Expression Of Autophagy Genes Research Articles

Class 3 phosphoinositide 3-kinase promotes hepatic glucocorticoid receptor stability and transcriptional activity.

AimLipid kinase class 3 phosphoinositide 3‐kinase (PI3K) and nuclear receptor transcription factor glucocorticoid receptor (GR) play essential physiological roles in metabolic adaptation to fasting by activating lysosomal degradation by autophagy and metabolic gene expression, yet their functional interaction is unknown. The requirement of class 3 PI3K for GR function was investigated in liver tissue.MethodsInactivation of class 3 PI3K was achieved through deletion of its essential regulatory subunit Vps15, by expressing Cre‐recombinase in the livers of Vps15f/f mice. The response to both 24‐h fasting and synthetic GR ligand, dexamethasone (DEX) was evaluated in control and mutant mice. Liver tissue was analysed by immunoblot, RT‐qPCR, and LC‐MS.ResultsVps15 mutant mice show decreased transcript levels of GR targets, coupled with lower nuclear levels of total and phosphorylated on Ser211, GR protein. Acute DEX treatment and 24‐h fasting both failed to re‐activate expression of GR targets in the livers of Vps15 mutant mice to the levels observed in controls. Decreased levels of endogenous GR ligand corticosterone and lower expression of 11β‐hydroxysteroid dehydrogenase 1 (11β‐HSD1), a metabolic enzyme that controls corticosterone availability, were found in the livers of Vps15 mutants. Hepatic Vps15 depletion resulted in the activation of nuclear Akt1 signalling, which was paralleled by increased polyubiquitination of GR.ConclusionIn the liver, class 3 PI3K is required for corticosterone metabolism and GR transcriptional activity.

Insulin-like growth factor 2 and autophagy gene expression alteration arise as potential biomarkers in Parkinson\u2019s disease

Insulin-like growth factor 2 (IGF2) and autophagy-related genes have been proposed as biomolecules of interest related to idiopathic Parkinson’s disease (PD). The objective of this study was to determine the IGF2 and IGF1 levels in plasma and peripheral blood mononuclear cells (PBMCs) from patients with moderately advanced PD and explore the potential correlation with autophagy-related genes in the same blood samples. IGF1 and IGF2 levels in patients' plasma were measured by ELISA, and the IGF2 expression levels were determined by real-time PCR and Western blot in PBMCs. The expression of autophagy-related genes was evaluated by real-time PCR. The results show a significant decrease in IGF2 plasma levels in PD patients compared with a healthy control group. We also report a dramatic decrease in IGF2 mRNA and protein levels in PBMCs from PD patients. In addition, we observed a downregulation of key components of the initial stages of the autophagy process. Although IGF2 levels were not directly correlated with disease severity, we found a correlation between its levels and autophagy gene profile expression in a sex-dependent pattern from the same samples. To further explore this correlation, we treated mice macrophages cell culture with α-synuclein and IGF2. While α-synuclein treatment decreased levels Atg5, IGF2 treatment reverted these effects, increasing Atg5 and Beclin1 levels. Our results suggest a relationship between IGF2 levels and the autophagy process in PD and their potential application as multi-biomarkers to determine PD patients' stages of the disease.

Upregulation of Unfolded Protein Response and ER Stress-Related IL-23 Production in M1 Macrophages from Ankylosing Spondylitis Patients.

The inflammatory interleukin (IL)-23/IL-17 axis plays an important role in the pathogenesis of ankylosing spondylitis (AS), but with an unknown regulatory mechanism. This study aimed to investigate the role of endoplasmic reticulum (ER) stress and autophagy pathway in the expression of IL-23 in peripheral blood-derived macrophages in AS patients. Peripheral blood samples were obtained from 15 AS and 15 healthy control subjects. MACS was used to isolate monocytes from PBMCs. Then, M-CSF was used to differentiate monocytes to M2 macrophages. IFN-γ and/or LPS were used to activate macrophages and M2 polarization towards M1 macrophages. Thapsigargin was used to induce ER stress and 3-MA to inhibit autophagy. The purity of extracted monocytes and macrophage markers was evaluated by flow cytometry. mRNA expression of HLA-B and-B27, ER stress-related genes, autophagy-related genes, and IL-23p19 was performed using RT-qPCR. Soluble levels of IL-23p19 were measured using ELISA. Significant increase in mRNA expression of HLA-B, HLA-B27, BiP, XBP1, CHOP, and PERK mRNAs was observed in macrophages of AS patients before and after stimulation with IFN-γ and LPS. No significant change in autophagy gene expression was detected. mRNA and soluble levels of IL-23p19 demonstrated a significant increase in macrophages of AS patients compared to healthy subjects. ER stress induction led to a significant increase in IL-23p19 in macrophages. Inhibition of autophagy did not affect IL-23 expression. ER stress, unlike autophagy, is associated with increased IL-23 levels in macrophages of AS patients.Key Messages ER stress in macrophages from AS patients plays a role in the increased production of IL-23. The autophagy pathway is not involved in the modulation of IL-23 production by AS macrophages.

