von Hippel-Lindau: Biological Overview | References
Gene name - von Hippel-Lindau
Cytological map position - 47E5-47E6
Function - signaling
Symbol - Vhl
FlyBase ID: FBgn0041174
Genetic map position - 2R:7,167,231..7,168,538 [+]
Classification - von Hippel-Landau (pVHL) tumor suppressor protein
Cellular location - cytoplasmic
|Recent literature||Nicholson, H. E., Tariq, Z., Housden, B. E., Jennings, R. B., Stransky, L. A., Perrimon, N., Signoretti, S. and Kaelin, W. G., Jr. (2019). HIF-independent synthetic lethality between CDK4/6 inhibition and VHL loss across species. Sci Signal 12(601). PubMed ID: 31575731
Inactivation of the VHL tumor suppressor gene is the signature initiating event in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, and causes the accumulation of hypoxia-inducible factor 2alpha (HIF-2alpha; see Drosophila Similar). HIF-2alpha inhibitors are effective in some ccRCC cases, but both de novo and acquired resistance have been observed in the laboratory and in the clinic. This study identified synthetic lethality between decreased activity of cyclin-dependent kinases 4 and 6 (CDK4/6) and VHL inactivation in two species (human and Drosophila) and across diverse human ccRCC cell lines in culture and xenografts. Although HIF-2alpha transcriptionally induced the CDK4/6 partner cyclin D1, HIF-2alpha was not required for the increased CDK4/6 requirement of VHL(-/-) ccRCC cells. Accordingly, the antiproliferative effects of CDK4/6 inhibition were synergistic with HIF-2alpha inhibition in HIF-2alpha-dependent VHL(-/-) ccRCC cells and not antagonistic with HIF-2alpha inhibition in HIF-2alpha-independent cells. These findings support testing CDK4/6 inhibitors as treatments for ccRCC, alone and in combination with HIF-2alpha inhibitors.
|Hwang, S. H., Bang, S., Kim, W. and Chung, J. (2020). Von Hippel-Lindau tumor suppressor (VHL) stimulates TOR signaling by interacting with phosphoinositide 3-kinase (PI3K). J Biol Chem. PubMed ID: 31959630
Cell growth is positively controlled by phosphoinositide 3-kinase (PI3K)-TOR signaling pathway under conditions of abundant growth factors and nutrients. To discover additional mechanisms that regulate cell growth, an RNAi-based mosaic analyses was performed in the Drosophila fat body, the primary metabolic organ in the fly. Unexpectedly, the knockdown of the Drosophila von Hippel-Lindau (VHL) gene markedly decreased cell size and body size. These cell growth phenotypes induced by VHL loss-of-function were recovered by activation of TOR signaling in Drosophila. Consistent with the genetic interactions between VHL and the signaling components of PI3K-TOR pathway in Drosophila, it was observed that VHL loss-of-function in mammalian cells causes decreased phosphorylation of ribosomal protein S6 kinase (S6K) and Akt, which represent the main activities of this pathway. It was further demonstrated that VHL activates TOR signaling by directly interacting with the p110 catalytic subunit of PI3K. On the basis of the evolutionarily conserved regulation of PI3K-TOR signaling by VHL observed in this study, it is proposed that VHL plays an important role in the regulation and maintenance of proper cell growth in metazoans.
Mutations in the human von Hippel-Lindau (VHL) genes are the cause of von Hippel-Lindau disease, which displays multiple benign and malignant tumors. The VHL gene has been shown to regulate angiogenic potential and glycolic metabolism via its E3 ubiquitin ligase function against the alpha subunit of hypoxia-inducible factor (HIF). However, many other HIF-independent functions of VHL have been identified and recent evidence indicates that the canonical function cannot fully explain the VHL mutant cell phenotypes. Many of these functions have not been verified in genetically tractable systems. Using an established follicular epithelial model in Drosophila, this study shows that the Drosophila VHL gene is involved in epithelial morphogenesis via stabilizing microtubule bundles and aPKC. Microtubule defects in VHL mutants lead to mislocalization of aPKC and subsequent loss of epithelial integrity. Destabilizing microtubules in ex vivo culture of wild-type egg chambers can also result in aPKC mislocalization and epithelial defects. Importantly, paclitaxel-induced stabilization of microtubules can rescue the aPKC localization phenotype in Drosophila VHL mutant follicle cells. The results establish a developmental function of the VHL gene that is relevant to its tumor-suppressor activity (Duchi, 2010).
Establishing and maintaining epithelial integrity is essential for embryonic development, organogenesis and tissue remodeling. The key characteristic of epithelial cells is asymmetrical specification of membrane domains marked by domain-specific proteins. The epithelial morphogenic mechanism, although with some variations in different epithelial tissues, is highly conserved from worm to mammal. The crucial initial step in establishing epithelial polarity is the specification of the apical domain, which is defined by the function of a complex containing atypical PKC (aPKC), Bazooka (Baz; mammalian and worm PAR-3) and PAR-6. The PAR complex is initially recruited by activated Cdc42 to the apical domain. The three proteins were originally thought to function as a complex; however, recent evidence indicates that Baz might be required first to recruit the aPKC-PAR-6 complex to the subapical domain juxtaposed to the future adherens junction (AJ) (Harris, 2007). The PAR complex is required for the localization of another apical complex containing Crumbs (Crb), Stardust (Std; mammalian Pals1) and Discs lost (Dlt; mammalian Patj). The PAR- and Crb-containing complexes occupy the apical-most region of the lateral membrane, just apical to the AJs (Duchi, 2010).
The apical complexes in turn restrict the localization of a third complex comprising Scribble (Scrib; mammalian Scribble/Vartul), Discs large (Dlg) and Lethal(2) giant larvae (Lgl) to the basolateral domain, while Lgl also antagonizes the apical components and prevents their spreading to the basolateral side. The antagonistic action of apical and basolateral complexes helps define the apicolateral loci eventually occupied by AJs. It is not yet completely clear how the initial localization of the apical complex is achieved (Duchi, 2010).
The VHL tumor-suppressor gene mutations are the genetic cause of the familial VHL disease. Germline mutations in VHL predispose the patients to several benign and malignant tumors, including renal cell carcinoma (RCC, kidney cancer), hemangioblastoma (overgrowth of blood vessels in the retina and central nervous system) and pheochromocytoma (tumors in the adrenal glands). VHL protein has been shown to function as an E3 ubiquitin ligase. Among its best-documented targets is the alpha subunit of the hypoxia-inducible factor (HIF-α). Therefore, the canonical tumor-suppressor function of VHL is modulation of the normal oxygen-sensing mechanism that regulates angiogenic response and metabolic switch to glycolysis (Kaelin, 2008a). However, how this function correlates with the origin of epithelial tumors such as RCC is unclear (Frew, 2007), although it is thought that HIF-independent mechanisms might be involved (Duchi, 2010).
