SENP3 is responsible for HIF-1 transactivation under mild oxidative stress via p300 de-SUMOylation
2009; Springer Nature; Volume: 28; Issue: 18 Linguagem: Inglês
10.1038/emboj.2009.210
ISSN1460-2075
AutoresChao Huang, Yan Han, Yumei Wang, Xuxu Sun, Shan Yan, Edward T.H. Yeh, Yuying Chen, Cang Hui, Hui Li, Guiying Shi, Jinke Cheng, Xueming Tang, Jing Yi,
Tópico(s)Mitochondrial Function and Pathology
ResumoArticle13 August 2009free access SENP3 is responsible for HIF-1 transactivation under mild oxidative stress via p300 de-SUMOylation Chao Huang Chao Huang Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, ChinaThese authors equally contributed to this work Search for more papers by this author Yan Han Yan Han Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, ChinaThese authors equally contributed to this work Search for more papers by this author Yumei Wang Yumei Wang Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Xuxu Sun Xuxu Sun Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Shan Yan Shan Yan Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Edward T H Yeh Edward T H Yeh Department of Cardiology, The University of Texas MD Anderson Cancer Center and Texas Heart Institute at St Luke's Episcopal Hospital, Houston, TX, USA Search for more papers by this author Yuying Chen Yuying Chen Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Hui Cang Hui Cang Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Hui Li Hui Li Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Guiying Shi Guiying Shi Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Jinke Cheng Jinke Cheng Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Xueming Tang Xueming Tang Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Jing Yi Corresponding Author Jing Yi Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Chao Huang Chao Huang Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, ChinaThese authors equally contributed to this work Search for more papers by this author Yan Han Yan Han Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, ChinaThese authors equally contributed to this work Search for more papers by this author Yumei Wang Yumei Wang Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Xuxu Sun Xuxu Sun Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Shan Yan Shan Yan Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Edward T H Yeh Edward T H Yeh Department of Cardiology, The University of Texas MD Anderson Cancer Center and Texas Heart Institute at St Luke's Episcopal Hospital, Houston, TX, USA Search for more papers by this author Yuying Chen Yuying Chen Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Hui Cang Hui Cang Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Hui Li Hui Li Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Guiying Shi Guiying Shi Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Jinke Cheng Jinke Cheng Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Xueming Tang Xueming Tang Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Jing Yi Corresponding Author Jing Yi Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China Search for more papers by this author Author Information Chao Huang1, Yan Han1, Yumei Wang1, Xuxu Sun1, Shan Yan1, Edward T H Yeh2, Yuying Chen1, Hui Cang1, Hui Li1, Guiying Shi1, Jinke Cheng1, Xueming Tang1 and Jing Yi 1 1Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China 2Department of Cardiology, The University of Texas MD Anderson Cancer Center and Texas Heart Institute at St Luke's Episcopal Hospital, Houston, TX, USA *Corresponding author. Department of Cell Biology, Shanghai Jiao Tong University School of Medicine, Institutes of Medical Sciences, 280 S. Chongqing Road, Shanghai, 200025, China. Tel.: +86 21 63846590; Fax: +86 21 64670177; E-mail: [email protected] The EMBO Journal (2009)28:2748-2762https://doi.org/10.1038/emboj.2009.210 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The physiological function of Sentrin/SUMO-specific proteases (SENPs) remains largely unexplored, and little is known about the regulation of SENPs themselves. Here, we show that a modest increase of reactive oxygen species (ROS) regulates SENP3 stability and localization. We found that SENP3 is continuously