Nicorandil alleviates cardiac remodeling and dysfunction post -infarction by up-regulating the nucleolin/autophagy axis

ObjectiveThe present study aimed to investigate whether the drug nicorandil can improve cardiac remodeling after myocardial infarction (MI) and the underlying mechanisms. MethodsMouse MI was established by the ligation of the left anterior descending coronary artery and H9C2 cells were cultured to investigate the underlying molecular mechanisms. The degree of myocardial collagen (Col) deposition was evaluated by Masson's staining. The expressions of nucleolin, autophagy and myocardial remodeling-associated genes were measured by Western blotting, qPCR, and immunofluorescence. The apoptosis of myocardial tissue cells and H9C2 cells were detected by TUNEL staining and flow cytometry, respectively. Autophagosomes were observed by transmission electron microscopy. ResultsTreatment with nicorandil mitigated left ventricular enlargement, improved the capacity of myocardial diastolic-contractility, decreased cardiomyocyte apoptosis, and inhibited myocardial fibrosis development post-MI. Nicorandil up-regulated the expression of nucleolin, promoted autophagic flux, and decreased the expressions of TGF-β1 and phosphorylated Smad2/3, while enhanced the expression of BMP-7 and phosphorylated Smad1 in myocardium. Nicorandil decreased apoptosis and promoted autophagic flux in H2O2-treated H9C2 cells. Autophagy inhibitors 3-methyladenine (3MA) and chloroquine diphosphate salt (CDS) alleviated the effects of nicorandil on apoptosis. Knockdown of nucleolin decreased the effects of nicorandil on apoptosis and nicorandil-promoted autophagic flux of cardiomyocytes treated with H2O2. ConclusionsTreatment with nicorandil alleviated myocardial remodeling post-MI through up-regulating the expression of nucleolin, and subsequently promoting autophagy, followed by regulating TGF-β/Smad signaling pathway.

Rapamycin Treatment Is Beneficial for the Generation of Rabbit-Induced Pluripotent Stem-Like Cells.

Autophagy could promote the generation of induced pluripotency stem cells (iPSCs) in humans and mice. However, little was known whether it had similar effects in other species, the detailed mechanism and the features of formed iPSC colonies were also not clear. In this study, we first established the doxycycline (DOX)-inducible tetO lentiviral vector system suitable for the generation of rabbit iPSCs. Rapamycin, a mechanistic target of rapamycin (mTOR) inhibitor, was added during rabbit embryonic fibroblasts induction to improve the autophagy level. The colony formation efficiency and the expression of autophagy- and pluripotent-related genes were detected. The results showed that the established DOX-inducible tetO lentiviral system was successfully used to induce rabbit iPS-like cells. Compared with the untreated group, the number of alkaline phosphatase (AP)-positive colonies was increased 5.5-fold, when 0.5 nM rapamycin was added on days 1-3 after transduction, the colony morphology was improved and the iPS-like cells could be passaged >10 generations. The expression of autophagy-related genes (ATG), ATG5, ATG7, LC3, and ULK1 was increased with different patterns during the induction process, expression of OCT4, SOX2, and KLF4 significantly increased (p < 0.05). The mentioned results indicate that rapamycin treatment is beneficial for the generation of rabbit iPSCs by regulating autophagy and pluripotency-related gene expression.

Hierarchical regulation of autophagy during adipocyte differentiation.

We previously showed that some adipogenic transcription factors such as CEBPB and PPARG directly and indirectly regulate autophagy gene expression in adipogenesis. The order and effect of these events are undetermined. In this study, we modeled the gene expression, DNA-binding of transcriptional regulators, and histone modifications during adipocyte differentiation and evaluated the effect of the regulators on gene expression in terms of direction and magnitude. Then, we identified the overlap of the transcription factors and co-factors binding sites and targets. Finally, we built a chromatin state model based on the histone marks and studied their relation to the factors' binding. Adipogenic factors differentially regulated autophagy genes as part of the differentiation program. Co-regulators associated with specific transcription factors and preceded them to the regulatory regions. Transcription factors differed in the binding time and location, and their effect on expression was either localized or long-lasting. Adipogenic factors disproportionately targeted genes coding for autophagy-specific transcription factors. In sum, a hierarchical arrangement between adipogenic transcription factors and co-factors drives the regulation of autophagy during adipocyte differentiation.

Protective effects of AdipoRon on the liver of Huoyan goose fed a high-fat diet

Adiponectin can participate in the regulation of glucose and lipid metabolism, energy regulation, immune response, resistance to inflammation, oxidative stress, and apoptosis. Studies in rodents demonstrated that the small molecule compound adiponectin receptor agonist AdipoRon could activate the adiponectin receptor and played the same biological role as adiponectin. To explore the influence and regulation of AdipoRon on lipid metabolism disorder in Huoyan goose liver, in this study, goslings were fed a high-fat diet and then administered different dosages of AdipoRon. Subsequently, goose body weight, liver index, liver histopathological changes, blood glucose, blood and liver lipid, biochemical indexes related to liver function and oxidative stress, and the expression levels of genes related to lipid metabolism, inflammation, apoptosis, and autophagy, adiponectin and its receptors, key molecules of adiponectin involved signal pathway, and transcription factors in the liver, were detected using H&E and Oil red O staining, ELISA, and qRT-PCR methods. The results indicated that AdipoRon could alter the expression of lipid metabolism-related genes, inflammatory factors, apoptosis and autophagy genes, and adiponectin and its receptor genes in liver tissues through signaling pathways such as AMPK and p38 MAPK, as well as the involvement of transcription factors such as PPARα, PPARγ, SIRT1, and FOXO1, reduce the lipid content in blood and liver tissues of geese fed high-fat diets, improve liver antioxidant capacity, regulate apoptosis and autophagy of hepatocytes, and reduce liver inflammatory injury. Our study suggests that AdipoRon has a protective effect on fatty liver injury in goslings fed a high-fat diet.

Exploring the Antiproliferative Mechanisms of Urtica dioica L. extract in Human Promyelocytic Leukemia Cell Line

Background: Nature is a source of therapeutic compounds which have fewer side effects compared to synthetic drugs. Stinging nettle extract, widely-used in Anatolia, has a strong antiproliferative effect on many types of cancer. However, the underlying molecular mechanisms of this effect is still not known. Materials and Methods: In this study, the interaction of Urtica dioica L. extract at different concentrations with apoptosis and autophagy pathways in the human promyelocytic cell line (HL-60) was studied to elucidate how it triggers the antiproliferative effects. In this context, firstly, the plant leaves were extracted in water with the Soxhlet extraction method. HL-60 cells were incubated with the extract at different concentrations for 24 hours, and the activated antitumoral effect pathway was investigated with advanced following molecular tests: MTT staining, Nitric oxide (NO) level, Annexin-V in flow cytometry, cell cycle, mitochondrial membrane potential measurement (MMP) and qPCR for evaluation of apoptosis and autophagy mediator genes. Results: It was determined that cell proliferation was suppressed at a concentration of 100 µg/mL and cells were kept in G0/G1 phase, MMP was impaired in cells and the rate of apoptotic cells increased. These apoptotic markers were confirmed by statistically increased expression of apoptotic and autophagy genes and NO level. Conclusions: Taken together, it is predicted that Urtica dioica L. water extract initiates apoptosis in HL-60 cells and could be promising compound candidate for cancer treatment.

Low Expression of SLC7A11 Confers Drug Resistance and Worse Survival in Ovarian Cancer via Inhibition of Cell Autophagy as a Competing Endogenous RNA.

