Hypoxia-inducible Factor-1 Activation in Nonhypoxic Conditions: The Essential Role of Mitochondrial-derived Reactive Oxygen Species

Abstract

Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor for responses to low oxygen. Different nonhypoxic stimuli, including hormones and growth factors, are also important HIF-1 activators in the vasculature. Angiotensin II (Ang II), the main effecter hormone in the renin-angiotensin system, is a potent HIF-1 activator in vascular smooth muscle cells (VSMCs). HIF-1 activation by Ang II involves intricate mechanisms of HIF-1␣ transcription, translation, and protein stabilization. Additionally, the generation of reactive oxygen species (ROS) is essential for HIF-1 activation during Ang II treatment. However, the role of the different VSMC ROS generators in HIF-1 activation by Ang II remains unclear. This work aims at elucidating this question. Surprisingly, repression of NADPH oxidase-generated ROS, using Vas2870, a specific inhibitor or a p22 phox siRNA had no significant effect on HIF-1 accumulation by Ang II. In contrast, repression of mitochondrial-generated ROS, by complex III inhibition, by Rieske Fe-S protein siRNA, or by the mitochondrial-targeted antioxidant SkQ1, strikingly blocked HIF-1 accumulation. Furthermore, inhibition of mitochondrial-generated ROS abolished HIF-1␣ protein stability, HIF-1-dependent transcription and VSMC migration by Ang II. A large number of studies implicate NADPH oxidase-generated ROS in Ang II-mediated signaling pathways in VSMCs. However, our work points to mitochondrial-generated ROS as essential intermediates for HIF-1 activation in nonhypoxic conditions. INTRODUCTIONHypoxia-inducible factor-1 (HIF-1) is a key hypoxia-inducible transcription factor responsible for the adaptation of cells and tissues to low oxygen by regulating such responses as cell metabolism, proliferation and survival, erythropoiesis, and angiogenesis (Semenza, 2003). HIF-1 binds to hypoxia-response elements (HRE) found in promoter or enhancer DNA regions of target hypoxia-inducible genes that include vascular endothelial growth factor (VEGF), glucose transporter-1 (Glut-1), nitric oxide synthases, and likely between 100 and 200 others (Kaelin and Ratcliffe, 2008).The active HIF-1 complex is a heterodimer consisting of an oxygen-sensitive HIF-1␣ subunit and a constitutively expressed HIF-1␤ subunit. HIF-1␣ possesses an oxygen-dependent degradation domain (ODDD) containing two key proline residues which are, in the presence of oxygen, hydroxylated by HIF prolyl-hydroxylases (PHDs; Kaelin and Ratcliffe, 2008). HIF-1␣ prolyl-hydroxylation allows for recognition by pVHL, the product of the von Hippel-Lindau tumor suppressor gene, the substrate recognition component of an E3 ubiquitin ligase complex that polyubiquitinates and targets HIF-1␣ for proteasomal degradation. In hypoxic conditions, low oxygen leads to HIF-1␣ stabilization due to inhibition of prolyl-hydroxylation and subsequent decreases in HIF-1␣ ubiquitination and degradation (Cockman et al., 2000;Epstein et al., 2001;Schofield and Ratcliffe, 2004).In addition to hypoxia, there are also different nonhypoxic HIF activators that include g...

Cells Lacking Rieske Iron-Sulfur Protein Have a Reactive Oxygen Species-Associated Decrease in Respiratory Complexes I and IV

“…Stable knockdown of RISP in 143B cells showed similar results, and when exogenous H 2 O 2 was added, hypoxic HIF1-␣ stabilization was restored, suggesting that ROS is required for stability of the transcription factor (33). Hypoxic abrogation of HIF1-␣ stabilization upon knockdown of RISP (15,50) and the role of ROS (46) have been reported in other cell types also. Interestingly, recent studies implicate another CIII subunit (UQCRB) in the oxygensensing role during hypoxia (37).…”

The biphasic redox sensing of SENP3 accounts for the HIF-1 transcriptional activity shift by oxidative stress

“…Our previous study proposed an additional mechanism in which mild oxidative stress induced by low doses of H 2 O 2 can rapidly stabilize SENP3, in turn promoting the transcriptional activity of HIF-1 through the deconjugation of SUMO2/3 from the co-activator p300. This activating mechanism functions independently of HIF-1α stabilization, and is required for ROS-mediated HIF-1 transcriptional activity under both hypoxia and normoxia [13] . To establish why the transcriptional activity of HIF-1 can be suppressed by a further dramatic increase of ROS, we hypothesized that SENP3 is a biphasic redox sensor that mediates the biphasic redox regulation of HIF-1.…”

Mitochondrial redox signalling at a glance

“…Since then, mitochondria have emerged as an important node of redox signalling in numerous biologically important areas. Among the most intriguing is the role of mitochondrial ROS in O 2 sensing, especially during hypoxia (Guzy and Schumacker, 2006;Guzy et al, 2008;Patten et al, 2010;Brunelle et al, 2005). In this process, it seems that the production of O 2 · -by the respiratory chain increases under conditions of low O 2 levels (Chandel et al, 1998;Chandel et al, 2000;Guzy et al, 2005).…”