Nitric Oxide Impairs Normoxic Degradation of HIF-1α by Inhibition of Prolyl Hydroxylases

Abstract

Hypoxia inducible factor-1 (HIF-1) is the master regulator of metabolic adaptation to hypoxia. It is appreciated that HIF-1␣ accumulation is achieved under normoxic conditions by e.g., nitric oxide. We determined molecular mechanisms of HIF-1␣ accumulation under the impact of S-nitrosoglutathione (GSNO). In human embryonic kidney cells GSNO provoked nuclear accumulation of HIF-1␣. This appeared unrelated to gene transcription and protein translation, thus pointing to inhibition of HIF-1␣ degradation. Indeed, GSNO as well as the hypoxia mimic CoCl 2 decreased ubiquitination of HIF-1␣ and GSNO-induced HIF-1␣ failed to coimmunoprecipitate with pVHL (von Hippel Lindau protein). Considering that HIF-1␣-pVHL interactions require prolyl hydroxylation of HIF-1␣, we went on to demonstrate inhibition of HIF-1␣ prolyl hydroxylases (PHDs) by GSNO. In vitro HIF-1␣-pVHL interactions revealed that GSNO dose-dependently inhibits PHD activity but not the interaction of a synthetic peptide resembling the hydroxylated oxygen-dependent degradation domain of HIF-1␣ with pVHL. We conclude that GSNO-attenuated prolyl hydroxylase activity accounts for HIF-1␣ accumulation under conditions of NO formation during normoxia and that PHD activity is subject to regulation by NO. INTRODUCTIONThe heterodimeric transcription factor hypoxia inducible factor-1 (HIF-1) plays a central role in adaptation to decreased oxygen availability (Semenza, 2002;Wenger, 2002;Brü ne and Zhou, 2003). HIF-1 is composed of the two basic helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) proteins HIF-1␣ and the aryl hydrocarbon receptor nuclear translocator (ARNT), also known as HIF-1␤ (Wang and Semenza, 1995). In many cell types, the mRNA of both HIF-1␣ and HIF-1␤ appear permanently expressed and HIF-1␤ protein is constitutively present. However, HIF-1␣ protein is kept at a low or undetectable level under normoxia. In hypoxia, HIF-1␣ is strikingly induced, translocates to the nucleus, and dimerizes with HIF-1␤ to form HIF-1, which binds to hypoxiaresponsive elements (HRE) in regulatory regions of an impressive array of target genes involved in angiogenesis, erythropoiesis, vasomotor control, and energy metabolism as well as in cell survival decisions (for references, see Maxwell et al., 2001;Wenger, 2002; Zhu et al., 2002). This makes HIF-1 the master regulator of oxygen homeostasis to meet cell and tissue requirements in a situation of oxygen deficiency.In normoxia HIF-1␣ is bound to the von Hippel Lindau protein (pVHL) (Maxwell et al., 1999;Hon et al., 2002;Min et al., 2002), which is the substrate recognizing component of an E3 ubiquitin ligase complex (Cockman et al., 2000;Ohh et al., 2000;Tanimoto et al., 2000). Consequently, HIF-1␣ is polyubiquitinated and degraded by the 26S proteasome system, thus accounting for its low normoxic level of expression (Salceda and Caro, 1997;Huang et al., 1998;Kallio et al., 1999). The requirement of pVHL for HIF-1␣ degradation is underscored in cells that do not contain a functional pVHL, which leads to high levels of HIF-1␣ in normox...

Hypoxia-Inducible Factor (HIF) 1α Accumulation and HIF Target Gene Expression Are Impaired after Induction of Endotoxin Tolerance

“…Under hypoxic conditions, prolyl hydroxylation is reduced, thus finally leading to an increase in HIF-1␣ protein and expression of HIF-1 target genes (9). In addition to hypoxia, which is also a typical feature of the tissue microenvironment during bacterial infection, multiple inflammatory stimuli lead to HIF-1␣ accumulation and activation (10,11). Along with this, an important role of HIF-1 for the host immune response during bacterial infection has recently been shown (12,13).…”

Reactive oxygen species and cellular oxygen sensing

“…In direct contrast to previous findings, some groups found that ρ° cell lines lacking functional mitochondria still stabilize HIFα under hypoxia [76,77]. Other studies suggested that cells with impaired mitochondrial function fail to stabilize HIFα under hypoxia, not because of defective ROS-generation, but because of intracellular O 2 re-distribution which leads to increased availability of O 2 as a substrate for the hydroxylation reaction [72,78]. Although these reports challenge a role for mitochondrial ROS in cellular O 2 sensing, they do not explain why antioxidants, including the recently described mitochondrially-targeted compound MitoQ, block hypoxic HIFα activity [79].…”

Role and regulation of prolyl hydroxylase domain proteins

“…19,20 Intracellular Fe(II) concentration. NO may inhibit PHD activity by chelating Fe(II) or other mechanisms in addition to the previously mentioned role in promoting PHD activity by oxygen redistribution 21,22 (Figure 2). The net outcome may depend on which effect happens to dominate and is more easily observed under specific experimental conditions.…”