genes associated with von Hippel-Lindau disease
studies of von Hippel-Lindau disease
Shmueli, M.D., Schnaider, L., Herzog, G., Gazit, E. and Segal, D. (2014). Computational and experimental characterization of dVHL establish a Drosophila model of VHL syndrome. PLoS One 9: e109864. PubMed ID: 25310726
Lower model organisms such as Drosophila melanogaster and Caenorhabditis elegans have often provided the first glimpse into the mechanism of action of human cancer-related proteins, thus making a substantial contribution to the elucidation of the molecular basis of the disease. More than 50% of the proteins that are associated with human diseases, including cancer, have orthologues in D. melanogaster. In many cases, the function of a given protein is also conserved evidenced by the fact that the corresponding human protein can rescue the loss of function of its Drosophila orthologue. Thus, studies of the fly orthologue as well as investigation of the transgenic human proteins in flies, which are highly amenable for genetic manipulations, provide important information on the human disease-associated protein and the pathways it is involved in. This is exemplified especially regarding to the contribution of Drosophila to basic and applied aspects of cancer research. The highly ordered, lattice-like, arrangement of the ommatidia in the fly eyes is especially suitable for large scale screens for genetic modifiers and for drug candidates. Flies have also greatly facilitated drug screening. Studies show that the Drosophila VHL (dVHL) can interact with Drosophila Elongin C in vitro and form a complex with the human Elongin CB complex and mouse Rbx-1, but not with the human Elongin A. Thus, the ubiquitin ligase function of pVHL is conserved in Drosophila. It is also known that dVHL is involved in degradation of hydroxylated oxygen-dependent degradation domain (ODD) of both the human HIF1-α or of the Drosophila homolog, SIMA. Studies of dVHL have suggested the involvement of the VHL protein in regulation of endocytosis and epithelial cell motility which was subsequently demonstrated for pVHL as well. Research on dVHL has also indicated a role for the VHL protein in microtubule stabilization during epithelial morphogenesis; yet, this remains to be demonstrated for pVHL as well (Shmueli, 2014).
The results presented in this study regarding the structural similarity between the dVHL and pVHL proteins provide insights into their functional homology. It is known that pVHL is a molten globule protein that it is intrinsically disordered but is functional as such. This study shows that dVHL, like pVHL, has a secondary structure but lacks well-defined tertiary structure and attains a molten globule conformation. Also, dVHL, like pVHL, is functional without being uniquely folded into a rigid 3D structure; thus, dVHL, like pVHL, is an intrinsically disordered protein (IDP). The demonstration of homology between IDPs is a major endeavor since they do not display sequence conservation. In addition, due to the lack of well-defined 3D structure, IDP regions fail to scatter X-rays coherently, hampering attempts to solve their structure by X-ray crystallography (Shmueli, 2014).
The in silico predicted homology in the conformation of the key functional amino acids of dVHL and pVHL, as well as their evolutionary conservation, supported by in vitro results in this study, provide a structural basis for understanding the homology in their function as well as their ability to interact with each other’s binding partners. dVHL can bind in vivo the hydroxylated ODD domain of the human HIF1-α and target it for degradation and it was shown that transgenic pVHL can do the same in flies. Moreover, it was demonstrated that transgenic pVHL can rescue the lethal phenotype of dVHL null mutants of Drosophila. These results suggest that pVHL is capable of interacting, in flies, with the Drosophila members of the VCB complex, thus regulating the fly HIF1-α homolog, SIMA. Indeed, this rescue involves regulation of the HIF1-α/SIMA dependent pathway, evidenced by the level of its target genes (Shmueli, 2014).
The Drosophila model described in this study should facilitate further studies on the function of pVHL and the pathways it regulates. The rough eye phenotype caused by over-expression of SIMA, which was shown to be suppressed by transgenic expression of normal pVHL, should be highly conducive for conducting screens for drug candidates for the VHL syndrome in Drosophila whole model organism. These, can be subsequently verified molecularly by examining the downstream HIF1-α dependent pathway which are conserved between flies and humans (Shmueli, 2014).
