The regulated expression of erythropoietin by two human hepatoma cell lines.
Goldberg M A,Glass G A,Cunningham J M,Bunn H F
Proceedings of the National Academy of Sciences of the United States of America
The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. We have screened multiple renal and hepatic cell lines (including MDCK, LLC-PK1, BHK, WRL 68, CLCL, A704, CRFK, A498, ACHN, TCMK-1, LLC-MK2, CaKi-2, HepG2, and Hep3B) for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10(6) cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities less than 3.3 X 10(5) cells per cm2, there was little constitutive release of Epo in the medium (less than 30 milliunits per 10(6) cells in 24 hr). With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 microM cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.
Effects of sorbitol and lactate on erythropoietin production in HepG2 cells.
Nishimura Kazuhiko,Katsuyama Hideaki,Ohishi Masahiro,Hirabayashi Arisa,Matsuda Kensyo,Nakagawa Hiroshi
Biochemical and biophysical research communications
Promotion of erythropoietin (EPO) production is important for erythropoiesis as well as cell viability. The most effective inducing factor for EPO production is hypoxia. Hypoxia inducible factor (HIF), a regulator of EPO production, is increased under hypoxic conditions and is also affected by various regulators such as sirtuin1 (SIRT1). SIRT1 is regulated by the cytoplasmic redox state, which is thought to affect EPO production. Therefore, we investigated the effects of sorbitol and lactic acid, which serve as substrates for cellular respiration and bring cells into a reduced state, on EPO production in HepG2 cells. The addition of low-concentration sorbitol to HepG2 cells produced a mildly reduced state similar to that of hypoxia and increased NAD, SIRT1, and HIF-α, and EPO mRNA expression. On the other hand, lactate suppressed EPO mRNA expression at all concentrations. Inhibition of lactate production from pyruvate abolished the effect of low sorbitol concentrations on EPO mRNA expression. When low-concentration sorbitol and a reducing agent were administered simultaneously, the effect of increasing EPO mRNA expression disappeared. It was suggested that SIRT1 and EPO production increased under conditions where lactate production was not suppressed, even under mildly reduced conditions similar to hypoxia.