Clear cell renal cell carcinoma (ccRCC) is an aggressive malignancy characterized by inactivation of the von Hippel-Lindau tumour suppressor gene, the protein product of which mediates degradation of the transcription factor hypoxia-inducible factor (HIF). GAL3ST1 is a sulfotransferase which catalyzes the production of sulfatide, a plasma membrane sulfolipid previously implicated in metastasis. We observed GAL3ST1 overexpression in primary ccRCC tumours relative to matched-normal tissue and subsequently asked if GAL3ST1 was a HIF-responsive gene that facilitates ccRCC metastasis. GAL3ST1 expression was suppressed in ccRCC cells by stable reconstitution of wild-type VHL and also siRNA-mediated knockdown of HIF1alpha and HIF2alpha. Dual luciferase assays and chromatin immunoprecipitation revealed a hypoxia-response element in the GAL3ST1 5’-UTR that appeared to be crucial for HIF-mediated upregulation. Finally, stable knockdown of GAL3ST1 significantly impeded ccRCC cell invasion through an in vitro basement membrane mimic. These results suggest GAL3ST1 is a HIF-responsive gene that promotes tumour cell invasion.

Activity of the hypoxia inducible HIF-alpha transcription factors drive the hypoxic response, resulting in enhancement of angiogenesis, tumour growth, invasion and metastasis. Seeking to uncover a role for Tid1 in control of HIF2-alpha, we used lentiviral shRNA to knock-down Tid1 in 786-0 RCC cells with and without pVHL. In 786-0 cells stably expressing pVHL30, Tid1 knock-down resulted in a dramatic reduction in HIF2-alpha levels relative to controls. Adenoviral-mediated overexpression of Tid1S rescued this decline in HIF2-alpha levels, while overexpression of Tid1L enhanced this decline. A protective role of Tid1S for HIF2-alpha was reproduced in a HEK293 cell model. Immunoprecipitations in HEK293 cells revealed a lack of direct binding between HIF2-alpha and Tid1 in vivo, while adenoviral-mediated overexpression of Tid1 in this model failed to alter in vitro binding between HIF2-alpha and pVHL30. We present a model in which Tid1 regulates HIF2-alpha stability through regulation of pVHL30 nuclear import.

SCIENTIFIC REPORTS へのhyperlink http://www.nature.com/articles/srep19118 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19926号 / 医博第4146号 / 新制||医||1017(附属図書館) / 33012 / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩井 一宏, 教授 井上 治久, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Die ständige Verfügbarkeit von molekularem Sauerstoff (O2) ist ein elementarer Bestandteil multizellulärer Lebensformen. Zur Aufrechterhaltung der Homöostase sind diese auf die Bildung des Energiesubstrates ATP durch oxidative Phosphorylierung angewiesen. Aus diesem Grunde mußten höhere Organismen während der Evolution komplexe Systeme entwickeln, die die Aufnahme und Verteilung von O2 in jede Zelle sicherstellen, sowie eine Adaptation in Phasen der Hypoxie erlauben. Mit der Identifikation des Transkriptionsfaktors "Hypoxia-inducible Factor-1" (HIF-1, 1995) wurde ein entscheidender Regulator der hypoxischen Adaptation gefunden. Unter anderem werden Prozesse wie die Erythropoiese, die Angiogenese, die Modulation des Gefäßtonus, des Glukosetransportes und der Glykolyse wesentlich durch HIF reguliert. HIF ist ein Heterodimer bestehend aus zwei Untereinheiten; einer konstitutiven beta- und einer regulativen alpha-Untereinheit. Letztere zeigt ein inverses Expressionsmuster zur perizellulären O2-Konzentration. Unter normoxischen Bedingungen ist HIFalpha instabil und wird mit einer Halbwertzeit von nur wenigen Minuten degradiert. Erst unter Hypoxie wird HIFalpha stabilisiert und ist transkriptionell aktiv. Es konnten bisher zwei funktionell relevante O2-abhängige alpha-Untereinheiten identifiziert werden: HIF-1 und HIF-2alpha. Die Bedeutung dieser beiden Systeme, der unterliegenden Regulationsmechanismen sowie die Relevanz dieses Systems in vivo waren weitgehend ungeklärt und sind wesentlicher Teil der hier zusammengefaßten Arbeiten. In den vorgelegten Studien ist es gelungen, die Expression und Regulation der beiden unterschiedlichen HIFalpha Isoformen sowohl in der Zellkultur, als auch in gesunden Geweben zu charakterisieren. In Zellkulturen zeigte sich ein sehr ähnliches