HYPOXIA-INDUCED ACCUMULATION OF ERYTHROPOIETIN MESSENGER-RNA IN ISOLATED HEPATOCYTES IS INHIBITED BY PROTEIN-KINASE-C

To define the role of protein kinase C (PKC) in oxygen-dependent produ ction of erythropoietin (EPO) in the liver, we have determined EPO mes senger ribonucleic acid (mRNA) expression in primary cultures of juven ile rat hepatocytes incubated at different oxygen tensions in the abse nce and presence of phorbol esters, vasopressin, and structurally diff erent kinase inhibitors. Upon reduction of oxygen concentrations from 40% to 3% EPO mRNA in cultured hepatocytes increased markedly within 1 .25 h, reached maximal values after 2.5 h and remained elevated for up to 72 h. Treatment of hepatocytes during 1.25-5 h of hypoxic exposure with phorbol 12-myristate-13 acetate (PMA) attenuated hypoxia-induced EPO mRNA levels dose-dependently by a maximum of approximately 50%. T his inhibitory effect of PMA disappeared upon treatment for more than 5 h and was completely lost after incubation for 9 and 18 h in the pre sence of 10(-6) M and 10(-7) M PMA, respectively. Phorbol 12,13-dibuty rate and vasopressin also inhibited EPO mRNA accumulation, whereas 4 a lpha-phorbol 12,13-didecanoate was ineffective. Western blot analysis of PKC isozymes revealed the presence of PKC alpha, beta II, delta, ep silon and zeta and provided no evidence that the PMA-induced inhibitio n of EPO expression was associated with depletion of any of these isoz ymes. Conversely, PMA-induced inhibition of EPO mRNA accumulation was paralleled by translocation of PKC alpha from cytosol to membranes and the time- and dose-dependent attenuation of the inhibitory effect of PMA on EPO mRNA levels was paralleled by down-regulation of PKC alpha. A dose-dependent inhibition of EPO mRNA formation, independent of eff ects on total RLNA synthesis, as determined by [H-3]uridine incorporat ion, was also found in the presence of the kinase inhibitor staurospor ine (ED(50) similar to 2 X 10(-8) M) and three structurally related de rivatives with increased selectivity for PKC (RO 317549, ED(50) simila r to 1 X 10(-6) M; RO 318220, ED(50) similar to 1 X 10(-6) M and CGP 4 1251, ED(50) similar to 4 X 10(-6) M). The markedly lower potency of t he latter three compounds as compared to staurosporine suggests that t his suppression of EPO gene induction was not mediated by inhibition o f PKC. In summary the data indicate that PKC alpha is a negative modul ator of EPO gene expression in hepatocytes. A kinase other than PKC, h owever, appears to be an essential element of hypoxic signalling.