Protein kinase C alpha protein expression is necessary for sustained erythropoietin production in human hepatocellular carcinoma (Hep3B) cells exposed to hypoxia

Although protein kinase C (PKC) has been implicated as an effector of eryth ropoietin (EPO) production, its exact role is still uncertain. Hep3B human hepatocellular carcinoma cells were used for this study and were depleted o f PKC in three different ways: long-term treatment with phorbol 12-myristat e 13-acetate (PMA), selective inhibition with calphostin C, and treatment w ith PKC alpha antisense oligonucleotides. When EPO-producing Hep3B cells we re incubated in 1% O-2 (hypoxia)for 24 h, PMA treatment resulted in signifi cant decreases in medium levels of EPO in Hep3B cell cultures at concentrat ions higher than 10 nM. The specific PKC inhibitor, calphostin C, significa ntly inhibited medium levels of EPO and EPO mRNA levels in Hep3B cells expo sed to 1% O-2. Western blot analysis revealed that Hep3B cells express the classical PKC alpha and gamma isoforms, as well as novel PKC epsilon and de lta and the atypical zeta isoform. Preincubation with PMA for 6 h specifica lly downregulated PKCa protein expression. Phosphorothioate modified antise nse oligonucleotides specific for PKC alpha also decreased EPO production i n Hep3B cells exposed to hypoxia for 20 h when compared to PKC alpha sense treatment. The translocation of PKCa from the soluble to particulate fracti ons was increased in Hep3B cells incubated under hypoxia compared with norm oxia (21% O-2) controls. These results suggest that the PKC alpha isoform p lays an important role in sustaining hypoxia-regulated EPO production. (C) 1999 Elsevier Science B.V. All rights reserved.