Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Free PMC article
Full text links

Actions

Share

.2008 Feb 29;40(1):71-83.
doi: 10.3858/emm.2008.40.1.71.

Nur77 upregulates HIF-alpha by inhibiting pVHL-mediated degradation

Affiliations

Nur77 upregulates HIF-alpha by inhibiting pVHL-mediated degradation

Bu Yeon Kim et al. Exp Mol Med..

Abstract

In this study, we investigated the role of Nur77, an orphan nuclear receptor, in HIF-alpha transcriptional activity. We found that Nur77 associates and stabilizes HIF-1alpha via indirect interaction. Nur77 was found to interact with pVHL in vivo via the alpha-domain of pVHL. By binding to pVHL, Nur77 competed with elongin C for pVHL binding. Moreover, Nur77-binding to pVHL inhibited the pVHL-mediated ubiquitination of HIF-1alpha and ultimately increased the stability and transcriptional activity of HIF-1alpha. The ligand-binding domain of Nur77 was found to interact with pVHL and the expression of this ligand-binding domain was sufficient to stabilize and transactivate HIF-1alpha. Under the conditions that cobalt chloride was treated or pVHL was knocked down, Nur77 could not stabilize HIF-alpha. Moreover, Nur77 could not further stabilize HIF-2alpha in A498/VHL stable cells, which is consistent with our finding that Nur77 indirectly stabilizes HIF-alpha by binding to pVHL. Thus, our results suggest that an orphan nuclear receptor Nur77 binds to pVHL, thereby stabilizes and increases HIF-alpha transcriptional activity under the non-hypoxic conditions.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Nur77 interacts with and accumulates HIF-1α. (A) HEK293 cells were transiently transfected with pCMV-HA-HIF-1α and pCS2+MT-Nur77. Cell lysates were immunoprecipitated with anti-HA antibody, and probed with anti-myc antibody. (B) Nur77 colocalizes with and accumulates HIF-1α in nucleus. HEK293 cells were transiently transfected with the same expression vectors as in (A), stained with anti-HA and anti-Nur77 antibodies, and then treated with anti-mouse-IgG-rhodamine and anti-rabbit IgG-FITC antibodies. After extensive washing, cells were observed under a confocal microscope.
Figure 2
Figure 2
Nur77 overexpression stabilized HIF-1α protein and increased HIF-1α transcription activity. (A) Nur77 overexpression stabilized HIF-1α at the protein level. HEK293 cells were transiently transfected with pCMV-HA-HIF-1α (0.1 µg) and/or increasing amount of pCS2+MT-Nur77. After 24 h of incubation, cell lysates were subjected to SDS-PAGE, and then immunoblotted with either anti-HA or anti-myc antibody. The same amount of cell lysate was probed with anti-β-actin antibody as a control. (B) Nur77 did not affect the mRNA level of HIF-1α. Total cellular RNA was prepared from HEK293 cells transfected with the indicated constructs. RT-PCR was performed as described in Materials and Methods. (C) Nur77 induced endogenous HIF-1α. HEK293 cells were transfected with pCS2+MT-Nur77. After 24 h of incubation, nuclear extracts were prepared as described previously (Kim et al., 2005b). Nuclear extracts were subjected to SDS-PAGE, and then immunoblotted with HIF-1α antibody or anti-lamin B antibody. As a positive control for detecting endogenous HIF-1α, HEK293 cells were treated with 200 µM of CoCl2 for 5 h. (D) Nur77 enhanced the transactivation of HIF-1α. SK-N-SH or PC-12 cells were transfected with luciferase reporter gene (3 × HRE_tk_luciferase) driven by 3 × HRE [+2,830 - +2,880 of human erythropoietin (EPO) promoter] and HSV-tk promoter region (-105 - +57), pCMV-HA-HIF-1α, and /or increasing amounts of pCS2+MT-Nur77. After 24 h of incubation, reporter activities were measured in cell lysates. Reporter activities were normalized with respect to lysate protein concentration. Means ± SDs forn = 3 are shown.P < 0.01 versus control.
Figure 3
Figure 3
Nur77 blocked the pVHL-mediated ubiquitination of HIF-1α. (A) HEK293 cells were transiently transfected with different combinations of pCS2+MT-Nur77, pCR3-VHL-HA, and pCMV-HA-HIF-1α. Protein expressions were detected using antibodies recognizing tagged sequences. This experiment is one representative of three independent experiments. (B) Nur77 reduced the ubiquitination of HIF-1α. HEK293 cells were transfected with the indicated constructs. After 24 h of incubation, cells were treated with MG132 (15 µM) (Calbiochem) for 5 h and then lysed. Cell lysates were immunoprecipitated with anti-HIF-1α antibody and then immunoblotted with anti-ubiquitin antibody (Santa Cruz Biotechnology). (C) Nur77 extends the half-life of HIF-1α. HEK293 cells were transiently transfected with pCMV-HA-HIF-1α with or without pCS2+MT-Nur77. After 24 h of incubation, cells were treated with cycloheximide (CHX, 25 µg/ml). Cell lysates were prepared at the shown time points, immunoprecipitated with anti-HA antibody, and immunoblotted with anti-HA antibody. The graph on the lower panel represents relative band intensities. Means ± SDs forn = 3 are shown.P < 0.01 versus control. Immunoblots represent one of three independent experiments.
Figure 4
Figure 4
