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Vol. 18, Issue 3, 1056-1063, March 2007
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*Institute of Molecular and Cell Biology, Singapore 138673, Singapore; and Department of Anatomy and Cell Biology, University of Oulu, 90014 Oulu, Finland
Submitted November 2, 2006;
Revised December 21, 2006;
Accepted January 2, 2007
Monitoring Editor: Vivek Malhotra
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Bonifacino and Glick 2004; Hong, 2005; Jahn and Scheller, 2006; McNiven and Thompson, 2006).
SNAREs mediate secretion of small molecules and polypeptides in many physiological processes. Synaptic vesicles of neurons store neurotransmitters and are triggered by the action potential to fuse with nerve terminals. The core SNARE complex mediating the release of neurotransmitters is well defined and is known to be regulated by many accessory proteins (Söllner et al., 1993; Jahn and Sudhof, 1999; Schoch et al., 2001; Rettig and Neher, 2002; Chandra et al., 2005; Rizo et al., 2006). VAMP2/synaptobrevin2 acts as the v-SNARE of synaptic vesicles, whereas syntaxin 1 and SNAP-25 interact to form the t-SNARE complex on the plasma membrane of nerve terminals. The interaction of VAMP2 with the syntaxin 1-SNAP-25 complex catalyzes the release of neurotransmitters. In contrast to the small synaptic vesicles used by neurons, endocrine and exocrine cells use larger containers called secretory granules to store proteins and bioactive components, and their fusion with the plasma membrane is regulated by various stimuli.
Despite the identification of 
38 SNAREs and the detailed studies of the SNARE complex mediating the synaptic transmission, the molecular mechanism and the SNARE complexes responsible for regulated exocytosis of the exocrine tissues remain elusive. Using the VAMP8 knockout mice generated recently (Wang et al., 2004), we have examined the hypothesis that VAMP8 may participate in regulated exocytosis of the entire exocrine system. Our study suggests that VAMP8 plays an important role in parotid and lacrimal acinar cells and that it functions in other exocrine tissues as well. This discovery will facilitate further analysis of the molecular mechanism responsible for regulated exocytosis of the exocrine system.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Other antibodies were purchased from commercial suppliers: rabbit anti-amylase from Calbiochem (La Jolla, CA); rabbit anti-KLK9 from Fitzgerald Industries (Concord, MA); rabbit anti-syntaxin4, syntaxin13, and rabbit anti-SNAP23 from Synaptic Systems (Göttingen, Germany); mAb to syntaxin6 from AbCam (Cambridge, United Kingdom).
Mice
The VAMP8 knockout mice have been described previously (Wang et al., 2004). In this study, we crossed the original VAMP8 knockout mouse line with the cre-transgenic strain (Schwenk et al., 1995) to remove the neomycin resistance cassette. The neo-deleted VAMP8 knockout line was then bred onto a 129/SvJ background for five generations. Sex- and age-matched 3–5-mo-old adult mice were used for experiments. Male mice were used unless otherwise indicated.
Histological Analysis and Immunofluorescence Microscopy
Organs were fixed with 4% paraformaldehyde (PFA) in PBS and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) or PAS. For immunofluorescence studies, PFA-fixed organs were embedded in OCT compound (Sakura Finetek USA, Torrance, CA). Cryosections were mounted on polylysine-coated slides and stained with primary antibodies followed by appropriate FITC-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). Nuclei were stained with DAPI or TO-PRO-3 (Molecular Probes, Eugene, OR).
For morphological electron microscopy (EM) study, fresh tissues were fixed with 4% PFA and 0.5% glutaraldehyde in PBS and embedded in Spurrs' resin. Ultrathin sections were stained with uranyl acetate and lead citrate before being observed with a transmission electronic microscope.
Immunoblot Analysis
Fresh tissues were homogenized in a modified RIPA buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mM DTT, 1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, 1 mM PMSF, and Complete proteinase inhibitors from Roche Diagnostics, Mannheim, Germany). Proteins were transferred onto Hybond-C extra membranes (Amersham, Piscataway, NJ) after being separated by SDS-polyacrylamide gels. Protein identities were determined with specific primary antibodies and appropriate peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Peroxidase activity was detected with the SuperSignal substrate and enhancer (Pierce, Rockford, IL) and visualized on x-ray films. Quantitative analysis was carried out with a GS-800 calibrated densitometer from Bio-Rad (Hercules, CA).
