Persistent mTORC1 activation due to loss of liver tuberous sclerosis complex 1 promotes liver injury in alcoholic hepatitis.

BACKGROUND AND AIMS: The aim of the study was to investigate the role and mechanisms of tuberous sclerosis complex 1 (TSC1) and mechanistic target of rapamycin complex 1 (mTORC1) in alcohol-associated liver disease. APPROACH AND RESULTS: Liver-specific Tsc1 knockout (L- Tsc1 KO) mice and their matched wild-type mice were subjected to Gao-binge alcohol. Human alcoholic hepatitis (AH) samples were also used for immunohistochemistry staining, western blot, and quantitative real-time PCR (q-PCR) analysis. Human AH and Gao-binge alcohol-fed mice had decreased hepatic TSC1 and increased mTORC1 activation. Gao-binge alcohol markedly increased liver/body weight ratio and serum alanine aminotransferase levels in L- Tsc1 KO mice compared with Gao-binge alcohol-fed wild-type mice. Results from immunohistochemistry staining, western blot, and q-PCR analysis revealed that human AH and Gao-binge alcohol-fed L- Tsc1 KO mouse livers had significantly increased hepatic progenitor cells, macrophages, and neutrophils but decreased HNF4α-positive cells. Gao-binge alcohol-fed L- Tsc1 KO mice also developed severe inflammation and liver fibrosis. Deleting Tsc1 in cholangiocytes but not in hepatocytes promoted cholangiocyte proliferation and aggravated alcohol-induced ductular reactions, fibrosis, inflammation, and liver injury. Pharmacological inhibition of mTORC1 partially reversed hepatomegaly, ductular reaction, fibrosis, inflammatory cell infiltration, and liver injury in alcohol-fed L- Tsc1 KO mice. CONCLUSIONS: Our findings indicate that persistent activation of mTORC1 due to the loss of cholangiocyte TSC1 promotes liver cell repopulation, ductular reaction, inflammation, fibrosis, and liver injury in Gao-binge alcohol-fed L- Tsc1 KO mice, which phenocopy the pathogenesis of human AH.

Loss of Hepatic Transcription Factor EB Attenuates Alcohol-Associated Liver Carcinogenesis.

Alcohol is a well-known risk factor for hepatocellular carcinoma. Autophagy plays a dual role in liver cancer, as it suppresses tumor initiation and promotes tumor progression. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy, which is impaired in alcohol-related liver disease. However, the role of TFEB in alcohol-associated liver carcinogenesis is unknown. Liver-specific Tfeb knockout (KO) mice and their matched wild-type (WT) littermates were injected with the carcinogen diethylnitrosamine (DEN), followed by chronic ethanol feeding. The numbers of both total and larger tumors increased significantly in DEN-treated mice fed ethanol diet than in mice fed control diet. Although the number of tumors was not different between WT and L-Tfeb KO mice fed either control or ethanol diet, the number of larger tumors was less in L-Tfeb KO mice than in WT mice. No differences were observed in liver injury, steatosis, inflammation, ductular reaction, fibrosis, and tumor cell proliferation in DEN-treated mice fed ethanol. However, the levels of glypican 3, a marker of malignant hepatocellular carcinoma, markedly decreased in DEN-treated L-Tfeb KO mice fed ethanol in comparison to the WT mice. These findings indicate that chronic ethanol feeding promotes DEN-initiated liver tumor development, which is attenuated by genetic deletion of hepatic TFEB.

Salvianolic acid B suppresses hepatic fibrosis by inhibiting ceramide glucosyltransferase in hepatic stellate cells.

