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.
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.
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.
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.
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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.
Cell death and cell survival are closely associated with alcoholic liver disease.
Ethanol metabolism leads to increased oxidative stress resulting in mitochondrial damage and apoptosis in hepatocytes.
Chronic alcohol consumption increases gut permeability and elevated systemic levels of gut‐derived endotoxins resulting in increased production of TNF‐α from activated Kupffer cells.
Depending on the cIAP levels and caspase‐8 activation in hepatocytes, TNF‐α either induces apoptosis or necroptosis by activating Bid‐mediated mitochondrial apoptotic pathway or ripoptosome or necrosome.
Advanced glycation end products (AGEs) boost the generation of reactive oxygen species (ROS) in glomerular mesangial cells (GMCs), and thereby play important roles in diabetic nephropathy (DN). ...Sirtuin 1 (Sirt1), a protein deacetylase, is known to markedly protect cells from oxidative stress (OSS) injury. Based on the critical involvements of AGEs and Sirt1 in OSS, Sirt1 is postulated to resist AGEs-induced diabetic renal fibrosis through its antioxidative effects. The current study was designed to explore the inhibitory effect of Sirt1 on the expressions of fibronectin (FN) and transforming growth factor-β1 (TGF-β1) induced by AGEs in GMCs. The molecular mechanism by which Sirt1 promoted the activation of the antioxidative pathway was further investigated. The following findings were obtained: (1) the treatment of GMCs with AGEs decreased Sirt1 levels in terms of protein expression and activity but increased FN and TGF-β1 levels in a dose- and time-dependent manner; (2) resveratrol or Sirt1 overexpression markedly increased Sirt1 levels and reduced FN and TGF-β1 expressions; (3) inhibition of Sirt1 activity further induced the productions of FN and TGF-β1; (4) Sirt1 promoted the nuclear accumulation, DNA binding, and transcriptional activities of Nrf2 and upregulated the expressions of Nrf2 downstream genes, heme oxygenase-1, and superoxide dismutase 1; ROS levels induced by AGEs eventually reduced in a deacetylase-dependent manner; and (5) with the deposition of AGEs in the kidneys, the diabetic rats suffered severe renal dysfunction and high OSS levels; resveratrol treatment evidently diminished the OSS levels, ameliorated renal injury, and prevented the expressions of FN and TGF-β1 in the kidneys of diabetic rats. This work supports a negative role of Sirt1 in AGE-induced overproductions of FN and TGF-β1. The molecular mechanisms that underlie the beneficial effects of Sirt1 on DN correlate well with the activation of the Nrf2/ARE antioxidative pathway.
Autophagy and liver cancer Chao, Xiaojuan; Qian, Hui; Wang, Shaogui ...
Clinical and molecular hepatology,
10/2020, Letnik:
26, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Autophagy is a highly conserved catabolic process that degrades cytosolic proteins and organelles via formation of autophagosomes that fuse with lysosomes to form autolysosomes, whereby autophagic ...cargos are degraded. Numerous studies have demonstrated that autophagy plays a critical role in the regulation of liver physiology and homeostasis, and impaired autophagy leads to the pathogenesis of various liver diseases such as viral hepatitis, alcohol associated liver diseases (AALD), non-alcoholic fatty liver diseases (NAFLD), and liver cancer. Recent evidence indicates that autophagy may play a dual role in liver cancer: inhibiting early tumor initiation while promoting progression and malignancy of already formed liver tumors. In this review, we summarized the progress of current understanding of how hepatic viral infection, alcohol consumption and diet-induced fatty liver diseases impair hepatic autophagy. We also discussed how impaired autophagy promotes liver tumorigenesis, and paradoxically how autophagy is required to promote the malignancy and progression of liver cancer. Understanding the molecular mechanisms underlying how autophagy differentially affects liver cancer development and progression may help to design better therapeutic strategies for prevention and treatment of liver cancer.
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.
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.
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.
Mitochondria damage plays a critical role in acetaminophen (APAP)-induced necrosis and liver injury. Cells can adapt and protect themselves by removing damaged mitochondria via mitophagy. ...PINK1-Parkin pathway is one of the major pathways that regulate mitophagy but its role in APAP-induced liver injury is still elusive. We investigated the role of PINK1-Parkin pathway in hepatocyte mitophagy in APAP-induced liver injury in mice. Wild-type (WT), PINK1 knockout (KO), Parkin KO, and PINK1 and Parkin double KO (DKO) mice were treated with APAP for different time points. Liver injury was determined by measuring serum alanine aminotransferase (ALT) activity, H&E staining as well as TUNEL staining of liver tissues. Tandem fluorescent-tagged inner mitochondrial membrane protein Cox8 (Cox8-GFP-mCherry) can be used to monitor mitophagy based on different pH stability of GFP and mCherry fluorescent proteins. We overexpressed Cox8-GFP-mCherry in mouse livers via tail vein injection of an adenovirus Cox8-GFP-mCherry. Mitophagy was assessed by confocal microscopy for Cox8-GFP-mCherry puncta, electron microscopy (EM) analysis for mitophagosomes and western blot analysis for mitochondrial proteins. Parkin KO and PINK1 KO mice improved the survival after treatment with APAP although the serum levels of ALT were not significantly different among PINK1 KO, Parkin KO and WT mice. We only found mild defects of mitophagy in PINK1 KO or Parkin KO mice after APAP, and improved survival in PINK1 KO and Parkin KO mice could be due to other functions of PINK1 and Parkin independent of mitophagy. In contrast, APAP-induced mitophagy was significantly impaired in PINK1-Parkin DKO mice. PINK1-Parkin DKO mice had further elevated serum levels of ALT and increased mortality after APAP administration. In conclusion, our results demonstrated that PINK1-Parkin signaling pathway plays a critical role in APAP-induced mitophagy and liver injury.
Renal tubulointerstitial fibrosis (TIF), characterized by epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells, is the typical pathological alteration in diabetic nephropathy. ...Gentiopicroside (GPS), a natural compound with anti-inflammatory activity, has been demonstrated to alleviate glomerulosclerosis, whereas whether GPS inhibits TIF
via
regulating inflammation remains unclear. In this study, diabetic db/db mice and high glucose (HG)-stimulated renal tubular epithelial cells (NRK-52E) were applied to explore the effects and mechanisms of GPS on TIF. The results
in vivo
showed that GPS effectively improves glycolipid metabolism disorder, renal dysfunction, and TIF. In particular, GPS treatment reversed the abnormal expressions of EMT marker proteins including elevated α-smooth muscle actin and vimentin and decreased E-cadherin in the kidney of db/db mice. Moreover, GPS treatment also inhibited protein expressions of angiotensinⅡ type 1 receptor (AT1R) and CK2α and the activation of the NF-κB pathway. Importantly, the aforementioned effects of GPS acted
in vivo
were further observed
in vitro
in HG-stimulated NRK-52E cells, which were independent of its effects on glucose and lipid-lowering activity but were reversed by AT1R over-expression. Together, our results indicate that GPS that directly inhibits the CK2/NF-κB inflammatory signaling pathway
via
AT1R may also contribute to the amelioration of TIF in diabetes.
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