Drug resistance is the main cause of chemotherapy failure in ovarian cancer (OC), and identifying potential druggable targets of autophagy is a novel and promising approach to overcoming drug resistance. In this study, 131 genes associated with autophagy were identified from three autophagy-related databases, and of these, 14 were differentially expressed in 90 drug-resistant OC tissues versus 197 sensitive tissues according to the Cancer Genome Atlas ovarian cancer cohort. Among these 14 genes, SLC7A11 was significantly decreased in two paclitaxel-resistant OC cells (HeyA8-R and SKOV3-R) and in 90 drug-resistant tissues compared with their controls. In vitro overexpression of SLC7A11 significantly increased the sensitivity of HeyA8-R cells to paclitaxel, inhibited colony formation, induced apoptosis, and arrested cell cycle. Further, low SLC7A11 expression was correlated with poor overall survival (OS), progression-free survival (PFS), and post-progression survival (PPS) in 1815 OC patients. Mechanistically, SLC7A11 strongly regulated cell autophagy as a competing endogenous RNA (ceRNA) based on pan-cancer analyses of 32 tumor types. Specifically, as a ceRNA for autophagy genes STX17, RAB33B, and UVRAG, SLC7A11 was strongly and positively co-expressed with these three genes in 20, 12, and 12 different tumors, respectively, in 379 OC tissues and in 90 drug-resistant OC tissues, and the former two were significantly upregulated in SLC7A11-overexpressed HeyA8-R cells. Further, SLC7A11 induced the protein expression of other autophagy genes, such as LC3, Atg16L1, and Atg7, and the expression of the respective proteins was further increased when the cells were treated with paclitaxel. The results strongly suggest that SLC7A11 regulates autophagy via ceRNA interactions with the three abovementioned genes in pan-cancer and in drug-resistant OC. Moreover, low expression of STX17 and UVRAG also significantly predicted low OS, PFS, and PPS. The combination of SLC7A11 with STX17 was more predictive of OS and PFS than either individually, and the combination of SLC7A11 with UVRAG was highly predictive of OS and PPS. The above results indicated that decreased SLC7A11 resulted in drug resistance and effected low rates of survival in OC patients, probably via ceRNA interactions with autophagy genes, and thus the gene could serve as a therapeutic target and potential biomarker in OC.

Dysregulated Autophagy Leads to Oxidative Stress and Aberrant Expression of ABC Transporters in Women with Early Miscarriage.

Early miscarriage (EMC) is a devastating obstetrical complication. ATP-binding cassette (ABC) transporters mediate cholesterol transfer across the placenta and enhance cell survival by effluxing substrates from target cells in the presence of stressors. Recent evidence reports an intricate interplay between autophagy and ABC transporters. We hypothesized that dysregulated autophagy and oxidative stress (OS) in the placenta leads to abnormal expression of membrane transporters contributing to poor pregnancy survival in EMC. We determined mRNA and protein expression of autophagy genes (Beclin-1/Bcl-2/LC3I/LC3II/p62) and ABC transporters (ABCA1/ABCG1/ABCG2) in placentae from EMC patients (n = 20), term controls (n = 19), first trimester (n = 6), and term controls (n = 5) controls. Oxidative/antioxidant status and biomarkers of oxidative damage were evaluated in maternal serum and placentae from EMC and healthy controls. In EMC, placental expression of LC3II/LC3I as well as of the key autophagy regulatory proteins Beclin-1 and Bcl-2 were reduced, whereas p62 was increased. Both in the serum and placentae of EMC patients, total OS was elevated reflected by increased oxidative damage markers (8-OHdG/malondialdehyde/carbonyl formation) accompanied by diminished levels of total antioxidant status, catalase, and total glutathione. Furthermore, we found reduced ABCG1 and increased ABCG2 expression. These findings suggest that a decreased autophagy status triggers Bcl-2-dependent OS leading to macromolecule damage in EMC placentae. The decreased expression of ABCG1 contributes to reduced cholesterol export to the growing fetus. Increasing ABCG2 expression could represent a protective feedback mechanism under inhibited autophagy conditions. In conclusion, dysregulated autophagy combined with increased oxidative toxicity and aberrant expression of placental ABC transporters affects materno-fetal health in EMC.

Inhibition of the NLRP3/IL-1β axis protects against sepsis-induced cardiomyopathy.

