An investigation into the ligand dependent regulation of the nuclear receptor PXR (NR1I2) and its target genes.

The study of absorption, distribution, metabolism and excretion (ADME) of compounds within the body is of vital importance to health. PXR is a nuclear receptor which regulates the expression of a great many ADME genes and is responsible for the regulation of all parts of the ADME of many compounds. It is believed that the nuclear receptors, including PXR, activate genes on a ligand dependent basis, a mechanism which is probably regulated by the recruitment of a different battery of co-regulator proteins. The ligand dependent selection of genes by recruitment of alternate co-regulator complexes has not yet been directly demonstrated. The co-regulator proteins of PXR are not well characterised although co-regulator complexes of other receptors, including the oestrogen receptor, growth hormone receptor and retinoic acid receptor are comparatively well characterised. In order to demonstrate that different co-regulator complexes can be recruited by different ligands of PXR is was first necessary to identify the co-regulator complexes of PXR. An immunoprecipitation assay was developed and used in conjunction with an half GFP interaction assay as validation. The immunoprecipitated samples gave interesting data by mass spectrometry although it was not possible to validate these results using the half GFP interaction assay. This thesis contributes significantly to the effort to identify PXR co-regulator proteins although further investigation using alternate techniques may yield more information. Conclusive demonstration of a differential gene expression by different ligands came in the form of a gene array based on primary rat hepatocytes dosed with pregnenalone-16?-carbonitrile (PCN) and lithocholic acid (LCA). Appropriate dose ranges of these compounds were established in a separate, pilot, study and were within appropriate literature dose ranges. This gene array generated some 2000+ significantly altered gene expression profiles, as determined by statistical analysis of the data. Venn diagram analysis shows a clear difference between PCN and LCA with a number of commonly and individually expressed genes. A number of significantly altered genes were identified by principal components analysis as being important drivers of difference between the effects of PCN and LCA. Taqman QPCR analysis of these genes gave strong evidence that these two compounds are exerting different transcriptional effects