Delgehyr, N., Wieland, U., Rangone, H., Pinson, X., Mao, G., Dzhindzhev, N.S., McLean, D., Riparbelli, M.G., Llamazares, S., Callaini, G., Gonzalez, C. and Glover, D.M. (2012). Drosophila Mgr, a Prefoldin subunit cooperating with von Hippel Lindau to regulate tubulin stability. Proc Natl Acad Sci U S A 109: 5729-5734. PubMed ID: 22451918
The idea that Vhl and Prefoldin can cooperate in regulating protein stability has been raised earlier by another study that identified the prefoldin subunit VBP1 as a binding partner of the HIV-1 viral integrase and suggested it to mediate the interaction of the integrase with the Cul2-Vhl E3-Ubiquitin ligase. This finding led to the suggestion for a role for prefoldin at a pivotal part of the pathway that would determine whether a protein was passed on to the CCT chaperonin for folding or to the proteasome for degradation. Similarly, this study speculates that prefoldin as a partner of Vhl may well serve a key role in regulating the equilibrium between tubulin targeted for destruction or for folding and incorporation into MTs. The concentration of assembly-competent tubulin must be tightly controlled because it affects cytoskeletal dynamics. Vhl might contribute to this influence by an effect on MT dynamics through interaction with MAPs on the MT lattice and by intervening in the regulation of tubulin folding. There is growing evidence for a critical function of Vhl in stabilizing cytoplasmic MTs and axonemal MTs in response to levels of soluble tubulin. Reciprocally, MT stability can contribute to regulating levels of proteins that are targets of the Cul2-Vhl E3-Ubiquitin ligase, such as the HIF proteins, the levels of which fall when their mRNAs accumulate in cytoplasmic P-bodies for translational repression following MT disruption. It will be important in future to consider the roles played by the Prefoldin complex and Vhl to understand the interrelationships between the machinery regulating tubulin levels in relation to MT stability, both in normal and tumor cells (Delgehyr, 2012).
Hsouna, A., Nallamothu, G., Kose, N., Guinea, M., Dammai, V. and Hsu, T. (2010). Drosophila von Hippel-Lindau tumor suppressor gene function in epithelial tubule morphogenesis. Mol Cell Biol 30: 3779-3794. PubMed ID: 20516215
The study also shows that dVHL genetically interacts with endocytic pathway components such as awd, shibire (shi) and Rab5 in both branch migration and lumen formation phenotypes but interacts with btl signaling pathway components only in branch migration phenotypes. The lumen defects in the dVHL mutant are similar to those found in the wurst mutant. wurst encodes a transmembrane protein that promotes clathrin-mediated endocytosis of lumen material. wurst function is specific for lumen maturation and, unlike dVHL, has little effect on branch migration. This indicates that wurst may be specific for pinocytosis (uptake of extracellular materials) and not involved in internalization of surface proteins, while the dVHL- and awd-regulated dynamin-Rab5 pathway is necessary for both (Hsouna, 2010).
It was earlier reported that some of the tracheal tubule migration defects generated by dVHL RNA interference could be attributed to upregulation of Sima, the Drosophila homolog of HIF-α, which in turn upregulates btl transcription. Hypoxia-induced, Sima-dependent btl transcription has also been demonstrated in terminal branching in the larval trachea. It is possible that the endocytic and the HIF-dependent functions of VHL are not mutually exclusive. On the other hand, it has been shown that the stereotypic tracheal branch migration pattern in the embryo (as opposed to the terminal branching in larvae) is normally not dependent on sima function. Also, overexpression of the wild-type btl transgene in the embryonic trachea using the cognate btl promoter, although exhibiting ectopic elevation of the btl transcript level, could not lead to embryonic tracheal defects. This indicates that posttranscriptional regulation is the major mechanism for modulating active Btl level and thus Btl signaling. Indeed, it was shown in this study that the exogenously expressed Btl::GFP protein is under stringent control at the protein level. Also, sima can only modestly suppress the dVHL1 branch migration phenotype. It is interesting to point out that a robust rescue of phenotypes in the btl promoter-driven dVHL RNA duplex by sima heterozygotes has been shown earlier. This may be because the exogenous btl promoter, presumably positively regulated by sima, is itself downregulated in the sima mutant, thereby diminishing the dVHL duplex expression, resulting in apparent rescue (Hsouna, 2010).
It should be noted that primary and secondary tracheal tubule development during embryogenesis is a stereotypic, genetically programmed process. Tissue microenvironmental factors such as hypoxia do not play a significant role in this process. On the other hand, in late embryonic and larval stages, when the trachea system extends to connect with internal organs, localized hypoxia in the target tissue is critical in inducing FGF/bnl expression and thus promoting terminal branching. HIF-α/Sima has been shown to also play an important role in the terminal tracheal tubule cells in sensing hypoxia and inducing FGFR/btl expression. This mechanism, in the absence of dVHL mutation, can contribute to tracheal branching probably because it is sensitized by the overproduced Bnl ligand in hypoxic conditions. It is conceivable that dVHL loss of function during embryogenesis can result in similar upregulation of the btl gene transcription. However, results in this study indicate that in nonhypoxic development of trachea in the embryonic stage, the additional function of dVHL in regulated protein internalization plays the major role in tracheal morphogenesis (Hsouna, 2010).
Most interestingly, the study demonstrates that the ectopic branching of the tubule epithelial cells and the malformation of lumen phenotypes are reproducible in the kidney tubules of mice with conditional Vhl knockout and in organoid culture using primary tubule cells. This is highly significant, since dilated tubules (minicysts) have been documented as preceding renal cell carcinoma. Thus, these findings provide a plausible mechanistic explanation, involving increased cell motility and disruption of tubule epithelium, for the etiology of VHL mutant kidney cancer. In addition, another study implicated dVHL in the morphogenesis of organ-associated epithelium, the follicle cells in the ovary. This function is mediated by another HIF-independent activity of dVHL that stabilizes microtubule bundles. Future studies should exploit further the Drosophila genetic system for elucidating how various VHL functions and a myriad of disease-related VHL mutations may differentially affect the pathophysiological roles of this interesting tumor suppressor gene (Hsouna, 2010).
Duchi, S., Fagnocchi, L., Cavaliere, V., Hsouna, A., Gargiulo, G. and Hsu, T. (2010). Drosophila VHL tumor-suppressor gene regulates epithelial morphogenesis by promoting microtubule and aPKC stability. Development 137: 1493-1503. PubMed ID: 20388653
Human VHL has been shown to translocate aPKC to MTs, thereby influencing MT reorganization. This study shows that the aPKC mutant can affect MT organization but not stability, whereas VHL can influence both. Conversely, disruption of MTs alone can result in aPKC mislocalization resembling that observed in VHL mutant cells. Importantly, paclitaxel-induced MT stabilization can rescue aPKC localization in VHL mutant follicle cells. The study therefore concludes that a major function of VHL in the follicular epithelium is regulation of MT stability. Loss of MTs leads to aPKC mislocalization and degradation. Conversely, part of the VHL epithelial functions might be mediated by its direct effect on aPKC stability, as exogenously expressed aPKC-GFP fusion protein can partially rescue (not statistically significant) the VHL mutant phenotype. Indeed, VHL can co-immunoprecipitate with tubulin or aPKC, and that, at least in S2 cells, aPKC levels can be affected by VHL levels without affecting tubulin. Taken together, it appears that the epithelial function of VHL is mediated through stabilization of MT, with an auxiliary role in directly stabilizing aPKC (Duchi, 2010).
It has been suggested that VHL interacts with MTs via the kinesin 2 family of motors. Future studies using the epithelial system should also address this issue in vivo. Also interestingly, it was shown that the VHLYH mutant (single amino acid substitution mutation of tyrosine to histidine at position 51, equivalent to human Y98) can associate with MT but has little MT-stabilizing activity. This suggests that the VHLYH mutant might be defective in recruiting other proteins, possibly including aPKC, that are important for regulating MT functions. In light of the role of Drosophila VHL in regulating MT stability, a function presumably important for all cells, it is curious that the tissue-specific btl-driven VHL expression can rescue the homozygous lethality of VHL1 (generated by replacing the wild-type copy, via homologous recombination, with a deletion that removes 81 codons encompassing the first two in-frame AUGs). It has been shown that tracheal defects are the major embryonic phenotype observed in VHL mutant. In the course of attempting to rescue the tracheal phenotype with btl-driven VHL, rescued homozygous adults are present. This indicates that the MT stabilizing function of VHL is not required in all tissues. It is possible that although VHL can enhance MT stability, by itself it is not an essential factor for MT polymerization. As such, some tissues might be less dependent on VHL levels. In the follicular environment, MT rearrangement, including depolymerization and repolymerization, is crucial when the entire epithelial sheet moves over the germ cell complex while the cells grow increasingly columnar. MT stabilization facilitated by VHL might be of particular importance during this process (Duchi, 2010).
The best-documented function of VHL is its E3 ubiquitin ligase activity that targets the alpha subunit of the HIF transcription factor. This activity provides an elegant mechanistic explanation for the hypervascularity of many of the VHL tumors and for a potential contributor to the metabolic switch to glycolysis, as HIF can upregulate pro-angiogenic factors such as vascular-endothelial growth factor and components in the glucose metabolic pathway. However, there is also evidence that VHL is a multifunctional protein. It can function as a regulator of matrix deposition, integrin assembly, endocytosis, kinase activity, senescence, protein stabilities and tight junction formation, among many others. Whether tight junction disassembly in VHL mutant cells is HIF-dependent is still unresolved; however, other – HIF-independent – functions appear to facilitate protein stability or activity instead of destabilizing them as a ubiquitin ligase. Such chaperon/adaptor function has also been implicated in promoting stability of MTs. The MT-stabilizing function, although potentially highly significant, has so far only been linked to cilium biogenesis and mitotic spindle orientation in cultured RCC and renal tubule cells. The physiological and developmental significance of this function has not been elucidated in vivo. Indeed, it is unclear how loss of many of these HIF-independent functions contributes to VHL tumor formation because of a lack of tractable genetic models (Duchi, 2010).
One crucial element in tumorigenesis is the breakdown of epithelial integrity that ultimately leads to epithelial-to-mesenchymal transition. This report provides the first demonstration of a potential tumor-suppressor function for VHL in regulating epithelial morphogenesis via its role in promoting MT stability. Future studies should exploit further this genetic system for elucidating how a myriad of disease-related VHL point mutations might differentially influence such function (Duchi, 2010).
Doronkin, S., Djagaeva, I., Nagle, M.E., Reiter, L.T. and Seagroves, T.N. (2010). Dose-dependent modulation of HIF-1alpha/sima controls the rate of cell migration and invasion in Drosophila ovary border cells. Oncogene 29: 1123-1134. PubMed ID: 19966858
It is noteworthy that ectopic expression of human VHL in the Drosophila ovary using either the slbo-GAL4 or c522-GAL4 driver also produces a similar range of phenotypes. VHL over-expression causes delays in migration of 39/29% of chambers, blocks migration in 7/6% of chambers, and accelerates migration in 5/8% of egg chambers, respectively. Together, these data demonstrate that BCM is more sensitive to ectopic expression of Sima than Vhl and provide additional evidence that the VHL pathway is highly conserved in Drosophila. Finally, expression of either Vhl or Hph increases BCM without damaging the cortical cytoskeleton. In a slbo–GAL4>UAS–Vhl egg chamber exhibiting the accelerated phenotype, the abnormal, posterior location of accelerated clusters is consistently accompanied by a stretching, but not complete disruption, of the oocyte/nurse cell actin network (Doronkin, 2010).
One of the key downstream effector molecules in collective BCM is Drosophila E-cadherin (DE-cadherin; encoded by shotgun). The VHL/HIF axis has been shown to regulate E-cadherin levels in human renal cancer cells. This study compared DE-cadherin expression levels by immunostaining fixed egg chambers dissected from UAS–sima, UAS–Vhl or sima mosaic clone border cell clusters. In wild-type clusters, DE-cadherin accumulates to the highest levels at the interface between the border cells and the polar cells, located at the center of the cluster. Accumulation is less pronounced between the cluster and the nurse cells. In UAS–sima clusters, expression of DE-cadherin is enhanced compared with control clusters at the boundaries between individual border cells. Similarly, in delayed UAS–Vhl clusters, DE-cadherin appears to more strongly accumulate at the interface of the cluster and the surrounding nurse cells rather than between the border cells and polar cells. Finally, in all accelerated UAS–Vhl clusters, DE-cadherin expression is potently reduced. It is possible that increased accumulation of DE-cadherin in the border cell clusters in response to Sima over-accumulation may have strengthened cell–cell adhesions leading to stalled migration. Similarly, reduction of the DE-cadherin levels in sima-mutant cells could have produced weaker contacts between the neighboring nurse cells and the migrating border cells, which would explain the accelerated cell migration observed in UAS–Vhl expressing cells (Doronkin, 2010).
Mortimer, N.T. and Moberg, K.H. (2009). Regulation of Drosophila embryonic tracheogenesis by dVHL and hypoxia. Dev Biol 329: 294-305. PubMed ID: 19285057
Since Trh and Sima/HIF-1α share a similar consensus DNA binding site, it is likely that the tracheal phenotypes elicited by either hypoxia or dVHL knockdown are to some degree the product of a combined ‘Trh/Sima-like’ transcriptional activity in tracheal cells. This conclusion is supported both by the general phenotypic similarity (i.e. migration and overgrowth defects) between hypoxia/dVHL knockdown and trh overexpression, by the modest ability of trh alleles to suppress dVHLi phenotypes, and by the overlap of transcriptional activity between Trh and human HIF-1α. Indeed, Trh is well-established as a required activator of developmental btl expression. However, because the excess Btl activity that occurs in hypoxia or in the absence of dVHL occurs independently of a change in Trh expression, it thus appears to be mediated largely by increased sima activity (Mortimer, 2009).
This study suggests that there are two distinct developmental ‘windows’ of embryogenesis during which hypoxia has opposite effects on tracheal branching. The first corresponds to a period immediately before and during primary branch migration that is relatively insensitive to hypoxia. Embryos in this stage show a minimal response to 1% O2, but show a nearly complete arrest of migration in 0.5% O2. Interestingly, a prior study found that similarly staged embryos (stage 11) respond to complete anoxia by prolonged developmental arrest, from which they can emerge and resume normal development. These somewhat paradoxical results — that acute hypoxia is more detrimental to development than chronic anoxia — might be explained by the observation that chronic exposure to low O2 induces Sima activity throughout the embryo while acute exposure activates Sima only in tracheal cells. The former scenario may result in coordinated developmental and metabolic arrest throughout the organism, while in the latter scenario developmental patterns of gene expression in non-tracheal cells may proceed such that tracheal cells emerging from an ‘early’ hypoxic response find an embryonic environment in which developmentally hard-wired migratory signals emanating from non-tracheal cells have ceased (Mortimer, 2009).
The second type of tracheal response occurs during a later ‘window’ of embryogenesis after btl/bnl-driven primary and secondary branch migration and fusion are largely complete. It involves sinuous overgrowth of the primary and secondary branches, and duplication of secondary branches. As in the ‘early’ response, ‘late’ hypoxic phenotypes are controlled by the dVHL/sima pathway, yet unlike the ‘early’ response, these phenotypes occur at high penetrance even at 1% O2. Thus the ‘late’ embryonic tracheal system is relatively sensitized to hypoxia and responds with increased branching in a manner similar to larval terminal cells. Indeed, much as larval branching increases with decreasing O2 levels, it was observed that dorsal trunk growth in the late embryo is graded to the degree of hypoxia. The mechanism underlying the differential sensitivity of the ‘early’ and ‘late’ tracheal system may be quite complex. However, it was found that tracheogenesis can be sensitized to hypoxia by reducing activity of ago, a ubiquitin ligase component that restricts btl transcription in tracheal cells via its role in degrading the Trh transcription factor. Increasing transcriptional input on the btl promoter thus appears to sensitize ‘early’ tracheal cells to hypoxia. As Sima also controls btl transcription, one explanation of the difference in sensitivity between different embryonic stages may thus lie in differences in the activation state of the btl promoter. If so then the activity of the endogenous btl regulatory network may be an important determinant of the threshold of hypoxia required to elicit changes in tracheal architecture (Mortimer, 2009).
