Low concentrations of nitric oxide (NO) induced cell death in PC12 cells through activation of p38 mitogen-activated protein kinase (p38 MAPK) but not via extracellular signal-regulated kinases (ERK1/2) or c-Jun N-terminal protein kinase (JNK)

Nitric oxide (NO), a highly reactive gaseous molecule, has been previously reported to induce apoptosis-like cell death even at a low concentration in PC12 cells. In this study, we examined NO-induced activation of members of the mitogen-activated protein kinase (MAPK) family, i.e., p38 MAPK, extracellular signal-regulated kinases (ERK1/2), and c-Jun N-terminal protein kinase (JNK). Following the exposure of PC12 cells to an NO donor, (+)-(E)-4-ethyl-2-[hydroxyimino]-5-nitro-3-hexenamide (NOR3; 100 muM), the phosphorylation level of p38 MAPK increased time dependently from 2 to 6 h, but that of both ERK1/2 and JNK did not. Treatment with a p38 MAPK inhibitor SB203580 partially blocked the NOR3-induced cell death. Neither PD98059, U0126 (inhibitors of ERK1/2) nor SP600125 (a specific inhibitor of JNK) treatments had any significant effect on the NOR3-induced cell death. These findings suggest that the activation of a p38 MAPK pathway, but not that of ERK1/2 or JNK, plays an essential role in the apoptosis-like cell death induced by low concentrations of NO.     

Secretory component is cleaved by neutrophil serine proteinases but its epithelial production is increased by neutrophils through NF-kappa B- and p38 mitogen-activated protein kinase-dependent mechanisms

We previously showed that expression of polymeric immunoglobulin receptor (pIgR)/secretory component (SC), the epithelial receptor assuming transport of polymeric IgA in mucosal secretions, is strongly decreased in severe chronic obstructive pulmonary disease. Here, we evaluated in vitro the effects of polymorphonuclear neutrophil (PMN) mediators on pIgR/SC. On polyacrylamide gel electrophoresis analysis, soluble SC was rapidly cleaved by supernatants from phorbol-myristate-acetate-activated PMN, through a serine proteinase activity. Moreover, purified PMN serine proteinases also cleaved SC. Similarly, polymeric IgA was rapidly cleaved in monomers by neutrophil elastase, whereas secretory immunoglobulin A was relatively resistant to neutrophil elastase. Surface pIgR on human bronchial epithelial cells was also cleaved by serine proteinases, as shown by immunofluorescence. In contrast, pIgR/SC production by cultured epithelial cells (quantified by enzyme-linked immunosorbent assay) was significantly increased by supernatants from interleukin-8/formylmethionylleucylphenylalanine-activated PMN (122.6 +/- 17.3 versus 70.9 +/- 9 ng/mg protein, P < 0.01). Upregulation of pIgR/SC production by bronchial epithelial cells was abolished by nuclear factor kappa B- and p38 mitogen-activated protein kinase (MAPK) inhibitors. Moreover, supernatants from interleukin-8/formylmethionylleucylphenylalanine-activated PMN induced the phosphorylation of I kappa B-alpha and p38 MAPK in epithelial cells, independently of serine proteinases. Thus, PMN serine proteinases cleave pIgR/SC, whereas activated PMN induce an increased pIgR/SC expression through epithelial activation of nuclear factor kappa B and p38 MAPK pathways.     

Retinoic acids acting through retinoid receptors protect hippocampal neurons from oxygen-glucose deprivation-mediated cell death by inhibition of c-jun-N-terminal kinase and p38 mitogen-activated protein kinase

Retinoic acids (RAs), including all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), play fundamental roles in a variety of physiological events in vertebrates, through their specific nuclear receptors: retinoic acid receptor (RAR) and retinoid X receptor (RXR). Despite the physiological importance of RA, their functional significance under pathological conditions is not well understood. We examined the effect of ATRA on oxygen/glucose-deprivation/reperfusion (OGD/Rep)-induced neuronal damage in cultured rat hippocampal slices, and found that ATRA significantly reduced neuronal death. The cytoprotective effect of ATRA was observed not only in cornu ammonis (CA) 1 but also in CA2 and dentate gyrus (DG), and was attenuated by selective antagonists for RAR or RXR. By contrast, in the CA3 region, no protective effects of ATRA were observed. The OGD/Rep also increased phosphorylated forms of c-jun-N-terminal kinase (P-JNK) and p38 (P-p38) in hippocampus, and specific inhibitors for these kinases protected neurons. ATRA prevented the increases in P-JNK and P-p38 after OGD/Rep, as well as the decrease in NeuN and its shrinkage, all of which were inhibited by antagonists for RAR or RXR. These findings suggest that the ATRA signaling via retinoid receptors results in the inhibition of JNK and p38 activation, leading to the protection of neurons against OGD/Rep-induced damage in the rat hippocampus.     

Activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) during hypoxia in cerebral cortical nuclei of guinea pig fetus at term: role of nitric oxide

Previously we have shown that cerebral tissue hypoxia results in generation of nitric oxide (NO) free radicals as well as increased expression of mitogen-activated protein kinase like extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK). The present study tested the hypothesis that administration of l-nitro-l-arginine methyl ester (L-NAME), a NOS inhibitor, prior to hypoxia prevents the hypoxia-induced activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) and in the cerebral cortex of the term guinea pig fetus. To test this hypothesis normoxic (Nx, n=6), hypoxic (Hx, n=7) and hypoxic pretreated with l-NAME (Hx+L-NAME, n=6) guinea pig fetuses at 60 days gestation were studied to determine the phosphorylated p38, ERK and JNK. Hypoxia was induced by exposing pregnant guinea pigs to FiO2 of 0.07 for 1h. l-NAME (30mg/kg i.p.) was administered to pregnant mothers 60min prior to hypoxia. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Neuronal nuclei were isolated, purified and proteins separated using 12% SDS-PAGE, and then probed with specific phosphorylated ERK, JNK and p38 antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by imaging densitometry and expressed as absorbance (ODxmm2). The relative level of p-p38 was 51.41+/-9.80 (Nx), 173.67+/-3.63 (Hx), 58.56+/-3.40 (Hx+L-NAME), p<0.05 vs. Hx. The relative level p-ERK was 44.91+/-4.20 (Nx), 135.12+/-17.02 (Hx), 58.37+/-9.5 (Hx+L-NAME), p<0.05 vs. Hx. The relative level of p-JNK was 34.86+/-6.77 (Nx), 97.36+/-19.24 (Hx), 46.65+/-12.81 (Hx+L-NAME), p<0.05 vs. Hx. The data show that administration of l-NAME prior to hypoxia decreased the relative level of phosphorylated p38, ERK and JNK at term gestation. Since a NOS inhibitor prevented the hypoxia-induced phosphorylation of p38, ERK and JNK, we conclude that the hypoxia-induced activation of p38, ERK and JNK in the cerebral cortical nuclei of guinea pig fetus at term is NO-mediated. We speculate that NO-mediated modification of cysteine residue leading to inhibition of MAP kinase phosphatases results in increased activation of p38, ERK and JNK in the guinea pig fetus at term.     

Inhibition of human rheumatoid arthritis synovial cell survival by hecogenin and tigogenin is associated with increased apoptosis, p38 mitogen-activated protein kinase activity and upregulation of cyclooxygenase-2

We conducted our study to assess the antiproliferative and proapoptotic potential of hecogenin and tigogenin, two saponins which are structurally similar to diosgenin. We particularly focused our attention on mitogen-activated protein kinase (MAPK) activation in relation to apoptosis but also with the COX-2 expression and activity. Rheumatoid arthritis (RA) synoviocytes were isolated from fresh synovial biopsies obtained from five RA patients undergoing hip arthroplasty. Measurement of cell proliferation was determined using the MTT assay. Apoptosis was evaluated by studying caspase-8, caspase-9 and caspase-3 activities but also by quantification of DNA fragmentation. Quantification of human phospho-MAPKs was realized by ELISA. COX-2 expression was demonstrated by Western blot analysis and COX-2 activity by assay of endogenous prostaglandin E2 (PGE2) production. Tigogenin was more effective than hecogenin in inducing apoptosis in human RA fibroblast-like synoviocytes (FLS) which was caspase dependent but poly(ADP-ribose) polymerase independent and characterized by DNA fragmentation. Our results demonstrated hecogenin- and tigogenin-induced apoptosis through activation of p38 without affecting the JNK and ERK pathways. Indeed, pretreatment with a p38 inhibitor decreased saponin-induced apoptosis with a significant decrease in DNA fragmentation. Furthermore, the rate of apoptosis induced by hecogenin or tigogenin was associated with overexpression of COX-2 correlated with overproduction of endogenous PGE2. These new results provide strong evidence that a family of structurally similar plant steroids is capable of inducing apoptosis in human RA FLS with different rates and different signalling pathways. This study also confirms the discussed appearance of the downregulation or upregulation of COX-2 in cell apoptosis as a function of cell type.     

Overexpression of cyclo-oxygenase-2 is an independent predictor of unfavourable outcome in node-negative breast cancer, but is not associated with protein kinase B (Akt) and mitogen-activated protein kinase (ERK1/2, p38) activation or with Her-2/neu signalling pathways

BACKGROUND AND AIM: The production of prostaglandins is regulated by cyclo-oxygenases (COXs), which also have a role in tumour development and progression in various malignancies, including breast cancer. The mechanisms by which COX-2 contributes to unfavourable prognosis are still poorly understood. The association between expression of COX-2 and possible linked signalling pathways-namely, Akt, extracellular regulated kinases (ERK1/2), the stress-activated kinase p38 or Her-2/neu-is assessed in a series of 113 node-negative breast cancers. RESULTS: COX-2 was identified as an independent prognostic factor (p = 0.034) in node-negative breast cancer by survival analysis. The lack of a relationship between COX-2 expression and activated Akt, Erk1/2, p38 and Her-2/neu was indicated by statistical analysis. CONCLUSIONS: The prognostic effect of COX-2 expression on lymph node-negative breast cancer is confirmed-COX-2 is probably not regulated by HER-2, Akt, Erk1/2 or p38. Further studies are necessary for the elucidation of the signalling pathways responsible for the modification of COX-2 expression and the increased aggressiveness of breast cancers overexpressing COX-2.