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 PKCalpha
antisense oligonucleotides. When EPO-producing Hep3B cells were incubated in 1% O2 (
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% O2. Western blot analysis revealed that Hep3B cells express the classical PKCalpha and gamma
isoforms, as well as novel PKCepsilon and delta and the atypical zeta
isoform. Preincubation with PMA for 6 h specifically down-regulated PKCalpha
protein expression. Phosphorothioate modified
antisense oligonucleotides specific for PKCalpha also decreased EPO production in Hep3B cells exposed to
hypoxia for 20 h when compared to PKCalpha sense treatment. The translocation of PKCalpha from the soluble to particulate fractions was increased in Hep3B cells incubated under
hypoxia compared with normoxia (21% O2) controls. These results suggest that the PKCalpha
isoform plays an important role in sustaining
hypoxia-regulated EPO production.