Snare protein expression and adenoviral transfection of amphicrine AR42J.

The amphicrine AR42J acinar cell line is an excellent model to study both exocrine and neuroendocrine exocytotic mechanisms. As a first step toward this goal, we determined the specific isoforms of the v- and t-SNARE and Munc18 families expressed in these cells. In addition, we show that dexamethasone-induced differentiation toward the exocrine phenotype causes an upregulation of several of these proteins. AR42J is notoriously difficult to transfect, limiting its usefulness as a model. However, we have now overcome this obstacle by acheiving high efficiency expression of a beta-galactosidase reporter gene and truncated SNAP-25 gene using adenoviral infection techniques. The AR42J cells can now be used to pursue and elucidate the distinct functions of individual SNARE isoforms used in endocrine and exocrine secretion within a single cell line.     

NF-kappaB p65 regulates nuclear translocation of Ku70 via degradation of heat shock cognate protein 70 in pancreatic acinar AR42J cells

Ku proteins such as Ku70 and Ku80 play key roles in multiple nuclear processes. Nuclear translocation of Ku70 is independent of Ku80 translocation and mediated by nuclear localization signal (NLS) receptors including importin-alpha. In the present study using pancreatic acinar AR42J cells, heat shock cognate protein 70 (Hsc70) was identified as the protein associated with NLS of Ku70. Interaction of Ku70 with importin-alpha and nuclear translocation of Ku70 was suppressed by overexpression of Hsc70, but enhanced by downregulation of Hsc70. The results suggest that the formation of Ku70 complex with Hsc70 prevents NLS of Ku70 from access of importin-alpha and inhibits nuclear translocation of Ku70. Since NF-kappaB p65 activation induced the decrease of Hsc70 level, the interaction of Ku70 with importin-alpha and nuclear translocation of Ku70 increased upon the activation of NF-kappaB p65. NF-kappaB p65 induced cell proliferation through decrease of Hsc70 levels and increase of nuclear translocation of Ku70. In the cells treated with cerulein as a physiological stimulus to activate NF-kappaB p65, nuclear translocation of Ku70 increased through NF-kappaB p65-mediated decrease of Hsc70 level. The results suggest that the involvement of NF-kappaB p65 in nuclear translocation of Ku70 may be mediated by Hsc70 degradation, which may play a key role in cell proliferation of pancreatic acinar AR42J cells.     

Manganese attenuates secretagogue-mediated phospholipid hydrolysis in AR42J cells

The effects of cholecystokinin octapeptide (CCK8), bombesin and manganese (Mn2+) on phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis were studied in AR42J cells. One-half maximal stimulation of inositol monophosphate (InsP1) accumulation occurred at either 5 nM CCK8 or 5 nM bombesin, and maximal stimulation occurred at 30 nM for each agonist. Mn2+ did not alter basal PIP2 hydrolysis. However, addition of Mn2+ 5 min prior to stimulation with either CCK8 or bombesin for 60 min significantly attenuated [3H]InsP1 accumulation. Following brief periods of incubation with CCK8 (15 sec) Mn2+ significantly reduced inositol tris- and tetrakisphosphate accumulation. These data suggest that Mn2+ may participate in the regulation of CCK8- and bombesin-mediated generation of phosphoinositides.     

Regulation of intracellular Ca2+ oscillation in AR42J cells.

Recordings of [Ca2+]i in single AR42J cells loaded with Fura 2 were used to study regulation of [Ca2+]i oscillation. Continuous stimulation with the cholecystokinin analogue, (t-butyloxycarbonyl-Tyr-(SO3)-norleucine-Gly-Trp-Nle-Asp-2-phenylethyl ester) or carbachol evoked long lasting oscillation in [Ca2+]i. Removal of CCK-JMV-180 after brief stimulation did not abruptly stop the oscillation. Rather, removal of CCK-JMV-180 resulted in time-dependent reduction in amplitude with little change in frequency of oscillation. The patterns of [Ca2+]i oscillation were affected by activation of protein kinase C and protein kinase A. However, down-regulation of protein kinase C activity did not prevent stimulation of [Ca2+]i oscillation. Hence, we conclude that an active protein kinase C pathway is not crucial for [Ca2+]i oscillation in this cell line. Variation in extracellular Ca2+ concentration (Ca2+out) was used to further characterize the oscillation. Reducing Ca2+out to approximately 10 microM resulted in a time dependent inhibition of [Ca2+]i oscillation. Subsequent step increases in Ca2+out up to 2-3 mM resulted in increased amplitude and frequency of oscillation. Further increase in Ca2+out or an increase in plasma membrane permeability to Ca2+, brought about by an increase in pHo, resulted in increased amplitude, decreased frequency, and modified shape of the [Ca2+]i spikes. These observations point to the existence of regulatory mechanisms controlling the duration of Ca2+ release and entry during [Ca2+]i oscillation.     