VHL is evolutionarily conserved. In Drosophila, the VHL gene has been implicated in tracheal tubule development and HIF-α regulation in the embryos based on biochemical and RNA interference-mediated phenotypic studies (Adryan, 2000; Arquier, 2006; Aso, 2000; Mortimer, 2009). In this report, the first genomic Drosophila VHL mutant was generated, and the function of VHL in epithelial morphogenesis was examined using a model epithelium -- the follicle cells in the egg chamber. VHL is shown to regulate the proper localization and stability of aPKC in the follicle cells, and this function is, at least in part, mediated by the action of VHL on microtubule (MT) stability. Without VHL function, MTs and aPKC are destabilized, resulting in epithelial defects. These results establish a developmental function of the VHL gene that is relevant to its tumor-suppressor activity (Duchi, 2010).
The VHL mutation was generated using the homologous recombination strategy. Homozygous VHL1 mutants are sluggish after hatching and die at the end of first instar larval stage. Wild-type, heterozygous and homozygous first instar larvae were hand-picked and subjected to genomic DNA PCR. The homozygous mutant animals show complete loss of the wild-type gene copy. The VHL1 allele, when paired with a deficiency chromosome encompassing the VHL locus (at 47E), shows the same late first instar lethality, suggesting that VHL1 is a null mutant. The lethal phenotype can be rescued by expressing a wild-type VHL cDNA under the control of the hsp70 promoter. Therefore, VHL gene truncation is the only major genetic defect in the VHL1 allele (Duchi, 2010).
The Drosophila follicle cells exhibit the typical epithelial polarity exemplified by markers such as the apical PAR complex, AJ components and the basolateral Lgl complex. Establishment of the epithelial polarity begins soon after follicle cells diverge from the somatic stem cells and encircle the germ cells (stage 1). The epithelium reaches maturity at mid-oogenic stages (after stage 6 at ~30 hours of egg chamber development; total developmental time ~70 hours), when follicle cells cease to proliferate. To examine the phenotype in the follicular epithelium, an adult tissue, mosaic mitotic mutant clones were generated using the Flp/FRT system. Mutant clones are identified by a lack of GFP expression. In an initial survey of potential egg chamber phenotypes, prominent epithelial defects were observed at stage 10 (about 50-55 hours into egg chamber development. The most notable morphological abnormalities were the piling-up of follicle cells and stretched follicular epithelium. The same phenotypes were observed with the VHL deficiency Df(2R)en-A. To quantify the phenotypes, 100 clones of various sizes at stage 9-10 were analyzed. Clone sizes were categorized based on the number of cells in an optical cross-section. As the penetrance in single-cell clones is variable, probably because of phenotypic rescue by the neighboring epithelial cells, only clones larger than two cells were considered. Thirty-eight percent of clones showed stacking of follicle cells and the more severe, multilayering phenotypes (more than three layers of cells); 42% showed flattening/stretching of epithelium; and the remaining 20% showed swelling. It was also determined that VHL loss-of-function mutation did not affect cell proliferation or viability. As mitotic recombination in a heterozygous progenitor cell (VHL1/ubi-GFP) generates one VHL homozygote (no GFP) and one wild type (two copies of GFP), the numbers of high GFP-expressing cells and GFP-negative cells should be equal if the mutant cells do not exhibit an altered rate of proliferation or viability. The ratio was calculated of the cell number within the VHL1 mutant clones versus the cell number within the sister wild-type clones, and a mean value of 0.96 was obtained. Importantly, the ratio between the cell number in mutant and wild-type sister clones did not change as a function of the clone size. Furthermore, staining for mitosis and apoptosis markers phospho-histone H3 and cleaved caspase 3, respectively, also showed that VHL mutation does not affect cell proliferation or caspase-mediated apoptosis (Duchi, 2010).
This report shows that Drosophila VHL is important for establishing and maintaining epithelial integrity via its regulation of MT and aPKC stability. MT disruption and epithelial phenotypes were observed early in oogenesis. This indicates that MT bundles in developing epithelial cells are crucial for epithelial development and are under pressure from dynamic instability. Without stabilizing activity provided by VHL, MTs are disorganized and ultimately disintegrate, resulting in loss of epithelial integrity. Disrupted MTs interfere with proper localization of aPKC, which in turn leads to mislocalization of downstream epithelial markers and epithelial defects. An ex vivo experiment also demonstrates that epithelial defects can occur within a short time (relative to the entire oogenesis time frame) after destabilizing MTs in non-proliferating epithelial cells. This indicates that the maintenance of epithelial integrity is a dynamic and continuous process even in a stable epithelium, for which MTs are crucially important. Previous studies using RNA interference-mediated knockdown demonstrated a morphogenic role of VHL in trachea development (Adryan, 2000; Mortimer, 2009). The tracheal phenotypes appear to be the result of elevated cell motility and ectopic chemotactic signaling. Therefore, the tracheal function of VHL might be mediated via different VHL targets in a tissue-specific context. Alternatively, regulation of MT stabilization might also be the underlying mechanism. A separate, tissue-specific function for VHL is favored since the tracheal defects in VHL knockdown can be relieved by decreased expression of breathless, which encodes the chemotactic signaling receptor in the trachea. The two VHL functions, however, are not necessarily mutually exclusive. These different organ systems might in the future serve as a model for testing whether the various functions assigned to VHL are tissue-specific and context-dependent (Duchi, 2010).
Human VHL has been shown to translocate aPKC to MTs, thereby influencing MT reorganization (Schermer, 2006). 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. It is therefore concluded 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, the results also indicate that 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, it was also demonstrated that 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 (Lolkema, 2007). Future studies using the Drosophila epithelial system should also address this issue in vivo. Also interestingly, this study showed that the YH mutant can associate with MT but has little MT-stabilizing activity. This suggests that the YH 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. This study has shown that tracheal defects are the major embryonic phenotype observed in VHL mutant (Adryan, 2000). In the course of attempting to rescue the tracheal phenotype with btl-driven VHL, the appearance of rescued homozygous adults was noted. 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, recent evidence has suggested that VHL is a multifunctional protein. It can function as a regulator of matrix deposition (Ohh, 1998), integrin assembly, endocytosis (Champion, 2008; Hsu, 2006), kinase activity (Yang, 2007), senescence (Young, 2008), protein stabilities (Chitalia, 2008; Roe, 2006) and tight junction formation (Calzada, 2006; Harten, 2009), among many others (Frew, 2007). 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 (Hergovich, 2003; Lolkema, 2004). 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 (Lolkema, 2008; Lutz, 2006; Schermer, 2006; Thoma, 2009). 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).
