Tanveer A. Javed

Bile acids induce pancreatic acinar cell injury and pancreatitis by activating calcineurin

Background: Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca2+. Results: Pharmacologic and genetic inhibition of the Ca2+-activated phosphatase calcineurin dramatically reduces acinar cell injury and in vivo pancreatitis resulting from bile acid exposure. Conclusion: Acinar cell calcineurin mediates acinar cell injury and pancreatitis resulting from bile acid exposure. Significance: Calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis. Biliary pancreatitis is the leading cause of acute pancreatitis in both children and adults. A proposed mechanism is the reflux of bile into the pancreatic duct. Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca2+. Thus, it would be clinically relevant to know the targets of this aberrant Ca2+ signal. We hypothesized that the Ca2+-activated phosphatase calcineurin is such a Ca2+ target. To examine calcineurin activation, we infected primary acinar cells from mice with an adenovirus expressing the promoter for a downstream calcineurin effector, nuclear factor of activated T-cells (NFAT). The bile acid taurolithocholic acid-3-sulfate (TLCS) was primarily used to examine bile acid responses. TLCS caused calcineurin activation only at concentrations that cause acinar cell injury. The activation of calcineurin by TLCS was abolished by chelating intracellular Ca2+. Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl ester) (BAPTA-AM) or the three specific calcineurin inhibitors FK506, cyclosporine A, or calcineurin inhibitory peptide prevented bile acid-induced acinar cell injury as measured by lactate dehydrogenase leakage and propidium iodide uptake. The calcineurin inhibitors reduced the intra-acinar activation of chymotrypsinogen within 30 min of TLCS administration, and they also prevented NF-κB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform Aβ (CnAβ) subunit had reduced pancreatitis severity after infusion of TLCS or taurocholic acid into the pancreatic duct. In summary, we demonstrate that acinar cell calcineurin is activated in response to Ca2+ generated by bile acid exposure, bile acid-induced pancreatic injury is dependent on calcineurin activation, and calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis.

Exposure to Radiocontrast Agents Induces Pancreatic Inflammation by Activation of Nuclear Factor-κB, Calcium Signaling, and Calcineurin

BACKGROUND & AIMS Radiocontrast agents are required for radiographic procedures, but these agents can injure tissues by unknown mechanisms. We investigated whether exposure of pancreatic tissues to radiocontrast agents during endoscopic retrograde cholangiopancreatography (ERCP) causes pancreatic inflammation, and studied the effects of these agents on human cell lines and in mice. METHODS We exposed mouse and human acinar cells to the radiocontrast agent iohexol (Omnipaque; GE Healthcare, Princeton, NJ) and measured intracellular release of Ca(2+), calcineurin activation (using a luciferase reporter), activation of nuclear factor-κB (NF-κB, using a luciferase reporter), and cell necrosis (via propidium iodide uptake). We infused the radiocontrast agent into the pancreatic ducts of wild-type mice (C57BL/6) to create a mouse model of post-ERCP pancreatitis; some mice were given intraperitoneal injections of the calcineurin inhibitor FK506 before and after infusion of the radiocontrast agent. CnAβ(-/-) mice also were used. This experiment also was performed in mice given infusions of adeno-associated virus 6-NF-κB-luciferase, to assess activation of this transcription factor in vivo. RESULTS Incubation of mouse and human acinar cells, but not HEK293 or COS7 cells, with iohexol led to a peak and then plateau in Ca(2+) signaling, along with activation of the transcription factors NF-κB and nuclear factor of activated T cells. Suppressing Ca(2+) signaling or calcineurin with BAPTA, cyclosporine A, or FK506 prevented activation of NF-κB and acinar cell injury. Calcineurin Aβ-deficient mice were protected against induction of pancreatic inflammation by iohexol. The calcineurin inhibitor FK506 prevented contrast-induced activation of NF-κB in pancreata of mice, this was observed by live imaging of mice given infusions of adeno-associated virus 6-NF-κB-luciferase. CONCLUSIONS Radiocontrast agents cause pancreatic inflammation in mice, via activation of NF-κB, Ca(2+) signaling, and calcineurin. Calcineurin inhibitors might be developed to prevent post-ERCP pancreatitis in patients.

