Acute pancreatitis (AP) is a potentially fatal disease with significant morbidity and mortality.1,2 Its pathogenesis remains obscure, and there are no specific or effective treatments. Acute pancreatitis is the third most common reason for hospital admissions in those with gastrointestinal disease and the sixth most common nonmalignant cause of death from digestive diseases in the United States, and is also a major risk factor for developing chronic pancreatitis and pancreatic cancer. The economic impact of AP is substantial, with total inpatient, outpatient, and long-term care costs of $2.8 billion annually.1
Key AP pathologies include the inappropriate/intrapancreatic zymogen activation (eg, conversion of trypsinogen to trypsin), reduced and dysregulated digestive enzyme secretion, vacuole accumulation, inflammation, and apoptotic and necrotic acinar cell death. Chronic pancreatitis is manifest by exocrine pancreatic atrophy, chronic inflammation (driven by macrophages and T cells), and fibrosis.2–5 The pancreatic acinar cell is a major disease participant, and it is generally held that disordering of its functions leads to both acute and chronic pancreatitis. Although considerable progress has been achieved during the past 15 years in elucidating the inflammatory responses, modulation of inflammation has not successfully treated pancreatitis.3,4,6 One reason for that could be the persistence of acinar cell damage sustaining the inflammatory and cell death responses.
The central physiologic function of the pancreatic acinar cell is to synthesize, transport, store, and secrete digestive enzymes.7,8 This is accomplished through coordinated and sequential actions of the endoplasmic reticulum (ER), Golgi apparatus, the endolysosomal system, storage and secretory organelles, and mitochondria. The work of Palade and colleagues from 50 years ago recognized distinct cellular structures seen by electron microscopy in the pancreatic acinar cell and predicted that they have special functional roles.9,10 Palade9 and his trainee Jim Jamieson worked to understand the physiologic role of these structures. They found that nascent proteins are vectorially transported through acinar cell organelles to the site of storage in mature zymogen granules (ZGs). Subsequent studies showed that specific steps in the biogenesis and maturation of export proteins occurred in distinct cellular compartments. Information relating to the roles of cytoplasmic organelle networks in physiologic and pathophysiologic processes in eukaryotic cells has greatly expanded during the last decade,11,12 prompting further investigations of the functions of cytoplasmic organelles in acinar cell physiology and pathophysiology.
Here, we first review key acinar cell organelles and their physiologic functions, then summarize findings from our laboratories on the pathogenic mechanisms of organelle dysfunction and their role in initiating and driving pancreatitis, emphasizing those that could be therapeutic targets.
CYTOPLASMIC ORGANELLES AND CELLULAR HOMEOSTASIS
The ER is a major site of protein and lipid synthesis; another important ER function is maintaining Ca2+ homeostasis. The ER also mediates protein posttranslational modifications (eg, glycosylation and disulfide bond formation), trafficking of newly synthesized proteins to the Golgi complex, and lipid synthesis (Fig. 1). To ensure the fidelity of protein synthesis, an ER quality control system regulates and monitors the folding and tagging of nascent proteins.13–17 Misfolded proteins can be toxic and can also sequester substrates that are needed for new protein synthesis. When unfolded and/or misfolded proteins accumulate at their site of synthesis, cells first respond by activating an “unfolded protein response” (UPR), which temporary reduces general translation and simultaneously upregulates the levels of ER/Golgi factors (chaperones and foldases) that mediate protein folding, trafficking, and degradation. If an adaptive UPR is unsuccessful to restore ER function, ER-stress signaling programs are activated leading to inflammation and cell death.14,16
Ca2+ homeostasis is central to both physiologic and pathologic acinar cell functions because many enzymes require calcium ion as a cofactor and because of the key role of Ca2+ in signal transduction. Ca2+ homeostasis is maintained by various organelles and at the same time impacts their function.18,19 The intracellular Ca2+ concentration in a typical eukaryotic cell is approximately 100 nM, which is ~10,000-fold lower than that in extracellular fluid. This gradient is maintained by membrane calcium pumps and channels as well as intracellular storage compartments that are primarily the ER and mitochondria. In response to physiologic hormonal or neurotransmitter stimulation, stored Ca2+ is released from ER into the cytosol and used by the cell to activate various Ca2+-dependent functions, such as exocytosis (see hereinafter). The ER can directly transport Ca2+ to mitochondria through specific modifications in both organelles' membranes (mitochondria-associated ER membranes, or MAMs) that regulate the synthesis and translocation of lipids, mitochondrial dynamics, autophagy, apoptosis, and energy metabolism. Ca2+ interchange through MAMs is critical to fuel mitochondrial metabolism and also modulates ER Ca2+ levels.15,20 Mitochondria can also directly uptake Ca2+ from cytosol; this is facilitated by the highly negative mitochondrial membrane potential and finely tuned by proteins in the mitochondrial Ca2+ uniporter complex.21
