TRANSCRIPTIONAL REGULATION OF THE JUNB PROMOTER - ANALYSIS OF STAT-MEDIATED SIGNAL-TRANSDUCTION

The product of the junB gene is a member of the AP-1 family of transcription factors that activate transcription by binding to TPA-responsive elements (TREs) within the promoters of target genes, Components of AP-1 are immediate-early genes whose expression is upregulated by a plethora of extracellular stimuli and are important in mediating cellular proliferation and differentiation, Such stimuli include the pleiotropic cytokine interleukin-6 (IL-6) which plays a role in immune and inflammatory responses and ciliary neurotrophic factor (CNTF) which enhances survival and differentiation of neurons and glia. We have analysed expression from junB promoter-CAT reporter constructs in HepG2 cells and found that a region between -196 and -91 can mediate response to IL-6 and CNTF and was able to confer responsiveness to a heterologous promoter, We further show by gel retardation analysis that distinct nuclear factors induced by IL-6 specifically bind to this interleukin-6 response element (IRE). This region contains both a putative ETS- and a STAT-transcription factor binding site, We show by mutational analysis and supershift data that the IL-6 induced complex indeed contains the transcription factor APRF/Stat3 that is both necessary and sufficient for activation, Interestingly this site does not appear to bind Stat1 itself, as shown by supershift analysis and a lack of response to IFN-gamma both at the DNA-binding and transcriptional level, Furthermore, we demonstrate that the junB IRE-binding activity induced by IL-6 requires tyrosine kinase activity, whereas induced transactivation of IRE-constructs additionally occurs through an H7-sensitive pathway that is p21ras-independent, implicating serine/threonine kinases in the transactivation of IRE-binding factors.