Autophagy-related 5 Research Articles

CTBP1‑AS2 inhibits proliferation and inducesautophagy in ox‑LDL‑stimulated vascular smooth musclecells by regulating miR‑195‑5p/ATG14.

Atherosclerosis (AS) is a chronic progressive disease caused by injury and functional changes in vascular smooth muscle cells (VSMCs). Long non‑coding RNAs (lncRNAs) are pivotal regulators in AS development. The present study aimed to explore the roles and molecular mechanisms of lncRNA CTBP1‑AS2 in AS progression. A dual‑luciferase reporter assay confirmed that miR‑195‑5p is a downstream target miRNA of lncRNA CTBP1‑AS2 and miR‑195‑5p was increased in AS. The expression levels of miR‑195‑5p and CTBP1‑AS2 in the serums of patients with AS and human aorta vascular smooth muscle cells was increased or decreased, respectively, following treatment with oxidized low‑density lipoprotein (ox‑LDL). Functional experiments showed that the overexpression of lncRNA CTBP1‑AS2 inhibited the proliferation of HA‑VSMCs and promoted their autophagy following ox‑LDL treatment. This effect could be reversed by treatment with ROC‑325, the inhibitor of autophagy, or miR‑195‑5p mimics. Autophagy related 14 (ATG14) was identified to be a target of miR‑195‑5p, and lncRNA CTBP1‑AS2 promoted ATG14 expression by serving as a competing endogenous RNA of miR‑195‑5p. The present study revealed that lncRNA CTBP1‑AS2 may serve a role in AS by inhibiting the proliferation and promoting the autophagy of VSMCs through ATG14 modulation via miR‑195‑5p. These data may provide a novel therapeutic target for AS.

Optimized low pH formulation of niacinamide enhances induction of autophagy marker ATG5 gene expression and protein levels in human epidermal keratinocytes.

Macromolecules in skin cells are damaged when exposed to environmental stressors, leading to disrupted cellular function and homeostasis. While epidermal turnover can eliminate some of this damage, autophagy can rapidly remove these defective components. Niacinamide (Nam) is known to induce autophagy and optimizing formulations to maximize this response could provide improved homeostasis in stressed skin. To determine (i) whether Nam can induce autophagy related 5 (ATG5), an autophagy marker, in human keratinocytes and (ii) whether optimized low pH Nam formulations can enhance the response in 3D skin models. Human keratinocytes treated with Nam were evaluated for autophagosome accumulation and induction of ATG5 by gene expression, immunoblotting and immune-fluorescence microscopy. 3D skin equivalents were topically treated with Nam formulations at pH 5.8 and 3.8. Gene expression profiling and immunoblot analysis of ATG5 were performed. Nam treatment of keratinocytes led to an accumulation of autophagosomes with a maximal signal at 48h. Gene expression of ATG5 was induced by Nam, and immunoblots stained for ATG5 showed a significant increase after 6h of treatment. Gene expression profiling of 3D epidermal skin equivalents treated with Nam at pH 3.8 showed stronger induction of autophagy-related genes, including ATG5, compared with pH 5.8 formulas. Enrichment for gene ontology terms on autophagy showed an increased linkage with Nam formulas at pH 3.8. We found that Nam induces autophagosome accumulation and ATG5 levels in keratinocytes. We also discovered that a Nam formulation at pH 3.8 can further increase levels of ATG5 in 3D skin models when compared to Nam at pH 5.8. These data support that Nam can induce autophagy in keratinocytes and formulations at pH 3.8 can enhance the impact. We hypothesize that optimized formulations at pH 3.8 can improve skin ageing appearance via autophagy induction.

Cellular evidence of autophagy in Sertoli cells during spermatogenesis in goats

Sertoli cells (SCs) play their nursing role as structural and functional supporting cells during spermatogenesis to ensure the production of highly specialized mature spermatozoa. Besides that, the role of SCs in autophagy during active (adult) and inactive (young) spermatogenesis in the caprine testis is still largely unknown. In this study, we investigated autophagy in goat SCs by light microscopy, immunohistochemistry (IHC), double immunofluorescence (double-IF), and transmission electron microscopy. Light microscopy showed active seminiferous tubules with SCs and layers of developing germ cells in the adult goat testis. In young goats, layer of germ cells and SCs was viewed on the basal membrane in the seminiferous tubule. IHC of autophagy-related 7 (ATG7) showed moderate expressions in the cytoplasmic extensions of SCs during inactive spermatogenesis, and strong expression was observed during active spermatogenesis in the testis of goat. Co-immunolabeling of p62 or light chain 3 (LC3) with vimentin showed increasing expression from the basal to the luminal compartment of the seminiferous tubule and stronger expression during active than inactive spermatogenesis in the testis of goat. Ultrastructure assessment of the cytoplasm in SCs showed phagophores, generated from the endoplasmic reticulum during active spermatocytogenesis. Numerous autophagosomes and autolysosomes were noted in the SCs cytoplasm, which surrounds the spermatogenic cells in the basal compartment of the seminiferous tubules. At a later stage, SCs showed autophagosomes and autolysosomes, together with multivesicular bodies (MVB), during spermiogenesis at different phases of the acrosome formation. Numerous embedded elongated spermatozoa were found in the cytoplasm of SCs, surrounded by autophagic components and MVB. Under TEM, the mean diameter of autophagosomes was 952.35 nm and that of autolysosomes was 504.38 nm. Collectively, these results suggest that autophagy is active in SCs during caprine spermatogenesis and that the level of autophagy becomes more evident as spermatogenesis advances from the basal to the luminal compartment of SC.

