Apoptosis In AR42J Cells Research Articles

Protective Effects of Baicalein on Lipopolysaccharide-Induced AR42J PACs through Attenuation of Both Inflammation and Pyroptosis via Downregulation of miR-224-5p/PARP1.

Baicalein has been used to treat inflammation-related diseases; nevertheless, its specific mechanism of action is unclear. Therefore, we examined the protective effects of baicalein on lipopolysaccharide-induced damage to AR42J pancreatic acinar cells (PACs) and determined its mechanism of action for protection. An in vitro cell model of acute pancreatitis (AP) was established using lipopolysaccharide (LPS) (1 mg/L)-induced PACs (AR42J), and the relative survival rate was determined using the 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) technique. Flow cytometry was applied to evaluate the apoptotic rates of AR42J PACs. The RNA and protein expression of miR-224-5p, poly ADP-ribose polymerase-1 (PARP1), nuclear transcription factor-κB65 (NF-κB65), phospho-kappa B alpha(p-IκB-α), interleukin(IL)-18R, NOD-like receptor thermal protein domain-associated protein 3 (NLRP3), gasdermin D (GSDMD), apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 was detected based on the WB and RT-PCR assays. IL-1β, IL-6, IL-18, and TNF-α expression levels in AR42J cells were measured via ELISA method. The cell morphology was examined using the AO/EB method. The experiment confirmed a significant increase in the activity of AR42J cells treated with various doses of baicalein. Moreover, IL-1β, IL-6, TNF-α, and IL-18 expression levels in AR42J cells were dramatically reduced (P < 0.05), while miR-224-5p level was obviously enhanced. The protein and gene expression of PARP1, NF-κB65, p-IκB-α, IL-18R, GSDMD, ASC, NLRP3, and caspase-1 was obviously decreased (P < 0.05). Apoptosis in AR42J cells was significantly reduced with significant improvement in cell morphology. Baicalein may significantly alleviate LPS-induced AR42J PAC damage by inhibiting the inflammatory response and pyroptosis. Its mode of action might be linked to higher miR-224-5p expression, which inhibits the PARP1/NF-κB and NLPR3/ASC/caspase-1/GSDMD pathways.

Phospholipase D2 targeted by miR-5132-5p alleviates cerulein-induced acute pancreatitis via the Nrf2/NFκB pathway.

Acute pancreatitis (AP) is an inflammatory process unexpectedly occurring in the pancreas, imposing a substantial burden on healthcare systems. Herein, we aimed to clarify the mechanism of action of phospholipase D2 (PLD2) in cerulein-treated AR42J cells, affording valuable insights into the treatment of AP. The levels of PLD2, miR-5132-5p, inflammatory factors (interleukin [IL]-10, IL-6, and tumor necrosis factor-α), caspase-3 activity, and apoptosis-related proteins (Bax and Bcl-2) in cerulein-treated AR42J cells were detected using reverse transcription-quantitative polymerase chain, caspase-3 activity, and Western blot analysis. Protein levels of nuclear Factor erythroid 2-Related Factor 2 (Nrf2) and nuclear factor-k-gene binding (NF-κB) were detected by Western blot analysis. TargetScan predicted upstream microRNAs (miRNAs) of PLD2, and the interaction between miR-5132-5p and PLD2 was verified using a luciferase assay. In cerulein-treated AR42J cells, PLD2 levels were downregulated, while miR-5132-5p expression was upregulated. Overexpression of PLD2 attenuated the cerulein-mediated facilitatory effect on inflammation and apoptosis in AR42J cells by regulating the Nrf2/NFκB pathway. Luciferase reporter analysis revealed that miR-5132-5p targeted PLD2, and miR-5132-5p negatively regulated PLD2. Upregulation of miR-5132-5p expression exacerbated inflammation and apoptosis and reversed the protective effect of PLD2 overexpression on AP. PLD2 targeted by miR-5132-5p can attenuate cerulein-induced AP in AR42J cells via the Nrf2/NFκB pathway, providing therapeutic targets for patients with AP.

