Impaired macroautophagy/autophagy has been implicated in experimental and human pancreatitis. However, the transcriptional control governing the autophagy-lysosomal process in pancreatitis is largely ...unknown. We investigated the role and mechanisms of TFEB (transcription factor EB), a master regulator of lysosomal biogenesis, in the pathogenesis of experimental pancreatitis. We analyzed autophagic flux, TFEB nuclear translocation, lysosomal biogenesis, inflammation and fibrosis in GFP-LC3 transgenic mice, acinar cell-specific tfeb knockout (KO) and tfeb and tfe3 double-knockout (DKO) mice as well as human pancreatitis samples. We found that cerulein activated MTOR (mechanistic target of rapamycin kinase) and increased the levels of phosphorylated TFEB as well as pancreatic proteasome activities that led to rapid TFEB degradation. As a result, cerulein decreased the number of lysosomes resulting in insufficient autophagy in mouse pancreas. Pharmacological inhibition of MTOR or proteasome partially rescued cerulein-induced TFEB degradation and pancreatic damage. Furthermore, genetic deletion of tfeb specifically in mouse pancreatic acinar cells increased pancreatic edema, necrotic cell death, infiltration of inflammatory cells and fibrosis in pancreas after cerulein treatment. tfeb and tfe3 DKO mice also developed spontaneous pancreatitis with increased pancreatic trypsin activities, edema and infiltration of inflammatory cells. Finally, decreased TFEB nuclear staining was associated with human pancreatitis. In conclusion, our results indicate a critical role of impaired TFEB-mediated lysosomal biogenesis in promoting the pathogenesis of pancreatitis.
Abbreviations: AC: acinar cell; AMY: amylase; ATP6V1A: ATPase, H+ transporting, lysosomal V1 subunit A; ATP6V1B2: ATPase, H+ transporting, lysosomal V1 subunit B2; ATP6V1D: ATPase, H+ transporting, lysosomal V1 subunit D; ATP6V1H: ATPase, H+ transporting, lysosomal V1 subunit H; AV: autophagic vacuole; CDE: choline-deficient, ethionine-supplemented; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; EM: electron microscopy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; H & E: hematoxylin and eosin; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MTORC1: mechanistic target of rapamycin kinase complex 1; ND: normal donor; NEU: neutrophil; PPARGC1A/PGC1α: peroxisome proliferator-activated receptor, gamma, coactivator 1 alpha; RIPA: radio-immunoprecipitation; RPS6: ribosomal protein S6; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TM: tamoxifen; WT: wild-type; ZG: zymogen granule
Lipid droplets (LDs) are intracellular organelles that store neutral lipids as energy reservoir. Recent studies suggest that autophagy is important in maintaining the homeostasis of intracellular LDs ...by either regulating the biogenesis of LDs, mobilization of fatty acids, or degradation of LDs in cultured cells. Increasing evidence also supports a role of autophagy in regulating glucose and lipid metabolism in vivo in mammals. In response to fasting/starvation, lipids are mobilized from the adipose tissue to the liver, which increases the number of intracellular LDs and stimulates fatty acid oxidation and ketogenesis. However, it is still controversial and unclear how impaired autophagy in hepatocytes affects the biogenesis of LDs in mouse livers. In the present study, it was demonstrated that hepatic autophagy-deficient (L-Atg)5 knockout mice had impaired adaptation to fasting-induced hepatic biogenesis of LDs. The maladaptation to fasting-induced hepatic biogenesis of LDs in L-Atg5 knockout mouse livers was not due to hepatic changes of de novo lipogenesis, secretion of very-low-density lipoprotein or fatty acid β-oxidation, but it was due to persistent nuclear factor-like 2 activation because biogenesis of LDs restored in L-Atg5/nuclear factor-like 2 double-knockout mice.
