Hypoxia-inducible factor 1 mediates increased expression of NADPH oxidase-2 in response to intermittent hypoxia

Sleep disordered breathing with recurrent apnea (transient, repetitive cessation of breathing) is a major cause of morbidity and mortality in adult humans (Nieto et al., 2000) and pre-term infants (Poets et al., 1994). Recurrent apneas are associated with periodic decreases in arterial blood oxygen or intermittent hypoxia (IH). Humans with recurrent apnea and rodents exposed to IH exhibit autonomic morbidities including persistent sympathetic activation, hypertension, and elevated circulating catecholamines (Narkiewicz and Somers, 1997; Peppard et al., 2000; Prabhakar and Kumar, 2010). Studies on humans (Narkiewicz and Somers, 1997) and rodent models of IH (Fletcher et al., 1992; Peng et al., 2006) have shown that the carotid body, which is the primary chemoreceptor for detecting changes in arterial PO2, responds to IH by triggering reflex activation of the sympathetic nervous system and enhanced catecholamine secretion from adrenal medullary chromaffin cells (AMC), leading to elevated circulating catecholamines and elevated blood pressure (Bao et al., 1997; Kumar et al., 2006; Souvannakitti et al., 2009). IH increases the levels of reactive oxygen species (ROS) in the carotid body (Peng et al., 2003) and adrenal medulla (Kumar et al., 2006; Souvannakitti et al., 2009). Anti-oxidant treatment prevents increased carotid body function (Peng et al., 2003; Peng and Prabhakar, 2004; Del Rio et al., 2010), augmented catecholamine secretion from AMC (Kumar et al., 2006; Kuri et al., 2007; Souvannakitti et al., 2009), and elevated blood pressure (Peng et al., 2006; Troncoso Brindeiro et al., 2007), indicating that ROS signaling is a critical cellular mechanism underlying IH-evoked autonomic morbidities. NADPH oxidase (Nox) activity is a major source of cellular ROS (Bedard and Krause, 2007). IH leads to increased expression of several Nox isoforms in the carotid body (Peng et al., 2009), AMC (Souvannakitti et al., 2010), and central nervous system (CNS; Zhan et al., 2005) as well as in cultured PC12 rat pheochromocytoma cells, which are derived from AMC (Yuan et al., 2008). Of the various Nox isoforms, Nox2 has been implicated in mediating the effects of IH on carotid body function (Peng et al., 2009), catecholamine secretion from AMC (Souvannakitti et al., 2010), and sleep behavior (Zhan et al., 2005). However, mechanisms underlying increased Nox2 gene expression in response to IH have not been elucidated. The transcriptional activator hypoxia-inducible factor 1 (HIF-1) is a master regulator of O2 homeostasis that controls multiple physiological processes by regulating the expression of hundreds of genes (Mole et al., 2009; Semenza, 2009). HIF-1 is a heterodimeric protein composed of a constitutively expressed HIF-1β subunit and an O2-regulated HIF-1α subunit (Wang et al., 1995). We previously reported that IH increases HIF-1α protein levels in PC12 cell cultures (Yuan et al., 2005, 2008) and in mice (Peng et al., 2006). In catecholamine-producing PC12 cells, the induction of HIF-1α protein levels by IH requires ROS-dependent activation of phospholipase Cγ (PLCγ), protein kinase C (PKC), and mammalian target of rapamycin (mTOR; Yuan et al., 2008). Physiological studies of wild-type (WT) mice exposed to IH revealed striking autonomic morbidities, including heightened carotid body activity, elevated plasma catecholamines, exaggerated hypoxic ventilatory response, and hypertension (Peng et al., 2006). In contrast, littermate Hif1a+/− mice, which are heterozygous for a null [knockout (KO)] allele at the locus encoding HIF-1α, do not display any of these IH-induced autonomic responses (Peng et al., 2006). Furthermore, ROS levels were significantly increased in the CNS of IH-exposed WT mice, but not in Hif1a+/− mice (Peng et al., 2006). Taken together, these results indicated that IH-induced ROS leads to HIF-1 activation, which leads to further ROS generation, thereby creating a feed-forward mechanism that is essential for the pathogenesis of cardiovascular and respiratory abnormalities. Establishing the molecular mechanism by which HIF-1 increases ROS generation is the critical next step in understanding the pathobiology of IH. Based on the observations described above, we tested the hypothesis that in response to IH, HIF-1 increases ROS generation by activation of the Nox2 gene.