Rutaecarpine alleviates acute pancreatitis in mice and AR42J cells by suppressing the MAPK and NF-κB signaling pathways via calcitonin gene-related peptide.

Acute pancreatitis (AP) is an acute inflammatory condition of the pancreas. Previous studies have shown that rutaecarpine (RUT), an important alkaloid component of Evodia rutaecarpa, exhibits certain protective effects against AP in rats by upregulating calcitonin gene-related peptide (CGRP). However, the molecular mechanism of RUT in AP remains unknown. This study aimed to investigate the effects of RUT on cerulein-induced AP in vivo and in vitro, and to explore the underlying molecular mechanisms. In cerulein/LPS-treated wild-type mice, but not CGRP gene knock-out mice, RUT significantly ameliorated pancreatic inflammation by alleviating histopathological changes, reducing IL-6 and TNF-α levels, and increasing in IL-10 levels. Moreover, RUT improved AP by suppressing the MAPK and NF-κB signaling pathways. These effects were mostly mediated through CGRP. Cell-based studies revealed that RUT significantly improved cell viability while suppressing the apoptosis of AR42J cells with cerulein-induced AP, downregulating IL-6 and TNF-α, stimulating IL-10 release, and inhibiting MAPK, NF-κB, and STAT3 signaling activation, all in a CGRP-dependent manner. RUT ameliorated cerulein/LPS-induced AP inflammatory responses in mice and AR42J cells in a CGRP-dependent manner and thus may represent a potential therapeutic option for AP patients. Our study provides valuable insights for AP drug development.

TNFAIP3-upregulated RIP3 exacerbates acute pancreatitis via activating NLRP3 inflammasome

Acute pancreatitis (AP) is an inflammatory disease of the pancreas. Accumulating studies have revealed the involvement of tumor necrosis factor alpha-induced protein 3 (TNFAIP3) in the progression of AP. Here, the current study was conducted to elucidate the role of TNFAIP3 and the underlying molecular mechanisms on the progression of AP. The in vivo animal model and in vitro cell model of AP were generated by retrograde injection of sodium taurocholate and stimulation of cerulein into AR42J cells, respectively. Relationships among TNFAIP3, receptor interacting protein 3 (RIP3) and nod-like receptor protein 3 (NLRP3) were predicted on bioinformatics websites and verified by co-immunoprecipitation. AR42J cells were transfected with overexpressing plasmid or shRNA to study the effects of TNFAIP3/RIP3/NLRP3 axis on cell proliferation and apoptosis, secretion of inflammatory cytokines and production of ROS. The effect of TNFAIP3/RIP3/NLRP3 axis in AP was further confirmed in vivo. High expression of TNFAIP3 was observed in AP pancreatic tissues and AP cell model. TNFAIP3 increased RIP phosphorylation through deubiquitination. RIP activated the NLRP3 inflammasome. Silencing of TNFAIP3 or RIP3T led to elevated proliferation and inhibited apoptosis in AR42J cells, accompanied by decreased inflammatory cytokine levels and ROS production. The protective role of inhibited TNFAIP3 in AP was confirmed evidenced by reduced levels of AMY, LIPA, and ROS in vivo. Collectively, overexpressed TNFAIP3 could contribute to the progression of AP by activating RIP3/NLRP3 axis, providing a potential therapeutic target for AP treatment.

Glucocorticoid receptor regulates expression of microRNA-22 and downstream signaling pathway in apoptosis of pancreatic acinar cells.