Diabetic nephropathy (DN) is the leading cause of end-stage renal failure and is characterized by excessive deposition of extracellular matrix (ECM) proteins such as fibronectin (FN), in the ...glomerular mesangium and tubulointerstitium. Betulinic acid (BA), a pentacyclic triterpene derived from the bark of the white birch tree, has been demonstrated to have many pharmacological activities. However, the effect of BA on DN has not been fully elucidated. To explore the possible anti-inflammatory effects of BA and their underlying mechanisms, we used streptozotocin-induced diabetic rat kidneys and high glucose-treated glomerular mesangial cells. Our study showed BA could inhibit the degradation of IκBα and the activity of NF-κB in diabetic rat kidneys and high glucose-induced mesangial cells, resulting in reduction of FN expression. In addition, BA suppressed the DNA binding activity and transcriptional activity of NF-κB in high glucose-induced glomerular mesangial cells (GMCs). Furthermore, BA enhanced the interaction between IκBα and β-arrestin2 in mesangial cells. Taken together, our data suggest BA inhibits NF-κB activation through stabilizing NF-κB inhibitory protein IκBα, thereby preventing diabetic renal fibrosis.
•Betulinic acid ameliorated diabetic nephropathy in the streptozocin-induced diabetic rats.•Betulinic acid suppressed the expression of fibronectin in both diabetic rat kidneys and high glucose-treated GMCs.•Betulinic acid blocked high glucose-induced NF-κB activity.
Tsc1 (Tuberous Sclerosis 1) and Tsc2 are tumor suppressor genes and negative regulators of mTORC1, and the mutation of Tsc1 or Tsc2 is associated with various cancers including hepatocellular ...carcinoma (HCC). mTORC1 activation promotes anabolic protein synthesis and inhibits catabolic autophagy process. Either hepatic persistent activation of mTORC1 due to the loss of Tsc1 or impaired hepatic autophagy due to the loss of Atg5 leads to spontaneous liver tumorigenesis in mice. However, how autophagy impacts mTORC1‐mediated liver metabolic changes and tumorigenesis is less clear. Here we show that deletion of Atg5 in liver‐specific Tsc1 knockout (L‐Tsc1 KO) mice inhibited liver tumorigenesis but increased mortality of L‐Tsc1 KO mice. Histological and unbiased RNA‐seq as well as metabolomic analysis revealed that lack of hepatic autophagy dramatically induced the remodeling of liver cell population and hepatic metabolic reprogramming in L‐Tsc1 KO mice. RNA‐seq analysis revealed increased cholangiocyte, hepatic stellate cell and macrophage but decreased hepatocyte gene signatures in liver‐specific Tsc1 and Atg5 double knockout (DKO) mice. Histologically, Atg5 and Tsc1 DKO mice had increased hepatomegaly, ductular reactions, increased number of macrophage/Kupffer cells and infiltrated neutrophils as well as massive fibrosis, and the DKO mice died at the age of approximately 8‐months‐old with no detectable liver tumors. Metabolomic analysis revealed marked increased hepatic glutathione and glutathione metabolites but decreased hepatic levels of glucose and lipid with accumulation of TCA cycle intermediate metabolites and fatty acid oxidation intermediates in Atg5 and Tsc1 DKO mice. Deletion of hepatic p62 partially but deletion of Nrf2 almost completely rescued the liver cell type remodeling and metabolic reprogramming and the mortality of Atg5 and Tsc1 DKO mice. Deletion of p62 promoted whereas deletion of Nrf2 suppressed the liver tumorigenesis of Atg5 and Tsc1 DKO mice. These results reveal a critical role of Nrf2 in regulating liver cell type remodeling and metabolic reprogramming in mice lack of hepatic autophagy with mTORC1 activation. Our data may provide a mechanistic basis for a precision medicine to target Nrf2 pathway for treating as subset of HCC, especially in HCV‐ or alcohol‐associated liver cancers that may have defective autophagy and mTOR activation.
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.
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
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.
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.
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.
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•Mouse livers with persistent mTOR activation and defective autophagy developed ductular reaction with metabolic reprogramming.•mTOR activation-induced liver tumorigenesis was inhibited in mice lacking hepatic autophagy and vice versa.•Loss of p62/Sqstm1 rescued ductular reaction, liver cell remodeling and metabolic reprograming in these mice.•p62/Sqstm1, a previously known oncogenic protein, paradoxically acted as a tumor suppressor and inhibited liver tumorigenesis in these mice.•Decreased p62 and increased mTOR activity was found in a subset of human patients with hepatocellular carcinoma.
