SENP1-mediated deSUMOylation of USP28 regulated HIF-1α accumulation and activation during hypoxia response
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The ubiquitin-specific protease 28 (USP28) is an oncogenic deubiquitinase, which plays a critical role in tumorigenesis via antagonizing the ubiquitination and degradation of tumor suppressor protein FBXW7-mediated oncogenic substrates. USP28 controls hypoxia-dependent angiogenesis and metastasis by preventing FBXW7-dependent hypoxia-inducible transcription factor-1α (HIF-1α) degradation during hypoxia. However, it remains unclear how USP28 activation and HIF-1α signaling are coordinated in response to hypoxia.
The in vitro deubiquitinating activity assay was used to determine the regulation of USP28 by hypoxia. The co-immunoprecipitation and GST Pull-down assays were used to determine the interaction between USP28 and SENP1. The in vivo deSUMOylation assay was performed to determine the regulation of USP28 by SENP1. The luciferase reporter assay was used to determine the transcriptional activity of HIF-1α.
Here, we report that USP28 is a SUMOylated protein in normoxia with moderate deubiquitinating activity towards HIF-1α in vitro, while hypoxia and HIF-1α activate USP28 through SENP1-mediated USP28 deSUMOylation to further accumulate HIF-1α protein in cells. In agreement with this, a SUMOylation mutant USP28 showed enhanced ability to increase HIF-1α level as well as control the transcriptional activity of HIF-1α.
Collectively, our results reveal a novel SENP1–USP28–HIF-1α positive feedback loop to maximize the concentration of HIF-1a protein and amplify its downstream effects during hypoxia response.
KeywordsUSP28 HIF-1α SENP1 Hypoxia
glycogen synthase kinase-3
hypoxia-inducible transcription factor-1α
hypoxia response elements
small hairpin RNA
SUMO1/sentrin specific peptidase
ubiquitin-specific protease 28
The hypoxia-inducible transcription factor-1α (HIF-1α) has central roles in angiogenesis, carcinogenesis, and cell adaptions to hypoxic conditions both transcriptionally dependent and independent [1, 2]. Thus, the protein levels of HIF-1α must be tightly regulated to prevent its inappropriate activation. Indeed, the E3 ubiquitin ligase, von Hippel–Lindau (VHL) mediates the ubiquitination and degradation of HIF-1α in normoxia condition . It has been demonstrated that HIF-1α is also degraded by glycogen synthase kinase-3 (GSK-3) and FBXW7-mediated ubiquitination, and could be antagonized by ubiquitin-specific protease 28 (USP28) in hypoxia condition [4, 5]. However, it is well-known that the HIF-1α protein was accumulated with increased protein stability during hypoxia, suggesting that either the elements that promoted HIF-1α degradation were repressed or that stabilized HIF-1α were enhanced.
SUMOylation is a highly dynamic process that could be reversed by SUMO1/sentrin specific peptidase (SENP) family members and appears to be dysregulated in diverse cancer types [6, 7]. HIF-1α is modified through different pathways, such as acetylation, phosphorylation, ubiquitination as well as SUMOylation during hypoxia [8, 9, 10]. Interestingly, as a transcriptional factor, HIF-1α could induce the expression of SENP1, which in turn deSUMOylates HIF1a, therefore forming a positive feedback loop to stabilize HIF-1α during hypoxia [11, 12]. USP28 has been also identified as a SUMOylation substrate, and SUMOylation at its N-terminal domain inhibits its deubiquitinating activity . Thus, it could be critical to investigate whether USP28 was involved in the HIF-1α positive feedback loop during hypoxia.
The aim of this study is to clarify the relationship between USP28 and HIF-1α during hypoxia. We found that USP28 is SUMOylated in normoxia but rapidly deSUMOylated under hypoxia condition. SENP1-mediated deSUMOylation of USP28 enhanced its deubiquitinating activity towards HIF-1α, and thereby prevented FBXW7-dependent degradation of HIF-1α during hypoxia. Our results indicate a novel mechanism by which SENP1-mediated deSUMOylation of USP28 stabilized HIF-1α during hypoxia.
