Abstract
The balance between cellular proliferation and apoptosis is a co-ordinated process. Rodent hepatocytes proliferate rapidly in response to non-genotoxic liver growth agents such as peroxisome proliferators, a result of both increased DNA synthesis and suppression of apoptosis (Roberts et al. , 1995). This study aimed to identify groups of genes that were co-regulated in the response of rodent liver to mitogenic stimuli, and used three compounds known to induce liver growth via different processes: cyproterone acetate (60 mg/kg/day), dexamethasone (100 mg/kg/day) and Wy-14,643 (50 mg/kg/day). Each compound or vehicle was administered to groups of five male rats for up to four days, with animals being sacrificed at time-points between 6 and 96 hours post-intial dose. Total RNA from liver samples was processed and analysed by DNA microarray (Affymetrix). The expression profile for each gene was first compared between the three compounds across all time points, identifying 38 genes that could be clustered into groups showing similar expression profiles. From these 38 genes, two groups were selected for further investigation and validation by quantitative real-time PCR analysis using a range of samples dosed with the same compounds over the same time-points. One group, composed of malic enzyme 1 (Me1) and rat brain acyl CoA hydrolase (rBACH), was found to respond only to peroxisome-proliferators, and hence was not investigated further. The second group, comprising seven genes, showed inhibition of gene expression in response to the compounds administered. A similar response was observed during quantitative real-time PCR validation as in the microarray analysis, therefore this group was investigated further using a reporter gene assay. Promoter sequences immediately upstream of the transcriptional start site (1 kilobase) were successfully cloned into a plasmid upstream of a reporter gene (SEAP) for four of the genes, and were transfected into the FaO rat hepatoma cell line. Exposure of transfected cells to liver growth agents produced a similar profile of SEAP expression to that seen in the original in vivo analysis, suggesting regulation of the genes was at the transcriptional level. A deletion construct series was prepared for two of the genes, HCaRG and Nob1p, and the transcriptional response localised to within the first ~100 bp of the proximal promoter in each case. Exposure of the ~100 bp reporter gene constructs to β-naphthoflavone (100 μM), which activates both the aryl hydrocarbon receptor (AhR) and the NF-E2-related factor 2 (Nrf2), elicited a significant ≥2-fold decrease in reporter gene expression, similar to the decrease elicited by the liver growth agents. Dosing with the AhR ligands 3-methylcholanthrene and benzo(a)pyrene failed to mimic the response of the promoter constructs to β-naphthoflavone indicating that AhR was not solely responsible for the inhibition of HCaRG and Nob1p expression observed. However, site-directed mutagenesis of individual potential AhR and Nrf2 response elements within the HCaRG and Nob1p promoter constructs failed to ablate the response to β-naphthoflavone or dexamethasone. This indicated either interplay between the two transcription factors in the response, or that the two transcription factors were capable of compensation. This study has identified and confirmed a link between two genes exhibiting co-ordinate regulation in their expression in response to liver growth agent administration, with the potential of coregulation with five other genes. It has identified the mediators of this response as being the AhR, Nrf2 or more likely a combination of the two transcription factors, with the potential of supporting the increasing observation that Nrf2 and the AhR co-operate in the regulation of genes in response to xenobiotic stimuli.