Mechanism of hypoxia-specific cytotoxicity of procaspase-3 fused with a VHL-mediated protein destruction motif of HIF-1alpha containing Pro564

Under normoxic conditions the alpha-subunit of hypoxia-inducible factor (HIF-1alpha) protein is targeted for degradation by the von Hippel-Lindau (VHL) tumor suppressor protein acting as an E3 ubiquitin ligase. Recently, we developed a hypoxia-targeting protein, TOP3, which consisted of procaspase-3 with the VHL-mediated protein destruction motif of HIF-1alpha. This design enables procaspase-3 to be regulated similarly with HIF-1alpha, being degraded under normoxia while stabilized under hypoxia. Furthermore, stabilized TOP3 was cleaved by the hypoxic stress-induced endogenous caspases and thus the procaspase-3 was converted to active caspase-3 specifically under hypoxic conditions. These data demonstrated that the VHL-mediated protein destruction motif of HIF-1alpha endowed procaspase-3 with hypoxia-specific cytotoxicity.     

Role of prolyl hydroxylation in oncogenically stabilized hypoxia-inducible factor-1alpha

Stabilization of the hypoxia-inducible factor-1 (HIF-1) protein is essential for its role as a regulator of gene expression under low oxygen conditions. Here, employing a novel hydroxylation-specific antibody, we directly show that proline 564 of HIF-1alpha and proline 531 of HIF-2alpha are hydroxylated under normoxia. Importantly, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 hydroxylation is diminished with the treatment of hypoxia, cobalt chloride, desferrioxamine, or dimethyloxalyglycine, regardless of the E3 ubiquitin ligase activity of the von Hippel-Lindau (VHL) tumor suppressor gene. Furthermore, in VHL-deficient cells, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 had detectable amounts of hydroxylation following transition to hypoxia, indicating that the post-translational modification is not reversible. The introduction of v-Src or RasV12 oncogenes resulted in the stabilization of normoxic HIF-1alpha and the loss of hydroxylated Pro-564, demonstrating that oncogene-induced stabilization of HIF-1alpha is signaled through the inhibition of prolyl hydroxylation. Conversely, a constitutively active Akt oncogene stabilized HIF-1alpha under normoxia independently of prolyl hydroxylation, suggesting an alternative mechanism for HIF-1alpha stabilization. Thus, these results indicate distinct pathways for HIF-1alpha stabilization by different oncogenes. More importantly, these findings link oncogenesis with normoxic HIF-1alpha expression through prolyl hydroxylation.     

Calpain mediates a von Hippel-Lindau protein-independent destruction of hypoxia-inducible factor-1alpha

Hypoxia-inducible factor 1 (HIF-1) is controlled through stability regulation of its alpha subunit, which is expressed under hypoxia but degraded under normoxia. Degradation of HIF-1alpha requires association of the von Hippel Lindau protein (pVHL) to provoke ubiquitination followed by proteasomal digestion. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1alpha under normoxia but destabilizes the protein under hypoxia. To understand the role of NO under hypoxia we made use of pVHL-deficient renal carcinoma cells (RCC4) that show a high steady state HIF-1alpha expression under normoxia. Exposing RCC4 cells to hypoxia in combination with the NO donor DETA-NO (2,2'-(hydroxynitrosohydrazono) bis-ethanimine), but not hypoxia or DETA-NO alone, decreased HIF-1alpha protein and attenuated HIF-1 transactivation. Mechanistically, we noticed a role of calpain because calpain inhibitors reversed HIF-1alpha degradation. Furthermore, chelating intracellular calcium attenuated HIF-1alpha destruction by hypoxia/DETA-NO, whereas a calcium increase was sufficient to lower the amount of HIF-1alpha even under normoxia. An active role of calpain in lowering HIF-1alpha amount was also evident in pVHL-containing human embryonic kidney cells when the calcium pump inhibitor thapsigargin reduced HIF-1alpha that was stabilized by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). We conclude that calcium contributes to HIF-1alpha destruction involving the calpain system.     

NO restores HIF-1alpha hydroxylation during hypoxia: role of reactive oxygen species

The activity of hypoxia-inducible factor 1 (HIF-1) is primarily determined by stability regulation of its alpha subunit, which is stabilized under hypoxia but degraded during normoxia. Hydroxylation of HIF-1alpha by prolyl hydroxylases (PHDs) recruits the von Hippel-Lindau (pVHL) E3 ubiquitin ligase complex to initiate proteolytic destruction of the alpha subunit. Hypoxic stabilization of HIF-1alpha has been reported to be antagonized by nitric oxide (NO). By using a HIF-1alpha-pVHL binding assay, we show that NO released from DETA-NO restored prolyl hydroxylase activity under hypoxia. Destabilization of HIF-1alpha by DETA-NO was reversed by free radical scavengers such as NAC and Tiron, thus pointing to the involvement of reactive oxygen species (ROS). Therefore, we examined the effects of ROS on HIF-1alpha stabilization. Treatment of cells under hypoxia with low concentrations of the superoxide generator 2,3-dimethoxy-1,4-naphthoquinone lowered HIF-1alpha protein stabilization. In vitro HIF-1alpha-pVHL interaction assays demonstrated that low-level ROS formation increased prolyl hydroxylase activity, an effect antagonized by ROS scavengers. While determining intracellular ROS formation we noticed that reduced ROS production under hypoxia was restored by the addition of DETA-NO. We propose that an increase in ROS formation contributes to HIF-1alpha destabilization by NO donors under hypoxia via modulation of PHD activity.