and lack of growth factor. Paradoxically, in autophagy under some conditions, non-apoptotic cell death can be observed via a caspase-independent pathway. Recent, studies indicated that there is a strong correlation between the composition of the oral microbiota and the pathogenesis of oral diseases, and autophagy plays a crucial role in the development of this process. This review is focused on the molecular mechanism of autophagy and its relationship with human oral diseases. In this review, the importance of the autophagy mechanism in the maintenance of oral tissue health is emphasized. After examining the studies, we observed the importance of autophagy in response to a variety of oral diseases and aimed to present this awareness. Thus, a better understanding of autophagy will be the basis for developing new therapeutic strategies in the treatment of oral diseases.

DR5 related autophagy can promote apoptosis in gliomas after irradiation

As a cancer treatment strategy, irradiation therapy is widely used that can cause DNA breakage and increase free radicals, which leads to different types of cell death. Among them, apoptosis and autophagy are the most important and the most studied cell death processes. Although the exploration of the relationship between apoptosis and autophagy has been a major area of focus, still the molecular mechanisms of autophagy on apoptosis remain unclear. Here, we have revealed that apoptosis was enhanced by the death receptor 5 (DR5) pathway, and the effect of autophagy on apoptosis was promoted by DR5 interacting with LC3B as well as Caspase8 in gliomas after irradiation. Interestingly, we observed that the addition of four different autophagy inducers, rapamycin (RAP), CCI779, ABT737 and temozolomide (TMZ), induced the differences of DR5 expression and cell apoptosis after irradiation. Unlike RAP and CCI779, ABT737 and TMZ were able to increase DR5 expression and further induce cell death. Therefore, we have concluded that DR5 plays a novel and indispensable role in promoting cell apoptosis under irradiation and suggest a potential therapeutic approach for glioblastoma treatment.

Relationship between molecular mechanism of autophagy and chronic myeloid leukemia

Autophagy is a common physiogical phenomenon in cells, which is closely related to cell growth, differentiation, apoptosis and carcinogenesis. Chronic myeloid leukemia (CML) is a malignant tumor formed by clonal proliferation of bone marrow hematopoietic stem cells. Autophagy may play an important role in occurrence, treatment and drug resistance of CML. Therefore, the study of the molecular mechanism of autophagy and its relationship with CML may provide a theoretical guide for the development of new drugs targeting autophagy in the treatment of CML. This paper reviews the molecular mechanism of autophagy and the relationship between autophagy and CML. Key words: Autophagy; Leukemia, myeloid, chronic; Therapeutics; Drug resistance

The role and molecular mechanism of autophagy in the development of prostate cancer

The role and molecular mechanism of autophagy in the development of prostate cancer

Molecular mechanisms of autophagic memory in pathogenic T cells in human arthritis

T-cell resilience is critical to the immune pathogenesis of human autoimmune arthritis. Autophagy is essential for memory T cell generation and associated with pathogenesis in rheumatoid arthritis (RA). Our aim here was to delineate the role and molecular mechanism of autophagy in resilience and persistence of pathogenic T cells from autoimmune arthritis.We demonstrated “Autophagic memory” as elevated autophagy levels in CD4+ memory T cells compared to CD4+ naive T cells and in Jurkat Human T cell line trained with starvation stress. We then showed increased levels of autophagy in pathogenic CD4+ T cells subsets from autoimmune arthritis patients. Using RNA-sequencing, transcription factor gene regulatory network and methylation analyses we identified MYC as a key regulator of autophagic memory. We validated MYC levels using qPCR and further demonstrated that inhibiting MYC increased autophagy. The present study proposes the novel concept of autophagic memory and suggests that autophagic memory confers metabolic advantage to pathogenic T cells from arthritis and supports its resilience and long term survival. Particularly, suppression of MYC imparted the heightened autophagy levels in pathogenic T cells. These studies have a direct translational valency as they identify autophagy and its metabolic controllers as a novel therapeutic target.

Inhibition of autophagy enhanced cobalt chloride‑induced apoptosis in rat alveolar typeII epithelial cells.

