Mechanisms of regulation of dioxin receptor function

Abstract: The dioxin receptor is a ligand-inducible transcription factor that belongs to the basic helix-loop- helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors. The dioxin receptor is regulated in a spatial manner, where the unliganded dioxin receptor resides in the cell cytoplasm in a complex with heat shock protein 90 (hsp90). Upon exposure to ligand, the receptor translocates to the nucleus where it binds to target sequences on DNA as a heterodimer with the partner bHLH/PAS factor Arnt. These heterodimeric factors upregulate transcription of target genes. Upon exposure to ligand the dioxin receptor protein, but not Arnt, becomes destabilized by enhanced ubiquitination and degradation by the 26S proteasome. In the present work, we have used a mammalian "hybrid interaction system" to study the ability of the dioxin receptor to interact with its partner protein Arnt via the PAS domain. This was done by replacement of the DNA-binding bHLH-motif of the dioxin receptor or Arnt with a heterologous DNA-binding domain. Using this approach, we could detect heterodimeric, but not homodimeric interactions mediated by the PAS domains of Arnt and the dioxin receptor. In addition, the PAS domain of Drosophila Period (Per) protein was able to interact with both the dioxin receptor and Arnt and thereby disrupt DNA-binding and abolish dioxin activity/ signalling on a reporter gene. Dioxin signalling is mediated by the dioxin receptor and Arnt, and one of the most studied responses to dioxins in intact cells is upregulation of genes encoding drug metabolizing enzymes such as Cythochrome P4501A1 and 1A2. Another effect of dioxin-exposure in intact cells is down- regulation of gap junction-mediated cell communication. We are here able to show that this effect is due to down-regulation of Connexin 43 (Cx 43) mRNA-levels in liver epithelial IAR 20 cells. As a consequence of ligand-induced activation of the dioxin receptor, the half-life of the receptor is drastically reduced. This effect is due to increased ubiquitination and subsequent degradation by the 26S proteasome. We show here that degradation of the receptor is due to Arnt-dependent disruption of the interaction between the dioxin receptor and hsp90. We also describe the varying kinetics of dioxin receptor degradation, induced by representatives of two distinct classes of ligands that bind to the dioxin receptor with high affinity. Degradation of the dioxin receptor induced by the metabolically unstable ligand indolo[3,2-b]carbazol, was more transient as compared with the stable ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Moreover, cells became refractory to further induction by ligand after a prolonged period of treatment with TCDD. This suggests that dioxin signalling is also regulated at the level of dioxin receptor protein stability.