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Rôle de la signalisation purinergique dans la régulation de la migration des kératinocytesFaure, Emilie 27 March 2012 (has links)
L'épiderme est un tissu stratifié, majoritairement constitué de kératinocytes qui forme la première barrière de l'organisme contre les agressions extérieures. Après blessure cutanée, la migration des kératinocytes est une phase cruciale de la cicatrisation. Le comportement des kératinocytes est placé sous le contrôle des molécules de la matrice extracellulaire ainsi que par les facteurs solubles (facteurs de croissance et cytokines..) sécrétés dans le microenvironnement. Les cellules résidentes ou recrutées sur le site de lésion libèrent également des nucléotides extracellulaires (ATP, UTP) dans l'environnement des kératinocytes. Dans ce travail de thèse, nous avons examiné l'impact des nucléotides extracellulaires et du récepteur purinergique P2Y2 sur la migration des kératinocytes et sur l'activité motogénique de deux facteurs de croissance, l'IGF-I et de l'EGF. Dans un premier temps, nous avons pu montrer que l'activation de P2Y2 et de la protéine hétérotrimérique Gαq inhibe l'activité de l'isoforme p110α de la PI3K sur des cellules stimulées par l'IGF. Cette inhibition de la voie PI3K/Akt aboutit à une perturbation de la mobilisation de la cortactine et de la formation des lamellipodes ainsi qu'une diminution de la vitesse de migration des kératinocytes. Dans un second temps, nous avons mis en évidence que l'activation de P2Y2 inhibe l'activation de la voie ERK1/2 par l'EGF en inhibant la phosphorylation des protéines MEK1/2, ERK1/2 et p90RSK. Nous avons établi que la conséquence de cette inhibition est la stabilisation des hémidesmosomes / The epidermis is a stratified tissue, mainly composed of keratinocytes, that forms the first barrier of the organism. When skin injury occurs, the epidermis structure is altered and many signalling pathways are activated in order to re-establish its homeostasis. Among these signalling pathways, the PI3K and MAPK ERK1/2 pathways play key roles by controlling keratinocyte migration and proliferation. The aim of this thesis was to analyse the regulation of these two signalling pathways by extracellular nucleotides, acting through purinergic receptors P2Y2 and the heterotrimeric Gαq protein and to evaluate the impact of this receptor on keratinocyte migration. Firstly, we showed that P2Y2 receptor activation inhibits PI3K p110α isoform and consequently alters keratinocyte cell shape and migration. Additionally, we showed that purinergic signalling activation inhibits EGF-induced ERK1/2 pathway activation by inhibiting the phosphorylation of MEK1/2, ERK1/2 and p90RSK proteins. As a consequence, P2Y2 stabilizes α6β4 integrin localisation into hemidesmosome-like structures and inhibits keratinocyte migration. The involvement of purinergic signalling pathway in regulation of different signalling events suggests that it may play a central role in regulation of cellular events that occurred during skin wound healing process. Moreover, our present data in association with those of the literature show that extracellular nucleotides can act as a double-edged sword in the regulation of cell migration: either activate or block cell migration in a striking cell-specific manner.
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An Integral Role of ARRDC3 in Stem Cell Migration and Breast Cancer Progression: A DissertationDraheim, Kyle M. 02 March 2010 (has links)
Despite the importance of integrins in epithelial cell biology surprisingly little is known about their regulation. It is known that they form hemidesmosomes (HDs), are actively involved in cell contacts during cell migration/invasion, and are key signaling molecules for survival and growth. However, there has been a distinct lack of understanding about what controls the dynamic integrin localization during cell activation and movement. Growth factors, such as EGF, are elevated during wound healing and carcinoma invasion leading to phosphorylation of ITGβ4 and the disassembly of the HD and mobilization of ITGβ4 to actin-rich protrusions. More recently the phosphorylation of a novel site on ITGβ4 (S1424) was found to be distinctly enriched on the trailing edge of migrating cells, suggesting a possible mechanism for the dissociation of ITGβ4 from HDs.
Arrestin family member proteins are involved in the regulation of cell surface proteins and vesicular trafficking. In this study, we find that over-expression of arrestin family member ARRDC3 causes internalization and proteosome-dependent degradation of ITGβ4, while decreased levels of ARRDC3 stabilizes ITGβ4 levels. These results lead us to a new mechanism of ITGβ4 internalization, trafficking and degradation. During migration, ARRDC3 co-localizes with ITGβ4 on the lagging edge of cells but has a distinct distribution on the leading edge of cells. Additional immuno co-precipitation experiments demonstrate that ARRDC3 preferentially binds to ITGβ4 when phosphorylated on S1424. Using confocal microscopy, we show that the expression pattern of ARRDC3 on the lagging edge of a migrating cell is identical to the expression pattern of ITGβ4-pS1424. We demonstrate that ARRDC3 expression represses cell proliferation, migration, invasion, growth in soft agar and tumorigenicity.
Collectively, our data reveals that ARRDC3 is a negative regulator of β4 integrin and demonstrates how this new pathway impacts biologic processes in stem cell and cancer biology. Additionally, as ARRDC3 is highly expressed in several tissues and conserved across species, our results are likely to be translated to other models.
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