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. 2007 Apr 13;26(1):63-74.
doi: 10.1016/j.molcel.2007.02.024.

Regulation of HIF-1alpha stability through S-nitrosylation

Fang Li  1 , Pierre Sonveaux, Zahid N Rabbani, Shanling Liu, Bin Yan, Qian Huang, Zeljko Vujaskovic, Mark W Dewhirst, Chuan-Yuan Li
Affiliations

Regulation of HIF-1alpha stability through S-nitrosylation

Fang Li et al. Mol Cell. .

Abstract

Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional factor. Under normal oxygen tension, HIF-1 activity is usually suppressed due to the rapid, oxygen-dependent degradation of one of its two subunits, HIF-1alpha. Here we report that normoxic HIF-1 activity can be upregulated through NO-mediated S-nitrosylation and stabilization of HIF-1alpha. In murine tumors, exposure to ionizing radiation stimulated the generation of NO in tumor-associated macrophages. As a result, the HIF-1alpha protein is S-nitrosylated at Cys533 (through "biotin switch" assay) in the oxygen-dependent degradation domain, which prevents its destruction. Importantly, this mechanism appears to be independent of the prolylhydroxylase-based pathway that is involved in oxygen-dependent regulation of HIF-1alpha. Selective disruption of this S-nitrosylation significantly attenuated both radiation-induced and macrophage-induced activation of HIF-1alpha. This interaction between NO and HIF-1 sheds new light on their involvement in tumor response to treatment as well as mammalian inflammation process in general.

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Figures

Figure 1
Figure 1
Nitric oxide is a key regulator of radiation-induced HIF-1α activation. (A) 4T1-ODD-luc or 4T1-luc tumors were established in the hind legs of nude mice and irradiated (at day 0) with or without the administration of L-NAME (at day −1). ODD-luc level were then monitored post irradiation. Tumors with the CMV-luc reporter were used as controls. Notice the significant inhibitions of ODD-luc expression by the use of L-NAME. Eight animals were used in each group and the error bars indicate standard error of the mean. P<0.05 from day 4 (two-way ANOVA). (B) Radiation-induced activation of endogenous HIF-1 binding activity to hypoxia responsive element (HRE) was measured by ELISA in tumors irradiated 5 days earlier. In each group average results from 4 tumor samples were shown (P<0.05, Student’s t test). Error bars represent standard deviation. (C) Radiation induced increase in intratumoral VEGF level as measured by ELISA. In each group, average results from 4 tumors were shown (P<0.05, Student’s t test). Error bars represent standard deviation.
Figure 2
Figure 2
The critical role of iNOS in radiation induced HIF-1α activation. (A) Effect of an iNOS specific inhibitor. Subcutaneous tumors were established in the hind legs of nude mice through the use of 4T1-ODD-luc cells and irradiated with or without the administration of 1400W, an (iNOS) inhibitor. ODD-luc level was then monitored daily post irradiation. Eight animals were used in each group. Significant inhibition of radiation-induced HIF-1 activation was observed in the group treated with 1400W(P<0.001 from day 4, two-way ANOVA). (B) Effect of genetic disruption of the iNOS gene in host animal on HIF-1α activation. Tumors were established from B16F10-ODD-luc cells in syngeneic wild type or iNOS−/− C57BL/6 mice. In some groups, mice received L-NAME one day before tumor irradiation (6 Gy) at day 0. Luciferase activities were determined every other day. Eight animals were used in each group and the error bar represents the standard error of the mean. In wild type C57BL/6 mice (solid lines), the difference between L-NAME treated and non-treated groups are statistically different (P<0.01 on days 1, 3 & 5, two-way ANOVA test). In iNOS−/− mice (broken lines), the difference between L-NAME treated and non-treated groups are not significant (P>0.05 at all time points, two-way ANOVA).
Figure 3
Figure 3
The role of macrophages in radiation induced HIF-1α activation. (A) Tumors were established from 4T1-ODD-luc cells in nude mice. In some mice, macrophages were depleted by injection of carrageenan. Selected groups of mice also received L-NAME one day before irradiation (6 Gy). Luciferase expression was determined every other day. Eight mice were included in each group. The error bar represents the standard error of the mean. (B) Immunohistochemistry analysis of HIF-1α., iNOS, and macrophages in tumors. Mice with irradiated 4T1 tumors were sacrificed and their tumors excised 5 days after localized 6-Gy or sham irradiation of tumors. Shown in the left panel are representative results from co-staining of CD68 (a marker for macrophages (Mφ)) and iNOS. Co-staining of HIF-1α and iNOS were shown on the right panel. In each case, merged pictures are provided. Orange color in both panels represents co-localization.
Figure 4
Figure 4
Prevention of normoxic HIF-1α degradation though S-nitrosylation of cysteine 533. (A) Amino acid sequence conservation across different species for murine HIF-1α Cys 533. (B) The effects of various stimuli (0.5% O2 for 24 hrs, 10 µM MG132, a proteasome in inhibitor, for 12 hrs, and CoCl2 at 240 µM for 12 hrs) on the activation of wild type ODD-luc and [C533S]-ODD-luc in 4T1 cells. The data shown are the results of triplicate experiments. The error bars represent standard deviation. In all treatment groups, P>0.05 between wild type and mutant ODD-luc expression levels (Student’s t test). (C) Wild type ODD-luc or [C533S] ODD-luc transduced 4T1 cells were treated with GSNO (1mM) and measured for luciferase activities. Note the significant attenuation of luc expression in [C533S]-ODD-luc transduced cells (P<0.01, Student’s t test). Each data point is the results of triplicate experiments and the error bars represent standard error. The unit for light output shown is p/sec/CM2/Sr. (D) Tumors were established from 4T1 cells transduced with wild type or [C533S]-ODD-luc and irradiated (6Gy). Luciferase expression was then monitored every other day. Note the significant attenuation of luciferase expression in [C533S]-ODD-luc-transduced 4T1 tumors (P<0.01 from day 5, two-way ANOVA). Each group has five animals and the error bar represents standard error of the mean. (E) S-nitrosylation of C533 in the ODD domain. 4T1 cells transduced with wild type ODD or [C533S]-ODD (both with a myc-tag at the 5’ end for western blot detection) were exposed to GSNO and then lysed. S-nitrosylation of ODD was determined through the biotin switch assay(Jaffrey and Snyder, 2001). Please note the clear nitrosylation signal for wild-type ODD after GSNO treatment and the absence of it in [C533S] ODD with or without GSNO treatment. (F) Lack of effect of C533 nitrosylation on proline hydroxylation in the ODD domain of HIF-1α. 4T tumor cells were transduced with pCMV-ODD-mycTag. Where indicated, the transfected cells were exposed to 1 mM GSNO for 8 hours. Western blot analyses of the lysates were then conduced for the total amount of ODD (though the use of anti-mycTag antibody) and the hydroxylated ODD (through the use of an antibody directed against the hydroxylated HIF-1α). No significant difference in the amount of hydroxylated ODD was observed between the wild type (WT) and the [C533S) mutant in non-treated (NT) or GSNO-treated cells. (G) Absence of binding between nitrosylated ODD and VHL. 4T1 tumor ells were transduced with CMV-ODD-mycTag CMV-[C533S]-ODD-mycTag, or CMV-HA-VHL. Where indicated, ODD-transfected cells were exposed to 1 mM GSNO for 8 hours. The lysate of ODD-transfected cells was admixed with lysate of cells expressing HA-VHL. Mixed lysates were immunoprecipitated with anti-HA antibody to pull down the VHL protein. The immunoprecipitate was then immunoblotted with antibody against mycTag to detect ODD bound to VHL. Total tagged ODD (Input ODD) and VHL (Input VHL) were detected using Western blot with antibodies against mycTag and HA-tag, respectively.
Figure 5
Figure 5
The effect of C533S mutation on activated macrophage-mediated HIF-1α stabilization by use of a full-length HIF-1α-luciferase fusion reporter gene. 4T1 cells stably transduced with either the wild-type full-length HIF-Luc reporter gene or its mutant version C533S HIF-luc gene were cultured in 24-well plates to 50% confluence (1.0×105 cell per well). About 1.5× 10 5 RAW 264.7 (mouse macrophage) were added after 24 hours. Two hrs later, 10 µg/ml of LPS and 0.1 ng/ml of IFN-γ were added to the medium. Twelve hours later, cells were imaged for luciferase activity in the Xenogen IVIS200 system. It is clear that the addition of LPS, which activated the wild type HIF-1α-luc fusion reporter significantly (p<0.005, t test), had minimal effect minimal effect on the C533S mutant HIF-1α fusion reporter.
Figure 6
Figure 6
Enhanced anti-tumor efficacy of radiotherapy in combination with L-NAME. B16F10 and 4T1 tumors were established in syngeneic C57BL/6 and Balb/C mice, respectively and irradiated with 3 fractions of X-rays at 6Gy/fraction (irradiation every other day). In some of the groups, L-NAME was administered in the drinking water one day before irradiation. Tumor sizes were monitored every other day. At least 6 animals were used in each treatment groups. Tumor sizes were then plotted against time for each tumor type. The error bars represent the standard error of the mean. (A) 4T1 tumor growth delay. (B) B16F10 melanoma growth delay.
Figure 7
Figure 7
A diagram summarizing radiotherapy induced HIF-1α stabilization through nitrosylation of C533 by macrophage-derived nitric oxide.
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