PROTEIN-KINASE-C ACTIVITY IS REQUIRED FOR ARYL-HYDROCARBON RECEPTOR PATHWAY-MEDIATED SIGNAL-TRANSDUCTION

The role of protein kinase C (PKC) in the human aryl hydrocarbon recep tor (hAhR) signal transduction pathway was examined in cell lines stab ly transfected with pGUDLUC6.1, in which luc(+) is solely controlled b y four dioxin-responsive elements (DREs). These cell lines, P5A11 and HG40/6, were derived from HeLa and HepG2 cells respectively. Simultane ous treatment of these cells with 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) and phorbol-12-myristate-13-acetate (PMA) enhanced transactiva tion of the reporter construct several-fold relative to cells treated with TCDD alone. PKC inhibitors block the PMA effect and hAhR-mediated signal transduction, demonstrating these processes require PKC activi ty. Examination of other independently generated, HeLa-derived cell li nes stably transfected with pGUDLUC6.1 demonstrates the PMA effect in P5A11 cells is not a clonal artifact. Transient transfections indicate the PMA effect is not due to a luciferase message/gene product stabil ization mechanism or stimulation of the basal transcription machinery. Examination of cytosolic preparations demonstrates PKC stimulation or inhibition does not alter hAhR and hAhR nuclear translocator protein levels or TCDD-induced down-regulation of hAhR levels. Similarly, exam ination of nuclear extracts indicated PKC stimulation or inhibition do es not alter nuclear AhR levels or hAhR/hAhR nuclear translocator prot ein heterodimer DRE-binding activity as assessed by electrophoretic mo bility shift assay. These results demonstrate a PKC-mediated event is required for the hAhR to form a functional transcriptional complex tha t leads to trans-activation and that the DRE is the minimal DNA elemen t required for PMA to enhance AhR-mediated trans-activation.