An amphicrine pancreatic cell line: AR42J cells combine exocrine and neuroendocrine properties.

The permanent cell culture line AR42J, derived from a rat pancreatic acinar carcinoma, is widely used for functional studies of exocrine pancreatic acinar cells. We now present evidence that these cells are amphicrine in that they contain zymogen granules as well as small (40-80 nm) neuroendocrine (NE) vesicles and typical neurotransmitters. Using the small NE vesicle-specific markers synaptophysin and "protein S.V.2", including synaptophysin cDNA probes, we have found that AR42J cells synthesize these proteins and contain vesicles harboring these proteins with biophysical properties similar to those of small NE vesicles. NE properties of these cells are further indicated by the presence of considerable amounts of stored amino acids (gamma-aminobutyric acid (GABA), glycine, glutamate) and by the presence of the GABA-synthesizing enzyme, glutamic acid decarboxylase. Finally, intermediate filament (IF) protein typing showed only cytokeratins 8 and 18, indicating that AR42J cells possess an IF protein complement indistinguishable from that of acinar and islet cells. Our results document the unusual case of a permanent cell line with combined exocrine and neuroendocrine properties that may be indicative of a derivation from a cell with multipotential character.     

Modulating zymogen granule formation in pancreatic AR42J cells

Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. To investigate ZG biogenesis, cargo sorting and packaging, suitable cellular model systems are required. Here, we demonstrate that granule formation in pancreatic AR42J cells, an acinar model system, can be modulated by altering the growth conditions in cell culture. We find that cultivation of AR42J cells in Panserin^? 401, a serum-free medium, enhances the induction of granule formation in the presence or absence of dexamethasone when compared to standard conditions including serum. Biochemical and morphological studies revealed an increase in ZG markers on the mRNA and protein level, as well as in granule size compared to standard conditions. Our data indicate that this effect is related to pronounced differentiation of AR42J cells. To address if enhanced expression of ZG proteins promotes granule formation, we expressed several zymogens and ZG membrane proteins in unstimulated AR42J cells and in constitutively secreting COS-7 cells. Neither single expression nor co-expression was sufficient to initiate granule formation in AR42J cells or the formation of granule-like structures in COS-7 cells as described for neuroendocrine cargo proteins. The importance of our findings for granule formation in exocrine cells is discussed.     

Carboxyl-methylation of Rab3D in the rat pancreatic acinar tumor cell line AR42J.

Rab3D is a small GTPase implicated in regulated exocytosis, and is a marker of secretory granules in exocrine cells. We have previously shown that rab3D undergoes reversible carboxyl-methylation in adult rat pancreatic acinar cells, and that carboxyl-methylation of rab3D is developmentally regulated concomitantly with the maturation of the regulated secretory apparatus in rat pancreas. We also observed that dexamethasone treatment of the rat pancreatic acinar tumor cell line, AR42J, led to a significant increase in the size of the unmethylated pool of a rab3-like protein. The current study was designed to further characterize this rab3-like protein. Here we show that AR42J cells express rab3D, and that the protein focuses on 2D gels as two spots with pI values of 4.9 and 5.0. Treatment of AR42J cells with N-acetyl-S-geranylgeranyl-l-cysteine, an inhibitor of carboxyl-methylation, led to a decrease in the basic form of rab3D and a proportional increase in the acidic form. In contrast, N-acetyl-S-farnesyl-l-cysteine, which inhibits carboxyl-methylation of farnesylated proteins, had no effect. Lovastatin, an inhibitor of geranylgeranylation, also induced an accumulation of the acidic form of rab3D. Taken together, these data indicate that rab3D can undergo reversible carboxyl-methylation in AR42J cells by a geranylgeranyl-specific methyltransferase. The 2D gel and immunoblotting analyses indicated that dexamethasone treatment of AR42J cells led to an increase in the proportion of the unmethylated form of rab3D concurrent to inducing a regulated secretory pathway, similar to the rab3D profile change in developing rat pancreas. Our data, along with previous studies done on developing rat pancreas, indicate that the tumor cell line AR42J represents a good model system for studying the regulated secretory pathway, and that carboxyl-methylation of rab3D may play a role in the acquisition of stimulus-secretion coupling.     

