Cytokine gene expression by central nervous system glia: Transcriptional activation of the TNFalpha and MuRantes genes by virus

Abstract

In this work, the signalling pathways regulating transcriptional activation of cytokine genes, specifically TNF{dollar}\alpha{dollar} and RANTES, in rat astrocytes by Newcastle Disease Virus (NDV) were explored and compared with those induced by LPS. Tyrosine kinase activation by NDV was directly demonstrated by a 2 to 3-fold transient increase in tyrosine phosphorylation of PLC-{dollar}\gamma{dollar}1. TNF{dollar}\alpha{dollar} gene transcription induced by NDV or the synergistic combination of LPS and IFN-{dollar}\gamma{dollar} was completely abolished by tyrosine kinase inhibitors while protein kinase C (PKC) inhibitors reduced transcription 40-50% for both stimuli. These results indicated that the TNF{dollar}\alpha{dollar} gene promoter has at least two virus and LPS inducible transcriptional elements, both of which are tyrosine kinase dependent and the other/s of which are also PKC dependent, such as NF-kB binding elements. While transcriptional activation of the TNF{dollar}\alpha{dollar} gene by both NDV and LPS involve PKC activation, only NDV induced PKC dependent stabilization of the TNF mRNA, making NDV a more potent inducer of mRNA expression. Although LPS does not efficiently induce TNF{dollar}\alpha{dollar} mRNA in astrocytes, MuRantes mRNA is potently induced by low doses of LPS and by NDV. MuRantes was induced by both LPS and NDV in astrocytes as an immediate early gene, and in a delayed response by TNF{dollar}\alpha{dollar}. NDV also induced MuRantes mRNA in microglia. Transcriptional activation of the MuRantes gene in astrocytes by NDV or LPS was similarly blocked by inhibition of tyrosine kinase or PKC activities, indicating overlapping signalling requirements for transcriptional activation. Activation of MuRantes transcription induced by LPS was further studied in the macrophage cell line RAW 264.7, where the MuRantes promoter minimal LPS response element (LRE) was previously characterized. Binding of nuclear proteins from LPS stimulated cells to LRE probe increased over 60 min as examined by electrophoretic mobility shift assay. Complex formation on the probe was inhibited by antibodies to CREB and c-Jun. SDS-Page analysis of complexes UV-cross linked to LRE probe revealed proteins of 38, 43, and 80 kD. The 38 and 43 kD proteins correspond to the molecular weights of c-Jun and CREB while the 80 kD protein is unidentified. Current efforts are focused on purification of the 80 kD protein.