How different cell death modes and cell survival pathways cross talk remains elusive. We determined the interrelation of apoptosis, necrosis, and autophagy in tumor necrosis factor ...(TNF)-α/actinomycin D (ActD) and lipopolysaccharide/D-galactosamine (GalN)-induced hepatotoxicity in vitro and in vivo . We found that TNF-α/ActD-induced apoptosis was completely blocked by a general caspase inhibitor ZVAD-fmk at 24 hours but hepatocytes still died by necrosis at 48 hours. Inhibition of caspases also protected mice against lipopolysaccharide/GalN-induced apoptosis and liver injury at the early time point, but this protection was diminished after prolonged treatment by switching apoptosis to necrosis. Inhibition of receptor-interacting protein kinase (RIP)1 by necrostatin 1 partially inhibited TNF-α/ZVAD-induced necrosis in primary hepatocytes. Pharmacologic inhibition of autophagy or genetic deletion of Atg5 in hepatocytes did not protect against TNF-α/ActD/ZVAD-induced necrosis. Moreover, pharmacologic inhibition of RIP1 or genetic deletion of RIP3 failed to protect and even exacerbated liver injury after mice were treated with lipopolysaccharide/GalN and a pan-caspase inhibitor. In conclusion, our results suggest that different cell death mode and cell survival pathways are closely integrated during TNF-α–induced liver injury when both caspases and NF-κB are blocked. Moreover, results from our study also raised concerns about the safety of currently ongoing clinical trials that use caspase inhibitors.
Rho kinase (ROCK) plays a critical role in actin cytoskeleton organization and is involved in diverse fundamental cellular functions such as contraction and gene expression. Fasudil, a ROCK ...inhibitor, has been clinically applied since 1995 for the treatment of subarachnoid hemorrhage (SAH) in Japan. Increasing evidences indicate that fasudil could exhibit markedly therapeutic effect on central nervous system (CNS) disorders, such as Alzheimer's disease.
This article summarizes results from supporting evidence for the potential therapy for fasudil against a variety of CNS diseases. And the properties of its analogs are also summarized.
Current therapies against CNS disorders are only able to attenuate the symptoms and fail in delaying or preventing disease progression and new approaches with disease-modifying activity are desperately needed. The dramatic effects of fasudil in animal models and/or clinical applications of CNS disorders make it a promising strategy to overcome CNS disorders in human beings. Given the complex pathology of CNS disorders, further efforts are necessary to develop multifunctional fasudil derivatives or combination strategies with other drugs in order to exert more powerful effects with minimized adverse effects in the combat of CNS disorders.
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Pancreatitis is a potentially fatal inflammatory disease with significant morbidity and mortality but the exact molecular mechanisms remain largely obscure. We investigated the role of ...vacuole membrane protein 1 (VMP1), an endoplasmic reticulum (ER) resident protein, in the pathogenesis of pancreatitis. We examined VMP1 expression and ER stress in human pancreatitis samples. We also determined VMP1 level and ER stress in experimental pancreatitis induced by cerulein as well as in pancreatic acinar cell‐specific VMP1 knockout (VMP1
Δacinar
) mice. We found that VMP1 expression was significantly lower in human pancreatitis samples associated with extensive ER stress. In a mouse experimental pancreatitis model, treatment with ceruleint markedly decreased VMP1 expression but increased levels of several ER stress markers such as p‐elf2α, XBP1s and CHOP. Western blot and immune histochemical analysis of human and experimental mouse pancreatitis samples showed impaired autophagy with accumulation of p62, an autophagy substrate protein that generally is degraded by autophagy. Intriguingly, VMP1
Δacinar
mice developed spontaneous pancreatitis with pathological features of chronic pancreatitis. In addition, VMP1
Δacinar
caused accumulation of p62 as well as selective ER‐phagy receptor FAM134B. Moreover, VMP1
Δacinar
mice had increased ER tress accompanied with massive cell death, suggesting VMP1 may serve as a key mediator of pancreatic ER homeostasis. Collectively, these data indicate a critical role of VMP1 in maintaining ER homeostasis in acinar cells and decreased VMP1 expression may contribute to the development of pancreatitis.
Support or Funding Information
R01 AA020518, R01 DK102142, U01 AA024733 and P30GM118247 (W.X.D).
