Diana D. H. Thomas

Pancreatic Acinar Cells Express Vesicle-associated Membrane Protein 2- and 8-Specific Populations of Zymogen Granules with Distinct and Overlapping Roles in Secretion

Previous studies have demonstrated roles for vesicle-associated membrane protein 2 (VAMP 2) and VAMP 8 in Ca2+-regulated pancreatic acinar cell secretion, however, their coordinated function in the secretory pathway has not been addressed. Here we provide evidence using immunofluorescence microscopy, cell fractionation, and SNARE protein interaction studies that acinar cells contain two distinct populations of zymogen granules (ZGs) expressing either VAMP 2 or VAMP 8. Further, VAMP 8-positive granules also contain the synaptosome-associated protein 29, whereas VAMP 2-expressing granules do not. Analysis of acinar secretion by Texas red-dextran labeling indicated that VAMP 2-positive ZGs mediate the majority of exocytotic events during constitutive secretion and also participate in Ca2+-regulated exocytosis, whereas VAMP 8-positive ZGs are more largely involved in Ca2+-stimulated secretion. Previously undefined functional roles for VAMP and syntaxin isoforms in acinar secretion were established by introducing truncated constructs of these proteins into permeabilized acini. VAMP 2 and VAMP 8 constructs each attenuated Ca2+-stimulated exocytosis by 50%, whereas the neuronal VAMP 1 had no effects. In comparison, the plasma membrane SNAREs syntaxin 2 and syntaxin 4 each inhibited basal exocytosis, but only syntaxin 4 significantly inhibited Ca2+-stimulated secretion. Syntaxin 3, which is expressed on ZGs, had no effects. Collectively, these data demonstrate that individual acinar cells express VAMP 2- and VAMP 8-specific populations of ZGs that orchestrate the constitutive and Ca2+-regulated secretory pathways.

Identification of annexin VI as a Ca2+-sensitive CRHSP-28-binding protein in pancreatic acinar cells

CRHSP-28 is a member of the tumor protein D52 protein family that was recently shown to regulate Ca2+-stimulated secretory activity in streptolysin-O-permeabilized acinar cells (Thomas, D. H., Taft, W. B., Kaspar, K. M., and Groblewski, G. E. (2001)J. Biol. Chem. 276, 28866–28872). In the present study, the Ca2+-sensitive phospholipid-binding protein annexin VI was purified from rat pancreas as a CRHSP-28-binding protein. The interaction between CRHSP-28 and annexin VI was demonstrated by coimmunoprecipitation and gel-overlay assays and was shown to require low micromolar levels of free Ca2+, indicating these molecules likely interact under physiological conditions. Immunofluorescence microscopy confirmed a dual localization of CRHSP-28 and annexin VI, which appeared in a punctate pattern in the supranuclear and apical cytoplasm of acini. Stimulation of cells for 5 min with the secretagogue cholecystokinin enhanced the colocalization of CRHSP-28 and annexin VI within regions of acini immediately below the apical plasma membrane. Tissue fractionation revealed that CRHSP-28 is a peripheral membrane protein that is highly enriched in smooth microsomal fractions of pancreas. Further, the content of CRHSP-28 in microsomes was significantly reduced in pancreatic tissue obtained from rats that had been infused with a secretory dose of cholecystokinin for 40 min, demonstrating that secretagogue stimulation transiently alters the association of CRHSP-28 with membranes in cells. Collectively, the Ca2+-dependent binding of CRHSP-28 and annexin VI, together with their colocalization in the apical cytoplasm, is consistent with a role for these molecules in acinar cell membrane trafficking events that are essential for digestive enzyme secretion.

Tumor protein D52 expression and Ca2+-dependent phosphorylation modulates lysosomal membrane protein trafficking to the plasma membrane.

Tumor protein D52 (also known as CRHSP-28) is highly expressed in multiple cancers and tumor-derived cell lines; however, it is normally abundant in secretory epithelia throughout the digestive system, where it has been implicated in Ca(2+)-dependent digestive enzyme secretion (41). Here we demonstrate, using site-specific mutations, that Ca(2+)-sensitive phosphorylation at serine 136 modulates the accumulation of D52 at the plasma membrane within 2 min of cell stimulation. When expressed in Chinese hamster ovary CHO-K1 cells, D52 colocalized with adaptor protein AP-3, Rab27A, vesicle-associated membrane protein VAMP7, and lysosomal-associated membrane protein LAMP1, all of which are present in lysosome-like secretory organelles. Overexpression of D52 resulted in a marked accumulation of LAMP1 on the plasma membrane that was further enhanced following elevation of cellular Ca(2+). Strikingly, mutation of serine 136 to alanine abolished the Ca(2+)-stimulated accumulation of LAMP1 at the plasma membrane whereas phosphomimetic mutants constitutively induced LAMP1 plasma membrane accumulation independent of elevated Ca(2+). Identical results were obtained for endogenous D52 in normal rat kidney and HeLA cells, where both LAMP1 and D52 rapidly accumulated on the plasma membrane in response to elevated cellular Ca(2+). Finally, D52 induced the uptake of LAMP1 antibodies from the cell surface in accordance with both the level of D52 expression and phosphorylation at serine 136 demonstrating that D52 altered the plasma membrane recycling of LAMP1-associated secretory vesicles. These findings implicate both D52 expression and Ca(2+)-dependent phosphorylation at serine 136 in lysosomal membrane trafficking to and from the plasma membrane providing a novel Ca(2+)-sensitive pathway modulating the lysosome-like secretory pathway.

Complexin 2 modulates vesicle associated membrane protein (VAMP) 2 regulated zymogen granule exocytosis in pancreatic acini

Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca2+-stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central α-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central α-helical domain did not alter SNARE binding and moreover, augmented Ca2+-stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca2+-sensitive regulatory pathway for zymogen granule exocytosis.

Tumor protein D52 controls trafficking of an apical endolysosomal secretory pathway in pancreatic acinar cells

Zymogen granule (ZG) formation in acinar cells involves zymogen cargo sorting from trans-Golgi into immature secretory granules (ISGs). ISG maturation progresses by removal of lysosomal membrane and select content proteins, which enter endosomal intermediates prior to their apical exocytosis. Constitutive and stimulated secretion through this mechanism is termed the constitutive-like and minor-regulated pathways, respectively. However, the molecular components that control membrane trafficking within these endosomal compartments are largely unknown. We show that tumor protein D52 is highly expressed in endosomal compartments following pancreatic acinar cell stimulation and regulates apical exocytosis of an apically directed endolysosomal compartment. Secretion from the endolysosomal compartment was detected by cell-surface antigen labeling of lysosome-associated membrane protein LAMP1, which is absent from ZGs, and had incomplete overlap with surface labeling of synaptotagmin 1, a marker of ZG exocytosis. Although culturing (16-18 h) of isolated acinar cells is accompanied by a loss of secretory responsiveness, the levels of SNARE proteins necessary for ZG exocytosis were preserved. However, levels of endolysosomal proteins D52, EEA1, Rab5, and LAMP1 markedly decreased with culture. When D52 levels were restored by adenoviral delivery, the levels of these regulatory proteins and secretion of both LAMP1 (endolysosomal) and amylase was strongly enhanced. These secretory effects were absent in alanine and aspartate substitutions of serine 136, the major D52 phosphorylation site, and were inhibited by brefeldin A, which does not directly affect the ZG compartment. Our results indicate that D52 directly regulates apical endolysosomal secretion and are consistent with previous studies, suggesting that this pathway indirectly regulates ZG secretion of digestive enzymes.

