Boorn, L.S.; (2010) The role of reactive oxygen species in the stabilisation of hypoxia-inducible factor-1α (HIF-1α). Doctoral thesis , UCL (University College London).

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Abstract

At physiological oxygen concentrations ([O2]) hypoxia-inducible factor-1α (HIF-1α) is constantly hydroxylated and thus prepared for proteosomal degradation through the action of the prolyl hydroxylases (PHDs) (Jiang et al., 1996). In hypoxia, however, the oxygen-sensitive PHDs are inhibited and HIF-1α is stabilised. Other agents, including cytokines and growth factors have been shown to stabilise HIF-1α at physiological [O2] through different mechanisms such as activation of the phosphatidylinositol 3-kinase (PI3K) or mitogen-activated protein kinase pathways (Semenza, 2003). Increased production of reactive oxygen species (ROS) during hypoxia have also been claimed to stabilise HIF-1α (Chandel et al., 1998) and we have now investigated the effect of endogenous ROS on HIF-1α stabilisation. HIF-1α stabilisation and ROS production in human embryonic kidney (HEK 293T) cells were determined by immunoblotting and the use of fluorescent probes, respectively. γGlutamyl cysteine synthetase (γGCS) is the rate limiting enzyme of glutathione (GSH) biosynthesis and therefore a crucial antioxidant. We used small interfering RNA (siRNA) to silence this enzyme and thus impair the capacity of the cells to detoxify ROS. In order to determine whether mitochondria are a major source of ROS we used cells depleted of mitochondrial DNA (Rho0); these were characterised in vitro by monitoring oxygen consumption. RT-PCR was used to determine mitochondrial DNA content and immunoblotting to assess mitochondrial-encoded protein expression. The effects of a Rho0 phenotype were then assessed in relation to HIF-1α stabilisation and ROS production. HIF-1α is stabilised in an oxygen-dependent manner. HIF-1α stabilisation at low [O2] (3%), but not at 0.5% O2 is prevented by treatment with antioxidants. Silencing γGCS augmented free radical production in HEK 293T cells. This was associated with HIF-1α stabilisation at ambient [O2] (21%) and could be prevented by treatment with antioxidants. Rho0 cells produced less ROS than wild-type cells and did not stabilise HIF-1α either at low [O2] (3%) in wild-type cells or at 21% O2 in γGCS silenced cells. The data suggest that HIF-1α can be stabilised by ROS generated by the mitochondria.

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