Background
The von Hippel-Lindau (VHL) disease is caused by mutation of
VHL tumor suppressor gene and classified into two types depend on genotype-phenotype correlation. The mutation of Type 1 VHL disease is truncation or exon deletion and type 2 VHL disease have missense mutation commonly. Type 2 VHL disease shows a high risk of pheochromocytoma (PCC) and germ line missense mutations is subdivided into high risk (2B), low risk (2A), or absence (2C) of Renal cell carcinoma (RCC) and heamangioblastoma is correlated with function of pVHL to impair HIF-1α activity [
1,
2]. Regarding to HIFs regulation, type 1 and type 2B VHL disease have high defect and type 2A relative low defect. In certain types 2VHL disease, mutations of
VHL gene retain their functionality to regulating HIFs but they exhibit instability of mutant VHL protein [
3‐
5]. However the mechanisms control the instability of missense mutant pVHLs are still under discovered.
Proteasome dependent proteolysis is efficient and powerful system for regulating half-life of cellular proteins. Ubiquitination is start signal for proteasomal degradation which is consisted by E1, E2 and E3 enzyme. pVHL is the substrate recognition component of an E3 ubiquitin ligase complex that also contains elongin B, elongin C, Cul2, and Rbx1 [
6‐
9]. pVHL has two functional domains that directly bind to elongin C and pVHL substrates, respectively and it targets the HIFs for ubiquitin-mediated degradation [
5,
10‐
13]. Prolyl-hydroxylated HIFs are recognized by pVHL, which results in it being polyubiquitinated and, thereby, targeted for proteasomal degradation [
14,
15]. The different domains of pVHL are also important for its stability because mutant pVHL which are defective in elongin C binding, are unstable and are rapidly degraded [
16]. pVHL also has role in maintaining extracellular matrix (ECM) thus pVHL-knock out cells like 786-O or RCC4 revealed loss of assembling fibronectin. The function of pVHL maintaining ECM is not depend on HIFs [
17].
Human E2-EPF UCP (UCP) was the first E2 family member to be cloned from epidermal tissue [
18]. Expression of UCP is five times higher in common human cancers than in normal tissues [
19,
20] Roos et al. has been reported that UCP implicated in papillary RCC which is second most common subtype of kidney cancer [
21]. Recombinant UCP is a bifunctional enzyme that is capable of catalyzing E3-independent and E3-dependent ligation of ubiquitin and UCP targets pVHL for ubiquitin-mediated degradation [
22,
23]. Since UCP impair to tumorigenesis, we examined whether UCP can degrade V155A, L158Q and Q164R missense mutant pVHLs which are linked to RCC. In this study, new biochemical mechanism of instability of missense mutant pVHL is provided and UCP can be served as a therapeutic target for RCC which is related missense mutation of
VHL gene.
Methods
Antibodies and reagents
Anti-Flag, anti-GST and anti-b-actin antibodies were purchased from SIGMA-Aldrich. Anti-HA antibody was purchased from AbFrontier, and anti-His antibody was purchased from Millipore. Human anti-HIF-1α was purchased from BD Pharmingen, and human anti-HIF-2α was purchased from Santa Cruz Biotechnology. The anti-UCP antibody was generated by protocol, as reported previously [
23]. The proteasome inhibitor MG132 was purchased from Boston Biochem, and cycloheximide was purchased from SIGMA-Aldrich. Luminol assay kit was purchased from Promega.
Plasmids
Human UCP, elongin C, HIF1a, and UbcH5C cDNA molecules were supplied by the 21C Frontier Human Gene-Bank, South Korea. Full-length UCP was cloned into pET28a (novagen) and pCMV-tag1 (Stratagene). Wild-type pVHL and point mutants were cloned by PCR amplification from pFlag-VHL (a gift from S. Cho, Chung-Ang University, Seoul, South Korea) into pCDNA3.1+ (Invitrogen), pEBG, pGEX-4 T1 and pET-28a. The shUCP (5′-AATGGCGATCTGCGTCAAC-3′) sequence was inserted into the pSUPER vector according to the manufacturer’s instructions (Invitrogen). The sequences of all plasmids were verified by direct sequencing before use. pTK-Hyg (Clontech) was used for producing HeLa -shUCP expressing constitutive cell line. Five repeat copies HRE derived from VEGF promoter cloned to pGL3 (Promega).
Cell culture and counting
786-O cells, HEK293T cells and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10 % fetal bovine serum (FBS, GIBCO) in a humidified incubator with 5 % CO2 at 37 °C. The 786–O cell lines stably expressing exogenous pVHL were transfected with the indicated plasmids or empty vector (pCDNA3.1), and were cultured with 1 mg/mL geniticin (G418, GIBCO) for 1 month for single colony selection. For the cell proliferation assay, the cells were plated at 5 × 103 cells/well in conditioned media on 24-well plates. At 24 h after seeding, the cells were trypsinized and counted by a hemocytometer. The viability of cells were observed by crystal violet staining (0.1 % w/v). Luminol assay for HRE-luc was performed as manufacturer’s indication.
Protein stability analysis
The 786-O cell lines stably expressing exogenous HA-tagged wild-type or mutant pVHLs were treated with 50 μg/ml cycloheximide for 0, 2, 4 and 6 h. At the indicated time points, the cells were harvested, and proteins were detected by western blot analysis with a VHL antibody (BD Pharmiongen). The signal intensity was determined using densitometer software.
Immunoblot analysis and pull down assay
Cells were lysed on ice using RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1 % NP40, 0.1 % SDS, 1 mM PMSF, 1X protease inhibitor) and were separated by 12 % SDS-PAGE. The proteins were transferred from the gel onto a PVDF membrane (polyvinylidene fluoride, Millipore), and the membrane was incubated with specific primary antibodies in PBS/0.1 % Tween20 (PBST) for 2 h at RT or overnight at 4 °C. Subsequently, the membrane was incubated with secondary antibody in PBST containing 0.5 % skim milk for 1 h at RT, and the proteins were visualized using a chemiluminescence kit (Intron). The cell lysate was prepared in NET gel buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 % NP-40, 1 mM EDTA, pH 8.0) supplemented complete proteinase inhibitor cocktail (Roche), and GST-tagged and His-tagged proteins were pulled-down with the glutathione sepharose beads (GE healthcare) and Ni-NTA agarose (Qiagen). Proteins were separated by SDS-PAGE and detected by immunoblot with antibody as indicated.
Purification of recombinant fusion proteins
GST fusion proteins were expressed and purified as described by the manufacturer (Amersham). pGEX-4 T1 vector-based GST fusion proteins were induced with 1 mM IPTG for 2 h at 37 °C. Cells were washed with PBS, resuspended in lysis buffer (PBS, protease inhibiter cocktail, 1 mM PMSF), and then sonicated on ice. Soluble protein extracts were added to glutathione sepharose 4B resin (Amersham) and incubated for 2 h at 4 °C. The columns were washed five times with PBS. Bead-bound proteins were eluted with elution buffer (50 mM Tris, pH 8.8, 1 mM EDTA, 20 mM glutathione reduced (GSH), 1 mM PMSF). His-fusion proteins were expressed and purified as described by the manufacturer (QIAGEN). pET28a vector-base His fusion proteins were induced with 1 mM IPTG for 2 h at 37 °C. The cells were resuspended in lysis buffer (0.5 M NaCl, 5 mM imidazole, 20 mM Tris, pH 7.9) and then sonicated on ice. The cell extracts were added to Ni-NTA resin (QIAGEN) and were incubated for 2 h at 4 °C. The columns were washed five times with wash buffer (0.5 M NaCl, 60 mM imidazole, 20 mM Tris, pH 7.9). Bead-bound proteins were eluted with elution buffer (0.25 M NaCl, 0.5 M imidazole, 10 mM Tris, pH 7.9). After dialysis, the purified proteins were stored at -70 °C.
Ubiquitination assay
In vivo ubiquitination assay was performed by protocol, as previously described [
23]. For the self-ubiquitination of UCP, the reaction mixture (50 μl) contained 0.3 μg of GST-UCP, 0.5 μg of His-E1 and 25 μg/ml Flag-ubiquitin in reaction buffer (25 mM Tris-Cl, pH 7.5, 1 mM ATP, 5 mm creatine phosphate, 0.5 μg/ml creatine phosphate kinase, 1 mM DTT, 5 mM MgCl
2, 0.5 μg/ml ubiquitin aldehyde) was used. The mixture was incubated for 1 h at 37 °C, and then a western blot analysis was performed using the indicated antibodies. For the ubiquitination of pVHL by UCP, the reaction mixture (50 μl) containing 0.3 μg of GST-UCP, 0.5 μg of His-E1, 3 μg of His-VHL, and 25 μg/ml Flag-ubiquitin in reaction buffer was used. After incubation at 37 °C for 1 h, GST-VHL was pulled down with glutathione sepharose 4B resin and was analyzed by SDS–PAGE. For the ubiquitination of HIF-ODD by the VHL-elongin B-elongin C (VCB) complex, the 786-O cells were washed and collected in PBS. The cells were disrupted, using a sonicator, in lysis buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.5 mM EDTA, 0.1 % NP40, 1 mM PMSF, 1X protease inhibitor). The cell lysates were centrifuged at 13000 rpm for 1 h at 4 °C. The total reaction volume was 50 μl and contained 50 μg of 786-O cell extracts, 3 μg of GST-ODD, 0.3 μg of His-VHL, 0.3 μg of UBCH5C, and 0.5 μg of His-E1 in reaction buffer. The mixtures were incubated for 2 h at 30 °C. After incubation, the reaction mixtures were pulled down with glutathione sepharose 4B resin and analyzed by SDS–PAGE.
RT-PCR and real time PCR
Total RNA was extracted from cells using an easy-spin RNA extraction kit (Intron). Complementary DNA (cDNA) was synthesized using 3–5 μg of total RNA, reverse transcriptase (TakaRa, Japan) and oligo (dT) primer. cDNA was amplified by polymerase chain reaction using primers specific for each gene (Additional file
5: Table S1). For the LightCycler (Roche Diagnostics) reaction, LightCycler mastermix and cDNA as the PCR template were filled in PCR tube. The mixtures were centrifuged and placed into the LightCycler rotor. The following LightCycler experimental run protocol was used: denaturation (95 °C for 10 min), amplification and quantification repeated 35 times (95 °C for 15 s, 60 °C for 10 s, and 72 °C for 60 s) with a single fluorescence measurement.
Animals and ex vivo xenograft assay
Seven-week-old female BALB/c nude mice were purchased from SLC japan and maintained in a accordance with guidelines and approval of Institutional Review Committees for Animal Care and Use, Korea Research Institute of Bioscience and Biotechnology (KRIBB-AEC-14024). 786-O and 786-VHL (WT and V155A) cells were transduced with adenoviral vectors (Ad.shUCP and Ad.shCont) at 200 MOI for 24 h. And then cells (10
7) are transplanted by subcutaneous injection into nude mice (Japan SLC, Inc.). Tumor size was measured for 44 days by following procedure, as reported previously [
23,
24].
Statistics
Statistical analysis was carried out using the unipolar, paired Student t test and the two-sided chi-square test. Data were considered statistically significant when the P value was less than 0.05.
Discussion
pVHL pocess E3 ubiquitin ligase activity to degrade HIFs which is related in tumor pomoting events but the mechanisms inducing instability of pVHL itself are not clarified clearly. Based on complex of VCB complex, folding and conformational chagnes of protein result in proteosomal degradation dependent on chaperones [
16,
25]. A recent findings supported that missense mutant pVHL was easily degraded, and therefore had shortened half-life in cell [
26,
27]. Missense mutation of
VHL gene is most frequent in type 2 VHL disease. Depend on ability to control HIFs, it is classified into 2A, 2B and 2C. In case of type 2C, mutant pVHL retains function as E3 ubiquitin ligase to HIFs which induce angiogenic factors and stimulate glucose metabolism in cancer cells. These information suggest that inhibition or retardation of degrading pVHL is crucial for gain of function of missense mutant pVHL.
Based on the correlation between functional loss of pVHL and missense mutations in the VHL disease-associated tumors, VHL disease was classified into three clusters [
24]. First cluster is formed by the surface residues are responsible for the interactions between elongin C and pVHL [
10,
13]. The residues V155, L158, Q164 and R167 are the most frequently mutated residues in VHL syndrome [
28]. The residue V155, L163 and V166 are associated in RCC [
29]. The second cluster of mutations are located in HIFs protein binding site of pVHL binding [
30]. Tyrosine 98 residue most popular mutated amino acid in this cluster that involved in tumorigenesis [
31‐
33]. Last cluster of mutations are located on the β-domain and residues R79, S80, R82, L89, D121, Q132, L135, F136, and P138 are reported [
34]. We characterized 7 VHL missense mutants as Y112H, R167Q, 188 V, V155A, L158Q, Q164R and N78S. Except V155A, 6 missense mutant pVHLs were discovered at nature and they are related with VHL disease [
35‐
39].
UCP has been revealed as a factor that reconganize and targeted wild type pVHL for proteosomal degradation thereby stabilize HIFs. Depletion of UCP inhibit tumor growth and metastasis in vitro and in vivo and it is highly expressed in various cancer [
23]. These findings lead us examine that UCP could recognize missense mutant pVHL and degrade it proteasome dependently like wild type pVHL and depletion of UCP level can rescue function of missense mutant pVHL. UCP was found to ubiquitinate all of missense mutant pVHL in vitro (Additional file
1: Figure S1) ubiquitination assay and these mutant pVHL interacted to HIF-1α (Additional file
2: Figure S2). These result suggested that tested mutant pVHL can regulate HIFs activity as far as it is stable. Taken together, UCP can be a critical factor for regulating HIFs via targeting missense mutant pVHL in RCC. In order to suggest meaning of UCP-VHL-HIFs axis, we characterized V155A, L158Q and Q164R missense mutant pVHLs which are most frequent in RCC. These three missense mutantations are located near the elongin C binding site of pVHL, which forms the pVHL-elongin complex to prevent the degradation of pVHL. UCP also recognized mutant pVHLs (V155A, L158Q and Q164R) and ubiquitinated them in vitro and in a cellular system. These missense mutations in
VHL gene do not cause structural changes to the UCP binding site. Thereby UCP ubiquitinated missense mutant pVHLs, it caused degradation by proteasomes in cell. Ubiquitination by UCP can be a critical factor to determine stability of missense mutant pVHLs (Figs.
1 and
2). In addition to ubiquitination, some post translational modifications of protein by ubiquitn-like molecules like SUMOylation or NEDDylation has been reported as strategy for regulating protein dynamics in cells. What are major factors for regulating stability of missense mutant pVHL is still under question.
The polymerization of ubiquitin occurred at between glycine of the ubiquitin and the lysine residues of target protein. Ployubiquitin chain which is attached on lysine, recognized by 26S proteasome. UCP possess E3 ubiquitin ligase activity to wild type pVHL and VHL protein has three lysine residues (K159, K171 and K196). We hypothesized that UCP recognize three lysine of pVHL for ubiquitination. As expectation, pVHL lysine zero mutant had longest half-life and K159 mutant pVHL was relative long half-life than the others (Fig.
3). Indeed UCP mostly did not ubiquitinated lysine zero mutant pVHL in cell (Additional file
3: Figure S3). But there is significant difference of ubiquitination between single lysine mutant (Additional file
3: Figure S3). Therefore, K159 mutant pVHL is regulated by another mechanism in addition to ubiquitination. These results suggest that the inhibition of UCP mediated poly-ubiquitin chain elongation at the lysine residues of pVHL increased the stability of pVHL in a cellular system. However, we did not determine whether these lysine mutants had the same functions as wild-type pVHL.
The effect of missense mutations in
VHL gene was examined by impairing E3 ligase activity. The E3 ubiquitin ligase complex, named as the VBC complex, is composed of elongin C, elongin B, Rbx1, cul2 and pVHL, and it functions as a substrate recognition molecule [
16]. Missense mutant pVHLs formed the VBC complex and directly ubiquitinated HIF-1 α in vitro even it is existed at RCC which is nearby Elongin binding site (Fig.
4). In fact, L158Q had very weaken interacting affinity with Elongin C. These data are collected from over expression system which might be different from endogenous expression level. Thus, the examined missense mutations did not impair the E3 ligase activity of pVHL. Instability is crucial for impairing the functionality of missense mutant pVHLs. Three missense mutants (V155A, L158Q, and Q164R) sustained E3 ligase activity as indicated by their ability for the VBC complex and degrade HIF-2 α thereby decreased the expression VEGF, Glut-1 and Epo, which are target genes of HIF-2α [
40] which promotes tumor cell growth, invasion and regulates glucose metabolism [
41]. Protein levels of missense mutant pVHLs were inversely correlated with HIF-2α and its functionality to cell growth (Fig.
5). Collectively in case of three missense mutations of
VHL gene (V155A, L158Q, and Q164R), they had function of tumor suppressor if they were protected from degradation. With regards to VHL disease, missense mutation of VHL gene induced pVHL instability, and the loss of function of pVHL caused an increase in the cellular level of HIFs, which promoted cell growth. Thus, we tested whether UCP depletion could rescue VHL disease particularly in RCC (Fig.
6). Depletion of UCP increased protein level of mutant pVHL and inhibited cell growth in vitro. Since depletion of UCP showed relative higher inhibition the growth of cell expressing V155A mutant pVHL than the others, we used cell expressing V155A mutant pVHL for ex vivo experiment. The UCP level was decreased and the pVHL level was increased in the tumor tissues and pVHL induced the expression of fibronectin and E-cadherin but HIF-2α was decreased in tumor nodules. Tumor microenvironments were composed with heterogeneous cells and molecules [
42]. pVHL enhanced extracellular matrix protein, thus prolonged the tissue morphology and inhibited tumor metastasis HIFs independently [
17]. V155A pVHL missense mutant is not existed in nature yet. Thus these data give biological prospect of new missense mutation of
VHL gene.
Consequently, UCP ubiquitinated missense mutant pVHLs (V155A, L158Q and Q164R) via proteasomal degradation. Therefore depletion of UCP can be the therapeutic method for type 2 VHL disease such as RCC.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
K-SP performed the experiment mainly. JHK and HWS supported the experiment with purification of recombinant proteins and construction of mutants. Dr. K-SC provided experimental comment. Dr. D-SI provided information and idea for writing revised paper. Dr. C-RJ and Dr. JHL wrote paper and designed experiments. All authors read and approved the final manuscript