Mutations in the human von Hippel-Lindau (VHL) gene are the cause of VHL disease that displays multiple benign and malignant tumors. The VHL gene has been shown to regulate angiogenic potential and glycolic metabolism via its E3 ubiquitin ligase function against the alpha subunit of hypoxia-inducible factor (HIF-alpha). However, many HIF-independent functions of VHL have been identified. Recent evidence also indicates that the canonical function cannot fully explain the VHL mutant cell phenotypes, although it is still unclear how many of these noncanonical functions relate to the pathophysiological processes because of a lack of tractable genetic systems. This study reports the first genomic mutant phenotype of Drosophila melanogaster VHL (dVHL) in the epithelial tubule network, the trachea, and shows that dVHL regulates branch migration and lumen formation via its endocytic function. The endocytic function regulates the surface level of the chemotactic signaling receptor Breathless and promotes clearing of the lumen matrix during maturation of the tracheal tubes. Importantly, the regulatory function in tubular morphogenesis is conserved in the mammalian system, as conditional knockout of Vhl in mouse kidney also resulted in similar cell motility and lumen phenotypes (Hsouna, 2010).
dVHL genetically interacts with endocytic pathway components such as awd, 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).
Recently, it was 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, this study shows that the exogenously expressed Btl::GFP protein is under stringent control at the protein level. Also, this study shows that sima can only modestly suppress the dVHL1 branch migration phenotype. It is interesting to point out that Mortimer (2009) reported a robust rescue of phenotypes in the btl promoter-driven dVHL RNA duplex by sima heterozygotes. 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, the results clearly 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).
This study shows that dVHL regulates tracheal tubule development in two aspects—branch migration and lumen formation. These two aspects of tracheal morphogenesis are regulated by the same endocytic function; one involves internalization of the Btl signaling receptor, and the other involves resorption of lumen materials (Hsouna, 2010).
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, this 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, previous work also 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).
Mutations in Drosophila merry-go-round (mgr) have been known for over two decades to lead to circular mitotic figures and loss of meiotic spindle integrity. However, the identity of its gene product has remained undiscovered. This study shows that mgr encodes the Prefoldin subunit counterpart of human von Hippel Lindau binding-protein 1. Depletion of Mgr from cultured cells also leads to formation of monopolar and abnormal spindles and centrosome loss. These phenotypes are associated with reductions of tubulin levels in both mgr flies and mgr RNAi-treated cultured cells. Moreover, mgr spindle defects can be phenocopied by depleting beta-tubulin, suggesting Mgr function is required for tubulin stability. Instability of beta-tubulin in the mgr larval brain is less pronounced than in either mgr testes or in cultured cells. However, expression of transgenic beta-tubulin in the larval brain leads to increased tubulin instability, indicating that Prefoldin might only be required when tubulins are synthesized at high levels. Mgr interacts with Drosophila von Hippel Lindau protein (Vhl). Both proteins interact with unpolymerized tubulins, suggesting they cooperate in regulating tubulin functions. Accordingly, codepletion of Vhl with Mgr gives partial rescue of tubulin instability, monopolar spindle formation, and loss of centrosomes, leading to the proposal of a requirement for Vhl to promote degradation of incorrectly folded tubulin in the absence of functional Prefoldin. Thus, Vhl may play a pivotal role: promoting microtubule stabilization when tubulins are correctly folded by Prefoldin and tubulin destruction when they are not (Delgehyr, 2012).
The finding that the Drosophila merry-go-round gene encodes a subunit of the Prefoldin complex has led to the accounting for aberrant structure and function of spindles and centrosomes in cells depleted of its gene product. The inability to correctly fold tubulins in Prefoldin-deficient cells leads to tubulin instability and, hence, defects that can be phenocopied by depleting β- or γ-tubulin. However, whereas β-tubulin depletion phenocopied all of the defects observed, γ-tubulin depletion only recapitulated some of them. The more dramatic phenotypes seen in Mgr-deficient cells expressing high levels of tubulin (primary spermatocytes and neuroblasts expressing a β-tubulin transgene) suggest that the Prefoldin complex is critical to maintain tubulin levels above a certain threshold of tubulin expression. This finding could be a consequence of the impact of an excess of tubulin upon its complex folding pathway. Interestingly, in mammalian cells, increased soluble tubulin, in response to a MT-destabilizing agent, leads to the rapid degradation of tubulin. In Drosophila, tubulins in the testes are the most affected by the absence of Mgr compared with other tissues. Indeed, it may be of particular importance to regulate tubulin levels at the late stages of spermatogenesis, where the very large meiotic cells are provided with proportionally large amounts of tubulin that are used in the meiotic spindle but have a major additional purpose: the building of the sperm tail. Similarly, in the mouse, the effects of depletion or mutation of prefoldin subunits are largely restricted to the brain, where tubulin levels are also very high. Whether this tissue specificity is a consequence of tubulin levels will be an interesting question to address. Finally, the demonstration that Vhl is required for tubulin destruction in the absence of Mgr and the ability of Vhl to interact with tubulin monomers and dimers raises the possibility that its role as an E3 ubiquitin-protein ligase could come to play in regulating tubulin levels (Delgehyr, 2012).
The idea that Vhl and Prefoldin can cooperate in regulating protein stability was also raised by another study that identified the prefoldin subunit VBP1 as a binding partner of the HIV-1 viral integrase and suggested this mediated the interaction of the integrase with the Cul2-Vhl E3-Ubiquitin ligase. This finding led to a suggestion of 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 speculatea 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).