Pancreatic Acinar Cell Nuclear Factor κB Activation Because of Bile Acid Exposure Is Dependent on Calcineurin

Background: Bile acids cause activation of NF-κB and lead to injury in pancreatic acinar cells, but the mechanism is unknown. Results: Pharmacologic and genetic inhibition of calcineurin reduces bile acid-induced NF-κB activation and PKC-δ translocation. Conclusion: Calcineurin facilitates bile-induced NF-κB activation. The mechanism is at the level of PKC activation. Significance: We have identified a novel mechanism by which bile acids facilitate NF-κB activation in pancreatic acinar cells. Biliary pancreatitis is the most common etiology of acute pancreatitis, accounting for 30–60% of cases. A dominant theory for the development of biliary pancreatitis is the reflux of bile into the pancreatic duct and subsequent exposure to pancreatic acinar cells. Bile acids are known to induce aberrant Ca2+ signals in acinar cells as well as nuclear translocation of NF-κB. In this study, we examined the role of the downstream Ca2+ target calcineurin on NF-κB translocation. Freshly isolated mouse acinar cells were infected for 24 h with an adenovirus expressing an NF-κB luciferase reporter. The bile acid taurolithocholic acid-3-sulfate caused NF-κB activation at concentrations (500 μm) that were associated with cell injury. We show that the NF-κB inhibitor Bay 11-7082 (1 μm) blocked translocation and injury. Pretreatment with the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, the calcineurin inhibitors FK506 and cyclosporine A, or use of acinar cells from calcineurin Aβ-deficient mice each led to reduced NF-κB activation with taurolithocholic acid-3-sulfate. Importantly, these manipulations did not affect LPS-induced NF-κB activation. A critical upstream regulator of NF-κB activation is protein kinase C, which translocates to the membranes of various organelles in the active state. We demonstrate that pharmacologic and genetic inhibition of calcineurin blocks translocation of the PKC-δ isoform. In summary, bile-induced NF-κB activation and acinar cell injury are mediated by calcineurin, and a mechanism for this important early inflammatory response appears to be upstream at the level of PKC translocation.

Cluster of differentiation 38 (CD38) mediates bile-acid induced acinar cell injury and pancreatitis through cyclic ADP ribose and intracellular calcium release

Background: Bile acids cause ryanodine receptor (RyR) Ca2+ release and lead to injury in pancreatic acinar cells, yet the mechanism is unknown. Results: Inhibition of the RyR activator cADPR reduces bile acid-induced acinar cell Ca2+ release, cell injury, and pancreatitis. Conclusion: CD38-cADPR facilitates bile-induced Ca2+ release, cell injury, and pancreatitis. Significance: The CD38-cADPR pathway may serve as a target for the treatment of some forms of pancreatitis. Aberrant Ca2+ signals within pancreatic acinar cells are an early and critical feature in acute pancreatitis, yet it is unclear how these signals are generated. An important mediator of the aberrant Ca2+ signals due to bile acid exposure is the intracellular Ca2+ channel ryanodine receptor. One putative activator of the ryanodine receptor is the nucleotide second messenger cyclic ADP-ribose (cADPR), which is generated by an ectoenzyme ADP-ribosyl cyclase, CD38. In this study, we examined the role of CD38 and cADPR in acinar cell Ca2+ signals and acinar injury due to bile acids using pharmacologic inhibitors of CD38 and cADPR as well as mice deficient in Cd38 (Cd38−/−). Cytosolic Ca2+ signals were imaged using live time-lapse confocal microscopy in freshly isolated mouse acinar cells during perifusion with the bile acid taurolithocholic acid 3-sulfate (TLCS; 500 μm). To focus on intracellular Ca2+ release and to specifically exclude Ca2+ influx, cells were perifused in Ca2+-free medium. Cell injury was assessed by lactate dehydrogenase leakage and propidium iodide uptake. Pretreatment with either nicotinamide (20 mm) or the cADPR antagonist 8-Br-cADPR (30 μm) abrogated TLCS-induced Ca2+ signals and cell injury. TLCS-induced Ca2+ release and cell injury were reduced by 30 and 95%, respectively, in Cd38-deficient acinar cells compared with wild-type cells (p < 0.05). Cd38-deficient mice were protected against a model of bile acid infusion pancreatitis. In summary, these data indicate that CD38-cADPR mediates bile acid-induced pancreatitis and acinar cell injury through aberrant intracellular Ca2+ signaling.

