Molecular and Cellular Changes in Acute Pancreatitis

Transcription

1 Molecular and Cellular Changes in Acute Pancreatitis Florin Zaharie, MD, PhD, Abstract Acute pancreatitis is a possibly lethal disease with high morbidity and mortality rates. The pathogenesis of acute pancreatitis is not easily understood, this condition having no specific or efficient treatment. It is thought to stem from acinar cells. Typical responses of pancreatitis include hyperamylasemia, intra-acinar activation of digestive enzymes (for example, trypsinogen converted into trypsin), agglomeration of large vesicles in acinar cells leading to generation of proinflammatory mediators and consequent acinar cell death induced by apoptosis and necrosis. These paper summarize the latest research in unveiling the cellular and molecular changes in acute pancreatitis. Keywords acute pancreatitis, molecular changes, acinar cells I. LYSOSOMAL AND AUTOPHAGIC DYSFUNCTIONS IN PANCREATITIS Based on experimental models, it has been that nonalcoholic pancreatitis has a double effect on autophagy.[1] It activates autophagy but impairs autophagic flux.[2-4] In pancreatitis, impairment of autophagy is determined by defective lysosomal degradation. Findings show that damaged lysosomal function is common in pancreatitis, indicating abnormal functioning of lysosomal hydrolases. There is mainly an early significant decrease in the enzymatic activity of cathepsins in the lysosome-enriched pancreatic subcellular fraction (first demonstrated by Steer and Saluja 20 years ago) in models of pancreatitis. Recent results[5] indicate an impaired cathepsin processing in experimental pancreatitis: decreasing fully mature ( double-chain ) cathepsin forms and accumulation of immature cathepsin forms. These results further note that pancreatitis determines an asymmetry between two major cathepsins, cathepsin B (CatB) and cathepsin L (CatL), with an increase in the ratio of their activities (CatB/CatL) in pancreatitis.[1] This work was funded by the Romanian Ministry of Education and Research. Gastroenterology Institute, Department of Nanomedicine, Iuliu Hatieganu University of Medicine and Pharmacy, Victor Babes nr.8, Cluj-Napoca, Romania. *Correspondence to Florin Zaharie ( zaharie.vasile@umfcluj.ro). 36 The dramatic decrease in pancreatic levels of lysosomal-associated membrane protein 1 (LAMP-1) and lysosomal-associated membrane protein 2 (LAMP-2) is another significant (and early) lysosomal defect.[6, 7]This decrease was found in 4 dissimilar in vivo models of nonalcoholic pancreatitis induced in rats and mice by CR, L-arginine and CDE diet, as well as in the ex vivo model. Moreover, pancreatic LAMP-2 recorded significant decreases in pancreatitis, generated by ethanol feeding combined with low-dose CR.[8]. Other significant decreases in LAMP-1 and -2 have been reported for human pancreatitis.[9]. The findings of this study show that LAMP damage in pancreatitis takes place in an acidic compartment, being mediated by cathepsins.[1] II. AUTOPHAGY PATHWAYS The continuous renewal of living cells recycles old components and replaces them with new ones. Cellular homeostasis can be maintained through macromolecule and organelle damage generating new building blocks. Cellular components are being damaged by two significant systems in eukaryotic cells: the ubiquitin-proteasome system and autophagy. Autophagy particularly alters short-lived proteins, which are tagged by ubiquitin to be recognized and degraded by the proteasome. [10] On the contrary, autophagy degrades long-lived proteins, lipids and cytoplasmic organelles through a lysosome-driven process.[11-14] Autophagy occurs at a basal rate in most cells, acting as a quality control mechanism to remove protein aggregates and damaged or unneeded organelles. Autophagy is also a significant protective mechanism that permits cell survival and adaption to fluctuations in external conditions. Nutrient deprivation is one of the strongest inducers of physiological autophagy, generating and recycling components (for instance, amino acids) which are crucial for cell survival.[15] Microautophagy is defined by the lysosome itself absorbing small parts of the cytoplasm through inward invagination of the lysosomal membrane.[16, 17] Similarly, in the case of crinophagy[18], lysosomes directly fuse with secretory granules and generate their degradation [insulin-containing granules in pancreatic β-cells.[19] There is no knowledge of the molecular signals and mechanisms mediating crinophagy. The main mechanism of autophagy is macroautophagy, which benefited from more extensive studies than the other pathways. It is here refered to as autophagy. Macroautophagy has more