Expression, localization, and functional role for synaptotagmins in pancreatic acinar cells

Secretagogue-induced changes in intracellular Ca(2+) play a pivotal role in secretion in pancreatic acini yet the molecules that respond to Ca(2+) are uncertain. Zymogen granule (ZG) exocytosis is regulated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. In nerve and endocrine cells, Ca(2+)-stimulated exocytosis is regulated by the SNARE-associated family of proteins termed synaptotagmins. This study examined a potential role for synaptotagmins in acinar secretion. RT-PCR revealed that synaptotagmin isoforms 1, 3, 6, and 7 are present in isolated acini. Immunoblotting and immunofluorescence using three different antibodies demonstrated synaptotagmin 1 immunoreactivity in apical cytoplasm and ZG fractions of acini, where it colocalized with vesicle-associated membrane protein 2. Synaptotagmin 3 immunoreactivity was detected in membrane fractions and colocalized with an endolysosomal marker. A potential functional role for synaptotagmin 1 in secretion was indicated by results that introduction of synaptotagmin 1 C2AB domain into permeabilized acini inhibited Ca(2+)-dependent exocytosis by 35%. In contrast, constructs of synaptotagmin 3 had no effect. Confirmation of these findings was achieved by incubating intact acini with an antibody specific to the intraluminal domain of synaptotagmin 1, which is externalized following exocytosis. Externalized synaptotagmin 1 was detected exclusively along the apical membrane. Treatment with CCK-8 (100 pM, 5 min) enhanced immunoreactivity by fourfold, demonstrating that synaptotagmin is inserted into the apical membrane during ZG fusion. Collectively, these data indicate that acini express synaptotagmin 1 and support that it plays a functional role in secretion whereas synaptotagmin 3 has an alternative role in endolysosomal membrane trafficking.

A role for tumor protein TPD52 phosphorylation in endo-membrane trafficking during cytokinesis.

Tumor protein D52 is expressed at high levels in exocrine cells containing large secretory granules where it regulates Ca(2+)-dependent protein secretion; however, D52 expression is also highly induced in multiple cancers. The present study investigated a role for the Ca(2+)-dependent phosphorylation of D52 at the single major phospho-acceptor site serine 136 on cell division. Ectopic expression of wild type D52 (D52wt) and the phosphomutants serine 136/alanine (S136A) or serine 136/glutamate (S136/E) resulted in significant multinucleation of cells. D52wt and S136/E each resulted in a greater than 2-fold increase in multinucleated cells compared to plasmid-transfected controls whereas the S136/A phospho-null mutant caused a 9-fold increase in multinucleation at 48h post-transfection. Electron microscopy revealed D52 expression induced a marked accumulation of vesicles along the mid-line between nuclei where the final stages of cell abscission normally occurs. Supporting this, D52wt strongly colocalized on vesicular structures containing the endosomal regulatory protein vesicle associated membrane protein 8 (VAMP 8) and this colocalization significantly increased with elevations in cellular Ca(2+). As VAMP 8 is known to be necessary for the endo-membrane fusion reactions that mediate the final stages of cytokinesis, these data indicate that D52 expression and phosphorylation at serine 136 play an important role in supporting the Ca(2+)-dependent membrane trafficking events necessary for cytokinesis in rapidly proliferating cancer cells.

Vesicle Associated Membrane Protein 8 (VAMP8)-mediated Zymogen Granule Exocytosis Is Dependent on Endosomal Trafficking via the Constitutive-Like Secretory Pathway