UDP-glucose ceramide glucosyltransferase (UGCG) is the first key enzyme in glycosphingolipid (GSL) metabolism that produces glucosylceramide (GlcCer). Increased UGCG synthesis is associated with cell proliferation, invasion and multidrug resistance in human cancers. In this study we investigated the role of UGCG in the pathogenesis of hepatic fibrosis. We first found that UGCG was over-expressed in fibrotic livers and activated hepatic stellate cells (HSCs). In human HSC-LX2 cells, inhibition of UGCG with PDMP or knockdown of UGCG suppressed the expression of the biomarkers of HSC activation (α-SMA and collagen I). Furthermore, pretreatment with PDMP (40 µM) impaired lysosomal homeostasis and blocked the process of autophagy, leading to activation of retinoic acid signaling pathway and accumulation of lipid droplets. After exploring the structure and key catalytic residues of UGCG in the activation of HSCs, we conducted virtual screening, molecular interaction and molecular docking experiments, and demonstrated salvianolic acid B (SAB) from the traditional Chinese medicine Salvia miltiorrhiza as an UGCG inhibitor with an IC50 value of 159 µM. In CCl4-induced mouse liver fibrosis, intraperitoneal administration of SAB (30 mg · kg-1 · d-1, for 4 weeks) significantly alleviated hepatic fibrogenesis by inhibiting the activation of HSCs and collagen deposition. In addition, SAB displayed better anti-inflammatory effects in CCl4-induced liver fibrosis. These results suggest that UGCG may represent a therapeutic target for liver fibrosis; SAB could act as an inhibitor of UGCG, which is expected to be a candidate drug for the treatment of liver fibrosis.

Decreased syntaxin17 expression contributes to the pathogenesis of acute pancreatitis in murine models by impairing autophagic degradation.

Acute pancreatitis (AP) is an inflammatory disease of the exocrine pancreas. Disruptions in organelle homeostasis, including macroautophagy/autophagy dysfunction and endoplasmic reticulum (ER) stress, have been implicated in human and rodent pancreatitis. Syntaxin 17 (STX17) belongs to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) subfamily. The Qa-SNARE STX17 is an autophagosomal SNARE protein that interacts with SNAP29 (Qbc-SNARE) and the lysosomal SNARE VAMP8 (R-SNARE) to drive autophagosome-lysosome fusion. In this study, we investigated the role of STX17 in the pathogenesis of AP in male mice or rats induced by repeated intraperitoneal injections of cerulein. We showed that cerulein hyperstimulation induced AP in mouse and rat models, which was characterized by increased serum amylase and lipase activities, pancreatic edema, necrotic cell death and the infiltration of inflammatory cells, as well as markedly decreased pancreatic STX17 expression. A similar reduction in STX17 levels was observed in primary and AR42J pancreatic acinar cells treated with CCK (100 nM) in vitro. By analyzing autophagic flux, we found that the decrease in STX17 blocked autophagosome-lysosome fusion and autophagic degradation, as well as the activation of ER stress. Pancreas-specific STX17 knockdown using adenovirus-shSTX17 further exacerbated pancreatic edema, inflammatory cell infiltration and necrotic cell death after cerulein injection. These data demonstrate a critical role of STX17 in maintaining pancreatic homeostasis and provide new evidence that autophagy serves as a protective mechanism against AP.

Hepatocytic p62 suppresses ductular reaction and tumorigenesis in mouse livers with mTORC1 activation and defective autophagy.