2 steps[20, 23], starting with the development of an autophagosome, a unique double-membrane vacuole that isolates organelles and proteins designed for deterioration. Autophagosomes merge with endosomes and further with lysosomes, producing single-membrane autolysosomes. In the end, the isolated material is deteriorated by lysosomal hydrolases, with the resulting degraded products (amino acids) being recycled back to the cytoplasm. The dynamic character is a major but undervalued aspect of autophagy[24, 25] Modifications of the steps described above alter autophagy progression and function. Therefore, it is of utmost importance to assess the flux by means of this pathway (the turnover rate of autophagic vacuoles), observing its alteration by various stresses and diseases.[15], [26, 27] III. PHYSIOLOGICAL AUTOPHAGY IN THE EXOCRINE PANCREAS Based on the abundance of GFP-LC3 puncta, the basal level of autophagy is higher in mouse exocrine pancreas than in liver, kidney, heart or endocrine pancreas. Moreover, the number of GFP-LC3 puncta 24 hours after starvation is bigger in the exocrine pancreas than in other organs.[28] The findings of our in vitro studies, based on the comparative analysis of LC3-II levels in both intact and blocked lysosomal deterioration situations, also notice efficient basal level of autophagy in mouse pancreatic acinar cells (Jia et al., unpublished observations). The significantly high rate of protein synthesis of the exocrine pancreas might lead to speculations regarding a greater need to eliminate defective (or excessive) proteins. The basic function of pancreatic acinar cell is to secrete digestive enzymes, mostly produced as inactive zymogens prepacked and stored in zymogen granules (ZGs) and generally activated after being secreted and reaching the intestine.[28] Therefore, macroautophagy might play a role in regulating the number of ZGs, adjusting to the needs of acinar cells and the whole organism. Nevertheless, the impact of autophagy on the regulation of the level and secretion of digestive enzymes has not been described yet. The interrelations between secretory granule (mainly ZGs) synthesis and secretion and their degradation are also not well understood.[15] Autophagosomes consist of various organelles (mitochondria, ER, ZGs) resulting in nonselective basal and starvation-induced autophagy in pancreatic acinar cells.[29, 30]. Starvation highly triggers autophagy in the pancreas, causing a significant decrease in the number of ZGs in acinar cells.[31] This finding is consistent with previous studies[32] showing a decrease in the number of ZGs in starved rodents. Nevertheless, crinophagy [direct fusion of secretory granules with lysosomes], and not autophagy, was believed to mediate ZG degradation during starvation.[33, 34] This aspect should be reconsidered using modern approaches.[15] IV. IMPACT OF PANCREATITIS ON AUTOPHAGOSOME FORMATION Tranmission electron microscopy (TEM) and immunogold-tem demonstrate the existence of autophagic vacuoles in acinar cells, accompanied by all their characteristics, that is double membrane, LC3-II and intact load. Genetic, control notes that Atg5 affects autophagosome formation in acinar cells[34, 35], proceeding via canonical pathway. Moreover, data indicate the implication of endoplasmic reticulum (ER) in autophagosome development in pancreatic acinar cells, as heterozygous deletion of X-box binding protein 1, a significant ER mediator of the adaptive Unfolded Protein Response, stimulating pancreatic autophagy in mice fed with ethanol.[36] A new autophagic pathway to selectively remove zymogen granules in pancreatitis is VMP1-mediated zymophagy, based on the selective isolation of ZGs (and not other organelles) by autophagosomes in VMP1-overexpressing mice with caerulein-induced pancreatitis.[36] These findings were obtained by immunoisolating autophagic vacuoles and require confirmation of the changes in load composition using quantitative TEM.