Nitric oxide reverses desferrioxamine- and hypoxia-evoked HIF-1α accumulation—Implications for prolyl hydroxylase activity and iron

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Abstract

Hypoxia inducible factor 1 (HIF-1) senses and coordinates cellular responses towards hypoxia. HIF-1 activity is primarily determined by stability regulation of its alpha subunit that is degraded by the 26S proteasome under normoxia due to hydroxylation by prolyl hydroxylases (PHDs) but is stabilized under hypoxia. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1alpha and promotes hypoxia-responsive target gene expression under normoxia. However, in hypoxia, NO attenuates HIF-1alpha stabilization and gene activation. It was our intention to explain the contrasting behavior of NO under hypoxia. We used the iron chelator desferrioxamine (DFX) or hypoxia to accumulate HIF-1alpha in HEK293 cells. Once the protein accumulated, we supplied NO donors and followed HIF-1alpha disappearance. NO-evoked HIF-1alpha destabilization was reversed by proteasomal inhibition or by blocking PHD activity. By using the von Hippel Lindau (pVHL)–HIF-1alpha capture assay, we went on to demonstrate binding of pVHL to HIF-1alpha under DFX/NO but not DFX alone. Showing increased intracellular free iron under conditions of hypoxia/NO compared to hypoxia alone, we assume that increased free iron contributes to regain PHD activity. Variables that allow efficient PHD activation such as oxygen availability, iron content, or cofactor accessibility at that end allow NO to modulate HIF-1alpha accumulation.

Introduction

HIF-1 plays a major role in sensing and coordinating proper responses of cells to low oxygen partial pressure [1], [2], [3], [4], [5]. HIF-1 is a heterodimer composed of one of the three roughly 120 kDa alpha subunits (HIF-1α, HIF-2α, or HIF-3α) and the 91–94 kDa HIF-1β subunit [6], [7]. Whereas HIF-1β is constitutively expressed, HIF-1α is continuously degraded by the 26S proteasome in the presence of oxygen due to polyubiquitination [8], [9] by an E3-ubiquitin ligase complex that contains the von Hippel Lindau protein (pVHL) [10]. Ubiquitination and binding of pVHL to HIF-1α depends upon hydroxylation of Pro402 and Pro564 within the oxygen-dependent degradation domain (ODD) of HIF-1α [11], [12], [13]. This reaction is facilitated by enzymes known as PH domain-containing enzymes (PHD), i.e., prolyl hydroxylases (PHD1, PHD2, PHD3, and PHD4) [14], [15], [16].

Oxygen, in addition to regulating HIF-1α protein stability, affects HIF transcriptional activity as well. This is explained by an additional hydroxylation of a critical Asn803 residue within the C-terminal transactivation domain (CTAD) of HIF-1α [17]. The asparagine hydroxylase also named factor inhibiting HIF [18] renders CTAD unable to bind the coactivator p300/CBP. Hypoxia attenuates Pro564/402 as well as Asn803 modifications which allows protein stabilization and coactivator recruitment which concomitantly culminates in HIF-1 transactivation [5].

Among the recent progress in the field of oxygen sensing are the discoveries that signals other than hypoxia such as nitric oxide (NO) and/or NO-derived species (RNI) participate in hypoxic signaling. The basic observation is that RNI inhibit hypoxia-induced HIF-1α accumulation while exposure of cells to RNI under normoxia stimulates HIF-1α accumulation. RNI-evoked HIF-1α stabilization and dimerization with HIF-1β allows occupation of hypoxia response element (HRE) sites within target genes to mimic a hypoxic response. These observations are compatible with the notion that RNI may block PHD activity, thus attenuating proline hydroxylation of HIF-1α which will result in its dissociation from pVHL with the consequence of protein accumulation due to decreased proteasomal degradation [19]. It is supposed that NO directly blocks PHDs based on the knowledge that they require Fe2+ for enzymatic activity and that NO binds to Fe2+ containing enzymes [19]. Opposite results are observed when RNI are generated under hypoxic conditions. RNI inhibit hypoxia-induced HIF-1α accumulation, HIF-1 DNA-binding, and HIF-1 transcriptional activation [20], [21], [22]. Although mechanistic details remained unclear, it has been proposed that NO derived from sodium nitroprusside enhanced the interaction between HIF-1α and pVHL in vitro through reactivation of PHDs [23]. Considering mitochondrial consumption of oxygen, it was shown more recently that NO, by blocking cytochrome c oxidase, leaves more oxygen available for PHD to regain its activity under hypoxic conditions [24]. Alternatively, reactive oxygen species including ONOO may contribute to destabilize HIF-1α under hypoxia by mechanisms to be identified [25], [26].

We approached the question of RNI actions in destabilizing HIF-1α by using the iron chelator desferrioxamine (DFX) to stabilize HIF-1α irrespective of decreased oxygen availability. Once HIF-1α was stabilized by DFX treatment, the addition of GSNO or DETA-NO decreased protein appearance. This required HIF-1α protein degradation via the proteasomal pathway and NO donors restored PHD activity in the presence of DFX. We went on to demonstrate that an increase of intracellular free iron correlated with HIF-1α disappearance and suggest that multiple variables that modulate PHD activity are affected by NO under hypoxia to explain how RNI decrease HIF-1α protein amount under hypoxia.

Section snippets

Materials

Medium and supplements were purchased from PAA (Linz, Austria). Fetal calf serum (FCS) was from Biochrom (Berlin, Germany). Desferrioxamine mesylate (DFX), 2,2′-(hydroxynitroso-hydrazono) bis-ethanimine (DETA-NO), Z-Leu-Leu-Leu-al (MG132), glutathione (GSH)-agarose, 2,2′-dipyridyl (2,2′-DPD), anti-actin, and anti-glutathione-S-transferase (GST) antibodies were ordered from Sigma (Schnelldorf, Germany). S-nitrosoglutathione (GSNO) was synthesized as described previously [27].

Impact of nitric oxide donors on desferrioxamine-induced HIF-1α accumulation

Incubations with 100 μM desferrioxamine (DFX) for 6 to 18 h provoked accumulation of HIF-1α protein compared to controls (Fig. 1). HIF-1α accumulation was significantly reduced when DFX was coincubated with NO donors.

In a first set of experiments, we compared two NO donors differing in the NO species being released, e.g., nitrosonium ion vs. NO radical and their estimated half-lives. DFX was added to HEK293 cells for 6 to 9 h to accumulate HIF-1α (Fig. 1A). Following a 6-h incubation period

Discussion

NO plays a dual role in regulating the stability of HIF-1α. In normoxia, NO provokes accumulation of HIF-1α whereas NO prevents stabilization of HIF-1α during hypoxia. In human embryonic kidney cells (HEK293), we show that the NO destabilizing effect on HIF-1α protein amount requires the proteasomal destructive pathway and is a result of a regained PHD activity. Considering that NO donors reactivate PHD activity that is attenuated under normoxic conditions by the iron chelator DFX makes oxygen

Acknowledgments

The technical assistance of Sandra Christmann and Andrea Trinkaus is highly appreciated. We thank Vadim Sumbayev for some initial experiments with iron determination. The work was supported by grants from Deutsche Forschungsgemeinschaft (BR999 to B.B. and SFB530/TPB1 to J.W.D.), Deutsche Krebshilfe (10-2008-Br 2), and Stiftung Rheinland-Pfalz für Innovation.

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