AIMTo elucidate the underlying mechanism that microRNA-22 (miR-22) promotes the apoptosis of rat pancreatic acinar cells (AR42J) and the elements that regulate the expression of miR-22.METHODSOne hundred nanomoles per liter of caerulein (Cae) was administrated to induce the apoptosis of AR42J cells and the apoptosis rate was detected by flow cytometry analysis. An amylase assay kit was used to measure the amylase expression level in the supernatant. Quantitative real-time PCR (qRT-PCR) was adopted to measure miR-22 expression. We used online tools to predict the potential transcription promoter of miR-22 and the binding sites, which was further identified by using luciferase reporter analysis, chromatin immunoprecipitation (ChIP) and ChIP-qPCR assays. Then, a mimic of miR-22, Nr3c1 plasmid encoding the glucocorticoid receptor (GR), and si-Nr3c1 were used to transfect AR42J cells, respectively. The mRNA expression of miR-22, Nr3c1, and Erb-b2 receptor tyrosine kinase 3 (ErbB3) was confirmed by qRT-PCR and the apoptosis rate of AR42J cells was detected by flow cytometry analysis. Western blot was used to detect the expression of ErbB3, GR, PI3k, PI3k-p85α, Akt, p-Akt, Bad, Bax, Bcl-xl, Bcl-2, and cleaved caspase3.RESULTSAfter inducing apoptosis of AR42J cells in vitro, the expression of miR-22 was significantly increased by 2.20 ± 0.26 and 4.19 ± 0.54 times, respectively, at 3 h and 6 h in comparison with the control group. As revealed by qRT-PCR assay, the expression of miR-22 was 78.25 ± 6.61 times higher in the miR-22 mimic group relative to the miRNA control group, accompanied with an obviously increased acinar cell apoptosis rate (32.53 ± 1.15 vs 18.07 ± 0.89, P = 0.0006). The upregulation of miR-22 could suppress its target gene, ErbB3, and the phosphorylation of PI3k and Akt. Furthermore, we predicted the potential transcription promoter of miR-22 and the binding sites using online tools. Luciferase reporter analysis and site-directed mutagenesis indicated that the binding site (GACAGCCATGTACA) of the GR, which is encoded by the Nr3c1 gene. Downregulation of the expression of GR could upregulate the expression of miR-22, which further promoted the apoptosis of AR42J cells.CONCLUSIONGR transcriptionally represses the expression of miR-22, which further promotes the apoptosis of pancreatic acinar cells by downregulating the downstream signaling pathway.

The interaction between ANXA2 and lncRNA Fendrr promotes cell apoptosis in caerulein-induced acute pancreatitis.

Annexin A2 (ANXA2) plays a crucial role in acute pancreatitis (AP). However, its potential mechanism remains unclear. In the present study, we used caerulein-treated AR42J rat pancreatic acinar cells as cell model of AP to investigate the potential functions of ANXA2 and its predicted long noncoding RNA (lncRNA) FOXF1 adjacent noncoding developmental regulatory RNA (lncRNA Fendrr). Cell apoptosis was evaluated by flow cytometry using annexinV-fluorescein isothiocyanate/propidium iodide staining. The expressions of ANAX2 and lncRNA Fendrr were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, Western blot analysis was performed to determine the protein levels of ANXA2, Bcl-2, and Bax. The association between lncRNA Fendrr and ANXA2 was disclosed by RNA pull-down, RNA immunoprecipitation, and electrophoretic mobility shift assays. ANXA2 was elevated in caerulein-induced AP model and promoted apoptosis of AR42J cells. LncRNA Fendrr was also upregulated in AP cell model and directly bound ANXA2 protein. Further studies indicated that the interaction between ANXA2 and lncRNA Fendrr contributed to the apoptosis of AR42J cells in AP cell model. Our study demonstrated that ANXA2 promoted AP progression via interacting with lncRNA Fendrr in vitro, which will provide a novel insight into the therapeutic target for AP.

Effect of long non-coding RNA RGD1566401 on apoptosis of rat pancreatic acinar cells in the rat acute pancreatitis model