SUN-564 ATG7 Overexpression Results in Reduction of Hepatocellular Lipid Content in Vitro

: Non-alcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide, paralleling the epidemic of obesity and type 2 diabetes. Despite the high prevalence of NAFLD, only a minority of patients progress to NASH and advanced fibrosis. The reasons for this inter-individual variability are not completely understood but can be partially accounted for by genetic risk factors (1). Although several common genetic variants associated with liver disease have been identified, there is still a proportion of NAFLD heritability that remains unknown. The rare rs143545741 C>T variant in the autophagy related 7 (ATG7) gene (P426L) has been associated with a higher risk of progressive NAFLD (2). Interestingly, ATG7 encodes a E1-like ubiquitin activating enzyme which is involved in hepatic lipophagy (3). We hypothesized that the unknown heritability of NAFLD might be partially explained by rare genetic variants, therefore not identified in the GWAS studies. Moreover, we assumed that loss-of-function variants in ATG7 might confer an increased susceptibility to NAFLD by reducing autophagic catabolism of lipid droplets in the liver. To examine the underlying mechanism of the low-frequency V471A variant and the rare T86I, L127I, Q170E, and P426L variants in ATG7, we performed in vitro experiments of HepaRG cells overexpressing the human V5-tagged ATG7. We observed a reduction in intracellular lipid content in HepaRG cells overexpressing the ATG7 wild type and the 86I mutant protein (p=0.029, n=4) but not the 127I, 170E, 426L, and 471A mutant proteins. Cells with the ATG7 127I, 170E, 426L, and 471A mutants had higher intracellular lipid content compared to cells overexpressing the wild type protein (p=0.029, n=4). Our data suggested that the low-frequency V471A variant and the rare L127I, Q170E, and P426L variants in ATG7 are loss-of-function, resulting in defective lipophagy, reduced hepatocellular lipid droplets turnover, and excessive lipid accumulation. More experiments are needed to clarify the underlying mechanism of the T86I variant. In conclusion, we highlighted a role for ATG7 in reducing hepatocellular lipid content. Furthermore, we provided evidence showing non-synonymous variants in ATG7 increase the risk of NAFLD and that these variants are loss-of-function. We speculate that ATG7 might be a new susceptibility risk genetic locus for liver disease development and progression.

Catabolic degradation of endothelial VEGFA via autophagy

Extracellular matrix-evoked angiostasis and autophagy within the tumor microenvironment represent two critical, but unconnected, functions of the small leucine-rich proteoglycan, decorin. Acting as a partial agonist of vascular endothelial growth factor 2 (VEGFR2), soluble decorin signals via the energy sensing protein, AMP-activated protein kinase (AMPK), in the autophagic degradation of intracellular vascular endothelial growth factor A (VEGFA). Here, we discovered that soluble decorin evokes intracellular catabolism of endothelial VEGFA that is mechanistically independent of mTOR, but requires an autophagic regulator, paternally expressed gene 3 (PEG3). We found that administration of autophagic inhibitors such as chloroquine or bafilomycin A1, or depletion of autophagy-related 5 (ATG5), results in accumulation of intracellular VEGFA, indicating that VEGFA is a basal autophagic substrate. Mechanistically, decorin increased the VEGFA clearance rate by augmenting autophagic flux, a process that required RAB24 member RAS oncogene family (RAB24), a small GTPase that facilitates the disposal of autophagic compartments. We validated these findings by demonstrating the physiological relevance of this process in vivo Mice starved for 48 h exhibited a sharp decrease in overall cardiac and aortic VEGFA that could be blocked by systemic chloroquine treatment. Thus, our findings reveal a unified mechanism for the metabolic control of endothelial VEGFA for autophagic clearance in response to decorin and canonical pro-autophagic stimuli. We posit that the VEGFR2/AMPK/PEG3 axis integrates the anti-angiogenic and pro-autophagic bioactivities of decorin as the molecular basis for tumorigenic suppression. These results support future therapeutic use of decorin as a next-generation protein therapy to combat cancer.

Paeonol prevents lipid metabolism dysfunction in palmitic acid-induced HepG2 injury through promoting SIRT1-FoxO1-ATG14-dependent autophagy

This study aimed to investigate the effects of paeonol (Pae) on lipid metabolism in palmitic acid (PA)-induced injury of HepG2, and to evaluate the protective mechanisms. Lipid metabolism dysfunction of HepG2 cells was produced by administration of palmitic acid (PA). The cells were pretreated with different concentrations of Pae. MTT method was used to detect the cell survival; lipid metabolism was evaluated based on total cholesterol (TC), triglycerides (TG); Western blotting was used to detect the expression of Sirtuin 1 (SIRT1), autophagy related 14 (ATG14), microtubule-associated protein 1A/1B-light chain 3 (LC3) and p62 proteins; immunoprecipitation was used to detect the expression of acetylated FoxO1. After treatment for 24 h, the inhibitory concentration 50 (IC50) of PA in HepG2 cells was about 566.8 μM. Pae at the concentration range from 7.5 to 30 μM did not affect cell viability. Thus, 500 μM PA was used to model metabolism dysfunction and Pae at the concentration range was selected to investigate the protective effect. Compared with the normal control group, the cell survival rate decreased, the number of lipid droplets, and TC and TG levels increased in the model group. Compared with model group, the cell survival rate of Pae (7.5, 15, 30 μM) group increased, the number of lipid droplets and content of TC and TG decreased, the ratio of LC3-II/I increased and p62 expression decreased with pretreatment of Pae. Additionally, Pae pretreatment promoted SIRT1 and ATG14 expression, but reduced acetylated FoxO1 levels in PA-treated cells. Most importantly, autophagy inhibitor, as well as SIRT1 inhibitor blocked the effects of Pae on PA-induced cell injury and metabolism dysfunction, respectively. Pae prevents lipid metabolism dysfunction in PA-induced HepG2 injury by promoting SIRT1-FoxO1-ATG14-dependent autophagy.