degraded through the ubiquitin-proteasome pathway under basal condition and that ROS inhibit this degradation. Furthermore, ROS causes SENP3 to redistribute from the nucleoli to the nucleoplasm, allowing it to regulate nuclear events. The stabilization and redistribution of SENP3 correlate with an increase in the transcriptional activity of the hypoxia-inducing factor-1 (HIF-1) under mild oxidative stress. ROS-enhanced HIF-1 transactivation is blocked by SENP3 knockdown. The de-SUMOylating activity of SENP3 is required for ROS-induced increase of HIF-1 transactivation, but the true substrate of SENP3 is the co-activator of HIF-1α, p300, rather than HIF-1α itself. Removing SUMO2/3 from p300 enhances its binding to HIF-1α. In vivo nude mouse xenografts overexpressing SENP3 are more angiogenic. Taken together, our results identify SENP3 as a redox sensor that regulates HIF-1 transcriptional activity under oxidative stress through the de-SUMOylation of p300. Introduction Small ubiquitin-like modifier (SUMO) modification is an important post-translational protein modification that has gained much prominence due to the large number of SUMO substrates (Hay, 2005). SUMOylation is catalysed by SUMO-specific E1, E2 and E3 ligases, and is reversed by a family of Sentrin/SUMO-specific proteases (SENPs) (Yeh et al, 2000). SUMOylation can regulate a broad spectrum of cellular processes, including DNA replication/repair, cell division, cell signalling and nuclear transport. De-SUMOylation mediated by SENPs has been shown to have an important function in many of these processes as well (Cheng et al, 2004, 2005, 2007; Degerny et al, 2005; Di Bacco et al, 2006; Deyrieux et al, 2007; Dorval and Fraser, 2007; Halliwell, 2007; Haindl et al, 2008). For example, SENP1 deconjugates SUMO1 from hypoxia-inducible factor-1α (HIF-1α) to control its stability and has a critical function in the regulation of hypoxic response (Cheng et al, 2007). SENP3 is required for rRNA processing through deconjugation of SUMO2/3 from nucleophosmin (Haindl et al, 2008). Importantly, the conjugation and deconjugation of SUMO modification is a highly dynamic event, and only a small fraction of a substrate is SUMOylated at a given time (Hay, 2005). Apparently, the SUMOylation/de-SUMOylation balance for a specific substrate is delicately regulated. What triggers de-SUMOylation and how SENPs are regulated under various physiological and pathological conditions are, therefore, intriguing questions. Oxidative stress, a common challenge to cellular homoeostasis, is mediated predominantly through the production of reactive oxygen species (ROS). Many extracellular insults, such as changes in temperature, pH, osmotic pressure, oxygen tension and sugar concentration, also lead to an increase in the production of intracellular ROS (Halliwell, 2003). Hypoxia itself paradoxically causes an increase in ROS (Chandel et al, 1998). The extent and duration of ROS increase usually determine the consequences of the cellular adaptive response to oxidative stress. Although severe oxidative stress causes cell senescence and even cell death, mild oxidative stress paradoxically promotes cell survival, during which global alterations of gene expression and protein post-translational modification occur (Halliwell, 2007). It has been reported that SUMO conjugation, in particular SUMO2/3 conjugation, is a major response to oxidative stress (Saitoh and Hinchey, 2000; Manza et al, 2004; Li et al, 2006; Dorval and Fraser, 2007; Cimarosti et al, 2008; Yang et al, 2008). Thus, it is highly likely that the balance between SUMOylation and de-SUMOylation may have a critical function in the cellular adaptive response to ROS production. It is unknown, however, whether SUMO2/3-specific protease, SENP3, is involved in the cellular response to oxidative stress (Tempe et al, 2008). HIF-1, consisting of an oxygen-regulated α-subunit and a constitutively expressed β-subunit, is a master transcriptional regulator of gene expression in response to hypoxia. HIF-1 activation is a multistep process involving the stabilization of HIF-1α protein, dimerization of the HIF-α and HIF-β subunits, translocation to the nucleus, binding to the HIF-1 responsive elements (HRE), and