Chelation of cellular calcium modulates hypoxia-inducible gene expression through activation of hypoxia-inducible factor-1alpha.
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.
Institut für Physiologie, Universität Duisburg-Essen, Hufelandstrasse 55, D-45122 Essen, Germany.
Utta
U
Berchner-Pfannschmidt
Frank
F
Petrat
Kathrin
K
Doege
Buena
B
Trinidad
Patricia
P
Freitag
Eric
E
Metzen
Herbert
H
de Groot
Joachim
J
Fandrey
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D002474:Cell-Free System; D002614:Chelating Agents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007477:Ions; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007834:Lasers; D008024:Ligands; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*
D002614:Chelating Agents; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007477:Ions; D008024:Ligands; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; D004533:Egtazic Acid; D019284:Thapsigargin; D007501:Iron; D011393:Procollagen-Proline Dioxygenase; D002256:Carbonic Anhydrases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium