Choline kinase catalyzes the phosphorylation of
choline, the first step of
phospholipid synthesis. Increased phosphorylation of
choline is a hallmark characteristic of the malignant phenotype in a variety of
neoplasms. However, in hypoxic
cancer cells,
choline phosphorylation is decreased. To understand the mechanism behind this altered metabolic state, we examined the expression and regulation of the major
choline kinase isoform,
choline kinase α (ChKα), in hypoxic PC-3 human
prostate cancer cells.
Hypoxia decreased
choline phosphorylation,
choline kinase activity, and ChKα
mRNA and
protein levels. Promoter analysis studies revealed a region upstream of the ChKα gene bearing a conserved
DNA consensus binding motif,
hypoxia response element-7 (HRE7), at position -222 relative to +1 translation start site, for binding the
hypoxia dependent master regulator
transcription factor,
hypoxia-inducible factor 1α (HIF-1α). Electrophoretic mobility shift competition/supershift assay and
chromatin immunoprecipitation assay confirmed binding of HIF-1α to HRE7. A putative promoter of ChKα was isolated from PC-3 genomic
DNA and cloned into a
luciferase-based reporter vector system. In PC-3 cells,
hypoxia decreased the expression of
luciferase under the control of the ChKα promoter. Mutation of HRE7 abrogated this
hypoxia effect, further demonstrating the involvement of HRE7 in
hypoxia-sensitive regulation of ChKα. The results strongly suggest that transcriptional control of
choline phosphorylation is largely mediated via HIF-1α binding to the newly identified HRE7.