Tryptophan metabolism and non-hypoxic induction of hypoxia-inducible factor (HIF)

Abstract

We studied the response of macrophages to activation of Hypoxia inducible factor 1 pathway triggered by hypoxia or, under normoxic conditions by Picolinic, a metabolite of tryptophan. We analyzed the expression of Glutamine: fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme in the hexosamine biosynthetic pathway controlling protein glycosylation. We obtained the first evidence that the GFAT mRNA and protein are constitutively present in mouse macrophages and we demonstrated that the expression is inducible by hypoxia and by the hypoxia-related stimuli picolinic acid (PA). The promoter of GFAT contains the consensus sequence of the Hypoxia responsive element (HRE) in position −74/−65 and we studied the role of HRE on the activation of the promoter by transfecting the macrophage cell lines with appropriate expression vectors containing fragments of the GFAT promoter. We found that GFAT HRE is essential for the transcriptional activation by hypoxia or PA and that HIF1α can augment this response activate GFAT expression. Moreover, we demonstrated that PA is a potent inducer of HIF 1 and HIF2. Comparison of the gene expression profile induced by hypoxia or PA revealed that only a small number of genes are induced by both stimuli like GFAT, despite the activation of HIF-dependent pathways by both stimuli.