In the U.K. deaths due to liver diseases, especially alcohol related diseases, have risen considerably over the last 20 years. In 2005 up to 13,000 people died from liver related diseases within the U.K., including alcohol and viral liver failure and liver cancers. Worldwide hepatitis B affects about 2 billion people, killing 500,000 to 1 million per year. An effective way to treat liver disease is often liver surgery, such as liver resection for cancers and liver transplant for failure. However, the failure of liver regeneration by hepatocyte proliferation after resection surgery leads to a high death rate, and a shortage of liver donors means most people with liver failure die without access to a transplant. Therefore, understanding hepatocyte proliferation is a key to improving survival after resection surgery and providing hepatocytes for cell therapy in place of organ donation. The mechanism of hepatocyte proliferation has been studied both in vivo and in culture by many groups. However, only limited proliferation and preservation of function of primary human and rat hepatocytes, not suitable for clinical use, has been achieved on stimulation with growth factors. This study focuses on the mechanism of epidermal growth factor (EGF) stimulation of rat hepatocyte cell cycle progression and proliferation, including the role of PI3K/Akt/mTOR and MEK/ERK signalling pathways, EGF receptor location after activation of downstream proteins such as protein kinase B (Akt) and extracellular signal-regulated kinases 1/2 (ERK1/2), and their effect on the cell cycle. Included in this study are some comparisons between the stimulation of the EGF receptor (a tyrosine kinase receptor) and the P2Y receptor (a G protein coupled receptor). The PI3K/Akt/mTOR signalling pathway appears to be necessary for the hepatocyte response to EGF, inducing progression to S phase and DNA synthesis, while the MEK/ERK pathway is important but not necessary. The P2Y2 agonist UTP, which also stimulates these two pathways, does not result in the cell entering S phase. This suggests that the activation of these two signalling pathways is not sufficient for cell cycle progression. Furthermore, infection of cells with adenovirus to express constitutively active Akt increases hepatocytes proliferation and induces cell cycle progression, which generates a window to obtain hepatocytes proliferation in culture. It has been shown in this thesis that EGF stimulation of ERK phosphorylation continues from endosomes, while the evidence suggests that UTP stimulation is restricted to signalling at the cell surface. Furthermore, endocytic EGF/EGFR alone (without stimulation from the cell surface) is sufficient to induce cell cycle progression. This endosomal signalling with EGF but not UTP may explain the absence of cell cycle progression following UTP. EGF stimulates the appearance of phospho-EGFR in the nucleus. Furthermore, nuclear EGFR has a different apparent molecule weight than the cytoplasmic receptor; this may be due to nuclear EGFR having fewer and/or different phosphates. In vivo work by others has shown that in liver regeneration following partial hepatectomy (PH) EGF and full-length activated-EGFR were showed to be present in proliferating hepatocytes. This thesis describes the mechanism of growth factor (EGF) stimulation of primary rat hepatocyte proliferation. It shows for the first time that endosomal EGF/EGFR alone is sufficient to stimulate cell cycle progression, and that EGF induces phospho-EGFR in the nucleus in cultured rat hepatocytes. This thesis also provides the possibility to obtain cultured hepatocytes proliferation including over-expression of constitutively active form of Akt and translocation to the nucleus of full-length EGFR in the phosphorylated form. These studies improve our understanding of growth factor (e.g. EGF) stimulation of hepatocyte proliferation in vitro and help to move closer to the goal of obtaining sufficient functional hepatocytes in culture for clinical use, and of drugs that will stimulate hepatocyte proliferation following resection surgery.

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Signalling through the EGF receptor is subject to a complex and multilayered regulation. One such mode of regulation is through control of ligand production which plays an important role in fine- tuning EGF receptor activation. In mammals, the production of soluble, biologically active forms of EGF receptor ligands relies on ADAM metalloproteases, predominantly ADAM10 and ADAM17. Recently, a pseudoprotease from the rhomboid-like family of intramembrane proteases, iRhom, emerged as a key positive regulator of ADAM17. However, Drosophila iRhom has also been implicated in the negative regulation of EGF receptor signalling by promoting the degradation of precursors of its ligands. Cell culture based assays suggest that mammalian iRhoms might also be involved in a similar process. In this thesis, the effect of mammalian iRhom overexpression on the levels of EGF receptor ligands has been investigated. Contrary to previous findings, the data presented in this thesis suggest that the observed effect might not be entirely iRhom specific, for the inactive mutants of rhomboid proteases also diminish the levels of EGF receptor ligands. Nor do we find the effect to be specific to EGF receptor ligands, as unrelated transmembrane proteins were also depleted by iRhom overexpression. The coexpression of ADAM17 was...