BACKGROUND & AIMS: Either activation of mTORC1 due to loss of Tsc1 (tuberous sclerosis complex 1) or defective hepatic autophagy due to loss of Atg5 leads to spontaneous liver tumorigenesis in mice. The purpose of this study was to investigate the mechanisms by which autophagy contributes to the hepatic metabolic changes and tumorigenesis mediated by mTORC1 activation. METHODS: Atg5 Flox/Flox (Atg5F/F) and Tsc1F/F mice were crossed with albumin-Cre mice to generate liver-specific Atg5 knockout (L-Atg5 KO), L-Tsc1 KO and L-Atg5/Tsc1 double KO (DKO) mice. These mice were crossed with p62/Sqstm1F/F (p62) and whole body Nrf2 KO mice to generate L-Atg5/Tsc1/p62 and L-Atg5/Tsc1-Nrf2 triple KO mice. These mice were housed for various periods up to 12 months, and blood and liver tissues were harvested for biochemical and histological analysis RESULTS: Deletion of Atg5 in L-Tsc1 KO mice inhibited liver tumorigenesis but increased mortality and was accompanied by drastically enhanced hepatic ductular reaction (DR), hepatocyte degeneration and metabolic reprogramming. Deletion of p62 reversed DR, hepatocyte degeneration and metabolic reprogramming as well as the mortality of L-Atg5/Tsc1 DKO mice, but unexpectedly promoted liver tumorigenesis via activation of a group of oncogenic signaling pathways. Nrf2 ablation markedly improved DR with increased hepatocyte population and improved metabolic reprogramming and survival of the L-Atg5/Tsc1 DKO mice without tumor formation. Decreased p62 and increased mTOR activity were also observed in a subset of human hepatocellular carcinomas. CONCLUSIONS: These results reveal previously undescribed functions of hepatic p62 in suppressing tumorigenesis and regulating liver cell repopulation and metabolic reprogramming resulting from persistent mTORC1 activation and defective autophagy. LAY SUMMARY: Metabolic liver disease and viral hepatitis are common chronic liver diseases and risk factors of hepatocellular carcinoma, which are often associated with impaired hepatic autophagy and increased mTOR activation. Using multiple genetically engineered mouse models of defective hepatic autophagy and persistent mTOR activation, we dissected the complex mechanisms behind this observation. Our results uncovered an unexpected novel tumor suppressor function of p62/Sqstm1, which regulated liver cell repopulation, ductular reaction and metabolic reprogramming in liver tumorigenesis.

Role of Mechanistic Target of Rapamycin and Autophagy in Alcohol-Induced Adipose Atrophy and Liver Injury.

Chronic alcohol consumption induces adipose tissue atrophy. However, the mechanisms for how alcohol induces lipodystrophy and its impact on liver steatosis and injury are not fully elucidated. Autophagy is a highly conserved lysosomal degradation pathway, which regulates cellular homeostasis. Mice with autophagy deficiency in adipose tissue have impaired adipogenesis. However, whether autophagy plays a role in alcohol-induced adipose atrophy and how altered adipocyte autophagy contributes to alcohol-induced liver injury remain unclear. To determine the role of adipose autophagy and mechanistic target of rapamycin (mTOR) in alcohol-induced adipose and liver pathogenesis, we generated adipocyte-specific Atg5 knockout (KO), adipocyte-specific mTOR KO, adipocyte-specific Raptor KO, and adipocyte-specific tuberous sclerosis complex 1 KO mice by crossing floxed mice with Adipoq-Cre. The KO mice and their matched wild-type mice were challenged with chronic-plus-binge alcohol mouse model. Chronic-plus-binge alcohol induced adipose atrophy with increased autophagy and decreased Akt/mTOR signaling in epididymal adipose tissue in wild-type mice. Adipocyte-specific Raptor KO mice experienced exacerbated alcohol-induced steatosis, but neither adipocyte-specific mTOR nor adipocyte-specific tuberous sclerosis complex 1 KO mice exhibited similar detrimental effects. Adipocyte-specific Atg5 KO mice had increased circulating levels of fibroblast growth factor 21 and adiponectin and were resistant to alcohol-induced adipose atrophy and liver injury. In conclusion, autophagy deficiency in adipose tissue leads to reduced sensitivity to alcohol-induced adipose atrophy, which ameliorates alcohol-induced liver injury in mice.

Trehalose activates hepatic transcription factor EB (TFEB) but fails to ameliorate alcohol-impaired TFEB and liver injury in mice.

