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

During adaptation to hypoxic and hyperoxic conditions, the genes involved i n glucose metabolism are upregulated. To probe involvement of the transcrip tion factor hypoxia-induced factor-1 (HIF-1) in hexokinase (HK) II expressi on in human pulmonary cells, A549 cells and small-airway epithelial cells ( SAECs) were exposed to stimuli such as hypoxia, deferoxamine (DFO), and met al ions. The largest increase in HK-II (20-fold for mRNA and 2.5-fold for e nzymatic activity) was observed in A549 cells when exposed to DFO. All stim uli selectively increased the 5.5-kb rather than 4-kb transcript in A549 ce lls. 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 tha t region. A549 cells transfected with the 4.0-kb construct and exposed to h ypoxia or DFO increased their luciferase activity 7- and 1.0-fold, respecti vely, indicating that HK-II induction is, at least in part, due to increase d 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, co transfection with a stable HIF-1 mutant and the 4.0-kb promoter construct r esulted in increased luciferase activity under normoxic conditions. These r esults strongly suggest that HK-II is selectively regulated in pulmonary ce lls by a HIF-1-dependent mechanism.