Hepatocyte Growth Factor (HGF)–induced Epithelial–Mesenchymal Transition (EMT) is a complex cellular pathway that causes epithelial cell scattering by breaking cell–cell contacts, eliminating apical–basal polarity, and replacing epithelial markers and characteristics with mesenchymal markers. Early EMT events include a brief period of cell spreading, followed by cell compaction and cell–cell contact breaks. A forward chemical genetics drug screen of 50,000 unique compounds measuring HGF–induced cell scattering identified 26 novel EMT inhibitors, including 2 proteolytic inhibitors. Here, we show that B5500–4, one of the EMT inhibitors from the screen, blocks HGF–induced EMT by a predicted blocking of the protease furin, in addition to secondarily blocking Beta–Secretase (BACE).We also show that MMP–12 and MMP–9 are required for HGF–induced EMT to progress. MMP–12 is required for cell contraction, and its inhibition produces a continuous cell spreading phenotype.We also demonstrate that both furin and BACE activity are required for HGF–induced EMT to proceed, but that they are involved in separate pathways. We show that BACE inhibition leads to a failure of cell spreading in early EMT, and that EphA2 is a member of this pathway. We also demonstrate that it is likely BACE2, and not BACE1 that is responsible for early cell spreading. Furin is also required for HGF–induced cell scattering, but does not play a role in the cell spreading process. These findings highlight the importance of proteolytic activity at the earliest stages of HGF–induced EMT.

Iron is an essential nutrient for energy and DNA replication. Its homeostasis is commonly perturbed by chronic inflammatory mechanisms. Chronic inflammation upregulates a cytokine, hepcidin, that degrades the iron export protein ferroportin. Without a way to export iron into the bloodstream iron availability in blood becomes depleted. Iron depletion in the blood stream hinders erythropoiesis and is termed anemia. Herein I investigate and inhibit the mechanism of hepcidin activation. Inhibition of hepcidin activation has released iron from tissues and alleviated anemic conditions in a cancer model. I have laid the foundation to investigate this pathway in a 3D spheroid model. The results show that hepcidin-25 inhibition is a promising treatment for anemia of cancer. More work needs to be done to confirm efficacy in an in vivo model. In addition to anemia of cancer I have also worked with diabetic rats and investigated their anemic state using common anemia diagnostic methods. I found that in this high fat high sugar diet Wistar rat model anemia was not induced. In addition to my studies on anemia I have investigated the use of portable x-ray fluorescence (pXRF) as an accessible and affordable elemental analysis technique for lateral flow immunoassays and biological samples such as cell lysates and animal tissue. While pXRF shows promising results more work needs to be done to increase its sensitivity and pixel size.

Abstract Calcified aortic valve disease represents a spectrum of disease spanning from mild aortic valve sclerosis to severe aortic valve stenosis (AS), being an actively regulated disease process and showing some hallmarks of atherosclerosis. The calcified aortic valve lesion develops endothelial injury and is characterized by inflammation, lipid accumulation, renin-angiotensin system activation and fibrosis. There is no approved pharmacological treatment available in AS. This study was aimed to characterize gene expression of endothelial factors in aortic valves in patients representing different stages of calcified aortic valve disease to reveal new targets for pharmacological interventions in AS. Aortic valves obtained from 75 patients undergoing valve replacement surgery were studied. Expression of natriuretic peptides (ANP, BNP and CNP), their processing enzymes (corin and furin), natriuretic receptors (NPR-A, NPR-B and NPR-C), endothelin-1 (ET-1), endothelin converting enzyme-1 (ECE-1), endothelin receptors A and B (ETA and ETB), and apelin pathway (apelin and its receptor APJ) was characterized by reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. AS was characterized by distinct downregulation of gene expression of CNP, its processing enzyme furin and the target receptor NPR-B. Furthermore, increased amount of ET-1 and its target receptor ETA as well as imbalance between ETA and ETB receptors and downregulated endothelial nitric oxide synthase (eNOS) gene expression were observed. Finally, gene expression of apelin and APJ receptor were significantly upregulated in stenotic valves when compared to controls in combination with disequilibrium between expression of angiotensin II receptors AT1 and AT2. The study provides a better understanding of molecular mechanisms associated with calcific aortic valve disease and suggest potential targets for novel therapeutic interventions.

Diabetes mellitus is presently recognized as the sixth leading cause of death in the United States, affecting over 20 million people. Diabetes is a condition characterized by high blood glucose due to an insulin deficiency or resistance. Type I, which comprises 5-10% of all cases, results from the destruction of pancreatic beta cells in the islets of Langerhans. The current treatment for type I diabetes is insulin administration through injection or pump. Purification, production, and storage of this insulin proves to be quite costly. By producing biologically functional insulin with oral delivery capabilities through chloroplast genetic engineering, many of these costs could be cut back. In addition, the possibility of providing the C-peptide currently lacking in commercially available insulin becomes available. The Daniell lab inserted cholera toxin B-subunit (CTB) fused proinsulin, containing three furin cleavage sites, (CTB-pins Fx3) into the tobacco chloroplasts of Petit Havana via particle gun bombardment. The insertion of three furin cleavage sites along with fusion to CTB will enable the insulin to sustain function when orally administered. Transgenic plants from the second generation were then analyzed and tested for quantification of the CTB-pins Fx3 gene. Tobacco leaves of varying ages were compared to determine expression levels. The resulting data is pertinent for future production of both orally deliverable insulin and more cost effective injectable insulin.

Les fonctions endocriniennes des os sont médiées par au moins deux hormones, l’ostéocalcine et le facteur de croissance fibroblastique 23 « Fibroblast growth factor 23 » (FGF23), ces derniers sont secrétés par les cellules osseuses, les ostéoblastes et les ostéocytes. L’ostéocalcine est produite par les ostéoblastes et régule le métabolisme du glucose et énergétique. Elle améliore ainsi la tolérance au glucose et la sensibilité à l’insuline. Également, elle favorise la sécrétion d’insuline et la prolifération des cellules β, elle augmente la dépense énergétique et réduit l’accumulation de graisse. L'ostéocalcine est gamma-carboxylée au niveau de trois résidus d'acide glutamique (Glu), un processus qui inhibe sa fonction endocrinienne chez la souris et l'humain. Le pH acide de la lacune de résorption décarboxyle l'ostéocalcine et libère sa forme non carboxylée (ucOCN), la forme active de cette hormone. Nos connaissances sur la régulation des fonctions endocriniennes d’ostéocalcine sont encore limitées à sa gamma-carboxylation. Puisque cette hormone est secrétée par les ostéoblastes et les ostéocytes, des cellules endocriniennes non classique, nous avons émis l’hypothèse que l'ostéocalcine pourrait être soumise à d'autres modifications post-traductionnelles (PTMs) au niveau de la voie de sécrétion contrôlant ses fonctions endocriniennes. Dans la première partie de cette thèse, nous avons montré que le propeptide de l'ostéocalcine pouvait être clivé dans son extrémité C-terminale au niveau du motif de base « RLRR » par la pro-protéine convertase furine, un processus qui se produit indépendamment de la gamma-carboxylation de l'ostéocalcine. L’inactivation du gène codant pour la furine, spécifiquement dans les ostéoblastes et les ostéocytes chez la souris, abolit totalement le clivage de la pro-ostéocalcine et altère son activation et sa libération lors de la résorption osseuse. Par conséquent, ces souris sont caractérisées par un niveau bas d'ucOCN dans le sérum, ce qui entraîne une altération de la tolérance au glucose, une diminution de la sécrétion d'insuline et de la dépense énergétique ainsi qu’une augmentation de l'accumulation de graisses. De plus, ces souris ont une perte d'appétit indépendamment de l'ostéocalcine. La restriction de la nourriture pour les souris contrôles ou « pair feeding » rend le phénotype des souris déficientes en furine plus apparent. Il apparait à un plus jeune âge avec une résistance à l'insuline. Dans la deuxième partie de cette thèse, nous avons découvert que l'ostéocalcine de souris est O-glycosylée au niveau de la sérine 8, un processus qui se produit indépendamment de sa gamma-carboxylation et de son clivage. Cette modification, qui n'est pas présente chez l'ostéocalcine humaine, augmente la demi-vie de l'ostéocalcine de souris dans le plasma ex vivo et in vivo. Il est intéressant de noter que la tyrosine 12 dans l'ostéocalcine humaine correspond à la sérine 8 dans la séquence de la souris, tandis que la mutation Tyr12Ser est suffisante pour générer une ostéocalcine humaine O-glycosylée et lui conférer une demi-vie plus longue dans le plasma de la souris comparativement à la forme native. FGF23 est une hormone secrétée par les ostéoblastes et les ostéocytes. Elle régule la réabsorption de phosphate et la production de vitamine D dans le tubule proximal du rein. Sa fonction endocrine est inhibée par un clivage endoprotéolytique qui libère ses fragments N- et C-terminaux. La mutation du motif « RHTR », un site de clivage consensus pour les proprotéines convertases PC(s), a été identifié chez les patients atteints du rachitisme hypophosphatémique génétiquement déterminés ou « Autosomal dominant hypophosphatemic rickets » (ADHR). Ces patients se caractérisent par une augmentation du taux de FGF23 intact, une hypophosphatémie et une ostéomalacie. Malgré l’importance de FGF23 dans plusieurs maladies, l’identité de l’enzyme responsable du clivage de FGF23 n’est pas encore connue, même si la furine et la proprotéine convertase subtilisine/kexine type 5 (PC5) peuvent cliver FGF23 in vitro. Dans la troisième partie de cette thèse, nous tentons de répondre à cette question en utilisant des souris déficientes en furine et/ou PC5 spécifiquement dans les ostéoblastes et les ostéocytes. Sous des conditions physiologiques, l’inactivation du gène de furine dans les ostéoblastes et les ostéocytes augmente le niveau du FGF23 intact par 25%. Malgré cette augmentation ces souris maintiennent une phosphatémie normale et elles ne montrent pas de signe d’ostéomalacie. On a aussi montré qu’une déficience en fer, une condition qui augmente la production de FGF23 au niveau de l’ARN messager et protéique, le FGF23 est totalement en forme intact dans les souris déficientes en furine, montrant que le clivage de FGF23 est totalement inhibé dans cette condition. En revanche, l’injection d’érythropoïétine ou d’interleukine 1-β, des conditions qui augmentent la production de FGF23, induit une augmentation significative du taux de FGF23 total dans le sérum des souris déficientes en furine et/ou PC5 dans les ostéoblastes et les ostéocytes, tandis que le niveau du FGF23 intact n’a pas augmenté de la même façon, suggérant que la FGF23 est correctement clivée chez ces souris. D’une façon intéressante et malgré les défauts développementaux et le retard dans la minéralisation osseuse observée dans les souris complètement déficientes en PC5, la suppression conditionnelle de PC5 dans les ostéoblastes et les ostéocytes chez la souris n'a entraîné aucun défaut osseux. Cependant, l’inactivation du gène codant pour la furine dans les ostéoblastes et les ostéocytes chez la souris a augmenté les paramètres osseux trabéculaires et a diminué l'épaisseur de l’os cortical. De plus, ces souris ont eu une diminution de la densité minérale et la rigidité des os reflétant une mauvaise qualité osseuse. En résumé, nous avons décrit pour la première fois que la furine est un régulateur multifonctionnel de la fonction des ostéoblastes et des ostéocytes in vivo. Elle régule le métabolisme du glucose en assurant le clivage de la pro-ostéocalcine, qui est nécessaire à la maturation et à la bio-activité de l'ostéocalcine, et en régulant l'appétit indépendamment de l'ostéocalcine. Ces résultats suggèrent la présence d'ostéokines supplémentaires régulant l'appétit et contrôlées par la furine. De plus, dans les ostéoblastes, la furine régule partiellement le clivage de FGF23 en assurant une phosphatémie normale, suggérant que la régulation de l'accumulation de masse osseuse par la furine est indépendante du FGF23. En outre, nous avons découvert que l'ostéocalcine de souris est soumise à l’O-glycosylation, une modification qui n'est pas conservée chez l'humain, ni chez d’autres espèces, et qui augmente la demi-vie de l'ostéocalcine de souris. La glycosylation artificielle confère à l'ostéocalcine humaine une demi-vie plus longue, offrant ainsi une approche permettant d'augmenter potentiellement la bio-activité de l'ostéocalcine humaine dans les futures applications thérapeutiques de l'ostéocalcine dans les maladies humaines. / Bone endocrine functions are mediated by at least two hormones, osteocalcin and fibroblast growth factor 23 (FGF23) which are secreted by the bone cells, osteoblasts and osteocytes. Osteocalcin is an osteoblast-derived hormone regulating glucose and energy metabolism. It improves glucose tolerance and insulin sensitivity, promotes insulin secretion and β-cell proliferation, increases energy expenditure and reduces fat accumulation. Osteocalcin is gamma-carboxylated on three of its glutamic acid residues (Glu), a process that inhibits its endocrine function in mice and humans. It is the acidic pH in the resorption lacuna which decarboxylates osteocalcin releasing the uncarboxylated osteocalcin (ucOCN), the active form of this hormone. Our knowledge on osteocalcin regulation by post-translational modifications is limited to its gamma-carboxylation. Since osteocalcin is secreted by differentiated osteoblasts, a non-classical endocrine cell, we hypothesized that osteocalcin may be subjected to additional post translational modifications (PTMs) in the secretory pathway that regulates its endocrine functions. In the first part of the thesis we showed that osteocalcin’s putative pro-peptide is cleaved in its C-terminus at the basic motif «RLRR», by the proprotein convertase furin. This process occurs independently of osteocalcin gamma-carboxylation. Furin inactivation specifically in osteoblasts in mice totally abolishes osteocalcin processing and impairs its activation and release during bone resorption. Consequently, these mice have decreased serum level of ucOCN resulting in impaired glucose tolerance, reduced insulin secretion and energy expenditure, and increased fat accumulation. Moreover, these mice have a decrease in the appetite independently of osteocalcin. Pair feeding of control mice resulted in more apparent phenotype in furin deficient mice, as it appears at younger age alongside with insulin resistance. In the second part of this thesis, we discovered that mouse osteocalcin is O-glycosylated on serine 8, a process that occurs independently of its gamma-carboxylation and processing. This modification is not conserved in human or any other species and it increases mouse osteocalcin half-life in plasma ex vivo and in vivo. Interestingly, tyrosine 12 in human osteocalcin corresponds to the serine 8 in the mouse sequence. Tyr12Ser mutation was sufficient to O-glycosylate human osteocalcin and to confer this hormone a longer half-life in mouse plasma compared to the native form. FGF23 is a hormone secreted by osteoblasts and osteocytes which regulates phosphate reabsorption and vitamin D production in the kidney proximal tubule. Its endocrine function is inhibited by endoproteolytic cleavage which releases its N-terminal and C-terminal fragments. Mutations in the «RHTR» motif, a consensus cleavage site for proprotein convertases (PCs), were found in patients with autosomal dominant hypophosphatemic rickets (ADHR). These patients are characterized by an increased intact FGF23 levels, hypophosphatemia and osteomalacia. Despite the importance of FGF23 in the pathology of multiple diseases, the identity of the enzyme(s) involved in FGF23 cleavage is yet unclear, even though furin and the proprotein convertase subtilisin/kexin type 5 (PC5) were shown to cleave FGF23 in vitro. In the third part of the thesis, we addressed this question using mice model deficient in furin and/or PC5 in osteoblasts and osteocytes in mice. Under physiological conditions, furin inactivation resulted in a 25% increase in intact FGF23; however, these mice maintained normal phosphate level and did not shown any sign of osteomalacia. We also showed that under iron restriction, a condition that induce FGF23 expression at the mRNA and protein level, FGF23 processing is totally impaired in furin deficient mice. However, the injection of erythropoietin or interleukin 1-β, two conditions that increase FGF23 production, induce FGF23 serum level while it is still properly processed in mice deficient in furin and/or PC5 in osteoblasts and osteocytes. Interestingly, despite the patterning defects observed in global inactivation of PC5, conditional inactivation of PC5 in osteoblasts and osteocytes in mice did not result in any bone defect. However, furin inactivation in osteoblasts and osteocytes in mice increases trabecular bone parameters and decreases cortical thickness. Moreover, these mice have decreased bone mineral density and bone strength reflecting a poor bone quality. In summary, we described for the first time that furin is a pleotropic regulator of osteoblast and osteocyte function in vivo. It regulates glucose and energy metabolism by mediating pro-osteocalcin processing which is required for osteocalcin maturation and bioactivity, and by regulating appetite independently of osteocalcin. These findings suggest the presence of additional osteokines controlling appetite and which are regulated by furin. Moreover, furin partially regulates FGF23 processing while maintaining normal phosphate homeostasis, suggesting that the regulation of bone mass accrual by furin occurs independently of FGF23. Additionally, we discovered that mouse osteocalcin is subjected to O-glycosylation, a species-specific modification that is not conserved in humans or any other species and increases mouse osteocalcin half-life. Artificial O-glycosylation confer human osteocalcin a longer half-life, thus providing an approach to increase human osteocalcin bioactivity in future therapeutic applications of osteocalcin in human diseases.