Saliva Collection under Resting and Pilocarpine-stimulation Conditions
Mice were anesthetized with Avertin (tribromoethanol from Morre Technology, Union, New Jersey), and saliva was then collected from the oral cavity with microcapillaries. Otherwise, mice were first anesthetized with Avertin and then injected with pilocarpine at 1 mg/kg i.p. (intraperitoneally) or 10 µl of 1 mg/ml pilocarpine at a location close to salivary glands. Saliva secreted into the oral cavity was collected with microcapillaries at 5-min intervals until 30-min after injection. Fifty to 100 µl of saliva was collected from each mouse. The saliva volume of mutant mice was not overtly different from that of normal mice.
GST Pulldown Analysis
The GST pulldown experiment was performed as previously described (Wang et al., 2004). Briefly, salivary glands were homogenized in homogenization buffer (20 mM HEPES, pH 7.4, 10 mM sucrose, 10 mM KCl, 2 mM EDTA, 2 mM EGTA, 6 mM MgCl2, 1 mM DTT, 1 mM PMSF, Complete proteinase inhibitors, and 1 mg/ml GST or GST-VAMP8 fusion protein). Nuclei were removed by centrifugation at 500 x g for 5 min, and total membranes were pelleted from the postnucleus supernatant by a spin at 100,000 x g for 1 h. Membranes were washed in washing buffer (500 mM KCl, 20 mM HEPES, 1 mM DTT, 1 mM EDTA, 1 mM PMSF, Complete proteinase inhibitors, 1 mg/ml GST or GST-VAMP8, pH 7.4) and then resuspended in 2 ml binding buffer (20 mM HEPES, 100 mM KCl, 1 mM DTT, 4 mM EGTA, 4 mM MgCl2, 2 mM ATP, 1 mM PMSF, Complete proteinase inhibitors, 1% BSA, 1 mg/ml GST or GST-VAMP8, pH 7.4) and incubated at 37°C for 5 min. After the incubation, the volume of the mixture was topped up to 12 ml with protein-free binding buffer before a spin at 100,000 x g for 1 h. Membranes were resuspended in extraction buffer (20 mM HEPES, 100 mM KCl, 1 mM DTT, 10 mM EDTA, 0.2 mM ATP, 2% Triton X-100, pH 7.4) and incubated at 4°C for 1 h with rotation. Triton-insoluble materials were removed by centrifugation at 200,000 x g for 30 min. Membrane extracts were incubated overnight with glutathione Sepharose 4B beads (Amersham). Beads were washed three times with extraction buffer containing 0.5% Triton followed by three times with Triton-free buffer. All the procedures were carried out at 4°C except binding. Proteins were eluted by boiling the beads for 5 min in SDS gel loading buffer and then were subjected to Western blotting analysis.
Isolation of Protein Aggregates from Lacrimal Glands
Lacrimal glands were homogenized in 280 mM sucrose supplemented with 10 mM HEPES, pH 7.4, 1 mM PMSF, and the Complete proteinase inhibitor (Roche Diagnostics) with a motor homogenizer (model T8.01; IKA Labortechnik, Staufen, Germany). The tissue suspension was then laid on top of a discontinuous sucrose gradient that consisted of 2, 1.5, and 1.0 M sucrose. Samples were centrifuged at 100,000 x g for 1 h. The black band at the interface between 2 and 1.5 M was retrieved and diluted with 2 volumes of 1% Triton X-100. Protein aggregates were pelleted after a spin at 10,000 x g for 5 min. The whole procedure was carried out at 4°C.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) prompted us to test the hypothesis that VAMP8 may have a more general role in the exocrine system. Salivary glands, which consist primarily of parotid, submandibular, and sublingual glands and secrete saliva into the mouth to moisten the mouth, initiate food digestion, and help protect the teeth from decay, were first examined. Immunofluorescence studies with an antibody specifically recognizing VAMP8 showed that VAMP8 was expressed in all acinar cells and lumen-lining duct epithelial cells of the three major salivary glands (Figure 1A). VAMP8 appeared to be enriched in the apical region of acinar cells and duct epithelial cells. The labeling is specific because no signal was detected in VAMP8-null sections. VAMP8 expression in salivary glands was confirmed by immunoblot analysis (Figure 1B).