BACKGROUND: Recent evidence demonstrates that alcohol activates the mechanistic target of rapamycin (mTOR) and impairs hepatic transcription factor EB (TFEB) reducing autophagy and contributing to alcohol-induced liver injury. Trehalose, a disaccharide, activates TFEB and protects against diet-induced nonalcoholic fatty liver disease in mice. The aim of the present study was to investigate whether trehalose would reverse the impairment of TFEB induced by alcohol and protect against alcohol-induced liver injury. METHODS: Male C57BL/6J mice were subjected to chronic-plus-binge (Gao-binge) alcohol feeding with and without trehalose supplementation. Some mice were also administrered Alda-1, an aldehyde dehydrogenase 2 agonist. RESULTS: We found that Alda-1 did not affect Gao-binge alcohol-induced mTOR activation and impaired TFEB in mouse livers. Trehalose increased TFEB nuclear translocation, elevated levels of LC3-II and lysosomal proteins in mouse livers and cultured AML12 cells, confirming the activation of TFEB by trehalose. However, trehalose did not improve the impairment in TFEB induced by Gao-binge alcohol. Both Alda-1 and trehalose failed to protect against Gao-binge alcohol-induced steatosis and liver injury, based on the serum levels of alanine aminotransferase (ALT), histological analysis, and levels of hepatic triglyceride. Interestingly, trehalose increased expression of pro-inflammatory genes in mouse macrophage RAW264.7 cells and slightly increased the infiltration of hepatic neutrophils and inflammatory cytokine gene expression in Gao-binge alcohol-fed mice livers. CONCLUSIONS: Trehalose fails to improve the impaired TFEB induced by Gao-binge alcohol and does not protect against alcohol-induced liver injury.

Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)-Mixed Lineage Kinase Domain-Like Protein (MLKL)-Mediated Necroptosis Contributes to Ischemia-Reperfusion Injury of Steatotic Livers.

Increased hepatic ischemia-reperfusion (IR) injury in steatotic livers is a major reason for rejecting the use of fatty livers for liver transplantation. Necroptosis is implicated in the pathogenesis of fatty liver diseases. Necroptosis is regulated by three key proteins: receptor-interacting serine/threonine-protein kinase (RIPK)-1, RIPK3, and mixed-lineage kinase domain-like protein (MLKL). Here, we found that marked steatosis of the liver was induced when a Western diet was given in mice; steatosis was associated with the inhibition of hepatic proteasome activities and with increased levels of key necroptosis-related proteins. Mice fed a Western diet had more severe liver injury, as demonstrated by increases in serum alanine aminotransferase and necrotic areas of liver, after IR than did mice fed a control diet. Although hepatic steatosis was not different between Mlkl knockout mice and wild-type mice, Mlkl knockout mice had decreased hepatic neutrophil infiltration and inflammation and were protected from hepatic IR injury, irrespective of diet. Intriguingly, Ripk3 knockout or Ripk3 kinase-dead knock-in mice were protected against IR injury at the late phase but not the early phase, irrespective of diet. Overall, our findings indicate that liver steatosis exacerbates hepatic IR injury via increased MLKL-mediated necroptosis. Targeting MLKL-mediated necroptosis may help to improve outcomes in steatotic liver transplantation.

Dynamin-1-Like Protein Inhibition Drives Megamitochondria Formation as an Adaptive Response in Alcohol-Induced Hepatotoxicity.

Despite the growing global burden of alcoholic liver diseases, therapeutic options are limited, and novel targets are urgently needed. Accumulating evidence suggests that mitochondria adapt in response to ethanol and formation of megamitochondria in the livers of patients is recognized as a hallmark of alcoholic liver diseases. The processes involved in ethanol-induced hepatic mitochondrial changes, the impact on mitochondria-shaping proteins, and the significance of megamitochondria formation remain unknown. In this study, we investigated the mitochondrial and cellular response to alcohol in hepatoma cell line VL-17A. The mitochondrial architecture rapidly changed after 3 or 14 days of ethanol exposure with double-pronged presentation of hyperfragmentation and megamitochondria, and cell growth was inhibited. Dynamin-1-like protein (Drp1) was identified as the main mediator driving these mitochondrial alterations, and its genetic inactivation was determined to foster megamitochondria development, preserving the capacity of the cells to grow despite alcohol toxicity. The role of Drp1 in mediating megamitochondria formation in mice with liver-specific inactivation of Drp1 was further confirmed. Finally, when these mice were fed with ethanol, the presentation of hepatic megamitochondria was exacerbated compared with wild type fed with the same diet. Ethanol-induced toxicity was also reduced. Our study demonstrates that megamitochondria formation is mediated by Drp1, and this phenomenon is a beneficial adaptive response during alcohol-induced hepatotoxicity.