Regulationsmuster hinsichtlich der O2-abhängigen Degradation, bzw. dem Induktionsverhalten unter Hypoxie, sowie der chemisch/pharmakologischen Modulation, so dass offensichtlich beide Isoformen über den gleichen O2-Sensing- und Transduktionsapparat reguliert werden. An Geweben von gesunden Ratten führten wir eine systematische Analyse der Expression und Regulation der beiden HIFalpha Isoformen durch. Nur unter systemischer Hypoxie konnten deutliche Signale für beide Isoformen gesehen werden. Interessanterweise zeigte sich, daß beide nur von spezifischen Zellpopulationen exprimiert werden. In vivo lassen sich also klare Unterschiede im Expressionsmuster der beiden Systeme feststellen. Über die unterschiedlichen zellulären Funktionen und different exprimierten Zielgene vermuten wir einen funktionell relevanten Unterschied. Mit der Identifikation des "von Hippel Lindau" Tumor Suppressor Gens als der bindende Anteil der E3 Ubiquitin Ligase, die für die HIFalpha Destruktion verantwortlich ist, konnte ein wichtiger Beitrag zu der späteren Klärung des O2-Sensing-Mechanismus geleistet werden. Diese Befunde wurden initial anhand von Zellkultur-Linien erhoben, liessen sich aber auf Nierenzellkarzinome aus einer klinischen Sammlung übertragen. Letzterer Befund ist daher für das Verständnis der Rolle von HIF für die Tumorbiologie, eventuell aber auch für die Entwicklung therapeutischer Ansätze von Bedeutung. / The permanent availability of molecular oxygen (O2) is an elemental need of multicellular life. For the maintenance of hemeostasis these are dependent on generation of the energy substrate ATP by oxidative phoshorylation. For this reason higher organisms had to develop complex systems during evolution that ensure the uptake and distribution of O2 into each cell, as well as permit adaptation to phases of hypoxia. With the identification of the transcription factor "Hypoxia-inducible Factor-1" (HIF-1, 1995) a master regulator of hypoxic adaptation has been found. Amongst others processes like erythropoiesis, angiogenesis, modulation of vascular tone, glucose transport and glycolysis are largely regulated by HIF. HIF is a heterodimer consisting of two subunits, a constitutive beta and a regulative alpha subunit. The latter shows an inverse relationship to the pericellular O2 concentration. HIFalpha is instable under normoxic conditions and degrades with a half life of only a few minutes. Under hypoxia the HIFalpha subunits are stabilised and are transcriptionally active. To date two functionally relevant O2-dependent alpha subunits have been identified: HIF-1 and HIF-2alpha. The importance of these two systems, the underlying regulatory mechanisms, as well as the relevance of this system in vivo were largely unknown and are a major part of the summarised studies. The presented work succeeded in characterising the expression and regulation of both HIFalpha isoforms in cell culture as well as healthy tissues. In tissue culture a very similar pattern of regulation was seen for oxygen dependent degradation, induction under hypoxia and chemical/pharmacological modulation, indicating that both subunits are regulated by the same O2-sensing and transduction apparatus. We undertook a systematic analysis of expression and regulation of both HIFalpha subunits in tissues of healthy rats. Signals for HIFalpha could only be seen under systemic hypoxia. Interestingly, both subunits were expressed by specific and different cell populations. Therefore, clear differences can be seen in expression pattern of both systems in vivo. We suspect that these differences will be functionally relevant through differing cellular functions and gene expression profile. With the identification of the "von Hippel Lindau" tumor suppressor gene as the binding part of the E3 ubiquitin ligase, which is responsible for HIF degradation, an important contribution to the clarification of the oxygen sensing mechanism was provided. Initially this data was generated in tissue culture lines, but could also be confirmed in renal cell carcinomas of a clinical collection. The latter finding is of importance for the understanding of the role of HIF in tumor biology, possibly also for the development of therapeutic strategies.