Objective To analyze the expression of RNA-RGD1566401 (long non-coding RNA-RGD1566401, lncR-RGD1566401) in rat pancreatic acinar cells (AR42J) and the effect of lncR-RGD1566401 on apoptosis of AR42J cells. Methods The GFP LncR-RGD1566401 overexpressed lentivirus and special long non-coding RNA (lncRNA) Smart Silencer were designed and transfected into AR42J cells repectively for 24 h. The apoptosis of AR42J cells in different groups was established by inducing cells with 100 nmol/L Caerulin for 24 h. The flow cytometry was used to detect the apoptosis of AR42J cells in different groups. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression level of lncR-RGD1566401 in AR42J cells of different groups after transfection. The expression of cysteinyl aspartate-specific protease (Caspase)-3 and p53 was detected by Western blotting. Results The apoptosis rate [(19.34±0.54)%] of Caerulin-induced AR42J cells was elevated as compared with the control group [(7.23±0.33)%]. The expression of lncR-RGD1566401 in the AR42J cells was increased by (5.43±0.31) times in the experimental group after treatment with Caerulin and the levels of Caspase-3 and p53 were increased obviously. LncR-RGD1566401 level was elevated by (8.24±0.22) times after AR42J cells were transfected with lenti-lncRNA as compared with the vehicle groups (P=0.006). The AR42J cells showed more tendency to apoptosis [(38.20±0.37)%] than the vehicle groups [(22.60±0.96)%, P=0.016] and the levels of Caspase-3 and p53 were increased obviously. The AR42J cells that were transfected with Silencer-lncRNA expressed the lower level of lncR-RGD1566401 about [(0.54±0.14), P=0.003] times and less apoptosis [(13.60±0.33)%] than the Silencer-NC groups [(19.60±0.46)%, P=0.012]. Conclusion LncR-RGD1566401 is associated with the apoptosis of AR42J cells, and up-regulation of LncR-RGD1566401 can promote the apoptosis of AR42J cells. Key words: Long non-coding RNA; Apoptosis; Pancreatic acinar cells

MiR-29a up-regulation in AR42J cells contributes to apoptosis via targeting TNFRSF1A gene.

To investigate the expression of miR-29a in rat acute pancreatitis and its functional role in AR42J cell apoptosis. Twelve SD rats were divided into a control group and an acute edematous pancreatitis (AEP) group randomly. AEP was induced by intraperitoneal injection of L-arginine (150 mg/kg) in the AEP group and equal volume of 0.9% NaCl was injected in the control group. The apoptosis of acinar cells in pancreatic tissue was determined by TUNEL assay. miRNA chip assay was performed to examine the expression of miRNAs in two groups. Besides, to further explore the role of miR-29a in apoptosis in vitro, recombinant rat TNF-α (50 ng/mL) was administered to treat the rat pancreatic acinar cell line AR42J for inducing AR42J cell apoptosis. Quantitative real-time PCR (qRT-PCR) was adopted to measure miR-29a expression. Then, miRNA mimic, miRNA antisense oligonucleotide (AMO) and control vector were used to transfect AR42J cells. The expression of miR-29a was confirmed by qRT-PCR and the apoptosis rate of AR42J cells was detected by flow cytometry analysis. Western blot was used to detect the expression of activated caspase3. Moreover, we used bioinformatics software and luciferase assay to test whether TNFRSF1A was the target gene of miR-29a. After transfection, qRT-PCR and Western blot was used to detect the expression of TNFRSF1A in AR42J cells after transfection. The expression of miR-29a was much higher in the AEP group compared with the control group as displayed by the miRNA chip assay. After inducing apoptosis of AR42J cells in vitro, the expression of miR-29a was significantly increased by 1.49 ± 0.04 times in comparison with the control group. As revealed by qRT-PCR assay, the expression of miR-29a was 2.68 ± 0.56 times higher in the miR-29a mimic group relative to the control vector group, accompanied with an obviously increased acinar cell apoptosis rate (42.83 ± 1.25 vs 24.97 ± 0.15, P < 0.05). Moreover, the expression of miR-29a in the miRNA AMO group was 0.46 ± 0.05 times lower than the control vector group, and the cell apoptosis rate was much lower accordingly (17.27 ± 1.36 vs 24.97 ± 0.15, P < 0.05). The results of bioinformatics software and luciferase assay showed that TNFRSF1A might be a target gene of miR-29a. TNFRSF1A expression was up-regulated in the miR-29a mimic group, while the miR-29a AMO group showed the reverse trend. miR-29a might promote the apoptosis of AR42J cells via up-regulating the expression of its target gene TNFRSF1A.

