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Role of Trypsinogen Activation in Genesis Of Role of trypsinogen activation in genesis of pancreatitis Ajay Dixit, Rajinder K Dawra, Vikas Dudeja and Ashok K Saluja Department of Surgery, Miller School of Medicine, University of Miami, Miami FL e-mail: [email protected] Version 1.0, August 24, 2016 [DOI: 10.3998/panc.2016.25] Abstract: especially trypsin, has become a major area of investigation in pathobiology of pancreatitis. Pancreatitis is an inflammatory disease that starts Premature activation of trypsin has been in pancreatic acinar cells and results in significant observed during both in vitro hyperstimulation and morbidity and mortality. Currently there is no in animal models of acute pancreatitis (16, 17, 22- therapy for pancreatitis because of lack of 24). In this review, we discuss our current complete understanding of the disease understanding of the role of trypsin in the mechanism. Premature trypsinogen activation is pathophysiology of pancreatitis. considered to be the key event in the disease development. However recent research in the 2. Physiology of Trypsinogen in field has pointed out that besides trypsinogen Health activation there are many other important factors that play important role in the disease. Our recent Trypsin is synthesized as trypsinogen, an inactive -/- study in Trypsinogen-7 knock out (T7 ) mice has precursor, in the rough endoplasmic reticulum and supported this and for the first time shown that transported to the Golgi apparatus for sorting. intra-acinar trypsinogen activation contributes only Trypsinogen is always co- synthesized and partially to acinar injury and local and systemic packed with a pancreatic secretory trypsin inflammation progress independently of trypsin inhibitor (PSTI) that inhibits its premature activation during pancreatitis. NF-κB activation activation. Once, it reaches the Golgi system, the that happens parallel and independent to trypsinogen and other digestive enzymes trypsinogen activation can still drive the condense into core particles and are packed in development of the acute or chronic pancreatitis zymogen granules. The condensed enzymes are even in absence of trypsin. stable and minimal activation happens within the zymogen granules. Once acini receive secretory 1. Introduction stimulus, these zymogen granules are released in to the lumen of pancreatic duct, which carries the Pancreatitis is an inflammatory disease of the digestive enzymes into the duodenum. Once in pancreas that starts in pancreatic acinar cells and duodenum, enteropeptidase activates trypsinogen results in significant morbidity and mortality (19). by removing 7-10 amino acid from N-terminal More than a century ago, pathologist Dr. Hans region known as trypsinogen activation peptide Chiari (6) proposed that the acute pancreatitis is a (TAP). Removal of TAP induces conformational disease rather than an infection, in which change resulting in active trypsin. TAP is pancreas destroy itself through autodigestion. immunologically distinct from the same sequence Since then the mechanism, site and importance of within trypsinogen, thereby allowing detection of premature activation of digestive enzymes, trypsinogen activation in situ (1, 10). This work is subject to an International Creative Commons Attribution 4.0 license. 3. Intra-acinar Location of Trypsin trypsinogen peptide was hydrolyzed (29). Activation During Acute Pancreatitis Interestingly, cathepsin B mediated trypsinogen activation seems not to be a crucial pathogenic While it is clear that there is intra-acinar activation step in hereditary pancreatitis patients with the of trypsin, the exact intracellular location where trypsinogen mutations D22G and K23R (29). trypsin is activated is a hotly debated area in pancreatitis. Subcellular fractionation and analysis 4. Role of Trypsin During Acute of pancreatic homogenate shortly after the Pancreatitis induction of pancreatitis have provided much insight about the location of trypsinogen For decades, intra-acinar trypsin activation has activation. In vitro studies on acinar cells show been considered the key event in acute that within 30 min of hyper stimulation most of the pancreatitis. This trypsin centric hypothesis has active trypsin localizes to a heavy, zymogen-rich been supported by various observations. pellet and after 60 min of hyperstimulation, the Inhibition of trypsin, by somewhat nonspecific trypsin activity shifts to the supernatant. This shift protease inhibitors, provides protection against was paralleled by appearance of immunoreactive injury during acute pancreatitis (31, 32). TAP and cathepsin B, a lysosomal enzyme Furthermore, inhibition of trypsinogen activation capable of activating trypsinogen, in the soluble by inhibiting the activity of cathepsin B or by fraction (14). This