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Nitric oxide and reactive oxygen species exert opposing effects on the stability of hypoxia-inducible factor-1 (HIF-1) in explants of human pial arteries¹

THERESA L. WELLMAN^,¶, JOSHUA JENKINS^,¶, PAUL L. PENAR^,,¶, BRUCE TRANMER^,¶, RIMA ZAHR and KAREN M. LOUNSBURY^,¶,2

Department of Pharmacology,
Department of Surgery, Division of Neurological Surgery, and
^¶ Totman Center for Cerebrovascular Research, University of Vermont, Burlington, Vermont, USA

²Correspondence: Department of Pharmacology, University of Vermont, Given Bldg., 89 Beaumont Ave., Burlington, VT 05405, USA. E-mail: Karen.Lounsbury{at}uvm.edu

SPECIFIC AIMS

Responses of cerebral vessels to hypoxia and nitric oxide (NO) are critical in physiological and pathological responses to ischemia. We tested the hypothesis that the effect of NO on hypoxia-inducible factor-1 (HIF-1) signaling in cerebrovascular smooth muscle cells is critically dependent on the form of NO and the presence on reactive oxygen species (ROS) in the environment of the responding cell. To investigate this, we used explants of human cerebrovascular smooth muscle cells to examine the regulation of HIF-1 stabilization and transcriptional activity after exposure to hypoxia, NO donors, and superoxide (O₂) generators/scavengers.

PRINCIPAL FINDINGS

1. NO donors exert opposing effects on HIF-1 stability and transcriptional activity, and the effect correlates with formation of ROS
To study the induction of HIF-1 in explanted human cerebral vascular smooth muscle cells (hcVSMCs), cells were treated with CoCl₂ or incubated at 2% O₂, followed by immunoblot or immunofluorescence (IMF) using anti-HIF-1 antibodies. The hcVSMCs responded to CoCl₂ and 2% O₂ by increasing HIF-1. To conserve hcVSMCs at low passage, HIF-1 protein levels were determined by quantifying HIF-1 immunofluorescence.

To determine the effect of NO donors on HIF-1 protein expression, cells were incubated in the presence of a variety of NO donors alone or in conjunction with exposure to CoCl₂ or 2% O₂. NO donors NOC-18 and GSNO stabilized HIF-1 alone and were synergistic with hypoxia, whereas NO donors SNP and SIN-1 significantly inhibited basal HIF-1 levels and HIF-1 stimulated by GSNO and NOC-18 (Fig. 1 A). The inhibition of HIF-1 protein stabilization by SNP and SIN-1 correlated with a dose-dependent inhibition of transcription of VEGF, but not HIF-1, detected by a VEGF promoter-luciferase assay and RT-PCR.

View larger version (48K): [in this window] [in a new window]   Figure 1. SIN-1 elicits a dose-dependent inhibition of NO-mediated HIF-1 stabilization and a dose-dependent increase in tyr-nitration. A) hcVSMCs were treated for 4 h with GSNO (filled circles) or NOC-18 (open circles) in the presence of SIN-1. HIF-1 was detected by IMF and quantified using image analysis. B) hcVSMCs were treated with GSNO (as in panel A) and SIN-1, followed by IMF for HIF-1 (red) and N-Tyr (green). Nuclei were stained with YoYo (blue).

To determine whether the loss of HIF-1 correlated with an increase in the production of nitrated species, cells treated with GSNO and increasing concentrations of SIN-1 were analyzed for the presence of nitrotyrosine-modified proteins. As shown in Fig. 1B , loss of HIF-1 occurred at lower concentrations of SIN-1 than that which produced detectable tyrosine nitration; tyrosine nitration was not detected in the nuclei of cells treated with SIN-1.

2. Inhibition of HIF-1 signaling by SNP and SIN-1 can be overcome by superoxide dismutase
Because SNP inhibited HIF-1 to a similar extent as SIN-1, it was plausible that its effects were mediated through generation of ROS, particularly peroxynitrite (ONOO). As shown in Fig. 2 A, the free radical scavenging system, superoxide dismutase (SOD) and catalase, overcame the inhibitory effect of SNP on hypoxia-induced HIF-1 expression. SOD alone was able to rescue the SNP effect; although catalase alone had no effect on HIF-1, it augmented the SOD effect. SOD/catalase was also able to reverse the effects of SIN-1 on HIF-1 stabilization (Fig. 2B ) and transcriptional activation (Fig. 2C ) stimulated by NOC-18, GSNO, and CoCl₂. SOD induced HIF-1 in the absence of hypoxia in a dose-dependent fashion unrelated to the addition of catalase (Fig. 2D ). These observations strongly suggest that the observed inhibition of HIF-1 signaling by SNP and SIN-1 is dependent on the production of ROS.