Expressions of miR-22 and miR-135a in acute pancreatitis

This study examined the expressions of miR-22 and miR-135a in rats with acute edematous pancreatitis (AEP) and their target genes in order to shed light on the involvement of miR-22 and miR-135a in the pathogenesis of acute pancreatitis (AP). The in vivo model of AEP was established by introperitoneal injection of L-arginine (150 mg/kg) in rats. The miRNA microarray analysis was used to detect the differential expression of miRNAs in pancreatic tissue in AEP and normal rats. The in vitro AEP model was established by inducing the rat pancreatic acinar cell line (AR42J) with 50 ng/mL recombinant rat TNF-α. Real-time quantitative RT-PCR was employed to detect the expression of miR-22 and miR-135a in AR42J cells. Lentiviruses carrying the miRNA mimic and anti-miRNA oligonucleotide (AMO) of miR-22 and miR-135a were transfected into the AR42J cells. The AR42J cells transfected with vehicle served as control. Western blotting was used to measure the expression of activated caspase3 and flow cytometry analysis to detect the apoptosis of AR42J cells. Targets of miR-22 and miR-135a were predicted by using TargetScan, miRanda, and TarBase. Luciferase reporter assay and quantitative real-time RT-PCR were performed to confirm whether ErbB3 and Ptk2 were the target gene of miR-22 and miR-135a, respectively. The results showed that the expression levels of miR-22 and miR-135a were obviously increased in AEP group compared with the control group in in-vivo and in-vitro models. The expression levels of miR-22 and miR-135a were elevated conspicuously and the expression levels of their target genes were reduced significantly in AR42J cells transfected with lentiviruses carrying the miRNA mimic. The apoptosis rate was much higher in the TNF-α-induced cells than in non-treated cells. The AR42J cells transfected with miRNA AMOs expressed lower level of miR-22 and miR-135a and had lower apoptosis rate, but the expression levels of ErbB3 and Ptk2 were increased obviously. It was concluded that the expression levels of miR-22 and miR-135a were elevated in AEP. Up-regulating the expression of miR-22 and miR-135a may promote the apoptosis of pancreatic acinar cells by repressing ErbB3 and Ptk2 expression in AEP.

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

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.

Role of parathyroid hormone-related protein in the pro-inflammatory and pro-fibrogenic response associated with acute pancreatitis

Pancreatitis is a common and potentially lethal necro-inflammatory disease with both acute and chronic manifestations. Current evidence suggests that the accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic disease, which is associated with an increased risk of pancreatic cancer. While parathyroid hormone-related protein (PTHrP) exerts multiple effects in normal physiology and disease states, its function in pancreatitis has not been previously addressed. Here we show that PTHrP levels are transiently elevated in a mouse model of cerulein-induced AP. Treatment with alcohol, a risk factor for both AP and chronic pancreatitis (CP), also increases PTHrP levels. These effects of cerulein and ethanol are evident in isolated primary acinar and stellate cells, as well as in the immortalized acinar and stellate cell lines AR42J and irPSCc3, respectively. Ethanol sensitizes acinar and stellate cells to the PTHrP-modulating effects of cerulein. Treatment of acinar cells with PTHrP (1–36) increases expression of the inflammatory mediators interleukin-6 (IL-6) and intracellular adhesion protein (ICAM-1), suggesting a potential autocrine loop. PTHrP also increases apoptosis in AR42J cells. Stellate cells mediate the fibrogenic response associated with pancreatitis; PTHrP (1–36) increases procollagen I and fibronectin mRNA levels in both primary and immortalized stellate cells. The effects of cerulein and ethanol on levels of IL-6 and procollagen I are suppressed by the PTH1R antagonist, PTHrP (7–34). Together these studies identify PTHrP as a potential mediator of the inflammatory and fibrogenic responses associated with alcoholic pancreatitis.

Effects of Arsenic Trioxide on the Cerulein-Induced AR42J Cells and Its Gene Regulation

To study the molecular mechanism of arsenic trioxide-induced cell death on cerulein-stimulated AR42J cells. AR42J cells were incubated for 24 hours, and cerulein (10 nmol/L) and different concentrations of arsenic trioxide were added for another 24 hours. The cells were collected and analyzed for apoptosis and oncosis by using rhodamine 123 and propidium iodide staining, and the changes in the genes that related to cell death were detected by a gene chip. After cerulein stimulation, apoptosis was significantly increased in the AR42J cells. The addition of arsenic trioxide increased the number of apoptotic cells, and the apoptotic index reached its peak in the 1-micromol/L group. Regarding oncosis, low concentrations of arsenic trioxide (0.5, 1, 2, and 4 micromol/L) reduced the development of oncosis, and in the 2-micromol/L group, it was most significant, whereas high concentration of arsenic trioxide (8 micromol/L) promoted the development of oncosis. A total of 96 genes related to apoptosis were detected, and 36 genes were differentially expressed. Appropriate concentrations of arsenic trioxide can induce apoptosis in AR42J cells that were induced by cerulein and lead to changes in the expressions of certain genes.

Orexin-Induced Apoptosis: The Key Role of the Seven-Transmembrane Domain Orexin Type 2 Receptor

Orexin-A and orexin-B are regulatory peptides involved in the control of feeding, sleep-wakefulness, and exerting various endocrine and metabolic actions. Recently we demonstrated that orexins, acting at OX(1) receptor (OX(1)R), are proapoptotic peptides. The aim of this study was to investigate the role of the receptor subtype OX(2)R in the control of apoptosis. Orexins caused a caspase-dependent cell death by apoptosis and a drastic cell growth inhibition in Chinese hamster ovary cells transfected with OX(2)R cDNA. On addition of either orexin (10(-6) m) for 48 h, apoptosis was demonstrated by DNA fragmentation, chromatin condensation, annexin-V binding, and activation of caspase-3 and caspase-9. Orexins were active on apoptosis and cell growth inhibition in the range of concentrations between 10(-10) and 10(-5) m with an EC(50) of 5 x 10(-8) m peptides. No effect of orexins could be detected in parental Chinese hamster ovary cells. A rat pancreatic acinar cell line, AR42J, which expresses OX(2)R but not OX(1)R, also underwent growth suppression and apoptosis on treatment with orexins. Suppression of AR42J cell growth by 10(-6) m orexin was more than 75% after 24 h. Induction of annexin-V-labeled AR42J cell number was dose dependent, with EC(50) of 5.1 x 10(-8) m orexin-A and 9.8 x 10(-8) m orexin-B. The OX(2)R agonist [Ala (11), d-Leu (15)]orexin-B promoted effects on cell growth and apoptosis, which were similar to those elicited by orexins. The OX(1)R antagonist SB33487 did not alter orexin-induced inhibition of growth or orexin-induced stimulation of apoptosis in AR42J cells. For the first time, we provide functional and pharmacological evidence for a role of the OX(2)R in orexin-induced apoptosis.

Ligands of peroxisome proliferator-activated receptor-gamma induce apoptosis in AR42J cells.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-activated transcription factor that controls growth, differentiation, and inflammation in different tissues. Roles of PPAR-gamma activation in pancreatic acinar cells are poorly characterized. To examine the effects of PPAR-gamma activation on the induction of apoptosis in rat pancreatic AR42J cells. AR42J cells were treated with ligands of PPAR-gamma, and induction of apoptosis was evaluated by cell viability, DNA-fragmentation, and flow cytometry. Treatment of the cells with ligands of PPAR-gamma (15-deoxy-open triangle12,14-prostaglandin J2 or troglitazone) induced apoptosis in a dose-dependent manner. Troglitazone-induced apoptosis was not blocked by inhibitors of caspases (acetyl-DEVD-aldehyde and benzoyloxycarbonyl-VAD-fluoromethylketone). Troglitazone induced the expression of pancreatitis-associated protein-1 and clusterin mRNAs. Troglitazone activated c-Jun NH2-terminal kinase/stress-activated protein kinase, but inhibited the activation of extracellular signal-regulated kinases 1/2. Troglitazone did not activate NF-kappaB, suggesting a role of NF-kappaB-independent pathways. In AR42J cells and isolated pancreatic acini, PPAR-gamma gene and protein were detected. In addition, troglitazone increased the PPAR-dependent transcriptional activity, suggesting that PPAR-gamma is functional in AR42J cells. These results indicate that activation of PPAR-gamma induces apoptosis in AR42J cells and imply that PPAR-gamma may be a potential therapeutic target of pancreatic inflammation, because of its anti-inflammatory effects in addition to its proapoptotic effects.