Dynamic Imaging of Pancreatic Nuclear Factor κB (NF-κB) Activation in Live Mice Using Adeno-associated Virus (AAV) Infusion and Bioluminescence

Background: NF-κB is an important signaling molecule in the development of acute pancreatitis. Results: Adeno-associated virus-NF-κB-luciferase-infused mice showed a 77- and 140-fold increase in pancreas-specific NF-κB bioluminescence following caerulein and caerulein + LPS pancreatitis, respectively. Conclusion: NF-κB activation can be examined in a live, dynamic fashion during pancreatic inflammation. Significance: This technique offers a valuable tool to study real-time activation of NF-κB in vivo. Nuclear factor κB (NF-κB) is an important signaling molecule that plays a critical role in the development of acute pancreatitis. Current methods for examining NF-κB activation involve infection of an adenoviral NF-κB-luciferase reporter into cell lines or electrophoretic mobility shift assay of lysate. The use of adeno-associated viruses (AAVs) has proven to be an effective method of transfecting whole organs in live animals. We examined whether intrapancreatic duct infusion of AAV containing an NF-κB-luciferase reporter (AAV-NF-κB-luciferase) can reliably measure pancreatic NF-κB activation. We confirmed the infectivity of the AAV-NF-κB-luciferase reporter in HEK293 cells using a traditional luciferase readout. Mice were infused with AAV-NF-κB-luciferase 5 weeks before induction of pancreatitis (caerulein, 50 μg/kg). Unlike transgenic mice that globally express NF-κB-luciferase, AAV-infused mice showed a 15-fold increase in pancreas-specific NF-κB bioluminescence following 12 h of caerulein compared with baseline luminescence (p < 0.05). The specificity of the NF-κB-luciferase signal to the pancreas was confirmed by isolating the pancreas and adjacent organs and observing a predominant bioluminescent signal in the pancreas compared with liver, spleen, and stomach. A complementary mouse model of post-ERCP-pancreatitis also induced pancreatic NF-κB signals. Taken together these data provide the first demonstration that NF-κB activation can be examined in a live, dynamic fashion during pancreatic inflammation. We believe this technique offers a valuable tool to study real-time activation of NF-κB in vivo.

Valproic Acid Limits Pancreatic Recovery after Pancreatitis by Inhibiting Histone Deacetylases and Preventing Acinar Redifferentiation Programs

The mechanisms by which drugs induce pancreatitis are unknown. A definite cause of pancreatitis is due to the antiepileptic drug valproic acid (VPA). On the basis of three crucial observations-that VPA inhibits histone deacetylases (HDACs), HDACs mediate pancreas development, and aspects of pancreas development are recapitulated during recovery of the pancreas after injury-we hypothesized that VPA does not cause injury on its own, but it predisposes patients to pancreatitis by inhibiting HDACs and provoking an imbalance in pancreatic recovery. In an experimental model of pancreatic injury, we found that VPA delayed recovery of the pancreas and reduced acinar cell proliferation. In addition, pancreatic expression of class I HDACs (which are the primary VPA targets) increased in the midphase of pancreatic recovery. VPA administration inhibited pancreatic HDAC activity and led to the persistence of acinar-to-ductal metaplastic complexes, with prolonged Sox9 expression and sustained β-catenin nuclear activation, findings that characterize a delay in regenerative reprogramming. These effects were not observed with valpromide, an analog of VPA that lacks HDAC inhibition. This is the first report, to our knowledge, that VPA shifts the balance toward pancreatic injury and pancreatitis through HDAC inhibition. The work also identifies a new paradigm for therapies that could exploit epigenetic reprogramming to enhance pancreatic recovery and disorders of pancreatic injury.

