Hypoxia signalling in the control of erythropoietin gene expression in rat hepatocytes

Abstract

This study aimed to investigate the sequence of events involved in the stimulation of erythropoietin (EPO) gene expression by hypoxia in hepatocytes. To this end, primary cultures of rat hepatocytes were kept at either high (40% O2) or low (3% O2) oxygen tensions for 2.5 h. Hypoxia increased EPO mRNA about fifteen-fold, whilst the divalent cation cobalt (50-100 μM) or the iron chelator desferrioxamine (10-200 μM) did not increase EPO mRNA levels. Addition of hydrogen peroxide (100-500 μM) to the culture medium did also not change EPO mRNA levels at high or low oxygen tension. Addition of catalase (50-200 μg/ml) to the culture medium resulted in a lower level of hypoxia-induced EPO mRNA. Inhibition of protein synthesis by cycloheximide (100 μM) completely abolished the increase of EPO mRNA in response to hypoxia. Hypoxia but not cobalt increased the appearance of the hypoxia-inducible factor 1 (HIF-1), and this increase was blunted by cycloheximide. Taken together, these findings suggest that a classic heme protein and a related oxygen-dependent production of oxygen radicals is less likely to be involved in the regulation of the EPO gene by oxygen in hepatocytes. On the other hand, intact protein synthesis is an absolute requirement for the hypoxia-induced appearance of HIF 1 and for hypoxia-stimulated expression of the EPO gene in hepatocytes.