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Tumor progression locus 2 (Tpl2)/cancer Osaka thyroid kinase is a newer member of MAP3K family that is now known for its essential role in tumor necrosis factor-aplha (TNFα) expression in macrophages, but its pro-inflammatory signaling, if any, in glia is unknown. When cultures of murine microglia and astrocytes were exposed to lipopolysaccharide, there was a rapid activation (i.e., phosphorylation) of Tpl2 in parallel to the activation of down-stream effector MAPKs, that is, extracellular signal regulated kinase (ERK), p38 MAPK and C-Jun N-terminal kinase (JNK). Pre-incubation of the cultures with a Tpl2 inhibitor selectively suppressed the activation of the primary down-stream target, that is, ERK relative to p38 MAPK and JNK. That Tpl2 activation was functionally involved in glial inflammatory response was indicated by a reduced release of the cytokines, i.e. TNFα and the expression of inducible nitric oxide synthase in the presence of the kinase inhibitor. Furthermore, over-expression of a wild-type Tpl2 construct in C-6 glia resulted in an enhanced transcriptional activation of inducible nitric oxide synthase, while transfection with a dominant negative form of Tpl-2 had the opposite effect. The findings assign an important pro-inflammatory signaling function for Tpl2 pathway in glial cells. [ABSTRACT FROM AUTHOR]

Abstract Inflammatory cell signaling leading to transcriptional activation is primarily mediated by signal transduction via mitogen-activated protein kinase (MAPK) and NFκB pathways. A common upstream kinase that signals the activation of these pathways is TGFβ–activated kinase 1 (TAK1), which itself becomes activated in response to cytokines and upon engagement of a class of cell surface receptors involved in innate immunity, that is Toll-like receptors (TLRs) by bacterial and viral pathogens. This study directly tests the role of TAK1 in the induction of inducible nitric oxide (NO) synthase (iNOS) in glial cells, which represent immune-regulatory cells of the CNS, by transient transfection assays. Transfection of C-6 glia, primary astrocytes and a rat microglial cell line with TAK1 (but not its inactive form) along with its activator protein, TAK1-binding protein 1 (TAB1) resulted in a marked stimulation of a co-transfected rat iNOS promoter-reporter construct (iNOS-Luc). TAK1-induced iNOS-Luc activity was substantially inhibited by pharmacological inhibitors of the known downstream kinases, p38 MAPK and JNK (SB203580 and SP620125), and was almost completely blocked by co-expression of a phosphorylation mutant of IκB. TAK1/TAB1 also induced the production of NO and the expression of iNOS in microglial cells in a p38 MAPK-, JNK- and NFκB-dependent manner. The results of these studies provide evidence for an important role for TAK1-mediated intracellular signaling, via p38 MAPK, JNK and NFκB, in the transcriptional activation of iNOS in glial cells. [ABSTRACT FROM AUTHOR]

Abstract : The induction of inducible nitric oxide synthase (iNOS) by proinflammatory cytokines was studied in an oligodendrocyte progenitor cell line in relation to mitogen-activated protein kinase (MAPK) activation and cytokine-mediated cytotoxicity. When introduced individually to cultures of CG4 cells, the cytokines, i.e., tumor necrosis factor-α (TNFα), interleukin-1 (IL-1), and interferon-γ (IFNγ), had either minimal (TNFα) or no (IL-1 and IFNγ) detectable stimulatory effect on the production of nitric oxide. However, combinations of these factors, in particular, TNFα plus IFNγ, elicited a strong enhancement of nitric oxide synthesis and, as revealed by western blot and RT-PCR analysis, the expression of iNOS. TNFα and IL-1 were able to activate p38 MAPK in a time- and dose-dependent manner and together showed a combinatorial effect. In contrast, IFNγ neither activated on its own nor enhanced the activation of p38 MAPK in response to TNFα and IL-1. However, a specific inhibitor of p38 MAPK, i.e., SB203580, inhibited the induction of iNOS in cytokine combination-treated cells in a dose-dependent manner, thereby suggesting a role for the MAPK cascade in regulating the induction of iNOS gene expression in cytokine-treated cells. Blocking of nitric oxide production by an inhibitor of iNOS, i.e., nitro-L-arginine methyl ester, had a minimal protective effect against cytokine-mediated cytotoxicity that occurred before the elevation of nitric oxide levels, thereby indicating temporal and functional dissociation of nitric oxide production from cell killing. [ABSTRACT FROM AUTHOR]

The enzyme nitric oxide synthase 2 (NOS2, or inducible NOS) is expressed by numerous cell types during inflammatory reactions in vivo and in vitro. The NO derived from NOS2 exerts beneficial actions such as the elimination of microorganisms, the reduction of thrombosis, and the increase of blood supply to injured tissues. However, NO released in excess can cause tissue damage, contributing to diseases like septic shock, rheumatoid arthritis, cerebral ischemia, multiple sclerosis, and diabetes. It is well established that induction of the NOS2 protein by inflammatory stimuli is primarily regulated at the level of gene transcription, and that the interaction of the transcription factor NFkB and its inhibitory protein, IkB, play a central role in this process. In addition, recent pharmacological evidence implicates the mitogen activated protein kinase (MAPK) pathways as a second key mediator of the cytokine-induced transcription of NOS2. In turn, the induction of the NOS2 gene can be either inhibited or potentiated, depending on the cell type, by cyclic AMP-dependent pathways, acting via transcription factors like CREB, NFkB and C/EBP. Finally, the heat shock response has recently emerged as a second potent means of suppressing both the cytokine and endotoxin-dependent activation of NOS2 transcription. The mechanism responsible for this suppression appears to be the prevention of NFkB activation. These observations suggest that the transcription of the NOS2 gene is tightly regulated by several cascades of cytoplasmic and nuclear reactions, thus providing a wide array of biological and therapeutical targets to control the production of NO to avoid its detrimental actions. [ABSTRACT FROM AUTHOR]