Bone tissue is continuously remodeled by bone-forming osteoblasts and bone-resorbing osteoclasts, in processes tightly regulated by hormones, cytokines and growth factors. CD47, a ubiquitously expressed protein, and one of its ligands, signal-regulatory protein alpha (SIRPα), are two cell-surface proteins belonging to the immunoglobulin (Ig)-superfamily. The interaction between CD47 and SIRPα is important for, amongst other processes, the fusion of macrophages into giant cells, which are closely related to osteoclasts. The aim of the present study was to gain knowledge about the role of CD47–SIRPα interaction and resultant downstream signaling pathways in bone cell differentiation, formation and function. The addition of antibodies against CD47 or SIRPα inhibited the formation of multinucleated osteoclasts from bone marrow monocytes (BMMs) in culture. Moreover, a significant decrease in the number of osteoclasts was detected in CD47-/- BMM cultures compared to CD47+/+ cultures. In line with these in vitro results, we found fewer osteoclasts in vivo in the trabecular bone of CD47-/- mice, as compared to CD47+/+ bone. Interestingly, an extended analysis of the trabecular bone of CD47-/- mice revealed that the bone volume, mineralizing surface, mineral apposition rate, bone formation rate and osteoblast number were also significantly reduced compared with CD47+/+ mice, indicating the importance of CD47 in osteoblast differentiation. In vitro studies of bone marrow stromal (BMS) cells from CD47-/- mice or SIRPα-mutant mice (mice lacking the signaling domain of SIRPa) showed a blunted expression of osteoblast-associated genes. Moreover, these altered genotypes were associated with reduced activity of the bone mineralization-associated enzyme alkaline phosphatase as well as a reduced ability to form mineral. To reveal the molecular mechanisms by which CD47 activation of SIRPα is important for BMS cell differentiation, we studied signaling downstream of SIRPα in the absence of CD47. In BMS cells lacking CD47, a considerable reduction in the levels of tyrosine phosphorylated SIRPα was detected, and the subsequent recruitment of the Src-homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP-2)–phosphoinositide 3-kinase (PI3K)–Akt2 signaling module was nearly abolished. In conclusion, the interaction between CD47 and SIRPα results in the activation of the SHP-2–PI3K–Akt2 pathway, which is necessary for normal osteoblast differentiation. In CD47-/- mice and SIRPα-mutant mice, this interaction is perturbed, which prevents normal osteoblast differentiation and subsequent mineral formation. In addition, the altered BMS cell phenotype results in an impaired ability to stimulate osteoclast differentiation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20976号 / 医博第4322号 / 新制||医||1026(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 岩田 想, 教授 中川 一路 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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SHP-2 est une tyrosine phosphatase impliquée dans la signalisation intracellulaire déclenchée par des facteurs de croissance, des cytokines pro-inflammatoires et des produits bactériens. Bien que cette phosphatase soit exprimée de manière ubiquiste et donc dans l’épithélium intestinal, son rôle dans ce tissu n'était pas connu. Afin de mieux comprendre les rôles joués par cette phosphatase dans l’intestin, nous avons généré un modèle murin de délétion conditionnelle de Shp-2 spécifiquement dans les cellules épithéliales intestinales (SHP-2[indice supérieur CEI-KO]). Nos résultats montrent que dès l'âge de 1 mois, toutes les souris expérimentales ont développé spontanément de l'inflammation au niveau du côlon. En fait, dans les cellules épithéliales intestinales, SHP-2 contrôle le niveau d’activation d’effecteurs de signalisation importants tels que les kinases ERK1/2 de même que les facteurs de transcription NFκB, STAT3 et β-caténine. En modulant ces voies de signalisation, SHP-2 contrôle des processus cellulaires primordiaux pour le maintien de l’homéostasie intestinale: la détermination des cellules à mucus et des cellules de Paneth, la composition de la flore, la perméabilité paracellulaire et la restitution épithéliale. La dérégulation de ces processus cellulaires peut expliquer l'apparition rapide d'inflammation colique chez les souris SHP-2C[indice supérieur EI-KO]. De plus, l'inflammation chronique observée chez les souris SHP-2[indice supérieur] CEI-KO entraîne avec l'âge le développement de cancer colorectal associé à la colite. Finalement, nos résultats chez l'humain montrent qu'il y a une diminution significative d'expression de SHP-2 chez les patients atteints de maladies inflammatoires intestinales comparativement aux patients témoins. Également, deux polymorphismes de PTPN11 sont retrouvés préférentiellement chez les patients atteints de colite ulcéreuse. En conclusion, nos résultats démontrent que la phosphatase SHP-2 protège l'épithélium intestinal contre l'inflammation et le cancer colorectal associé à la colite.

