Marking proteins for degradation by the proteasome is a classical function of ubiquitination. This process of covalent attachment of a chain of ubiquitin molecules to target proteins is governed by the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and the ubiquitin ligase (E3). The von Hippel-Lindau (VHL) tumour suppressor protein forms an E3 ubiquitin ligase, ECV (Elongins BC/Cul2/VHL), which targets the alpha subunit of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction under normal oxygen tension. Tumour hypoxia promotes accumulation of HIFalpha, whose expression is associated with cancer progression, poor prognosis and resistance to conventional therapies, thus establishing HIF as a therapeutic target. Notably, VHL is functionally inactivated in VHL disease, a hereditary cancer syndrome characterized by the formation of tumours in multiple organs, as well as in the majority of sporadic clear-cell renal cell carcinomas (CCRCC) and haemangioblastomas. Recently, certain VHL mutations have been shown to cause the congenital disorder Chuvash polycythemia. Work contained in this thesis describes the temporally coordinated activation of the ECV, whereby oxygen-dependent recognition of HIFalpha by VHL triggers Cul2 modification by the ubiquitin-like molecule NEDD8, which enhances ECV ubiquitin ligase activity by recruiting the E2. In addition, the feasibility of ‘bio-tailored’ enzymes in the treatment of cancer is introduced by creating a bioengineered VHL capable of targeting HIFalpha for degradation irrespective of oxygen tension, which leads to the dramatic inhibition of CCRCC tumour growth and angiogenesis in a xenograft model. Furthermore, a ubiquitin ligase composed of two F-box proteins, VHL and suppressor of cytokine signalling 1 (SOCS1), was identified and shown to be paramount for the negative regulation of erythropoiesis by targeting phosphorylated Janus kinase 2 (JAK2) for ubiquitin-mediated destruction. The malfunction of this ubiquitin ligase explains the excessive erythrocytosis observed in Chuvash polycythemia patients and reveals a novel genetic link between the seemingly distinct genes VHL and JAK2 in the development of polycythemia.

Marking proteins for degradation by the proteasome is a classical function of ubiquitination. This process of covalent attachment of a chain of ubiquitin molecules to target proteins is governed by the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and the ubiquitin ligase (E3). The von Hippel-Lindau (VHL) tumour suppressor protein forms an E3 ubiquitin ligase, ECV (Elongins BC/Cul2/VHL), which targets the alpha subunit of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction under normal oxygen tension. Tumour hypoxia promotes accumulation of HIFalpha, whose expression is associated with cancer progression, poor prognosis and resistance to conventional therapies, thus establishing HIF as a therapeutic target. Notably, VHL is functionally inactivated in VHL disease, a hereditary cancer syndrome characterized by the formation of tumours in multiple organs, as well as in the majority of sporadic clear-cell renal cell carcinomas (CCRCC) and haemangioblastomas. Recently, certain VHL mutations have been shown to cause the congenital disorder Chuvash polycythemia. Work contained in this thesis describes the temporally coordinated activation of the ECV, whereby oxygen-dependent recognition of HIFalpha by VHL triggers Cul2 modification by the ubiquitin-like molecule NEDD8, which enhances ECV ubiquitin ligase activity by recruiting the E2. In addition, the feasibility of ‘bio-tailored’ enzymes in the treatment of cancer is introduced by creating a bioengineered VHL capable of targeting HIFalpha for degradation irrespective of oxygen tension, which leads to the dramatic inhibition of CCRCC tumour growth and angiogenesis in a xenograft model. Furthermore, a ubiquitin ligase composed of two F-box proteins, VHL and suppressor of cytokine signalling 1 (SOCS1), was identified and shown to be paramount for the negative regulation of erythropoiesis by targeting phosphorylated Janus kinase 2 (JAK2) for ubiquitin-mediated destruction. The malfunction of this ubiquitin ligase explains the excessive erythrocytosis observed in Chuvash polycythemia patients and reveals a novel genetic link between the seemingly distinct genes VHL and JAK2 in the development of polycythemia.

Cancer is responsible for one fourth of the total deaths and is the second leading cause of death, behind heart disease, in the United States. However, there are as many approaches to curing cancer as there are types of cancer. One important issue in solid tumors is hypoxia, a lack of oxygen, which promotes angiogenesis and anaerobic metabolism, which can increase cancer progression and metastasis. The HIF transcription factor is responsible for the mediation of many processes involved during hypoxia and is linked to poor patient prognosis, increased cancer progression, and invasiveness of tumors. With this in mind, the HIF pathway has become an attractive target for small molecule inhibition. Herein, we describe the design and synthesis of small molecules that inhibit the HIF pathway. These compounds are based off an initial “hit” compound, KCN-1, from screening of a 10,000 compound library. KCN1 is both highly effective and has a low toxicity profile. Over 200 compounds have been synthesized by the Wang lab, with the best compound IVSR64b having an IC50 of 0.28 μM. Of special interest is that these compounds do not appear to have any inherent toxicity toward healthy tissues, but only affect cancer cells. Moreover, x-ray crystal structures for both KCN-1 and IVSR64b were obtained and used as the basis for computational modeling, which is still in progress.