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.     

Effects of heat shock on gene expression and subcellular protein distribution in Chinese hamster ovary cells.

Incubation of Chinese Hamster Ovary (CHO) cells for one hour at 43 degrees C results in several obvious changes in protein distribution and protein synthesis. One major protein of the cytoplasm (molecular weight 45,000 daltions), also present as a minor component in the nucleus, rapidly disappeared while several proteins, especially high molecular weight peptides, were induced by heat shock. Localization of the proteins in the cytoplasm, extra-nucleolar chromatin and nucleolar bodies has been carried out. Different sets of induced proteins appear in each subcellular compartment. Four hours after restoration of the normal temperature, the normal pattern of protein synthesis was observed. The 45,000 dalton protein reappeared first. Relations between structural and functional alterations and changes in protein distribution are suggested.     

Characterization of a 42 degrees C-specific heat shock protein of mammalian cells

HeLa cells synthesize a particular heat shock protein that is induced only by heat shock at 42 degrees C, and not at 45 degrees C or by other stresses that induce major heat shock proteins (Hatayama et al. (1986) Biochem. Biophys. Res. Commun. 137, 957-963). We further characterized the 42 degrees C-specific protein. This protein was induced in mouse FM 3A cells as well as in human HeLa cells. In both cell lines, the protein was resolved into two spots, a basic polypeptide and an acidc one. The mRNA of the protein was induced during the incubation of these cells at 42 degrees C, and the in vitro translation product of mRNA corresponded to the basic, not to the acidic, polypeptide. During the chase period for cells that were labeled with [35S]-methionine, the basic polypeptide of the protein decreased, and the acidic one increased, indicating that the protein was synthesized as the basic polypeptide and then somehow modified to become the acidic one. The 42 degrees C-specific protein was found only in the cytosol fraction, and not in the nuclear or other particulate fractions, in both HeLa and FM 3A cells. The results suggested that the 42 degrees C-specific protein may have some function in the cytoplasm of mammalian cells during mild heat shock.     

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.     

Effect of acute heat shock on gene expression by human peripheral blood mononuclear cells.

We studied the effect of heat shock on gene expression by normal human cells. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy adults. Paired samples from each subject were subjected to either 20 min of heat shock (43 degrees C) or control (37 degrees C) conditions and then returned to 37 degrees C. RNA was isolated 160 min later, and five representative samples were analyzed on Affymetrix gene chip arrays containing approximately 12,600 probes. A biologically meaningful effect was defined as a statistically significant, twofold or greater difference in expression of sequences that were detected in all five experiments under control (downregulated sequences) or heat shock (upregulated sequences) conditions. Changes occurred in 395 sequences (227 increased by heat shock, 168 decreased), representing 353 Unigene numbers, in every functional category previously implicated in the heat shock response. By RT-PCR, we confirmed the findings for one upregulated sequence (Rad, a G protein) and one downregulated sequence (osteopontin, a cytokine). We conclude that heat shock causes extensive gene expression changes in PBMCs, affecting all functional categories of the heat shock response.     

Vasoactive intestinal peptide receptors on AR42J rat pancreatic acinar cells.

VIP receptors on AR42J rat pancreatic cells were analyzed by competition binding, affinity labeling and by N-glycanase digestion analyses. These studies revealed the presence of specific, high affinity (Kd approximately 1 nM) VIP receptors with a mass of 67 kDa or 59 kDa under reducing or non-reducing conditions, respectively. N-glycanase digestion of affinity labeled membranes generated a core receptor protein of approximately 44 kDa and evidence for at least two N-linked glycans on the mature receptor. The receptor lacked O-linked oligosaccharides but contained terminal sialic acid residues on its N-linked glycan(s) based on digestions with O-glycanase and neuraminidase. The similarity of the AR42J VIP receptor to the recently cloned cDNA for human VIP receptors makes this cell line an attractive model for further analysis of VIP receptor signal transduction events.     