Dual Roles of Mammalian Target of Rapamycin in Regulating Liver Injury and Tumorigenesis in Autophagy-Defective Mouse Liver.

Autophagy is a lysosomal degradation pathway that degrades cytoplasmic proteins and organelles. Absence of autophagy in hepatocytes has been linked to promoting liver injury and tumorigenesis; however, the mechanisms behind why a lack of autophagy induces these complications are not fully understood. The role of mammalian target of rapamycin (mTOR) in impaired autophagy-induced liver pathogenesis and tumorigenesis was investigated by using liver-specific autophagy related 5 knockout (L-ATG5 KO) mice, L-ATG5/mTOR, and L-ATG5/Raptor double knockout (DKO) mice. We found that deletion of mTOR or Raptor in L-ATG5 KO mice at 2 months of age attenuated hepatomegaly, cell death, and inflammation but not fibrosis. Surprisingly, at 6 months of age, L-ATG5/mTOR DKO and L-ATG5/Raptor DKO mice also had increased hepatic inflammation, fibrosis, and liver injury, similar to the L-ATG5 KO mice. Moreover, more than 50% of L-ATG5/mTOR DKO and L-ATG5/Raptor DKO mice already developed spontaneous tumors, but none of the L-ATG5 KO mice had developed any tumors at 6 months of age. At 9 months of age, all L-ATG5/mTOR DKO and L-ATG5/Raptor DKO had developed liver tumors. Mechanistically, L-ATG5/mTOR DKO and L-ATG5/Raptor DKO mice had decreased levels of hepatic ubiquitinated proteins and persistent nuclear erythroid 2 p45-related factor 2 activation but had increased Akt activation compared with L-ATG5 KO mice. Conclusion: Loss of mTOR signaling attenuates the liver pathogenesis in mice with impaired hepatic autophagy but paradoxically promotes tumorigenesis in mice at a relatively young age. Therefore, the balance of mTOR is critical in regulating the liver pathogenesis and tumorigenesis in mice with impaired hepatic autophagy.

Impaired TFEB-Mediated Lysosome Biogenesis and Autophagy Promote Chronic Ethanol-Induced Liver Injury and Steatosis in Mice.

BACKGROUND & AIMS: Defects in lysosome function and autophagy contribute to the pathogenesis of alcoholic liver disease. We investigated the mechanisms by which alcohol consumption affects these processes by evaluating the functions of transcription factor EB (TFEB), which regulates lysosomal biogenesis. METHODS: We performed studies with GFP-LC3 mice, mice with liver-specific deletion of TFEB, mice with disruption of the transcription factor E3 gene (TFE3-knockout mice), mice with disruption of the Tefb and Tfe3 genes (TFEB and TFE3 double-knockout mice), and Tfebflox/flox albumin cre-negative mice (controls). TFEB was overexpressed from adenoviral vectors or knocked down with small interfering RNAs in mouse livers. Mice were placed on diets of regular ethanol feeding plus an acute binge to induce liver damage (ethanol diet); some mice also were given injections of torin-1, an inhibitor of the kinase activity of the mechanistic target of rapamycin (mTOR). Liver tissues were collected and analyzed by immunohistochemistry, immunoblots, and quantitative real-time polymerase chain reaction to monitor lysosome biogenesis. We analyzed levels of TFEB in liver tissues from patients with alcoholic hepatitis and from healthy donors (controls) by immunohistochemistry. RESULTS: Liver tissues from mice on the ethanol diet had lower levels of total and nuclear TFEB compared with control mice, and hepatocytes had decreased lysosome biogenesis and autophagy. Hepatocytes from mice on the ethanol diet had increased translocation of mTOR into lysosomes, resulting in increased mTOR activation. Administration of torin-1 increased liver levels of TFEB and decreased steatosis and liver injury induced by ethanol. Mice that overexpressed TFEB in the liver developed less severe ethanol-induced liver injury and had increased lysosomal biogenesis and mitochondrial bioenergetics compared with mice carrying a control vector. Mice with knockdown of TFEB and TFEB-TFE3 double-knockout mice developed more severe liver injury in response to the ethanol diet than control mice. Liver tissues from patients with alcohol-induced hepatitis had lower nuclear levels of TFEB than control tissues. CONCLUSIONS: We found that ethanol feeding plus an acute binge decreased hepatic expression of TFEB, which is required for lysosomal biogenesis and autophagy. Strategies to block mTOR activity or increase levels of TFEB might be developed to protect the liver from ethanol-induced damage.

