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 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.

17β-Estradiol decreases hypoxic induction of erythropoietin gene expression

“…NO regulation of EPO synthesis is also much debated. NO has been reported to attenuate EPO gene expression under multiple conditions (20,57), although some investigators have demonstrated that NO can induce EPO synthesis during hypoxia in Hep3B cells (37,68). We therefore hypothesized that E2-␤ increases NO production to attenuate EPO gene expression.…”

Superoxide dismutase-3 promotes full expression of the EPO response to hypoxia

“…NO is reported to inhibit HIF-1 and EPO expression 53,54 yet supports EPO synthesis during hypoxia in Hep3B cells and cultured renal tissue. 55,56 Therefore, we evaluated renal eNOS expression and plasma NO x production in the mice during hypoxia. Hypoxia increased plasma NO x only slightly more in Wt than SOD3-deficient mice, and Western analysis demonstrated modest increases in renal eNOS protein expression, which were not significantly different between strains.…”

Protein Kinase C (PKC) Isozymes and Cancer

“…TPA-induced growth arrest in the G 0 /G 1 phase, upregulation of cancer suppressor gene miR-101, and downregulation of enhancer of zeste homolog 2 during embryonic ectoderm development in HepG2 cells are all PKC -dependent [274]. Downregulation of PKC by adding TPA into Hep3B cells also decreases the production of erythropoietin [263], which is a glycoprotein hormone that is stimulated under the hypoxic environment [275]. On the other hand, expression of PKC and suppresses HGFinduced phosphorylation of ERK and paxillin, resulting in the reduction of HepG2 cell migration, whereas PKC and are required for phosphorylation of paxillin [276].…”

“…In Hep3B cells, different patterns of PKC isozymes have been reported, such as PKC , , , , and [262] and PKC , , , , and [263]. Furthermore, PKC II and are downregulated in human HCC tissues and are correlated with the grade of HCC and hepatitis B virus infection, respectively [264].…”