Mitochondria are responsible for a range of cellular functions22,23 (Fig. 1). The major one is generation of ATP through consecutive redox reactions carried out by a series of linked protein complexes within the inner mitochondrial membrane, called the electron transport chain (ETC). Electron flow through ETC generates a pH gradient and an electrical potential used by ATP synthase to transform adenosine diphosphate into adenosine triphosphate in a phosphorylation reaction. Mitochondria also mediate cell survival; a universal trigger of cell death is mitochondrial membrane permeabilization.24 A major mechanism of mitochondrial permeabilization is through persistent opening of the mitochondrial permeability transition pore (MPTP), a multiprotein nonselective channel traversing both the inner and outer mitochondrial membranes.25,26 In its “open” conformation, MPTP allows for unregulated entry of solutes <1500 Da (including water) into the matrix, resulting in mitochondrial depolarization, inhibition of ATP synthesis, swelling, and, ultimately, necrosis. Various stresses, such as mitochondrial Ca2+ overload, overproduction of reactive oxygen species (ROS), and NAD/NADH shift, can cause MPTP opening. The MPTP backbone is organized around the mitochondrial resident protein cyclophilin D (CypD); genetic, molecular, or pharmacologic inactivation of CypD blocks MPTP opening.25,26
Individual mitochondria form a tubular-circular dynamic network, which tailors ATP production to cellular demand.27 When energy demand is low, most mitochondria are present in a circular form characterized by short ETC length and low ATP synthesis. In response to increased energy demand, mitochondria fuse, forming a tubular configuration to increase ATP synthesis. Imbalance in mitochondrial fission-fusion is associated with various diseases and can result in inadequate ATP production, increased mitochondrial ROS, and impaired Ca2+ transport.28
Vesicular Transporters and the Endosomal System
Newly synthesized proteins are moved to specific cellular organelles by vesicular transporters; related transport vesicles also mediate import of the material from outside the cell (through endocytosis). Organelles of endosomal system control trafficking of these vesicles to distinct cellular destinations such as lysosomes, the plasma membrane, or recycling back to Golgi, as well as cargo sorting and concentration (Fig. 1). Endosomal compartments are composed of highly dynamic membrane-enclosed tubulovesicular structures and include early/sorting, recycling, and late endosomes.29–31 Endosomes of each subtype differ in their functions, which are largely determined by the presence on their surface of distinct Rab proteins, members of the Rab family of small GTPases.31 For example, Rab4 and Rab5 are associated with early endosomes, which mediate endocytosis and recycling; whereas Rab7 and Rab9 are markers of late endosomes, responsible for delivery of both endocytic and autophagic cargo to lysosomes for degradation. Rab proteins function as molecular switches that alternate between the active GTP-bound form and the inactive GDP-bound form.29–32
Lysosomes are a final destination for select endosomal trafficking and are primarily responsible for protein, lipid, and carbohydrate degradation33 (Fig. 1). Normal lysosomal activity requires several key elements. First, a unique mechanism for the sorting and delivery of a range of lysosomal enzymes to this compartment34,35 involves a covalent mannose 6-phosphate (M6P) addition to asparagine residues of many lysosomal hydrolases in the cis-Golgi network. The modified hydrolases then bind to 1 of 2 transmembrane M6P receptors (M6P-Rs) on vesicles that are subsequently directed toward endosomes to deliver their cargo to lysosomes.36–38 A second characteristic feature of lysosomes is their acidic pH, which matches the pH optimum of most lysosomal enzymes and is modulated by a vacuolar H+-ATPase that mediates proton influx.39 Lysosomal hydrolases, such as cathepsins, are often synthesized as proproteins and undergo proteolytic processing first in endosomes and then in lysosomes where acidic pH is required for their maturation to reach full catalytic activity.40 A third critical feature of lysosomes is the presence of essential membrane proteins, including lysosome-associated membrane proteins LAMP1 and LAMP2.41,42 LAMPs are transmembrane glycoproteins whose luminal domain stabilizes content proteins. LAMP2, in particular, is important for fusion of the lysosome with autophagosomes and lysosomal proteolytic activity.