BackgroundSeptic cardiomyopathy worsens the prognosis of critically ill patients. Clinical data suggest that interleukin‐1β (IL‐1β), activated by the NLRP3 inflammasome, compromises cardiac function. Whether or not deleting Nlrp3 would prevent cardiac atrophy and improve diastolic cardiac function in sepsis was unclear. Here, we investigated the role of NLRP3/IL‐1β in sepsis‐induced cardiomyopathy and cardiac atrophy.Methods Male Nlrp3 knockout (KO) and wild‐type (WT) mice were exposed to polymicrobial sepsis by caecal ligation and puncture (CLP) surgery (KO, n = 27; WT, n = 33) to induce septic cardiomyopathy. Sham‐treated mice served as controls (KO, n = 11; WT, n = 16). Heart weights and morphology, echocardiography and analyses of gene and protein expression were used to evaluate septic cardiomyopathy and cardiac atrophy. IL‐1β effects on primary and immortalized cardiomyocytes were investigated by morphological and molecular analyses. IonOptix and real‐time deformability cytometry (RT‐DC) analysis were used to investigate functional and mechanical effects of IL‐1β on cardiomyocytes.ResultsHeart morphology and echocardiography revealed preserved systolic (stroke volume: WT sham vs. WT CLP: 33.1 ± 7.2 μL vs. 24.6 ± 8.7 μL, P < 0.05; KO sham vs. KO CLP: 28.3 ± 8.1 μL vs. 29.9 ± 9.9 μL, n.s.; P < 0.05 vs. WT CLP) and diastolic (peak E wave velocity: WT sham vs. WT CLP: 750 ± 132 vs. 522 ± 200 mm/s, P < 0.001; KO sham vs. KO CLP: 709 ± 152 vs. 639 ± 165 mm/s, n.s.; P < 0.05 vs. WT CLP) cardiac function and attenuated cardiac (heart weight–tibia length ratio: WT CLP vs. WT sham: −26.6%, P < 0.05; KO CLP vs. KO sham: −3.3%, n.s.; P < 0.05 vs. WT CLP) and cardiomyocyte atrophy in KO mice during sepsis. IonOptix measurements showed that IL‐1β decreased contractility (cell shortening: IL‐1β: −15.4 ± 2.3%, P < 0.001 vs. vehicle, IL‐1RA: −6.1 ± 3.3%, P < 0.05 vs. IL‐1β) and relaxation of adult rat ventricular cardiomyocytes (time‐to‐50% relengthening: IL‐1β: 2071 ± 225 ms, P < 0.001 vs. vehicle, IL‐1RA: 564 ± 247 ms, P < 0.001 vs. IL‐1β), which was attenuated by an IL‐1 receptor antagonist (IL‐1RA). RT‐DC analysis indicated that IL‐1β reduced cardiomyocyte size (P < 0.001) and deformation (P < 0.05). RNA sequencing showed that genes involved in NF‐κB signalling, autophagy and lysosomal protein degradation were enriched in hearts of septic WT but not in septic KO mice. Western blotting and qPCR disclosed that IL‐1β activated NF‐κB and its target genes, caused atrophy and decreased myosin protein in myocytes, which was accompanied by an increased autophagy gene expression. These effects were attenuated by IL‐1RA.ConclusionsIL‐1β causes atrophy, impairs contractility and relaxation and decreases deformation of cardiomyocytes. Because NLRP3/IL‐1β pathway inhibition attenuates cardiac atrophy and cardiomyopathy in sepsis, it could be useful to prevent septic cardiomyopathy.

Toxoplasma gondii profilin and tachyzoites RH strain may manipulate autophagy via downregulating Atg5 and Atg12 and upregulating Atg7.

Autophagy process is an important defense mechanism against intracellular infection. This process plays a critical role in limiting the development of Toxoplasma gondii. This study aimed to investigate the effects of T. gondii profilin and tachyzoites on the expression of autophagy genes. PMA-activated THP-1 cell line was incubated with T. gondii profilinand tachyzoites for 6h. After RNA extraction and cDNA synthesis, the expression of Atg5, Atg7, Atg12, and LC3b was evaluated using real-time PCR. The results revealed statistically significant downregulation of Atg5 for 1.43 (P-value = 0.0062) and 4.15 (P-value = 0.0178) folds after treatment with T. gondii profilin and tachyzoites, respectively. Similar to Atg 5, Atg 12 revealed a statistically significant downregulation for profilin (1.41 fold; P-value = 0.0047) and T. gondii tachyzoites (3.25 fold; P-value = 0.011). The expression of Atg7 elevated in both T. gondii profilin (2.083 fold; P-value = 0.0087) and tachyzoites (1.64 fold; P-value = 0.206). T. gondii profilin and tachyzoites downregulated (1.04 fold; P-value = 0.0028) and upregulated (twofold; P-value = 0.091) the expression of LC3b, respectively. Our findings suggest that T. gondii and profilin may manipulate autophagy via preventing from the formation of Atg5-12-16L complex to facilitate replication of T. gondii and development of toxoplasmosis.