Lysophosphatidylcholine induces apoptosis in AR42J cells.

Phospholipase A2 (PLA2) has been suggested to play an important role in the pathogenesis of acute pancreatitis, in part through the PLA2-generated phospholipid by-products, most notably lysophosphatidylcholine (lyso-PC). The effects of lyso-PC on pancreatic acinar cells, other than the induction of necrosis, are poorly characterized. Here we examined the effects of lyso-PC on the induction of apoptosis in rat pancreatic AR42J cells. Lyso-PC induced apoptosis in a dose-dependent manner at 10 and 25 microM, but induced cell lysis at > or = 50 microM. Lyso-PC-induced (at 25 microM) apoptosis was not blocked by a protein kinase C inhibitor (staurosporine) or by inhibitors of caspases (acetyl-DEVD-aldehyde and benzoyloxycarbonyl-VAD-fluoromethylketone). Lyso-PC at 10 and 25 microM induced the expression of clusterin mRNA and wild-type p53. Apoptosis induction by lyso-PC (at 25 microM) was not inhibited by a specific antagonist of platelet-activating factor (PAF) receptor, suggesting that the action was independent of th

P8 expression is induced in acinar cells during chronic pancreatitis.

The p8 gene is barely expressed in the normal pancreas, but is overexpressed in acute pancreatitis. To elucidate the dynamic expression of p8 mRNA and its significance in the course of chronic pancreatitis, we investigated the p8 expression in spontaneous chronic pancreatitis in the WBN/Kob rat as well as in humans and arginine-treated rat pancreatic acinar AR4-2J cells. p8 mRNA was significantly increased at 12 weeks when chronic pancreatitis first appeared in the WBN/Kob rats. p8 was immunolocalized in the acinar cell nuclei. Acinar cell apoptosis was significantly increased at 12 and 20 weeks in the WBN/Kob rats. In AR4-2J cells, p8 mRNA was significantly induced at 4 hr after arginine addition. Apoptosis of AR4-2J cells was not increased during the strong expression of p8 mRNA. These results suggest that p8 is induced in the acinar cells during chronic pancreatitis as the self-defence mechanism against proapoptotic insults.

Tumor necrosis factor α triggers antiapoptotic mechanisms in rat pancreatic cells through pancreatitis-associated protein I activation

Background & Aims: Tumor necrosis factor (TNF)-α contributes to the development of acute pancreatitis. Because TNF-α is involved in the control of apoptosis, we studied its interaction with the pancreatic apoptotic pathway. Methods: Pancreatic acinar AR4-2J cells were used. Apoptosis was monitored by morphologic and biochemical criteria. Results: TNF-α induced apoptosis in AR4-2J cells. Induction was strongly enhanced in cells treated with actinomycin D, suggesting that TNF-α activated concomitantly an antiapoptotic mechanism through newly synthesized proteins. This mechanism involved activation of nuclear factor–κB (NF-κB) and mitogen-activated protein (MAP) kinases because their inhibition worsened TNF-α–induced apoptosis. The antiapoptotic pancreatitis-associated protein (PAP) I is a candidate for mediating TNF-α activity. Its expression is induced by TNF-α, and cells overexpressing PAP I show significantly less apoptosis on exposure to TNF-α. We examined whether TNF-α induction of PAP I expression was mediated by NF-κB or MAP kinases by using specific inhibitors of both pathways. Inhibition of NF-κB had no effect. However, inhibitors of MEK1 eliminated PAP I induction. Conclusions: TNF-α induces concomitantly proapoptotic and antiapoptotic mechanisms in pancreatic AR4-2J cells. Antiapoptotic mechanisms are mediated by NF-κB and MAP kinases, and PAP I is one of the effectors of apoptosis inhibition.GASTROENTEROLOGY 2000;119:816-828

Lysophosphatidylcholine activates transcription factors and induces apoptosis in AR42J cells

Lysophosphatidylcholine activates transcription factors and induces apoptosis in AR42J cells

Supraphysiologic concentrations of cerulein induce apoptosis in the rat pancreatic acinar cell line AR4-2J.

