Abstract
Hypoxia-inducible factor 1 (HIF-1) mediates many of the systemic and cellular responses to intermittent hypoxia (IH), which is an experimental model that simulates O2 saturation profiles occurring with recurrent apnea. IH-evoked HIF-1α synthesis and stability are due to increased reactive oxygen species (ROS) generated by NADPH oxidases, especially Nox2. However, the mechanisms by which IH activates Nox2 are not known. We recently reported that IH activates xanthine oxidase (XO) and the resulting increase in ROS elevates intracellular calcium levels. Since Nox2 activation requires increased intracellular calcium levels, we hypothesized XO-mediated calcium signaling contributes to Nox activation by IH. We tested this possibility in rat pheochromocytoma PC12 cells subjected to IH consisting alternating cycles of hypoxia (1.5% O2 for 30 sec) and normoxia (21% O2 for 5 min). Kinetic analysis revealed that IH-induced XO preceded Nox activation. Inhibition of XO activity either by allopurinol or by siRNA prevented IH-induced Nox activation, translocation of the cytosolic subunits p47phox and p67phox to the plasma membrane and their interaction with gp91phox. ROS generated by XO also contribute to IH-evoked Nox activation via calcium-dependent protein kinase C stimulation. More importantly, silencing XO blocked IH-induced upregulation of HIF-1α demonstrating that HIF-1α activation by IH requires Nox2 activation by XO.
Highlights
Sleep disordered breathing with recurrent apnea is characterized by transient (~10–15 sec in adults) repetitive cessations of breathing, resulting in periodic decreases in arterial blood O2 or intermittent hypoxia (IH)
Analysis of the time course revealed that xanthine oxidase (XO) activity increased progressively with increasing number of IH cycles with a significant activation after 5 cycles, whereas NADPH oxidase (Nox) activation required a minimum of 10 cycles of IH (Fig. 1A)
XO activation by IH is mediated by proteolytic conversion of xanthine dehydrogenase (XDH) to XO which is blocked by inhibiting proteolysis with a trypsin inhibitor [13]
Summary
Sleep disordered breathing with recurrent apnea is characterized by transient (~10–15 sec in adults) repetitive cessations of breathing, resulting in periodic decreases in arterial blood O2 or intermittent hypoxia (IH). Hypoxia-inducible factor-1 (HIF-1) is the master transcriptional activator that regulates gene expression during hypoxia [3]. Intermittent Hypoxia and HIF-1α Activation by NADPH Oxidase activates HIF-1 mediated transcription in cell cultures and rodents [4,5,6]. Mice with heterozygous deficiency of HIF-1α exhibit remarkable absence of IH-induced hypertension, and breathing abnormalities [5, 6], suggesting that activation of HIF-1 contributes to the cardiorespiratory abnormalities caused by IH. Analysis of the mechanisms underlying HIF-1 activation showed that IH activates NADPH oxidase (Nox), especially Nox2 [7]. Disruption of Nox function, prevents HIF-1 activation by IH in cell cultures and in mice [4, 8], suggesting that Nox is critical for HIF-1 activation by IH. The mechanism by which IH activates Nox has not been examined
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