Rapidly progressive glomerulonephritis (RPGN) is a clinical syndrome characterized by loss of renal function within days to weeks and by glomerular crescents on biopsy. The pathogenesis of this disease is unclear, but circulating factors such as antineutrophil cytoplasmic antibodies (ANCA) are believed to play a major role. In this thesis, we show that deletion of the Von Hippel-Lindau gene (Vhlh) from intrinsic glomerular cells of mice is sufficient to initiate a necrotizing crescentic glomerulonephritis and the clinical features that accompany RPGN. Loss of Vhlh leads to stabilization of hypoxia-inducible factor alpha subunits (HIFαs). Using gene expression profiling, we identified de novo expression of the HIFα target gene Cxcr4. In glomeruli from mice with RPGN, the course of RPGN is markedly improved in mice treated with a blocking antibody to Cxcr4, whereas overexpression of Cxcr4 alone in podocytes of transgenic mice is sufficient to cause glomerular disease. Despite the development of glomerular disease in mice that overexpress Cxcr4, their disease was milder and lacked features of full-blown RPGN. The Vhlh gene encodes VHL protein (pVHL, product of the Von Hippel-Lindau gene) that functions as the substrate recognition component of an E3 ubiquitin ligase. Although HIFα subunits are the best characterized substrates for pVHL, additional non-HIF mediated targets have been identified. To determine the role of HIF stabilization in this RPGN model, we generated double mutants that lack aryl hydrocarbon receptor nuclear translocator gene (Arnt, also called HIF1beta), an obligate dimerization partner for HIFα subunit function. Podocyte-selective deletion of Arnt in Vhlh mutant mice completely rescued the RPGN phenotype and mice survived longer than 8 months of age. Furthermore, stabilization of HIF2α alone led to glomerular disease characterized by crescentic transformation. Collectively, these results indicate an alternative mechanism for the pathogenesis of RPGN and glomerular disease in an animal model and suggest novel molecular pathways for intervention in this disease. In addition, we demonstrate a key role for VHL-HIF-Cxcr4 molecular pathway for the integrity of the glomerular barrier.

The von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is transmitted in an autosomal dominant manner. The disease is characterized by the formation of highly angiogenic tumors in many organs but the main causes of mortality are renal cell carcinomas and hemangioblastomas. Mutations in the VHL protein are responsible for the pathogenesis of the disease. VHL associates with elongin Band C to form the VBC complex. The cullin 2 protein (CUL2) and ring box protein 1 (RBX1) also associate with the VBC complex to form an E3 ubiquitin ligase involved in the ubiquitination and subsequent degradation of the hypoxia inducible transcription factor (HIF2alpha). Mutations in VHL that abrogate its E3 ligase activity lead to increased levels ofHIF2alpha and the subsequent accumulation of pro-proliferative and pro-angiogenic HIF2alpha target genes. VHL also has an important function in the regulation of extracellular matrix (ECM) assembly which is independent of its HIF2alpha regulation pathway. VHL's regulation of ECM assembly was shown to have important consequences for tumor angiogenesis and cell invasion. It was shown to be necessary for the proper assembly of a fibronectin matrix and was most recently found to interact with collagen IV alpha 2 (COL4A2). The aim of this thesis is to further characterize the VHL-COL4A2 interaction. VHL was shown to interact directly and specifically to COL4A2 and is necessary for proper COL4A2 matrix assembly. The association of VHL with COL4A2 appears to be independent of its functions as an E3 ubiquitn ligase and CUL2 was identified as part of the VBC complex that associates with collagen IV (COL4). Furthermore, a strategy to identify the binding site of VHL on COL4A2 has been employed and is in progress. These experiments represent the beginning of investigations into the novel interaction between VHL and COL4A2.

