Abstract
A common reaction to xenobiotic exposure is the modulation of genes that mediate the response to such insult, including the drug metabolising enzymes and drug transporters. This transcriptional response is typically mediated by the nuclear receptor family of ligand activated transcription factors, and the translocation of these key regulators into the nucleus, whether before or after ligand binding, is a pre-requisite for their activity. The karyopherin a family of adapter proteins (6 in man) form a molecular bridge between nuclear cargoes and the nuclear import machinery. Previous work demonstrated that the rat karyopherin a genes are themselves responsive to a number of xenobiotics, including classical nuclear receptor ligands. The aim of this study was to delineate the molecular mechanism underlying transcriptional regulation of human karyopherin a2 (KPNA2) gene. In silico analysis of the 2.6kb immediately upstream and including the first exon of human KPNA2 promoter revealed several putative transcription factor binding sites, most notably for the nuclear receptors vitamin D receptor (VDR), progesterone receptor (PR), retinoid related orphan receptor a (RORa), peroxisome proliferated activated receptor a (PPARa), glucocorticoid receptor a (GRa), pregnane X receptor (PXR) and hepatic nuclear factor 4 (HNF4) and the anti-oxidant response protein, Nrf2. Using a reporter gene assay in the human hepatoma cell line Huh7, preliminary experiments demonstrated that the KPNA2 gene was responsive to cognate ligands for many of these nuclear receptors, as well as oxidative stress-mediated activation of Nrf2. In-depth analysis including promoter deletion, site directed mutagenesis and electrophoretic mobility shift assay were carried out to determine the molecular mechanism underlying these transcriptional effect. The down-regulation of human KPNA2 expression by cyproterone acetate was mediated via GRa and not PXR whereas the down-regulation of human KPNA2 expression by Wy-14,643 was elicited not by its cognate nuclear receptor, PPARa but most likely via Nrf2 through the oxidative stress signaling pathway. In conclusion, these findings provide a rational explanation for the mechanistic basis of human KPNA2 gene regulation by a number of endobiotics and xenobiotics, and postulate the importance of this phenomenon for optimizing cellular response to these stimuli.