Estrogenic regulation of murine uterine 90-kilodalton heat shock protein gene expression.

Murine uterine steady-state protein levels of the 90-kilodalton heat shock protein (HSP90) have been demonstrated recently to be increased by estrogen in a target tissue- and steroid-specific manner (C. Ramachandran, M.G. Catelli, W. Schneider, and G. Shyamala, Endocrinology 123:956-961, 1988). We now report that this regulation occurred with both the HSP86 and HSP84 forms of HSP90 as well as with the 94-kilodalton glucose-regulated protein. At the mRNA level, this response was greatest for HSP86 (15-fold). In contrast, estradiol had no significant effect on HSP70.     

Recombinant expression and in vitro refolding of the yeast small heat shock protein Hsp42

Small Hsps represent a variation on the theme of protection of proteins from irreversible aggregation by reversible interaction with chaperone proteins. While different sHsps are highly heterogeneous in sequence and size, the common trait is the presence of a conserved alpha-crystallin domain. In addition sHsps assemble into large oligomeric complexes where dimers represent the basic building blocks. Hsp42, a member of the sHsp family in the cytosol of S. cerevisiae, forms ordered oligomers with a barrel-like structure. Here, we present the recombinant expression and purification of Hsp42. We demonstrate, that Hsp42 is expressed in inclusion bodies and can be resolubilized and folded to correct, active oligomers. This indicates that in contrast to thermal unfolding, the chemical disassembly and unfolding of Hsp42 is fully reversible. In comparison to the purification of mature Hsp42 from yeast, its recombinant expression leads to a substantial increase in the yield of the protein and to a reduction of contamination caused by aggregation prone proteins complexed by Hsp42. In addition, the recombinant Hsp42 is fully active as a chaperone in an energy independent manner.     

Modulation of protein phosphorylation and stress protein expression by okadaic acid on heat shock cells

We have demonstrated that pretreatment but not post-treatment with okadaic acid (OA) can aggravate cytotoxicity as well as alter the kinetics of stress protein expression and protein phosphorylation in heat shocked cells. Compared to heat shock, cells recovering from 1 hr pretreatment of OA at 200 nM and cotreated with heat shock at 45°C for the last 15 min of incubation (OA→HS treatment) exhibited enhanced induction of heat shock proteins (HSPs) 70 and 110. In addition to enhanced expression, the attenuation of HSC70 and HSP90 after the induction peaks was also delayed in OA→HS-treated cells. The above treatment also resulted in the rapid induction of the 78 kDa glucose-regulated protein (GRP78), which expression remained constant in cells recovering from treatment with 200 nM OA for 1 hr, heat shocked at 45°C for 15 min, or in combined treatment in reversed order (HS→OA treatment). Enhanced phosphorylation of vimentin and proteins with molecular weights of 65, 40, and 33 kDa and decreased phosphorylation of a protein with a molecular weight of 29 kDa were also observed in cells recovering from OA→HS treatment. Again, protein phosphorylation in cells recovering from HS→OA treatment did not differ from those in cells treated only with heat shock. Since the alteration in the kinetics of stress protein expression and protein phosphorylation was tightly correlated, we concluded that there is a critical link between induction of the stress proteins and phosphorylation of specific proteins. Furthermore, the rapid induction of GRP78 under the experimental condition offered a novel avenue for studying the regulation of its expression. © 1996 Wiley-Liss, Inc.     

NADPH oxidase and apoptosis in cerulein-stimulated pancreatic acinar AR42J cells

Apoptosis linked to oxidative stress has been implicated in pancreatitis. We investigated whether NADPH oxidase mediates apoptosis in cerulein-stimulated pancreatic acinar AR42J cells. We report here that cerulein treatment resulted in the activation of NADPH oxidase, as determined by ROS production, translocation of cytosolic subunits p 47(phox) and p 67(phox) to the membrane, and interaction between NADPH oxidase subunits. Cerulein induced Ca(2+) oscillation, the expression of apoptotic genes p53 and bax, and apoptotic indices (DNA fragmentation, TUNEL staining, caspase 3 activity, decrease in cell viability) in AR42J cells. Treatment with a Ca(2+) chelator, BAPTA-AM, or transfection with antisense oligonucleotides for NADPH oxidase subunits p22(phox) and p 47(phox) inhibited cerulein-induced ROS production, translocation of NADPH oxidase cytosolic subunits p 47(phox) and p 67(phox) to the membrane, and the expression of apoptotic genes and apoptotic indices, as compared to the cells without treatment and those transfected with the corresponding sense oligonucleotides. These results indicate that NADPH oxidase may mediate ROS-induced apoptosis in pancreatic acinar cells in a Ca(2+)-dependent manner.     

