Abstract
Increased erythropoietin (EPO) production is important for erythropoiesis as well as cell viability. The most effective factor for promoting EPO production is hypoxia, which alters the redox state and produces a reducing environment in the cell. In this study, we examined the influence of ethanol on EPO production in HepG2 cells to investigate the effect of increasing the free NADH/NAD+ ratio in the cytosol during normoxia. Ethanol treatment increased the lactate/pyruvate ratio, an index of the cytosolic redox state, in a dose-dependent manner, with maximal promotion of EPO production observed at 300 μM ethanol. These results suggest that altering the cytosolic NADH/NAD+ redox state to the same degree as hypoxia is effective in promoting EPO production. Ethanol (300 μM) increased mRNA expression and protein levels of sirtuin1, which is a transcription factor, related to both hypoxia inducible factor and cytosolic redox state, whereas 2000 μM ethanol did not produce these effects. Although the sirtuin1 inhibitorEX-527 did not affect the lactate/pyruvate ratio, EX-527 inhibited the induction of EPO mRNA expression by 300 μM ethanol. In rat primary hepatocytes and kidney cells, 300 μM ethanol increased sirtuin1 and EPO mRNA expression, as well as EPO concentrations in media. In conclusion, we showed low concentrations of ethanol promote EPO production by increasing sirtuin1 in HepG2 cells, as well as primary liver and kidney cells. The use of ethanol represents a hypoxia-independent method to promote EPO production.
Highlights
Erythropoietin (EPO) is a hematopoietic cytokine that is best known for its role in promoting red blood cell formation and survival [1,2,3,4]
Page 3 of 7 Since the effect of ethanol on EPO mRNA expression was not observed at high ethanol concentrations, the effects of 2000 μM ethanol were compared to 300 μM ethanol, which showed increased EPO mRNA expression
We showed that low concentrations of ethanol increased EPO production in HepG2 cells and rat primary hepatocytes or kidney cells
Summary
Erythropoietin (EPO) is a hematopoietic cytokine that is best known for its role in promoting red blood cell formation and survival [1,2,3,4]. EPO production is regulated by hypoxia inducible factor (HIF), which is a heterodimer consisting of HIF-α and β subunits [5,6]. Both HIF-1 and 2 promote EPO production, and the role of each HIF subtype in EPO production differs according to cell type and the duration of exposure [7,8]. Hypoxia increases the content of HIF-1α and/or 2α by reducing the activity of PHDs, which hydrolyze HIF-α, and increases transcription of EPO mRNA in the nucleus [9]. Hypoxia, caused by anemia or relocation to mountainous elevations, decreases PHD activity and increases HIF-α content, resulting in elevated EPO production [5,10]
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