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

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, long-term (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.     

Pancreatic tau related maps: Biochemical and immunofluorescence analysis in a tumoral cell line

In the present study, we report the existence of four tau-related microtubule-associated proteins (MAPs) of 48, 50, 55 and 58 kDa in a pancreatic exocrine cell line (AR4-2J). Using immunofluorescence, we demonstrate that these tau-related MAPs are associated with microtubules in AR4-2J cells. That colocalization is particularly striking on microtubules bundles in cellular extensions and is the first evidence for tau-related MAPs colocalization with microtubules in non-neuronal cells. As it has been often discussed for neuronal tau, the localization of tau-related proteins in AR4-2J cells suggests that these proteins may be involved in microtubule bundling.     

The amphicrine pancreatic cell line, AR42J, secretes GABA and amylase by separate regulated pathways.

Treatment of AR42J cells with dexamethasone leads to an enhanced formation of amylase-containing granules and facilitates their regulated secretion. Besides the exocrine properties, AR42J cells possess a specific uptake system for [3H]GABA. The stored GABA can be released upon potassium depolarisation in a Ca(2+)-dependent manner. After treatment with dexamethasone, potassium depolarisation fails to release GABA, but instead causes a Ca(2+)-dependent secretion of amylase. Since vesicles similar to small synaptic vesicles of neurons have been identified in AR42J cells, we suggest that the regulated GABA release is mediated by this vesicle type. It is tentatively speculated that other epithelial cells, which also contain small synaptic vesicles and amino acid neurotransmitters, may release them in a similar fashion.     

Membrane lipid composition of pancreatic AR42J cells: modification by exposure to different fatty acids

Dietary fat type influences fatty acids in rat pancreatic membranes, in association with modulation of secretory activity and cell signalling in viable acini. We aimed to confirm whether AR42J cells are a valid model to study the interactions between lipids and pancreatic acinar cell function. For this purpose we have (i) compared the baseline fatty acid composition of AR42J cells with that of pancreatic membranes from rats fed a standard chow; (ii) investigated if fatty acids in AR42J membranes can be modified in culture; and (iii) studied if similar compositional variations that can be evoked in rats when dietary fat type is altered occur in AR42J cells. Weaning Wistar rats were fed for 8 weeks either a commercial chow (C) or semi-purified diets containing virgin olive oil (VOO) or sunflower oil (SO) as fat source. AR42J cells were incubated for 72 hrs in medium containing unmodified fetal calf serum (FCS, AR42J-C cells), FCS enriched with 18:1 n-9 (AR42J-O cells), or FCS enriched with 18:2 n-6 (AR42J-L cells). Fatty acids in crude membranes from rat pancreas and AR42J cells were determined by gas-liquid chromatography. Differences in membrane fatty acids between C rats and AR42J-C cells can be explained in part by variations in the amount of fatty acids in the extracellular environment. Supplementation of FCS with 18:1 n-9 or 18:2 n-6 changed the fatty acid spectrum of AR42J cells in a manner that resembles the pattern found, respectively, in VOO and SO rats, although AR42J-L cells were unable to accumulate 20:4 n-6. The AR42J cell line can be a useful tool to assess the effect of membrane compositional changes on acinar cell function. However, differences in baseline characteristics, and perhaps fatty acid metabolism, indicate that results obtained in AR42J cells should be confirmed with experiments in the whole animal.     

Subcellular distribution and function of Rab3A-D in pancreatic acinar AR42J cells