the formation of active transcriptional complexes with the accessory proteins p300/CBP (Arany et al, 1996; Ebert and Bunn, 1998; Kallio et al, 1998; Yamashita et al, 2001; Freedman et al, 2002; Lando et al, 2002). Recent studies show that the ROS generated in mitochondria under hypoxia are both necessary and sufficient for HIF-1 activation (Ebert and Bunn, 1998; Chandel et al, 2000; Brunelle et al, 2005; Guzy et al, 2005; Mansfield et al, 2005; Guzy and Schumacker, 2006). In addition, HIF-1 activation occurs in response to a variety of environmental stimuli other than hypoxia (Zelzer et al, 1998; Chandel et al, 2000; Haddad and Land, 2001; Harris, 2002; Lu et al, 2002; Jung et al, 2003; Semenza, 2003; Wang et al, 2004; Kamat et al, 2005; Thomas and Kim, 2005; De Ponti et al, 2007), for instance, during inflammation (Cramer et al, 2003; Jung et al, 2003; Melillo, 2004; Walmsley et al, 2005) and insulin administration (Zelzer et al, 1998; Roth et al, 2004; Carroll and Ashcroft, 2006; Treins et al, 2006; Glassford et al, 2007; Zhou et al, 2007), in which ROS generated by NADPH oxidase are required (Shiose et al, 2001; Biswas et al, 2007; Xia et al, 2007). An immediate stabilization of HIF-1α is often observed on exposure to exogenous hydrogen peroxide (H2O2) or accompanying endogenous ROS generation (Chandel et al, 2000; Guzy et al, 2005). Moreover, HIF-1α accumulation induced by normoxic ROS generation is responsible for initiating the expression of an HRE-controlled luciferase reporter, as well as HIF-1 target genes that may relate to tumourigenesis and malignant phenotypes of cancer cells (Biswas et al, 2007; Gao et al, 2007; Xia et al, 2007; Guzy et al, 2008). Therefore, how ROS regulate HIF-1 activation under both hypoxia and normoxia is an important question. To date, the stabilization of HIF-1α is suggested as a mechanism by which ROS activate HIF-1 (Brunelle et al, 2005; Guzy et al, 2005; Mansfield et al, 2005). Whether other steps in HIF-1 activation may be promoted by ROS has not been addressed. We herein propose a new mechanism in which a mild oxidative stress induced by low doses of H2O2 can rapidly stabilize the SUMO2/3-specific protease, SENP3, and SENP3 in turn promotes the transcriptional activity of HIF-1 through deconjugation of SUMO2/3 from p300, the co-activator of HIF-1α. This mechanism has a critical function in HIF-1 activation under both normoxia and hypoxia, working independently of HIF-1α stabilization. Results Mild oxidative stress induces a rapid stabilization of SENP3 protein HeLa cells were exposed to various concentrations of H2O2 and the expression of SUMO2/3-specific protease, SENP3, was evaluated. The increase in SENP3 protein was seen after treatment with 50 μM H2O2, and this increase occurred in a dose-dependent manner (Figure 1A, left). A similar SENP3 increase could be observed in some non-tumour cells, for instance, human umbilical vein endothelial cells (HUVEC), but the required doses of H2O2 were much lower (2.5 μM) than in HeLa cells (data not shown). The obvious increase in SENP3 protein on H2O2 exposure was seen at 30 min, reached a plateau at 60 min, and remained stable over time (Figure 1A, right). Interestingly, other stresses, such as UV radiation, low pH, hypotonic and hypertonic, or hypothermal and hyper-thermal stimuli that increase ROS, and even hypoxia, also increased SENP3 protein levels (data not shown). As ROS generation is common to these stress inducers, we tested whether the increase in SENP3 protein level could be blocked by anti-oxidants such as N-acetyl cysteine (NAC), or the thiol-reducing agent dithiothreitol (DTT). Indeed, increase of SENP3 was blocked by NAC and DTT (Figure 1B), suggesting that the level of SENP3 protein is regulated by changes in redox state. Figure 1.Mild oxidative stress induces a rapid stabilization of SENP3 protein. (A) HeLa cells were treated with the indicated concentrations of hydrogen peroxide (H2O2) for 1 h (left panel) or 100 μM H2O2 for the indicated time (right panel). SENP3 protein level was evaluated by immunoblotting (IB) using SENP3 antibody. β-actin was used as a loading control. (B) HeLa cells were pre-treated with 5 mM NAC for 4 h or 10 mM DTT for 1 h, before H2O2 was added