Signalling through the EGF receptor is subject to a complex and multilayered regulation. One such mode of regulation is through control of ligand production which plays an important role in fine- tuning EGF receptor activation. In mammals, the production of soluble, biologically active forms of EGF receptor ligands relies on ADAM metalloproteases, predominantly ADAM10 and ADAM17. Recently, a pseudoprotease from the rhomboid-like family of intramembrane proteases, iRhom, emerged as a key positive regulator of ADAM17. However, Drosophila iRhom has also been implicated in the negative regulation of EGF receptor signalling by promoting the degradation of precursors of its ligands. Cell culture based assays suggest that mammalian iRhoms might also be involved in a similar process. In this thesis, the effect of mammalian iRhom overexpression on the levels of EGF receptor ligands has been investigated. Contrary to previous findings, the data presented in this thesis suggest that the observed effect might not be entirely iRhom specific, for the inactive mutants of rhomboid proteases also diminish the levels of EGF receptor ligands. Nor do we find the effect to be specific to EGF receptor ligands, as unrelated transmembrane proteins were also depleted by iRhom overexpression. The coexpression of ADAM17 was...

The Rab family of proteins are low molecular weight GTPases that have the ability to switch between GTP- (active) and GDP- (inactive) bound form, and in that sense act as molecular switches. Through distinct localization on various vesicles and organelles and by cycling through GTP/GDP bound forms, Rabs are able to recruit and activate numerous effector proteins, both spatially and temporally, and hence behave as key regulators of trafficking in both endocytic and biosynhtetic pathways. The Rab5 protein has been shown to regulate transport from plasma membrane to the early endosome as well as activate signaling pathways from the early endosome. This dissertation focused on understanding Rab5 activation via endocytosis of receptor tyrosine kinases (RTKs). First, tyrosine kinase activity of RTKs was linked to endosome fusion by demonstrating that tyrosine kinase inhibitors block endosome fusion and activation of Rab5, and a constitutively active form of Rab5 is able to rescue endosome fusion. However, depending on how much ligand is available at the cell surface, the receptor-ligand complexes can be internalized via a number of distinct pathways. Similarly, Rab5 was activated in a ligand-dependent concentration dependent manner via clathrin- and caveolin-mediated pathways, as well as a pathway independent of both. However, overexpression Rabex-5, a nucleotide exchange factor for Rab5, is able to rescue activation even when all of the pathways of EGF-receptor internalization were blocked. Next, the three naturally occurring splice variants of Rabex-5 selectively activated Rab5. Lastly, Rabex-5 inhibits differentiation of 3T3-L1 and PC12 cells through 1) degradation of signaling endosome via Rab5-dependent fusion with the early endosome, 2) and inhibition of signaling cascade via ubiquitination of Ras through the ZnF domain at the N-terminus of Rabex-5. In conclusion, these data shed light on complexity of the endosomal trafficking system where tyrosine kinase activity of the receptor is able to affect endosome fusion; how different endocytic pathways affect activation of one of the key regulators of early endocytic events; and how selective activation of Rab5 via Rabex-5 can control adipogenesis and neurogenesis.

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Receptor-tyrosine kinases (RTKs) are membrane bound receptors characterized by their intrinsic kinase activity. RTK activities play an essential role in several human diseases, including cancer, diabetes and neurodegenerative diseases. RTK activities have been regulated by the expression or silencing of several genes as well as by the utilization of small molecules. Ras Interference 1 (Rin1) is a multifunctional protein that becomes associated with activated RTKs upon ligand stimulation. Rin1 plays a key role in receptor internalization and in signal transduction via activation of Rab5 and association with active form of Ras. This study has two main objectives: (1) It determines the role of Rin1 in the regulation of several RTKs focusing on insulin receptor. This was accomplished by studying the Rin1-insulin receptor interaction using a variety of biochemical and morphological assays. This study shows a novel interaction between the insulin receptor and Rin1 through the Vps9 domain. Two more RTKs (epidermal growth factor receptor and nerve growth factor receptor) also interacted with the SH2 domain of Rin1. The effect of the Rin1-RTK interaction on the activation of both Rab5 and Ras was also studied during receptor internalization and intracellular signaling. Finally, the role of Rin1 was examined in two differentiation processes (adipogenesis and neurogenesis). Rin1 showed a strong inhibitory effect on 3T3-L1 preadipocyte differentiation but it seems to show a modest effect in PC12 neurite outgrowth. These data indicate a selective function and specific interaction of Rin1 toward RTKs. (2) It examines the role of the small molecule Dehydroleucodine (DhL) on several key signaling molecules during adipogenesis. This was accomplished by studying the differentiation of 3T3-L1 preadipocytes exposed to different concentrations of DhL in different days of the adipocyte formation process. The results indicate that DhL selectively blocked adipocyte formation, as well as the expression of PPARγ, and C/EBPα. However, DhL treatment did not affect Rin1 or Rab5 expression and their activities. Taken together, the data indicate a potential molecular mechanism by which proteins or small molecules regulate selective and specific RTK intracellular membrane trafficking and signaling during cell growth and differentiation in normal and pathological conditions.

