Evidence against a major role for Ca2+ in hypoxia-induced gene expression in human hepatoma cells (Hep3B).
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.
Metzen
E
E
Institute of Physiology, Medical University of Lubeck, Germany. metzen@physio.mu-luebeck.de
Fandrey
J
J
Jelkmann
W
W
D015152:Blotting, Northern; D002118:Calcium / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D006528:Carcinoma, Hepatocellular; D015687:Cell Hypoxia / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D016228:Endothelial Growth Factors / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004921:Erythropoietin / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008113:Liver Neoplasms; D008222:Lymphokines / Q000235:genetics*; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D002614:Chelating Agents; D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; D008222:Lymphokines; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D004921:Erythropoietin; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D015759:Ionomycin; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium