Modulators of protein kinase C inhibit hypoxia-induced erythropoietin production.
The human hepatoma cell line, Hep 3B, produces biologically active erythropoietin (Epo) in response to normal physiologic stimuli and thus provides a model system for the study of Epo regulation. The addition of phorbol 12-myristate 13-acetate (PMA) to Hep 3B cells subsequently grown under hypoxic conditions resulted in a dose-dependent inhibition of hypoxia-induced Epo production by as much as 95 +/- 1% with half-maximal inhibition at 8 ng/mL. By Northern blot analysis, Epo mRNA levels were correspondingly decreased after treatment with PMA. Direct measurement of both membrane and cytosolic protein kinase C activity in Hep 3B cells following treatment with PMA demonstrated a biphasic response as a function of time. Membrane-associated protein kinase C activity initially increased but subsequently decreased to baseline levels by 12 hours. The PMA-induced inhibition of hypoxia-induced Epo production was shown to occur as early as 3 hours after PMA addition, suggesting that the initial activation, rather than the subsequent decrease in protein kinase C activity, is of primary importance. The relative specificity of the PMA-induced inhibition of Epo production was demonstrated by 1) the finding that overall protein and RNA synthesis were not similarly decreased as measured by 3H-leucine and 3H-uridine pulse labeling studies and 2) the observation that the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, failed to have any effect on hypoxia-induced Epo production. In addition, the synthetic analog of diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionophore, A23187, inhibited hypoxia-induced Epo production up to 85 +/- 3% and 82 +/- 4%, respectively, in a dose-dependent manner. Taken together, these findings suggest that hypoxia-induced Epo production may be negatively regulated by activators of a protein kinase C-mediated pathway.
Faquin
W C
WC
Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115.
Schneider
T J
TJ
Goldberg
M A
MA
D015152:Blotting, Northern; D000001:Calcimycin / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D000860:Hypoxia / Q000378:metabolism / Q000503:physiopathology*; D007930:Leucine / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D012313:RNA / Q000032:analysis / Q000235:genetics; D011863:Radioimmunoassay; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014316:Tritium; D014407:Tumor Cells, Cultured; D014529:Uridine / Q000378:metabolism
D004075:Diglycerides; D014316:Tritium; D004921:Erythropoietin; D000001:Calcimycin; D012313:RNA; C038237:1-oleoyl-2-acetylglycerol; D004247:DNA; D011493:Protein Kinase C; D007930:Leucine; D013755:Tetradecanoylphorbol Acetate; D014529:Uridine