No description available.

Óxido nítrico (NO) obtido a partir de fonts exógenas estimula a via de sinalização Ras/MAP cinases ERK1/2 em células endoteliais de coelho (RAEC). A ativação desta via também envolve a transativação do receptor de EGF (EGFR) mediada por ERK1/2 (Oliveira, 2003). Agora, nós avaliamos os efeitos de NO gerado endogenamente por bradicinina e \"shear stress\" sobre a fosforilação de resíduos de tirosina em EGFR e no processo de angiogênese; Nós encontramos que após estímulo com bradicinina (1 M) ou \"shear stress\" (16 dynes/cm2) a isoforma endotelial de NO sintetase (eNOS) foi ativada em células RAEC e HUVEC. Além disso, o aumento na produção de NO correlaciona-se com um aumento na fosforilação em resíduos de tirosina de EGFR conforme verificado por imunoprecipitação e western blot. Para determinar a capacidade angiogênica da via de sinalização NO-EGFR, nós usamos um ensaio in vitro baseado em Matrigel®. Em adição nós analisamos as vias de sinalização envolvidas no processo. Nós mostramos que bradicina e \"shear stress\" induz a formação de estruturas semelhantes a capilares em células HUVEC cultivadas em Matrigel®. Células HUVEC expressando um mutante de EGFR com atividade de tirosina cinase defective não forma estruturas semelhantes a capilares após estímulo com bradicinina ou \"shear stress\". Reunidos, estes achados nos sugerem que a ativação da via de NO e EGFR é necessária na promoção de angiogênese em células HUVEC / Nitric oxide (NO) obtained from exogenous sources stimulated the Ras/MAP kinases ERK1/2 signaling pathway in rabbit endothelial cells (RAEC). Activation of this pathway also involved the transactivation of the EGF receptor (EGF-R) mediated by ERK1/2 (FRBM 35:381; 2003). Now, we evaluate the effects of endogenously generated NO elicited by bradykinin and fluid laminar \"shear stress\" on tyrosine phosphorylation of the EGF receptor and in the process of angiogenesis. We found that upon stimulation with bradykinin (1 M) or under shear stress conditions (16 dynes/cm2) the endothelial isoform of NO synthase was activated in RAEC and in human endothelial cells (HUVEC). Furthermore, increase in NO production correlated with enhanced phosphorylation of tyrosine residues of the EGF-R as seen by immunoprecipitation and western blot analysis. To determine the importance of the NO-EGFR signaling pathway in angiogenesis, we used the Matrigel®-based in vitro assay for angiogenesis. In addition, we analyzed the signaling pathway involved in the process. We showed that bradykinin and shear stress induced the formation of capillary-like structures in HUVEC cultures grown in Matrigel®. HUVEC expressing a mutant of the EGF-R lacking tyrosine kinase activity did not form capillary-like structures upon stimulation with bradykinin or shear stress conditions. Taken together, these findings suggest that the activation of the NO-EGFR signaling pathway is necessary to promote angiogenesis in HUVEC

Le récepteur à l’EGF (Epidermal Growth Factor) est exprimé, entre autres, par les cellules inflammatoires et vasculaires. Il est impliqué dans la survie et la migration cellulaire. L’EGF-R et ses ligand sont exprimés dans les plaques d’athérosclérose. L’objectif de cette étude est d’évaluer les effets de l’inhibition de l’EGF-R sur les fonctions des lymphocytes T CD4+ et sur les macrophages au cours du développement de l’athérosclérose expérimentale. L’EGFR est exprimé par les lymphocytes T CD4+, ainsi que par les macrophages au sein des plaques d’athérosclérose murines. L’inhibition pharmacologique de l’EGFR (Erlotinib) chez des souris Ldlr-/- sous régime riche en matières grasses réduit le développement et la progression des lésions athéromateuses. Afin d’étudier le rôle spécifique de l’EGFR dans les lymphocytes T CD4+, nous avons généré des souris Cd4Cre Egfrlox/lox. Des souris Ldlr-/- ont été irradiées et retransplantées avec une moelle Cd4Cre Egf-r+/+ ou Cd4Cre Egf-rlox/lox puis mise sous régime riche en matières grasses. L’invalidation spécifique de l’EGFR dans les lymphocytes T CD4+ induit une diminution de la prolifération lymphocytaire T CD4+ in vitro et in vivo, une diminution de la production d’IFN-γ, d’IL-4 et d’IL-2. La transplantation de la moelle Cd4Cre Egf-rlox/lox induit une réduction de la taille des lésions d’athérosclérose sans différence concernant la cholestérolémie, et une diminution de l’infiltration lymphocytaire T dans les plaques. Nous avons ensuite généré des souris LysMCre Egf-rlox/lox pour étudier le rôle spécifique de l’EGF-R exprimé par les cellules myéloides. Des souris Ldlr-/- ont été irradiées et retransplantées avec une moelle LysMCre-Egf-rlox/lox ou LysMCre+Egfrlox/lox. La transplantation de la moelle LysMCre+Egf-rlox/lox induit une réduction de la taille des lésions après 4, 7 et 12 semaines de régime riche en matière grasses. Les plaques des souris chimères Ldlr-/-/LysMCre+Egf rlox/lox sont caractérisées par une diminution significative de l’infiltration macrophagique ainsi qu’une diminution de la taille du noyau nécrotique. L’invalidation génétique de l’EGFR dans la lignée myéloide réduit significativement la production du TNF-α et d’IL-6. Par ailleurs, l’inhibition pharmacologique et l’invalidation génétique d’EGFR réduit la formation des cellules spumeuses par une « down-régulation » du CD36. L’inhibition pharmacologique l’EGF-R diminue l’activité pro-inflammatoire pro-athérogène des lymphocytes T CD4+ et des macrophages, et in fine réduit le développement et la progression de l’athérosclérose expérimentale. Nos résultats suggèrent que l’inhibition de l’EGFR pourrait être une nouvelle stratégie thérapeutique pour le traitement de l’athérosclérose. / Background: Several Epidermal Growth Factor receptor (EGF-R) inhibitors have been successfully developed for the treatment of cancer, inhibiting tumor cell survival, proliferation and migration. EGF-R is expressed by leucocytes, but little is known about its role in the modulation of the immune response. The first part of the projet is to determine whether EGFR expressed on myeloid cells is functional, and to address the consequences of EGFR inhibition specifically in myeloid cells on atherosclerosis. The second part is to explore the expression of EGF-R on CD4+ T cells, and to study the effects of the specific EGF-R invalidation on CD4+ T cells during atherosclerosis development. Methods and results: Ldlr-/- mice were orally treated with a specific EGFR inhibitor (Erlotinib, 15mg/kg) for 6 weeks, under a high fat diet. EGFR pharmacological inhibition reduced T cell infiltration, decreases macrophage accumulation within atherosclerotic lesions, and thus, protected against atherosclerosis development in the aortic sinus. In parallel, we generated chimeric Ldlr-/- mice. Ldlr-/- mice were lethally irradiated and reconstituted with LysMCre+ EgfrLox/lox or LysM Cre- EgfrLox/lox bone marrow cells. In addition, irradiated Ldlr-/- mice were also reconstituted with bone marrow from Cd4Cre Egfrlox/lox , or Cd4Cre Egfr+/+ and put under a high fat diet. Animal weight and cholesterolemia were not different between groups. We observed a decrease of atherosclerosis plaque size in the aortic sinus in chimeric Ldlr-/-/LysMCre+ EgfrLox/lox and Ldlr-/-Cd4Cre Egfrlox/lox mice in comparison with chimeric Ldlr-/-/LysMCre- EgfrLox/lox, and Ldlr-/-Cd4Cre Egfr+/+ respectively. Myeloid invalidation of EGFR and pharmacological inhibition using AG-1478, a specific tyrosine kinase inhibitor, affected cytoskeleton reorganization limiting macrophage adhesion, spreading and migration. EGF-R blockage significantly reduced lipid uptake and foam cell formation through the down-regulation of CD36 expression. Selective deletion of Egfr in CD4+ T cells resulted in decreased T cell proliferation and activation both in vitro and in vivo, as well as reduced IFN-γ, IL-17A, IL-4 and IL-10 production. Finally, human blood T cells expressed EGFR and EGFR inhibition reduced T cell proliferation both in vivo and in vitro. Conclusion. EGFR is expressed by human and mouse CD4+ T cells. EGFR pharmacological inhibition or genetic invalidation induced T cell anergy in vitro and in vivo, blocked macrophage activity, and limited atherosclerosis initiation and progression. Our results suggest that targeting EGFR may be a novel strategy to combat atherosclerosis.