ABSTRACT
Expression of inducible heat shock protein (HSP70) requires activation of heat shock transcription factor-1 (HSF-1). Recent evidence suggests that interleukin-6 (IL-6) can modify the response of HSF-1 to heat. We hypothesized that IL-6 would prime the HSP response by causing de-repression of HSF-1 resulting in augmented HSP expression in stressed cells. In this study we show that IL-6 has no direct effect on HSP70 expression at 37 degrees C but does augment HSP70 expression in response to heat. IL-6 treatment decreased active MAPK/pERK and glycogen synthase kinase 3beta (GSK3beta) expression and GSK3beta kinase activity. In IL-6-treated cells, monomeric HSF-1 accumulated in the cytoplasm and nucleus, bound DNA but was transcriptionally inactive. On exposure to heat shock this modified monomer assumed the transcriptionally active phenotype with trimerization and hyperphosphorylation evident. The increased induction of HSP70 in IL-6 and heat-treated cells was inhibited using PI3-kinase inhibitors or Akt inhibition and was HSF-1 dependent. IL-6, via the PI3-kinase/Akt pathway leads to inhibition of the repressive kinases MAPK/pERK and GSK3beta, and this converts inactive HSF-1 to an intermediate DNA-binding form augmenting transcriptional activation in the presence of a second stressor.
Subject(s)
DNA-Binding Proteins/metabolism , Down-Regulation/drug effects , Glycogen Synthase Kinase 3/metabolism , Hepatocytes/drug effects , Hepatocytes/enzymology , Interleukin-6/pharmacology , Mitogen-Activated Protein Kinase 3/metabolism , Transcription Factors/metabolism , Androstadienes/pharmacology , Cell Nucleus/drug effects , Cells, Cultured , Chromones/pharmacology , Cytoplasm/drug effects , DNA-Binding Proteins/chemistry , Enzyme Activation/drug effects , Glycogen Synthase Kinase 3 beta , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Heat Shock Transcription Factors , Hepatocytes/cytology , Humans , Hyperthermia, Induced , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Protein Isoforms/metabolism , Protein Structure, Quaternary/drug effects , Protein Transport/drug effects , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/chemistry , Transcription, Genetic/drug effects , Up-Regulation/drug effects , WortmanninABSTRACT
In addition to various roles in membrane structure and metabolism, polyunsaturated fatty acids have effects on signal transduction and on the regulation of gene expression. Eicosapentaenoic acid (EPA) is an omega-3 fatty acid which is known to induce cell cycle arrest and apoptosis in pancreatic tumour cells. NFkappaB is a key transcription factor regulating genes involved in the immune response and has been implicated in apoptotic pathways. In this study we investigated the effect of eicosapentanoic acid on the NFkappaB pathway in pancreatic tumour cells. The pancreatic cell line MIA PaCa2 was incubated in the presence of the fatty acids EPA (n-3), arachidonic acid (AA, n-6) or oleic acid (OA, n-9) before pulsing with TNF to provide a kinetic assessment of NFkappaB activation and IkappaBalpha degradation. Pre-incubation of pancreatic cells with EPA or AA for 2 h before pulsing with TNF preserved IkappaBalpha but did not prevent NFkappaB activation. Indeed, NFkappaB activation was prolonged after exposure to EPA. N-acetyl-L-cysteine did not influence the effect of EPA on TNF-stimulated IkappaBalpha degradation. These results suggest that the omega-3 fatty acid EPA perturbs the NFkappaB pathway by a novel mechanism. This mechanism may be important in delineating alternative pathways to NFkappaB activation.