MODULATORS OF PROTEIN-KINASE-C INHIBIT HYPOXIA-INDUCED ERYTHROPOIETINPRODUCTION

The human hepatoma cell line, Hep 3B, produces biologically active ery thropoietin (Epo) in response to normal physiologic stimuli and thus p rovides a model system for the study of Epo regulation. The addition o f phorbol 12-myristate 13-acetate (PMA) to Hep 3B cells subsequently g rown under hypoxic conditions resulted in a dose-dependent inhibition of hypoxia-induced Epo production by as much as 95 +/- 1% with half-ma ximal inhibition at 8 ng/mL. By Northern blot analysis, Epo mRNA level s were correspondingly decreased after treatment with PMA. Direct meas urement of both membrane and cytosolic protein kinase C activity in He p 3B cells following treatment with PMA demonstrated a biphasic respon se as a function of time. Membrane-associated protein kinase C activit y initially increased but subsequently decreased to baseline levels by 12 hours. The PMA-induced inhibition of hypoxia-induced Epo productio n was shown to occur as early as 3 hours after PMA addition, suggestin g that the initial activation, rather than the subsequent decrease in protein kinase C activity, is of primary importance. The relative spec ificity of the PMA-induced inhibition of Epo production was demonstrat ed by 1) the finding that overall protein and RNA synthesis were not s imilarly decreased as measured by H-3-leucine and H-3-uridine pulse la beling studies and 2) the observation that the biologically inactive p horbol ester, 4 alpha-phorbol didecanoate, failed to have any effect o n hypoxia-induced Epo production. In addition, the synthetic analog of diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionop hore, A23187, inhibited hypoxia-induced Epo production up to 85 +/- 3% and 82 +/- 4%, respectively, in a dose-dependent manner. Taken togeth er, these findings suggest that hypoxia-induced Epo production may be negatively regulated by activators of a protein kinase C-mediated path way.