Activation of C-jun N-terminal kinase/stress-activated protein kinase in primary glial cultures.
Glial cells in the mammalian CNS are subject to environmental stress resulting from a variety of neuro-pathological conditions. In this study, we have examined the activation of a stress signal responsive kinase, i.e., stress-activated protein kinase (SAPK) or c-Jun N-terminal kinase (JNK), in primary cultures of rat brain glial cells (i.e., astrocytes and oligodendrocytes) and an oligodendrocyte progenitor cell line, CG4, in response to cytokines and other stress inducers. JNK/SAPK activity was measured by an immune complex kinase assay using polyclonal anti-JNK antibodies along with GST c-Jun (1-79) as the substrate. Among the cytokines tested, TNF-alpha had the strongest effect on JNK activation followed by TNF-beta in both the glial cell types while a substantial level of kinase activation was observed in response to IL-1 in astrocytes. JNK activation by TNF-alpha in astrocytes, but not in oligodendrocytes, showed a biphasic response. An in-gel kinase assay of cell extracts and immunoprecipitated JNK confirmed the activation of JNK1 in cells treated with TNF-alpha. JNK was also activated by several other stress-inducing factors including. UV light, heat shock, inhibitors of protein synthesis, and mechanical injury. Incubation of cells with bacterial sphingomyelinase and a cell-permeable ceramide stimulated JNK activity, suggesting that the ceramide pathway may play a role in JNK activation, although the time course of activation did not correspond to that of TNF-alpha. The results are discussed in terms of possible roles of JNK activation in signaling for gliosis in astrocytes and as a protective/toxic response in oligodendrocytes.
Department of Neurology, Medical University of South Carolina, Charleston 29425, USA.
P
P
Zhang
B S
BS
Miller
S A
SA
Rosenzweig
N R
NR
Bhat
D000818:Animals; D001253:Astrocytes / Q000201:enzymology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000502:physiology; D005347:Fibroblasts / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D009457:Neuroglia / Q000201:enzymology*; D009836:Oligodendroglia / Q000201:enzymology; D051381:Rats; D013108:Sphingomyelin Phosphodiesterase; D013234:Stem Cells / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016207:Cytokines; D014409:Tumor Necrosis Factor-alpha; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D013108:Sphingomyelin Phosphodiesterase