Abstract

Oxygen-dependent prolyl hydroxylation of hypoxia-inducible factor (HIF) by a set of closely related prolyl hydroxylase domain enzymes (PHD1, 2 and 3) regulates a range of transcriptional responses to hypoxia. This raises important questions about the role of these oxygen-sensing enzymes in integrative physiology. We investigated the effect of both genetic deficiency and pharmacological inhibition on the change in ventilation in response to acute hypoxic stimulation in mice. Mice exposed to chronic hypoxia for 7 days manifest an exaggerated hypoxic ventilatory response (HVR) (10.8 ± 0.3 versus 4.1 ± 0.7 ml min−1 g−1 in controls; P < 0.01). HVR was similarly exaggerated in PHD2+/− animals compared to littermate controls (8.4 ± 0.7 versus 5.0 ± 0.8 ml min−1 g−1; P < 0.01). Carotid body volume increased (0.0025 ± 0.00017 in PHD2+/− animals versus 0.0015 ± 0.00019 mm3 in controls; P < 0.01). In contrast, HVR in PHD1−/− and PHD3−/− mice was similar to littermate controls. Acute exposure to a small molecule PHD inhibitor (PHI) (2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid) did not mimic the ventilatory response to hypoxia. Further, 7 day administration of the PHI induced only modest increases in HVR and carotid body cell proliferation, despite marked stimulation of erythropoiesis. This was in contrast with chronic hypoxia, which elicited both exaggerated HVR and cellular proliferation. The findings demonstrate that PHD enzymes modulate ventilatory sensitivity to hypoxia and identify PHD2 as the most important enzyme in this response. They also reveal differences between genetic inactivation of PHDs, responses to hypoxia and responses to a pharmacological inhibitor, demonstrating the need for caution in predicting the effects of therapeutic modulation of the HIF hydroxylase system on different physiological responses.

Highlights

  • Acute exposure to hypoxia elicits a rapid increase in ventilation that occurs within seconds, followed by a further progressive increase in ventilation developing over a period of hours to days

  • In contrast to PHD2+/− mice, the hypoxic ventilatory response to this gas mixture was unaltered in both PHD1−/− and PHD3−/− mice (Fig. 6A and B; Supplemental Table 2). These results indicate that partial deficiency of PHD2 – the principal enzyme acting on hypoxia-inducible factor (HIF)-1α – had the clearest effects in mimicking chronic hypoxia by inducing ventilatory sensitivity

  • This study demonstrates that heterozygous inactivation of the ‘oxygen-sensing’ enzyme PHD2 is associated with markedly enhanced ventilatory sensitivity to hypoxia

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Summary

Introduction

Acute exposure to hypoxia elicits a rapid increase in ventilation that occurs within seconds, followed by a further progressive increase in ventilation developing over a period of hours to days. It is recognised that many cellular and systemic responses to hypoxia are mediated by the HIF hydroxylase system, in which the oxygen-sensitive signal is generated by impaired catalysis of a set of 2-oxoglutarate-dependent dioxygenases (Kaelin & Ratcliffe, 2008; Prabhakar & Semenza, 2012) These enzymes catalyse the oxygen-dependent post-translational hydroxylation of HIF-α subunits to regulate both the stability and activity of the transcriptional complex. Despite binding a similar DNA consensus, HIF-1α and HIF-2α have only partially overlapping transcriptional targets and have distinct biological actions in many settings (Hu et al 2003; Schodel et al 2011) The stability of both HIF-1α and HIF-2α is regulated by prolyl hydroxylation which promotes degradation by the von Hippel–Lindau (VHL) ubiquitin E3 ligase and degradation by the ubiquitin–proteasome pathway (Kaelin & Ratcliffe, 2008; Prabhakar & Semenza, 2012). This has generated widespread interest in the pharmaceutical development of such inhibitors for anaemia, ischaemic diseases and other conditions where augmentation of hypoxic signalling may be beneficial (Hsieh et al 2007; Fraisl et al 2009; Bernhardt et al 2010; Yan et al 2010; Rose et al 2011)

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