A Mechanistic Review of Cell Death in Alcohol-Induced Liver Injury.

Alcoholic liver disease (ALD) is a major health problem in the United States and worldwide without successful treatments. Chronic alcohol consumption can lead to ALD, which is characterized by steatosis, inflammation, fibrosis, cirrhosis, and even liver cancer. Recent studies suggest that alcohol induces both cell death and adaptive cell survival pathways in the liver, and the balance of cell death and cell survival ultimately decides the pathogenesis of ALD. This review summarizes the recent progress on the role and mechanisms of apoptosis, necroptosis, and autophagy in the pathogenesis of ALD. Understanding the complex regulation of apoptosis, necrosis, and autophagy may help to develop novel therapeutic strategies by targeting all 3 pathways simultaneously.

RhoA/rho kinase signaling reduces connexin43 expression in high glucose-treated glomerular mesangial cells with zonula occludens-1 involvement.

RhoA/Rho kinase (ROCK) signaling has been suggested to be involved in diabetic nephropathy (DN) pathogenesis. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. Both of them have been found to regulate nuclear factor kappa-B (NF-κB) activation in high glucose-treated glomerular mesangial cells (GMCs). The aim of this study was to investigate the relationship between RhoA/ROCK signaling and Cx43 in the DN pathogenesis. We found that upregulation of Cx43 expression inhibited NF-κB p65 nuclear translocation induced by RhoA/ROCK signaling in GMCs. Inhibition of RhoA/ROCK signaling attenuated the high glucose-induced decrease in Cx43. F-actin accumulation and an enhanced interaction between zonula occludens-1 (ZO-1) and Cx43 were observed in high glucose-treated GMCs. ZO-1 depletion or disruption of F-actin formation also inhibited the reduction in Cx43 protein levels induced by high glucose. In conclusion, activated RhoA/ROCK signaling induces Cx43 degradation in GMCs cultured in high glucose, depending on F-actin regulation. Increased F-actin induced by RhoA/ROCK signaling promotes the association between ZO-1 and Cx43, which possibly triggered Cx43 endocytosis, a mechanism of NF-κB activation in high glucose-treated GMCs.

Recent insights about autophagy in pancreatitis.

Acute pancreatitis is a common inflammatory gastrointestinal disease without any successful treatment. Pancreatic exocrine acinar cells have high rates of protein synthesis to produce and secrete large amounts of digestive enzymes. When the regulation of organelle and protein homeostasis is disrupted, it can lead to endoplasmic reticulum (ER) stress, damage to the mitochondria and improper intracellular trypsinogen activation, ultimately resulting in acinar cell damage and the onset of pancreatitis. To balance the homeostasis of organelles and adapt to protect themselves from organelle stress, cells use protective mechanisms such as autophagy. In the mouse pancreas, defective basal autophagy disrupts ER homoeostasis, leading to ER stress and trypsinogen activation, resulting in spontaneous pancreatitis. In this review, we discuss the regulation of autophagy and its physiological role in maintaining acinar cell homeostasis and function. We also summarise the current understanding of the mechanisms and the role of defective autophagy at multiple stages in experimental pancreatitis induced by cerulein or alcohol.

Natural compounds modulating mitophagy: Implications for cancer therapy.

Cancer is considered as the second leading cause of mortality, and cancer incidence is still growing rapidly worldwide, which poses an increasing global health burden. Although chemotherapy is the most widely used treatment for cancer, its effectiveness is limited by drug resistance and severe side effects. Mitophagy is the principal mechanism that degrades damaged mitochondria via the autophagy/lysosome pathway to maintain mitochondrial homeostasis. Emerging evidence indicates that mitophagy plays crucial roles in tumorigenesis, particularly in cancer therapy. Mitophagy can exhibit dual effects in cancer, with both cancer-inhibiting or cancer-promoting function in a context-dependent manner. A variety of natural compounds have been found to affect cancer cell death and display anticancer properties by modulating mitophagy. In this review, we provide a systematic overview of mitophagy signaling pathways, and examine recent advances in the utilization of natural compounds for cancer therapy through the modulation of mitophagy. Furthermore, we address the inquiries and challenges associated with ongoing investigations concerning the application of natural compounds in cancer therapy based on mitophagy. Overcoming these limitations will provide opportunities to develop novel interventional strategies for cancer treatment.

High-throughput screening of novel TFEB agonists in protecting against acetaminophen-induced liver injury in mice.

Macroautophagy (referred to as autophagy hereafter) is a major intracellular lysosomal degradation pathway that is responsible for the degradation of misfolded/damaged proteins and organelles. Previous studies showed that autophagy protects against acetaminophen (APAP)-induced injury (AILI) via selective removal of damaged mitochondria and APAP protein adducts. The lysosome is a critical organelle sitting at the end stage of autophagy for autophagic degradation via fusion with autophagosomes. In the present study, we showed that transcription factor EB (TFEB), a master transcription factor for lysosomal biogenesis, was impaired by APAP resulting in decreased lysosomal biogenesis in mouse livers. Genetic loss-of and gain-of function of hepatic TFEB exacerbated or protected against AILI, respectively. Mechanistically, overexpression of TFEB increased clearance of APAP protein adducts and mitochondria biogenesis as well as SQSTM1/p62-dependent non-canonical nuclear factor erythroid 2-related factor 2 (NRF2) activation to protect against AILI. We also performed an unbiased cell-based imaging high-throughput chemical screening on TFEB and identified a group of TFEB agonists. Among these agonists, salinomycin, an anticoccidial and antibacterial agent, activated TFEB and protected against AILI in mice. In conclusion, genetic and pharmacological activating TFEB may be a promising approach for protecting against AILI.

Connexin43 mediates NF-κB signalling activation induced by high glucose in GMCs: involvement of c-Src.

BACKGROUND: Nuclear factor kappa-B (NF-κB) signalling plays an important role in diabetic nephropathy. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. The aim of the current study was to investigate the role of Cx43 in the activation of NF-κB induced by high glucose in glomerular mesangial cells (GMCs) and to determine whether c-Src is involved in this process. RESULTS: We found that downregulation of Cx43 expression induced by high glucose activated NF-κB in GMCs. Orverexpression of Cx43 attenuated NF-κB p65 nuclear translocation induced by high glucose. High glucose inhibited the interaction between Cx43 and c-Src, and enhanced the interaction between c-Src and IκB-α. PP2, a c-Src inhibitor, also inhibited the tyrosine phosphorylation of IκB-α and NF-κB p65 nuclear translocation induced by high glucose. Furthermore, overexpression of Cx43 or inhibition of c-Src attenuated the upregulation of intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-ß1) and fibronectin (FN) expression induced by high glucose. CONCLUSIONS: In conclusion, downregulation of Cx43 in GMCs induced by high glucose activates c-Src, which in turn promotes interaction between c-Src and IκB-α and contributes to NF-κB activation in GMCs, leading to renal inflammation.