Members of the Rab3 subfamily have been linked to the regulation of exocytosis in secretory cells. We have recently shown by Northern blot analysis that pancreatic acinar-like AR42J cells express all four Rab3 isoforms (Rab3A-D). In the present study, we examined the subcellular distribution of endogenously expressed Rab3 proteins and their relation to the amylase-containing secretory compartment in dexamethasone-differentiated AR42J cells. Rab3A and Rab3C were enriched in the cytosol, Rab3B and Rab3D in the membrane fraction. Accordingly, confocal immunocytochemistry revealed that Rab3B and Rab3D were located in a compartment close to the plasma membrane, whereas anti-Rab3A and Rab3C mainly stained the cytosol. Sucrose density gradient centrifugation showed overlapping, but distinct localization of each Rab3 isoform. The order of banding from lighter to more dense fractions was Rab3C < Rab3A < Rab3B < Rab3D. All Rab3 proteins at least partially colocalized with amylase immunoreactivity. Transient overexpression of Rab3 proteins showed that Rab3A inhibited cholecystokinin (CCK)-induced amylase secretion, whereas overexpression of other Rab3 isoforms had no significant effect. In conclusion, our data indicate that the different Rab3 proteins show distinct subcellular distribution, suggesting different impact on exocrine secretory response in dexamethasone-differentiated AR42J cells.     

Palmitate induced lipoapoptosis of exocrine pancreas AR42J cells

Chronic surplus of dietary consumption, typical to obesity, results in overflow of fat to non-adipose tissues. Intracellular accumulation of fat in non-adipose tissues is associated with cellular dysfunction and cell death and ultimately contributes to the pathogenesis of chronic diseases. The influence of fat overflow on the exocrine pancreas is not known. The purpose of this research was to study the lipotoxic and lipoapoptotic effect of prolonged (72 h) long chain saturated palmitic fatty acid (0.1 mM) on the survival of exocrine pancreas AR42J cells. We demonstrate that chronic exposure of AR42J cells to palmitic acid results in significant increase in triglycerides accumulation (up to 25% of cells area), compared to untreated cultures. Lipid accumulation prompted a typical apoptotic process, demonstrated by both DNA fragmentation and condensed chromatin appearance (DAPI staining). Quantitative real-time PCR studies demonstrated that prolonged palmitic acid supplementation induced down-regulation of the anti-apoptotic Bcl2 mRNA levels (22%) and up-regulation of the pro-apoptotic Bax mRNA levels (300%), leading to disruption of the pro/anti apoptotic balance (Bax/Bcl2=3). No major change was detected in iNOS mRNA expression. In conclusion, prolonged exposure to saturated palmitic acid induces lipoapoptosis in exocrine pancreatic AR42J cells, through disturbance of the Bax/Bcl-2 balance.     

Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells

Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes.     

Exocrine granule specific packaging signals are present in the polypeptide moiety of the pancreatic granule membrane protein GP2 and in amylase: implications for protein targeting to secretory granules.

The mechanisms for segregation of secretory and membrane proteins incorporated into storage granules from those transported constitutively have been thought to be conserved in diverse cell types, including exocrine and endocrine cells. However, GP2, the major protein of pancreatic zymogen granule membranes, in its native glycosyl phosphatidylinositol (GPI)-linked form, is incorporated into secretory granules when expressed in exocrine pancreatic AR42J cells, but not in the endocrine cells such as pituitary AtT20. To determine whether the protein moiety of GP2 contains the cell-type specific information for packaging into granules, a secretory form of GP2 (GP2-GPI-), with the GPI attachment site deleted, was generated and introduced into AR42J and AtT20 cells. Like native GP2, GP2-GPI- localized to the zymogen-like granules of AR42J cells and underwent regulated secretion. In AtT20 cells expressing GP2-GPI-, however, the protein was secreted by the constitutive pathway. Thus, a granule packaging signal is present in the luminal portion of GP2 that is functional only in the exocrine cells. However, this cell-type dependent sorting process is not limited to GP2 or membrane proteins. Amylase, a major content protein of pancreatic acinar and serous salivary gland granules, was also secreted exclusively by the constitutive pathway when expressed in AtT20 cells. The cell-type specific targeting of GP2 to granules correlated with its behavior in an in vitro aggregation assay where it co-aggregated more effectively with content proteins from pancreatic zymogen granules than with those from pituitary granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucocorticoids have opposite effects on ornithine decarboxylase and cell growth in pancreatic acinar AR42J cells.