Globular CTRP9 protects cardiomyocytes from palmitic acid-induced oxidative stress by enhancing autophagic flux

BackgroundOxidative stress in cardiac myocytes is an important pathogenesis of cardiac lipotoxicity. Autophagy is a cellular self-digestion process that can selectively remove damaged organelles under oxidative stress, and thus presents a potential therapeutic target against cardiac lipotoxicity. Globular CTRP9 (gCTRP9) is a newly identified adiponectin paralog with established metabolic regulatory properties. The aim of this work is to investigate whether autophagy participates the protection effects of gCTRP9 in neonatal rat cardiac myocytes (NRCMs) under oxidative stress and the underlying mechanism. ResultsNRCMs were treated with PA of various concentrations for indicated time period. Our results showed that PA enhanced intracellular ROS accumulation, decreased mitochondrial membrane potential (Δψm) and increased activation of caspases 3. These changes suggested lipotoxicity due to excessive PA. In addition, PA was observed to impair autophagic flux in NRCMs and impaired autophagosome clearance induced by PA contributes to cardiomyocyte death. Besides, we found that gCTRP9 increased the ratio of LC3II/I and the expression of ATG5 which was vital to the formation of autophagosomes and decreased the level of P62, suggesting enhanced autophagic flux in the absence or presence of PA. The result was further confirmed by the methods of infection with LC3-mRFP-GFP lentivirus and blockage of autophagosome–lysosome fusion by BafA1. Moreover, gCTRP9 reestablished the loss of mitochondrial membrane potential, suppressed ROS generation, and reduced PA -induced myocyte death. However, the protective effect of gCTRP9 on the cardiac lipotoxicity was partly abolished by blockade of autophagy by autophagy-related 5 (ATG5) siRNA, indicating that the effect of gCTRP9 on cell survival is critically mediated through regulation of autophagy. ConclusionAutophagy induction by gCTRP9 could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes.

Endurance exercise-induced expression of autophagy-related protein coincides with anabolic expression and neurogenesis in the hippocampus of the mouse brain.

Autophagy and neurogenesis play a pivotal role in maintaining cellular homeostasis of neurons in the brain. Endurance exercise (EXE) serves as a potent regulator of both autophagy and neurogenesis in the hippocampus of the brain; however, the underlying molecular mechanisms of the dual expression remains unclear. Thus, we examined the signaling pathways of EXE-induced autophagy and neurogenesis-associated protein expression in the hippocampus. C57BL/6 male mice (10 weeks old) were randomly divided into two groups: control group (n = 10) and EXE group (EXE, n = 10). Our results showed that EXE increased expression of autophagy-related protein [LC3 II, BECLIN1, autophagy-related 7 (ATG7), p62, LAMP2, CATHEPSIN L and transcription factor EB] in the presence of anabolic signaling expression (AKT-mammalian target of rapamycin-ribosomal S6 kinase). Intriguingly, long-term EXE-mediated neurogenesis in the hippocampus was observed despite the downregulated expressions of canonical neurotrophic factors (e.g. brain-derived neurotrophic factor, glial cell line-derived neurotrophic factors and nerve growth factor); instead, upregulation of neuregulin-1 (NRG1)-mediated signaling cascades (e.g. NRG1-extracellular signal-regulated kinase-ribosomal s6 kinase-cyclic adenosine mono-phosphate response element-binding protein) were associated with EXE-induced hippocampal neurogenesis and synaptic plasticity. Our data, for the first time, show that EXE-mediated expression of autophagy-related protein coincides with anabolic expression and that NRG1 is involved in EXE-mediated neurogenesis and synaptic plasticity. Taken together, this study provides a novel mechanism of hippocampal autophagy and neurogenesis, which may provide potential insight into developing therapeutic neuroprotective strategies.

Prevention of Doxorubicin-Induced Autophagy Attenuates Oxidative Stress and Skeletal Muscle Dysfunction.

Clinical use of the chemotherapeutic doxorubicin (DOX) promotes skeletal muscle atrophy and weakness, adversely affecting patient mobility and strength. Although the mechanisms responsible for DOX-induced skeletal muscle dysfunction remain unclear, studies implicate the significant production of reactive oxygen species (ROS) in this pathology. Supraphysiological ROS levels can enhance protein degradation via autophagy, and it is established that DOX upregulates autophagic signaling in skeletal muscle. To determine the precise contribution of accelerated autophagy to DOX-induced skeletal muscle dysfunction, we inhibited autophagy in the soleus via transduction of a dominant negative mutation of the autophagy related 5 (ATG5) protein. Targeted inhibition of autophagy prevented soleus muscle atrophy and contractile dysfunction acutely following DOX administration, which was associated with a reduction in mitochondrial ROS and maintenance of mitochondrial respiratory capacity. These beneficial modifications were potentially the result of enhanced transcription of antioxidant response element-related genes and increased antioxidant capacity. Specifically, our results showed significant upregulation of peroxisome proliferator-activated receptor gamma co-activator 1-alpha, nuclear respiratory factor-1, nuclear factor erythroid-2-related factor-2, nicotinamide-adenine dinucleotide phosphate quinone dehydrogenase-1, and catalase in the soleus with DOX treatment when autophagy was inhibited. These findings establish a significant role of autophagy in the development of oxidative stress and skeletal muscle weakness following DOX administration.

Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

ABSTRACT Although the treatment of brain tumors by targeting kinase-regulated macroautophagy/autophagy, is under investigation, the precise mechanism underlying autophagy initiation and its significance in glioblastoma (GBM) remains to be defined. Here, we report that PAK1 (p21 [RAC1] activated kinase 1) is significantly upregulated and promotes GBM development. The Cancer Genome Atlas analysis suggests that the oncogenic role of PAK1 in GBM is mainly associated with autophagy. Subsequent experiments demonstrate that PAK1 indeed serves as a positive modulator for hypoxia-induced autophagy in GBM. Mechanistically, hypoxia induces ELP3-mediated PAK1 acetylation at K420, which suppresses the dimerization of PAK1 and enhances its activity, thereby leading to subsequent PAK1-mediated ATG5 (autophagy related 5) phosphorylation at the T101 residue. This event not only protects ATG5 from ubiquitination-dependent degradation but also increases the affinity between the ATG12–ATG5 complex and ATG16L1 (autophagy related 16 like 1). Consequently, ELP3-dependent PAK1 (K420) acetylation and PAK1-mediated ATG5 (T101) phosphorylation are required for hypoxia-induced autophagy and brain tumorigenesis by promoting autophagosome formation. Silencing PAK1 with shRNA or small molecule inhibitor FRAX597 potentially blocks autophagy and GBM growth. Furthermore, SIRT1-mediated PAK1-deacetylation at K420 hinders autophagy and GBM growth. Clinically, the levels of PAK1 (K420) acetylation significantly correlate with the expression of ATG5 (T101) phosphorylation in GBM patients. Together, this report uncovers that the acetylation modification and kinase activity of PAK1 plays an instrumental role in hypoxia-induced autophagy initiation and maintaining GBM growth. Therefore, PAK1 and its regulator in the autophagy pathway might represent potential therapeutic targets for GBM treatment. Abbreviations: 3-MA: 3-methyladenine; Ac-CoA: acetyl coenzyme A; ATG5: autophagy related 5; ATG16L1, autophagy related 16 like 1; BafA1: bafilomycin A1; CDC42: cell division cycle 42; CGGA: Chinese Glioma Genome Atlas; CHX, cycloheximide; ELP3: elongator acetyltransferase complex subunit 3; GBM, glioblastoma; HBSS: Hanks balanced salts solution; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAP2K1: mitogen-activated protein kinase kinase 1; MAPK14, mitogen-activated protein kinase 14; PAK1: p21 (RAC1) activated kinase 1; PDK1: pyruvate dehydrogenase kinase 1; PGK1, phosphoglycerate kinase 1; PTMs: post-translational modifications; RAC1: Rac family small GTPase 1; SQSTM1: sequestosome 1; TCGA, The Cancer Genome Atlas.

Anti-fibrosis activity of quercetin attenuates rabbit tracheal stenosis via the TGF-β/AKT/mTOR signaling pathway

AimsThis study aimed to explore the possible mechanism of trauma-induced laryngotracheal stenosis and potential protective and therapeutic efficacy of quercetin on trauma-induced laryngotracheal stenosis. Main methodsThe expression and activity of fibrotic factors [interleukin (IL)-6, IL-8, autophagy related 5 (ATG5), collagen (COL)-1, tumor growth factor (TGF)-β COL-3, microtubule-associated proteins 1A/1B light chain 3A (LC3), and vascular endothelial growth factor (VEGF)] and fibrotic signaling mediators [mammalian target of rapamycin (mTOR) and phosphorylated AKT (pAKT)] were detected by real-time quantitative PCR (qRT-PCR), ELISA, Western blot, and immunohistochemical staining, respectively, in the lipopolysaccharide (LPS)-induced WI-38 (a human embryonic lung fibroblast cell line) cellular fibrotic model and a trauma-induced rabbit tracheal stenosis model, with and without quercetin treatment. Key findingsPre-treatment with quercetin significantly reversed the LPS-induced upregulation of pro-fibrotic factors (IL-6, IL-8, COL-1, COL-3, LC3) and fibrotic signaling mediators (mTOR and AKT), and it induced the downregulation of ATG5 in the WI-38 cells. Furthermore, the anti-fibrotic activity of quercetin was confirmed in the trauma-induced rabbit tracheal stenosis model. Thus, the nasogastric administration of quercetin attenuated the tracheal stenosis of the rabbit tracheal stenosis model, in addition to effectively reversing an increase in pro-fibrotic factors (VEGF, IL-6, TGF-β, COL-1, and COL-3) and fibrotic signaling mediators (mTOR and AKT), as well as downregulating ATG5 of the rabbit tracheal stenosis model. SignificanceQuercetin exhibits anti-fibrotic activity by inhibiting pro-fibrotic factors and AKT/mTOR signaling pathway, in addition to activating autophagy activity. This study provided experimental evidence supporting the application of quercetin in tracheal stenosis, clinically.

ADAM12 silencing promotes cellular apoptosis by activating autophagy in choriocarcinoma cells.