Alcohol-related liver disease (ALD) is a major health problem and a leading cause for liver disease-related mortality worldwide, claiming more than 3 million deaths, or 5.9% of all deaths globally ...per year. ALD comprises a range of disorders and pathologic changes in individuals with acute and chronic alcohol consumption, ranging from simple steatosis to severe forms of liver injury including steatohepatitis, liver fibrosis/cirrhosis, and hepatocellular carcinoma (HCC). While decades of research have enriched our understanding on the pathogenesis of ALD, therapeutic options for ALD treatment are still very limited. So far, alcohol abstinence achieved by psychosomatic intervention is the best treatment for all stages of ALD. No successful treatment for advanced ALD, such as cirrhosis, alcoholic hepatitis, or HCC, is available other than liver transplantation, which is largely due to the lack of suitable animal models to help to identify therapeutic targets. Autophagy is a lysosomal degradation pathway, which is activated as a protective mechanism for cells against various adverse conditions. We previously demonstrated that acute alcohol exposure activates autophagy as a protective mechanism in acute alcohol-induced liver injury However, acute and chronic alcohol exposure may differentially regulate hepatic autophagy. Animals with chronic ethanol exposure and human heavy drinkers develop hepatomegaly with increased hepatic protein accumulation, suggesting a possible defect in hepatic autophagy in chronic alcohol conditions. However, the mechanisms by which chronic alcohol consumption impairs autophagy and/or lysosomal functions in the liver are largely unknown. We characterized the autophagy status in a chronic feeding plus acute binge alcohol (hereafter referred to as “Gao-binge”) mouse model according to the guidelines of autophagy research (Chapter 3). We investigated the role and mechanisms of transcription factor EB (TFEB), which is a master regulator of autophagy and lysosomal biogenesis, in Gao-binge alcohol-induced liver injury. Compared with control mice, liver tissues from mice fed on the ethanol diet had lower levels of total and nuclear TFEB, and hepatocytes had decreased lysosome biogenesis and autophagy. Mechanistically, hepatocytes from mice fed on the ethanol diet had increased translocation of mechanistic target of rapamycin (mTOR) onto lysosomes, resulting in increased activation of mTOR complex 1 (mTORC1). Increased mTORC1 activation phosphorylates and inactivates TFEB resulting in decreased hepatic lysosomal biogenesis and insufficient autophagy. Administration of torin1, a specific mTOR kinase inhibitor, increased liver levels of TFEB and decreased steatosis and liver injury in mice fed with Gao-binge alcohol. Overexpression of TFEB in mouse livers increased lysosomal biogenesis and mitochondrial bioenergetics and protected against Gao-binge alcohol-induced liver injury. In contrast, genetic knockdown of Tfeb or double deletion of Tfeb and Tfe3 (another homologue of Tfeb in mammals) exacerbated Gao-binge alcohol-induced liver injury. More importantly, liver tissues from alcoholic hepatitis patients also had lower nuclear levels of TFEB than control normal human liver tissues. Results from my research presented in Chapter 3 demonstrated that chronic alcohol consumption impairs TFEB-mediated lysosomal biogenesis resulting in insufficient liver autophagy. Strategies to boost TFEB activation such as the use of mTOR inhibitor may pave a novel avenue for treating/preventing ALD. Tuberous sclerosis 1 (TSC1) forms a TSC1/2 complex with TSC2, which is a key inhibitor of mTORC1 that functions as a GTPase-activating protein (GAP) for the small Ras-related GTPase Rheb (Ras homolog enriched in brain). The active, GTP-bound form of Rheb directly interacts with and activates mTORC1. As a Rheb-specific GAP, TSC1/2 negatively regulates mTORC1 signaling by converting Rheb into its inactive GDP-bound state. In Chapter 4, we established a novel mouse model that can phenocopy the typical pathogenesis of human alcoholic hepatitis. We found Gao-binge alcohol markedly increased hepatomegaly, ductular reactions (DRs), inflammation, and liver injury in liver-specific Tsc1 knockout (L-Tsc1) KO mice compared to either Gao-binge alcohol fed WT mice or control diet-fed L-Tsc1 KO mice. Mechanistic studies revealed increased YAP and Notch activation as well as endoplasmic reticulum (ER) stress markers in alcohol-fed L-Tsc1 KO mice. In addition, we observed decreased nuclear translocation of TFEB in hepatocytes of alcohol-fed mice. In contrast, we found that nuclear TFEB translocation was significantly increased in cholangiocytes in alcohol-fed