During development of the mammalian nervous system, neural stem cells generate neurons first and glia second, thereby allowing the initial establishment of neuronal circuitry, and subsequent matching of glial numbers and position to that circuitry. Multiple molecular mechanisms act in concert to control neural precursor expansion prior to neurogenesis, and to allow for an exponential generation of neurons while ensuring the maintenance of sufficient precursors to produce later-born neurons, glial cells and adult neural stem cells. Throughout cortical development, these processes are regulated in part by the precursors’ environment as well as intrinsic changes in precursors and their modes of division, which regulate the fate of daughter cells and the balance between self-renewal and differentiation. In the first part of this thesis, the protein tyrosine phosphatase SHP-2 was identified as a novel signaling protein that regulates the neurogenic to gliogenic switch by potentiating neurogenic signals and suppressing gliogenic signals until the appropriate developmental time point for astrogenesis, providing one mechanism whereby precursors integrate conflicting environmental cues. A Noonan Syndrome (NS)-associated activated SHP-2 mutation causes perturbations in neural cell genesis, which may contribute to the mild mental retardation and learning disabilities observed in NS patients. In the second part of this thesis, a novel Rho-regulatory pathway which includes the Rho-GEF Lfc and its negative regulator Tctex-1 were also found to regulate neurogenesis, potentially by directing mitotic spindle orientation during precursor divisions, thereby regulating the symmetric and asymmetric nature of radial precursor divisions.

During development of the mammalian nervous system, neural stem cells generate neurons first and glia second, thereby allowing the initial establishment of neuronal circuitry, and subsequent matching of glial numbers and position to that circuitry. Multiple molecular mechanisms act in concert to control neural precursor expansion prior to neurogenesis, and to allow for an exponential generation of neurons while ensuring the maintenance of sufficient precursors to produce later-born neurons, glial cells and adult neural stem cells. Throughout cortical development, these processes are regulated in part by the precursors’ environment as well as intrinsic changes in precursors and their modes of division, which regulate the fate of daughter cells and the balance between self-renewal and differentiation. In the first part of this thesis, the protein tyrosine phosphatase SHP-2 was identified as a novel signaling protein that regulates the neurogenic to gliogenic switch by potentiating neurogenic signals and suppressing gliogenic signals until the appropriate developmental time point for astrogenesis, providing one mechanism whereby precursors integrate conflicting environmental cues. A Noonan Syndrome (NS)-associated activated SHP-2 mutation causes perturbations in neural cell genesis, which may contribute to the mild mental retardation and learning disabilities observed in NS patients. In the second part of this thesis, a novel Rho-regulatory pathway which includes the Rho-GEF Lfc and its negative regulator Tctex-1 were also found to regulate neurogenesis, potentially by directing mitotic spindle orientation during precursor divisions, thereby regulating the symmetric and asymmetric nature of radial precursor divisions.

La protéine tyrosine phosphatase SHP-2 est connue pour jouer un rôle important dans le maintien de l’homéostasie de plusieurs tissus et organes par ses effets régulateurs sur plusieurs voies de signalisation intracellulaire. Notre laboratoire a récemment démontré que la délétion embryonnaire de SHP-2 (SHP-2CEI-KO) à l’épithélium intestinal entraîne le développement rapide d’une inflammation colique sévère. La délétion ayant lieu au stade embryonnaire, une altération dans le développement intestinal pourrait être en cause dans l’initiation de cette inflammation. Afin d’éliminer cette possibilité, un modèle de délétion conditionnelle inductible de SHP-2 dans les cellules épithéliales intestinales (SHP-2CEI-KOER) a été généré, la délétion ayant été induite par injection de tamoxifène trois mois après la naissance. Ce modèle a permis d’éliminer la composante développementale, la délétion ayant lieu chez la souris adulte. De manière intéressante, les souris subissant la délétion de SHP-2 développent aussi une inflammation colique suite à 18 jours de traitement au tamoxifène. Cette inflammation s’accompagne d’une diminution de l’activité de la signalisation MEK/ERK MAPK et d’une activation de la signalisation JAK/STAT. Des altérations dans la différenciation des cellules caliciformes et de Paneth sont observées, les souris expérimentales présentant une diminution du nombre de cellules caliciformes ainsi qu’une présence aberrante de cellules intermédiaires (précurseurs des cellules caliciformes) dans leur côlon. Cette diminution du nombre de cellules caliciformes est présente dès le 10e jour de traitement au tamoxifène, précédant donc l’apparition des signes d’inflammation colique, ceux-ci débutant seulement au 12e jour de traitement au tamoxifène. Nos résultats sur une lignée cellulaire capable de se différencier dans un phénotype caliciforme, les LS174T, montrent une activation de la voie Notch suite à l’inactivation de la voie ERK/MAPK, cette activation étant associée à une augmentation de l’expression des gènes associés aux cellules caliciformes. Ces résultats corrèlent avec l’observation que MEK/ERK est inhibée dans l’épithélium déficient pour l’expression de SHP-2 alors que celle de Notch est activée. Ces résultats nous indiquent donc un rôle important de la tyrosine phosphatase SHP-2 dans le maintien de l’homéostasie intestinale, et ce, même chez l’adulte. L’inactivation de SHP-2 résulte en effet dans la perte de l’intégrité de la muqueuse colique amenant l’inflammation. De manière intéressante, des polymorphismes (SNP) dans le gène encodant SHP-2 ont été récemment associés à une susceptibilité accrue à développer une colite ulcéreuse chez des patients. Pris ensemble, ces résultats suggèrent donc que SHP-2 pourrait constituer une nouvelle cible dans le traitement des maladies inflammatoires intestinales.

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No description available.

<p>x</p> / <p>Platelet-derived growth factors (PDGF), a family of potent mitogens and chemoattractants for cells of mesenchymal origin, elicit their biological effects through the binding of two related receptor tyrosine kinases, denoted α- and β-receptors. The binding of PDGF to the receptors causes receptor dimerization and autophosphorylation on tyrosine residues. Src homology 2 (SH2) domain-containing proteins then bind the phosphorylated receptors, mediating further propagation of the signal. This thesis describes how the interaction between the PDGF receptors and some of their downstream targets can modify the cellular response to PDGF.</p><p>The tyrosine phosphatase SHP-2 has been implicated in activation of the Ras/MAPK pathway downstream of several receptor tyrosine kinases. We found that SHP-2 binds to phosphorylated Y763 in the PDGF β-receptor, in addition to the already reported binding to Y1009. Cells expressing PDGF β-receptors with Y763 and Y1009 mutated to phenylalanine exhibited decreased Ras-GTP loading and reduced activation of Erk2 in response to PDGF. Whereas these cells did not show any change in the mitogenic response to PDGF, the PDGF-induced chemotaxis was significantly reduced in cells expressing mutant compared to wild-type receptor.</p><p>The phosphorylation of Y771 of the PDGF β-receptor had been shown to be significantly lower in the αβ-heterodimeric receptor compared to in the ββ-homodimer, causing reduced binding of RasGAP to the heterodimer and increased Ras/MAPK activation. We could demonstrate that the reduced phosphorylation of Y771 is due to dephosphorylation by tyrosine phosphatases, including SHP-2.</p><p>SHP-2 had been shown to associate with the docking protein Gab1 after growth factor stimulation. We showed that the adaptor protein Grb2 was required for PDGF mediated phosphorylation of Gab1, and that phosphorylated Gab1, Grb2 and SHP-2 create a complex upon PDGF stimulation. Using a cell system with an inducible Gab1 expression, we further demonstrated that Gab1 increased SHP-2 activity in response to PDGF, without affecting the interaction between SHP-2 and the b-receptor. Induction of Gab1 correlated with an increase in both PDGF-induced Erk and p38 MAPK activation, whereas Akt activation was unaffected. The latter finding was in line with our observation that PDGF had no effect on the interaction between Gab1 and p85 of PI3’-kinase. The increase in MAPK activity after Gab1 induction and PDGF treatment did not correlate with an increase in PDGF-induced mitogenicity; instead these cells displayed more pronounced actin reorganization in response to PDGF.</p><p>In conclusion, our data indicate that SHP-2 regulates the PDGF response both through direct dephosphorylation of the receptor and through its interaction with Gab1. PDGF stimulated activation of SHP-2 seems to be correlated not only with mitogenesis, but also with reorganization of the actin cytoskeleton and cell migration.</p>