Decreased autophagy impairs osteogenic differentiation of adipose-derived stem cells via Notch signaling in diabetic osteoporosis mice

BackgroundThe osteogenic differentiation ability of adipose-derived stem cells (ASCs) is attenuated in type 2 diabetic osteoporosis (Dop) mice. Several studies suggest autophagy and Notch signaling pathway play vital roles in cell proliferation, differentiation, and osteogenesis. However, the mechanisms of autophagy and Notch signaling in the osteogenic differentiation of Dop ASCs were unclear. Thus, it is meaningful to reveal potential correlations between autophagy, Notch signaling, and osteogenesis, and explore involved molecular mechanisms in Dop ASCs. Materials and methodsThe diabetic osteoporosis C57BL/6 mouse model, which was confirmed by micro-CT and HE & Masson staining, was established through high-sugar and high-fat diet and streptozotocin injection. ASCs were obtained from the inguinal subcutaneous fat of Dop mice. The multi-differentiation potential of ASCs was evaluated by staining with Alizarin Red (osteogenesis), Oil Red O (adipogenesis), and Alcian blue (chondrogenesis). Cell viability was assessed by Cell Counting Kit-8 assay. Torin1, an inhibitor of mTOR, was used to stimulate the autophagy signaling pathway. DAPT, a γ-secretase inhibitor, was used to suppress Notch signaling pathway activity. Gene and protein expression of autophagy, Notch signaling pathway, and osteogenic factors were detected by real-time quantitative PCR, western blot, and immunofluorescence microscopy. ResultsOur findings showed autophagy and osteogenic differentiation ability of Dop ASCs exhibited downward trends that were both rescued by Torin1. Notch signaling was suppressed in Dop ASCs, but upregulated when autophagy was activated. After activation of autophagy, DAPT treatment led to decreased Notch signaling pathway activation and attenuated osteogenic differentiation ability in Dop ASCs. ConclusionsDownregulated autophagy suppressed Notch signaling, leading to a reduced osteogenic differentiation capacity of Dop ASCs, and Torin1 can rescue this process by activating autophagy. Our findings contribute to understanding the mechanism underlying impairment of the osteogenic differentiation ability of Dop ASCs.

Zmiz1a zebrafish mutants have defective erythropoiesis, altered expression of autophagy genes, and a deficient response to vitamin D

ZMIZ1 is a transcriptional coactivator that is related to members of the protein inhibitor of activated STAT (PIAS) family. ZMIZ1 regulates the activity of various transcription factors including the androgen receptor, p53, and Smad3. ZMIZ1 also interacts with Notch1 and selectively regulates Notch1 target genes relevant for T cell development and leukemogenesis in mammals. Human ZMIZ1 is additionally characterized as a latitude-dependent autoimmune disease (LDAD) risk gene, as it is responsive to vitamin D and has been associated with at least eleven blood cell traits.To address the function of ZMIZ1 in fish, we introduced CRISPR/Cas9 mutations in the zmiz1a gene in zebrafish. We observed that inactivation of zmiz1a in developing zebrafish larvae results in lethality at 15 days post fertilization (dpf) and delayed erythroid maturation. Differential gene expression analysis indicated that 15 dpf zmiz1a-null larvae had altered expression of autophagy genes, and erythrocytes that lacked Zmiz1a function exhibited an accumulation of mitochondrial DNA. Furthermore, we observed that autophagy gene expression was dysregulated at earlier stages of development, which suggests the involvement of Zmiz1a in the regulation of autophagy genes beyond the process of red blood cell differentiation. Finally, we showed that the loss of Zmiz1a decreased the capacity of the embryos to respond to vitamin D, indicating additional participation of Zmiz1a as a mediator of vitamin D activity.

Calpain 6 inhibits autophagy in inflammatory environments: A preliminary study on myoblasts and a chronic kidney disease rat model.