Little is known as yet about the role of apoptosis in pancreatic damage. This study evaluated the effects of supraphysiologic concentrations of the cholecystokinin (CCK) analog, cerulein, which causes cell damage in vitro and acute pancreatitis in vivo, on cell proliferation and DNA fragmentation in the rat pancreatic acinar cell line AR4-2J. Cerulein inhibited the cell proliferation of AR4-2J time- and dose-dependently to approximately 60% of the control level at 10(-6) M after 72 h. DNA fragmentation, as assessed by both electrophoresis and enzyme-linked immunosorbent assay (ELISA), occurred at cerulein concentrations > or = 10(-8) M. The maximal DNA fragmentation as measured by ELISA was reached after 24 h. Cerulein at concentrations > or = 10(-9) M induced wild-type p53. Glutathione (1 mM) diminished the effects of cerulein on both cell proliferation and DNA fragmentation, whereas spermine (100 microM), which partially attenuated DNA fragmentation, did not have an effect on cell proliferation. The CCK-A-receptor antagonist loxiglumide completely abolished the effect of cerulein on DNA fragmentation. The serine-protease inhibitor FUT-175 (10 microM), the cysteine-protease inhibitor NCO-700 (5 mM), and ethylene glycol tetraacetic acid (EGTA; 500 microM) all had no effects on the changes in cell proliferation and DNA fragmentation induced by cerulein. The data suggest that supraphysiologic concentrations of cerulein rapidly induce apoptosis in AR4-2J cells and only later inhibit cell proliferation. These effects are mediated by CCK-A receptors. Cerulein-induced apoptosis may involve the induction of wild-type p53 or glutathione depletion or both.

Clusterin overexpression in rat pancreas during the acute phase of pancreatitis and pancreatic development.

Molecular mechanisms associated with apoptosis in pancreas remain largely unknown. Clusterin mRNA is induced in several tissues in response to most apoptotic stimuli. In these tissues, clusterin has an antiapoptotic activity. The aim of this work was to test whether clusterin, which is not expressed in normal pancreas, was induced in pancreas during pancreatitis and pancreatic development. Clusterin mRNA levels were strongly increased 6 h after pancreatitis induction. Maximal expression happened between 24-48 h and decreased progressively to undetectable levels at day 5. Clusterin mRNA was expressed with similar intensity in oedematous caerulein-induced pancreatitis and in response to various degrees of necrohaemorrhagic taurocholate-induced pancreatitis, indicating a maximal gene activity in all types of pancreatitis; in situ hybridization showed that the acinar cells and some ducts expressed clusterin mRNA. A single band of about 35-38 kDa was detected by western blot in pancreatic homogenates and in pancreatic juice from rats with acute pancreatitis, but not from control rats. Clusterin mRNA expression was strong in late fetal life and remains high until day 11 post-partum, then decreased progressively with a minimum from 35 to 90 days post-partum. Clusterin mRNA levels were strongly induced in pancreatic acinar AR4-2J cells in response to various apoptotic stimuli (i.e., cycloheximide, staurosporine, ceramide and H2O2) but not with interleukin (IL)-1, IL-4 or IL-6 or heat shock, which do not induce apoptosis in AR4-2J cells. In conclusion, we demonstrated that clusterin is synthesized and released by the pancreas. Its strong expression during acute pancreatitis suggests its involvement in the pancreatic response to injury. Clusterin is also induced during pancreatic development. Because these situations are associated with apoptosis and clusterin was shown to protect against apoptosis, we speculate that clusterin could be involved in the control of acinar cell apoptosis.