The role of the hypoxic response during metastasis was analysed in migrating border cells of the Drosophila ovary. Acute exposure to 1% O2 delayed or blocked border cell migration (BCM), whereas prolonged exposure resulted in the first documented accelerated BCM phenotype. Similarly, manipulating the expression levels of sima, the Drosophila hypoxia-inducible factor (HIF)-1alpha ortholog, revealed that Sima can either block or restore BCM in a dose-dependent manner. In contrast, over-expression of Vhl (Drosophila von Hippel-Lindau) generated a range of phenotypes, including blocked, delayed and accelerated BCM, whereas over-expression of hph (Drosophila HIF prolyl hydroxylase) only accelerated BCM. Mosaic clone analysis of sima or tango (HIF-1beta ortholog) mutants revealed that cells lacking Hif-1 transcriptional activity were preferentially detected in the leading cell position of the cluster, resulting in either a delay or acceleration of BCM. Moreover, in sima mutant cell clones, there was reduced expression of nuclear slow border cells (Slbo) and basolateral DE-cadherin, proteins essential for proper BCM. These results show that Sima levels define the rate of BCM in part through regulation of Slbo and DE-cadherin, and suggest that dynamic regulation of Hif-1 activity is necessary to maintain invasive potential of migrating epithelial cells (Doronkin, 2010). In addition to other investigations, this study also investigated whether increasing Vhl expression, which would also be expected to decrease accumulation of Sima, would affect BCM. Stocks were created in which expression of full-length, FLAG epitope-tagged Vhl or human VHL was driven in border cells. A broad spectrum of phenotypes was observed ranging from normal to accelerated to blocked migration using either driver. Compared with wild-type chambers dissected at stage 10, in which migration was complete, up to 40% of egg chambers show a delay in migration. However, up to 10% of chambers exhibit accelerated migration, moving beyond the border between the nurse cells and the oocyte. Although 50–60% of UAS–Vhl egg chambers display normal BCM, a small fraction (7%) show complete migration failure. Similar results were obtained for the c522–GAL4 driver at stages 9 and 10 (Doronkin, 2010).
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).
The tracheal system of Drosophila is an interconnected network of gas-filled epithelial tubes that develops during embryogenesis and functions as the main gas-exchange organ in the larva. Larval tracheal cells respond to hypoxia by activating a program of branching and growth driven by HIF-1α/sima-dependent expression of the breathless (btl) FGF receptor. By contrast, the ability of the developing embryonic tracheal system to respond to hypoxia and integrate hard-wired branching programs with sima-driven tracheal remodeling is not well understood. This study shows that embryonic tracheal cells utilize the conserved ubiquitin ligase dVHL to control the HIF-1 α/sima hypoxia response pathway, and two distinct phases of tracheal development with differing hypoxia sensitivities and outcomes were identified: a relatively hypoxia-resistant 'early' phase during which Sima activity conflicts with normal branching and stunts migration, and a relatively hypoxia-sensitive 'late' phase during which the tracheal system uses the dVHL/sima/btl pathway to drive increased branching and growth. Mutations in the archipelago (ago) gene, which antagonizes btl transcription, re-sensitize early embryos to hypoxia, indicating that their relative resistance can be reversed by elevating activity of the btl promoter. These findings reveal a second type of tracheal hypoxic response in which Sima activation conflicts with developmental tracheogenesis, and identify the dVHL and ago ubiquitin ligases as key determinants of hypoxia sensitivity in tracheal cells. The identification of an early stage of tracheal development that is vulnerable to hypoxia is an important addition to models of the invertebrate hypoxic response (Mortimer, 2009).
The development and survival of an organism are dependent on its ability to adapt to changing environmental conditions. Responses to some environmental changes, for example in nutrient availability, temperature, or oxygen concentration, involve alterations in patterns of gene expression that allow the organism to survive periods of environmental stress. In metazoan cells, the cellular response to reduced oxygen is mediated primarily by the HIF (hypoxia inducible factor) family of transcription factors, which are heterodimers composed of α and β subunits belonging to the bHLH Per-ARNT-Sim (bHLH-PAS) protein family. The HIF-1 αβ heterodimer is the primary oxygen-responsive HIF in mammalian cells and binds to a specific DNA sequence termed hypoxia response element (HRE) present in the promoters of target genes involved in energy metabolism, angiogenesis, erythropoiesis, and autophagy. HIF-1 activity is inhibited under normoxic conditions by two hydroxylase enzymes that use dioxygen as a substrate for catalysis to hydroxylate specific proline or aspartate residues in the HIF-1α subunit (reviewed in Kaelin, 2008b). These modifications limit HIF-1 activity by either reducing HIF-1α levels or inhibiting its ability to activate HRE-containing target promoters. One of these inhibitory mechanisms involves the 2-oxoglutarate/Fe(II)-dependent HIF-1 prolyl hydroxylase (HPH), which attaches a hydroxyl group onto each of two conserved proline residues in the oxygen-dependent degradation domain (ODD) of mammalian HIF-1α. These modifications create a binding site in the HIF-1α ODD for the Von Hippel-Lindau (VHL) protein, the substrate adaptor component of a ubiquitin ligase that subsequently polyubiquitinates HIF-1α and targets it for degradation by the proteasome (reviewed in Kaelin, 2005). This degradation mechanism operates constitutively in normoxia and is epistatic to otherwise wide spread expression of HIF-1α mRNA. HIF-1α protein is also modified by a second oxygen-dependent hydroxylase termed Factor Inhibiting HIF (FIH) that hydroxylates an asparagine residue in the HIF-1α C-terminal activation domain (reviewed in Kaelin, 2005). This blocks interaction with the CBP/p300 transcriptional co-factor and thus further restricts expression of HIF-1 responsive genes. These parallel O2-dependent hydroxylation mechanisms by HPH and FIH ensure that HIF-1α levels and activity remain low in normoxic conditions. However as oxygen levels become limiting in the cellular environment, rates of hydroxylation decline and HIF-1α is rapidly stabilized in a form that dimerizes with HIF-1β, translocates to the nucleus, and promotes transcription of HRE-containing target genes (Mortimer, 2009).
Evidence suggests that invertebrate homologs of HIF-1 are also regulated in response to changes in oxygen availability (reviewed by Gorr, 2006). In the fruit fly Drosophila melanogaster, the HPH homolog fatiga (fga) has been shown to genetically antagonize the HIF-1α homolog similar (sima) during development (Centanin, 2005). The Drosophila VHL homolog dVHL has also been shown to be capable of binding to human HIF-1α and stimulating its proteasomal turnover in vitro (Aso, 2000). In addition, the Drosophila genome encodes a well-characterized HIF-1β homolog tango (tgo), and two potential FIH homologs (CG13902 and CG10133; Berkeley Drosophila Genome Project) that have yet to be analyzed functionally. Spatiotemporal analysis of sima activation using sima-dependent hypoxia-reporter transgenes has shown that exposure to an acute hypoxic stress induces Sima most strongly in cells of the larval and embryonic tracheal system, while induction of reporter activity in other tissues requires more chronic exposure to low oxygen. The larval tracheal system is composed of an interconnected network of polarized, epithelial tubes that duct gases through the organism. As the trachea acts as the primary gas-exchange organ in the larva, it is thus a logical site of hypoxia sensitivity. During larval stages, specific cells within the tracheal system called 'terminal cells' respond to hypoxia by initiating new branching and growth that results in the extension of fine, unicellular, gas-filled tubes toward hypoxic tissues in a manner somewhat analogous to mammalian angiogenesis . Studies have shown that sima and its upstream antagonist fga function within terminal cells to regulate this process (Centanin, 2008). sima is necessary for terminal cell branching in hypoxia and its ectopic activation, by either transgenic overexpression or loss of fga, is sufficient to induce excess branching even in normoxia. These phenotypes have been linked to the ability of sima to promote expression of the breathless (btl) gene (Centanin, 2008), which encodes an FGF receptor that is activated by the branchless (bnl) FGF ligand. This receptor/ligand pair is known to act via a downstream MAP-kinase signaling cascade to promote cell motility and tubular morphogenesis in a variety of systems. Excessive activation of this pathway within tracheal cells by transgenic expression of btl is sufficient to drive excess branching. Reciprocally, misexpression of the bnl ligand in certain peripheral tissues is sufficient to attract excess terminal cell branching. Indeed production of secreted factors such as Bnl may be a significant part of the physiologic mechanism by which hypoxic cells attract new tracheal growth. Sima-driven induction of btl in conditions of hypoxia thus allows larval terminal cells to enter what has been termed an 'active searching' mode (Centanin, 2008) in which they are hyper-sensitized to signals emanating from nearby hypoxic non-tracheal cells (Mortimer, 2009 and references therein).
The role of the btl/bnl pathway in tracheal development is not restricted to hypoxia-induced branching of larval terminal cells. It also plays a critical, earlier role in the initial development of the embryonic tracheal system from the tracheal placodes, groups of post-mitotic ectodermal cells distributed along either side of the embryo that undergo a process of invagination, polarization, directed migration, and fusion to create a network of primary and secondary tracheal branches . btl and bnl are each required for this process via a mechanism in which restricted expression of bnl in cells outside the tracheal placode represents a directional cue for the migration of btl-expressing cells within the placode. Accordingly, btl expression is normally highest in pre-migratory and migratory embryonic fusion cells. In contrast to the larval hypoxic response, sima does not appear to be required for morphogenesis of the embryonic tracheal system. Rather, developmentally programmed signals in the embryo dictate a stereotyped pattern of btl and bnl expression that leads to a similarly stereotyped pattern of primary and secondary tracheal branches. The btl/bnl pathway thus responds to developmental signals to drive a fixed pattern of branching in the embryo, while in the subsequent larval stage it responds to hypoxia-dependent sima activity to facilitate the homeostatic growth of larval terminal cells and tracheal remodeling (Mortimer, 2009 and references therein).
Under normal circumstances, developing Drosophila tissues do not begin to experience hypoxia until the first larval stage, when organismal growth and movement begin to consume more oxygen than can be provided by passive diffusion alone. As a consequence, the first hypoxic challenge normally occurs after the btl/bnl-dependent elaboration of the primary and secondary embryonic branches is complete. Thus, the ability of the larval tracheal system to drive new branching and remodeling via sima and btl represents the response of a developed 'mature' tracheal system to reduced oxygen availability. By contrast the effect of hypoxia on embryonic tracheal development, which requires tight spatiotemporal control of Btl signaling to pattern the tracheal network, is not as well understood. Given that the trachea does not function as a gas-exchange organ until after fluid is cleared from the tubes at embryonic stage 17, it may be that the transcriptional response of embryonic tracheal cells to hypoxia leads to mainly metabolic changes rather than to a btl-driven program of tubulogenesis and remodeling. However, if the embryonic tracheal system does utilize the sima pathway to induce hypoxia-dependent changes in btl gene transcription, then hypoxic exposure of embryos might be predicted to produce a situation of competing developmental and homeostatic inputs that converge on the btl/bnl pathway. The ability of tracheal cells to integrate such signals may then determine whether or not the embryonic tracheal system is able to adapt to oxygen stress, or whether embryonic tracheal development represents a sensitive period during which the organism's ability to respond to changes in oxygen levels is inherently limited by a pre-programmed pattern of developmental gene expression (Mortimer, 2009).
This study shows that the embryonic tracheal system utilizes the dVHL/sima pathway to respond to hypoxia, but that the type and severity of resulting phenotypes depend on the developmental stage of exposure. Hypoxic challenge while embryonic tracheal cells are responding to developmentally programmed btl/bnl migration signals disrupts tracheal development and results in fragmented and unfused tracheal metameres. In contrast, hypoxic challenge at a somewhat later embryonic stage after fusion is complete results in overgrowth of the primary tracheal branches and the production of extra secondary branches. Interestingly, it was found that the threshold of hypoxia required to induce tracheal phenotypes in the early embryo is higher than that required to induce excess branching phenotypes in later embryonic stages, indicating that tracheal patterning events in the embryo are relatively resistant to hypoxia. Genetic analysis indicates that both types of hypoxic tracheal phenotypes -- stunting and overgrowth -- require sima and can be phenocopied in normoxia by reducing expression of the HIF-1α ubiquitin ligase gene dVHL specifically within tracheal cells. Moreover, it was found that reduced dVHL expression in the larval trachea leads to excess terminal cell branching in a manner quite similar to that observed in fga mutants. Molecular and genetic data indicate that excess btl transcription is a major cause of hypoxia-induced tracheal phenotypes. Consistent with this, mutations in the archipelago (ago) gene, which antagonizes btl transcription in tracheal fusion cells, synergize strongly with dVHL inactivation to disrupt tracheal migration and branching. Interestingly, ago mutations also lower the threshold of hypoxia required to elicit tracheal phenotypes in the 'early' embryo, suggesting that the relative activity of the btl promoter can affect hypoxic sensitivity. These findings show that the dVHL/sima pathway plays an important role in tracheal development, and identify two distinct phases of embryonic development that show different phenotypic outcomes of activating this pathway: an early phase during which sima activity conflicts with developmental control of tracheal branching and migration, and a later phase during which the tracheal system uses the dVHL/sima/btl pathway to adapt to hypoxia by increasing its future capacity to deliver oxygen to target tissues (Mortimer, 2009).
