Involvement of autophagy in trypsinogen activation within the pancreatic acinar cells (original) (raw)
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Impaired autophagy and organellar dysfunction in pancreatitis
Journal of Gastroenterology and Hepatology, 2012
Recent findings from our group, obtained on experimental in vivo and ex vivo models of pancreatitis, reveal that this disease causes a profound dysfunction of key cellular organelles, lysosomes and mitochondria. We found that autophagy, the main cellular degradative, lysosomedriven process, is activated but also impaired in acute pancreatitis because of its' inefficient progression/resolution (flux) resulting from defective function of lysosomes. One mechanism underlying the lysosomal dysfunction in pancreatitis is abnormal processing (maturation) and activation of cathepsins, major lysosomal hydrolases; another is a decrease in pancreatic levels of key lysosomal membrane proteins LAMP-1 and LAMP-2. Our data indicate that lysosomal dysfunction plays an important initiating role in pancreatitis pathobiology. The impaired autophagy mediates vacuole accumulation in acinar cells; furthermore, the abnormal maturation and activation of cathepsins leads to increase in intra-acinar trypsin, the hallmark of pancreatitis; and LAMP-2 deficiency causes inflammation and acinar cell necrosis. Thus, the autophagic and lysosomal dysfunctions mediate key pathologic responses of pancreatitis. On the other hand, we showed that pancreatitis causes acinar cell mitochondria depolarization, mediated by the permeability transition pore (PTP). Genetic (via deletion of cyclophilin D) inactivation of PTP prevents mitochondrial depolarization and greatly ameliorates the pathologic responses of pancreatitis. Further, our data suggest that mitochondrial damage, by stimulating autophagy, increases the demand for efficient lysosomal degradation and therefore aggravates the pathologic consequences of lysosomal dysfunction. Thus, the combined autophagic, lysosomal and mitochondrial dysfunctions are key to the pathogenesis of pancreatitis.
Activation of trypsinogen in large endocytic vacuoles of pancreatic acinar cells
Proceedings of the National Academy of Sciences, 2007
The intracellular activation of trypsinogen, which is both pH-and calcium-dependent, is an important early step in the development of acute pancreatitis. The cellular compartment in which trypsinogen activation occurs currently is unknown. We therefore investigated the site of intracellular trypsinogen activation by using an established cellular model of acute pancreatitis: supramaximal stimulation of pancreatic acinar cells with cholecystokinin. We used fluorescent dextrans as fluid phase tracers and observed the cholecystokininelicited formation and translocation of large endocytic vacuoles. The fluorescent probe rhodamine 110 bis-(CBZ-L-isoleucyl-L-prolyl-Larginine amide) dihydrochloride (BZiPAR) was used to detect trypsinogen activation. Fluid phase tracers were colocalized with cleaved BZiPAR, indicating that trypsinogen activation occurred within endocytic vacuoles. The development of BZiPAR fluorescence was inhibited by the trypsin inhibitor benzamidine. Fluorescein dextran and Oregon Green 488 BAPTA-5N were used to measure endosomal pH and calcium, respectively. The pH in endocytic vacuoles was 5.9 ؎ 0.1, and the calcium ion concentration was 37 ؎ 11 M. The caged calcium probe o-nitrophenyl EGTA and UV uncaging were used to increase calcium in endocytic vacuoles. This increase of calcium caused by calcium uncaging was followed by recovery to the prestimulated level within Ϸ100 s. We propose that the initiation of acute pancreatitis depends on endocytic vacuole formation and trypsinogen activation in this compartment.
American Journal of Physiology-Gastrointestinal and Liver Physiology, 2012
Acute pancreatitis is an inflammatory disease of the exocrine pancreas that carries considerable morbidity and mortality; its pathophysiology remains poorly understood. Recent findings from experimental models and genetically altered mice summarized in this review reveal that autophagy, the principal cellular degradative pathway, is impaired in pancreatitis and that one cause of autophagy impairment is defective function of lysosomes. We propose that the lysosomal/autophagic dysfunction is a key initiating event in pancreatitis and a converging point of multiple deranged pathways. There is strong evidence supporting this hypothesis. Investigation of autophagy in pancreatitis has just started, and many questions about the “upstream” mechanisms mediating the lysosomal/autophagic dysfunction and the “downstream” links to pancreatitis pathologies need to be explored. Answers to these questions should provide insight into novel molecular targets and therapeutic strategies for treatment o...
Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis
Journal of Clinical Investigation, 2000
Autodigestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. The mechanism responsible for the intrapancreatic activation of digestive zymogens is unknown, but a recent hypothesis predicts that a redistribution of lysosomal cathepsin B (CTSB) into a zymogen-containing subcellular compartment triggers this event. To test this hypothesis, we used CTSB-deficient mice in which the ctsb gene had been deleted by targeted disruption. After induction of experimental secretagogue-induced pancreatitis, the trypsin activity in the pancreas of ctsb-/animals was more than 80% lower than in ctsb +/+ animals. Pancreatic damage as indicated by serum activities of amylase and lipase, or by the extent of acinar tissue necrosis, was 50% lower in ctsb-/animals. These experiments provide the first conclusive evidence to our knowledge that cathepsin B plays a role in intrapancreatic trypsinogen activation and the onset of acute pancreatitis.
Trypsinogen activation in rat pancreatic acinar cells hyperstimulated by caerulein
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1997
Inappropriate trypsinogen activation is discussed as an early intracellular event in the secretagogue-induced model of acute pancreatitis. However, the mechanisms by which trypsinogen is activated are not well characterized. In the present Ž. wŽ. work, trypsinogen activation was studied in intact acinar cells using bis-CBZ-arginyl-Rhodamine 110 CBZ-Arg-Rho 2 x 110 as a cell-permeant substrate for trypsin and also independently via the formation of trypsinogen activation peptide Ž. TAP. Preincubation with 10 nM caerulein increased the Rho 110-substrate cleavage more than threefold. This proteolytic Ž. activity was fully sensitive to a benzamidine BA-type serine protease inhibitor. The appearance of enzymatic activity was paralleled by the formation of TAP. The lack of effect of the high-molecular soybean trypsin inhibitor indicates an intracellular substrate cleavage. The cathepsin B inhibitor CA-074 prevented neither the caerulein-induced formation of Ž. TAP nor the CBZ-Arg-Rho 110-cleaving activity. BA inhibited the Rho 110-substrate cleavage and significantly reduced 2 the TAP formation. These results show that trypsinogen activation in caerulein-hyperstimulated acinar cells may occur independently of the activity of cathepsin B. On the contrary, the effect of BA suggests the role of a serine protease in trypsinogen activation. q 1997 Elsevier Science B.V.
Trypsin activity is not involved in premature, intrapancreatic trypsinogen activation
American journal of physiology. Gastrointestinal and liver physiology, 2002
A premature and intracellular activation of digestive zymogens is thought to be responsible for the onset of pancreatitis. Because trypsin has a critical role in initiating the activation cascade of digestive enzymes in the gut, it has been assumed that trypsin also initiates intracellular zymogen activation in the pancreas. We have tested this hypothesis in isolated acini and lobules from rat pancreas. Intracellular trypsinogen activation was induced by supramaximal secretagogue stimulation and measured using either specific trypsin substrates or immunoreactivity of the trypsinogen activation peptide (TAP). To prevent a trypsin-induced trypsinogen activation, we used the cell-permeant, highly specific, and reversible inhibitor Nalpha-(2-naphthylsulfonyl)-3-amidinophenylalanine-carboxymethylpiperazide (S124), and to prevent cathepsin-induced trypsinogen activation, we used the cysteine protease inhibitor E-64d. Incubation of acini or lobules in the presence of S124 completely preven...
Intracellular Trypsin Induces Pancreatic Acinar Cell Death but Not NF- B Activation
Journal of Biological Chemistry, 2009
Premature intracellular activation of the digestive enzyme trypsinogen is considered to be the initiating event in pancreatitis. However, the direct consequences of intracellular trypsin activity have not previously been examined. In the current study, a mutant trypsinogen (paired basic amino acid cleaving enzyme (PACE)-trypsinogen), which is activated intracellularly by the endogenous protease PACE, was developed. This new construct allowed for the first time direct examination of the effects of intracellular trypsin on pancreatic acinar cells. We found that PACE-trypsinogen was expressed in the secretory pathway and was activated within acinar cells.
Gastroenterology, 2011
The role of trypsinogen activation in the pathogenesis of acute pancreatitis (AP) has not been clearly established. METHODS: We generated and characterized mice lacking trypsinogen isoform 7 (T7) gene (T Ϫ/Ϫ ). The effects of pathologic activation of trypsinogen were studied in these mice during induction of AP with cerulein. Acinar cell death, tissue damage, early intra-acinar activation of the transcription factor nuclear factor B (NF-B), and local and systemic inflammation were compared between T Ϫ/Ϫ and wild-type mice with AP. RESULTS: Deletion of T7 reduced the total trypsinogen content by 60% but did not affect physiologic function. T Ϫ/Ϫ mice lacked pathologic activation of trypsinogen, which occurs within acinar cells during early stages of AP progression. Absence of trypsinogen activation in T Ϫ/Ϫ mice led to near complete inhibition of acinar cell death in vitro and a 50% reduction in acinar necrosis during AP progression. However, T Ϫ/Ϫ mice had similar degrees of local and systemic inflammation during AP progression and comparable levels of intra-acinar NF-B activation, which was previously shown to occur concurrently with trypsinogen activation during early stages of pancreatitis. CONCLUSIONS: T7 is activated during pathogenesis of AP in mice. Intra-acinar trypsinogen activation leads to acinar death during early stages of pancreatitis, which accounts for 50% of the pancreatic damage in AP. However, progression of local and systemic inflammation in AP does not require trypsinogen activation. NF-B is activated early in acinar cells, independently of trypsinogen activation, and might be responsible for progression of AP.
The Pancreatitis-induced Vacuole Membrane Protein 1 Triggers Autophagy in Mammalian Cells
Journal of Biological Chemistry, 2007
Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression colocalizes with LC3 in pancreas tissue undergoing pancreatitisinduced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process.