Background: Acinar cells contain two vesicle-associated membrane proteins (VAMP) 2 and 8 for secretion. Results: WT and VAMP8 knock-out acini expressing various regulatory protein constructs demonstrate VAMP2 regulates early and VAMP8 late phases of secretion. Conclusion: VAMP8- but not VAMP2-mediated secretion is dependent on anterograde endosomal trafficking. Significance: Results provide mechanistic insight into how different zymogen granule VAMPs shape secretion. Acinar cell zymogen granules (ZG) express 2 isoforms of the vesicle-associated membrane protein family (VAMP2 and -8) thought to regulate exocytosis. Expression of tetanus toxin to cleave VAMP2 in VAMP8 knock-out (−/−) acini confirmed that VAMP2 and -8 are the primary VAMPs for regulated exocytosis, each contributing ∼50% of the response. Analysis of VAMP8−/− acini indicated that although stimulated secretion was significantly reduced, a compensatory increase in constitutive secretion maintained total secretion equivalent to wild type (WT). Using a perifusion system to follow secretion over time revealed VAMP2 mediates an early rapid phase peaking and falling within 2–3 min, whereas VAMP8 controls a second prolonged phase that peaks at 4 min and slowly declines over 20 min to support the protracted secretory response. VAMP8−/− acini show increased expression of the endosomal proteins Ti-VAMP7 (2-fold) and Rab11a (4-fold) and their redistribution from endosomes to ZGs. Expression of GDP-trapped Rab11a-S25N inhibited secretion exclusively from the VAMP8 but not the VAMP2 pathway. VAMP8−/− acini also showed a >90% decrease in the early endosomal proteins Rab5/D52/EEA1, which control anterograde trafficking in the constitutive-like secretory pathway. In WT acini, short term (14–16 h) culture also results in a >90% decrease in Rab5/D52/EEA1 and a complete loss of the VAMP8 pathway, whereas VAMP2-secretion remains intact. Remarkably, rescue of Rab5/D52/EEA1 expression restored the VAMP8 pathway. Expressed D52 shows extensive colocalization with Rab11a and VAMP8 and partially copurifies with ZG fractions. These results indicate that robust trafficking within the constitutive-like secretory pathway is required for VAMP8- but not VAMP2-mediated ZG exocytosis.

Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

Background & Aims Pancreatic acinar cells have an expanded apical endosomal system, the physiologic and pathophysiologic significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is an essential phospholipid generated by phosphatidylinositol 3-phosphate 5-kinase (PIKfyve), which phosphorylates phosphatidylinositol-3-phosphate (PI3P). PI(3,5)P2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Methods Inhibition of EE to LE trafficking was achieved using pharmacologic inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to supramaximal cholecystokinin (CCK-8), bile acids, and cigarette toxin was determined. Results PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to supramaximal CCK-8, tobacco toxin, and bile salts in both rodent and human acini. Conclusions These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens, leading to the pathogenicity of acute pancreatitis.

Acute acinar pancreatitis blocks vesicle-associated membrane protein 8 (VAMP8)-dependent secretion, resulting in intracellular trypsin accumulation

Zymogen secretory granules in pancreatic acinar cells express two vesicle-associated membrane proteins (VAMP), VAMP2 and -8, each controlling 50% of stimulated secretion. Analysis of secretion kinetics identified a first phase (0–2 min) mediated by VAMP2 and second (2–10 min) and third phases (10–30 min) mediated by VAMP8. Induction of acinar pancreatitis by supramaximal cholecystokinin (CCK-8) stimulation inhibits VAMP8-mediated mid- and late-phase but not VAMP2-mediated early-phase secretion. Elevation of cAMP during supramaximal CCK-8 mitigates third-phase secretory inhibition and acinar damage caused by the accumulation of prematurely activated trypsin. VAMP8−/− acini are resistant to secretory inhibition by supramaximal CCK-8, and despite a 4.5-fold increase in total cellular trypsinogen levels, are fully protected from intracellular trypsin accumulation and acinar damage. VAMP8-mediated secretion is dependent on expression of the early endosomal proteins Rab5, D52, and EEA1. Supramaximal CCK-8 (60 min) caused a 60% reduction in the expression of D52 followed by Rab5 and EEA1 in isolated acini and in in vivo. The loss of D52 occurred as a consequence of its entry into autophagic vacuoles and was blocked by lysosomal cathepsin B and L inhibition. Accordingly, adenoviral overexpression of Rab5 or D52 enhanced secretion in response to supramaximal CCK-8 and prevented accumulation of activated trypsin. These data support that acute inhibition of VAMP8-mediated secretion during pancreatitis triggers intracellular trypsin accumulation and loss of the early endosomal compartment. Maintaining anterograde endosomal trafficking during pancreatitis maintains VAMP8-dependent secretion, thereby preventing accumulation of activated trypsin.