ADAM metallopeptidase domain 12 (ADAM12) has been demonstrated to mediate cell proliferation and apoptosis resistance in several types of cancer cells. However, the effect of ADAM12 silencing on the proliferation and apoptosis of choriocarcinoma cells remains unknown. The present study revealed that ADAM12 silencing significantly inhibited cellular activity and proliferation in the human choriocarcinoma JEG3 cell line and increased the rate of apoptosis. In addition, ADAM12 silencing significantly increased the expression levels of the autophagy proteins microtubule-associated protein-light-chain 3 (LC3B) and autophagy related 5 (ATG5) and the fluorescence density of LC3B in JEG-3 cells. However, the suppression of autophagy by 3-methyladenine could block ADAM12 silencing-induced cellular apoptosis. ADAM12 silencing reduced the levels of the inflammatory factors interleukin-1β, interferon-γ and TNF-α, and inactivated nuclear p65-NF-κB and p-mTOR in JEG-3 cells. The downregulation of p-mTOR expression by ADAM12 silencing was rescued in 3-methyladenine-treated JEG-3 cells, indicating that mTOR might participate in the autophagy-mediated pro-apoptotic effect of ADAM12 silencing. In conclusion, ADAM12 silencing promoted cellular apoptosis in human choriocarcinoma JEG3 cells, which might be associated with autophagy and the mTOR response. These findings indicate that ADAM12 silencing might be a potential novel therapeutic target for choriocarcinoma.

Involvement of the JNK signaling in granular corneal dystrophy by modulating TGF-β-induced TGFBI expression and corneal fibroblast apoptosis.

Granular corneal dystrophy (GCD) is featured by corneal deposits of transforming growth factor beta-induced gene (TGFBI) mediated by the TGF-β (transforming growth factor-β)/Smad signaling. However, the roles of c-Jun amino-terminal kinase (JNK) pathway in GCD pathogenesis remains unexplored, which was investigated in this study. JNK signaling activation and inhibition in primary corneal fibroblasts were obtained by treatments with anisomycin and SP600125, respectively. Protein abundance and phosphorylation were detected by immunoblotting. Cell viability and apoptosis were analyzed by CCK-8 and flow cytometry respectively. TGFBI deposit and autophagy progression were assessed by immunofluorescence. The results found that JNK1 expression and phosphorylation were greatly increased in corneal tissues from GCD2 patients. JNK signaling activation impaired the viability and promoted apoptosis and autophagy processes in primary corneal fibroblasts, along with Smad2/3 phosphorylation, TGFBI accumulation and Bcl-2 suppression. Autophagy related proteins, such as ATG5 (autophagy related 5), ATG12 (autophagy related 12) and LC3B (microtubule-associated protein 1 light chain 3 beta), were also increased in anisomycin or TGF-β1 treated corneal fibroblasts. However, SP600125 effectively reversed the above effect induced by TGF-β1 treatment in corneal fibroblasts, including the TGF-β-induced autophagy progression. The results suggested that JNK signaling was activated in GCD2 corneal tissues, and it mediated the TGF-β-induced TGFBI protein accumulation and apoptosis of corneal fibroblasts during GCD2 pathogenesis.

Autophagy-based unconventional secretion of HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation

ABSTRACT The precise mechanism through which macroautophagy/autophagy affects psoriasis is poorly understood. Here, we found that keratinocyte (KC) autophagy, which was positively correlated with psoriatic severity in patients and mouse models and could be inhibited by mitogen-activated protein kinase (MAPK) family inactivation. The impairment of autophagic flux alleviated psoriasisform inflammation. We also found that an autophagy-based unconventional secretory pathway (autosecretion) dependent on ATG5 (autophagy related 5) and GORASP2 (golgi reassembly stacking protein 2) promoted psoriasiform KC inflammation. Moreover, the alarmin HMGB1 (high mobility group box 1) was more effective than other autosecretory proteins in regulating psoriasiform cutaneous inflammation. HMGB1 neutralization in autophagy-efficient KCs eliminated the differences in psoriasiform inflammation between Krt14+/+-Atg5f/f KCs and Krt14Cre/+-atg5f/f KCs, and conversely, recombinant HMGB1 almost completely restored psoriasiform inflammation in Krt14Cre/+-atg5f/f KCs in vivo. These results suggest that HMGB1-associated autosecretion plays a pivotal role in cutaneous inflammation. Finally, we demonstrated that Krt14Cre/+-hmgb1f/f mice displayed attenuated psoriatic inflammation due to the essential crosstalk between KC-specific HMGB1-associated autosecretion and γδT cells. Thus, this study uncovered a novel autophagy mechanism in psoriasis pathogenesis, and the findings imply the clinical significance of investigating and treating psoriasis. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AGER: advanced glycosylation end-product specific receptor; Anti-HMGB1: anti-HMGB1 neutralizing antibody; Anti-IL18: anti-IL18 neutralizing antibody; Anti-IL1B: anti-IL1B neutralizing antibody; ATG5: autophagy related 5; BAF: bafilomycin A1; BECN1: beclin 1; CASP1: caspase 1; CCL: C-C motif chemokine ligand; CsA: cyclosporine A; ctrl shRNA: lentivirus harboring shRNA against control; CXCL: C-X-C motif chemokine ligand; DCs: dendritic cells; DMEM: dulbecco’s modified Eagle’s medium; ELISA: enzyme-linked immunosorbent assay; EM: electron microscopy; FBS: fetal bovine serum; GORASP2 shRNA: lentivirus harboring shRNA against GORASP2; GORASP2/GRASP55: golgi reassembly stacking protein 2; GR1: a composite epitope between LY6 (lymphocyte antigen 6 complex) locus C1 and LY6 locus G6D antigens; H&E: hematoxylin and eosin; HMGB1: high mobility group box 1; HMGB1 shRNA: lentivirus harboring shRNA against HMGB1; IFNG/IFN-γ: interferon gamma; IL17A: interleukin 17A; IL18: interleukin 18; IL1A/IL-1α: interleukin 1 alpha; IL1B/IL-1β: interleukin 1 beta; IL22/IL-22: interleukin 22; IL23A: interleukin 23 subunit alpha; IL23R: interleukin 23 receptor; IMQ: imiquimod; ITGAM/CD11B: integrin subunit alpha M; ITGAX/CD11C: integrin subunit alpha X; IVL: involucrin; KC: keratinocyte; KD: knockdown; KO: knockout; Krt14+/+-Atg5f/f mice: mice bearing an Atg5 flox allele, in which exon 3 of the Atg5 gene is flanked by two loxP sites; Krt14+/+-Hmgb1f/f : mice bearing an Hmgb1 flox allele, in which exon 2 to 4 of the Hmgb1 gene is flanked by two loxP sites; Krt14Cre/+-atg5f/f mice: keratinocyte-specific atg5 knockout mice generated by mating Atg5-floxed mice with mice expressing Cre recombinase under the control of the promoter of Krt4; Krt14Cre/+-hmgb1f/f mice: keratinocyte-specific hmgb1 knockout mice generated by mating Hmgb1-floxed mice with mice expressing Cre recombinase under the control of the promoter of Krt14; Krt14-Vegfa mice: mice expressing 164-amino acid Vegfa splice variant recombinase under the control of promoter of Krt14; LAMP1: lysosomal associated membrane protein 1; LDH: lactate dehydrogenase; LORICRIN: loricrin cornified envelope precursor protein; M5: TNF, IL1A, IL17A, IL22 and OSM in combination; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MKI67: marker of proliferation Ki-67; MTT: thiazolyl blue tetrazolium bromide; NFKB/NF-κB: nuclear factor kappa B; NHEKs: primary normal human epidermal keratinocytes; NS: not significant; OSM: oncostatin M; PASI: psoriasis area and severity index; PtdIns3K: class III phosphatidylinositol 3-kinase; qRT-PCR: quantitative RT-PCR; RELA/p65: RELA proto-oncogene, NF-kB subunit; rHMGB1: recombinant HMGB1; rIL18: recombinant interleukin 18; rIL1B: recombinant interleukin 1 beta; S100A: S100 calcium binding protein A; SQSTM1/p62: sequestosome 1; T17: IL17A-producing T; TCR: T-cell receptor; tcrd KO mice: tcrd (T cell receptor delta chain) knockout mice, which show deficient receptor expression in all adult lymphoid and epithelial organs; TLR: toll-like receptor; TNF/TNF-α: tumor necrosis factor; WOR: wortmannin; WT: wild-type; γδT17 cells: IL17A-producing γδ T cells.

CD5L-induced activation of autophagy is associated with hepatoprotection in ischemic reperfusion injury via the CD36/ATG7 axis.

Hepatic ischemia/reperfusion (I/R) injury is a side effect of major liver surgery that is difficult to prevent. I/R injury induces metabolic strain on hepatocytes and limits the tolerable ischemia during liver resection, as well as preservation times during transplantation. Additionally, I/R injury induces apoptosis in hepatocytes. CD5-like (CD5L), an inducer of autophagy, is a soluble scavenger cysteine-rich protein that modulates hepatocyte apoptosis. The aim of the present study was to determine if pharmacologic CD5L was protective against hepatic ischemia-reperfusion injury. Hepatocytes were subjected to I/R culture conditions, and apoptosis and caspase family activity were measured after I/R to model hepatic injury. Treatment with recombinant CD5L significantly suppressed apoptosis and caspase activity through modulating cellular autophagy to maintain activation of the cluster of differentiation 36 (CD36)-dependent autophagy-related 7 (ATG7) signaling pathway. The regulation loop between CD5L and the autophagy signaling pathway was identified to be associated with the inhibition of oxidative stress. Treatment with CD5L significantly inhibited cellular oxidative stress, which was confirmed by silencing the CD36 receptor or the autophagy related protein ATG7 using small interfering RNA, which reversed the antiapoptotic and antioxidative effects of CD5L on hepatocytes under I/R conditions. The results of the present study suggested that CD5L-mediated attenuation of hepatic I/R injury occurs through the CD36-dependent ATG7 pathway, accompanied by the inhibition of oxidative stress, which is associated with enhanced autophagy. In conclusion, the present study identifies CD5L as a novel therapeutic agent for hepatic I/R injury.

