HIF1α stabilization in hypoxia is not oxidant-initiated.

Amit Kumar,John T Pinto,Irina Gazaryan,Saravanan S Karuppagounder,John W Cave,Austin M Rountree,Anatoly A Starkov,Manisha Vaish,Elizabeth T Anderson,Wang Wang,Ian R Sweet,Sheng Zhang,Rajiv R Ratan

doi:10.7554/elife.72873

Abstract

Hypoxic adaptation mediated by HIF transcription factors requires mitochondria, which have been implicated in regulating HIF1α stability in hypoxia by distinct models that involve consuming oxygen or alternatively converting oxygen into the second messenger peroxide. Here, we use a ratiometric, peroxide reporter, HyPer to evaluate the role of peroxide in regulating HIF1α stability. We show that antioxidant enzymes are neither homeostatically induced nor are peroxide levels increased in hypoxia. Additionally, forced expression of diverse antioxidant enzymes, all of which diminish peroxide, had disparate effects on HIF1α protein stability. Moreover, decrease in lipid peroxides by glutathione peroxidase-4 or superoxide by mitochondrial SOD, failed to influence HIF1α protein stability. These data show that mitochondrial, cytosolic or lipid ROS were not necessary for HIF1α stability, and favor a model where mitochondria contribute to hypoxic adaptation as oxygen consumers.

Highlights

Over the past decade, our ability to monitor and manipulate reactive oxygen species (ROS) has grown enormously
Seminal studies have supported the notion that mitochondria are essential regulators of hypoxic adaptation possibly acting via their ability to generate peroxide (Agani et al 2000, Chandel et al 1998)
We show that HIF1α stability mediated by HIF PHDs during hypoxia does not require peroxide

Summary

Introduction

Our ability to monitor and manipulate reactive oxygen species (ROS) has grown enormously. Iron Sulfur cluster proteins in complex III and this leads to an increase in mitochondrial ROS generation via the ubiquinone binding site near the outer aspect of the inner mitochondrial membrane (the Qo site) (Bell et al 2007) Peroxide generated at this site could diffuse through the outer mitochondrial membrane to inhibit HIF PHDs via either direct redox modulation of HIF PHDs (Bell et al 2007) or activation of established redox sensitive p38 MAP kinase signaling (Emerling et al 2005) to stabilize HIF1α. Understanding the role of ROS in HIF1α mediated adaptation has numerous clinical implications (Weidemann et al 2008, Conde et al 2012, Semenza 2014, Sheldon et al 2014, Semenza 2012, Bryant et al 2016) In this manuscript, we leverage a host of complementary approaches that support the conclusion that peroxide is dispensable in regulated HIF1α stability in hypoxia. Our results suggest that HIF1α stability in hypoxia is not oxidant-initiated

Results

Discussion

Materials and Methods