[37] The existence of a selective autophagic pathway for ZG removal, induced by pancreatitis, is an appealing aspect. Nevertheless, autophagosomes isolate all types of organelles, including mitochondria, ER and ZGs, in wild-type mice with caerulein-induced pancreatitis, as well as in other experimental models[38] and human diseases. Zymophagy is also not triggered by VMP1 overexpression alone, with stimulation of autophagosome formation, but mainly occurs after caerulein-induced pancreatitis in VMP1-overexpressing mice.[39] Therefore, the occurrence of zymophagy implies that ZGs in pancreatitis receive a kind of pathological signal that generates their selective isolation. [40] There is still need to identify these signals. Eventually, the impact of VMP1 overexpression can be more complex, causing cell death in (nonpancreatic) cell lines[39], and VMP1 has other functions besides autophagy, as indicated in Dictyostelium discoideum.[15] V. MECHANISMS OF INFLAMMATORY PATHWAY ACTIVATION Activation of nuclear factor kappa beta (NFkB) has been noticed in experimental models complementing trypsinogen activation in terms of time course.[48, 49] Present data indicate that pathologic Ca 2+ i and activation of novel isoforms of protein kinase C cause NFkB activation. The indication for the role of pathologic Ca 2+ i comes from experiments using pharmacologic agents to block Ca 2+ i inhibiting NFkB.[50] The activation of novel protein kinase C (PKC) isoforms ε and ζ has been shown to influence NFkB activation in studies employing caerulein and ethanol based models of pancreatitis. These new PKC isoforms also seem to be involved in zymogen activation.[51, 52] There is not enough knowledge of the effect of pathologic Ca 2+ i and PKC in the activation of these early pancreatitis responses, and whether they are independent or not. The signal transduction steps causing PKC activation and pathologic Ca 2+ i are described in depending on the caerulein model of pancreatitis. Downstream targets of PKC and Ca 2+ i are still not known. Protein kinase D1 has been suggested as the downstream target of PKC isoforms[52] even if PKD1 activation may also take place through other PKC independent mechanisms.[53] Pharmacologic inhibition of PKD1 decreased trypsinogen activation and NFkB activation without affecting cell injury.[54] Calcinerin has also been suggested as 37

3 downstream target of Ca 2+ i even without fully understanding its effects on NFkB activation during pancreatic injury.[1] Besides Calcium or PKC dependent pathways other mechanisms have also been stated, including angiotensin 2 acting through AT2 receptors[55] and p38/mapk pathways.[54] The role of acinar cell injury alone in the activation inflammatory cascades has been examined in a recent study. Cell injury was found to cause activation of damage-associated molecular pattern (DAMP) receptors (TLR-9 and P2X-7 being the most important). DAMP activation triggers the formation of a cytosolic complex, the inflammasome, involved in the initiation of inflammatory cascade during pancreatic injury.[56] VI. CELL SIGNALING The initiation of acute pancreatitis is correlated with two pathologic intracellular signals in the acinar cell. Several forms of acute pancreatitis have been strongly associated with acute changes in cytosolic Ca 2+ signaling. This abnormal signal is characterized by loss of Ca 2+ oscillations and emergence of continuous high peak levels of cytosolic Ca 2+. Even if more mechanisms act to develop this pathologic signal, the ryanodine receptor (RyRs) Ca 2+ release channel in the endoplasmic reticulum seems to have a notable early contribution.[57] Other studies imply that Ca 2+ mainly targets calcineurin (CaN), a protein phosphatase 2B.[58] As both RyRs and CaN can be targeted by specific drugs, they might be significant therapeutic targets. Acute pancreatitis responses are also correlated with another pathologic signal, the activation of specific protein kinase C isoforms, including the stimulation of NFκβ and zymogen activation.[59] Activation of protein kinase C (PKC)-δ is noticed following physiologic stimulation. Nevertheless, the activation of both PKC-δ and PKC-ε is observed under acute pancreatitis generating conditions. The stimulating effects of ethanol might be connected with PKC activation. Therefore, in combination with physiologic CCK activation of PKC-δ, ethanol leads to the activation of PKC-ε.