Molecular mechanisms implicated in bone resorption

Le remodelage osseux est un processus physiologique de renouvellement de l’os ancien par de l’os nouveau. Les ostéoclastes sont des cellules multinucléées géantes dont la fonction principale est de dégrader la matrice osseuse, première étape de ce remodelage. Le travail réalisé s’inscrit dans une thématique d’expertise de notre laboratoire, celle de l’organisation du cytosquelette d’actine dans les ostéoclastes résorbants. Nous avons pu élucider le rôle de la formation des podosomes et de leur organisation collective sur l’adhérence et la migration ostéoclastique. Nos résultats ont démontré que l’assemblage de podosomes sous forme de structures circulaires dites « anneaux » exerce une force centripète sur le substrat et ainsi déclenche la migration de l’ostéoclaste. L’alternance entre apparition et disparition de ces anneaux au sein de la cellule résulte en une migration saltatoire universelle pour tous les ostéoclastes.L’objectif principal de cette thèse était de trouver de nouveaux gènes impliqués dans l’organisation des podosomes. Nous avons mis en place une analyse transcriptomique comparant les ostéoclastes avec d’autres cellules multinucléées géantes qui présentent des podosomes mais sont incapables de résorber l’os. Parmi la liste de six gènes établie par cette méthode, nous avons étudié RhoE. En exploitant la culture d’ostéoclastes primaires déplétés de RhoE, nous avons démontré que ce gène est essentiel pour la migration ostéoclastique et la résorption osseuse. Nous avons ensuite établi que RhoE agit comme antagoniste de la voie de Rock pour assurer le renouvellement d’actine au sein des podosomes, ce qui entretien la fonction ostéoclastique.Bone remodeling is a physiological process by which old bone is replaced by new bone. Osteoclasts are multinucleated giant cells of the monocytic lineage. Their function is bone resorption, the first step of bone remodeling. The work of this thesis is in continuity with a theme long developed in our laboratory, that of the actin cytoskeleton organization in bone-resorbing osteoclasts. Our first study investigated the role of the podosome organization in osteoclast spreading, adhesion and migration. Our results showed that podosome patterning into rings exerted outward tension upon the substrate and thereby triggered cell migration. Through cycles of assembly, growth and alternating disassembly, rings promote a saltatory mode of migration universal to all osteoclasts.The main objective of this thesis, however, was dedicated to finding new genes that govern podosome patterning in resorption-related processes such as osteoclast migration and sealing zone formation. To find such new genes, we employed a differential transcriptomic analysis of osteoclasts and osteoclast-like cells that exhibit podosomes but are unable to resorb bone. Among a list of six genes highly and exclusively expressed in osteoclasts, we chose to investigate RhoE, a constitutively active GTP-binding protein known for its regulation of actin structures. We provided evidence, using primary RhoE-deficient osteoclasts, that RhoE activity is essential to bone resorption. We unveiled a new role for RhoE in the control of actin turnover in podosomes through a Rock-antagonistic function. Finally, we demonstrated that the role of RhoE in osteoclasts is essential to their migration and sealing zone formation

Mécanismes moléculaires impliqués dans la résorption osseuse

Bone remodeling is a physiological process by which old bone is replaced by new bone. Osteoclasts are multinucleated giant cells of the monocytic lineage. Their function is bone resorption, the first step of bone remodeling. The work of this thesis is in continuity with a theme long developed in our laboratory, that of the actin cytoskeleton organization in bone-resorbing osteoclasts. Our first study investigated the role of the podosome organization in osteoclast spreading, adhesion and migration. Our results showed that podosome patterning into rings exerted outward tension upon the substrate and thereby triggered cell migration. Through cycles of assembly, growth and alternating disassembly, rings promote a saltatory mode of migration universal to all osteoclasts.The main objective of this thesis, however, was dedicated to finding new genes that govern podosome patterning in resorption-related processes such as osteoclast migration and sealing zone formation. To find such new genes, we employed a differential transcriptomic analysis of osteoclasts and osteoclast-like cells that exhibit podosomes but are unable to resorb bone. Among a list of six genes highly and exclusively expressed in osteoclasts, we chose to investigate RhoE, a constitutively active GTP-binding protein known for its regulation of actin structures. We provided evidence, using primary RhoE-deficient osteoclasts, that RhoE activity is essential to bone resorption. We unveiled a new role for RhoE in the control of actin turnover in podosomes through a Rock-antagonistic function. Finally, we demonstrated that the role of RhoE in osteoclasts is essential to their migration and sealing zone formation.Le remodelage osseux est un processus physiologique de renouvellement de l’os ancien par de l’os nouveau. Les ostéoclastes sont des cellules multinucléées géantes dont la fonction principale est de dégrader la matrice osseuse, première étape de ce remodelage. Le travail réalisé s’inscrit dans une thématique d’expertise de notre laboratoire, celle de l’organisation du cytosquelette d’actine dans les ostéoclastes résorbants. Nous avons pu élucider le rôle de la formation des podosomes et de leur organisation collective sur l’adhérence et la migration ostéoclastique. Nos résultats ont démontré que l’assemblage de podosomes sous forme de structures circulaires dites « anneaux » exerce une force centripète sur le substrat et ainsi déclenche la migration de l’ostéoclaste. L’alternance entre apparition et disparition de ces anneaux au sein de la cellule résulte en une migration saltatoire universelle pour tous les ostéoclastes.L’objectif principal de cette thèse était de trouver de nouveaux gènes impliqués dans l’organisation des podosomes. Nous avons mis en place une analyse transcriptomique comparant les ostéoclastes avec d’autres cellules multinucléées géantes qui présentent des podosomes mais sont incapables de résorber l’os. Parmi la liste de six gènes établie par cette méthode, nous avons étudié RhoE. En exploitant la culture d’ostéoclastes primaires déplétés de RhoE, nous avons démontré que ce gène est essentiel pour la migration ostéoclastique et la résorption osseuse. Nous avons ensuite établi que RhoE agit comme antagoniste de la voie de Rock pour assurer le renouvellement d’actine au sein des podosomes, ce qui entretien la fonction ostéoclastique

