Chelation of cellular calcium modulates hypoxia-inducible gene expression through activation of Hypoxia-Inducible Factor-1α

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-1α- 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-1α 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 trans-activation domain of HIF-1α and greatly enhanced expression of the HIF-1 target gene CA9. Detailed analysis of HIF-1α accumulation revealed that BAPTA attenuated the interaction of HIF-1α with von-Hippel-Lindau protein thus decreasing proteasomal degradation of HIF-1α. Knock down of HIF-1α mRNA and protein by small interference RNA for HIF-1α revealed that both hypoxia and the BAPTA-induced gene expression of CA9 were strictly dependent on HIF-1α. 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-1α protein through inhibition of HIF-prolyl hydroxylases and activated HIF-1-dependent gene expression under normoxic conditions.