Rapidly progressive glomerulonephritis (RPGN) is a clinical syndrome characterized by loss of renal function within days to weeks and by glomerular crescents on biopsy. The pathogenesis of this disease is unclear, but circulating factors such as antineutrophil cytoplasmic antibodies (ANCA) are believed to play a major role. In this thesis, we show that deletion of the Von Hippel-Lindau gene (Vhlh) from intrinsic glomerular cells of mice is sufficient to initiate a necrotizing crescentic glomerulonephritis and the clinical features that accompany RPGN. Loss of Vhlh leads to stabilization of hypoxia-inducible factor alpha subunits (HIFαs). Using gene expression profiling, we identified de novo expression of the HIFα target gene Cxcr4. In glomeruli from mice with RPGN, the course of RPGN is markedly improved in mice treated with a blocking antibody to Cxcr4, whereas overexpression of Cxcr4 alone in podocytes of transgenic mice is sufficient to cause glomerular disease. Despite the development of glomerular disease in mice that overexpress Cxcr4, their disease was milder and lacked features of full-blown RPGN. The Vhlh gene encodes VHL protein (pVHL, product of the Von Hippel-Lindau gene) that functions as the substrate recognition component of an E3 ubiquitin ligase. Although HIFα subunits are the best characterized substrates for pVHL, additional non-HIF mediated targets have been identified. To determine the role of HIF stabilization in this RPGN model, we generated double mutants that lack aryl hydrocarbon receptor nuclear translocator gene (Arnt, also called HIF1beta), an obligate dimerization partner for HIFα subunit function. Podocyte-selective deletion of Arnt in Vhlh mutant mice completely rescued the RPGN phenotype and mice survived longer than 8 months of age. Furthermore, stabilization of HIF2α alone led to glomerular disease characterized by crescentic transformation. Collectively, these results indicate an alternative mechanism for the pathogenesis of RPGN and glomerular disease in an animal model and suggest novel molecular pathways for intervention in this disease. In addition, we demonstrate a key role for VHL-HIF-Cxcr4 molecular pathway for the integrity of the glomerular barrier.

No description available.

Le syndrome von Hippel Lindau (VHL) prédispose au développement de multiples tumeurs hautement vascularisées, telles que des hémangioblastomes rétiniens ou du système nerveux central, des phéochromocytomes et des carcinomes rénaux à cellules claires (CCRCC). Les patients atteints de ce syndrome sont porteurs d’une mutation du gène VHL. Ce gène, composé de trois exons, est transcrit en deux ARN messagers par épissage alternatif de l’exon 2. L’ARNm composé des 3 exons (variant #1) est la forme majoritairement exprimée par rapport à l’ARNm dépourvu de l’exon 2 (variant #2). Toutefois, une diminution du ratio variant #1/variant #2 a été essentiellement décrite dans deux situations : (i) dans les tissus embryonnaires humains et en particulier le rein, et (ii) dans certains CCRCC. Ces données suggèrent un rôle potentiel de ce variant #2 dans la tumorigenèse rénale. Deux protéines, pVHL213 et pVHL160, sont produites à partir du variant #1 et elles agissent comme suppresseurs de tumeur. Au début de ce travail, l’expression de l’isoforme protéique pVHL172 produite à partir du variant #2 restait à démontrer et sa fonction était inconnue. Les travaux effectués au cours de cette thèse ont permis de mettre en évidence l’expression de pVHL172 dans des lignées cellulaires et dans des tissus tumoraux grâce à un nouvel anticorps monoclonal de souris dirigé contre les trois isoformes protéiques humaines de pVHL. Pour savoir si l’isoforme pVHL172 a un rôle de suppresseur de tumeur, des lignées cellulaires tumorales rénales exprimant stablement cette protéine ont été établies puis des expériences de xénogreffes de