Targeted Inhibition of Pancreatic Acinar Cell Calcineurin Is a Novel Strategy to Prevent Post-ERCP Pancreatitis

Background & Aims There is a pressing need to develop effective preventative therapies for post–endoscopic retrograde cholangiopancreatography pancreatitis (PEP). We showed that early PEP events are induced through the calcium-activated phosphatase calcineurin and that global calcineurin deletion abolishes PEP in mice. A crucial question is whether acinar cell calcineurin controls the initiation of PEP in vivo. Methods We used a mouse model of PEP and examined the effects of in vivo acinar cell-specific calcineurin deletion by either generating a conditional knockout line or infusing a novel adeno-associated virus–pancreatic elastase improved Cre (I–iCre) into the pancreatic duct of a calcineurin floxed line. Results We found that PEP is dependent on acinar cell calcineurin in vivo, and this led us to determine that calcineurin inhibitors, infused within the radiocontrast, largely can prevent PEP. Conclusions These results provide the impetus for launching clinical trials to test the efficacy of intraductal calcineurin inhibitors to prevent PEP.

Pancreatitis-Induced Depletion of Syntaxin 2 Promotes Autophagy and Increases Basolateral Exocytosis

BACKGROUND & AIMS Pancreatic acinar cells are polarized epithelial cells that store enzymes required for digestion as inactive zymogens, tightly packed at the cell apex. Stimulation of acinar cells causes the zymogen granules to fuse with the apical membrane, and the cells undergo exocytosis to release proteases into the intestinal lumen. Autophagy maintains homeostasis of pancreatic acini. Syntaxin 2 (STX2), an abundant soluble N-ethyl maleimide sensitive factor attachment protein receptor in pancreatic acini, has been reported to mediate apical exocytosis. Using human pancreatic tissues and STX2-knockout (KO) mice, we investigated the functions of STX2 in zymogen granule-mediated exocytosis and autophagy. METHODS We obtained pancreatic tissues from 5 patients undergoing surgery for pancreatic cancer and prepared 80-μm slices; tissues were exposed to supramaximal cholecystokinin octapeptide (CCK-8) or ethanol and a low concentration of CCK-8 and analyzed by immunoblot and immunofluorescence analyses. STX2-KO mice and syntaxin 2+/+ C57BL6 mice (controls) were given intraperitoneal injections of supramaximal caerulein (a CCK-8 analogue) or fed ethanol and then given a low dose of caerulein to induce acute pancreatitis, or saline (controls); pancreata were isolated and analyzed by histology and immunohistochemistry. Acini were isolated from mice, incubated with CCK-8, and analyzed by immunofluorescence microscopy or used in immunoprecipitation experiments. Exocytosis was quantified using live-cell exocytosis and Ca2+ imaging analyses and based on formation of exocytotic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes. Dysregulations in autophagy were identified using markers, electron and immunofluorescence microscopy, and protease activation assays. RESULTS Human pancreatic tissues and dispersed pancreatic acini from control mice exposed to CCK-8 or ethanol plus CCK-8 were depleted of STX2. STX2-KO developed more severe pancreatitis after administration of supramaximal caerulein or a 6-week ethanol diet compared with control. Acini from STX2-KO mice had increased apical exocytosis after exposure to CCK-8, as well as increased basolateral exocytosis, which led to ectopic release of proteases. These increases in apical and basolateral exocytosis required increased formation of fusogenic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes, mediated by STX3 and STX4. STX2 bound ATG16L1 and prevented it from binding clathrin. Deletion of STX2 from acini increased binding of AT16L1 to clathrin, increasing formation of pre-autophagosomes and inducing autophagy. Induction of autophagy promoted the CCK-8-induced increase in autolysosome formation and the activation of trypsinogen. CONCLUSIONS In studies of human pancreatic tissues and pancreata from STX2-KO and control mice, we found STX2 to block STX3- and STX4-mediated fusion of zymogen granules with the plasma membrane and exocytosis and prevent binding of ATG16L1 to clathrin, which contributes to induction of autophagy. Exposure of pancreatic tissues to CCK-8 or ethanol depletes acinar cells of STX2, increasing basolateral exocytosis and promoting autophagy induction, leading to activation of trypsinogen.