Calpain mediates caspase-dependent apoptosis initiated by hydrogen peroxide in pancreatic acinar AR42J cells

Abstract

Several studies have shown that oxidative stress induces apoptosis in many cellular systems including pancreatic acinar cells. However, the exact molecular mechanisms leading to apoptosis remain partially understood. This study aimed to investigate the role of the cytosolic cysteine protease calpain in H₂O₂-induced apoptosis in pancreatic AR42J cells. Apoptosis was evaluated using flow cytometric analysis of sub-G1 DNA populations, electron-microscopic analysis, caspase-3-specific αII-spectrin breakdown, and measuring the proteolytic activities of the initiator caspase-12 and caspase-8, and the executioner caspase-3. H₂O₂ induced an increase in the calpain proteolytic activity immediately after starting the experiments that tended to return to a nearly normal level after 8 h and could be attributed to m-calpain. Whereas no caspase-12, caspase-8 and caspase-3 activations could be detected within the first 0.5 h, significantly increased proteolytic activities were observed after 8 h compared with the control. At the same time, the cells showed first ultrastructural hallmarks of apoptosis and a decreased viability. In addition, αII-spectrin fragmentation was identified using immunoblotting that could be attributed to both calpain and caspase-3. Calpain inhibition reduced the activities of caspase-12, caspase-8, and caspase-3 leading to a decrease in the number of apoptotic cells. Immunoblotting analyses of caspase-12 and caspase-8 indicate that calpain may be involved in the activation process of both proteases. The results suggest that H₂O₂-induced apoptosis of AR42J cells requires activation of m-calpain initiating the endoplasmic reticulum stress-induced caspase-12 pathway and a caspase-8-dependent pathway. The findings also suggest that calpain may be involved in the execution phase of apoptosis.     

人热休克蛋白70的原核高效表达

将人热休克蛋白基因hsp70片段克隆到高效原核表达载体pMAL-c2X中,酶切鉴定并进行DNA测序。将该重组表达载体转化大肠杆菌DH50α,用IPTG在不同温度及时间下进行诱导表达。收集细菌,菌体裂解后进行SDS-PAGE及Western blot检测,并以凝胶薄层扫描分析表达水平。结果表明,成功地构建了含人hsp70基因的表达载体pMAL-c2X／hsp70,该载体能在大肠杆菌中表达相对分子质量为110000并具有抗原活性的融合蛋白;改变诱导温度和时间,目的蛋白表达总量及可溶性部分所占比例不同。对人hsp70基因的克隆、表达,并对其进行表达条件的优化,为研究HSP70的结构、功能与临床应用提供了必要条件。     

Glucagon-like peptide-1 does not mediate amylase release from AR42J cells.

In this study, AR42J pancreatic acinar cells were used to investigate if glucagon-like peptide-1 (GLP-1) or glucagon might influence amylase release and acinar cell function. We first confirmed the presence of GLP-1 receptors on AR42J cells by reverse trasncriptase-polymerase chain reaction (RT-PCR), Western blotting, and partial sequencing analysis. While cholecystokinin (CCK) increased amylase release from AR42J cells, GLP-1, alone or in the presence of CCK, had no effect on amylase release but both CCK and GLP-1 increased intracellular calcium. Similar to GLP-1, glucagon increased both cyclic adenosine monophosphate (cAMP) and intracellular calcium in AR42J cells but it actually decreased CCK-mediated amylase release (n = 20, P < 0.01). CCK stimulation resulted in an increase in tyrosine phosphorylation of several cellular proteins, unlike GLP-1 treatment, where no such increased phosphorylation was seen. Instead, GLP-1 decreased such protein phosphorylations. Genestein blocked CCK-induced phosphorylation events and amylase secretion while vanadate increased amylase secretion. These results provide evidence that tyrosine phosphorylation is necessary for amylase release and that signaling through GLP-1 receptors does not mediate amylase release in AR42J cells. J. Cell. Physiol. 181:470-478, 1999. Published 1999 Wiley-Liss, Inc.