Neuronal survival activity of s100betabeta is enhanced by calcineurin inhibitors and requires activation of NF-kappaB.

S100betabeta is a calcium binding, neurotrophic protein produced by nonneuronal cells in the nervous system. The pathway by which it enhances neuronal survival is unknown. Here we show that S100betabeta enhances survival of embryonic chick forebrain neurons in a dose-dependent manner. In the presence of suboptimal amounts of S100betabeta, neuronal survival is enhanced by the immunosuppressants FK506 and cyclosporin A at concentrations that inhibit calcineurin, which is present in these cells. Rapamycin, an immunosuppressant that does not inhibit calcineurin, did not enhance cell survival. Cypermethrin, a direct and highly specific calcineurin inhibitor, mimicked the immunophilin ligands in its neurotrophic effect. None of the drugs stimulated neuronal survival in the absence of S100betabeta. In the presence of suboptimal amounts of S100betabeta, FK506, cyclosporin A, and cypermethrin (but not rapamycin) also increased NF-kappaB activity, as measured by immunofluorescence of cells stained with antibody to the active subunit (p65) and by immunoblotting of nuclear extracts. Antioxidant and glucocorticoid inhibitors of NF-kappaB decreased both the amount of active NF-kappaB and the survival of neurons caused by S100betabeta alone or in the presence of augmenting drugs. We conclude that S100betabeta enhances the survival of chick embryo forebrain neurons through the activation of NF-kappaB.     

Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity

We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1beta (IL-1beta) led to ROI production and NF-kappaB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-kappaB activation in these cells. IL-1beta stimulation of epithelial cells did not generate any ROIs and NF-kappaB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-kappaB activation. In monocytic cells, IL-1beta treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-kappaB activation by IL-1beta in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-kappaB activation by IL-1beta: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells.     

ASC-mediated NF-kappaB activation leading to interleukin-8 production requires caspase-8 and is inhibited by CLARP

ASC is an adaptor molecule that mediates apoptotic and inflammatory signals from several Apaf-1-like molecules, including CARD12/Ipaf, cryopyrin/PYPAF1, PYPAF5, PYPAF7, and NALP1. To characterize the signaling pathway mediated by ASC, we established cell lines in which muramyl dipeptide, the bacterial component recognized by another Apaf-1-like molecule, Nod2, induced an interaction between a CARD12-Nod2 chimeric protein and ASC, and elicited cell autonomous NF-kappaB activation. This response required caspase-8, and was suppressed by CLARP/FLIP, an inhibitor of caspase-8. The catalytic activity of caspase-8 was required for the ASC-mediated NF-kappaB activation when caspase-8 was expressed at an endogenous level, although it was not essential when caspase-8 was overexpressed. In contrast, FADD, the adaptor protein linking Fas and caspase-8, was not required for this response. Consistently, ASC recruited caspase-8 and CLARP but not FADD and Nod2 to its speck-like aggregates in cells. Finally, muramyl dipeptide induced interleukin-8 production in MAIL8 cells. These results are the first to indicate that caspase-8 plays an important role in the ASC-mediated NF-kappaB activation, and that the ASC-mediated NF-kappaB activation actually induces physiologically relevant gene expression.