La production de cytokines et chimiokines par les polymorphonucléaires (PMN) est une fonction importante dans la réponse inflammatoire. La phagocytose et la migration, ainsi que d’autres fonctions des PMN changent en milieu hypoxique. II est bien connu que la régulation par l’hypoxie dépend principalement de l'activation du facteur de transcription HIF, cependant, l’effet de l’hypoxie sur la production des cytokines n’est pas encore établi. Notre hypothèse est que l’hypoxie change le profil de production des cytokines et chimiokines dans les neutrophiles humains en réponse aux agonistes en impliquant HIF. Dans ce travail, nous avons tout d’abord démontré que les PMN expriment constitutivement HIF-2? et HIF-3?. De plus, les agonistes G-CSF, GM-CSF, TNF? ou LPS augmentent l’expression de HIF-1? en hypoxie. D’autre part, nous avons démontré que l’hypoxie seule induit la sécrétion de TNF? et MIP-3a et modifie les niveaux des MIP-1?/1?, IL-8 et MIP-3? produites en réponse au GM-CSF, LPS et PGN. Ceci suggère que l'hypoxie oriente la production des cytokines dans les PMN de façon dépendante du stimulus et témoigne d’une mobilisation des voies de signalisation et des facteurs de transcription différente de celle connue en normoxie. Par la suite, nous avons étudié les mécanismes qui pourraient être à l’origine de ces modifications tels que la voie des MAPK p42/p44, STAT3, ERK, JNK et C/EBP-?. D’autre part, nous avons montré que la production inédite d’IL-8 par G-CSF en hypoxie dépend de STAT3 et p38 et que cette production met en jeu l'action autocrine des cytokines endogènes IL-18, IL-1ra et TNF?. De plus, l’utilisation d’une lignée cellulaire PLB-985 différenciée en PMN portant une mutation sur les sites consensus du NF-?B ou HIF, nous a permis de démontrer que non seulement l’hypoxie seule ou associé au G-CSF, GMCSF, TNF? ou au LPS régule l’activité de ce promoteur, mais le HIF régule aussi cette activité en normoxie. Finalement, les travaux présentés dans ce mémoire démontrent que l’hypoxie modifie l’expression et la production des cytokines par les neutrophiles humains de façon différente de la normoxie. Si le rôle crucial des neutrophiles dans l’inflammation physiologique et pathologique basé sur leur production des cytokines a été largement documenté en normoxie, il est primordial de réaliser des études pour approfondir ce rôle en considérant l’effet de l’hypoxie. [symboles non conformes]

No description available.

Loss of glucose-stimulated insulin secretion (GSIS) from the pancreatic beta-cells is one of the earliest detectable defects in the pathogenesis of type 2 diabetes. However, despite its relevance, the mechanisms that govern GSIS are still not completely understood. ARNT/HIF-1β is a member of the bHLH-PAS family of transcription factors, with a prominent role in the transcriptional regulation of enzymes required for the metabolism of xenobiotics as well as regulation of genes that are critical for cellular responses to hypoxia. Recent research has uncovered a previously unknown function for ARNT/HIF-1β in the pancreatic beta-cells, where the gene was found to be 90% down-regulated in human type 2 diabetic islets and loss of ARNT/HIF-1β protein leads to defective GSIS in pancreatic beta-cells of mice. The main focus of this thesis was to understand the mechanisms by which ARNT/HIF-1β maintains normal GSIS from pancreatic beta-cells and understand how loss of ARNT/HIF-1β leads to beta-cell dysfunction and type 2 diabetes in mice. ARNT/HIF-1β was found to positively regulate GSIS in both INS-1 derived 832/13 cell line and mice islets. In the 832/13 cells, loss of ARNT/HIF-1β leads to a reduction in glycolysis without affecting the glucose oxidation and the ATP/ADP ratio suggesting that the regulation of GSIS takes place in a manner that is independent of the KATP channels. In order to further assess the mechanism of lowered GSIS in the absence of ARNT/HIF-1β in the 832/13 cells, a metabolite profiling was performed which revealed a significant reduction in the metabolite levels of glycolysis and the TCA cycle intermediates and glucose-induced fatty acid production, suggesting the involvement of ARNT/HIF-1β in regulating glucose-stimulated anaplerosis, which is believed to play a key role in the regulation of GSIS from the pancreatic beta-cells. The changes in metabolite levels in the absence of ARNT/HIF-1β were associated with corresponding changes in the gene expression pattern of key enzymes regulating glycolysis, the TCA cycle and fatty acid synthesis in beta-cells. In an attempt to understand how loss of ARNT/HIF-1β leads to beta-cell dysfunction and type 2 diabetes in mice, a pancreatic beta-cell specific ARNT/HIF-1β knock out mouse (β-ARNT KO) was generated using the Cre-loxP technology. Functional characterization of islets from both male and female β-ARNT KO mice revealed a significant impairment in GSIS, which was attributed due to a small, but significant reduction in rise in intracellular calcium upon glucose stimulation. Further analysis revealed reduced secretory response to glucose in the presence of KCl and diazoxide indicating a defect in the amplifying pathway of GSIS in β-ARNT KO islets. Expression of pyruvate carboxylase (PC) was significantly reduced in β-ARNT KO islets suggesting possible impairments in anaplerosis and consistent with this, defect in GSIS in β-ARNT KO islets could be almost completely rescued by treatment with membrane permeable TCA intermediates. Surprisingly, both male and female β-ARNT KO mice have normal glucose homeostasis. In an attempt to assess how β-ARNT KO mice maintained normal blood glucose levels, indirect calorimetry was used to understand changes in whole-body energy expenditure. This investigation revealed that β-ARNT KO mice exhibited a small but significant increase in respiratory exchange ratio (RER), suggesting a preference in utilizing carbohydrates as a fuel source, possibly leading to improved glucose uptake from the blood stream. Response to exogenous insulin was completely normal in β-ARNT KO mice suggesting intact functioning of the skeletal muscles. To conclude, based on our in vitro data, we believe that ARNT/HIF-1β plays an indispensable role in maintaining normal beta-cell secretory function, however, results from β-ARNT KO mice indicates that these mice are protected from the adverse effects of hyperglycemia. Although loss of ARNT/HIF-1β alone is not sufficient for the genesis of type 2 diabetes, it creates a perfect storm in the pancreatic beta-cells that may eventually lead to an imbalance in the whole body glucose homeostasis. Our study provides significant information to the scientific community that engages in assessing the pharmacological potential of gene targets for the treatment of type 2 diabetes.

Cancer develops in many organs and tissues in the body through genetic and environmental modifications to acquire the hallmarks of cancer. The hallmarks of cancer allow the cells to become malignant and progress to a tumorigenic state. It has previously been shown in various carcinomas that HIF-2a, a key component in hypoxia adaptation, has a role in autonomous growth, the first hallmark of cancer. Ovarian cancer is the most lethal of the gynecological malignancies and accounts for 3% of new cases in women annually but is the fifth most common cause of death due to cancer. Here, it is shown in two ovarian carcinoma cell lines that HIF-2a is involved in in vitro and in vivo growth. It is also shown that the effect of HIF-2a is due to its role in autonomous growth and not vascularization with the use of in vitro spheroids. From recent findings in the laboratory the oxygen-stimulated translation initiation complex was discovered and HIF-2a is one of its components. In the absence of HIF-2a there is a downregulation in translation in hypoxia in ovarian carcinoma. This is also seen in a HIF-2a translational target, IGF1R and its downstream signaling pathway, which may be involved in autonomous growth as well as other hallmarks of cancer. Taken together, the data in this thesis presents the importance of HIF-2a in autonomous growth and cancer progression in ovarian carcinoma, as well as verifying its role in translation.

L’hypoxie est une caractéristique majeure des glioblastomes (GB). Elle est la cause principale de la résistance aux traitements observée dans ces tumeurs. Les conséquences de la baisse en oxygène au niveau tumoral, sont médiées par les facteurs de transcription Hypoxia Inducible Factors (HIF). Ces facteurs sont des protéines hétérodimériques HIF-α/HIF-1β responsables de la transcription de nombreux gènes cibles. Certaines de ces cibles participent à la progression tumorale et à la mise en place d’un phénotype agressif. L’une des cibles de l’isoforme HIF-2α, est la βIII-tubuline (βIII-t). Cette protéine, qui compose les microtubules, est décrite comme surexprimée dans les gliomes de haut grade, comme les GB. Ces travaux de thèse s’intéressent au rôle de la βIII-t dans la progression tumorale ainsi qu’au développement de stratégies permettant d’inhiber l’expression de HIF-2α. Les résultats obtenus montrent que la βIII-t a une importance centrale dans le développement tumoral. En effet, les tumeurs issues de l’implantation de cellules humaines de GB invalidées pour la βIII-t, se développent significativement moins vite comparées aux tumeurs contrôles. In vitro, nous avons montré que cette protéine est impliquée dans la prolifération, la migration et l’invasion cellulaires. En revanche, nous n’avons pas pu confirmer que la βIII-t est impliquée dans la résistance aux traitements (chimio- ou radiothérapeutiques). Les deux composés testés comme inhibiteurs de HIF-2α (SR2933 et PT2385) ont montré des résultats prometteurs sur la βIII-t, gène cible spécifique de HIF-2α. Cependant, malgré les stratégies développées, nous n’avons pas pu évaluer l’efficacité directe de ces deux composés sur l’hétérodimérisation de HIF-2α avec HIF-1β. / Hypoxia is a major feature of glioblastoma (GB). It is the main cause of the resistance to treatments observed in these tumors. The consequences of the decrease in oxygen at the tumor level, is mediated by the Hypoxia Inducible Factors (HIF). These transcription factors are heterodimeric proteins HIF-α/HIF-1β responsible for the transcription of many target genes. Some of these targets are responsible for setting up an aggressive phenotype and in tumor progression. One of the targets of the HIF-2α isoform is βIII-tubulin (βIII-t). This protein, which is a constituent of microtubules, is described as overexpressed in high grade gliomas, such as GB. In the present thesis, we examined the role of βIII-t in tumor progression and we developed strategies to inhibit the expression of HIF-2α. Our results show that βIII-t is of central importance in tumor development. Indeed, tumors resulting from the implantation of GB human cells invalidated for βIII-t, developed significantly less rapidly compared to control tumors. In vitro, we have shown that this protein is involved in cell proliferation, migration and invasion. However, we could not confirm that βIII-t is involved in resistance to treatments (chemotherapeutic or radiotherapeutic).The two compounds tested as inhibitors of HIF-2α (SR2933 and PT2385) showed promising results on βIII-t, a specific target gene of HIF-2α. However, despite the different tested strategies, we could not evaluate the direct inhibitory action of these two compounds on the heterodimerization of HIF-2α with HIF-1β.

Indiana University-Purdue University Indianapolis (IUPUI) / FGF23 is an osteocyte produced hormone necessary for maintaining systemic phosphate handling, and thus bone structure and function in both rare and common disorders such as chronic kidney disease (CKD). FGF23 is a critical factor in CKD, with elevated levels causing alterations in mineral metabolism and increased odds for mortality. However, the mechanisms directing the production of key modulators of skeletal homeostasis and biomineralization within osteocytes, and how this is altered in chronic kidney disease, remain unclear. The experimental focus of this dissertation was to dissect the molecular systems and role of oxygen sensing in the regulated production of FGF23. In CKD, up to 75% of patients have anemia and concomitant marked elevations in FGF23, increasing mortality odds. Anemia is a potent driver of FGF23 secretion, therefore, current and emerging therapies, including recombinant EPO and the hypoxia inducible factorprolyl hydroxylase inhibitors (HIF-PHI) FG-4592 and BAY 85-3934, were used to improve anemia in the adenine diet-induced mouse model of CKD. In the mice with CKD, iFGF23 was markedly elevated in control mice but was attenuated by 65-85% after delivery of EPO or HIF-PHI, with no changes in serum phosphate. This was associated with improved systemic iron utilization and reductions in mRNA markers of renal fibrosis. In osteocyte-like cell cultures treated with HIF-PHI, integrative RNAseq and ATACseq analysis identified candidate genes upregulated in response to mimicked hypoxia, concomitant with elevated Fgf23 expression. These genes were found to be downregulated in CKD bone, therefore, knock-out cells were generated using CRISPR/Cas9 technology. These cells were found to be functionally similar to in vivo conditional knockout models that have enhanced bone mass and elevated FGF23. Taken together, these results further define novel factors involved in the regulation of FGF23 and identify new therapeutic targets. / 2023-05-26

The control of oxygen delivery to cells in the body is the result of a small group of primary oxygen sensors, one of the most important of which is the hypoxia-inducible transcription factor-1 (HIF-1). Two alpha-ketoglutarate dependent non-heme iron dioxygenases are responsible for the regulation of HIF-1 through hydroxylation of residues on the HIF-1a subunit. One of these enzymes, known as the factor inhibiting HIF-1 (FIH-1) is responsible for hydroxylating residue Asn803 on HIF-1a, preventing the transcription of hypoxia related genes controlled by HIF-1. It was hypothesized that there would be a difference in inhibition of FIH-1 from the other HIF-1 regulating enzyme, the prolyl hydroxylase domain-2 (PHD2), when testing a series of ten small molecule inhibitors. The ten inhibitors chosen fell into three classes: pyrones, pyridines, and catechols. Of these inhibitors, it was found that catechols produced a significant inhibitory difference between PHD2 and FIH, and may provide useful in further inhibitor design and synthesis work.