ces cellules chez la souris ont été réalisées. Non seulement pVHL172 n’inhibe pas la formation de tumeurs mais son expression induit un phénotype tumoral plus agressif avec une composante sarcomatoïde plus importante ainsi qu’une vascularisation immature plus conséquente que dans les tumeurs contrôles (n’exprimant pas pVHL). De plus, pVHL172 augmente l’expression des métalloprotéases de matrice MMP1 et MMP13, en partie via l’activation de la voie de signalisation Smad-dépendante du TGF-β. Par ailleurs, des partenaires protéiques de cette protéine ont été recherchés par une analyse protéomique différentielle. Les réseaux d’interaction réalisés à partir des protéines identifiées concernent entre autres la régulation de la matrice extracellulaire et le contrôle qualité des protéines. En conclusion, ce travail a montré que le gène VHL produit des isoformes protéiques avec des fonctions distinctes voire antagonistes, ce qui implique que la balance de leur expression influencerait la progression tumorale rénale. Chez certains patients, une augmentation de l’expression de pVHL172 pourrait être corrélée à une pathologie plus sévère. Ce travail montre l’intérêt de poursuivre l’étude des fonctions de cette protéine pour une meilleure compréhension de son implication dans le cancer du rein et dans la maladie VHL afin d’envisager de nouvelles approches thérapeutiques. / VHL disease predisposes to the development of multiple and highly vascularized tumors, including central nervous system and retinal haemangioblastomas, phaeochromocytomas and clear cell renal cell carcinomas (ccRCCs). Patients with VHL disease harbor a mutant allele of the VHL gene. This gene is transcribed into two mRNAs by alternative splicing of the exon 2. The mRNA variant #1 composed of 3 exons usually predominates over the mRNA variant #2 lacking exon 2. A decrease of the variant #1/variant #2 ratio was however described in 2 situations: (i) in embryonic tissues, particularly in the kidney, and (ii) in some ccRCCs. These data suggest a potential role for the variant #2 in kidney tumorigenesis. pVHL213 and pVHL160 are the two proteins encoded by the mRNA variant #1 and act as tumor suppressors. At the beginning of this Ph.D. project, the expression of pVHL172 isoform encoded by the mRNA variant #2 remained to be established and its function was unknown. The experiments performed during this Ph.D. shed light on pVHL172 expression in cell lines and in tumor tissues using a newly produced mouse monoclonal antibody recognizing the three human pVHL isoforms. To examine if pVHL172 had a tumor suppressor function, human kidney tumor cell lines stably expressing this isoform were established, characterized and then grafted in mice. pVHL172 not only inhibits tumor formation, but its expression also induces a more aggressive phenotype with a higher sarcomatoid component and a more immature vasculature compared to control tumors (that do not express any pVHL). Moreover, pVHL172 increases the matrix metalloproteases MMP1 and MMP13 expression, partly by the activation of the Smad-dependent TGF-β signalling pathway. Besides, we looked for protein partners of pVHL172 by a differential proteomic analysis and showed that interaction networks obtained with the identified proteins are related to extracellular matrix regulation and protein quality control. To conclude, this work demonstrated that the VHL gene encodes protein isoforms with distinct and even antagonistic functions. The balance of expression of these isoforms is likely to influence kidney tumor progression. For some patients, an increase of pVHL172 expression could be correlated with a more severe pathology. This work shows the importance of further studying this isoform’s functions to better understand its involvement in kidney cancer and in VHL disease, so that new therapeutic approaches could be developed.