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     

Atherogenic role of lysophosphatidylcholine in low-density lipoprotein modified by phospholipase A2 and in diabetic patients: protection by nitric oxide donor

The aim of our study was to investigate the atherogenic role of lysophosphatidylcholine (lyso-PC) in low-density lipoprotein (LDL) under diabetic environment. Expression of monocyte chemoattractant protein-1 (MCP-1) mRNA and nuclear factor-kappa B (NF-kappaB)-DNA binding activity were determined in human umbilical vein endothelial cells (HUVEC) incubated with native or glycoxidized LDL, LDL modified by phospholipase A2 (PLA2) and LDL isolated from diabetic patients. Lyso-PC contents in LDL were measured using electrospray ionization-liquid chromatography/mass spectrometry (ESI-LC/MS). Lyso-PC contents were higher in glycoxidized LDL and PLA2-treated LDL compared with native LDL. Glycoxidized LDL and enrichment of lyso-PC by PLA2 treatment resulted in upregulation of MCP-1 mRNA expression through increased NF-kappaB activity in HUVEC. Moreover, LDL isolated from diabetics contained more lyso-PC than that from nondiabetic subjects, and induced higher MCP-1 mRNA expression and NF-kappaB activity in HUVEC. In both in vitro and human studies, palmitoyl- and stearoyl-lyso-PC contents correlated with MCP-1 expression and NF-kappaB activity. Preincubation with 4-ethyl-2-hydroxyimino-5-nitro-3-hexenamide, a NO donor, abrogated increased expression of MCP-1 mRNA and high NF-kappaB activity induced by PLA2-treated LDL and by LDL isolated from diabetic patients. Our results suggest that lyso-PC contents in LDL play an important role in atherogenesis under diabetic condition, which could be prevented by increased availability of vascular NO.     

Reactive oxygen species activate mitogen-activated protein kinases in pancreatic acinar cells

It has been recently reported that kinases that belong to the mitogen-activated protein kinase (MAPK) family are rapidly activated by cholecystokinin (CCK) in rat pancreas both in vitro and in vivo. It is known that reactive oxygen species (ROS) play an important role in the pathogenesis of acute pancreatitis induced by supraphysiologic stimulation with CCK analogue, cerulein. The aim of our study was to evaluate whether MAPKs are activated by ROS in pancreatic acini. The activity of MAPK, c-Jun amino-terminal kinase (JNK), and p38 MAPK was determined in isolated rat pancreatic acinar cells by means of Western blotting, with the use of specific antibody that recognizes active, dually phosphorylated kinases. Incubation of acini with ROS donors, hydrogen peroxide (H2O2) and/or menadione (MND), strongly activated all three kinases. Activation of these kinases by ROS, but not by CCK, was substantially inhibited by pretreatment of acini with antioxidant N-acetylo-L-cysteine (NAC). Whereas CCK-induced activation of MAPK or JNK was totally or partially blocked by protein kinase C (PKC) inhibitor GF-109203X, ROS-induced activation of MAPK, JNK, and p38 MAPK was PKC independent. In conclusion, ROS strongly activate MAPK, JNK, and p38 MAPK in pancreatic acinar cells. It may be of importance in acute pancreatitis, because ROS are involved in the pathogenesis of this disease.     

Cellular immune reaction in the pancreas is induced by constitutively active IkappaB kinase-2

BACKGROUND: Activation of the nuclear factor kappaB (NF-kappaB) system is a major event in acute and chronic inflammatory processes. NF-kappaB cascades are comprised of IkappaB kinases, IkappaBs and NF-kappaB dimers. Little is known of the individual roles of these proteins in organ specific inflammation. The aim of the present study was to analyse the consequences of ectopic IkappaB kinase-2 (IKK2) activation in the pancreas of mice. METHODS: Transgenic mice were generated using an inducible genetic system (tet system) to conditionally overexpress a gain of function mutant of IKK2 (tetO-IKK2-EE) in the pancreas. To achieve transgene expression in the pancreas, these animals were crossed with CMV-rtTA mice that are known to express the rtTA protein in the pancreas. RESULTS: In these double transgenic animals, doxycycline treatment induced expression of IKK2-EE (IKK2(CA)) in pancreatic acinar cells resulting in moderate activation of the IkappaB kinase complex, as measured by the immune complex kinase assay, and up to 200-fold activation of the transgene expression cassette, as detected by luciferase assay. IKK2(CA) expression in the pancreas had a mosaic appearance. Ectopic IKK2(CA) mostly activated the classical NF-kappaB pathway. The activation level of the NF-kappaB cascade induced by IKK2(CA) was considerably lower compared with that observed after supramaximal caerulein stimulation but still led to the formation of leucocyte infiltrates first observed after 4 weeks of doxycycline stimulation with a maximum after 8-12 weeks. The infiltrates were mainly composed of B lymphocytes and macrophages. Increased mRNA levels of tumour necrosis factor alpha and RANTES were detected in pancreatic acinar cells. However, only minor damage to pancreatic tissue was observed. A combination of supramaximal caerulein stimulation with induction of IKK2(CA) caused increased tissue damage compared with either IKK2(CA) or caerulein alone. CONCLUSIONS: Our observations suggest that the role of IKK2 activation in pancreatic acini is to induce leucocyte infiltration, but at a moderate level of activation it is not sufficient to induce pancreatic damage in mice. The IKK2(CA) induced infiltrations resemble those observed in autoimmune pancreatitis, indicating a role for IKK2/NF-kappaB in this disease. IKK2(CA) in pancreatic acinar cells increases tissue damage of secretagogue induced experimental pancreatitis underlining the proinflammatory role of the IKK/NF-kappaB pathway in this disease.     

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

BACKGROUND & AIMS: Tumor necrosis factor (TNF)-alpha contributes to the development of acute pancreatitis. Because TNF-alpha 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-alpha induced apoptosis in AR4-2J cells. Induction was strongly enhanced in cells treated with actinomycin D, suggesting that TNF-alpha activated concomitantly an antiapoptotic mechanism through newly synthesized proteins. This mechanism involved activation of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein (MAP) kinases because their inhibition worsened TNF-alpha-induced apoptosis. The antiapoptotic pancreatitis-associated protein (PAP) I is a candidate for mediating TNF-alpha activity. Its expression is induced by TNF-alpha, and cells overexpressing PAP I show significantly less apoptosis on exposure to TNF-alpha. We examined whether TNF-alpha induction of PAP I expression was mediated by NF-kappaB or MAP kinases by using specific inhibitors of both pathways. Inhibition of NF-kappaB had no effect. However, inhibitors of MEK1 eliminated PAP I induction. CONCLUSIONS: TNF-alpha induces concomitantly proapoptotic and antiapoptotic mechanisms in pancreatic AR4-2J cells. Antiapoptotic mechanisms are mediated by NF-kappaB and MAP kinases, and PAP I is one of the effectors of apoptosis inhibition.