Endothelial autophagy deficiency induces IL6 - dependent endothelial mesenchymal transition and organ fibrosis

ABSTRACT Macroautophagy/autophagy plays a vital role in the homeostasis of diverse cell types. Vascular endothelial cells contribute to vascular health and play a unique role in vascular biology. Here, we demonstrated that autophagy defects in endothelial cells induced IL6 (interleukin 6)-dependent endothelial-to-mesenchymal transition (EndMT) and organ fibrosis with metabolic defects in mice. Inhibition of autophagy, either by a specific inhibitor or small interfering RNA (siRNA) for ATG5 (autophagy related 5), in human microvascular endothelial cells (HMVECs) induced EndMT. The IL6 level was significantly higher in ATG5 siRNA-transfected HMVECs culture medium compared with the control HMVECs culture medium, and neutralization of IL6 by a specific antibody completely inhibited EndMT in ATG5 siRNA-transfected HMVECs. Similar to the in vitro data, endothelial-specific atg5 knockout mice (Atg5 Endo; Cdh5-Cre Atg5 flox/flox mice) displayed both EndMT-associated kidney and heart fibrosis when compared to littermate controls. The plasma level of IL6 was higher in Atg5 Endo compared to that of control mice, and fibrosis was accelerated in Atg5 Endo treated with a HFD; neutralization of IL6 by a specific antibody inhibited EndMT and fibrosis in HFD-fed Atg5 Endo associated with the amelioration of metabolic defects. These results revealed the essential role of autophagy in endothelial cell integrity and revealed that the disruption of endothelial autophagy could lead to significant pathological IL6-dependent EndMT and organ fibrosis. Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; EndMT: endothelial-to-mesenchymal transition; HES: hematoxylin and eosin stain; HFD: high-fat diet; HMVECs: human microvascular endothelial cells; IFNG: interferon gamma; IL6: interleukin 6; MTS: Masson’s trichrome staining; NFD: normal-fat diet; siRNA: small interfering RNA; SMAD3: SMAD family member 3; TGFB: transforming growth factor β; TNF: tumor necrosis factor; VEGFA: vascular endothelial growth factor A

Autophagy drives fibroblast senescence through MTORC2 regulation

ABSTRACT Sustained macroautophagy/autophagy favors the differentiation of fibroblasts into myofibroblasts. Cellular senescence, another means of responding to long-term cellular stress, has also been linked to myofibroblast differentiation and fibrosis. Here, we evaluate the relationship between senescence and myofibroblast differentiation in the context of sustained autophagy. We analyzed markers of cell cycle arrest/senescence in fibroblasts in vitro, where autophagy was triggered by serum starvation (SS). Autophagic fibroblasts expressed the senescence biomarkers CDKN1A/p21 and CDKN2A/p16 and exhibited increased senescence-associated GLB1/beta-galactosidase activity. Inhibition of autophagy in serum-starved fibroblasts with 3-methyladenine, LY294002, or ATG7 (autophagy related 7) silencing prevented the expression of senescence-associated markers. Similarly, suppressing MTORC2 activation using rapamycin or by silencing RICTOR also prevented senescence hallmarks. Immunofluorescence microscopy showed that senescence and myofibroblast differentiation were induced in different cells, suggesting mutually exclusive activation of senescence and myofibroblast differentiation. Reactive oxygen species (ROS) are known inducers of senescence and exposing fibroblasts to ROS scavengers decreased ROS production during SS, inhibited autophagy, and significantly reduced the expression of senescence and myofibroblast differentiation markers. ROS scavengers also curbed the AKT1 phosphorylation at Ser473, an MTORC2 target, establishing the importance of ROS in fueling MTORC2 activation. Inhibition of senescence by shRNA to TP53/p53 and shRNA CDKN2A/p16 increased myofibroblast differentiation, suggesting a negative feedback loop of senescence on autophagy-induced myofibroblast differentiation. Collectively, our results identify ROS as central inducers of MTORC2 activation during chronic autophagy, which in turn fuels senescence activation and myofibroblast differentiation in distinct cellular subpopulations. Abbreviations: 3-MA: 3-methyladenine; ACTA2: actin, alpha 2, smooth muscle, aorta; AKT1: AKT serine/threonine kinase 1; p-AKT1: AKT1 Ser473 phosphorylation; t-AKT1: total AKT serine/threonine kinase 1; ATG4A: autophagy related 4A cysteine peptidase; ATG7: autophagy gene 7; C12FDG: 5-dodecanoylaminofluorescein Di-β-D-Galactopyranoside; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; Ctl: control; DAPI: 4ʹ,6-diamidino-2-phenylindole, dilactate; ECM: extracellular matrix; GSH: L-glutathione reduced; H2O2: hydrogen peroxide; HLF: adult human lung fibroblasts; Ho: Hoechst 33342 (2′‐[4‐ethoxyphenyl]‐5‐[4‐methyl‐1‐piperazinyl]‐2.5′‐bi‐1H‐benzimidazole); HSC: hepatic stellate cells; LY: LY294002; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTORC1/2: mechanistic target of rapamycin kinase complex 1/2; N: normal growth medium; NAC: N-acetyl-L-cysteine; PBS: phosphate-buffered saline; PDGFA: platelet derived growth factor subunit A; PRKCA/PKCα: protein kinase C alpha; PtdIns3K: class III phosphatidylinositol 3-kinase; PTEN: phosphatase and tensin homolog; R: rapamycin; RICTOR: RPTOR independent companion of MTOR complex 2; ROS: reactive oxygen species; RPTOR: regulatory associated protein of MTOR complex 1; SA-GLB1/β-gal: senescence-associated galactosidase beta 1; SGK1: serum/glucocorticoid regulated kinase 1; shRNA: short hairpin RNA; siCtl: control siRNA; siRNA: small interfering RNA; SQSTM1: sequestosome 1; SS: serum-free (serum starvation) medium; TP53: tumor protein p53; TUBA: tubulin alpha; V: vehicle.

HIF2A overexpression reduces cisplatin sensitivity in cervical cancer by inducing excessive autophagy.

