Protein Kinase C Activity Is Required for Aryl Hydrocarbon Receptor Pathway-Mediated Signal Transduction

Abstract

The role of protein kinase C (PKC) in the human aryl hydrocarbon receptor (hAhR) signal transduction pathway was examined in cell lines stably transfected with pGUDLUC6.1, in which luc ϩ is solely controlled by four dioxin-responsive elements (DREs). These cell lines, P5A11 and HG40/6, were derived from HeLa and HepG2 cells respectively. Simultaneous treatment of these cells with 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) and phorbol-12-myristate-13-acetate (PMA) enhanced transactivation 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 activity. Examination of other independently generated, HeLa-derived cell lines 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 stabilization 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 downregulation of hAhR levels. Similarly, examination of nuclear extracts indicated PKC stimulation or inhibition does not alter nuclear AhR levels or hAhR/hAhR nuclear translocator protein heterodimer DRE-binding activity as assessed by electrophoretic mobility shift assay. These results demonstrate a PKCmediated event is required for the hAhR to form a functional transcriptional complex that leads to trans-activation and that the DRE is the minimal DNA element required for PMA to enhance AhR-mediated trans-activation.

Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the FrogXenopus Laevis: Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

“…Cells were seeded in 48-well plates at 2 Â 10 4 cells/well and cultured overnight. Cells were transiently transfected with the reporter construct pGudLuc6.1, which is driven by a region of the mouse CYP1A1 enhancer containing 4 xenobiotic response elements (XREs; Long et al, 1998). Transfection was carried out with Lipofectamine 2000 (Invitrogen).…”

“…To further interrogate the function of FICZ as an AHR1a agonist for CYP1A induction, we monitored reporter gene induction by each AHR following 18-h exposure to 100 nM FICZ. In this heterologous context, AHR1a readily mediated transcription of luciferase expression in pGudLuc6.1, driven by the mouse CYP1A1 enhancer (Long et al, 1998), in the absence of AHR1b Figure 7). Thus, endogenous enhancer elements and a native chromatin context, lacking in the transfected reporter gene, likely play an important role in the distinct activities of the paralogous frog AHRs.…”

The Aryl Hydrocarbon Receptor Complex and the Control of Gene Expression

“…333,334 Indeed, studies suggest that AhR transcriptional activity is repressed on inhibition of PKC activity as measured in cell-based DRE-driven luciferase reporter studies. 335 However, there are also other possible mechanisms that may explain the ability of PMA to repress CYP1A1, and that is through its ability to induce an immediate early gene response. PMA is able to dramatically induce AP-1 activity both through the transcriptional upregulation of the Fos and Jun genes and direct phosphorylation of these transcription factors.…”

Functional properties of the four Atlantic salmon (Salmo salar) aryl hydrocarbon receptor type 2 (AHR2) isoforms

“…The rainbow trout AHR2 α and β cDNAs were subcloned into the pcDNA 3.1 expression vector using EcoRI/NotI. The plasmid pGudLuc 6.1 containing the firefly luciferase reporter under the control of an MMTV promoter regulated by four AHREs from the murine CYP1A1 promoter (Long et al 1998), was a gift from Dr. M. Denison (University of California, Davis, CA, USA). The rainbow trout ARNTb was a gift from Dr. Pollenz (University of South Florida, FL, USA).…”