Studies from the last decade have dramatically changed our view of lysosomes from that of a simple “garbage disposal” to a dynamic organelle that regulates basic cellular processes such as nutrient sensing, vesicular protein trafficking, endocytosis, and autophagy33,43,44 (Fig. 1). Recently, members of the MiT/TEE family of transcription factors (a major one is TFEB) have been shown to play a pivotal role in organelle biogenesis and metabolic processes by globally regulating lysosomal and autophagic functions.33,44
Autophagy is a collective term for several pathways through which cytoplasmic materials, in particular organelles and long-lived proteins, are delivered to the lysosome to be degraded by lysosomal hydrolases.3,4,45,46 The degradation serves 2 purposes: (1) removal of damaged or dysfunctional cellular components, such as uncoupled mitochondria or ubiquitinated protein aggregates, and (2) the delivery of recycled substrates, such as amino acids and lipids, for critical cellular processes. The major and best-studied pathway, macroautophagy (hereon referred to as autophagy), requires de novo formation of double-membraned structures termed autophagosomes, which sequester cargo and ultimately fuse with lysosomes to form autolysosomes, where degradation occurs (Fig. 2). The process begins with the formation of so-called isolation membrane, or phagophore, followed by its elongation and closure to form the mature autophagosome. These steps are mediated by sequentially recruited complexes of evolutionary conserved autophagy-related (ATG) proteins.46 Autophagy initiation is controlled by ULK1/ATG1-mediated complex, followed by the formation of another multiprotein complex involving phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34) and Beclin1/ATG6, which nucleates the phagophore. Phagophore expansion and elongation are controlled by the ubiquitin-like conjugation systems involving the ATG5-ATG12-ATG16 complex, and the microtubule-associated protein 1 light chain 3a (LC3). LC3, the mammalian paralog of yeast ATG8, is necessary for phagophore closure; during this process, ATG7 and ATG3 mediate lipidation of its cytosolic form (LC3-I) to become LC3-II, which specifically translocates to the autophagosome membrane. Autophagosomes then fuse with lysosomes (or with late endosomes before lysosomal fusion) to form single-membraned autolysosomes where cargo breakdown occurs.
Autophagy can be both nonselective or selective in its targeting specific cellular constituents for degradation.47 Thus, in conditions of high-nutrient and high-energy demand, such as starvation, macroautophagy is often nonselective. Other conditions, including organelle injury, can induce highly select organelle degradation. Examples include selective degradation of mitochondria (“mitophagy”) or lipid droplets (“lipophagy”).47,48 Dysfunction of selective autophagy can lead to reduced protein turnover, accumulation of damaged/dysfunctional organelles, and shortages of substrates for energy metabolism or protein and lipid synthesis.
ROLE OF ACINAR CELL ORGANELLES IN PHYSIOLOGIC SECRETION OF DIGESTIVE ENZYMES
The acinar cell secretory apparatus relies on cytoplasmic organelles to maintain a complex matrix of membrane trafficking pathways and coordinate polarized secretion according to cell demands. Physiologic secretion pathway starts in the ER where protein synthesis and folding occur; nascent proteins then travel to the Golgi complex, where they are packed in secretory vesicles and through post-Golgi trafficking delivered to the plasma membrane for exocytosis7,8 (Fig. 3). Cytosolic Ca2+ is a major mediator of ZG exocytosis.18,49 Secretagogues (hormones and neurotransmitters) cause a release of calcium from ER stores though IP3 (inositol 1,4,5-trisphosphate)- and RyR (ryanodine receptor)-sensitive channels. This release causes a transient increase in free cytosolic Ca2+ concentration ([Ca2+]i), which is immediately offset by removal of Ca2+ from the cytosol by Ca2+-ATPases that pump Ca2+ from the cytosol to the ER lumen and into the extracellular space. Therefore, increases in cytosolic Ca2+ during physiologic cellular functions are transient and oscillatory. This prevents a sustained increase in [Ca2+]i, which can cause aberrant activation of various calcium-dependent proteins resulting in cell damage. Further depletion of luminal ER calcium may disrupt secretion by decreasing the activities of UPR mediators, such as calreticulin.50 Thus, maintaining low cytosolic Ca2+ and constantly replenished ER Ca2+ pools is necessary for normal acinar cell secretion.
Our recent data (unpublished) show that the UPR factor X-box binding protein 1 (XBP1) is a key regulator required for maintaining the secretory phenotype of the acinar cell. As discussed hereinafter, XBP1 and the UPR transcription factor CCAAT/enhancer binding protein homologous protein (CHOP) both play a role in the acinar cell pathobiology during pancreatitis.