Nur77 binds to pVHL and competes with elongin C. (A) Nur77 interacts with pVHL. Cell lysates prepared from HEK293 cells transfected with pCR3-VHL-HA and/or pCS2+MT-Nur77, were immunoprecipitated and immunoblotted with appropriate antibodies as indicated in the figure. (B) Nur77 binding to the α subdomain of pVHL. Cell lysates were prepared from HEK293 cells transfected with pCS2+MT-Nur77 or pCMV-HA-HIF-1α and precipitated with GST-fused α (amino acids 155-213) or β (amino acids 2-154) domains of pVHL. Precipitates were probed with anti-myc or anti-HA antibodies. (C, D) Nur77 competed with elongin C for binding to pVHL. In (C), HEK293 cells were transiently transfected with pCR3-VHL-HA and pcDNA3-myc-Gal4-elongin C in the presence of increasing amounts of pCS2+MT-Nur77. Cell lysates were immunoprecipitated with anti-HA and elongin C was detected using anti-myc antibody. In (D), HEK293 cells were transiently transfected with pCR3-VHL-HA and pCS2+MT-Nur77 in the presence of increasing amounts of pcDNA3-myc-Gal4-elongin C. Graph data are expressed as the means ± SD for three independent experiments. Immunoblots represent one of three independent experiments. (E) Nur77 did not disrupt the pVHL-HIF interaction. HEK 293 cells were transiently transfected with pcDNA3-VHL-flag and pCMV-HA-HIF-1α in the presence of increasing amounts of pCS2+MT-Nur77. Cell lysates were immunoprecipitated with anti-flag antibody and HIF-1α was detected using anti-HA antibody.
Figure 5
Figure 5
Binding of LBD-Nur77 to pVHL leads to stabilize and transactivate HIF-1α. (A) Schematic diagram of Nur77. (B) Mapping of the pVHL-binding site on Nur77. Three different pCS2+MT constructs of Nur77 truncated mutants (TAD; amino acid 1-265, DBD; amino acid 266-400, LBD; amino acid 401-601) and pCR3-VHL-HA were transiently transfected into HEK293 cells. Cell lysates were immunoprecipitated with anti-flag antibody and immunoblotted with anti-myc antibody. (C) LBD-Nur77 stabilizes HIF-1α and increases its transcriptional activity. HEK293 cells were transfected with different Nur77 truncated mutants along with 3 × HRE-tk-luciferase plasmid. HIF-1α stabilization was detected with anti-HA antibody, and Nur77 mutants were probed using anti-myc antibody. (D) Incremental increases in LBD enhanced HIF-1α transcriptional activity. The experimental conditions in (D) are the same as (C). In both (C) and (D), data are expressed as the mean ± SD for three independent experiments. Immunoblots represent one of three independent experiments. (E) The expression of endogenous BNIP3. The ectopic expression of LBD induces HIF-downstream genes. mRNAs were isolated from transfected HEK293 cells. The expressions of BNIP3, BNIP3-like, Glut1 and β-actin were detected by RT-PCR. The primers used in this experiment are described inMaterials and Methods.
Figure 6
Figure 6
pVHL is required for the Nur77-mediated HIF-1α stabilization. (A) The treatment of cobalt chloride abolishes the Nur77-mediated HIF-1α stabilization. HEK293 cells were transfected with HA-HIF-1α along with or without myc-Nur77. After 24 h of incubation, cells were treated with 200 µM of cobalt chloride for further 5 h. Cell lysates were immunoblotted with anti-HA and anti-myc antibodies for detecting HIF-1α and Nur77, respectively. β-actin was used as a control. (B) HEK293 cells transfected with indicated plasmids were incubated in the presence or absence of CoCl2. Cell lysates were immunoprecipitated with anti-myc antibody and immunoblotted with anti-HA antibody. (C) HEK293 cells were transfected with wild type or proline mutated (P402A/P564A) HIF-1α along with or without myc-Nur77. Cell lysates were immunoblotted with indicated antibodies. (D) Stable expression of HA-tagged VHL and endogenous expression of HIF-2α in established A498 cells was verified by immunoblot using specific antibodies. (E) Retrovirus expressing GFP (pMT-GFP), empty vector (pMT) or myc-tagged Nur77 was infected to A498/pBabe or A498/VHL stable cells as indicated. After 48 h of incubation, cell lysates were subjected to SDS-PAGE and immunoblotted with suitable antibodies.
Figure 7
Figure 7
A schematic model of HIF-1α stabilzation by Nur77. HIF-1α; hypoxia inducible factor-1α, pVHL; von Hippel-Lindau protein, ECB-UL; elongin C/B-ubiquitin ligase complex, UB; ubiquitin.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bardos JI, Chau NM, Ashcroft M. Growth factor-mediated induction of HDM2 positively regulates hypoxia-inducible factor 1alpha expression. Mol Cell Biol. 2004;24:2905–2914. - PMC - PubMed
    1. Bemis L, Chan DA, Finkielstein CV, Qi L, Sutphin PD, Chen X, Stenmark K, Giaccia AJ, Zundel W. Distinct aerobic and hypoxic mechanisms of HIF-alpha regulation by CSN5. Genes Dev. 2004;18:739–744. - PMC - PubMed
    1. Berra E, Benizri E, Ginouves A, Volmat V, Roux D, Pouyssegur J. HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1alpha in normoxia. EMBO J. 2003;22:4082–4090. - PMC - PubMed
    1. Chen C, Pore N, Behrooz A, Ismail-Beigi F, Maity A. Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia. J Biol Chem. 2001;276:9519–9525. - PubMed
    1. Chen D, Li M, Luo J, Gu W. Direct interactions between HIF-1 alpha and Mdm2 modulate p53 function. J Biol Chem. 2003;278:13595–13598. - PubMed

Publication types

MeSH terms

Substances

Related information

LinkOut - more resources

Full text links
Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp