Signaling via the bHLH-PAS proteins AhR and HIF

Abstract

This thesis concerns some mechanistic properties of the basic helix-loop-helix/PAS (bHLH-PAS) factors aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1 alpha (HIF-1α). The bHLH-PAS family of proteins is a family of factors that controls a variety of developmental and physiological events. A common feature for this family of proteins is that they act mainly as intracellular transcription factors. They bind to DNA as a heterodimeric complex, usually together with a bHLH-PAS protein belonging to the aryl hydrocarbon receptor nuclear translocator (ARNT) subfamily. The AhR bind ligands that are environmental pollutants, as well as possibly physiological compounds occurring in the diet. Known functions of the ligand-activated AhR include activation of genes involved in xenobiotic metabolism and an ubiquitin ligase activity targeting nuclear receptors (such as the estrogen receptor) and beta-catenin. HIF-1α mediates signal transduction and gene regulation in cells exposed to deprived oxygen conditions (hypoxia). In paper I, we have shown that the Ah-receptor can be activated by stimulus other than xenobiotics, e.g dioxin. AhR is recruited to target genes in both ligand treated and in suspension culture, suggesting a common mechanism of activation between these two routes of AhR activation. The gene expression profiles critically differ between xenobiotic and suspension activated AhR signaling. The classical xenobiotic metabolizing AhR targets such as Cyp 1a1, Cyp 1b1 and Nqo were regulated by both ligand and suspension conditions. Sequence analysis coupled with ChIP assays and reporter gene analysis identified a functional xenobiotic response element (XRE) within the mouse TIPARP gene that features a concatamer of 4 XRE cores residing in the first intron. In paper II we have shown that ectopic expression of ARNT, in mammalian cells and yeast cells, was sufficient to promote nuclear accumulation of the Ah-receptor in a ligand-independent manner. We further observed that overexpression of ARNT promotes derepression of Ah-receptor function in the absence of ligand, thereby possibly representing an alternative mechanism of activation that is distinct from activation by xenobiotic ligands and thus may be of physiological relevance. We also describe that an excess of ARNT in relation to the Ah-receptor and HIF-1α promotes derepression of the receptor and stabilization of HIF-1α in vivo and in vitro, representing a possible alternative mechanism of activation of bHLH-PAS proteins