Materials and methods
HCT116, A549 and 293T cells were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (CAS, Shanghai, China), and cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 IU/ml penicillin and 100 mg/ml streptomycin, maintained at 37 °C in a humidified atmosphere with 5% CO2.
Plasmids and stable cell line construction
Wild type USP28 construct (#41948) was obtained from addgene and was used for subcloning into pcDNA-Flag vector. USP28KR was created with site-directed mutagenesis (Stratagene). Wild type HIF-1a construct (#21101) was obtained from addgene and was used for subcloning into pcDNA-HA vector. SUMO2 and SENP1 were amplified from 293T cells by PCR and cloned into the pbabe- or pEGFP-vector, respectively. The stable cell line construction has been described previously. For brief, viral supernatants were produced in HEK293T cells co-transfected with the pBabe-control or pBabe-his-SUMO2 constructs and packaging vectors. Viral supernatants were collected 48 h after transfection, filter-sterilized, and added to the HCT116 cells with 10 μg/ml Polybrane for 48 h and selected with puromycin (1 μg/ml) for 3 days.
Cells were harvested and lysed with cold lysis buffer (150 mM Tris–HCl, pH 6.8, 100 mM DTT, 2% SDS and 10% glycerol). Proteins were separated by 10–12% SDS-PAGE, transferred to NC membranes. Western blot assay was then performed by using the following antibodies: anti-USP28, anti-SENP1, anti-GST, anti-His, anti-GFP, anti-GAPDH (Santa Cruz, CA, USA), anti-HIF1a (H1alpha67, abcam), anti-Myc (Cell Signaling, Boston, MA, USA), anti-Flag M2 and anti-HA (Sigma, St Louis, MI, USA).
The immunoprecipitation process has been described previously with some modifications [14, 15]. Briefly, cells transfected with Flag-USP28 and GFP-SENP1 were lysed in cold NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris–HCl, pH 8.0, 0.5% NP-40) for 45 min at 4 °C. Cell lysates were centrifuged, and the supernatants were incubated with M2 beads for 2 h at 4 °C. The beads were washed with NETN buffer, and the bound proteins were eluted with Flag peptide and subjected to western blot with indicated antibodies.
SUMOylation and Ubiquitination assays
HCT116 cells were co-transfected with his-SUMO2 or his-Ubiquitin and other indicated plasmids for 36 h. Cells were sonicated in lysis buffer containing 8 M urea and 10 mM imidazole. His-SUMO2 or his-Ubiquitin conjugated proteins were recovered with Ni–NTA resin (Qiagen), washed with urea lysis buffer containing 20 mM imidazole, and eluted with buffer containing 5% SDS, 0.72 M DTT, and 200 mM imidazole. Proteins were separated and visualized by western blot assay.
The whole procedure of GST pull-down has been described previously . Briefly, the purified GST or GST-SENP1 proteins were incubated with the purified USP28 protein for 1 h at 30 °C. Then the precipitations were eluted by 2Χ SDS loading buffer and followed by western blot with previously described antibody.
For luciferase reporter assays, cells were seeded in 24-well plates and transfected with HA-HIF-1α, pGL3-HRE-Luc, pSV40-renilla and USP28 plasmids. Cells were then harvested 36 h after transfection. Luciferase activities were measured and assessed using the Dual Luciferase Reporter Assay System (Promega, USA).
Data were shown as mean ± standard deviation (SD). Differences between groups were analyzed using Student’s t-test, and P < 0.05 was considered statistically significant. Statistical significance is displayed as *(P < 0.05), **(P < 0.01) or ***(P < 0.001).
Hypoxia enhanced the deubiquitinating activity of USP28 on HIF-1α
Hypoxia and HIF-1α promoted USP28 deSUMOylation
SUMOylation of USP28 impaired its deubiquitinating activity on HIF-1α
SENP1 directly interacts with USP28
SENP1 deSUMOylated and enhanced USP28 deubiquitinating activity towards HIF-1α
Expression of USP28 K99R induces an increase in HIF-1α protein and HIF-1α transcriptional activation
In the present study, we proved that USP28 was SUMOylated under normoxic condition but rapidly deSUMOylated during hypoxia. We further showed that SUMOylation at K99 regulates the deubiquitinating activity of USP28 towards HIF-1α. We also demonstrated that SENP1 directly interacts with and also deSUMOylates USP28. In USP28 knockdown cells, the HIF-1α stabilization activity of SENP1 was largely impeded. Thus, our data revealed a previous unknown SENP1–USP28–HIF-1α positive feedback loop to maximize the accumulation of HIF-1α protein and amplify its downstream effects during hypoxia response (Fig. 6c). In line with these observations, cells with K99R mutant USP28 overexpression exhibited increased HIF-1α protein level and enhanced HIF-1α transcriptional activity.
Previous studies have revealed that expression of USP28 was strongly elevated in human colon and breast carcinomas, and overexpression of USP28 enhanced HIF-1α-dependent angiogenesis and promoted colorectal cancer [4, 17, 18], suggesting that it might play a critical role in tumor cellular pathways. SENP1 has also been shown to be essential for HIF-1α accumulation during hypoxia by directly interacting and deSUMOylating HIF-1α protein . Our data further elucidated that SENP1-mediated USP28 deSUMOylation was indispensable for its deubiquitinating activity towards HIF-1α, indicating SENP1 associated with USP28 to fully activate the HIF-1α pathway. Thus, the results in this manuscript filled a mysterious gap in the accumulation and activation of HIF-1α during hypoxia response. Moreover, it will be interested to further explore whether the activity of other deubiquitination enzymes were also regulated by SUMOylation and deSUMOylation circle, and whether other SENP family members also participated in this regulatory process.
Furthermore, our data have extended the understanding of the mechanisms mediating the accumulation of HIF-1α during hypoxia and introduced a new cross-talk between SUMOylation and ubiquitination in controlling HIF-1α stabilization. The posttranscriptional regulation by SENP1–USP28–HIF-1α axis may constitute a novel cellular adaptive response to allow HIF-1α maximal accumulation in response to hypoxia signals that affect cells survival, angiogenesis, and even tumorgenesis. Thus, further functional works are warranted to further clarify the exact roles of this axis in the board biological processes. Nevertheless, our studies offer the opportunity to shutdown HIF-1α-dependent downstream effects during hypoxia response by targeting SUMO modified USP28, and setup the stage for relevant drug discoveries in future researches.
The present study found that the deubiquitinase USP28 was SUMOylated at K99 under normoxic condition but rapidly deSUMOylated by SENP1 during hypoxia. USP28 SUMOylation regulates its deubiquitinating activity against HIF-1α. Overexpression of a K99R USP28 mutant in cells significantly increased HIF-1α protein level and enhanced HIF-1α transcriptional activity. These founding reveals a previous unknown SENP1–USP28–HIF-1α positive feedback loop and offers a novel target for cancer therapy.
SD, LZ and YW conducted most of the biochemical and cellular experiments with the help of JL, DZ, YC and QP under the supervision of WL and BL. SD wrote most of the manuscript with the guidance and help of WL and BL. All authors read and approved the final manuscript.
We thank Dr. Wei Wu for her technical support.
The authors declare that they have no competing interests.
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Ethics approval and consent to participate
This study is supported by grants from the National Natural Science Foundation of China (81773018, 3140118, 81600702, 81502381), Research Foundation of Hubei Polytechnic University for Talented Scholars (16xjz01R, 17xjz06A), open foundation from Hubei Key Laboratory for Kidney Disease Pathogenesis and Intervention (SB201606), Min Hang District Science and Technology Committee (2015MHZ064), Fund of the Central Hospital of Minhang District (2016MHLC08), the National Institutes of Health grants R21 DA042298 (W.L.) and R01 GM124152 (W.L.), the National Science Foundation STC award 1231306 (W.L.), and the Flinn Foundation Seed Grant (W.L.).
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