Hypoxia induces hexokinase II gene expression in human lung cell line A549

Abstract

During adaptation to hypoxic and hyperoxic conditions, the genes involved in glucose metabolism are upregulated. To probe involvement of the transcription factor hypoxia-induced factor-1 (HIF-1) in hexokinase (HK) II expression in human pulmonary cells, A549 cells and small-airway epithelial cells (SAECs) were exposed to stimuli such as hypoxia, deferoxamine (DFO), and metal ions. The largest increase in HK-II (20-fold for mRNA and 2.5-fold for enzymatic activity) was observed in A549 cells when exposed to DFO. All stimuli selectively increased the 5.5-kb rather than 4-kb transcript in A549 cells. Cycloheximide and actinomycin D inhibited these responses. In addition, cells were transfected with luciferase reporter constructs driven by the full-length HK-II 5′-regulatory region (4.0 kb) or various deletions of that region. A549 cells transfected with the 4.0-kb construct and exposed to hypoxia or DFO increased their luciferase activity 7- and 10-fold, respectively, indicating that HK-II induction is, at least in part, due to increased gene transcription. Sixty percent of the inducible activity of the 4.0-kb construct was shown to reside within the proximal 0.5 kb. Additionally, cotransfection with a stable HIF-1 mutant and the 4.0-kb promoter construct resulted in increased luciferase activity under normoxic conditions. These results strongly suggest that HK-II is selectively regulated in pulmonary cells by a HIF-1-dependent mechanism.