Protein synthesis, trafficking, and secretion require energy; Jamieson and Palade51 showed that mitochondrial inhibitors block post-ER trafficking and secretion of digestive enzymes, indicating that export of digestive enzymes to the plasma membrane for exocytosis requires normally functioning mitochondria. [Ca2+]i increase by secretagogues stimulates mitochondrial ETC activity, resulting in an increase in ATP production necessary for protein trafficking,52 and at the same time causes transient mitochondrial depolarization53–55 limiting ATP generation. The balance between these 2 processes maintains ATP homeostasis during physiologic secretory responses.
Recent studies revealed a critical role of the autophagy/lysosomal pathway in maintaining acinar cell homeostasis, particularly its secretory function.34,45,56,57 Blockade of autophagosome formation by Atg5 genetic ablation inhibits hormone-induced secretion in acinar cells.58,59 Cholecystokinin-8 (CCK)–induced amylase secretion is also inhibited in acinar cells from mice with genetic ablation of IKKα (the inhibitor of the nuclear factor κB [IκB] kinase α), which causes impaired completion of autophagy (unrelated to nuclear factor κB activity).60 Similarly, inhibiting autophagic flux by disrupting lysosomal function in LAMP2-null mice decreases pancreatic digestive enzyme content and inhibits CCK-induced secretion.61 Thus, acinar cell secretion is impaired if quality control system mediated by autophagy is not working.
Most digestive enzymes are trafficked to their cellular storage sites and concentrated many-fold throughout the secretory pathway.62 Such concentration begins at the ER exit sites in vesicles anterogradely transported to the Golgi complex, and it continues in post-Golgi compartments. Final maturation, resulting in concentration of the digestive enzyme content, is achieved by budding of small vesicles from immature secretory granules, which removes both membrane and content to form ZG.63 The M6P-Rs also extract some, but not all, lysosomal enzymes from immature secretory granules.63 In addition to the major, ZG-mediated secretory pathway, a minority (~5%) of digestive enzymes are directly released in constitutive and constitutive-like secretory pathways (also termed the minor secretory pathway [MSP]).64–66 Some fraction of small vesicles that bud from immature secretory granules participates in secretion of nascent export proteins through MSP (Fig. 3). A unique feature of this pathway66,67 is that secretion through MSP can be enhanced (~2-fold) by very low concentrations of secretagogues (10-fold less than those that trigger secretion from the ZG storage compartment). There is evidence that physiologic secretion through MSP is controlled by endosomes; in particular, we find that the soluble protein D52 has a major role in the regulation of both exocytic and endocytic pathways.7,68,69 In this context, D52 interacts with Rab5, a key regulator of early endosomal compartment.66,70,71 We also find68,72 that the MSP transports a portion of LAMP1 to the apical membrane before LAMP1 is directed to its final residence in the lysosome, demonstrating another unexpected link between organelles in the secretory and lysosomal pathways.
Zymogen granule exocytosis at the apical membrane involves assembly of SNARE protein complexes required for vesicle docking and subsequent membrane fusion. VAMP2 and VAMP8 (vesicle-associated membrane proteins) are SNAREs found on ZGs that seem to mediate the release of distinct ZG populations.7,8,66,73 Our studies using VAMP2 and VAMP8 genetic ablation showed that VAMP2, but not VAMP8, mediates initial acinar cell secretion during the first minutes after stimulation; whereas VAMP8 mediates the subsequent phase of secretion.66 These data indicate the existence of distinct ZG pools. VAMP8 localizes to both exocytic and endocytic compartments (as does D52), suggesting that some proteins may regulate both exocytosis and endocytosis. Supporting this, loss of the endosomal proteins Rab5 and EEA1 during a 16-h acinar culture is accompanied by a loss of VAMP8-mediated (but not VAMP2-mediated) ZG secretion and is fully prevented by maintaining Rab5 and EEA1 expression via adenoviral vectors.66,72
Collectively, the studies discussed earlier reveal critical and interconnected roles of ER, mitochondria, endolysosomes, and autophagy in maintaining physiologic secretion in acinar cells.