This paper reviews the relationships between the effects of glucocorticoids on rat pancreatic acinar AR42J cell polyamine levels and cellular growth and differentiation. Glucocorticoids inhibit the growth of AR42J cells. Glucocorticoids either stimulate or inhibit the formation of polyamines in a variety of cell types. Cells require polyamines for normal growth. Therefore, we tested the hypothesis that polyamines mediate the effects of glucocorticoids on AR42J cells. First, to confirm that AR42J cells required polyamines for growth we examined the effects of inhibiting ornithine decarboxylase (ODC). ODC is the most important and generally rate-limiting enzyme in the synthesis of the polyamines. As expected, the ODC inhibitor difluoromethylornithine (DFMO) inhibited AR42J cell DNA synthesis, and the addition of exogenous putrescine reversed this effect. The levels of growth inhibition by glucocorticoids and DFMO treatment were similar. Second, we examined the effects of glucocorticoids on ODC. Surprisingly, glucocorticoids increased levels of AR42J cell ODC mRNA, ODC activity, and putrescine. Glucocorticoids increased these parameters over a similar time-course as they decreased DNA synthesis. Analog specificity studies indicated that a glucocorticoid receptor mediated both the growth inhibitory and ODC stimulatory effects. Dose-response studies indicated, however, that growth inhibition was more sensitive to dexamethasone (DEX) than were ODC levels. Therefore, polyamines do not account for the effects of glucocorticoids on AR42J cell growth. In these cells, glucocorticoids have opposite and independent effects on ODC and growth.     

Activation of p-ERK1/2 by nicotine in pancreatic tumor cell line AR42J: effects on proliferation and secretion

The objectives of the present study were to determine the effect of nicotine on MAPK signaling and on the proliferation of AR42J cells as well as to assess the relationship between MAPK activation and exocrine secretion in these cells. AR42J cells were incubated with nicotine and analyzed for the activation of MAPK by Western blot analysis using their respective antibodies and confirmed by immunohistochemistry. The effect of nicotine on cell proliferation was determined by the spectrophotometric method, and cell function was assessed by cholecystokinin (CCK)-stimulated amylase release into the culture medium. Nicotine at a dose of 100 microM induced phospho-ERK1/2 activation maximally in 3 min compared with untreated cells. Furthermore, immunofluorescence study confirmed the nicotine-induced increase in translocation of phospho-ERK1/2 to the nucleus. Activation of phospho-ERK1/2 was inhibited by an ERK1/2 pathway inhibitor but not by a nicotine receptor antagonist. At the same dose, there was significantly enhanced proliferation of AR42J cells until 72 h without toxic effect, as the percentage of lactate dehydrogenase release remained unchanged. Other MAPKs, c-Jun NH2-terminal kinase 1/2 and p38 MAPK, were not affected by nicotine treatment. At a nicotine dose of 100 microM, the CCK-stimulated release of amylase was maximal at 6 min, and, although a nicotinic receptor antagonist inhibited this response, it was not inhibited by the ERK1/2 pathway inhibitor. We conclude that nicotine treatment induced activation of ERK1/2 and increased the proliferation of AR42J cells. The data further indicate that MAPK signaling by nicotine is independent of the secretory response.     

Transdifferentiation of pancreas to liver

Transdifferentiation is the name used to describe the direct conversion of one differentiated cell type into another. Cells which have the potential to interconvert by transdifferentiation generally arise from adjacent regions in the developing embryo. For example, the liver and pancreas arise from the same region of the endoderm. The transdifferentiation of pancreas to liver (and vice versa) has been observed in animal experiments and in certain human pathologies. Understanding transdifferentiation is important to developmental biologists because it will help elucidate the cellular and molecular differences that distinguish neighbouring regions of the embryo. While the in vivo models for the transdifferentiation of liver to pancreas have been valuable, it is more difficult to extrapolate from these studies to individual changes at the cellular or molecular levels. The recent development of two in vitro systems (AR42J cells and embryonic pancreatic cultures) for the transdifferentiation of pancreas to liver has shown that an environmental change in the form of an exogenous glucocorticoid can cause the conversion of pancreatic exocrine cells into hepatocytes. The AR42J cell system has been used to elucidate the cell lineage and the molecular basis of transdifferentiation of pancreas to liver.