[59, 60] This pattern states again the cause generated by supraphysiologic concentrations of CCK. Protein kinase D (PKD), which is downstream from PKC-δand PKC-ε, seems to mediate some of these responses (Thrower et al., unpublished findings). Even if these observations offer fascinating evidence that specific PKC isoforms may mediate responses in early acute pancreatitis, there is still little knowledge of the molecular targets of these enzymes. References [1] Gukovsky I, Pandol SJ, Mareninova OA, Shalbueva N, Jia W, Gukovskaya AS. Impaired autophagy and organellar dysfunction in pancreatitis. J Gastroenterol Hepatol 2012; 27: [2] Mareninova O A, Hermann K, French SW, O Konski MS, Pandol SJ, Webster P et al. Impaired autophagic flux mediates acinar cell vacuole formation and trypsinogen activation in rodent models of acute pancreatitis. J Clin Invest 2009; 119:3340. [3] Czaja M J. Functions of autophagy in hepatic and pancreatic physiology and disease. Gastroenterology 2011; 140: [4] Pandol S J, Lugea A, Mareninova OA, Smoot D, Gorelick FS, Gukovskaya AS et al. Investigating the pathobiology of alcoholic pancreatitis. Alcoholism: Clinical and Experimental Research 2011; 35: [5] Gukovsky I, Pandol SJ, Gukovskaya AS. Organellar dysfunction in the pathogenesis of pancreatitis. Antioxidants & redox signaling 2011; 15: [6] Adler G, Kern HF. Alteration of membrane fusion as a cause of acute pancreatitis in the rat. Dig Dis Sci 1982; 27: [7] Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell 2011; 147: [8] Pandol S J, Lugea A, Mareninova OA, Smoot D, Gorelick FS, Gukovskaya AS et al. Investigating the pathobiology of alcoholic pancreatitis. Alcoholism: Clinical and Experimental Research 2011; 35: [9] Fortunato F, Kroemer G. Impaired autophagosome-lysosome fusion in the pathogenesis of pancreatitis. Autophagy 2009; 5: [10] Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nature reviews Molecular cell biology 2007; 8: [11] Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell 2008; 132: [12] Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene 2004; 23: [13] Mathew R, Karantza-Wadsworth V, White E. Role of autophagy in cancer. Nature Reviews Cancer 2007; 7: [14] Yang Z, Klionsky DJ. An overview of the molecular mechanism of autophagy. In: Anonymous Autophagy in Infection and Immunity: Springer; p [15] Gukovskaya A S, Gukovsky I. Autophagy and pancreatitis. American Journal of Physiology-Gastrointestinal and Liver Physiology 2012; 303:G993-G1003. [16] Cuervo A M. Autophagy: many paths to the same end. Mol Cell Biochem 2004; 263: [17] Raben N, Wong A, Ralston E, Myerowitz R. Autophagy and mitochondria in Pompe disease: nothing is so new as what has long been forgotten 2012; 160: [18] Seehafer S S, Pearce DA. You say lipofuscin, we say ceroid: defining autofluorescent storage material. Neurobiol Aging 2006; 27: [19] Cano D A, Rulifson IC, Heiser PW, Swigart LB, Pelengaris S, German M et al. Regulated β-cell regeneration in the adult mouse pancreas. Diabetes 2008; 57: [20] Glick D, Barth S, Macleod KF. Autophagy: cellular and molecular mechanisms. J Pathol 2010; 221:3-12. [21] Rabinowitz J D, White E. Autophagy and metabolism. Science 2010; 330: [22] Levine B, Yuan J. Autophagy in cell death: an innocent convict?. J Clin Invest 2005; 115:

4 [23] Scarlatti F, Granata R, Meijer A, Codogno P. Does autophagy have a license to kill mammalian cells?. Cell Death & Differentiation 2008; 16: [24] Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH et al. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death & Differentiation 2009; 16: [25] Mizushima N. Methods for monitoring autophagy. Int J Biochem Cell Biol 2004; 36: [26] Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature 2008; 451: [27] Meijer A J, Codogno P. Regulation and role of autophagy in mammalian cells. Int J Biochem Cell Biol 2004; 36: [28] Hartleben B, Gödel M, Meyer-Schwesinger C, Liu S, Ulrich T, Köbler S et al. Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice. J Clin Invest 2010; 120:1084. [29] Gardner T B, Vege SS, Chari ST, Petersen BT, Topazian MD, Clain JE et al. Faster rate of initial fluid resuscitation in severe acute pancreatitis diminishes in-hospital mortality. Pancreatology 2009; 9: [30] Mizushima N. Methods for monitoring autophagy. Int J Biochem Cell Biol 2004; 36: [31] Masini M, Bugliani M, Lupi R, Del Guerra S, Boggi U, Filipponi F et al. Autophagy in human type 2 diabetes pancreatic beta cells. Diabetologia 2009; 52: [32] Gukovsky I, Gukovskaya AS. Impaired autophagy underlies key pathological responses of acute pancreatitis. Autophagy 2010; 6: [33] Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 2004; 15: [34] Gukovsky I, Gukovskaya AS. Impaired autophagy underlies key pathological responses of