DISORDERING OF ACINAR CELL ORGANELLAR NETWORK IN PANCREATITIS
Experimental Models of AP
Because of the lack of access to human tissue until very recently, most studies addressing pancreatitis disease mechanisms make use of animal models or freshly isolated acinar cells (or pancreatic fragments) to study AP pathogenesis.74–76 These models reproduce the spectrum of human disease severity and have greatly advanced our understanding of the cell biology of pancreatitis and the molecular factors involved; they also allowed testing of potential therapeutic approaches. The most widely used in vivo AP models of pancreatitis include those induced in rodents by administering supramaximal doses of cerulein (CER; an ortholog of CCK), bile salts, or L-arginine (Arg) and by feeding young female mice choline-deficient, ethionine-supplemented diet. Incubation of isolated acinar cells with supramaximal CCK/CER or bile salts triggers early pathologic responses of AP (such as trypsinogen activation, dysregulated secretion, and vacuole accumulation) and recapitulates the earliest stages of disease. These ex vivo models have been helpful in elucidating acinar cell organelle dysfunction in AP.76
Persistent/pathologic ER stress occurs in experimental and genetic models of pancreatitis,77–84 indicating that pancreatitis impairs homeostatic protein folding mechanisms. Furthermore, evidence indicates that hereditary pancreatitis is caused by mutations in human cationic trypsinogen (PRSS1),85 and that mutations in PRSS1, carboxypeptidase A1, the endogenous trypsin inhibitor SPINK1 (serine protease inhibitor Kazal-type 1) and the trypsinogen-degrading enzyme chymotrypsinogen C that are associated with protein misfolding, resulting in ER stress, predispose to pancreatitis development.86,87 For example, the PRSS1 mutation p.L104P is found to markedly reduce secretion of the mutated protein because of its retention and aggregation associated with ER stress.77
Endoplasmic reticulum stress in pancreatitis manifests by increased phosphorylation of PKR-like ER kinase, splicing of XBP1, and expression of CHOP.77–81 We showed that increase in spliced (s)XBP1 may protect the pancreas against injury, whereas activation of CHOP is associated with acinar cell injury and pancreatitis responses.81,88 For example, chronic ethanol exposure in mice selectively enhances spliced X-box binding protein 1 (sXBP1) levels81; this response seems to protect the pancreas against injury.81,89 We reported redox and other changes in the pancreatic acinar cell ER proteome induced by ethanol feeding in sXBP1+/− mice.90 We also reported88 that a combination of ethanol and smoking results in inhibition of the sXBP1 response, associated with an increase in CHOP and pancreatitis responses, in particular acinar cell death. These findings help explain epidemiologic studies indicating that smoking promotes alcoholic pancreatitis.91,92
Dysregulation of Ca2+ Transport
The common effect seen in the first minutes of acinar cell injury in ex vivo AP models induced by supramaximal CCK/CER or bile salts is the loss of normal cytosolic Ca2+ oscillations, which are replaced by a “peak-plateau” response resulting in a sustained increase in [Ca2+]i.18,93,94 This occurs because pancreatitis-causing stimuli largely deplete ER calcium stores, and the depletion promotes massive Ca2+ entry into the acinar cell through so-called store-operated Ca2+ channels, which sense the reduced levels of calcium in the ER lumen. This sensing is mediated by stromal interaction molecule proteins at the ER/plasma membrane interface, which activate the plasma membrane calcium transporter ORAI1.95 Another type of a channel that mediates excessive Ca2+ influx and acinar cell damage is the transient receptor potential (canonical) channel TRPC3.96
Sustained increases in [Ca2+]i promote Ca2+ uptake by mitochondria resulting in mitochondrial Ca2+ overload, which in turn causes mitochondrial depolarization leading to decreased ATP synthesis and ultimately necrosis.54,55,97,98 Furthermore, increase in cytosolic Ca2+ results in activation of the phosphatase calcineurin, which promotes acinar cell injury through several pathways.99,100
Mitochondrial dysfunction is prominent in both in vivo and ex vivo experimental and genetic models of pancreatitis.54,55,98,101,102 Its main manifestation is the sustained opening of MPTP resulting in loss of mitochondrial membrane potential, which in turn causes mitochondrial fragmentation.101 Our results show that the mechanism of MTPT opening in experimental pancreatitis is model specific. In AP models induced by supramaximal CCK/CER or bile salts, increases in [Ca2+]i lead to mitochondrial Ca2+ overload and MPTP opening.54,98,101 However, MPTP opening in L-arginine–induced acute pancreatitis (Arg-AP) is through inhibition of ATP synthase in the absence of Ca2+ overload.101 In alcohol-mediated pancreatitis, MPTP opening is caused by a decrease in the NAD+/NADH ratio resulting from oxidative ethanol metabolism.55 Notably, the MPTP opening in all models of pancreatitis is CypD dependent, and genetic or pharmacologic inactivation of CypD prevents mitochondrial depolarization and largely restores mitochondrial dynamics in pancreatitis.98,101
Mitochondrial permeability transition pore–mediated pancreatic injury has been investigated in detail,54,55,98,101 but whether pancreatitis also causes mitochondrial damage through MPTP-independent pathways is unknown. The effects of pancreatitis on mitochondrial biogenesis, activities of ETC complexes, ROS generation, and mitochondrial antioxidant systems remain largely unexplored.
Dysregulation of Endosomal System
Our studies indicate that endosomal system is dysregulated in AP. We showed, in particular, that secretion through the MSP, which is mediated by endosomes (as discussed previously), is rapidly inhibited in AP models induced by supramaximal CCK, bile salts, or cigarette smoke toxin, leading to intracellular trypsin accumulation and acinar damage.72 When inhibition of MSP secretion was prevented by molecular or pharmacological approaches, basal secretion was normalized and intracellular trypsin accumulation was abolished. Further evidence for disordered endosomal function in AP is provided by our recent data103 that experimental pancreatitis causes marked reduction in the levels of Rabs controlling the early/recycling endosomes (such as Rab5 and Rab11) and alters their cellular localization. In addition, tissue fractionation data have shown significant increases in the density of early endosomes in cerulein-induced acute pancreatitis (CER-AP). Functionally, we find dramatic defects in receptor-mediated endocytosis (measured by transferrin endocytosis) in CCK-hyperstimulated acinar cells.103 The data suggest dysregulation of early/recycling endosomes in experimental AP, in particular, defective transition from early to late endosomes.
Impairment of Lysosomal and Autophagy Pathways
Experimental pancreatitis is associated with severe defects in the lysosomal pathway.3,4,45,56,61,101,104,105 These include defective processing/maturation of cathepsins, major lysosomal proteases, manifested by reduced levels of fully processed, and accumulation of intermediate forms of cathepsins.45,101,105 Concomitantly, cathepsins' enzymatic activities decrease in lysosome-enriched pancreatic subcellular fractions from rodents with pancreatitis,45,105 as noted long ago for cathepsin B.106 Experimental pancreatitis alters lysosomal vacuolar H+-ATPase localization (which maintains acidic pH in the lysosomal lumen): its soluble component is driven to assemble on acinar cell membranes.107 Finally, the levels of LAMPs, the integral membrane proteins critical for maintaining both the structure and function of lysosomes, dramatically decrease across various experimental models of nonalcoholic and alcoholic pancreatitis.61,108 Although defects in lysosomal function in pancreatitis were observed 30 years ago,106 the underlying mechanisms remain poorly understood. We posit that a critical defect is the incomplete processing/maturation of cathepsins. In normally functioning lysosomes, hydrolases are thought to form large luminal complexes, ensuring their spatial separation from lysosome membrane proteins.109,110 In pancreatitis, accumulation of the intermediate forms of cathepsins may compromise this separation resulting in LAMP proteolysis by cathepsins. Indeed, we showed that cathepsin B can cleave the intralysosomal part of LAMP molecule and that LAMPs' degradation in CER-AP is prevented by genetic ablation of cathepsin B.61 Dysregulation of endosomal system could also play a role in lysosomal dysfunction in pancreatitis, as suggested by a study of the effects of Rab7 genetic ablation in the pancreas.111
Accumulation in acinar cells of cytoplasmic vacuoles, often filled with cellular debris, has long been recognized as an early marker of AP, both in various experimental models and in human disease.45,105,112–117 Such vacuoles are also observed in the pancreas of mice with genetic ablation of key mediators of autophagy or lysosomal pathways that spontaneously develop pancreatitis (see hereinafter). Our studies45,61,101,105 show that these are autophagic vacuoles and their accumulation in acinar cells is caused by impaired autophagy. We showed that pancreatitis increases autophagosome formation but at the same time inhibits autophagic degradation, resulting in impaired autophagic flux.3,45,101,105 The most direct evidence for this is the finding that acinar cell vacuolization in experimental AP is associated with decreased rates of long-lived protein degradation.105 Furthermore, electron microscopy data show that the autophagic vacuoles accumulating in experimental pancreatitis are predominantly large autolysosomes, indicating that the fusion of autophagosomes with lysosomes is not blocked (although it could be impaired).61,101,105 The consequences of inefficient autophagic degradation are likely exacerbated by the concomitant increase in autophagosome formation in pancreatitis.45,101
Recent studies using genetic models that specifically target autophagy/lysosomal pathways provide further mechanistic insights into the role of these pathways in the initiation of pancreatitis. Mice with pancreas-specific knockouts of key autophagy mediators Atg5 or Atg7118,119 develop spontaneous pancreatitis, with trypsinogen activation, inflammation, fibrosis, and acinar-to-ductal metaplasia. Disrupting lysosomal functions by LAMP2 genetic ablation in mice causes impaired pancreatic autophagy, similar to that observed in experimental AP, and the development of spontaneous pancreatitis.61 We also found that disruption of the Gnptab gene, which encodes key enzyme mediating the addition of M6P moieties onto acid hydrolases (a critical step for their delivery to the lysosome), caused complete block of the autophagic flux in the pancreas. These knockout mice developed severe pancreatitis.120 In addition, Atg5 or LAMP2 deficiency worsened CER-AP and Arg-AP compared with wild type.58,59,61 Interestingly, CER-AP and Arg-AP responses, such as hyperamylasemia, were worsened in transgenic GFP-LC3 mice in which the increased autophagosome formation (due to overexpression of LC3) is not balanced by increased lysosomal degradation, resulting in retarded autophagic flux.121,122
Collectively, the findings in experimental and genetic models3,4,45,56–61,76,101,105,118–120,122 show the essential role of autophagy/lysosomal pathways in maintaining pancreatic acinar cell homeostasis and strongly implicate the disordered pathways in initiation and development of pancreatitis. Of note, pancreatitis develops regardless of whether these pathways are disrupted at the level of autophagosome formation, as in Atg5 and Atg7 knockout mice, or at the completion of autophagy, as in LAMP2, IKKα, or Gnptab knockout mice.
Interrelations Between the Dysfunctions of Individual Organelles in the Exocrine Pancreas
It is increasingly clear that in eukaryotic cells, cytoplasmic organelles are integrated into a highly dynamic, cooperative network that exchanges signals and material to maintain and balance cellular homeostasis, metabolism, and survival,15,123 and that each organelle functions in a milieu of coordinated exchanges with other organelles through both vesicular trafficking pathways and membrane contact sites.124 Little is known about interorganellar interactions in the exocrine pancreas, but our data indicate that disordering of a particular type of organelle in acinar cells results in the failure of the whole network, whereas restoring the function of one type of organelle improves work of others.
The most detailed information on interrelations between organelle disorders in AP has been obtained for the autophagy/lysosomal and mitochondria-mediated pathways (Fig. 4). For example, blocking autophagy by genetic ablation of Atg5, Atg7, or IKKα all caused ER stress and accumulation of dysfunctional mitochondria that overproduce ROS and generate less ATP.60,118,119 Conversely, mitochondrial damage in Arg-AP and CER-AP caused activation of pancreatic autophagy (in particular, mitophagy) and, at the same time, its impairment.98,101 Restoring mitochondrial function by CypD genetic ablation largely normalized pancreatic autophagy.101 Endoplasmic reticulum stress caused by dysregulation of UPR in XBP1-deficient pancreas led to mitochondrial dysfunction manifest by reduced oxidative phosphorylation and pathologic alterations in autophagy.81 Restoring mitochondrial function with CypD genetic ablation and enhancing autophagic efficiency with disaccharide trehalose both alleviated ER stress in experimental pancreatitis.101
Reduced acinar cell secretion is a hallmark of AP. Studies discussed earlier in relation to the role of organelles in secretion reveal that disordering of autophagy/lysosomal60,61 or endosomal69 pathways causes inhibition of secretion in acinar cells. Cholecystokinin-8–induced amylase secretion is inhibited in Atg5-null acinar cells.58,59 D52 and Rab5, proteins that regulate MSP and the endosomal compartment, are depleted from CCK-hyperstimulated acinar cells and in vivo in mouse CER-AP and pancreatitis induced with choline deficiency.69,72 Inhibition of VAMP8-dependent secretion is associated with inhibition of autophagy.69 Finally, the SNARE protein syntaxin 2 has been shown to regulate both autophagy and exocytosis in acinar cells in pancreatitis.125 Whether and how these effectors maintain homeostatic balance between autophagy and secretion in acinar cells remains to be investigated.