View larger version (33K): [in this window] [in a new window]   Figure 2. SOD overcomes the inhibition of HIF-1 by SNP or SIN-1 and alone is able to induce HIF-1 stability. A) hsVMCs were exposed to hypoxia (2% O2) for 4 h in the presence or absence of SNP. Where indicated, SOD and/or catalase (Cat) was included. Compiled fluorescence intensities normalized to the hypoxia response are shown; n = 4, *different from control, P < 0.001, #different from hypoxia+SNP, P < 0.001. B) hsVMCs were treated with NO donors or CoCl2. SOD and catalase were included as indicated. HIF-1 was detected by IMF and quantified as in panel A; n = 3, * different from control, P< 0.001, #different from condition+SIN-1, P< 0.001. C) raVSMCs expressing a VEGF-driven luciferase construct were treated in duplicate with the indicated agents for 24 h, followed by lysis and detection of luciferase activity. Values were normalized to Ranilla luciferase; averaged data representative of 3 experiments. D) Cells were treated with the indicated concentrations of SOD in the absence (filled circles) or presence (open circles) of catalase. HIF-1 was detected by IMF and quantified as in panel A. Data are representative of 3 experiments.

3. A O₂ generating system of xanthine/xanthine oxidase is inhibitory to HIF-1 stability
Although the data pointed to a role for ROS in the inhibition of HIF-1 signaling, a clear role for nitrating species was not evident. To explore a role for O₂ independent of NO, the effect of an O₂ generating system of xanthine/xanthine oxidase on HIF-1 signaling was tested. Xanthine/xanthine oxidase significantly inhibited HIF-1 stability and transcriptional activity in control cells and cells exposed to hypoxia, CoCl₂, or NOC-18, suggesting that O₂ alone is inhibitory to HIF-1 signaling. The NO scavenger PTIO exhibited a dose-dependent inhibition of HIF-1 stabilization by NOC-18 but was not able to reverse the inhibitory effect of SIN-1 on CoCl₂-mediated HIF-1 stability. These results suggest that the balance of NO/ ONOO is likely more important in HIF-1 signaling than a direct effect of protein nitration.

CONCLUSIONS AND SIGNIFICANCE

The role of NO in arterial hypoxia signaling pathways remains elusive. Because NO is highly redox sensitive, the dichotomy of effects in response to NO donors may be due to differences in the mechanisms of NO release and their ability to produce ROS. We observed an inhibition of HIF-1 stability and transcriptional activity when cells were exposed to SNP or to SIN-1 (known to produce NO and O₂). A negative role for NO on HIF-1 signaling in O₂ generation was supported by a reversal of the inhibitory effect of SIN-1 by SOD. The inability of SIN-1 to inhibit HIF-1 signaling under hypoxic conditions further confirms the requirement for O₂ in the inhibitory response, as molecular oxygen is required for its production. Removal of NO with PTIO reduced the HIF-1 stabilization by GSNO but was unable to reverse the inhibition by SIN-1, indicating that NO is required for the stimulatory effect but that O₂ is key to the inhibitory effect. ROS are known to serve as second messengers to activate multiple intracellular proteins and enzymes. SNP and SIN-1 thus may offset the balance of normal HIF-1 signaling by contributing ROS directly to the system. We propose that NO-mediated signaling that occurs in an oxidative environment leads to degradation of HIF-1, whereas NO in a reducing environment enhances the stability of HIF-1 (Fig. 3 ).

View larger version (22K): [in this window] [in a new window]   Figure 3. A model for the regulation of HIF-1 signaling by NO and O2. NO shifts the HIF-1 equilibrium toward stability and induction of gene transcription whereas O2 shifts the HIF-1 equilibrium toward ubiquitylation and degradation.

We observed an increase in tyrosine nitration after SIN-1 exposure localized to the extracellular matrix. Restricted to the extracellular matrix, detection of tyrosine nitration is similar to that seen in intact coronary arteries and endothelial cells. Nitrotyrosine modifications were not detected in nuclei and were observed only at concentrations of SIN-1 in which HIF-1 was ablated. It is therefore not likely that HIF-1 is a direct target for tyrosine nitration unless modification results in immediate degradation of HIF-1.

Stabilization of HIF-1 by NO compounds has been proposed to depend on protein S-nitration. Hif-1 has been shown to be a substrate for S-nitration in solution and in cells. It is not likely that S-nitration is involved in the inhibition of HIF-1 by SIN-1 because inhibition was also observed in the presence of ROS produced independent of NO. Other protein targets of NO and ROS that could modulate HIF-1 stability include proline hydroxyl transferase, proteins involved in ubiquitylation, including von Hippel-Lindau protein, and components of the proteosome.

These results have important implications for signaling in response to acute and chronic hypoxia in cerebral arteries. Identification of HIF-1 stability as a molecular target for NO in cerebrovascular smooth muscle cells provides more evidence for the importance of endothelial cell communication with the smooth muscle layer in preconditioning and recovery from ischemic events or responses to tumors. NO has been shown to enhance VEGF expression during cerebral ischemia. However, ROS have been detected in the reperfused (but not ischemic) brain; thus, rapid loss of HIF-1 after reoxygenation would be predicted by our results. Significant levels of HIF-1 have been detected for extended periods after in vivo ischemia and reperfusion, suggesting a role for other signaling pathways in maintaining HIF-1 signaling.

Our study provides strong evidence that NO promotes HIF-1 stability; however, the presence ROS prevents basal and NO-mediated HIF-1 signaling. These data provide a rationale for the variety of biological effects seen after the application of NO donors to hypoxic cell systems and confirm the complexities associated with the potential use of NO donors in the treatment of ischemia-associated diseases.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0143fje

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