Human Molecular Genetics, 2001, Vol. 10, No. 10 1029-1038
© 2001 Oxford University Press
Contrasting effects on HIF-1
regulation by disease-causing pVHL mutations correlate with patterns of tumourigenesis in von Hippel-Lindau disease
1Section of Medical and Molecular Genetics, Department of Paediatrics and Child Health, University of Birmingham, The Medical School, Edgbaston, Birmingham B15 2TT, UK, 2Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK and 3Cambridge University Department of Pathology, Tennis Court Road, Cambridge CB1 4QP, UK
Received 16 December 2000; Revised and Accepted 2 March 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The von Hippel-Lindau tumour suppressor gene product (pVHL) associates with the elongin B and C and Cul2 proteins to form a ubiquitin-ligase complex (VCBC). To date, the only VCBC substrates identified are the hypoxia-inducible factor
subunits (HIF-1 and HIF-2). However, pVHL is thought to have multiple functions and the significance of HIF-1 and HIF-2 regulation for tumour suppressor activity has not been defined. VHL disease is characterized by distinct clinical subtypes. Thus haemangioblastomas (HABs) and renal cell carcinoma (RCC) but not phaeochromocytoma (PHE) occur in type 1 VHL disease. Type 2 subtypes are characterized by PHE susceptibility but differ with respect to additional tumours (type 2A, PHE+HAB but not RCC; type 2B, PHE+ HAB+RCC; type 2C, PHE only). We investigated in detail the effect of 13 naturally occurring VHL mutations (11 missense), representing each phenotypic subclass, on HIF- subunit regulation. Consistent effects on pVHL function were observed for all mutations within each subclass. Mutations associated with the PHE-only phenotype (type 2C) promoted HIF- ubiquitylation in vitro and demonstrated wild-type binding patterns with pVHL interacting proteins, suggesting that loss of other pVHL functions are necessary for PHE susceptibility. Mutations causing HAB susceptibility (types 1, 2A and 2B) demonstrated variable effects on HIF- subunit and elongin binding, but all resulted in defective HIF- regulation and loss of p220 (fibronectin) binding. All RCC-associated mutations caused complete HIF- dysregulation and loss of p220 (fibronectin) binding. Our findings are consistent with impaired ability to degrade HIF- subunit being required for HAB development and RCC susceptibility. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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von Hippel-Lindau disease (VHL, MIM 193300) is a dominantly inherited familial cancer syndrome characterized by the development of vascular tumours [haemangioblastomas (HABs)] in the retina and central nervous system, clear-cell renal cell carcinoma (RCC) and phaeochromocytoma (PHE) (1–3). In addition, somatic inactivation of the VHL tumour suppressor gene (TSG) is frequent in sporadic clear cell RCC and sporadic central nervous system HABs (4–7). Expression of wild-type pVHL in VHL-null RCC cells suppresses tumour formation in vivo in nude mice (8,9).
Although the sequence of the VHL TSG product itself did not immediately suggest a function, two lines of evidence provided early clues. First, hypoxia-inducible mRNAs, including vascular endothelial growth factor (VEGF), were noted to be constitutively upregulated in VHL-null cells (10–12). Second, following the identification of elongins B, C and Cul2 as pVHL interacting proteins, sequence and structural similarities between the tetrameric pVHL/elongin C/elongin B/Cul2 (VCBC) complex and the yeast Skp1-Cdc53/Cul1-F-box (SCF) complex led to suggestions that pVHL may have a role in targeting oncogenic proteins for ubiquitin-dependent proteolysis (13,14).
Recently, we have linked these two lines of investigation, showing that pVHL interacts with the regulatory -subunits of hypoxia-inducible factor (HIF-1) and targets them for oxygen-dependent proteolysis (15). HIF-1 is a heterodimeric transcription complex consisting of and ß subunits. It is central to a broad range of cellular responses to hypoxia and modulates expression of genes involved in energy metabolism, angiogenesis and apoptosis (e.g. GLUT-1 and VEGF). Under normoxic conditions, the HIF- subunits are degraded rapidly by the proteasome, a process which involves physical association with pVHL. Accordingly, constitutively high HIF- levels are observed in VHL-defective RCC lines, with oxygen-dependent instability restored by re-expression of wild-type pVHL (15). In the absence of pVHL, HIF-1 is activated in normoxia, resulting in upregulation of an extensive range of hypoxia-inducible mRNAs. Taken together with the sequence homology to SCF complexes, these observations suggested that pVHL acts as a component of an E3 ubiquitin-ligase complex which targets HIF- subunits for destruction (15). Structural analysis of the pVHL/elonginC/elonginB complex has indicated that pVHL has two domains; an approximately 100-residue N-terminal ß-sheet domain containing a putative macromolecular binding site and a smaller C-terminal -helical domain which interacts directly with elongin C (16). Under the SCF-like model for VCBC function, the putative ß-domain surface binding site on pVHL would be predicted to bind proteins targeted for ubquitylation, with pVHL thus acting as the recognition component of the E3 ligase complex. Following this, we and others have provided direct evidence establishing that pVHL targets HIF subunits for ubiquitylation via interaction with the ß-domain surface (17–19).
Other functions of pVHL have been reported, including a role in cell cycle exit control (20), fibronectin binding and extracellular matrix assembly (21) and post-transcriptional regulation of target gene expression through mRNA stability effects (22–24). To date however, only HIF- regulation has been directly linked to an SCF-like function for pVHL and the molecular basis for other putative pVHL functions are unknown. A major current objective is to define the link between the range of putative pVHL functions and tumourigenesis.
VHL disease displays complex genotype–phenotype correlations, and the phenotype has been subclassified on the basis of the predominant manifestations (Fig. 1). Type 1 mutations (more commonly deletions and truncating mutations) predispose to HAB and RCC but not (or rarely) PHE. Type 2 mutations predispose to PHE and are more commonly missense mutations. They are subdivided as follows; type 2A, HAB, PHE and rarely RCC [e.g. Tyr98His (25)]; type 2B, HAB, RCC and PHE (e.g. Arg167Gln); type 2C, PHE without other manifestations [e.g. Leu188Val (26)]. This mutation-dependency of tumour susceptibility in VHL disease suggests multiple and/or tissue-specific functions for pVHL, and the existence of specific mutations in VHL disease which are associated with differing tumour risks provides tools to dissect the relationships between pVHL functions and tumour susceptibility.
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To pursue this we have analysed the effects of pVHL mutations which are associated with different disease patterns in a series of in vitro and in vivo assays of HIF regulation, and have displayed the pattern of binding-proteins interacting with each mutant pVHL by immunoprecipitation following stable transfection into a VHL-defective RCC cell line.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutation selection
For analysis of the effect of different pVHL mutations on regulation of the HIF system, a panel of 13 disease-associated germline mutations was selected to represent each VHL disease phenotype (six type 1 and seven type 2 mutations) (Fig. 1). Since not all these mutations are common, the extent of phenotypic information on which classification of the selected mutations [11 missense, one single amino acid deletion (76
Phe) and a 41 amino acid deletion (114–154)] was variable. Some mutations have been described in several independent families or large single kindreds, whereas others have occurred in only a single small family. Thus, although Leu188Val provided the best example of a PHE-only (type 2C) mutation (26), further mutations associated with a PHE-only phenotype have been identified only in smaller families, and hence the functional behaviour of two such mutations (Ser80Gln and Val84Leu) was assessed for comparison with Leu188Val. In addition to the type 2C Leu188Val mutation, an alternative substitution, Leu188Gln, has been reported to produce a type 1 phenotype. To understand further the basis of this substitution-dependent variation in phenotype, we undertook functional studies of both mutations.
In vitro analysis of mutant pVHL
We first assessed the impact of these pVHL mutations on regulation of the HIF system using in vitro assays of HIF- subunit binding and ubiquitylation. For functional assays of HIF-1 ubiquitylation we used an in vitro system based on the ability of wild-type pVHL to restore HIF-1 ubiquitylation activity to a cytoplasmic extract derived from a pVHL-deficient RCC line (17). 35S-labelled HIF-1 substrate and the pVHL species to be tested were generated in reticulocyte lysates which were added to the ubiquitylation reaction and product-assayed for the appearance of low mobility polyubiquitylated HIF-1 species by SDS–PAGE (Fig. 2). Three independent replicate assays were performed on all of the mutants with consistent results. All assays were negatively controlled using reticulocyte lysate programmed with empty vector (pcDNA3.1). In each assay, the activity of mutant pVHLs was also directly compared with positive controls provided by the addition of full-length wild-type pVHL (1–213) and/or wild-type p19 VHL (54–213), both of which resulted in the appearance of ubiquitylated HIF-1 species.
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In vitro HIF-1
ubiquitylation activity could not be re-constituted using any of the mutations associated with types I, 2A or 2B phenotypes. Indeed each of these mutations gave results comparable to those obtained by addition of lysate programmed with empty vector (pcDNA3.1). In contrast the prototypic type 2C mutation (Leu188Val) behaved like the wild-type (1–213) pVHL product. Furthermore, the other two type 2C mutants also promoted HIF-1 ubiquitylation in vitro similarly to wild-type pVHL, suggesting that these mutations belong to a class that is functionally distinct from types 1, 2A and 2B mutants.
The mutant pVHLs were also assayed for the ability to interact with HIF- subunits in vitro using co-immunoprecipitation assays of proteins produced in the reticulocyte translation system (Fig. 3). In keeping with the results of ubiquitylation assays, the type 2C mutations were all able to interact with both HIF-1 and HIF-2 subunits, exhibiting similar efficiency to wild-type pVHL. Among the remaining mutations, two patterns were observed. The majority showed a major reduction in capture of HIF- subunits. Generally, similar patterns of interaction were observed for the HIF-1 and HIF-2 subunits, although HIF-2 did retain a low but detectable interaction with certain mutants, which failed to bind HIF-1. However, two mutants (Arg167Gln and Gln195Ter) were able to bind HIF- subunits, although not as strongly as the wild-type pVHL. Thus, the HIF- subunit interaction appeared necessary but not sufficient for pVHL competence in promoting HIF- ubiquitylation. Inspection of the ubiquitylation assays revealed no difference in ubiquitylation between the seven defective mutants that failed to interact with HIF- and the two that retained this ability. In all cases there was essentially no activity. Thus the in vitro functional assays demonstrated a marked difference between mutations associated with the PHE-only type 2C phenotype and other classes of mutation, but there were no discernible differences between the types 1, 2A and 2B. Though the type 2B-associated mutations contrasted with types 1 and 2A in being able to bind HIF- subunits, they were equally disabling in HIF-1 ubiquitylation assays. This indicated that if the differences in clinical tumor predisposition between types 1, 2A and 2B are due to effects on HIF regulation, then the differences were not reflected in these in vitro assays.
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Protein interaction profiles and HIF-
subunit regulation by pVHL mutants in vivoWe therefore performed in vivo studies to determine the effects of these mutations on HIF-
subunit regulation using stable transfection of VHL-defective RCC4 cells. These studies also enabled comparative display of labelled pVHL co-immunoprecipitated species in wild-type and mutant pVHL transfectants. We postulated that this more complete display of interacting species might provide a better understanding of defective HIF ubiquitylation by the pVHL mutants that were competent in HIF- binding assays, and might provide further insights into the clinical behaviour of different classes of mutant that were all defective in in vitro assays of HIF-1 ubiquitylation.
Stable RCC4 transfectants were generated expressing wild-type or mutant haemagglutinin (HA) epitope-tagged pVHL and assayed for effects on the suppression of HIF- subunit levels in normoxic cells. As reported previously, HIF- subunits are regulated in a pVHL-dependent fashion. RCC4 cells transfected with pcDNA3.1 demonstrated constitutively elevated HIF- protein levels under normoxic conditions, whereas the normal normoxic suppression of HIF- subunits was completely restored upon stable transfection with wild-type pVHL. In contrast, all type 1 pVHL mutants failed to restore HIF- subunit regulation. Both type 2A and 2B mutants were also impaired in their ability to regulate HIF- subunits, though the defect in type 2A mutants appeared less complete. In concordance with the in vitro assays all three type 2C mutations restored HIF- subunit regulation behaving similarly to wild-type pVHL (Fig. 4).
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To display pVHL interacting species, transfectants were labelled with [35S]methionine, lysed and subjected to anti-HA immunoprecipitation. Proteasomal blockade was used to permit the display of HIF-
subunits and other potential proteolytic substrates that might otherwise be degraded following interaction with pVHL. In anti-HA immunoprecipitations of pVHL(wild-type 1–213.HA) transfected RCC4 cells (Fig. 5, lanes 2, 8 and 16) pVHL was observed as three species of 
30, 25 and 21 kDa, comparable to the pattern observed by immunoblotting (Fig. 4). Immunoprecipitation of RCC4 transfectants overexpressing the internally initiated (codon 54) form of wild-type pVHL produced an identical pattern of interacting proteins to the full-length gene product, although the 25 and 30 kDa pVHL species were absent (Fig. 5, lanes 2 and 3). Hence, the 21 kDa pVHL species most likely represents the epitope-tagged version of the internally translated wild-type pVHL species (27,28) while the 25 and 30 kDa bands represent species arising from the full-length (1–213) protein. In addition to pVHL, specific species of 220, 135, 120, 18 and 15 kDa were precipitated. We have previously assigned the 135 and 120 kDa species as HIF-1 and HIF-2 (17), whereas the 220–240, 18 and 15 kDa species are consistent with pVHL-interacting proteins previously identified as fibronectin, elongin B and elongin C, respectively (21,29,30). Under our labelling conditions, the elongin C (15 kDa) species was consistently more intense than the 18 kDa elongin B species, and the species assigned by others as CUL2 was not reliably identified. For the purpose of these experiments, interaction of pVHL mutants with elongin C was therefore taken as indicative of VCBC complex recruitment.
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Results for the immunoprecipitation studies are shown in Figure 5 and summarized in Table 1. All types 1, 2A and 2B mutants behaved abnormally. Type 1 mutants failed to capture either HIF-
subunit or significant levels of p220/fibronectin. Though most were also defective in elongin C capture, two type 1 mutants (Asn78Ser, 114–154) (Fig. 5 and Table 1) retained near normal capture. Types 2A and 2B mutants also showed much reduced levels or absent interactions with HIF-1, HIF-2 and the p220/fibronectin species. Interestingly the two type 2B mutants (Arg167Gln and Gln197Ter), which failed to ubiquitylate HIF-1 in the in vitro assays but retained HIF- binding in vitro, failed to interact with elongin C in these assays and also showed much reduced HIF- subunit interaction in the in vivo assay. The two type 2A mutants that showed incomplete suppression of normoxic HIF- levels in these transfectants retained elongin C binding and some HIF- subunit interactions. In contrast to the other mutants, the three type 2C mutants behaved as wild-type, with equivalent binding of HIF-, p220/fibronectin and the elongins. No novel species were observed in the co-immunoprecipitates of any of the mutant pVHL molecules.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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To better understand the mechanisms underlying VHL disease, we have analysed a large series of different disease-associated germline VHL mutations for effects on protein interaction and regulation of the HIF system. Previous studies have indicated that pVHL associates/interacts with elongins B/C and Cul2 to form a multicomponent ubiquitin ligase (VCBC). HIF-
subunits have been identified as targets of this complex. Structural studies of the pVHL elongin B/C complex predict that elongin C and substrate interactions involve distinct binding interaction surfaces on pVHL encoded by and ß domains, respectively. In keeping with this model we found that mutations in pVHL could disable HIF regulation by interfering with either elongin C complex recruitment, HIF- capture or both. Nevertheless, the effects of mutation within each domain were residue- and substitution-specific. Thus, within the ß-domain Asn78Ser abrogated HIF- capture but retained elongin C binding, whereas Ser65Trp ablated binding of both proteins, most probably because of major structural effects on pVHL. Similarly, within the -domain, Arg167Gln greatly reduced elongin C binding but retained HIF- binding in some assays, whereas Leu158Pro abrogated both interactions. Overall, the findings demonstrate that it is difficult to predict the functional effects of point mutations from site alone, and that direct testing in functional assays will be necessary to understand the syndrome. The spectrum of pVHL mutations analysed was chosen to permit comparison of functional assays with aspects of the clinical phenotype–genotype correlation. In familial cancer such correlations are confounded by variability in the occurrence of additional stochastic events necessary for tumourigenesis. We have also reported evidence for non-allelic inherited modifiers of HAB and RCC risk in VHL disease (31). Nevertheless, the described phenotype–genotype correlations at the VHL locus require explanation in terms of the functions of the pVHL molecule itself, raising a question as to the extent that specific disease patterns are reflected in the known function of pVHL in regulation of HIF. When the effects of pVHL mutations on the HIF system are considered from the perspective of each of the major disease manifestations, several patterns are observed.
Mutations associated with HAB predisposition showed a clear association with dysregulation of the HIF system. Thus types 1, 2A and 2B mutations all showed defective HIF- ubiquitylation and regulation when assayed both in vitro and in vivo, whereas type 2C (PHE-only) mutations that are not associated with HAB behave as wild-type in analyses of the HIF system. This tight association strongly suggests that upregulation of HIF contributes causally to HAB development, and upregulation of HIF target genes such as VEGF in the stromal cells of these tumours would provide a plausible mechanism. The association may also be of clinical utility in predicting the pathogenetic significance of novel VHL missense mutations. The prototypic type 2C mutant, Leu188Val, was described in two large (probably related) German families containing nine individuals with PHE and no other evidence of VHL disease despite careful screening (26). We observed wild-type activity of Leu188Val in several assays of HIF- regulation and our results are consistent with a recent independent report showing activity of this mutation in a similar HIF- ubiquitylation assay (19). We also analysed two other putative PHE-only (type 2C) mutations that have been described in small single families only. Results for these mutations (Val84Leu and Ser80Gln) were similar to those for Leu188Val and wild-type pVHL. Thus it seems likely that substitutions at multiple sites may produce PHE susceptibility without predisposing to HAB or RCC, and that assays of HIF ubiquitylation may predict this phenotype.
For mutations that predispose to RCC a relationship with HIF dysregulation was also apparent. All pVHL mutations associated with a high risk of RCC (types 1 and 2B) showed complete loss of HIF- ubiquitylation and regulation, whereas two pVHL mutations associated with lower risk of RCC showed an apparently incomplete defect in HIF regulation in the in vivo assays. In addition, we and others have assayed pVHL mutations from sporadic RCC and observed HIF dysregulation in every case (17–19). Thus the data are consistent with a role for HIF dysregulation in RCC. Although the results for the two type 2A (lower RCC risk) mutations might be taken to indicate that a more complete defect in HIF regulation results in a stronger predisposition to RCC, such a conclusion requires caution and will need testing across a larger array of clinically characterized mutations.
In contrast there appeared to be no relationship between dysregulation of the HIF system and mutational predisposition to PHE. HIF regulation was normal in PHE-predisposing type 2C mutations and abnormal in the remainder, irrespective of whether the risk of PHE was low (type 1) or high (type 2). These data indicate that the PHE-only type 2C mutations are functionally distinct and suggest that pVHL-mediated effects other than the dysregulation of HIF are central to the pathogenesis of VHL-associated PHE. Nevertheless, there is one important caveat to this conclusion. Because of the absence of a VHL-defective cell line derived from PHE, all studies have been conducted in RCC cell lines. Thus it is difficult to be sure that the type 2C mutations would have no effect on HIF regulation in the PHE cell background. For instance, it is possible that these mutations prevent interaction with a cell type-specific factor which is required for HIF regulation in PHE but not in RCC. Interestingly, PHE is rare in patients with germline VHL deletions, suggesting that total loss of function mutations are incompatible with PHE development (32–34). These findings are, however, also difficult to accommodate with a role for HIF in VHL-associated PHE, as our assays did not demonstrate consistent differences in HIF regulation between low PHE risk type 1 mutations and high PHE risk type 2 mutations. In vivo modelling of genotype–phenotype relationships in VHL disease might further inform mechanisms of site-specific tumourigenesis. Since homozygous disruption of vhl in mice causes embryonic lethality from defective placental vasculogenesis (35), this would require tissue-specific inactivation of the wild-type vhl allele in transgenic mice with germline vhl missense mutations (36).
Our results are therefore compatible with a major role for HIF dysregulation in the development of VHL-associated HAB and RCC, but they suggest that pVHL has other functions which are critically involved in the development of PHE. One other clearly defined function of pVHL is in fibronectin/extracellular matrix assembly (21). Although our studies were not designed to assess fibronectin assembly in detail, the display of metabolically labelled pVHL-associated proteins did enable us to assess the effect of pVHL mutations on capture of the p220 species which has been assigned as fibronectin. We found an unexpectedly clear concordance between mutational effects on HIF- binding and p220/fibronectin capture. These findings make it difficult to distinguish between mutational effects on one or other of these systems, and raise the possibility that the two systems could be functionally connected. In an accompanying paper, Hoffman et al. (37) report that the type 2C mutation L188V displayed a reduction in fibronectin binding under high stringency conditions and in defective fibronectin matrix assembly. Our own preliminary observations indicate that, in functional assays, certain type 2C mutants display defects in fibronectin matrix deposition (S.C. Clifford, unpublished data). Thus, our limited data concerning fibronectin are compatible with the study of Hoffman et al. (37). Further investigation of the effects of other type 2C mutants on fibronectin metabolism will be of interest. In addition, as hypoxia and the HIF system have been implicated in extracellular matrix metabolism (38,39), it will be of interest to determine whether, and in what way, these two functions of pVHL are interrelated.
What other functions might pVHL possess? Based on precedent derived from analyses of other ubiquitin ligases one might expect a broader range of substrates for the pVHL ligase complex. Somewhat unexpectedly, HIF- subunits were the only substrates in the pVHL co-immunoprecipitation experiments revealed in proteasomally blocked cells. Nevertheless, using a different approach, others have provided evidence for an as yet unidentified 200 kDa ubiquitylation substrate (40). Equally, other quite different functions in transcriptional regulation and cell cycle control have been proposed (20,22–24). In understanding the pathogenesis of VHL disease it will be important to assess the mutational spectrum in different functional assays. Nevertheless, the current data suggest that, at least for HAB and RCC, involvement of the HIF/fibronectin system(s) is important and might reasonably be considered as a target for the development of therapeutic interventions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutation cloning and plasmid construction
Construction of pcDNA3.1-VHL(1–213).HA, comprising the full-length wild-type pVHL coding sequence (amino acids 1–213) with a C-terminal HA epitope tag cloned into the pcDNA3.1 vector (Invitrogen), together with the derivation of pcDNA3.1-VHL(54–213).HA from pcDNA3.1-VHL(1–213).HA has been described previously (17). Missense pVHL mutations were generated in pcDNA3.1-VHL.HA by PCR-based site-directed mutagenesis (QuickChange, Stratagene) performed according to the manufacturer’s instructions. The
114-154 and 76PHE deletion mutations were cloned by PCR amplification from cDNA derived from patient material, followed by restriction digestion and ligation into the appropriate position in pcDNA3-VHL(1–213).HA. The empty pcDNA3.1 vector was used to represent a whole gene deletion of pVHL (VHL). Plasmids based on pcDNA3.1 expressing the full coding sequences of HIF-1 and HIF-2 have been described previously (17). Sequences of all plasmids were verified by both direct sequencing and restriction digestion analysis before use.
Cell culture
RCC4 cells (originally a gift from C.H.M.C. Buys) were maintained in DMEM with 10% fetal calf serum (FCS). Series of stable transfectants were generated from RCC4 by transfection with wild-type or mutant pVHL expression plasmids (see above) or empty vector (pcDNA3.1), using Fugene6 (Roche), followed by selection in G418 (1 mg/ml). Clones were picked as individual colonies and maintained in G418. Expression of stably transfected VHL genes was verified by immunoblotting with anti-pVHL and anti-HA antibodies and at least two independent clones were studied for each expression plasmid. Twenty-four hours before the experimental exposures, cells were subdivided onto 100 cm2 dishes in medium lacking G418. Parallel exposures to control and test conditions were for 4 h. Hypoxic incubation was in an atmosphere of 1% oxygen/5% CO2 /balance nitrogen in a Napco 7001 incubator (Jouan). For radio-isotopic labelling, cells were first incubated for 1 h in serum-free medium lacking methionine and cystine, which was replaced with 4.5 ml medium lacking methionine and cystine containing 2% dialysed FCS and 200 µCi/ml [35S]methionine/cysteine (Pro-mix, Amersham Pharmacia). Proteasomal inhibition was with 10 µM N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal and 100 µM calpain inhibitor 1 (Sigma).
In vitro transcription-translation.
[35S]methionine-labelled proteins were prepared by coupled transcription and translation reactions of expression plasmids in rabbit reticulocyte lysate (TNT, Promega), were performed according to the manufacturer’s instructions. All pVHL products contain three methionine residues except for pcDNA3-VHL(54–213).HA and pcDNA3-VHL(GLN 195 TER).HA, which contain two.
HIF-1 in vitro ubiquitylation assay
Background, validation and determination of optimal conditions for this assay are described in Cockman et al. (17). Briefly, ubiquitylation assays were performed in a 40 µl final volume. For pVHL reconstitution experiments, [35S]methionine-labelled wild-type or mutant pVHL (4 µl programmed reticulocyte lysate) was added to 26 µl RCC4 cell extract, 4 µl 10x ATP-regenerating system (20 mM Tris pH 7.5, 10 mM ATP, 10 mM magnesium acetate, 300 mM creatine phosphate and 0.5 mg/ml creatine phosphokinase) and 4 µl ubiquitin (5 mg/ml, Sigma), and the reaction mixture pre-incubated at room temperature for 5 min. [35S]methionine-labelled HIF-1 substrate (2 µl programmed reticulocyte lysate) was then added and the reaction allowed to proceed at 30°C for 90 min. Aliquots were removed after this time, mixed with SDS sample buffer and analysed by SDS–PAGE and autoradiography.
In vitro protein interaction assay
[35S]methionine-labelled proteins were produced in reticulocyte lysates programmed with plasmids encoding HA epitope-tagged pVHL and HIF- sequences. One microlitre of each of the indicated lysates was mixed in 100 µl NETN buffer (150 mM NaCl, 0.5 mM EDTA, 20 mM Tris–HCl pH 8.0, 0.5% v/v Igepal CA630). After 90 min at 4°C, 0.25 µg anti-HA antibody (12CA5, Roche) was added, followed after a further 1 h by 10 µl pre-blocked protein G–Sepharose beads. After 30 min mixing on a rotator, beads were washed five times with NETN buffer. Proteins were analysed by SDS–PAGE followed by fluorography.
Antibodies
Anti-HA antibodies (12CA5) were purchased from Roche, anti-HIF-1 antibody (clone 54) was purchased from Transduction Laboratories, antibody to SV40 T antigen (PAb419) was a gift from E. Harlow. Anti-HIF-2 antibody (190b) was described previously (41).
Immunoprecipitation
Immunoprecipitations were performed as described previously (15). Briefly, cells were lysed in 100 µl 100 mM NaCl, 0.5% Igepal CA630 (Sigma), 20 mM Tris–HCl pH7.6, 5 µM MgCl2, 1 mM sodium orthovanadate, 5 mM levamisole with aprotinin (1 µg/ml), 1x ‘Complete’ protease inhibitor (Roche) and 0.5 mM 4-(2-aminoethyl)benzene sulphonyl fluoride for 1 h on ice with occasional mixing. Lysates were then centrifuged at 10 000 g for 30 min at 4°C. 200 µg of cell extract was pre-cleared overnight at 4°C with 10 µl of protein G–Sepharose beads pre-blocked with phosphate-buffered saline containing 20 mg/ml bovine serum albumin. 1 µg antibody was then added and samples incubated at 4°C for 2 h, followed by 2 h incubation with 10 µl pre-blocked protein G–Sepharose beads on a rotator. Beads were washed five times in lysis buffer. Samples were resolved by SDS–PAGE using discontinuous gels (8% acylamide upper portion, 13% lower portion) and were detected by fluorography (Amplify, Amersham Pharmacia).
Immunoblotting
Whole cell protein extracts were prepared on ice by lysis in 8 M urea containing 10% (v/v) glycerol, 1% (w/v) SDS, 0.01 M Tris pH 6.8, 5 mM DTT, 10 µg/ml aprotinin, 1x ‘Complete’ protease inhibitor, 1 mM 4-(2-aminoethyl)benzene sulphonyl fluoride and 5 mM levamisole, followed by repeated passage through a fine gauge needle and centrifugation (10 000 g, 30 min, 4°C). Thirty-five micrograms of total protein per sample was then resolved by SDS–PAGE and transferred to PVDF membrane by electroblotting. For immunodetection of specific proteins, membranes were pre-blocked in PBS/0.1% Tween 20 containing 5% (w/v) dried skimmed milk for 1 h, incubated with primary antibody for 1 h, followed by horseradish peroxidase (HRP)-conjugated secondary antibody for 45 min (both diluted in PBS/0.1% Tween 20 containing 5% (w/v) dried skimmed milk). Membranes were washed in PBS/0.1% Tween 20 between all incubations. HRP activity was visualized by chemoluminescense (ECL+, Amersham) and autoradiography.
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ACKNOWLEDGEMENTS |
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We thank the Medical Research Council, Cancer Research Campaign (CRC), Wellcome Trust and the VHL Family Alliance for financial support.
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FOOTNOTES |
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+ Present address: Cancer Research Unit, University of Newcastle, Newcastle-upon-Tyne NE2 4HH, UK
Present address: Department of Medicine, Hammersmith Hospital, London W12, UK
¶ To whom correspondence should be addressed. Tel: +44 121 627 2741; Fax: +44 121 414 2538; Email: e.r.maher@bham.ac.uk
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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W. K. Rathmell, M. M. Hickey, N. A. Bezman, C. A. Chmielecki, N. C. Carraway, and M. C. Simon In vitro and In vivo Models Analyzing von Hippel-Lindau Disease-Specific Mutations Cancer Res., December 1, 2004; 64(23): 8595 - 8603. [Abstract] [Full Text] [PDF]
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R. I. Sufan, M. A. S. Jewett, and M. Ohh The role of von Hippel-Lindau tumor suppressor protein and hypoxia in renal clear cell carcinoma Am J Physiol Renal Physiol, July 1, 2004; 287(1): F1 - F6. [Abstract] [Full Text] [PDF]
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 V. R. Gordeuk, A. I. Sergueeva, G. Y. Miasnikova, D. Okhotin, Y. Voloshin, P. L. Choyke, J. A. Butman, K. Jedlickova, J. T. Prchal, and L. A. Polyakova Congenital disorder of oxygen sensing: association of the homozygous Chuvash polycythemia VHL mutation with thrombosis and vascular abnormalities but not tumors Blood, May 15, 2004; 103(10): 3924 - 3932. [Abstract] [Full Text] [PDF]
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N. H. Stickle, J. Chung, J. M. Klco, R. P. Hill, W. G. Kaelin Jr., and M. Ohh pVHL Modification by NEDD8 Is Required for Fibronectin Matrix Assembly and Suppression of Tumor Development Mol. Cell. Biol., April 15, 2004; 24(8): 3251 - 3261. [Abstract] [Full Text] [PDF]
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M. I. Zhou, H. Wang, R. L. Foy, J. J. Ross, and H. T. Cohen Tumor Suppressor von Hippel-Lindau (VHL) Stabilization of Jade-1 Protein Occurs through Plant Homeodomains and Is VHL Mutation Dependent Cancer Res., February 15, 2004; 64(4): 1278 - 1286. [Abstract] [Full Text] [PDF]
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L. Gunaratnam, M. Morley, A. Franovic, N. de Paulsen, K. Mekhail, D. A. E. Parolin, E. Nakamura, I. A. J. Lorimer, and S. Lee Hypoxia Inducible Factor Activates the Transforming Growth Factor-{alpha}/Epidermal Growth Factor Receptor Growth Stimulatory Pathway in VHL-/- Renal Cell Carcinoma Cells J. Biol. Chem., November 7, 2003; 278(45): 44966 - 44974. [Abstract] [Full Text] [PDF]
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W. G. Kaelin Jr. The von Hippel-Lindau Gene, Kidney Cancer, and Oxygen Sensing J. Am. Soc. Nephrol., November 1, 2003; 14(11): 2703 - 2711. [Abstract] [Full Text] [PDF]
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P. Maxwell HIF-1: An Oxygen Response System with Special Relevance to the Kidney J. Am. Soc. Nephrol., November 1, 2003; 14(11): 2712 - 2722. [Full Text] [PDF]
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J. Bryant, J. Farmer, L. J. Kessler, R. R. Townsend, and K. L. Nathanson Pheochromocytoma: The Expanding Genetic Differential Diagnosis J Natl Cancer Inst, August 20, 2003; 95(16): 1196 - 1204. [Abstract] [Full Text] [PDF]
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E. Metzen, J. Zhou, W. Jelkmann, J. Fandrey, and B. Brune Nitric Oxide Impairs Normoxic Degradation of HIF-1{alpha} by Inhibition of Prolyl Hydroxylases Mol. Biol. Cell, August 1, 2003; 14(8): 3470 - 3481. [Abstract] [Full Text] [PDF]
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 J C C Rocha, R L A Silva, B B Mendonca, S Marui, A J G Simpson, and A A Camargo High frequency of novel germline mutations in the VHL gene in the heterogeneous population of Brazil J. Med. Genet., March 1, 2003; 40(3): e31 - 31. [Full Text] [PDF]
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S. J. Welsh, R. R. Williams, A. Birmingham, D. J. Newman, D. L. Kirkpatrick, and G. Powis The Thioredoxin Redox Inhibitors 1-Methylpropyl 2-Imidazolyl Disulfide and Pleurotin Inhibit Hypoxia-induced Factor 1{alpha} and Vascular Endothelial Growth Factor Formation Mol. Cancer Ther., March 1, 2003; 2(3): 235 - 243. [Abstract] [Full Text] [PDF]
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Y. D. Pastore, J. Jelinek, S. Ang, Y. Guan, E. Liu, K. Jedlickova, L. Krishnamurti, and J. T. Prchal Mutations in the VHL gene in sporadic apparently congenital polycythemia Blood, February 15, 2003; 101(4): 1591 - 1595. [Abstract] [Full Text] [PDF]
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G. Weirich, B. Klein, T. Wohl, D. Engelhardt, and H. Brauch VHL2C Phenotype in a German von Hippel-Lindau Family with Concurrent VHL Germline Mutations P81S and L188V J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 5241 - 5246. [Abstract] [Full Text] [PDF]
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 P. T. Schumacker Hypoxia, anoxia, and O2 sensing: the search continues Am J Physiol Lung Cell Mol Physiol, November 1, 2002; 283(5): L918 - L921. [Full Text] [PDF]
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M. I. Zhou, H. Wang, J. J. Ross, I. Kuzmin, C. Xu, and H. T. Cohen The von Hippel-Lindau Tumor Suppressor Stabilizes Novel Plant Homeodomain Protein Jade-1 J. Biol. Chem., October 11, 2002; 277(42): 39887 - 39898. [Abstract] [Full Text] [PDF]
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E. R. Maher and C. Eng The pressure rises: update on the genetics of phaeochromocytoma Hum. Mol. Genet., October 1, 2002; 11(20): 2347 - 2354. [Abstract] [Full Text] [PDF]
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 R. H. WENGER Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression FASEB J, August 1, 2002; 16(10): 1151 - 1162. [Abstract] [Full Text] [PDF]
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 A. O. Fedele, M. L. Whitelaw, and D. J. Peet Regulation of Gene Expression by the Hypoxia-Inducible Factors Mol. Interv., July 1, 2002; 2(4): 229 - 243. [Abstract] [Full Text] [PDF]
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M. Zatyka, N. F. da Silva, S. C. Clifford, M. R. Morris, M. S. Wiesener, K.-U. Eckardt, R. S. Houlston, F. M. Richards, F. Latif, and E. R. Maher Identification of Cyclin D1 and Other Novel Targets for the von Hippel-Lindau Tumor Suppressor Gene by Expression Array Analysis and Investigation of Cyclin D1 Genotype as a Modifier in von Hippel-Lindau Disease Cancer Res., July 1, 2002; 62(13): 3803 - 3811. [Abstract] [Full Text] [PDF]
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D E McNeil, W M Linehan, and G M Glenn Comorbid VHL and SCA2 mutations in a large kindred: confounding diagnosis of neurological dysfunction caused by CNS VHL vascular tumours versus SCA2 atrophic neurodegeneration J. Med. Genet., July 1, 2002; 39(7): e37 - 37. [Full Text] [PDF]
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M. S. Wiesener, M. Seyfarth, C. Warnecke, J. S. Jurgensen, C. Rosenberger, N. V. Morgan, E. R. Maher, U. Frei, and K.-U. Eckardt Paraneoplastic erythrocytosis associated with an inactivating point mutation of the von Hippel-Lindau gene in a renal cell carcinoma Blood, May 15, 2002; 99(10): 3562 - 3565. [Abstract] [Full Text] [PDF]
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 M. S. Wiesener and K.-U. Eckardt Erythropoietin, tumours and the von Hippel-Lindau gene: towards identification of mechanisms and dysfunction of oxygen sensing Nephrol. Dial. Transplant., March 1, 2002; 17(3): 356 - 359. [Full Text] [PDF]
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 P. C. Mahon, K. Hirota, and G. L. Semenza FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity Genes & Dev., October 15, 2001; 15(20): 2675 - 2686. [Abstract] [Full Text] [PDF]
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E. A. Shoubridge Nuclear genetic defects of oxidative phosphorylation Hum. Mol. Genet., October 1, 2001; 10(20): 2277 - 2284. [Abstract] [Full Text] [PDF]
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 H. Yang and W. G. Kaelin Jr. Molecular Pathogenesis of the Von Hippel-Lindau Hereditary Cancer Syndrome: Implications for Oxygen Sensing Cell Growth Differ., September 1, 2001; 12(9): 447 - 455. [Full Text] [PDF]
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