Chelation of cellular calcium modulates hypoxia-inducible gene expression through activation of hypoxia-inducible factor-1alpha.

Abstract

Hypoxia-Inducible Factor-1 (HIF-1) is the key transcription factor in control of the expression of hypoxia-inducible genes needed by cells to adapt to decreased oxygen availability. Herein, we investigated the HIF-1alpha-mediated gene expression of carbonic anhydrase 9 (CA9) in response to hypoxia and changes of intracellular calcium levels in the neuroblastoma cell line SH-SY5Y. Decreasing the intracellular calcium level by BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) induced HIF-1alpha nuclear accumulation and enhanced HIF-1 DNA binding within 1 h of incubation. Like hypoxia, BAPTA stimulated HIF-1-dependent transcription by increasing the activity of the C-terminal transactivation domain of HIF-1alpha and greatly enhanced expression of the HIF-1 target gene CA9. Detailed analysis of HIF-1alpha accumulation revealed that BAPTA attenuated the interaction of HIF-1alpha with von-Hippel-Lindau protein thus decreasing proteasomal degradation of HIF-1alpha. Knock down of HIF-1alpha mRNA and protein by small interference RNA for HIF-1alpha revealed that both hypoxia and the BAPTA-induced gene expression of CA9 were strictly dependent on HIF-1alpha. In contrast, elevation of cytosolic calcium level by thapsigargin reduced the BAPTA-mediated effects. Measurements of intracellular calcium under hypoxia revealed a change in the cellular calcium distribution. BAPTA-dependent induction of HIF-1 activity was not caused by its in vitro capability to chelate iron. Instead, effective chelation of cellular calcium caused the accumulation of HIF-1alpha protein through inhibition of HIF-prolyl hydroxylases and activated HIF-1-dependent gene expression under normoxic conditions.

• Authors: Utta Berchner-Pfannschmidt, Frank Petrat, Kathrin Doege, Buena Trinidad, Patricia Freitag, Eric Metzen, Herbert de Groot, Joachim Fandrey
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 279 Issue 43 Pg. 44976-86 (Oct 22 2004) ISSN: 0021-9258 [Print] United States
• PMID: 15322093 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Chelating Agents
• HIF1A protein, human
• Hypoxia-Inducible Factor 1, alpha Subunit
• Ions
• Ligands
• RNA, Messenger
• RNA, Small Interfering
• Transcription Factors
• Egtazic Acid
• Thapsigargin
• Iron
• Procollagen-Proline Dioxygenase
• Carbonic Anhydrases
• 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
• Calcium

Topics

• Blotting, Western
• Calcium (metabolism)
• Carbonic Anhydrases (metabolism)
• Cell Line, Tumor
• Cell Nucleus (metabolism)
• Cell Survival
• Cell-Free System
• Chelating Agents (pharmacology)
• Dose-Response Relationship, Drug
• Egtazic Acid (analogs & derivatives, pharmacology)
• Humans
• Hypoxia
• Hypoxia-Inducible Factor 1, alpha Subunit
• Ions
• Iron (chemistry, metabolism)
• Lasers
• Ligands
• Microscopy, Confocal
• Microscopy, Fluorescence
• Plasmids (metabolism)
• Procollagen-Proline Dioxygenase (metabolism)
• Protein Binding
• Protein Structure, Tertiary
• RNA Interference
• RNA, Messenger (metabolism)
• RNA, Small Interfering (metabolism)
• Reverse Transcriptase Polymerase Chain Reaction
• Thapsigargin (pharmacology)
• Time Factors
• Transcription Factors (metabolism)