Hypoxia is a type of cellular stress that may result in apoptosis and autophagy. The molecular mechanisms underlying the association between autophagy and apoptosis remain unclear, particularly in hypoxic conditions. Transmission electron microscope, AO-PI staining, flow cytometry and western blot were used to examine the crosstalk between autophagy and apoptosis in hypoxic conditions. Rat alveolar type II epithelial RLE-6TN cells were cultured in a long-term hypoxic environment established by cobalt (II) chloride. It was demonstrated that autophagy and apoptosis occurred in RLE-6TN cells under hypoxic conditions. Treatment of RLE-6TN cells with the autophagy inhibitor 3-methyladenine increased the generation of reactive oxygen species, mitochondrial damage and hypoxia-induced apoptosis. The expression of caspases, particularly caspase-9, increased and may have participated in these processes. The data indicated that the inhibition of autophagy enhanced apoptosis through the mitochondria-mediated intrinsic pathway. These findings provide important insight into the molecular mechanism of autophagy and apoptosis crosstalk. This may provide new insights into pulmonary disease surveillance, diagnosis and treatment.

Autophagy: novel applications of nonsteroidal anti-inflammatory drugs for primary cancer.

In eukaryotic cells, autophagy is a process associated with programmed cell death. During this process, cytoplasmic proteins and organelles are engulfed by double‐membrane autophagosomes, which then fuse with lysosomes to form autolysosomes. These autolysosomes then degrade their contents to recycle the cellular components. Autophagy has been implicated in a wide variety of physiological and pathological processes that are closely related to tumorigenesis. In recent years, an increasing number of studies have indicated that nonsteroidal anti‐inflammatory drugs, such as celecoxib, meloxicam, sulindac, aspirin, sildenafil, rofecoxib, and sodium salicylate, have diverse effects in cancer that are mediated by the autophagy pathway. These nonsteroidal anti‐inflammatory drugs can modulate tumor autophagy through the PI3K/Akt/mTOR, MAPK/ERK1/2, P53/DRAM, AMPK/mTOR, Bip/GRP78, CHOP/ GADD153, and HGF/MET signaling pathways and inhibit lysosome function, leading to p53‐dependent G1 cell‐cycle arrest. In this review, we summarize the research progress in autophagy induced by nonsteroidal anti‐inflammatory drugs and the molecular mechanisms of autophagy in cancer cells to provide a reference for the potential benefits of nonsteroidal anti‐inflammatory drugs in cancer chemotherapy.

TM9SF4 is a novel factor promoting autophagic flux under amino acid starvation.

Autophagy is a highly complicated process with participation of large numbers of autophagy-related proteins. Under nutrient starvation, autophagy promotes cell survival by breaking down nonessential cellular components for recycling use. However, due to its high complexity, molecular mechanism of autophagy is still not fully understood. In the present study, we report a novel autophagy-related protein TM9SF4, which plays a functional role in the induction phase of autophagic process. TM9SF4 proteins were abundantly expressed in the kidney, especially in renal proximal tubular epithelial cells. At subcellular cells, TM9SF4 proteins were mostly localized in lysosome, Golgi, late endosome and autophagosome. Knockdown of TM9SF4 with TM9SF4-shRNAs markedly reduced the starvation-induced autophagy in HEK293 cells, the effect of which persisted in the presence of bafilomycin A1. TM9SF4-shRNAs also substantially attenuated the starvation-induced mTOR inactivation. In animal model, starvation was able to induce LC3-II accumulation and cause mTOR inactivation in renal cortical tissue in wild-type mice, the effect of which was minimal/absent in TM9SF4 knockout (TM9SF4-/-) mice. Co-immunoprecipitation and proximity ligation assay demonstrated physical interaction of TM9SF4 proteins with mTOR. In addition, knockdown or knockout of TM9SF4 reduced the starvation-induced cell death in HEK293 cells and animal model. Taken together, the present study identifies TM9SF4 as a novel autophagy-related protein. Under nutrient starvation, TM9SF4 functions to facilitate mTOR inactivation, resulting in an enhanced autophagic flux, which serves to protect cells from apoptotic cell death.