Depletion of the membrane-fusion regulator Munc18c attenuates caerulein hyperstimulation–induced pancreatitis

Epithelial pancreatic acinar cells perform crucial functions in food digestion, and acinar cell homeostasis required for secretion of digestive enzymes relies on SNARE-mediated exocytosis. The ubiquitously expressed Sec1/Munc18 protein mammalian uncoordinated-18c (Munc18c) regulates membrane fusion by activating syntaxin-4 (STX-4) to bind cognate SNARE proteins to form a SNARE complex that mediates exocytosis in many cell types. However, in the acinar cell, Munc18cs functions in exocytosis and homeostasis remain inconclusive. Here, we found that pancreatic acini from Munc18c-depleted mice (Munc18c+/−) and human pancreas (lenti-Munc18c-shRNA–treated) exhibit normal apical exocytosis of zymogen granules (ZGs) in response to physiologic stimulation with the intestinal hormone cholecystokinin (CCK-8). However, when stimulated with supraphysiologic CCK-8 levels to mimic pancreatitis, Munc18c-depleted (Munc18c+/−) mouse acini exhibited a reduction in pathological basolateral exocytosis of ZGs resulting from a decrease in fusogenic STX-4 SNARE complexes. This reduced basolateral exocytosis in part explained the less severe pancreatitis observed in Munc18c+/− mice after hyperstimulation with the CCK-8 analog caerulein. Likely as a result of this secretory blockade, Munc18c-depleted acini unexpectedly activated a component of the endoplasmic reticulum (ER) stress response that contributed to autophagy induction, resulting in downstream accumulation of autophagic vacuoles and autolysosomes. We conclude that Munc18cs role in mediating ectopic basolateral membrane fusion of ZGs contributes to the initiation of CCK-induced pancreatic injury, and that blockade of this secretory process could increase autophagy induction.

Pancreatic gene expression during recovery after pancreatitis reveals unique transcriptome profiles

It is well known that pancreatic recovery after a single episode of injury such as an isolated bout of pancreatitis occurs rapidly. It is unclear, however, what changes are inflicted in such conditions to the molecular landscape of the pancreas. In the caerulein hyperstimulation model of pancreatitis, the murine pancreas has the ability to recover within one week based on histological appearance. In this study, we sought to characterize by RNA-sequencing (RNA-seq) the transcriptional profile of the recovering pancreas up to two weeks post-injury. We found that one week after injury there were 319 differentially expressed genes (DEGs) compared with baseline and that after two weeks there were 53 DEGs. Forty (12.5%) of the DEGs persisted from week one to week two, and another 13 DEGs newly emerged in the second week. Amongst the top up-regulated DEGs were several trypsinogen genes (trypsinogen 4, 5, 12, 15, and 16). To our knowledge, this is the first characterization of the transcriptome during pancreatic recovery by deep sequencing, and it reveals on a molecular basis that there is an ongoing recovery of the pancreas even after apparent histological resolution. The findings also raise the possibility of an emerging novel transcriptome upon pancreatic recovery.