to the medium, for an additional 1 h. SENP3 protein levels were evaluated by IB. (C) HeLa cells were treated with the indicated concentrations of H2O2 for 1 h. SENP3 mRNA levels were evaluated by real-time PCR and shown as folds of control. (D) SENP3 protein levels were evaluated by IB after HUVEC cells were treated with cycloheximide (CHX) for different times (upper panel), or pre-treated with H2O2 for 2 h before CHX was added and co-incubated for the indicated time (lower panel). β-actin was used as a loading control. (E) SENP3 protein levels were evaluated by IB after HUVEC cells were treated with H2O2 as indicated for 1 h with or without 30 μM MG132. (F) HEK293T cells were transfected with RH-SENP3 or/and HA-Ubiquitin (HA-Ub) for 36 h, and MG132 was then added for additional 10 h as indicated. Co-IP was carried out with RH antibody and IB was carried out as indicated. The blots with long time of exposure (long expo) and short time of exposure (short expo) are displayed to show the differences in quantity between ubiquitin-conjugated and non-conjugated SENP3, respectively. (G) HEK293T cells were transfected with RH-SENP3 for 36 h. MG132, H2O2 and DTT were added as indicated for an additional 1 h. RH-SENP3 was pulled down with Talon beads and IB was carried out using anti-ubiquitin and anti-RGS antibodies, respectively. Download figure Download PowerPoint The rapid increase in the SENP3 protein level by H2O2 could not be due to an increase in transcription, as the SENP3 messenger RNA remained unchanged following exposure to H2O2 (Figure 1C). We then tested whether SENP3 was regulated through a post-transcriptional mechanism. After exposure to the protein synthesis inhibitor, cycloheximide (CHX), SENP3 protein rapidly decreased and became undetectable after 6 h in HUVEC (Figure 1D, upper panel). However, if H2O2 was pre-incubated with cells for 2 h before CHX addition, the decrease in SENP3 protein level was blocked (Figure 1D, bottom panel), suggesting that the SENP3 protein was stabilized by H2O2. To test whether turnover of SENP3 protein was regulated by the ubiquitin–proteasome pathway, the proteasome inhibitor, MG132, was added to medium. The SENP3 protein level was increased when HUVEC cells were treated with MG132 alone and remained stable when exposed to a low concentration of H2O2 (Figure 1E). Immunoprecipitation (IP) was then carried out on HEK293T cells co-transfected with RGS-SENP3 and HA-ubiquitin, in the presence or absence of MG132. As shown in Figure 1F, addition of MG132 led to an accumulation of ubiquitin-conjugated SENP3 and an increase in total SENP3. These results indicate that SENP3 protein was being constantly ubiquitin-conjugated and degraded by the proteasome. Furthermore, endogenous ubiquitin conjugation of SENP3 was attenuated by H2O2 and partially reversed by DTT (Figure 1G). Taken together, these results indicate that the H2O2-mediated increase in SENP3 protein stability is because of the inhibition of the ubiquitin–proteasome pathway. SENP3 redistributes between the nucleolus and the nucleoplasm on H2O2 exposure SENP3 is reported to be preferentially localized in the nucleoli (Nishida et al, 2000). We, therefore, check whether the localization of SENP3 can be altered when cells are exposed to H2O2. RGS-His (RH)-tagged SENP3 was overexpressed and detected using antibody against RH. We showed that most of the RH-tagged SENP3 co-localized with the nucleolus marker B23, in untreated cells as expected, and was almost invisible in the nucleoplasm (Figure 2A). However, SENP3 became visible in the nucleoplasm when cells were exposed to H2O2, seen as a dim dispersion with numerous bright foci, whereas SENP3 staining in the nucleoli did not decrease (Figure 2B). A similar redistribution of endogenous SENP3 was also observed by immunofluorescence in cells exposed to H2O2 (Supplementary Figure S1). Furthermore, SENP3 redistribution from the nucleoli to the nucleoplasm was reversed by pre-treating cells with NAC or DTT (Figure 2B), indicating that the localization of SENP3 was regulated by ROS. To determine the association between SENP3 localization and the ubiquitin–proteasome pathway, we then used MG132 to inhibit SENP3 degradation by the proteasome. Strikingly, MG132 