Hypoxia-induced remodeling of tracheal terminal cells represents the response of a developed larval tracheal system to reduced levels of O2 in the environment. By contrast, the response of the developing embryonic tracheal system to systemic hypoxia has not been as well characterized. In light of the observation that embryonic tracheal cells display hypoxia-induced activation of a Sima-reporter) and that sima promotes btl expression in larval tracheal cells, embryonic exposure to hypoxia may thus produce a situation in which hard-wired btl/bnl patterning signals in the embryo come into conflict with the type of sima/btl-driven plasticity of tracheal cell branching seen in the larva. This study examined the effect of hypoxia on embryonic tracheal branching and migration. It was found that hypoxia has dramatic effects on the patterns of morphogenesis of the primary and secondary tracheal branches. Surprisingly, varying the timing and severity of hypoxic challenge is able to shift the outcome from severely stunted tracheal branching to excess branch number and enhanced branch growth. Genetic and molecular data indicate that both classes of phenotypes, stunting and overgrowth, involve regulation of sima activity and btl transcription by dVHL, and that the effects of hypoxia on tracheal development can be mimicked in normoxia by tracheal-specific knockdown of dVHL. This observation confirms a central role for dVHL in restricting the hypoxic response in vivo, and identifies a role for dVHL as a required inhibitor of sima and btl during normal tracheogenesis (Mortimer, 2009).
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 (Mortimer, 2007), by the modest ability of trh alleles to suppress dVHLi phenotypes, and by the previously demonstrated 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 analysis 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 (Mortimer, 2007). 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 (Centanin, 2008; Jarecki, 1999) 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 (Centanin, 2008). 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 (Jarecki, 1999). 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).
tHE data 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 disrupted normal tracheogenesis (Adryan, 2000), but was technically limited in its ability to conduct a detailed analysis of dVHL function in development and homeostasis. The current study found 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 (Centanin, 2005). 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 (Centanin, 2008) 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 (this study; Mortimer, 2007), 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).
Biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes human patients to the development of highly vascularized neoplasms in multiple organ systems. This study shows that zebrafish vhl mutants display a marked increase in blood vessel formation throughout the embryo, starting at 2 days post-fertilization. The most severe neovascularization is observed in distinct areas that overlap with high vegfa mRNA expression, including the vhl mutant brain and eye. Real-time quantitative PCR revealed increased expression of the duplicated VEGFA orthologs vegfaa and vegfab, and of vegfb and its receptors flt1, kdr and kdr-like, indicating increased vascular endothelial growth factor (Vegf) signaling in vhl mutants. Similar to VHL-associated retinal neoplasms, diabetic retinopathy and age-related macular degeneration, this study showed, by tetramethyl rhodamine-dextran angiography, that vascular abnormalities in the vhl-/- retina leads to vascular leakage, severe macular edema and retinal detachment. Significantly, vessels in the brain and eye express cxcr4a, a marker gene expressed by tumor and vascular cells in VHL-associated hemangioblastomas and renal cell carcinomas. VEGF receptor (VEGFR) tyrosine kinase inhibition (through exposure to sunitinib and 676475) blocked vhl-/--induced angiogenesis in all affected tissues, demonstrating that Vegfaa, Vegfab and Vegfb are key effectors of the vhl-/- angiogenic phenotype through Flt1, Kdr and Kdr-like signaling. Since it was shown that the vhl-/- angiogenic phenotype shares distinct characteristics with VHL-associated vascular neoplasms, zebrafish vhl mutants provide a valuable in vivo vertebrate model to elucidate underlying mechanisms contributing to the development of these lesions. Furthermore, vhl mutant zebrafish embryos carrying blood vessel-specific transgenes represent a unique and clinically relevant model for tissue-specific, hypoxia-induced pathological angiogenesis and vascular retinopathies. Importantly, they will allow for a cost-effective, non-invasive and efficient way to screen for novel pharmacological agents and combinatorial treatments (van Rooijen, 2010).
In early neonates, the fetal circulatory system undergoes dramatic transition to the adult circulatory system. Normally, embryonic connecting vessels, such as the ductus arteriosus and the foramen ovale, close and regress. In the neonatal retina, hyaloid vessels maintaining blood flow in the embryonic retina regress, and retinal vessels take over to form the adult-type circulatory system. This process is regulated by a programmed cell death switch mediated by macrophages via Wnt and angiopoietin 2 pathways. This study seeks other mechanisms that regulate this process, and focuses on the dramatic change in oxygen environment at the point of birth. The von Hippel-Lindau tumor suppressor protein (pVHL) is a substrate recognition component of an E3-ubiquitin ligase that rapidly destabilizes hypoxia-inducible factor alphas (HIF-αs) under normoxic, but not hypoxic, conditions. To examine the role of oxygen-sensing mechanisms in retinal circulatory system transition, retina-specific conditional-knockout mice for VHL (Vhlα-CreKO mice) were generated. These mice exhibit arrested transition from the fetal to the adult circulatory system, persistence of hyaloid vessels and poorly formed retinal vessels. These defects are suppressed by intraocular injection of FLT1-Fc protein [a vascular endothelial growth factor (VEGF) receptor-1 (FLT1)/Fc chimeric protein that can bind VEGF and inhibit its activity], or by inactivating the HIF-1αα gene. These results suggest that not only macrophages but also tissue oxygen-sensing mechanisms regulate the transition from the fetal to the adult circulatory system in the retina (Kurihara, 2010).
The von Hippel-Lindau (VHL) cancer syndrome is associated with mutations in the VHL gene. The pVHL protein is involved in response to changes in oxygen availability as part of an E3-ligase that targets the Hypoxia-Inducible Factor (see Drosophila Similar for degradation. pVHL has a molten globule configuration with marginal thermodynamic stability. The cancer-associated mutations further destabilize it. The Drosophila homolog, dVHL, has relatively low sequence similarity to pVHL, and is also involved in regulating HIF1-α. Using in silico, in vitro and in vivo approaches this study demonstrates high similarity between the structure and function of dVHL and pVHL. These proteins have a similar fold, secondary and tertiary structures, as well as thermodynamic stability. Key functional residues in dVHL are evolutionary conserved. This structural homology underlies functional similarity of both proteins, evident by their ability to bind their reciprocal partner proteins, and by the observation that transgenic pVHL can fully maintain normal dVHL-HIF1-alpha downstream pathways in flies. This novel transgenic Drosophila model is thus useful for studying the VHL syndrome, and for testing drug candidates to treat it (Shmueli, 2014: 25310726). The von Hippel-Lindau (VHL) syndrome is a rare hereditary cancer, associated with mutations in the VHL tumor suppressor gene. It is characterized by increased susceptibility to various tumors, both benign and malignant, including central nervous system haemangioblastomas, renal cysts and renal cell carcinoma (RCC) and phaeochromocytoma. The pVHL tumor suppressor protein is the substrate recognition subunit of a complex, comprising pVHL as well as Elongin C and B, termed VCB complex. This complex functions as part of an SCF-like ubiquitin-ligase that promotes the degradation of target proteins required for growth and vascularization of solid tumors. The pVHL protein has been implicated in a variety of cellular processes, most notably in response to changes in oxygen availability, due to its role as part of an E3-ligase which targets the Hypoxia-Inducible Factor (HIF1-α) for proteasomal degradation. Upon decrease in oxygen levels residues P564 and/or P402, in the oxygen dependent degradation domain (ODD, residues 401–603) of HIF1-α, are hydroxylated and interact with Y98 in pVHL leading to VCB-mediated E3 degradation of HIF1-α. This hydroxylation reaction is mediated by the prolyl-hydroxylase domain protein (PHD). Under hypoxic conditions, the prolyl hydroxylase is inactive and the HIF1-α subunit is not hydroxylated. HIF1-α then dimerizes with the β subunit (HIF1-β), which is constitutively expressed. The HIF heterodimer translocates to the nucleus where it functions as a transcription factor. The best known target genes of HIF encode proteins involved in glycolysis, glucose transport (Glut-1), angiogenesis (vascular endothelial growth factor (VEGF)) and erythropoiesis (erythropoietin), i.e., proteins that mediate the cellular response and adaptation to hypoxic conditions. The VHL gene encodes two biologically active isoforms of pVHL (19 kDa and 30 kDa) as a result of in-frame alternative AUG codon usage. However, no functional significance has been assigned to the extra 53 amino acids at the N-terminus of the large isoform. pVHL shuttles back and forth between the nucleus and the cytoplasm. The short isoform of pVHL is localized primarily to the nucleus whereas the long isoform is more frequently associated tightly with microtubules in the cytoplasm (Shmueli, 2014).
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).
The mechanism by which hypoxia induces gene transcription involves the inhibition of HIF-1alpha (hypoxia-inducible factor-1 alpha subunit) PHD (prolyl hydroxylase) activity, which prevents the VHL (von Hippel-Lindau)-dependent targeting of HIF-1alpha to the ubiquitin/proteasome pathway. HIF-1alpha thus accumulates and promotes gene transcription. The present study provides direct biochemical evidence for the presence of a conserved hypoxic signalling pathway in Drosophila melanogaster. An assay for 2-oxoglutarate-dependent dioxygenases was developed using Drosophila embryonic and larval homogenates as a source of enzyme. Drosophila PHD has a low substrate specificity and hydroxylates key proline residues in the ODD (oxygen-dependent degradation) domains of human HIF-1alpha and Similar, the Drosophila homologue of HIF-1alpha. The enzyme promotes human and Drosophila [(35)S]VHL binding to GST (glutathione S-transferase)-ODD-domain fusion protein. Hydroxylation is enhanced by proteasomal inhibitors and was ascertained using an anti-hydroxyproline antibody. Secondly, by using transgenic flies expressing a fusion protein that combined an ODD domain and the green fluorescent protein (ODD-GFP), the hypoxic cascade was analyzed in different embryonic and larval tissues. Hypoxic accumulation of the reporter protein was observed in the whole tracheal tree, but not in the ectoderm. Hypoxic stabilization of ODD-GFP in the ectoderm was restored by inducing VHL expression in these cells. These results show that Drosophila tissues exhibit different sensitivities to hypoxia (Arquier, 2006).
Mutations of the von Hippel-Lindau (VHL) tumor suppressor gene predispose individuals to a variety of human tumors, including renal cell carcinoma, hemangioblastoma of the central nervous system, and pheochromocytoma. This study reports on the identification and characterization of the Drosophila homolog of VHL. The predicted amino acid sequence of Drosophila VHL protein shows 29% identity and 44% similarity to that of human VHL protein. Biochemical studies have shown that Drosophila VHL protein binds to Elongins B and C directly, and via this Elongin BC complex, associates with Cul-2 and Rbx1. Like human VHL, Drosophila VHL complex containing Cul-2, Rbx1, Elongins B and C, exhibits E3 ubiquitin ligase activity. In addition, evidence is provided that hypoxia-inducible factor (HIF)-1alpha is the ubiquitination target of both human and Drosophila VHL complexes (Aso, 2000).
