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

Abstract

Although protein kinase C (PKC) has been implicated as an effector of erythropoietin (EPO) production, its exact role is still uncertain. Hep3B human hepatocellular carcinoma cells were used for this study and were depleted of PKC in three different ways: long-term treatment with phorbol 12-myristate 13-acetate (PMA), selective inhibition with calphostin C, and treatment with PKCα antisense oligonucleotides. When EPO-producing Hep3B cells were incubated in 1% O 2 (hypoxia) for 24 h, PMA treatment resulted in significant decreases in medium levels of EPO in Hep3B cell cultures at concentrations higher than 10 nM. The specific PKC inhibitor, calphostin C, significantly inhibited medium levels of EPO and EPO mRNA levels in Hep3B cells exposed to 1% O 2. Western blot analysis revealed that Hep3B cells express the classical PKCα and γ isoforms, as well as novel PKCϵ and δ and the atypical ζ isoform. Preincubation with PMA for 6 h specifically down-regulated PKCα protein expression. Phosphorothioate modified antisense oligonucleotides specific for PKCα also decreased EPO production in Hep3B cells exposed to hypoxia for 20 h when compared to PKCα sense treatment. The translocation of PKCα from the soluble to particulate fractions was increased in Hep3B cells incubated under hypoxia compared with normoxia (21% O 2) controls. These results suggest that the PKCα isoform plays an important role in sustaining hypoxia-regulated EPO production.