DISORDERING OF ACINAR CELL ORGANELLES IN HUMAN PANCREATITIS
Two approaches have been applied to detect acinar cell organelle disordering in human pancreatitis. The first is to analyze pancreatic tissue specimens from patients with pancreatitis. These have shown patterns of organelle dysfunction in human disease similar to those in rodent models, including the dramatically reduced levels of LAMP1 and LAMP261,108 and impaired autophagy evidenced by large autophagic vacuoles (seen in acinar cells with both electron and light microscopy),105 the accumulation of LC3-positive autophagic vacuoles,101 and increased levels of both LC3-II and the autophagy substrate p62/SQSTM1 (sequestosome 1).60,101 Mitochondrial dysfunction in human pancreatic tissue specimens is manifest by massive mitochondrial fragmentation.101
The second approach is to use human acinar cells (obtained as byproducts of islet isolation from cadaveric pancreata of organ donors) and subject them to ex vivo pancreatitis insults. We and others showed that healthy human acinar cells isolated from cadaveric tissue retain their morphology and functional characteristics, which has allowed for examination of early pancreatitis responses to a number of stressors, for example, bile salts.72,98,100,113,126,127 We characterized physiologic and pathophysiologic responses of the human acinar cell preparations and found them similar to those observed in mouse ex vivo models.126 In particular, disordered organelle responses—the ER stress, aberrant Ca2+ signal, mitochondrial depolarization, and impaired autophagy—are all prominent in ex vivo pancreatitis on human acinar cells.98,126 Similar results were obtained on human pancreatic slices.125,128
RESTORING THE FUNCTION OF ORGANELLAR NETWORK TO TREAT PANCREATITIS
Potential Pharmacologic Approaches
Studies discussed in this review indicate that restoring organellar homeostasis is a promising strategy for pancreatitis treatment (Fig. 5).129 Recently, the development of small-molecule enhancers of autophagy has become a major approach for various neurologic diseases.130 One such agent, the natural disaccharide trehalose, was shown to reduce injury in animal models of neurodegenerative diseases131 and is now in clinical trials for spinocerebellar ataxia type 3. We found that trehalose enhanced autophagic degradation and alleviated essentially all pancreatitis responses in both Arg-AP and CER-AP.101 Although the mechanism of trehalose action, as well as the treatment regimens to obtain a therapeutic response, needs to be further elucidated, it may provide a valuable therapeutic option.
Restoring mitochondrial potential in pancreatitis with CypD genetic ablation alleviated pancreatitis in multiple experimental models. In particular, experimental pancreatitis in CypD null mice showed lesser or no trypsinogen activation, reduced necrosis, inflammatory infiltration, and improved pancreatic histopathology (compared with wild type).55,98,101 Furthermore, small-molecule inhibitors of CypD restored mitochondrial function and prevented or markedly alleviated AP responses.98,132 Several new CypD inhibitors were recently synthesized and shown to protect mitochondrial functions and reduce necrosis in ex vivo pancreatitis,132 and thus should be considered for pancreatitis treatment.
Finally, abnormal (global and sustained) increases in cytosolic Ca2+ associated with several AP models cause both mitochondrial dysfunction and defects in the endosomal system. Modifying acinar cell Ca2+ signaling has been used in animal models to reduce AP injury, in particular, inhibition of excessive Ca2+ entry by ORAI1 inhibitors133 or TRPC3 inhibitor.96 Some of the Ca2+ damaging effects are through activating the phosphatase calcineurin. Calcineurin inhibition by genetic or pharmacologic means largely alleviated pancreatitis responses in Ca2+-dependent models of AP.100 The results suggest that pharmacologic approaches aimed to reduce [Ca2+]i can be developed for the treatment for patients with pancreatitis.133
Despite the recent progress, detailed analyses of organelle disorders in the exocrine pancreas have just begun, and there is much to be learned about their role in pancreatitis and the underlying mechanisms.3,4,134–143 One important area is the interrelations between different types of organelles in acinar cells and how these are altered by pancreatitis. A key question is whether there is a critical pathologic defect common for all types of pancreatitis and leading to failure of the whole organellar network, or whether organelle dysfunction is mediated through different mechanisms and more than one organelle disorder should be restored (eg, normalizing both autophagy and mitochondrial function at the same time) to reestablish the organellar network homeostasis.136 Another important research direction is the mechanisms that link disordering of cellular organelles to “classic” pancreatitis responses such as inflammation and cell death. In particular, recent studies began elucidating the mechanisms whereby impaired autophagy and mitochondrial dysfunction in acinar cells cause inflammation in pancreatitis.3,4,141–143
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