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

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-1alpha (HIF-1alpha). 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.

Figure 1. The effect of BAPTA-AM (A), EGTA-AM in combination with EGTA (B), and thapsigargin (C) on EPO and VEGF production in hypoxic Hep3B cells

EPO (□) and VEGF () levels were determined by RIA and ELISA, respectively, after an incubation period of 24 h. All experiments were carried out on at least 4 separate cell cultures. Data are given as means ±s.d.* Statistically significant difference (i.e. P < 0.05; Dunnett's post hoc test) compared with control values.

Figure 2. Northern blot analysis after incubation of Hep3B cells with BAPTA-AM and thapsigargin

Fifteen micrograms of total RNA were loaded per lane and hybridized with an EPO or VEGF cDNA probe. Equal loading was confirmed by ethidium bromide staining of 28S and 18S rRNA.

Figure 4. Western blot analysis of HIF-1α expression in Hep3B cells

Normoxic control was incubated for 4 h in an atmosphere of 20 % O₂, 5 % CO₂, balance N₂. Hypoxia was induced by placing the cultures in an atmosphere of 3 % O₂, 5 % CO₂, balance N₂ for 4 or 24 h as indicated. Protein extraction and Western blotting were done as described in the Methods section. Reactive proteins were detected using the ECL system (Amersham).

Figure 3. [Ca²⁺]_i monitored in Hep3B cells by measurement of fura2 fluorescence

A, the effect of 10 nm thapsigargin on [Ca²⁺]_i. B, superimposed Ca²⁺ traces from 3 different cultures. * Effect of 1 μm ionomycin without pretreatment. † Effect of 1 μm ionomycin in Ca²⁺-free buffer, i.e. after 2 min of incubation with excess EGTA. The increase of [Ca²⁺]_i was caused solely by release of Ca²⁺ from intracellular stores. ‡ Effect of 1 μm ionomycin in Ca²⁺-free buffer after a 24 h incubation with 10 μm thapsigargin in the presence of Ca²⁺.