A non-classical calpain, calpain 6 (CAPN6), can inhibit skeletal muscle differentiation and regeneration. In the present study, the role of CAPN6 in the regulation of the autophagy of myoblasts in vitro was investigated. The underlying molecular events and the CAPN6 level in atrophic skeletal muscle in a rat model of chronic kidney disease (CKD) were also investigated. In vitro, CAPN6 was overexpressed, or knocked down, in rat L6 myoblasts to assess autophagy and related gene expression and co-localization. Subsequently, myoblasts were treated with a mixture of cytokines, and relative gene expression and autophagy were assessed. A rat model of CKD for muscle atrophy was established, and blood chemical level and the expression of CAPN6 in muscle were assessed. The data revealed that the knockdown of CAPN6 in rat myoblasts resulted in increased microtubule-associated protein 1 light chain 3 (LC3) levels, while its overexpression decreased LC3 levels and impaired autophagy. Additionally, it was observed that the co-localization of mammalian target of rapamycin (mTOR) and lysosomal-associated membrane protein 1 (LAMP1), a lysosomal marker, proteins was increased. In addition, mTOR, Raptor and α-tubulin (a marker of microtubules) increased in the CAPN6 overexpression group. However, inflammatory cytokines, such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, interferon (INF)-γ and lipopolysaccharides upregulated CAPN6 expression, inhibited L6 myoblast autophagy and stabilized mTOR activity. Furthermore, the animal model successfully mimicked human disease as regards an increase in body weight, and a reduction in muscle mass, cross-sectional area and blood biomarker concentrations; a slight increase in CAPN6 mRNA and protein levels in muscles was observed. Finally, the data of the present study suggested that CAPN6 reduced autophagy via the maintenance of mTOR signaling, which may play a role in CKD-related muscle atrophy. However, future studies are required to determine whether CAPN6 may be used as an intervention target for CKD-related skeletal muscle atrophy.

Methamphetamine-Triggered Neurotoxicity in Human Dorsolateral Prefrontal Cortex.

Background:Methamphetamine (MA), is an extremely addictive stimulant that adversely affects the central nervous system. Accumulating evidence indicates that molecular mechanisms such as oxidative stress, apoptosis, and autophagy are involved in the toxicity of MA. Considering experimental animal studies exhibiting MA-induced neurotoxicity, the relevance of these findings needs to be evidently elucidated in human MA users. It is generally assumed that multiple chemical substances released in the brain following MA-induced metabolic activation are primary factors underlying damage of neural cells. Hence, this study aimed to investigate the role of autophagy and apoptosis as well as oxidative stress in the brain of postmortem MA-induced toxicity.Materials and Methods:In this study, we determine the gene expression of autophagy and apoptosis, including BECN1, MAP1ALC3, CASP8, TP53, and BAX genes in ten healthy controls and ten chronic users of MA postmortem dorsolateral prefrontal cortex (DLPFC) by real-time polymerase chain reaction. Also, we applied immunohistochemistry in formalin-fixed and paraffin-embedded human brain samples to analyze brain-derived neurotrophic factor (BDNF). Also, spectrophotometry was performed to measure glutathione (GSH) content.Results:The expression level of apoptotic and autophagic genes (BECN1, MAP1ALC3, CASP8, TP53, and BAX) were significantly elevated, while GSH content and BDNF showed substantial reductions in DLPFC of chronic MA users. Discussion: Our data showed that MA addiction provokes transduction pathways, namely apoptosis and autophagy, along with oxidative mechanisms in DLPFC. Also, MA induces multiple functional and structural perturbations in the brain, determining its toxicity and possibly contributing to neurotoxicity. Discussion:Our study showed BDNF-positive cells as well as GSH amount, displayed significant declines in DLPFC of MA user. MA addiction provokes transduction pathways, namely apoptosis and autophagy, along with oxidative mechanisms in DLPFC. However, further investigations are needed to throw light on the cellular and molecular mechanisms that act in the various regions of the addicted brain, especially in DLPFC.

Methamphetamine-Triggered Neurotoxicity in Human Dorsolateral Prefrontal Cortex

Background: Methamphetamine (MA), is an extremely addictive stimulant that adversely affects the central nervous system. Accumulating evidence indicates that molecular mechanisms such as oxidative stress, apoptosis, and autophagy are involved in the toxicity of MA. Considering experimental animal studies exhibiting MA-induced neurotoxicity, the relevance of these findings needs to be evidently elucidated in human MA users. It is generally assumed that multiple chemical substances released in the brain following MA-induced metabolic activation are primary factors underlying damage of neural cells. Hence, this study aimed to investigate the role of autophagy and apoptosis as well as oxidative stress in the brain of postmortem MA-induced toxicity. Materials and Methods: In this study, we determine the gene expression of autophagy and apoptosis, including BECN1, MAP1ALC3, CASP8, TP53, and BAX genes in ten healthy controls and ten chronic users of MA postmortem dorsolateral prefrontal cortex (DLPFC) by real-time polymerase chain reaction. Also, we applied immunohistochemistry in formalin-fixed and paraffin-embedded human brain samples to analyze brain-derived neurotrophic factor (BDNF). Also, spectrophotometry was performed to measure glutathione (GSH) content. Results: The expression level of apoptotic and autophagic genes (BECN1, MAP1ALC3, CASP8, TP53, and BAX) were significantly elevated, while GSH content and BDNF showed substantial reductions in DLPFC of chronic MA users. Conclusion: Our data showed that MA addiction provokes transduction pathways, namely apoptosis and autophagy, along with oxidative mechanisms in DLPFC. Also, MA induces multiple functional and structural perturbations in the brain, determining its toxicity and possibly contributing to neurotoxicity. [GMJ.2021;10:e2016]