Molecular mechanisms implicated in bone resorption

Le remodelage osseux est un processus physiologique de renouvellement de l os ancien par de l os nouveau. Les ostéoclastes sont des cellules multinucléées géantes dont la fonction principale est de dégrader la matrice osseuse, première étape de ce remodelage. Le travail réalisé s inscrit dans une thématique d expertise de notre laboratoire, celle de l organisation du cytosquelette d actine dans les ostéoclastes résorbants. Nous avons pu élucider le rôle de la formation des podosomes et de leur organisation collective sur l adhérence et la migration ostéoclastique. Nos résultats ont démontré que l assemblage de podosomes sous forme de structures circulaires dites anneaux exerce une force centripète sur le substrat et ainsi déclenche la migration de l ostéoclaste. L alternance entre apparition et disparition de ces anneaux au sein de la cellule résulte en une migration saltatoire universelle pour tous les ostéoclastes.L objectif principal de cette thèse était de trouver de nouveaux gènes impliqués dans l organisation des podosomes. Nous avons mis en place une analyse transcriptomique comparant les ostéoclastes avec d autres cellules multinucléées géantes qui présentent des podosomes mais sont incapables de résorber l os. Parmi la liste de six gènes établie par cette méthode, nous avons étudié RhoE. En exploitant la culture d ostéoclastes primaires déplétés de RhoE, nous avons démontré que ce gène est essentiel pour la migration ostéoclastique et la résorption osseuse. Nous avons ensuite établi que RhoE agit comme antagoniste de la voie de Rock pour assurer le renouvellement d actine au sein des podosomes, ce qui entretien la fonction ostéoclastique.Bone remodeling is a physiological process by which old bone is replaced by new bone. Osteoclasts are multinucleated giant cells of the monocytic lineage. Their function is bone resorption, the first step of bone remodeling. The work of this thesis is in continuity with a theme long developed in our laboratory, that of the actin cytoskeleton organization in bone-resorbing osteoclasts. Our first study investigated the role of the podosome organization in osteoclast spreading, adhesion and migration. Our results showed that podosome patterning into rings exerted outward tension upon the substrate and thereby triggered cell migration. Through cycles of assembly, growth and alternating disassembly, rings promote a saltatory mode of migration universal to all osteoclasts.The main objective of this thesis, however, was dedicated to finding new genes that govern podosome patterning in resorption-related processes such as osteoclast migration and sealing zone formation. To find such new genes, we employed a differential transcriptomic analysis of osteoclasts and osteoclast-like cells that exhibit podosomes but are unable to resorb bone. Among a list of six genes highly and exclusively expressed in osteoclasts, we chose to investigate RhoE, a constitutively active GTP-binding protein known for its regulation of actin structures. We provided evidence, using primary RhoE-deficient osteoclasts, that RhoE activity is essential to bone resorption. We unveiled a new role for RhoE in the control of actin turnover in podosomes through a Rock-antagonistic function. Finally, we demonstrated that the role of RhoE in osteoclasts is essential to their migration and sealing zone formation.LYON-ENS Sciences (693872304) / SudocSudocFranceF

Podosome organization drives osteoclast-mediated bone resorption

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Twist1-positive epithelial cells retain adhesive and proliferative capacity throughout dissemination.

Dissemination is the process by which cells detach and migrate away from a multicellular tissue. The epithelial-to-mesenchymal transition (EMT) conceptualizes dissemination in a stepwise fashion, with downregulation of E-cadherin leading to loss of intercellular junctions, induction of motility, and then escape from the epithelium. This gain of migratory activity is proposed to be mutually exclusive with proliferation. We previously developed a dissemination assay based on inducible expression of the transcription factor Twist1 and here utilize it to characterize the timing and dynamics of intercellular adhesion, proliferation and migration during dissemination. Surprisingly, Twist1(+) epithelium displayed extensive intercellular junctions, and Twist1(-) luminal epithelial cells could still adhere to disseminating Twist1(+) cells. Although proteolysis and proliferation were both observed throughout dissemination, neither was absolutely required. Finally, Twist1(+) cells exhibited a hybrid migration mode; their morphology and nuclear deformation were characteristic of amoeboid cells, whereas their dynamic protrusive activity, pericellular proteolysis and migration speeds were more typical of mesenchymal cells. Our data reveal that epithelial cells can disseminate while retaining competence to adhere and proliferate

Twist1-positive epithelial cells retain adhesive and proliferative capacity throughout dissemination.

Dissemination is the process by which cells detach and migrate away from a multicellular tissue. The epithelial-to-mesenchymal transition (EMT) conceptualizes dissemination in a stepwise fashion, with downregulation of E-cadherin leading to loss of intercellular junctions, induction of motility, and then escape from the epithelium. This gain of migratory activity is proposed to be mutually exclusive with proliferation. We previously developed a dissemination assay based on inducible expression of the transcription factor Twist1 and here utilize it to characterize the timing and dynamics of intercellular adhesion, proliferation and migration during dissemination. Surprisingly, Twist1(+) epithelium displayed extensive intercellular junctions, and Twist1(-) luminal epithelial cells could still adhere to disseminating Twist1(+) cells. Although proteolysis and proliferation were both observed throughout dissemination, neither was absolutely required. Finally, Twist1(+) cells exhibited a hybrid migration mode; their morphology and nuclear deformation were characteristic of amoeboid cells, whereas their dynamic protrusive activity, pericellular proteolysis and migration speeds were more typical of mesenchymal cells. Our data reveal that epithelial cells can disseminate while retaining competence to adhere and proliferate

Twist1-positive epithelial cells retain adhesive and proliferative capacity throughout dissemination

Dissemination is the process by which cells detach and migrate away from a multicellular tissue. The epithelial-to-mesenchymal transition (EMT) conceptualizes dissemination in a stepwise fashion, with downregulation of E-cadherin leading to loss of intercellular junctions, induction of motility, and then escape from the epithelium. This gain of migratory activity is proposed to be mutually exclusive with proliferation. We previously developed a dissemination assay based on inducible expression of the transcription factor Twist1 and here utilize it to characterize the timing and dynamics of intercellular adhesion, proliferation and migration during dissemination. Surprisingly, Twist1(+) epithelium displayed extensive intercellular junctions, and Twist1(–) luminal epithelial cells could still adhere to disseminating Twist1(+) cells. Although proteolysis and proliferation were both observed throughout dissemination, neither was absolutely required. Finally, Twist1(+) cells exhibited a hybrid migration mode; their morphology and nuclear deformation were characteristic of amoeboid cells, whereas their dynamic protrusive activity, pericellular proteolysis and migration speeds were more typical of mesenchymal cells. Our data reveal that epithelial cells can disseminate while retaining competence to adhere and proliferate

Twist1-positive epithelial cells retain adhesive and proliferative capacity throughout dissemination.

Dissemination is the process by which cells detach and migrate away from a multicellular tissue. The epithelial-to-mesenchymal transition (EMT) conceptualizes dissemination in a stepwise fashion, with downregulation of E-cadherin leading to loss of intercellular junctions, induction of motility, and then escape from the epithelium. This gain of migratory activity is proposed to be mutually exclusive with proliferation. We previously developed a dissemination assay based on inducible expression of the transcription factor Twist1 and here utilize it to characterize the timing and dynamics of intercellular adhesion, proliferation and migration during dissemination. Surprisingly, Twist1(+) epithelium displayed extensive intercellular junctions, and Twist1(-) luminal epithelial cells could still adhere to disseminating Twist1(+) cells. Although proteolysis and proliferation were both observed throughout dissemination, neither was absolutely required. Finally, Twist1(+) cells exhibited a hybrid migration mode; their morphology and nuclear deformation were characteristic of amoeboid cells, whereas their dynamic protrusive activity, pericellular proteolysis and migration speeds were more typical of mesenchymal cells. Our data reveal that epithelial cells can disseminate while retaining competence to adhere and proliferate