The ubiquitin-conjugating enzyme UBE2O modulates c-Maf stability and induces myeloma cell apoptosis
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UBE2O is proposed as a ubiquitin-conjugating enzyme, but its function was largely unknown.
Mass spectrometry was applied to identify c-Maf ubiquitination-associated proteins. Immunoprecipitation was applied for c-Maf and UBE2O interaction. Immunoblotting was used for Maf protein stability. Luciferase assay was used for c-Maf transcriptional activity. Lentiviral infections were applied for UBE2O function in multiple myeloma (MM) cells. Flow cytometry and nude mice xenografts were applied for MM cell apoptosis and tumor growth assay, respectively.
UBE2O was found to interact with c-Maf, a critical transcription factor in MM, by the affinity purification/tandem mass spectrometry assay and co-immunoprecipitation assays. Subsequent studies showed that UBE2O mediated c-Maf polyubiquitination and degradation. Moreover, UBE2O downregulated the transcriptional activity of c-Maf and the expression of cyclin D2, a typical gene modulated by c-Maf. DNA microarray revealed that UBE2O was expressed in normal bone marrow cells but downregulated in MGUS, smoldering MM and MM cells, which was confirmed by RT-PCR in primary MM cells, suggesting its potential role in myeloma pathophysiology. When UBE2O was restored, c-Maf protein in MM cells was significantly decreased and MM cells underwent apoptosis. Furthermore, the human MM xenograft in nude mice showed that re-expression of UBE2O delayed the growth of myeloma xenografts in nude mice in association with c-Maf downregulation and activation of the apoptotic pathway.
UBE2O mediates c-Maf polyubiquitination and degradation, induces MM cell apoptosis, and suppresses myeloma tumor growth, which provides a novel insight in understanding myelomagenesis and UBE2O biology.
Keywordsc-Maf UBE2O Ubiquitin proteasome pathway Multiple myeloma
Affinity purification-coupled mass spectrometry
BRCA1 associated protein-1
C-C chemokine receptor type 1
Gene Expression Omnibus
Kyoto Encyclopedia of Genes and Genomes
Liquid chromatography tandem mass spectrometry
c-Maf recognition element
Monoclonal gammopathy of undetermined significance
Smoldering multiple myeloma
TNF receptor associated factor 6
Ubiquitin-conjugating enzyme E2 O
The ubiquitin-conjugating enzymes (E2s) transfer activated ubiquitin molecules to a ubiquitin ligase (E3) or directly mediate substrate ubiquitination. Therefore, the E2 enzymes are critical for protein ubiquitination and stability. There are 35 E2s, of which most are small proteins with molecular weights from 14 to 35 kDa, and these E2s fail to exhibit intrinsic affinity for physiological substrates ; however, there is an unusually large E2 called E2-230K or UBE2O that can ubiquitinate proteins in an E3-independent manner . UBE2O is ubiquitously expressed and has been proposed to play a role in erythroid differentiation [2, 3]. Recently, it is found to mediate monoubiquitination of SMAD6  and polyubiquitination of BAP1  and AMPKα2 . UBE2O also inhibits TRAF6 K63-polyubiquitination . Therefore, UBE2O might exhibit various functions upon the contexts.
Multiple myeloma (MM) is an incurable malignancy derived from plasma cells. Although the detailed mechanisms are not fully understood, the Maf family transcription factors raise high attention . This family is comprised of seven members of which c-Maf, MafA, and MafB belong to the large Maf subfamily because these transcription factors contain the complete structure and functional domains including the DNA-binding domain and the transcription activation domain . All c-Maf, MafA, and MafB are found to be highly overexpressed in MM cells in association with chromosomal translocations and other unknown mechanisms . Notably, c-Maf is reported in more than 50% of MM cell lines and primary MM patient samples . Overexpression of c-Maf has been regarded as a key factor in MM pathophysiology because interference with c-Maf blocks MM tumor growth in immunodeficient mice , while c-Maf-transgenic mice develop MM-like symptoms at old age around 50–60 weeks old . Therefore, c-Maf is proposed as a target for MM therapy. Our recent study found that c-Maf undergoes degradation via the ubiquitin-proteasome pathway under the direction of ubiquitin ligase HERC4 . However, its ubiquitin-conjugating enzyme is yet to know.
In the study of c-Maf ubiquitination-associated enzymes by the affinity purification-coupled mass spectrometry (AP/MS) strategy, UBE2O was identified in the c-Maf co-immunoprecipitates and it interacts with c-Maf protein and mediates c-Maf ubiquitination and degradation. More importantly, UBE2O induces c-Maf expressing MM cell apoptosis and delays MM tumor growth in mice.
Primary bone marrow cells
Primary bone marrow species were collected from the Department of Hematology, the First Affiliated Hospital of Soochow University. The use of primary bone marrow cells was approved by the Review Board and Ethical Committee of Soochow University, and each patient provided written informed consent to donate 2–5 ml of bone marrow for this study after diagnostic and clinical procedures in accordance with the Declaration of Helsinki. Mono-nuclear cells were isolated by Lympholyte® Cell Separation (Cedarlane, Canada) .
c-Maf and MafA were cloned as described previously . UBE2O cDNA (Genebank accession No. BC051868.2) was obtained from the SPARC BioCentre, The Hospital for Sick Children, Toronto, Canada.
To generate UBE2O-expressing lentivirus, the cDNA fragment of UBE2O was generated by using the following primers: 5′-TCGAGCTCAAGCTTATGACCTCAGCCGACGTGATG-3′ (forward) and 5′-CTCACCATGACCCATGACCGGTGGATCCTCCTTGTCCTCTGTGCACTCCG-3′ (reverse) and inserted into pLVX-AcGFP lentiviral vector (Clontech) within the EcoRI and BamHI sites. The specific protocol for packaging of the virus and preparation of lentiviral particles were described previously .
Affinity purification-coupled mass spectrometry (AP/MS)
The AP/MS process and data analysis were performed as described previously .
HEK293T cells were transfected with c-Maf and UBE2O plasmids for 24–48 h followed by immunoprecipitation as described as previously  using specific primary antibodies as needed followed by incubation with 40 μl of a 50% slurry of protein A+G agarose beads with gentle rotation at 4 °C for 2 h. Agarose beads were collected and washed five times with the lysis buffer , followed by re-suspension in 20 μl of 2× SDS loading buffer. Samples were then boiled before being subjected to fractionation on sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and immunoblotting (IB) analysis.
In-tube ubiquitination assay
This assay was adapted from a previous report . Briefly, HA-c-Maf and Flag-UBE2O plasmids were transfected into HEK293T cells, respectively. Forty-eight hours later, cells were treated with MG132 for 2 h, followed by cell lysate preparation. To enrich and purify c-Maf and UBE2O proteins, individual cell lysates were subjected to immunoprecipitation with HA- (for c-Maf) or Flag- (for UBE2O) antibody-conjugating agarose beads, respectively, at 4 °C for overnight. After that, the beads were washed four times with an immunoprecipitation lysis buffer, twice with 1× ubiquitin reaction buffer (Boston Biochem, Boston, MA) and then re-suspended in 20 μl of 1× ubiquitin reaction buffer containing 200 ng of recombinant E1, 250 ng of recombinant UbcH5c, 10 μg of ubiquitin, 0.5 mM ATP, and 1× Energy Restoration System (Boston Biochem, Boston, MA). The reaction was carried out at 30 °C for 2 h and then terminated by boiling in the 2× SDS loading buffer. Ubiquitinated products were resolved by SDS-PAGE and detected by immunoblotting analysis.
Immunoblotting (IB) analyses and cycloheximide (CHX) chase analysis
All the IB and CHX chase assays were performed as described previously .
Cell proliferation assay
Cell cycle analysis
MM cells were infected with lentiviral UBE2O for 48 h; cells were collected and treated with cold 70% ethanol and stained with propidium iodide (PI). Cell cycle was analyzed on a cytometer (FACSCalibur; BD Biosciences, San Jose, CA) as described previously .
Cell apoptosis by cytometry
MM cells were infected with lentiviral UBE2O at various periods; cells were then stained with 7-ADD and Annexin V-PE (MultiSciences BiotechCo., Ltd., Hangzhou, China) followed by analysis on a cytometer as described previously .
The luciferase reporter plasmid pGL4-CCND2-Luc was constructed as described previously . The DNA sequence of CCND2 promoter contained a c-Maf recognition element (MARE) which could be recognized by c-Maf protein [10, 21]. To examine the effect of UBE2O on c-Maf biological function, HEK293T cells were co-transfected with pGL4-CCND2, c-Maf, and UBE2O, and a β-gal expression vector (100 ng). Luciferase and β-gal expression were measured 36 h after transfection according to the manufacturer’s protocols (Promega, Madison, WI, USA). Firefly luciferase activity was normalized to β-gal expression for each sample . All transfection experiments were performed in duplicates.
GEO dataset analyses
The DNA microarray dataset from primary MM patients and healthy donors was retrieved from Gene Expression Omnibus (GEO) databases (http://www.ncbi.nlm.nih.gov/gds) . Log2(UBE2O mRNA level) was reported.
Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted using Trizol® (Transgene, Beijing, China). RNA (2.5 μg) was reversely transcribed using a SuperscriptTM-III kit (Invitrogen) according to the manufacturer’s instruction. PCR amplification was carried out in 25 μL of PCR reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 20 pmol of each primer set, two units of Taq DNA polymerase (Transgene, Beijing, China), 0.2 mM dNTPs, and 2 μL cDNA. The primers for UBE2O were 5′-ACATCCGCTCCAACGAC-3′ and 5′-GCTGGTGCTGCCTTCTAC-3′, and the primers for GAPDH were 5′-CCAGCCGAGCCACATCGCTC-3′ and 5′-ATGAGCCCCAGCCTTCTCCAT-3′. PCR products were visualized by ethidium bromide staining after electrophoresis on 1.5% agarose gels. The optical densities of the genes were quantified using the ImageJ software (National Institutes of Health, Bethesda, MD) .
Xenografts in nude mice
LP1 cells (2 × 107) were s.c. inoculated into the right flanks of nude mice (The SLAC Experimental Animal Co., Shanghai, China). When the tumors were palpable, 10 μg of UBE2O plasmids or empty vectors in 100 μl of In Vivo-jetPEI® Delivery Reagent (N/P = 6) (Polyplus-transfection Inc., New York, USA) [23, 24] were injected into tumors twice a week for continued 3 weeks . Tumor sizes and mice body weights were monitored every other or 3 days. This xenograft study was approved by the Review Board of Animal Ethics of Soochow University. At the end of the experiment, all tumor species were excised for immunoblotting analysis against specific antibodies as indicated.
Student’s t test was used to calculate P values for differences. Differences were considered significant at P < 0.05.
UBE2O interacts with c-Maf
The mass spectrometry data of UBE2O
To verify the interaction between UBE2O and c-Maf, a UBE2O and a c-Maf plasmid were co-transfected into HEK293T cells, from which the lysates were co-immunoprecipitated with a specific anti-HA antibody followed by immunoblotting. As shown in Fig. 1c, UBE2O was found in the c-Maf precipitates but not in controls. To confirm this finding, a reciprocal Co-IP was performed with a specific antibody against UBE2O, followed by immunoblotting against c-Maf and the result showed that c-Maf was present in the UBE2O precipitates (Fig. 1d). To further check this finding in MM cells, lentiviral UBE2O was infected in a MM cell line LP1. In this experiment, c-Maf was used as a baitor. As shown in Fig. 1e, UBE2O was identified in the c-Maf precipitates. Therefore, these immunoprecipitation/immunoblotting assays suggested that c-Maf interacted with UBE2O.
UBE2O mediates c-Maf polyubiquitination
UBE2O mediates c-Maf turnover in the proteasomes
K331 and K345 are critical for c-Maf stability mediated by UBE2O
UBE2O suppresses c-Maf transcriptional activity
To further evaluate the effects of UBE2O on c-Maf activity, a luciferase reporter assay was performed after UBE2O was transfected into HEK293T cells along with pCCND2.Luci with or without c-Maf plasmids. The results showed that c-Maf markedly increased the luciferase activity under the control of CCND2 promoter containing a c-Maf recognition element (Fig. 5d) . To confirm this conclusion, we next measured c-Maf and cyclin D2 levels in MM cells after MM cell lines LP1 and RPMI-8226 were infected with lentiviral UBE2O. As shown in Fig. 5e, both c-Maf and CCND2 were downregulated when UBE2O was re-expressed (Fig. 5e). However, when these MM cell lines infected with lentiviral UBE2O were subjected to cell cycle analysis, the results showed that UBE2O arrested both RPMI-8226 and LP1 cells at the S or S/M phases (Fig. 5f), which was out of expectation because cyclin D2 is a key player in control the progress of cell cycle from G1 to S phase.
UBE2O suppresses proliferation and induces apoptosis of MM cells expressing c-Maf but not the MM cells lacking c-Maf
UBE2O delays myeloma tumor growth in nude mice
The above results demonstrated that UBE2O is a ubiquitin-conjugating enzyme that mediates c-Maf polyubiquitination in the absence of ubiquitin E3 ligases. UBE2O induces K48-linked polyubiquitinaition of c-Maf and mediates its degradation in the proteasomes. Moreover, UBE2O displays as a tumor suppressor against MM.
There are more than 35 E2s, of which most are small proteins with molecular weight from 14 to 35 kDa, and these E2s do not exhibit intrinsic affinity for physiological substrates but usually coordinate with E3s to mediate protein ubiquitination . As the only large-size E2, UBE2O has been reported to modify several important proteins in an atypical ubiquitination manner, such as K63-polyubiquitination  and monoubiquitination . Accordingly, UBE2O modulates the biological functions of its target proteins. For example, UBE2O mediates multiple mono-ubiquitination of BAP1, a nuclear localization signal, and leads to its cytoplasmic sequestration . Recently, UBE2O was found to inhibit, rather than to induce, TRAF6 polyubiquition . In the present study, we found that UBE2O induces c-Maf polyubiquitination. This is consistent with that finding that UBE2O mediates the K48-linked polyubiquitination of c-Maf, a typical ubiquitination manner in modulating protein stability, because when K48 was mutated, c-Maf failed to be ubiquitinated by UBE2O. Furthermore, we demonstrated that UBE2O also functions as an E3 ubiquitin ligase because it alone suffices to mediate c-Maf ubiquitination in tube in the absence of any E3 ligases. Therefore, we can conclude that UBE2O probably modifies protein with various ubiquitination manners including monoubiquitination, K48-linked ubiquitination and K63-linked ubiquitination.
In terms of the biological functions, UBE2O is originally believed to participate in erythroid differentiation because it is highly expressed in reticulocytes and can ubiquitinate endogenous proteins involved in erythroid cells [2, 3]. Recent evidence shows that UBE2O is involved in adipogenesis , actin polymerization , inflammation , nuclear trafficking of chromatin-associated proteins , and tumorigenesis . Different from previous findings, the present study suggests that UBE2O acts as a negative modulator in MM because it is downregulated in MM cells which was confirmed by both DNA microarray and RT-PCR. When it is restored, UBE2O induces MM cell apoptosis and delays MM tumor growth in nude mice (Fig. 6), suggesting UBE2O is probably a tumor suppressor protein against MM. This action of UBE2O suppressing MM is highly associated with c-Maf expression, c-Maf protein ubiquitination, and degradation because UBE2O fails to induce apoptosis of MM cells lacking c-Maf. c-Maf is a frequently expressed basic leucine zipper transcription factors in MM. As a transcription factor, c-Maf binds to the Maf recognition element (MARE) of downstream genes and triggers their transcription. These downstream genes mainly include cyclin D2, integrin β7, CCR1, and ARK5, which are responsible for cell cycle progress, bone marrow stromal cell adhesion, MM cell proliferation, and myeloma cell invasion, respectively . However, out of expectation, UBE2O fails to arrest MM cell cycles at the G0/G1phase. This finding probably suggests that UBE2O also targets other proteins that also modulate MM cell cycle and cell proliferation, but these proteins act on other cell cycle phases, such as S or G2/M phases.
In the present study, we also found that UBE2O is downregulated in MM cells, how does this happens is not clear. The probable mechanisms include suppression of gene transcription modulated by small interfering RNA or non-coding RNA or other factors in gene expression regulation. In addition, c-Maf is a basic zipper family transcription factor and this class of transcription factors displays various regulations in gene transcription, including both promotion and suppression cell differentiation or cell proliferation . Whether UBE2O is negatively modulated by c-Maf is not known; it will be very interesting if UBE2O-targeted therapy towards MM can be developed.
The present study showed that UBE2O inhibits proliferation and induces apoptosis of MM cells expressing c-Maf but not those lacking c-Maf, because MM is highly heterogeneous. Our previous studies showed that the c-Maf status is a significant factor for MM cell response to dexamethasone, a mainstay of MM treatment . This study showed that targeting UBE2O could be a promising strategy for the treatment of a subset of c-Maf expressing MM.
Taken together, the present study identifies that UBE2O as a novel regulator modulates c-Maf protein stability by mediating its polyubiquitination and subsequent degradation in proteasomes. UBE2O specifically induces apoptosis and inhibits proliferation of a subset of MM cells that express c-Maf. This study provides a novel insight in understanding c-Maf biological function and targeted MM therapy.
The authors thanked Dr. Guoqiang Xu for his discussion on this project.
This work was partly supported by the National Natural Science Foundation of China (81320108023 and 81600171), by the Natural Science Foundation of Jiangsu Province (BE2014630), by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and by the Jiangsu Key Laboratory for Translational Research and Therapeutics of Neuro-Psycho-Diseases (BK2013003). The study was also partly supported by the Suzhou Key Laboratory for Pediatric Leukemia (SZS201615).
XM designed the study; ZZ, YX, JL, TJ, BC, ZX, and PT conducted experiments; XM, XT, DW, YZ, and MM analyzed data; XM and YZ wrote the manuscript. All authors read and approved the final manuscript.
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This study was approved by the Review Board and Ethical Committee of Soochow University, and each patient provided written informed consent to donate bone marrows for this study after diagnostic and clinical procedures.
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