Stabilization Of Hypoxia-inducible Factor-1α Research Articles

Prostaglandin E2 Induces Hypoxia-inducible Factor-1α Stabilization and Nuclear Localization in a Human Prostate Cancer Cell Line

Hypoxia-induced up-regulation of vascular endothelial growth factor (VEGF) expression is a critical event leading to tumor neovascularization. Hypoxia stimulates hypoxia-inducible factor-1alpha (HIF-1alpha), a transcriptional activator of VEGF. Cyclooxygenase (COX)-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, is also induced by hypoxia. We reported previously that COX-2 inhibition prevents hypoxic up-regulation of VEGF in human prostate cancer cells and that prostaglandin E(2) (PGE(2)) restores hypoxic effects on VEGF. We hypothesized that PGE(2) mediates hypoxic effects on VEGF by modulating HIF-1alpha expression. Addition of PGE(2) to PC-3ML human prostate cancer cells had no effect on HIF-1alpha mRNA levels. However, PGE(2) significantly increased HIF-1alpha protein levels, particularly in the nucleus. This effect of PGE(2) largely results from the promotion of HIF-1alpha translocation from the cytosol to the nucleus. PGE(2) addition to PC-3 ML cells transfected with a GFP-HIF-1alpha vector induced a time-dependent nuclear accumulation of the HIF-1alpha protein. Two selective COX-2 inhibitors, meloxicam and NS398, decreased HIF-1alpha levels and nuclear localization, under both normoxic and hypoxic conditions. Of several prostaglandins tested, only PGE(2) reversed the effects of a COX-2 inhibitor in hypoxic cells. Finally, PGE(2) effects on HIF-1alpha were specifically inhibited by PD98059 (a MAPK inhibitor). These data demonstrate that PGE(2) production via COX-2-catalyzed pathway plays a critical role in HIF-1alpha regulation by hypoxia and imply that COX-2 inhibitors can prevent hypoxic induction of HIF-mediated gene transcription in cancer cells.

Hypoxic but not anoxic stabilization of HIF-1alpha requires mitochondrial reactive oxygen species.

The molecular mechanisms by which cells detect hypoxia (1.5% O2), resulting in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) protein remain unclear. One model proposes that mitochondrial generation of reactive oxygen species is required to stabilize HIF-1alpha protein. Primary evidence for this model comes from the observation that cells treated with complex I inhibitors, such as rotenone, or cells that lack mitochondrial DNA (rho(0)-cells) fail to generate reactive oxygen species or stabilize HIF-1alpha protein in response to hypoxia. In the present study, we investigated the role of mitochondria in regulating HIF-1alpha protein stabilization under anoxia (0% O2). Wild-type A549 and HT1080 cells stabilized HIF-1alpha protein in response to hypoxia and anoxia. The rho(0)-A549 cells and rho(0)-HT1080 cells failed to accumulate HIF-1alpha protein in response to hypoxia. However, both rho(0)-A549 and rho(0)-HT1080 were able to stabilize HIF-1alpha protein levels in response to anoxia. Rotenone inhibited hypoxic, but not anoxic, stabilization of HIF-1alpha protein. These results indicate that a functional electron transport chain is required for hypoxic but not anoxic stabilization of HIF-1alpha protein.

Cobalt- and Nickel-Binding Property of Cullin-2

Treatment with divalent metal ions such as cobalt (Co2+) or nickel (Ni2+) result in the stabilization of hypoxia-inducible factor-1α (HIF1α). Recently, HIF1α was shown to be ubiquitinated by an E3-ligase complex and be subsequently targeted for proteasomal degradation. In this study, we demonstrated that Co2+ and Ni2+ specifically bind to cullin-2. Mutant analysis revealed that cullin-2 possesses at least three sites for the binding. Furthermore, fluorescence spectroscopy revealed that only Co2+ and Ni2+ have the binding activity to cullin-2, but other metal ions, including Cu2+, Ca2+, Mg2+, Mn2+, and Zn2+, did not. Finally, we found that Co2+ and Ni2+ do not bind to any components of the E3-ligase other than cullin-2, suggesting that cullin-2 is a key target of Co2+ and Ni2+. Interestingly, Co2+ did not affect the complex formation of the ligase, suggesting that the metal binding to cullin-2 affects the function, but not the assembly of the E3-ligase.