von Hippel-Lindau disease is a hereditary cancer syndrome. Mutations in the VHL tumor suppressor gene predispose individuals to highly vascularized tumors. However, VHL-deficient mice die in utero due to a lack of vascularization in the placenta. To resolve the contradiction, the Drosophila VHL homologue (d-VHL) was cloned and its function was studied. It showed an overall 50% similarity to the human counterpart and 76% similarity in the crucial functional domain: the elongin C binding site. The putative d-VHL protein can bind Drosophila elongin C in vitro. During embryogenesis, d-VHL is expressed in the developing tracheal regions where tube outgrowth no longer occurs. Reduced d-VHL activity (using RNA interference methodology) caused breakage of the main vasculature accompanied by excessive looping of smaller branches, whereas over-expression caused a general lack of vasculature. Importantly, human VHL can induce the same gain-of-function phenotypes. VHL is likely involved in halting cell migration at the end of vascular tube outgrowth. Loss of VHL activity can therefore lead to disruption of major vasculature (as in the mouse embryo), which requires precise cell movement and tube fusion, or ectopic outgrowth from existing secondary vascular branches (as in the adult tumors) (Adryan, 2002)
Search PubMed for articles about Drosophila Vhl
Adryan B., et al. (2000). Tracheal development and the von Hippel-Lindau tumor suppressor homolog in Drosophila. Oncogene 19: 2803-2811. PubMed ID: 10851083
Arquier, N., et al. (2006). Analysis of the hypoxia-sensing pathway in Drosophila melanogaster. Biochem. J. 393: 471-480. PubMed ID: 16176182
Aso T., et al. (2000). Drosophila von Hippel-Lindau tumor suppressor complex possesses E3 ubiquitin ligase activity. Biochem. Biophys. Res. Commun. 276: 355-361. PubMed ID: 11006129
Calzada M. J., et al. (2006). von Hippel-Lindau tumor suppressor protein regulates the assembly of intercellular junctions in renal cancer cells through hypoxia-inducible factor-independent mechanisms. Cancer Res. 66: 1553-1560. PubMed ID: 16452212
Centanin, L., Ratcliffe, P. J. and Wappner, P. (2005). Reversion of lethality and growth defects in Fatiga oxygen-sensor mutant flies by loss of Hypoxia-Inducible Factor-alpha/Sima. EMBO Rep. 6: 1070-1075. PubMed ID: 16179946
Centanin, L., et al. (2008). Cell autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce terminal branch sprouting. Dev. Cell. 14: 547-558. PubMed ID: 18410730
Champion, K. J., et al. (2008). Endothelial function of von Hippel-Lindau tumor suppressor gene: control of fibroblast growth factor receptor signaling. Cancer Res. 68: 4649-4657. PubMed ID: 18559510
Chitalia, V. C., et al. (2008). Jade-1 inhibits Wnt signalling by ubiquitylating beta-catenin and mediates Wnt pathway inhibition by pVHL. Nat. Cell Biol. 10: 1208-1216. PubMed ID: 18806787
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
Doronkin, S., et al. (2010). Dose-dependent modulation of HIF-1alpha/sima controls the rate of cell migration and invasion in Drosophila ovary border cells. Oncogene 29(8): 1123-34. PubMed ID: 19966858
Duchi, S., et al. (2010). Drosophila VHL tumor-suppressor gene regulates epithelial morphogenesis by promoting microtubule and aPKC stability. Development 137(9): 1493-503. PubMed ID: 20388653
Frew I. J. and Krek W. (2007). Multitasking by pVHL in tumour suppression. Curr. Opin. Cell Biol. 19: 685-690. PubMed ID: 18006292
Gorr, T. A., Gassmann, M. and Wappner, P. (2006). Sensing and responding to hypoxia via HIF in model invertebrates. J. Insect Physiol. 52: 349-364. PubMed ID: 16500673
Harris T. J. and Peifer M. (2007). aPKC controls microtubule organization to balance adherens junction symmetry and planar polarity during development. Dev. Cell 12: 727-738. PubMed ID: 17488624
Harten S. K., et al. (2009). Regulation of renal epithelial tight junctions by the von Hippel-Lindau tumor suppressor gene involves occludin and claudin 1 and is independent of E-cadherin. Mol. Biol. Cell 20: 1089-1101. PubMed ID: 19073886
Hergovich A., et al. (2003). Regulation of microtubule stability by the von Hippel-Lindau tumour suppressor protein pVHL. Nat. Cell Biol. 5: 64-70. PubMed ID: 12510195
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
Hsu, T., et al. (2006). Endocytic function of von Hippel-Lindau tumor suppressor protein regulates surface localization of fibroblast growth factor receptor 1 and cell motility. J. Biol. Chem. 281: 12069-12080. PubMed ID: 16505488
Jarecki, J., Johnson, E. and Krasnow, M. A. (1999). Oxygen regulation of airway branching in Drosophila is mediated by branchless FGF. Cell 99: 211-220. PubMed ID: 10535739
Kaelin, W. G. (2005). The von Hippel-Lindau protein, HIF hydroxylation, and oxygen sensing. Biochem. Biophys. Res. Commun. 338: 627-638. PubMed ID: 15952883
Kaelin W. G. (2008a). The von Hippel-Lindau tumour suppressor protein: O2 sensing and cancer. Nat. Rev. Cancer 8: 865-873. PubMed ID: 18923434
Kaelin, W. G. and Ratcliffe, P. J. (2008b). Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. Mol. Cell. 30: 393-402. PubMed ID: 18498744
Kurihara, T., et al. (2010). von Hippel-Lindau protein regulates transition from the fetal to the adult circulatory system in retina. Development 137(9): 1563-71. PubMed ID: 20388654
Lolkema, M. P., et al. (2004). The von Hippel-Lindau tumor suppressor protein influences microtubule dynamics at the cell periphery. Exp. Cell Res. 301(2): 139-46. PubMed ID: 15530850
Lolkema M. P., et al. (2007). The von Hippel-Lindau tumour suppressor interacts with microtubules through kinesin-2. FEBS Lett. 581: 4571-4576. PubMed ID: 17825299
Lolkema M. P., et al. (2008). Allele-specific regulation of primary cilia function by the von Hippel-Lindau tumor suppressor. Eur. J. Hum. Genet. 16: 73-78. PubMed ID: 17912253
Lutz M. S. and Burk R. D. (2006). Primary cilium formation requires von hippel-lindau gene function in renal-derived cells. Cancer Res. 66: 6903-6907. PubMed ID: 16849532
Mortimer, N. T. and Moberg, K. H. (2009). Regulation of Drosophila embryonic tracheogenesis by dVHL and hypoxia. Dev. Biol. 329(2): 294-305. PubMed ID: 19285057
Ohh, M., et al. (1998). The von Hippel-Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix. Mol. Cell 1: 959-968. PubMed ID: 9651579
Roe, J. S. and Youn, H. D. (2006). The positive regulation of p53 by the tumor suppressor VHL. Cell Cycle 5: 2054-2056. PubMed ID: 16969113
Schermer, B., et al. (2006). The von Hippel-Lindau tumor suppressor protein controls ciliogenesis by orienting microtubule growth. J. Cell Biol. 175: 547-554. PubMed ID: 17101696
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
Thoma, C. R., et al. (2009). VHL loss causes spindle misorientation and chromosome instability. Nat. Cell Biol. 11: 994-1001. PubMed ID: 19620968
van Rooijen, E., et al. (2010). von Hippel-Lindau tumor suppressor mutants faithfully model pathological hypoxia-driven angiogenesis and vascular retinopathies in zebrafish. Dis. Model Mech. 3(5-6): 343-53. PubMed ID: 20335444
Yang, H., et al. (2007). pVHL acts as an adaptor to promote the inhibitory phosphorylation of the NF-kappaB agonist Card9 by CK2. Mol. Cell 28: 15-27. PubMed ID: 17936701
date revised: 10 February 2015
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