Evidence against a major role for Ca2+ in hypoxia‐induced gene expression in human hepatoma cells (Hep3B)

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Abstract
1 The human hepatoma cell line Hep3B is a widely used model for studies of hypoxia‐related gene expression. Cytosolic free calcium concentration ([Ca2+]i) has been implicated in cellular oxygen‐sensing processes. We investigated whether calcium ions have a significant impact on the production of erythropoietin (EPO) and vascular endothelial growth factor (VEGF). 2 We found that the calcium ionophore ionomycin induced a rapid and sustained increase of [Ca2+]i while thapsigargin, an inhibitor of endoplasmic reticulum calcium ATPase, only caused a 20 % elevation of [Ca2+]i within 10 min after application. However, the calcium content of intracellular stores was considerably reduced by thapsigargin after an incubation period of 24 h. 3 Variations in [Ca2+]o did not result in altered EPO or VEGF secretion rates. Ionomycin decreased EPO production while the lowering of VEGF production was not statistically significant. In the presence of extracellular Ca2+ the membrane permeant calcium chelator BAPTA‐AM stimulated the production of EPO (P < 0.05) but not of VEGF while EGTA‐AM, a closely related agent, affected neither EPO nor VEGF formation under these conditions. Incubation with thapsigargin resulted in decreased EPO synthesis (P < 0.05) but stimulated VEGF secretion (P < 0.05). 4 In the absence of extracellular calcium, EGTA‐AM led to an accumulation of hypoxia‐inducible factor‐1α (HIF‐1α). This treatment significantly stimulated VEGF synthesis but also decreased EPO secretion (P < 0.05). 5 Our data suggest that the calcium transient and the cytosolic Ca2+ concentration do not play a key role in hypoxia‐induced EPO and VEGF production in Hep3B cells.