Contrôle de l' ostéogénèse membranaire par le facteur de transcription Twist (implication de FGFR2 et de l'ubiquitine ligase Cbl)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Roles of FGFR2 and Twist in Human Craniosynostosis: Insights from Genetic Mutations in Cranial Osteoblasts

International audienceRecent advances in molecular genetics have led to a better understanding of the role of specific genes such as fibroblast growth factor receptor (FGFR) and Twist in cranial bone formation. Specifically, the analysis of osteoblast abnormalities induced by FGFR2 and Twist genetic mutations inducing craniosynostosis in humans has provided some insights into the role of these genes in the premature cranial suture formation in syndromic craniosynostosis. This also led to a better understanding of the cellular and molecular mechanisms that control osteoblast biology and pathology in humans. In this review paper, we summarize the effects of FGFR2 and Twist genetic mutations resulting in altered osteoblast phenotype and premature cranial fusion based on our analysis in human syndromic craniosynostosis

The endothelium, a key actor in organ development and hPSC-derived organoid vascularization

International audienceAbstract Over the last 4 decades, cell culture techniques have evolved towards the creation of in vitro multicellular entities that incorporate the three-dimensional complexity of in vivo tissues and organs. As a result, stem cells and adult progenitor cells have been used to derive self-organized 3D cell aggregates that mimic the morphological and functional traits of organs in vitro. These so-called organoids were first generated from primary animal and human tissues, then human pluripotent stem cells (hPSCs) arose as a new tool for organoid generation. Due to their self-renewal capacity and differentiation potential, hPSCs are an unlimited source of cells used for organoids. Today, hPSC-derived small intestinal, kidney, brain, liver, and pancreas organoids, among others, have been produced and are promising in vitro human models for diverse applications, including fundamental research, drug development and regenerative medicine. However, achieving in vivo-like organ complexity and maturation in vitro remains a challenge. Current hPSC-derived organoids are often limited in size and developmental state, resembling embryonic or fetal organs rather than adult organs. The use of endothelial cells to vascularize hPSC-derived organoids may represent a key to ensuring oxygen and nutrient distribution in large organoids, thus contributing to the maturation of adult-like organoids through paracrine signaling. Here, we review the current state of the art regarding vascularized hPSC-derived organoids (vhPSC-Orgs). We analyze the progress achieved in the generation of organoids derived from the three primary germ layers (endoderm, mesoderm and ectoderm) exemplified by the pancreas, liver, kidneys and brain. Special attention will be given to the role of the endothelium in the organogenesis of the aforementioned organs, the sources of endothelial cells employed in vhPSC-Org protocols and the remaining challenges preventing the creation of ex vivo functional and vascularized organs

Cbl-mediated ubiquitination of α5 integrin subunit mediates fibronectin-dependent osteoblast detachment and apoptosis induced by FGFR2 activation

International audienceFibroblast growth factor receptor signaling is an important mechanism regulating osteoblast function. To gain an insight into the regulatory role of FGF receptor-2 (FGFR2) signaling in osteoblasts, we investigated integrin-mediated attachment and cell survival in human calvarial osteoblasts expressing activated FGFR2. FGFR2 activation reduced osteoblast attachment on fibronectin. This was associated with reduced expression of the α5 integrin subunit normally expressed in human calvarial osteoblasts in vivo. Treatment with lactacystin, a potent inhibitor of proteasome, restored α5 integrin levels in FGFR2 mutant osteoblasts. Immunoprecipitation analysis showed that α5 integrin interacts with both the E3 ubiquitin ligase Cbl and ubiquitin. Immunocytochemistry revealed that α5 integrin colocalizes with FGFR2 and Cbl at the leading edge in membrane ruffle regions. Transfection with the 70Z-Cbl mutant lacking the RING domain required for Cbl-ubiquitin interaction, or with the G306E Cbl mutant that abolishes the binding ability of Cbl phosphotyrosine-binding domain restored α5 integrin levels. This suggests that Cbl-mediated ubiquitination plays an essential role in α5 integrin proteasome degradation induced by FGFR2 activation. Reduced α5 integrin expression was associated with an increased Bax/Bcl-2 ratio and increased caspase-9 and -3 activities in FGFR2 mutant osteoblasts. Forced expression of α5 integrin rescued cell attachment and corrected both the Bax/Bcl-2 ratio and caspase-3 and caspase-9 activities in FGFR2 mutant osteoblasts. We show that Cbl recruitment induced by FGFR2 activation triggers α5 integrin degradation by the proteasome, which results in reduced osteoblast attachment on fibronectin and caspase-dependent apoptosis. This identifies a functional role of the α5 integrin subunit in the induction of apoptosis triggered by FGFR2 activation in osteoblasts, and reveals that a Cbl-dependent mechanism is involved in the coordinated regulation of cell apoptosis induced by α5 integrin degradation

FGFR2-Cbl interaction in lipid rafts triggers attenuation of PI3K/Akt signaling and osteoblast survival.

International audienceFibroblast growth factor receptor (FGFR) signaling plays an important role in skeletogenesis. The molecular mechanisms triggered by activated FGFR in bone forming cells are however not fully understood. In this study, we identify a role for phosphatidylinositol 3-kinase (PI3K) signaling in cell apoptosis induced by FGFR2 activation in osteoblasts. We show that FGFR2 activation leads to decrease PI3K protein levels, resulting in attenuation of PI3K signaling in human osteoblasts. Biochemical and molecular analyses revealed that the attenuated PI3K signaling induced by FGFR2 activation is due to increased Cbl-PI3K molecular interaction mediated by the Cbl Y731 residue, which results in increased PI3K ubiquitination and proteasome degradation. Biochemical and immunocytochemical analyses showed that FGFR2 and Cbl interact in raft micro-domains at the plasma membrane. FGFR2 activation increases FGFR2 and Cbl recruitment in micro-domains, resulting in increased molecular interactions. Consistently, functional analyses showed that the attenuation of PI3K/Akt signaling triggered by FGFR2 activation results in increased osteoblast apoptosis. These results identify a functional molecular mechanism by which activated FGFR2 recruits Cbl in raft micro-domains to trigger PI3K ubiquitination and proteasome degradation, and reveal a novel role for PI3K/Akt attenuation in the control of osteoblast survival by FGFR2 signaling

Human embryonic stem cells derivatives enable full reconstruction of the pluristratified epidermis.

Background: Cell therapy for large burns has successfully made use of autologous epidermis reconstructed in vitro for more than two decades. One limit to the efficacy of the current procedures is the need for a long lasting expansion of the patient's own keratinocytes. Immediate access to unlimited amounts of human epidermis may contribute to protect patients awaiting autologous grafts. The keratinocyte progeny of human embryonic stem cells is an interesting candidate for such a role, provided they are fully capable of reconstructing a pluristratified epidermis. Methods: Human embryonic stem cells were seeded on fibroblasts feeder cells during forty days in a medium supplemented with BMP4 (0.5 nM) and ascorbic acid (0.3 mM). A molecular characterization of cell differentiation was performed all along the process by quantitative-PCR, fluorescence activated cell sorting and immunocytochemical techniques. Keratinocyte molecular differentiation and functional capacity to construct a human epidermis were assessed in vitro and in vivo. Finding: Using a protocol that respected the chronobiology of epidermis formation during human ontogenesis, we have generated a homogenous population of cells exhibiting all phenotypic characteristics of basal keratinocytes from hESC. These cells readily constructed a pluristratified epidermis displaying normal human characteristics after seeding on an artificial matrix, and either secondarily grown in vitro or else transplanted in immunodeficient mice. Interpretation: Human embryonic stem cells can be coaxed to differentiate into basal keratinocytes that are fully functional, i.e. construct a pluristratified epidermis. This resource may be developed to provide temporary skin substitutes to patients awaiting autologous grafts

Endothelial and hematopoietic hPSCs differentiation via a hematoendothelial progenitor

International audienceAbstract Background hPSC-derived endothelial and hematopoietic cells (ECs and HCs) are an interesting source of cells for tissue engineering. Despite their close spatial and temporal embryonic development, current hPSC differentiation protocols are specialized in only one of these lineages. In this study, we generated a hematoendothelial population that could be further differentiated in vitro to both lineages. Methods Two hESCs and one hiPSC lines were differentiated into a hematoendothelial population, hPSC-ECs and blast colonies (hPSC-BCs) via CD144 + -embryoid bodies (hPSC-EBs). hPSC-ECs were characterized by endothelial colony-forming assay, LDL uptake assay, endothelial activation by TNF-α, nitric oxide detection and Matrigel-based tube formation. Hematopoietic colony-forming cell assay was performed from hPSC-BCs. Interestingly, we identified a hPSC-BC population characterized by the expression of both CD144 and CD45. hPSC-ECs and hPSC-BCs were analyzed by flow cytometry and RT-qPCR; in vivo experiments have been realized by ischemic tissue injury model on a mouse dorsal skinfold chamber and hematopoietic reconstitution in irradiated immunosuppressed mouse from hPSC-ECs and hPSC-EB-CD144 + , respectively. Transcriptomic analyses were performed to confirm the endothelial and hematopoietic identity of hESC-derived cell populations by comparing them against undifferentiated hESC, among each other’s ( e.g. hPSC-ECs vs. hPSC-EB-CD144 + ) and against human embryonic liver (EL) endothelial, hematoendothelial and hematopoietic cell subpopulations. Results A hematoendothelial population was obtained after 84 h of hPSC-EBs formation under serum-free conditions and isolated based on CD144 expression. Intrafemorally injection of hPSC-EB-CD144 + contributed to the generation of CD45 + human cells in immunodeficient mice suggesting the existence of hemogenic ECs within hPSC-EB-CD144 + . Endothelial differentiation of hPSC-EB-CD144 + yields a population of > 95% functional ECs in vitro. hPSC-ECs derived through this protocol participated at the formation of new vessels in vivo in a mouse ischemia model. In vitro, hematopoietic differentiation of hPSC-EB-CD144 + generated an intermediate population of > 90% CD43 + hPSC-BCs capable to generate myeloid and erythroid colonies. Finally, the transcriptomic analyses confirmed the hematoendothelial, endothelial and hematopoietic identity of hPSC-EB-CD144 + , hPSC-ECs and hPSC-BCs, respectively, and the similarities between hPSC-BC-CD144 + CD45 + , a subpopulation of hPSC-BCs, and human EL hematopoietic stem cells/hematopoietic progenitors. Conclusion The present work reports a hPSC differentiation protocol into functional hematopoietic and endothelial cells through a hematoendothelial population. Both lineages were proven to display characteristics of physiological human cells, and therefore, they represent an interesting rapid source of cells for future cell therapy and tissue engineering

Down-Regulation of Ubiquitin Ligase Cbl Induced by Twist Haploinsufficiency in Saethre-Chotzen Syndrome Results in Increased PI3K/Akt Signaling and Osteoblast Proliferation

Genetic mutations of Twist, a basic helix-loop-helix transcription factor, induce premature fusion of cranial sutures in Saethre-Chotzen syndrome (SCS). We report here a previously undescribed mechanism involved in the altered osteoblastogenesis in SCS. Cranial osteoblasts from an SCS patient with a Twist mutation causing basic helix-loop-helix deletion exhibited decreased expression of E3 ubiquitin ligase Cbl compared with wild-type osteoblasts. This was associated with decreased ubiquitin-mediated degradation of phosphatidyl inositol 3 kinase (PI3K) and increased PI3K expression and PI3K/Akt signaling. Increased PI3K immunoreactivity was also found in osteoblasts in histological sections of affected cranial sutures from SCS patients. Transfection with Twist or Cbl abolished the increased PI3K/Akt signaling in Twist mutant osteoblasts. Forced overexpression of Cbl did not correct the altered expression of osteoblast differentiation markers in Twist mutant cells. In contrast, pharmacological inhibition of PI3K/Akt, but not ERK signaling, corrected the increased cell growth in Twist mutant osteoblasts. The results show that Twist haploinsufficiency results in decreased Cbl-mediated PI3K degradation in osteoblasts, causing PI3K accumulation and activation of PI3K/Akt-dependent osteoblast growth. This provides genetic and biochemical evidence for a role for Cbl-mediated PI3K signaling in the altered osteoblast phenotype induced by Twist haploinsufficiency in SCS