led to SENP3 accumulation in the nucleoplasm in a pattern similar to that caused by H2O2 (Figure 2B, bottom panel). This implies that in resting cells, SENP3 is frequently transported from the nucleoli to the nucleoplasm where it is degraded. On oxidative stress, this degradation is blocked by inhibition of the ubiquitination of SENP3, which stabilizes SENP3 as it accumulates in the nucleoplasm. Figure 2.SENP3 redistributes between the nucleolus and the nucleoplasm on H2O2 exposure. (A) HeLa cells were transfected with RH-SENP3 for 36 h. Immunofluorescence was carried out with RH and B23 antibodies. B23 is a nucleolar marker. DAPI was used for counterstaining the nuclei. Scale bar=9 μm. (B) HeLa cells were transfected with RH-SENP3 for 36 h, and the cells were treated with H2O2 (100 μM) or MG132 (10 mM), respectively, for 1 h, or pretreated with 5 mM NAC for 4 h or 10 mM DTT for 1 h before H2O2 exposure for another 1 h. Immunofluorescence was carried out using RH antibody. DAPI was used for counterstaining the nuclei. Scale bar=9 μm. Bar charts showed the difference in the rhodamine intensity that reflected SENP3 quantity in the nucleoplasm in eighty cells with or without H2O2 treatment. Download figure Download PowerPoint SENP3 participates in H2O2-induced global changes in SUMO2/3 modification and can regulate the SUMOylation status of HIF-1α As SENP3 is a de-SUMOylating enzyme, its increase and redistribution following H2O2 exposure may affect the balance of protein SUMOylation/de-SUMOylation. Global SUMOylation patterns were examined in cells with intact or knocked-down SENP3 on exposure to H2O2 at a very low dose range. Knock down of SENP3 by siRNA was validated (Figure 3A, upper panel). In cells exposed to H2O2, SUMO2/3-conjugated proteins were gradually increased (Figure 3A, middle panel, left). However, in samples with SENP3 knocked down, SUMO2/3 conjugation was markedly increased following treatment with escalating doses of H2O2 (Figure 3A, middle panel, right). This suggests that in SENP3-intact cells, SENP3 normally antagonizes SUMO2/3 conjugation induced by ROS. In contrast, SUMO-1 conjugation is not changed by H2O2 in the same dose range (Figure 3A, bottom panel). Figure 3.SENP3 participates in H2O2-induced changes in SUMO2/3 modification and can interact with HIF-1. (A) HeLa cells were transfected with non-specific siRNA and SENP3 siRNA for 72 h, and SENP3 expression was markedly knocked down (upper panel). The cells were then treated with various concentrations of H2O2 for 1 h before global protein SUMOylation was evaluated using the SUMO2/3 antibody (middle panel) and the SUMO1 antibody (bottom panel). (B) After being transfected with HA-HIF-1α and RH-SENP3 for 48 h, HeLa cells were exposed to 100 μM H2O2 for 1 h. Cell monolayers were fixed and immunofluorescence was carried out using anti-HIF-1α and anti-RH antibodies. Cell nuclei were stained with DAPI. Scale bar=9 μm. (C) After HeLa cells were transfected with RH-HIF-1α with or without HA-SUMO3 and Myc-SENP3 as indicated for 48 h, cells were lysed and RH-HIF-1α was pulled down using Ni beads. The pulled-down HIF-1α and cell lysates were analysed by IB as indicated. Download figure Download PowerPoint As our data (Figure 2 and Supplementary Figure 1) show that SENP3 accumulates in the nucleoplasm on H2O2 exposure, allowing for SENP3 de-SUMOylation activity in the milieu outside the nucleoli, and also because our previous studies have indicated the association of ROS with HIF-1, we checked whether HIF-1, an important nuclear protein, could be affected by SENP3. Immunoprecipitation showed that SENP3 could have physical interaction with HIF-1α (Supplementary Figure S2). Immunofluorescence staining showed that, although in resting cells SENP3 was mainly localized in the nucleolus and only slightly dispersed in the nucleoplasm, and HIF-1α was localized in the nucleoplasm, the co-existence of the two proteins in nucleoplasm was greatly enhanced by treatment with H2O2 (Figure 3B). Indeed, SENP3 could remove SUMO3 (Figure 3C), but not SUMO1 (data not shown), from HIF-1α. Collectively, these data indicate that SENP3 participates in the removal of SUMO2/3 from a number of proteins in the nucleoplasm