An organism can have its hypoxic response triggered in two ways, either by systemic exposure of the whole organism to a reduced O2 environment or by localized hypoxia produced by increased O2 consumption in metabolically active tissues. Data from this study and others suggests there may be distinctions between these two triggers. Exposing larvae or embryos to a systemic pulse of hypoxia results in a ‘btl-centric’ response specifically in tracheal cells. Outside of an ‘early’ vulnerable period which corresponds to embryonic branch migration and fusion, elevated Btl activity in embryonic tracheal cells promotes branch duplications and overgrowth similar to that seen in larvae. By contrast, tracheal growth induced by localized hypoxia in the larva has been suggested to involve a ‘bnl-centric’ model in which the hypoxic tissue secretes Bnl and recruits new tracheal branching. Whether this type of mechanism operates in embryos, or whether embryos ever experience localized hypoxia in non-tracheal cells, has not been established (Mortimer, 2009).
Data from this study also indicate that dVHL is a central player in the hypoxic response pathway in embryonic and larval tracheal cells. A prior study found that injection of dVHL dsRNA into syncytial embryos disrupts normal tracheogenesis, but was technically limited in its ability to conduct a detailed analysis of dVHL function in development and homeostasis. This study shows that dVHL knockdown specifically in tracheal cells mimics the effect of systemic hypoxia on embryonic tracheal architecture and larval terminal cell branching. dVHL knockdown thus phenocopies loss of the HPH gene fga, which normally functions to target Sima to the dVHL ubiquitin ligase in normoxia. Moreover, all phenotypes that result from reduced dVHL expression can be rescued by reducing sima activity, suggesting that Sima is the major target of dVHL in the tracheal system. These data support a model in which dVHL, fga, and sima function as part of a conserved VHL/HPH/HIF-1α pathway to control tracheal morphogenesis in embryos and larvae. The btl receptor appears to be an important target of this pathway in embryonic (this study) and larval tracheal cells. Knockdown of dVHL elevates btl transcription in embryonic placodes and tracheal branches, and removal of a copy of the gene effectively suppresses dVHL tracheal phenotypes. Reciprocally, overexpression of wild type btl in embryonic tracheal cells can produce migration defects and sinuous overgrowth, while expression of a constitutively active btl chimera (btlλ) also leads to primary branch stunting and duplication of secondary branches. Interestingly, pupal lethality associated with tracheal-specific knockdown of dVHL is not sensitive to the dose of btl, but is dependent on sima. Thus the dVHL/sima pathway may have btl independent effects on tracheal cells in later stages of development (Mortimer, 2009).
In addition to sima and Btl/FGF pathway mutants, dVHL also shows very strong genetic interactions with alleles of the ago ubiquitin ligase subunit. The interactions are consistent with the ability of ago to modulate hypoxia sensitivity in the embryo, and suggest a speculative model in which each ligase acts through its own target — Sima or Trh — to regulate btl transcription in tracheal cells. Given that the human orthologs of dVHL and ago are significant tumor suppressor genes, it is intriguing to consider whether their ability to co-regulate tubular morphogenesis in the Drosophila embryo is conserved in mammalian development and disease (Mortimer, 2009).
Aso, T., Yamazaki, K., Aigaki, T. and Kitajima, S. (2000). Drosophila von Hippel-Lindau tumor suppressor complex possesses E3 ubiquitin ligase activity. Biochem Biophys Res Commun 276: 355-361. PubMed ID: 11006129
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von Hippel-Lindau tumor suppressor protein, a component of an E3 ubiquitin ligase complex, is required for degradation of HIF-1
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Date revised: 20 June 2015
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