HYPOXIA SIGNALING IN THE CONTROL OF ERYTHROPOIETIN GENE-EXPRESSION INRAT HEPATOCYTES

This study aimed to investigate the sequence of events involved in the stimulation of erythropoietin (EPO) gene expression by hypoxia in hep atocytes. To this end, primary cultures of rat hepatocytes were kept a t either high (40% O-2) or low (3% O-2) oxygen tensions for 2.5 h. Hyp oxia increased EPO mRNA about fifteen-fold, whilst the divalent cation cobalt (50-100 mu M) or the iron chelator desferrioxamine (10-200 mu M) did not increase EPO mRNA levels. Addition of hydrogen peroxide (10 0-500 mu M) to the culture medium did also not change EPO mRNA levels at high or low oxygen tension. Addition of catalase (50-200 mu g/ml) t o the culture medium resulted in a lower level of hypoxia-induced EPO mRNA. Inhibition of protein synthesis by cycloheximide (100 mu M) comp letely abolished the increase of EPO mRNA in response to hypoxia. Hypo xia but not cobalt increased the appearance of the hypoxia-inducible f actor 1 (HIF-1), and this increase was blunted by cycloheximide. Taken together, these findings suggest that a classic heme protein and a re lated oxygen-dependent production of oxygen radicals is less likely to be involved in the regulation of the EPO gene by oxygen in hepatocyte s. On the other hand, intact protein synthesis is an absolute requirem ent for the hypoxia-induced appearance of HIF-1 and for hypoxia-stimul ated expression of the EPO gene in hepatocytes. (C) 1996 Wiley-Liss, I nc.