BackgroundHypoxia-induced autophagy is a crucial factor that induces chemotherapy resistance in tumor cells. As a key regulator facilitating the adaptation of solid tumors to hypoxia, the role of hypoxia-inducible factors (HIFs) in regulating hypoxia-induced chemotherapy resistance and autophagy has been extensively studied. However, the majority of studies have mainly focused on HIF-1. Direct evidence concerning the role of HIF2A in cisplatin resistance is sparse, and its underlying mechanism is not yet known.MethodsAnimal models were constructed by subcutaneously injecting cervical cancer cells stably overexpressing HIF2A (LV-HIF2A) with or without intraperitoneal injection of cisplatin. Tumor size and weight were evaluated to determine tumor growth. Apoptosis was detected by TUNEL assay and protein expression by western blotting.ResultsNude mice injected with cells overexpressing HIF2A showed larger and heavier tumors than those in mice injected with negative control lentivirus (LV-NC)-infected cells, with or without cisplatin. Fewer apoptotic cells were noted in tumor tissues from the LV-HIF2A group than from the LV-NC group, with or without cisplatin. Additionally, expression of the anti-apoptotic protein, B-cell lymphoma 2 (BCL2), and autophagy-related proteins, beclin 1 and autophagy related 5 (ATG5), were found to be higher in the LV-HIF2A group than in the LV-NC group, regardless of cisplatin treatment. Moreover, expression of the pro-apoptotic protein, BCL2-associated X (BAX), was lower in tumor tissues from the LV-HIF2A group than from the LV-NC group. Effect of HIF2A overexpression on cisplatin sensitivity was found to be alleviated in vivo by the autophagy inhibitor, 3-methyladenine (3-MA).ConclusionsHIF2A overexpression promoted tumor growth and autophagy but suppressed apoptosis in vivo, with or without cisplatin. The HIF2A overexpression-affected cisplatin sensitivity was alleviated by 3-MA. Therefore, we suggest that HIF2A overexpression reduces cisplatin sensitivity in cervical cancer by inducing excessive autophagy.

Rubicon in pancreatic beta cells plays a limited role in maintaining glucose homeostasis following increased insulin resistance.

Autophagy has been reported to play a crucial role in the maintenance of intracellular homeostasis, including in pancreatic beta cells. Rubicon, which interacts with the phosphoinositide 3-kinase (PI3K) complex, through autophagy-related 14 (ATG14), is among the few autophagy regulators that have been reported to inhibit autophagic flux to date and the deletion of Rubicon has been shown to increase autophagic flux. Based on previous results showing a causal relationship between autophagic dysfunction and pancreatic beta-cell impairment, we hypothesized that the deletion of Rubicon in pancreatic beta cells would improve cell integrity and confer protective effects. To test this hypothesis, we first confirmed that Rubicon knockdown (KD) promoted autophagic flux in βTC3 pancreatic beta-cell line. Next, we generated pancreatic beta-cell-specific Rubicon knockout (βKO) mice, by administering tamoxifen to Rubiconflox/flox:MIP-Cre-ERT mice, which showed normal glucose tolerance and insulin secretion under a normal chow diet, despite successful gene recombination. We also attempted to increase insulin resistance by feeding the mice with a high-fat diet for an additional 2 months to find little differences among the parameters evaluated for glucose metabolism. Finally, severe insulin resistance was induced with insulin receptor antagonist treatment, which resulted in comparable glucose homeostasis measurements between Rubicon βKO and control mice. In summary, these results suggest that in pancreatic beta cells, Rubicon plays a limited role in the maintenance of systemic glucose homeostasis.

Multifunctional N-P-doped carbon dots for regulation of apoptosis and autophagy in B16F10 melanoma cancer cells and in vitro imaging applications

Rationale: The present study reports the multifunctional anticancer activity against B16F10 melanoma cancer cells and the bioimaging ability of fluorescent nitrogen-phosphorous-doped carbon dots (NPCDs).Methods: The NPCDs were synthesized using a single-step, thermal treatment and were characterized by TEM, XPS, fluorescence and UV-Vis spectroscopy, and FTIR analysis. The anticancer efficacy of NPCDs was confirmed by using cell viability assay, morphological evaluation, fluorescent live-dead cell assay, mitochondrial potential assay, ROS production, RT-PCR, western-blot analysis, siRNA transfection, and cellular bioimaging ability.Results: The NPCDs inhibited the proliferation of B16F10 melanoma cancer cells after 24 h of treatment and induced apoptosis, as confirmed by the presence of fragmented nuclei, reduced mitochondrial membrane potential, and elevated levels of reactive oxygen species. The NPCDs treatment further elevated the levels of pro-apoptotic factors and down-regulated the level of Bcl2 (B-cell lymphoma 2) that weakened the mitochondrial membrane, and activated proteases such as caspases. Treatment with NPCDs also resulted in dose-dependent cell cycle arrest, as indicated by reduced cyclin-dependent kinase (CDK)-2, -4, and -6 protein levels and an enhanced level of p21. More importantly, the NPCDs induced the activation of autophagy by upregulating the protein expression levels of LC3-II and ATG-5 (autophagy-related-5) and by downregulating p62 level, validated by knockdown of ATG-5. Additionally, owing to their excellent luminescence property, these NPCDs were also applicable in cellular bioimaging, as evidenced by the microscopic fluorescence imaging of B16F10 melanoma cells.Conclusion: Based on these findings, we conclude that our newly synthesized NPCDs induced cell cycle arrest, autophagy, and apoptosis in B16F10 melanoma cells and presented good cellular bioimaging capability.