acute pancreatitis. Autophagy 2010; 6: [35] Wek R C, Cavener DR. Translational control and the unfolded protein response. Antioxidants & redox signaling 2007; 9: [36] Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 2011; 27: [37] Ylä Anttila P, Vihinen H, Jokitalo E, Eskelinen E. Monitoring autophagy by electron microscopy in Mammalian cells. Meth Enzymol 2009; 452: [38] Reiser J, Adair B, Reinheckel T. Specialized roles for cysteine cathepsins in health and disease. J Clin Invest 2010; 120:3421. [39] Vaccaro M I, Grasso D, Ropolo A, Iovanna JL, Cerquetti MC. VMP1 expression correlates with acinar cell cytoplasmic vacuolization in arginine-induced acute pancreatitis. Pancreatology 2003; 3: [40] Shaid S, Brandts C, Serve H, Dikic I. Ubiquitination and selective autophagy. Cell Death & Differentiation 2012; 20: [41] Gardner T B, Vege SS, Chari ST, Pearson RK, Clain JE, Topazian MD et al. The effect of age on hospital outcomes in severe acute pancreatitis. Pancreatology 2008; 8: [42] Masini M, Bugliani M, Lupi R, Del Guerra S, Boggi U, Filipponi F et al. Autophagy in human type 2 diabetes pancreatic beta cells. Diabetologia 2009; 52: [43] Schneider A, Whitcomb DC, Singer MV. Animal models in alcoholic pancreatitis what can we learn?. Pancreatology 2002; 2: [44] Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane permeabilization in cell death. Physiol Rev 2007; 87: [45] Schild L, Keilhoff G, Augustin W, Reiser G, Striggow F. Distinct Ca2 thresholds determine cytochrome c release or permeability transition pore opening in brain mitochondria. The FASEB Journal 2001; 15: [46] Kanki T, Klionsky DJ. Mitophagy in yeast occurs through a selective mechanism. J Biol Chem 2008; 283: [47] Mareninova O A, Hermann K, French SW, O Konski MS, Pandol SJ, Webster P et al. Impaired autophagic flux mediates acinar cell vacuole formation and trypsinogen activation in rodent models of acute pancreatitis. J Clin Invest 2009; 119:3340. [48] Rakonczay Z, Hegyi P, Takacs T, McCarroll J, Saluja AK. The role of NF-κB activation in the pathogenesis of acute pancreatitis. Gut 2008; 57: [49] Zhang X, Zhang L, Chen L, Cheng Q, Wang J, Cai W et al. Influence of dexamethasone on inflammatory mediators and NF-kappaB expression in multiple organs of rats with severe acute pancreatitis. World Journal of Gastroenterology 2007; 13:548. [50] Perides G, Sharma A, Gopal A, Tao X, Dwyer K, Ligon B et al. Secretin differentially sensitizes rat pancreatic acini to the effects of supramaximal stimulation with caerulein. American Journal of Physiology-Gastrointestinal and Liver Physiology 2005; 289:G713-G721. [51] Sah R P, Saluja A. Molecular mechanisms of pancreatic injury. Curr Opin Gastroenterol 2011; 27:444. [52] Rozengurt E. Protein kinase D signaling: multiple biological functions in health and disease. Physiology 2011; 26: [53] Yuan J, Lugea A, Zheng L, Gukovsky I, Edderkaoui M, Rozengurt E et al. Protein kinase D1 mediates NF-κB activation induced by cholecystokinin and cholinergic signaling in pancreatic acinar cells. American Journal of Physiology-Gastrointestinal and Liver Physiology 2008; 295:G1190-G1201. [54] Satoh A, Gukovskaya AS, Nieto JM, Cheng JH, Gukovsky I, Reeve JR et al. PKC-δ and-ε regulate NF-κB activation induced by cholecystokinin and TNF-α in pancreatic acinar cells. American Journal of Physiology-Gastrointestinal and Liver Physiology 2004; 287:G582-G591. [55] Sah R P, Saluja A. Molecular mechanisms of pancreatic injury. Curr Opin Gastroenterol 2011; 27:444. [56] Awla D, Abdulla A, Regnér S, Thorlacius H. TLR4 but not TLR2 regulates inflammation and tissue damage in acute pancreatitis induced by retrograde infusion of taurocholate. Inflammation Res 2011; 60:

5 [57] Petersen O H, Sutton R. Ca< sup> 2 </sup> signalling and pancreatitis: effects of alcohol, bile and coffee. Trends Pharmacol Sci 2006; 27: [58] Williams J A. Intracellular Signaling Mechanisms Activated by Cholecystokinin-Regulating Synthesis and Secretion of DigestiveEnzymes in Pancreatic Acinar Cells. Annu Rev Physiol 2001; 63: [59] Baumann B, Wagner M, Aleksic T, von Wichert G, Weber CK, Adler G et al. Constitutive IKK2 activation in acinar cells is sufficient to induce pancreatitis in vivo. J Clin Invest 2007; 117: [60] Husain S Z, Grant WM, Gorelick FS, Nathanson MH, Shah AU. Caerulein-induced intracellular pancreatic zymogen activation is dependent on calcineurin. American Journal of Physiology-Gastrointestinal and Liver Physiology 2007; 292:G1594-G