Isoproterenol stimulates transient SNAP23-VAMP2 interaction in rat parotid glands.

The exocytosis of salivary proteins is mainly regulated by cAMP, although soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), which mediate cAMP-dependent exocytic membrane fusion, have remained unidentified. Here we examined the effect of isoproterenol (ISO) and cytochalasin D (CyD) on the level of SNARE complexes in rat parotid glands. When SNARE complexes were immunoprecipitated by anti-SNAP23, the coprecipitation of VAMP2 was significantly increased in response to ISO and/or CyD, although the coprecipitation of VAMP8 or syntaxin 4 was scarcely augmented. These results suggest that the SNAP23-VAMP2 interaction plays a key role in cAMP-mediated exocytosis from parotid glands.

SNARE proteins are not excessive for the formation of post-Golgi SNARE complexes in HeLa cells.

To evaluate the role of SNARE proteins in the constitutive exocytosis, we knocked down syntaxin 3, 4, 5, 6, 7, and VAMP3, 5, 7, 8 with their siRNAs, and determined the cell-to-medium ratio of CLuc, a secreted luciferase of Cypridina noctiluca. Although the protein level of SNAREs in HeLa cells was markedly reduced by the siRNA treatment, the cell/medium ratio was scarcely increased by any siRNAs except for syntaxin 5. The accumulation of GFP-tagged human growth hormone was also visible only by the knockdown of syntaxin 5. To examine whether the residual amount of SNAREs are sufficient for maintaining normal constitutive exocytosis, we estimated the effect of siRNAs on the level of post-Golgi SNARE complexes containing syntaxin 4, SNAP23, and VAMP3 or VAMP8. The amount of SNARE complexes was robustly decreased by siRNAs and was well correlated with the residual amount of SNAREs in the lysates, suggesting that SNAREs are unnecessarily excessive for the formation of post-Golgi SNARE complexes in HeLa cells.

Role of VAMP8/endobrevin in constitutive exocytotic pathway in HeLa cells.

To evaluate the role of VAMP8/endobrevin in constitutive exocytosis, we have examined the exocytotic pathways of VAMP8 and human growth hormone, both GFP-tagged, by total internal reflection fluorescence microscopy (TIRF-M). Human GH-GFP and VAMP8-GFP were similarly expressed in small round vesicles and elongated tubular vesicles in HeLa cells, and were mostly exocytosed at the peripheral area of the cells. VAMP8-GFP gave 2 types of exocytotic images: a burst type and a non-burst type. The burst type showed a sharp transient increase in the peak fluorescence intensity and a much slower decrease in the average intensity in the active windows, where exocytosis took place, as observed in the "full-fusion" type of exocytosis. The non-burst type showed a relatively long-lasting fusion to the plasma membrane with little transfer of VAMP8-GFP to the plasma membrane, as observed in the so-called "kiss-and-run" type of exocytosis. Endogenous VAMP8 and hGH-GFP were colocalized on the same vesicles at least in part. However, the constitutive exocytosis of hGH-GFP and CLuc, a secreted luciferase from Cypridina noctiluca, was normal, even when siRNAs for VAMP8 and VAMP3 robustly decreased their proteins. These results suggest that VAMP8 is not essential for constitutive exocytosis, although it can be involved in the exocytosis.

Keratocystic odontogenic tumor: a retrospective study of 183 cases.

In 2005, the WHO Working Group considered odontogenic keratocyst (OKC) to be a tumor and recommended the term keratocystic odontogenic tumor (KCOT), separating the lesion from the orthokeratinizing variant, which is now considered an odontogenic cyst. We analyzed the clinicopathological features of KCOTs encountered over a period of 28 years at Meikai University Hospital. The diagnosis was confirmed by reevaluation of hematoxylin and eosin-stained slides on the basis of the 2005 WHO Classification. Clinical history was also taken into consideration. A total of 183 KCOTs were found, and the two genders were affected almost evenly (51.3% male; 48.7% female; male to female ratio 1.05 to 1). Patient age at the time of diagnosis ranged from 6 to 78 years, with a peak in the third decade of life (mean age: 32.8 years). The mandible was the site of occurrence of 70.5% of tumors; 16.4% occurred in the maxilla and 13.1% in both. Association with the nevoid basal cell carcinoma syndrome (NBCCS) was found in 6.0% of all tumors, and recurrence was found in 13.1% of patients. We found that tumors that initially appeared in the maxilla alone had a higher recurrence rate than those that first appeared in the mandible alone. Pathological examination of KCOT is important to avoid misdiagnosis and provide appropriate treatment and follow-up.

SNAP-23 is not essential for constitutive exocytosis in HeLa cells.

We applied the small interfering RNA (siRNA) technique and over-expression of a dominant-negative mutant to evaluate the role of SNAP-23, a non-neuronal isoform of SNAP-25, in constitutive exocytosis from HeLa cells. Although the protein level of SNAP-23 was reduced to less than 10% of the control value by siRNA directed against SNAP-23, exocytosis of SEAP (secreted alkaline phosphatase) was normal. Double knockdown of SNAP-23 and syntaxin-4 also failed to inhibit the secretion. Furthermore, over-expression of deltaC8-SNAP-23, a dominant-negative mutant of SNAP-23, did not abrogate SEAP secretion. These results suggest that SNAP-23 is not essential for constitutive exocytosis of SEAP.

Role of VAMP-2, VAMP-7, and VAMP-8 in constitutive exocytosis from HSY cells.

We evaluated the role of VAMP-2/synaptobrevin, VAMP-7/TI-VAMP, and VAMP-8/endobrevin in exocytic pathways of HSY cells, a human parotid epithelial cell line, by coexpressing these VAMP proteins tagged with green fluorescent protein (GFP) and human growth hormone (hGH) as a secretory cargo. Exocytosis of hGH was constitutive and the fluorescent signal of hGH-GFP was observed in the Golgi area and small vesicles quickly moving throughout the cytoplasm. The cytoplasmic vesicles containing hGH overlapped well with VAMP-7-GFP, but did so scarcely with VAMP-2-GFP or VAMP-8-GFP. However, when the vesicle transport from the trans-Golgi network to the plasma membrane was arrested by incubation at 20 degrees C for 2 h and then released by warming up to 37 degrees C; VAMP-2-GFP and hGH were clearly colocalized together in small cytoplasmic vesicles. Neither VAMP-7-GFP nor hGH-GFP was colocalized with LAMP-1, a marker for lysosomes and late endosomes. These results suggest that (1) VAMP-2 can be one of the v-SNAREs for constitutive exocytosis; (2) VAMP-7 is involved in the constitutive exocytosis as a slow, minor v-SNARE, but not in the lysosomal transport; and (3) VAMP-8 is unlikely to be a v-SNARE for constitutive exocytosis in HSY cells.

Trafficking of green fluorescent protein-tagged SNARE proteins in HSY cells.

SNARE proteins are widely accepted to be involved in the docking and fusion process of intracellular vesicle trafficking. VAMP-2, syntaxin-4, and SNAP-23 are plausible candidate SNARE proteins for non-neuronal exocytosis. Thus, we examined the localization, protein-protein interaction, and intracellular trafficking of these proteins by expressing them as green fluorescent protein (GFP)- and FLAG-tagged fusion proteins in various cells, including HSY cells, a human parotid epithelial cell line. GFP-VAMP-2 was ex-pressed strongly in the Golgi area and weakly on the plasma membrane. Although GFP-SNAP-23 seemed to be expressed universally in the cytosol, the GFP signal was clearly seen on the plasma membrane, when soluble GFP-SNAP-23 was removed by treatment with saponin. GFP-syntaxin-4 was undetectable on the plasma membrane but was strongly expressed on unidentified unusually large vesicles. GFP-syntaxin-4 without its transmembrane domain was still incompletely soluble and observed as aggregates. When syntaxin-4 and munc18c were coexpressed, syntaxin-4 was translocated at least in part to the plasma membrane. The protein-protein interaction between syntaxin-4 and VAMP-2 with their transmembrane domains was markedly inhibited on coexpression of munc18c. These results suggest that munc18c plays an important role in the trafficking of syntaxin-4 to its proper destination by preventing premature interactions with other proteins, including SNARE proteins.