Microarray analyses of hypoxia-regulated genes in an aryl hydrocarbon receptor nuclear translocator (Arnt)-dependent manner

We investigated hypoxia-inducible factor (HIF)-dependent changes in the expression of 5592 genes in response to hypoxia (0.1% O(2), 16 h) by performing cDNA microarray analyses of mouse hepa1c1c7 and BpRc1 cells. BpRc1 cells are a hepa1c1c7 variant defective in HIF-beta/aryl hydrocarbon receptor nuclear translocator (Arnt), and are therefore unable to induce HIF target genes in response to hypoxia. By comparing hepa1c1c7 cells with BpRc1 cells, we were able to investigate hypoxia-regulated gene expression as well as the role played by HIF in regulating the hypoxic-dependent response of gene expression. This study identified 50 hypoxia-induced genes and 36 hypoxia-repressed genes. Quantitative PCR analysis of nine genes confirmed our ability to accurately analyze changes in hypoxia-induced gene expression by microarray analysis. By comparing quantitative PCR analyses of these nine genes in BpRc1 and hepa1c1c7 cells, we determined that eight of the nine hypoxia-induced genes are Arnt dependent. Additional quantitative PCR analyses of eight hypoxia-repressed genes confirmed, with a 50% probability, that microarray analysis was able to predict hypoxia-repressed gene expression. Only two of the four confirmed genes were found to be repressed in an Arnt-dependent manner. Collectively, six of these 13 genes (46.2% probability) showed a pattern of expression consistent with the microarray analysis with regard to Arnt dependence. Finally, we investigated the HIF-1 alpha dependence of these 13 genes by quantitative PCR analysis in HIF-1 alpha knockdown 3T3-L1 cells. These analyses identified novel hypoxia-regulated genes and confirmed the role of Arnt and HIF-1 alpha in regulating their expression. These results identify additional HIF target genes and provide a more complete understanding of hypoxia signaling.