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.

• Authors: E Metzen, J Fandrey, W Jelkmann
• Journal: The Journal of physiology (J Physiol) Vol. 517 ( Pt 3) Pg. 651-7 (Jun 15 1999) ISSN: 0022-3751 [Print] England
• PMID: 10358107 (Publication Type: Journal Article)

Chemical References

• Chelating Agents
• Endothelial Growth Factors
• Enzyme Inhibitors
• Lymphokines
• Vascular Endothelial Growth Factor A
• Vascular Endothelial Growth Factors
• Erythropoietin
• 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester
• Egtazic Acid
• Ionomycin
• Thapsigargin
• Calcium-Transporting ATPases
• Calcium

Topics

• Blotting, Northern
• Calcium (physiology)
• Calcium-Transporting ATPases (antagonists & inhibitors)
• Carcinoma, Hepatocellular
• Cell Hypoxia (physiology)
• Chelating Agents (pharmacology)
• Egtazic Acid (analogs & derivatives, pharmacology)
• Endothelial Growth Factors (genetics)
• Enzyme Inhibitors (pharmacology)
• Erythropoietin (genetics)
• Gene Expression Regulation, Neoplastic (drug effects)
• Humans
• Ionomycin (pharmacology)
• Kinetics
• Liver Neoplasms
• Lymphokines (genetics)
• Thapsigargin (pharmacology)
• Tumor Cells, Cultured
• Vascular Endothelial Growth Factor A
• Vascular Endothelial Growth Factors