experimental evidence led to deleting the cathepsin B gene also decreases development of the “co-localization hypothesis” pancreatic injury during acute pancreatitis, again which purports that during acute pancreatitis suggesting that trypsinogen activation is important lysosomal enzymes and zymogens fuse to form for pancreatic damage (12, 31). Halangk et al (12) structures named co-localization organelles. showed that cathepsin B knock out (CB-KO) mice Further, it has been proposed that in these co- have less necrosis when compared to WT type localization organelles lysosomal enzyme mice. However, the degree of leukocyte infiltration cathepsin B activates trypsinogen to trypsin. in the pancreas or lungs during pancreatitis was Studies have shown that premature trypsinogen not affected by the absence of cathepsin B activation occurs in membrane-bound indicating cathepsin B, and thus trypsin, compartments resembling autophagic vesicles independent evolution of local and systemic formed by co-localization of zymogen and inflammation. lysosomes (22). In these co-localized vacuoles, the lysosomal protease cathepsin B activates The strongest support for the trypsin centric trypsinogen. It is proposed that the active trypsin theory comes from identification of mutations in further activates other digestive enzymes with in the cationic trypsinogen gene PRSS1 in acinar cells, presumably in the same manner, as it hereditary pancreatitis, an uncommon form of normally would do in the duodenum. The pancreatitis with autosomal dominant inheritance presence of these co-localization vacuoles has (34). The biochemical studies of the pancreatitis- been observed in all models of experimental associated p.R122H mutations of human cationic pancreatitis as wells as in pathological specimens trypsinogen in vitro show that this trypsinogen of human pancreatitis. The studies, which have variant has an increased propensity for auto- demonstrated that cathepsin B can activate activation and is resistant to degradation by trypsinogen in vitro, further support this theory chymotrypsin C (30). However, this mutation has (25, 29) and it seems that in the absence of pleiotropic effects rather than being exclusively an cathepsin B, less than 1 % of the trypsinogen activating mutation, and there is no direct peptide is hydrolyzed but after incubation with evidence for increased intracellular trypsin activity cathepsin B for 30 min at pH 5, 96% of in patients with hereditary pancreatitis due to this 2 mutation (3). Furthermore, patients with this observed during acute pancreatitis. Contrary to mutation do not suffer continuous pancreatitis but these findings, a study by Wartmann et al rather experience episodic attacks of the disease. reported that cathepsin L knockout mice have Also, in mouse model of hereditary pancreatitis much higher trypsinogen activation but generated by transgenic expression of R122H significantly reduced pancreatic injury suggesting trypsinogen, no increased trypsinogen activation trypsinogen activation may even have a protective was observed indicating involvement of other role during pancreatitis by degrading trypsinogen factors. R122H mutation has been shown to and other proteases (33). increase the frequency of trypsin auto-activation (3, 9, 15). Study of another PRSS1 mutant To obtain further insight into the role of trypsin in p.R116C deciphered an entirely novel mechanism acute pancreatitis, we have generated a novel of acinar cell injury, which is unrelated to knockout mouse lacking trypsinogen isoform-7 trypsinogen activation. The mutation induces (mouse paralog of human cationic trypsinogen, proenzyme misfolding leading to endoplasmic PRSS1) (7). In this mouse strain, we do not reticulum (ER) stress and unfolded protein observe intra-acinar pathologic trypsin activation response (UPR) activation (15). The trypsinogen during acute pancreatitis suggesting that the activation hypothesis of hereditary pancreatitis trypsinogen isoform-7 (T7) is responsible for the also does not explain incomplete penetrance, the pathologic trypsin activation. Intriguingly, in these intermittent nature of the disease, and lack of novel trypsinogen-7 knockout mice (T-/-) mice we progression to chronic pancreatitis in some observed that the acinar cell necrosis during individuals despite recurrent episodes. While, caerulein (Figure 1A) as well as L-arginine emerging epidemiologic and genetic data induced acute pancreatitis is reduced to about continue to link pancreatitis to trypsinogen half of that observed in mice with intact T7 (7). In activation, it is becoming increasingly clear that vitro we observe that acini lacking T7 do not with the exception of hereditary pancreatitis and undergo necrosis (as measured by LDH release) cystic fibrosis, a direct
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