Alteration in autophagy gene expression profile correlates with low sperm quality

AimsLow sperm quality, a crucial factor of male infertility, is becoming a public health issue all over the world. In male reproductive system, autophagy plays an important role in maintaining physiological functions. There exist conjectures that disordered autophagy autophagy might be related to low sperm quality. However, there is no evidence can confirm that. This study aims to investigate the association between autophagy-associated genes and low sperm quality. MethodsSperm samples of low sperm quality cases and matched controls were included to select differential expressed genes (DE genes) by autophagy-related functional gene microarray analysis. Then, 104 cases and 250 controls were included to validate the expression of four important autophagy genes (CXCR4, ESR1, PTEN and LC3B). Based on the obtained DE gene, gene Ontology and pathway analyses were conducted. ResultsChip results showed that expression of all 18 DE genes were decreased in the cases compared to the controls (P < 0.05). Expression of ESR1 were verified to be significantly decreased (P < 0.05). ConclusionOur results provided clues with the association among down-regulated expression of autophagy regulating and associated genes and low sperm quality. These findings revealed possible role of impaired autophagy in the mechanism of low sperm quality. Moreover, these may also provide potential targets for the treatment of low sperm quality.

Invasive Pathobionts Contribute to Colon Cancer Initiation by Counterbalancing Epithelial Antimicrobial Responses.

Background & AimsMicrobiota dysbiosis and mucosa-associated bacteria are involved in colorectal cancer progression. We hypothesize that an interaction between virulent pathobionts and epithelial defense promotes tumorigenesis.MethodsChemical-induced CRC mouse model was treated with antibiotics at various phases. Colonic tissues and fecal samples were collected in a time-serial mode and analyzed by gene microarray and 16S rRNA sequencing. Intraepithelial bacteria were isolated using a gentamicin resistance assay, and challenged in epithelial cultures.ResultsOur study showed that antibiotic treatment at midphase but not early or late phase reduced mouse tumor burden, suggesting a time-specific host–microbe interplay. A unique antimicrobial transcriptome profile showing an inverse relationship between autophagy and oxidative stress genes was correlated with a transient surge in microbial diversity and virulence emergence in mouse stool during cancer initiation. Gavage with fimA/fimH/htrA-expressing invasive Escherichia coli isolated from colonocytes increased tumor burden in recipient mice, whereas inoculation of bacteria deleted of htrA or triple genes did not. The invasive E.coli suppressed epithelial autophagy activity through reduction of microtubule-associated protein 1 light-chain 3 transcripts and caused dual oxidase 2–dependent free radical overproduction and tumor cell hyperproliferation. A novel alternating spheroid culture model was developed for sequential bacterial challenge to address the long-term changes in host–microbe interaction for chronic tumor growth. Epithelial cells with single bacterial encounter showed a reduction in transcript levels of autophagy genes while those sequentially challenged with invasive E.coli showed heightened autophagy gene expression to eliminate intracellular microbes, implicating that bacteria-dependent cell hyperproliferation could be terminated at late phases. Finally, the presence of bacterial htrA and altered antimicrobial gene expression were observed in human colorectal cancer specimens.ConclusionsInvasive pathobionts contribute to cancer initiation during a key time frame by counterbalancing autophagy and oxidative stress in the colonic epithelium. Monitoring gut microbiota and antimicrobial patterns may help identify the window of opportunity for intervention with bacterium-targeted precision medicine.

Epigenetic Control of Autophagy Related Genes Transcription in Pulpitis via JMJD3.

Autophagy is an intracellular self-cannibalization process delivering cytoplasmic components to lysosomes for digestion. Autophagy has been reported to be involved in pulpitis, but the regulation of autophagy during pulpitis progression is largely unknown. To figure out the epigenetic regulation of autophagy during pulpitis, we screened several groups of histone methyltransferases and demethylases in response to TNFα treatment. It was found JMJD3, a histone demethylase reducing di- and tri-methylation of H3K27, regulated the expression of several key autophagy genes via demethylation of H3K27me3 at the gene promoters. Our study highlighted the epigenetic regulation of autophagy genes during pulpitis, which will potentially provide a novel therapeutic strategy.