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). H&E staining of parotid gland sections showed that the cytoplasm of normal acinar cells was stained purple, whereas that of VAMP8-null cells appeared pink (Figure 2A), indicating possible accumulation of secretory granules by mutant cells. To verify this possibility, we examined the distribution of secretory granules in parotid glands by immunolabeling their constituents such as amylase and carbonic anhydrase VI. As shown in Figure 2A, both amylase and carbonic anhydrase VI were restricted to the apical region in normal cells. In contrast, they were evenly distributed throughout the entire cytoplasm in VAMP8-null cells. These results are similar to those observed earlier in the pancreas and can be explained by the accumulation of secretory granules in the acinar cells due to defective exocytosis. The overall accumulation of secretory granules by VAMP8-null acinar cells was confirmed by EM analysis (Figure 2B). However, we also noticed that the dense granules were reduced in mutant cells. The cause for this reduction is unknown. We speculate that it might be due to premature activation of enzymes, which we have previously found in the pancreas (Wang et al., 2004). Consistent with a possible exocytosis defect, amylase and carbonic anhydrase VI were substantially increased in mutant parotid glands (Figure 2C). These enzymes were also increased in VAMP8-null submandibular glands, but the difference was not as obvious as in parotid glands (Figure 2C). Because VAMP8 was detected in all the three primary salivary glands, we speculated that VAMP8 might be required not only for secretion of enzymes by serous acinar cells but also for secretion of mucin by mucous cells. PAS staining for carbohydrates showed that VAMP8-null mucous cells in sublingual glands and submandibular glands contained more mucin than their wild-type counterpart (Figure 2D). Taken together, these results suggest that the absence of VAMP8 caused a defect in exocytosis, leading to the accumulation of secretory granules in acinar cells.
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Previous studies of pancreatic acinar cells indicates that VAMP8 may act as a vesicular SNARE (v-SNARE) of zymogen granules and mediate exocytosis by interacting with the target SNAREs (t-SNARE) syntaxin 4 and SNAP-23 on the plasma membrane. To assess whether a similar mechanism exists in salivary glands, we used a GST-VAMP8 fusion protein in affinity coisolation analysis to test if VAMP8 could interact with syntaxin 4 and SNAP-23. As shown in Figure 3D, syntaxin 4 and SNAP-23 were efficiently corecovered with GST-VAMP8, whereas syntaxin6 and syntaxin13 were not corecovered.
Ablation of VAMP8 Leads to Accumulation of Secretory Granules and Protein Aggregates in Lacrimal Glands
Lacrimal (tear) glands are responsible for secretion of lubrication material for the eyes. VAMP8 expression was also detected in the acinar cells of this gland (Figure 4A). VAMP8 was primarily distributed in the apical pole of acinar cells. EM revealed accumulation of secretory granules in VAMP8-null acinar cells (Figure 4B). Unlike normal acinar cells whose secretory granules were restricted to the apical region, mutant cells had secretory granules distributed all over the cytoplasm. The overall number of secretary granules was obviously increased in VAMP8-null cells. This phenotype suggests that VAMP8 might be required for exocytosis in lacrimal glands.
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). Hemoglobin probably has escaped from detection because of its low concentration and/or propensity to form aggregates with histone. The source of tear plasma proteins is unclear. We suspect that plasma proteins and hemoglobin are probably derived from the blood. These proteins might somehow be selectively picked up by acinar cells and/or duct cells and then be transported into the lumen of acini and ducts. Whatever the source of hemoglobin is, VAMP8 did not seem to be involved in the trafficking of this protein because the protein deposits were found outside cells. Under normal condition, most hemoglobin might be flushed out of the lacrimal gland, whereas a small portion forms fine deposits and provides the gland with a light gray color. These tiny aggregates can hardly be observed on H&E-stained sections. When secretion from acinar cells is reduced, hemoglobin may accumulate and form large aggregates in the lumen of acini and ducts. The defects described above suggest that exocytosis of lacrimal acinar cells is compromised in the absence of VAMP8, indicating a role for VAMP8 in tear secretion.
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Our findings that VAMP8 is crucially involved in the regulated exocytosis of the parotid and tear glands and it is widely expressed in all the exocrine tissues examined together with its established function in the pancreatic exocrine cells (Wang et al., 2004), suggest that VAMP8 may play a role in regulated exocytosis of the entire exocrine system.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The focus of the SNARE field has recently shifted to the regulation of SNARE functions (Giraudo et al., 2006). Another challenge in the field is to define the physiological role of individual SNAREs. This kind of studies will advance our understanding of human diseases. Actually, mutations in trafficking proteins have already been shown to be the bases of many human diseases. However, of the some 38 SNAREs identified, only a few have been studied in whole animals. It has been shown by gene-targeting that synaptobrevin/VAMP2 is important for neurotransmitter release (Schoch et al., 2001), whereas syntaxin4 is required for surface deployment of GLUT4 (Yang et al., 2001a). Epimorphin/syntaxin2 negatively regulates epithelium growth (Wang et al., 2006). Despite its wide expression, VAMP3 seems to be nonessential (Yang et al., 2001b), and Vti1b plays only a minor role in the endocytic pathway (Atlashkin et al., 2003). VAMP8 has recently been shown to play an important role in regulated secretion of pancreas and platelets (Wang et al., 2004; Ren et al., 2006). In this study, we extend our investigation into the role of VAMP8 in the whole exocrine system.