L-Tsc1 KO mice, which was independent of mTORC1 activation. Moreover, deleting Tsc1 in cholangiocyte but not in hepatocyte aggravated alcohol-induced liver injury, hepatomegaly, DR, fibrosis, and inflammation in mouse livers. Administration of torin1 partially reversed hepatic TSC1 deletion-induced hepatomegaly, DR, fibrosis, inflammatory cell infiltration and liver injury in Gao-binge alcohol-fed mice. Therefore, in Chapter 4, we established a novel advanced mouse ALD model that phenocopy human ASH. The most severe form of ALD is alcohol associated HCC wherein the role of autophagy in HCC is still controversial. Activation of cellular stress response pathways, such as autophagy, unfolded protein response in endoplasmic reticulum and mitochondria, DNA damage response, etc. to maintain metabolic homeostasis is a critical growth and survival mechanism in many cancer cells. As discussed in Chapter 3, activating TFEB protects against alcohol-induced liver injury and steatosis in early stages of ALD. However, the exact role of TFEB in alcohol-associated liver carcinogenesis in the later stages of ALD is still unknown. In Chapter 5, we investigated the role of TFEB in a diethylnitrosamine (DEN)-alcohol-induced HCC mouse model. Mice fed chronically with ethanol diet developed bigger and more liver tumors than that fed with control diet- in DEN-treated mice. There is no significant difference in the tumor numbers between WT and L-Tfeb KO mice fed with control diet or ethanol diet. But the size of the liver tumors was smaller in L-Tfeb KO mice than that in WT mice fed either the control or ethanol diets. Additionally, chronic alcohol consumption increased cell proliferation in both WT and L-Tfeb KO mouse livers. There were no significant changes in the DR, fibrosis, or inflammation in WT or L- Tfeb KO mice fed with either control diet or ethanol diet. In this chapter, we found that chronic alcohol consumption promotes carcinogen-induced liver tumor development in mice. Hepatic deletion of TFEB in mice attenuates alcohol-associated liver tumorigenesis. In summary, studies in this dissertation discovered that chronic plus binge ethanol activates hepatic mTORC1 that leads to impaired TFEB-mediated lysosomal biogenesis resulting in insufficient autophagy and liver injury. We also established a novel advanced ALD mouse model by using mice with persistent hepatic mTORC1 activation fed with Gao-binge alcohol. This mouse model phenocopied the pathogenesis of human alcoholic hepatitis including significant liver injury, hepatomegaly, DR, inflammation, fibrosis, and liver cell repopulation, which will be beneficial for the development of targeted therapy and further understanding the pathogenesis of severe ALD. Finally, we determined the role of TFEB in an alcohol-associated HCC mouse model. We found that hepatic TFEB deficiency slightly attenuates alcohol-associated liver tumorigenesis in mice, suggesting autophagy-lysosomal pathway may be critical for liver tumor development and progression. Overall, the findings from the dissertation may provide important insights on targeting the TFEB-autophagy-lysosomal pathway in treating/preventing ALD. In the early stage of ALD before the HCC development, activation of TFEB-lysosomal pathway may be beneficial to attenuate steatosis, inflammation, DR, and liver injury of ALD. However, once HCC has been developed, perhaps, inhibition of TFEB-lysosomal pathway may only slow down alcohol-associated HCC progression.
Immunotherapy has shown robust efficacy in treating a broad spectrum of hematological and solid cancers. Despite the transformative impact of immunotherapy on cancer treatment, several outstanding ...challenges remain. These challenges include on-target off-tumor toxicity, systemic toxicity, and the complexity of achieving potent and sustainable therapeutic efficacy. Synthetic biology has emerged as a promising approach to overcome these obstacles, offering innovative tools for engineering living cells with customized functions. This review provides an overview of the current landscape and future prospects of cancer immunotherapy, particularly emphasizing the role of synthetic biology in augmenting its specificity, controllability, and efficacy. We delineate and discuss two principal synthetic biology strategies: those targeting tumor surface antigens with engineered immune cells and those detecting intratumoral disease signatures with engineered gene circuits. This review concludes with a forward-looking perspective on the enduring challenges in cancer immunotherapy and the potential breakthroughs that synthetic biology may contribute to the field.
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