Rab3D regulates amylase levels, not agonist-induced amylase release, in AR42J cells

Abstract

Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone (Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D and a constitutively active form of Rab3D (Rab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, longterm (48 hr) basal amylase release or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may regulate amylase content in these cells.

Polymeric immunoglobulin receptor traffics through two distinct apically targeted pathways in primary lacrimal gland acinar cells

“…There is also evidence that regulation of secretion by Rab3D may be more complex. In the AR42J cell line derived from rat pancreatic tumor, Rab3D may regulate the level of amylase but is not involved in exocytosis induced by cholecystokinin (Limi et al, 2012). In addition, Rab3D has been shown to regulate secretory granule maturation in neuroendocrine PC12 cells by regulating the removal of cargo from immature secretory granules (Kogel and Gerdes, 2010).…”

RAB26 coordinates lysosome traffic and mitochondrial localization

“…We next decided to investigate RAB26 scalability in a cell culture system that would facilitate analysis of RAB26 expression level relative to its subcellular distribution and function. First, we analyzed the wellestablished secretory pancreatic cell line, AR42J, which expresses MIST1 (Jia et al, 2008) and can be differentiated with dexamethasone treatment to upregulate MIST1 target gene expression (Limi et al, 2012;Qiu et al, 2001) and increase amylase-containing secretory vesicles (Logsdon, 1986;Rinn et al, 2012) (Fig. 1D).…”

Variations in the multimerization region of the Helicobacter pylori cytotoxin CagA affect virulence

“…Cell pellets were resuspended in lysing solution [20 mM Tris-HCL (pH 7.2), 150 mM NaCl, 1% TX-100, 1 mM NaF, 2 mM Na 3 VO 4 and 1X Halt protease inhibitor cocktail (Thermo Fisher Scientific, Inc.)]. Protein assays were performed using the Coomassie Protein assay reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, and immunoblotting was performed as previously described (15). Primary antibodies included anti-GFP (#PA1980A; Thermo Fisher Scientific, Inc.), anti-Caspase-3 (#9662; Cell Signaling Technology, Inc., Danvers, MA, USA) and Ki-67 (#RB9043; Thermo Fisher Scientific, Inc.) and were diluted at 1:1,000.…”