Saliva has lubricative, protective, and digestive functions and is produced by various salivary glands associated with the oral cavity. The parotid, submandibular, and sublingual glands represent the major salivary glands with independent excretory ducts, whereas minor salivary glands are located throughout the oral mucosa and the tongue. Based on secreted materials, salivary glands are described as serous or mucous glands. Serous cells secret a thin watery fluid rich in enzymes, whereas mucous cells produce a thick viscid secretion containing a large amount of glycoproteins. The parotid gland is predominantly composed of serous acini, whereas the sublingual gland is mainly mucous and the submandibular gland is a mixture of serous and mucous cells. VAMP8 was expressed in all three major salivary glands. Two lines of evidence support a role for VAMP8 in exocytosis of salivary glands. First, mucin and enzymes such as amylase and carbonate anhydrase VI accumulated in VAMP8-null glands due to accumulation of secretory granules. Second, mutant saliva protein levels were substantially reduced, especially in pilocarpine (PLP)-stimulated saliva. In our study, PLP-induced secretion was measured within a period of 30 min compared with a period of 4 h for basal secretion. As such, we were measuring acute regulated exocytosis by the major salivary glands. Under this condition, basal secretion is less significant than PLP-stimulated secretion. The result is the key evidence suggesting that VAMP8 is more important for regulated secretion than for constitutive secretion. This interpretation is supported by other morphological (including EM) analysis. In line with earlier analysis of pancreatic acinar cells (Wang et al., 2004), VAMP8 is likely to act as the v-SNARE of the secretory granule in salivary glands as well. Because the difference between normal and VAMP8-null saliva amylase and CA VI are more significant in PLP-stimulated secretion than in constitutive secretion, we conclude that VAMP8 is more important for regulated exocytosis than for basal secretion.
KLK9 was molecularly identified in 2000 (Diamandis et al., 2000; Yousef and Diamandis, 2000), but its function remains to be determined. Our results here suggest that KLK9 is likely a male-specific salivary protein whose secretion is strictly dependent on VAMP8. In contrast to the partially reduced secretion of well-characterized salivary proteins such as amylase and carbonic anhydrase VI, KLK9 in saliva was essentially abolished in the absence of VAMP8. This disparity between KLK9 and amylase (or carbonic anhydrase VI) suggests that KLK9 and amylase (or carbonic anhydrase VI) might be present in different secretary granules that express distinct sets of v-SNAREs. In fact, it is well known that there are different types of secretory granules that response to different stimuli (Castle et al., 2002). An interesting question to be answered in future studies is whether the secretion of other KLKs in salivary glands and other tissues is also dependent on VAMP8.
The cornea of the eye is covered by a tear film which keeps the eye moist and washes away dirty and small particles. The tear film consists of three layers, with the middle aqueous layer of tear fluid being composed of water, salt, proteins and other substances that help to lubricate the eye (Zhou et al., 2006). Tear fluid is primarily formed in the lacrimal tear glands; it is secreted by the tubuloalveolar acinar cells and transported via a number of ducts to the eye's surface. In the absence of VAMP8, tear secretion might be compromised, although no overt injury has been observed. Consistent with this is the cellular observation that acinar cells accumulated secretory granules, indicating that exocytosis was defective. Furthermore, the lacrimal glands of VAMP8 knockout mice accumulate dark brown protein aggregates in the lumen of the acinus and duct, which may partially affect the flow of tear. These defects ultimately result from the lack of VAMP8 that is important for fusion of secretory granules with the plasma membrane. VAMP2, VAMP7, and VAMP8 have been proposed to be candidate v-SNAREs in lacrimal glands (Wu et al., 2006). Our study shows that VAMP8 is a major if not the only v-SNARE of lacrimal glands.
In human, there is a disease called Sjögren's syndrome. Patients of this syndrome suffer from dry mouth and dry eyes. Because the PLP-stimulated saliva volume was not significantly different between normal mice and VAMP8 knockout mice, despite the difference in protein composition, we do not believe the VAMP8 knockout mouse line is a perfect model for human Sjögren's syndrome. But human with defective VAMP8 might suffer from a mild symptom of dry mouth and dry eyes.
Our current study suggests that VAMP8 is important for regulated exocytosis of salivary glands and lacrimal glands, in addition to its role in regulated exocytosis of pancreatic acinar cells (Wang et al., 2004). In the context of its wide expression in all other exocrine tissues examined, we believe that VAMP8 may also participate in regulated secretion of additional exocrine tissues. It is reasonable to speculate that VAMP8 is likely a common v-SNARE responsible for regulated exocytosis in the entire exocrine system.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Wanjin Hong (mcbhwj{at}imcb.a-star.edu.sg)
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