under mild oxidative stress. Mild oxidative stress enhances the transcriptional activity of HIF-1 through SENP3, but this action is not attributed to the de-SUMOylation of HIF-1α by SENP3 We then examined the association of SENP3 with HIF-1 activation under oxidative stress. On the basis of the above data, low concentrations of H2O2 that stabilize SENP3 (i.e. 50–100 μM in HeLa cells) were selected to mimic mild oxidative stress, and HIF-1 transactivation was assessed by luciferase reporter assay. When cells were exposed to 50 μM H2O2, the endogenous and ectopically expressed HIF-1α was immediately stabilized and the transcriptional activity of HIF-1 was induced. However, the transcriptional activity of HIF-1 continuously increased with increasing doses of H2O2, whereas the quantities of HIF-1α remained unchanged (Figure 4A and B). H2O2-enhanced HIF-1 transactivation could be blocked by NAC (Figure 4C). Moreover, HIF-1 transactivation induced by cobalt chloride (CoCl2) could also be enhanced by H2O2 exposure (Figure 4D). Immunofluorescent staining confirmed that when H2O2 was added, HIF-1α accumulated in the nucleus, but the quantity in the nucleus remained the same when H2O2 was further increased (Figure 4E). These results indicate that the transcriptional activity of HIF-1 can be enhanced by a modest ROS increase and this action is independent of HIF-1α protein level. Figure 4.Mild oxidative stress enhances the transcriptional activity of HIF-1 through SENP3, but this action is not attributed to the de-SUMOylation of HIF-1α by SENP3. (A–D) HeLa cells were co-transfected with the constructs of luciferase reporters for HRE or HRE mutant, and Renilla, with or without RH-HIF-1α. At 40 h post-transfection, cells were exposed to the indicated concentrations of H2O2 and CoCl2, as described in Materials and methods section, before cells were lysed, and the relative luciferase activity (RLA) for HIF-1 was assayed. NAC, when used, was pre-incubated with cells for 4 h before H2O2 was added to the medium. The cell lysates of one of the representative experiments were subjected to IB with anti-HIF-1α antibody. The results of the RLA are given as the mean±s.d. of three independent experiments, and samples were duplicated in each experiment. (E) After HeLa cells were exposed to the indicated concentration of H2O2 for 1 h, cell monolayers were fixed and immunofluorescence was carried out using anti-HIF-1α antibody. Scale bar=10 μm. (F) HeLa cells were co-transfected with the constructs of luciferase reporters for HRE or HRE mutant, and Renilla, along with RH-HIF-1α, RH-SENP3 or RH-SENP3 mutant were additionally co-transfected as indicated. At 48 h post-transfection, cells were lysed and HIF-1 RLA was assayed. Download figure Download PowerPoint As the same concentrations of H2O2 can enhance HIF-1 transactivation and concomitantly stabilize SENP3, the causal relationship between SENP3 and HIF-1 transactivation needs to be determined. As shown in Figure 4F, the HIF-1 transactivation was enhanced by overexpression of wild-type SENP3, but not by the SENP3 mutant lacking de-SUMOylation activity, indicating that the enhancement of HIF-1 transcriptional activity required SENP3 de-SUMOylation activity. The quantity of HIF-1α was not changed by the overexpression of SENP3. Furthermore, expression of HIF-1 target genes, including vascular endothelial growth factor (VEGF), Glut-1 and CA-9 was also upregulated by the overexpression of SENP3, and this upregulation could be reversed by ablation of endogenous HIF-1α using shRNA (Figure 4G). Strikingly, when endogenous SENP3 was depleted by siRNA, H2O2 could no longer boost the transcriptional activities and the target gene expression of HIF-1 at all, even if the accumulation of HIF-1α remained unchanged (Figure 4H). These results suggest that H2O2-enhanced HIF-1 transcriptional activity is mediated by SENP3, and this led us to hypothesize that SENP3 may de-SUMOylate HIF-1α to achieve this effect. However, to our surprise, enhancement of HIF-1 transcriptional activity by overexpression of SENP3 did not disappear in cells co-expressing mutant HIF-1α that had mutated SUMOylation sites; the increase in tra
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