27 research outputs found

    LoXL4 is induced by transforming growth factor β1 through Smad and JunB/Fra2 and contributes to vascular matrix remodeling

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    Transforming growth factor β1 (TGF-β1) is a pleiotropic factor involved in the regulation of extracellular matrix (ECM) synthesis and remodeling. In search for novel genes mediating the action of TGF-β1 on vascular ECM, we identified the member of the lysyl oxidase family of matrix-remodeling enzymes, lysyl oxidase-like 4 (LOXL4), as a direct target of TGF-β1 in aortic endothelial cells, and we dissected the molecular mechanism of its induction. Deletion mapping and mutagenesis analysis of the LOXL4 promoter demonstrated the absolute requirement of a distal enhancer containing an activator protein 1 (AP-1) site and a Smad binding element for TGF-β1 to induce LOXL4 expression. Functional cooperation between Smad proteins and the AP-1 complex composed of JunB/Fra2 accounted for the action of TGF-β1, which involved the extracellular signal-regulated kinase (ERK)- dependent phosphorylation of Fra2. We furthermore provide evidence that LOXL4 was extracellularly secreted and significantly contributed to ECM deposition and assembly. These results suggest that TGF-β1-dependent expression of LOXL4 plays a role in vascular ECM homeostasis, contributing to vascular processes associated with ECM remodeling and fibrosis.This work was supported by grants from the Ministerio de Economía y Competitividad (Plan Nacional de I+D+I: SAF2009-09085, SAF2012-34916), Comunidad Autónoma de Madrid (2010-BMD2321, FIBROTEAM Consortium), Fundación Genoma España (MEICA project), Consejo Superior de Investigaciones Científicas (Proyecto Intramural de Incorporación, 200920I158), and Fundación Renal Iñigo Alvárez de Toledo. O.B. is a recipient of a fellowship from the Ministerio de Economía y Competi- tividad (Formación de Personal Investigador)Peer Reviewe

    The Cellular Prion Protein PrPc Is Involved in the Proliferation of Epithelial Cells and in the Distribution of Junction-Associated Proteins

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    BACKGROUND: The physiological function of the ubiquitous cellular prion protein, PrP(c), is still under debate. It was essentially studied in nervous system, but poorly investigated in epithelial cells. We previously reported that PrP(c) is targeted to cell-cell junctions of polarized epithelial cells, where it interacts with c-Src. METHODOLOGY/FINDINGS: We show here that, in cultured human enterocytes and in intestine in vivo, the mature PrP(c) is differentially targeted either to the nucleus in dividing cells or to cell-cell contacts in polarized/differentiated cells. By proteomic analysis, we demonstrate that the junctional PrP(c) interacts with cytoskeleton-associated proteins, such as gamma- and beta-actin, alpha-spectrin, annexin A2, and with the desmosome-associated proteins desmoglein, plakoglobin and desmoplakin. In addition, co-immunoprecipitation experiments revealed complexes associating PrP(c), desmoglein and c-Src in raft domains. Through siRNA strategy, we show that PrP(c) is necessary to complete the process of epithelial cell proliferation and for the sub-cellular distribution of proteins involved in cell architecture and junctions. Moreover, analysis of the architecture of the intestinal epithelium of PrP(c) knock-out mice revealed a net decrease in the size of desmosomal junctions and, without change in the amount of BrdU incorporation, a shortening of the length of intestinal villi. CONCLUSIONS/SIGNIFICANCE: From these results, PrP(c) could be considered as a new partner involved in the balance between proliferation and polarization/differentiation in epithelial cells

    SoxF factors induce Notch1 expression via direct transcriptional regulation during early arterial development.

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    Arterial specification and differentiation are influenced by a number of regulatory pathways. While it is known that the Vegfa-Notch cascade plays a central role, the transcriptional hierarchy controlling arterial specification has not been fully delineated. To elucidate the direct transcriptional regulators of Notch receptor expression in arterial endothelial cells, we used histone signatures, DNaseI hypersensitivity and ChIP-seq data to identify enhancers for the human NOTCH1 and zebrafish notch1b genes. These enhancers were able to direct arterial endothelial cell-restricted expression in transgenic models. Genetic disruption of SoxF binding sites established a clear requirement for members of this group of transcription factors (SOX7, SOX17 and SOX18) to drive the activity of these enhancers in vivo Endogenous deletion of the notch1b enhancer led to a significant loss of arterial connections to the dorsal aorta in Notch pathway-deficient zebrafish. Loss of SoxF function revealed that these factors are necessary for NOTCH1 and notch1b enhancer activity and for correct endogenous transcription of these genes. These findings position SoxF transcription factors directly upstream of Notch receptor expression during the acquisition of arterial identity in vertebrates.This work was supported by the National Health and Medical Research Council of Australia (NHMRC) (APP1107643); The Cancer Council Queensland (1107631) (M.Fran.); the Australian Research Council Discovery Project (DP140100485) and a Career Development Fellowship (APP1111169) (M.Fran.); the Ludwig Institute for Cancer Research (M.Frit., A.N., I.R., S.D.V.); the Medical Research Council (MR/J007765/1) (K.L., G.B.-G., S.D.V.); the Fondazione Cariplo (2011-0555) (M.B., B.H., M.Fran.); and the Biotechnology and Biological Sciences Research Council (BB/L020238/1) (A.N., K.L., G.B.-G., S.D.V.)

    Rôle de la Lysyl oxidase-like-2 dans l'assemblage de la matrice extracellulaire et l'angiogenèse

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    Au cours de l angiogenèse, la matrice extracellulaire (MEC) est profondément remodelée. Les mécanismes moléculaires impliqués dans l assemblage de MEC et leur rôle dans l angiogenèse sont encore peu connus. L équipe a identifié LOXL2 comme étant une cible majeure de l hypoxie dans la MEC des cellules endothéliales, exprimée dans les capillaires au cours du développement et dans un modèle d ischémie. Dans des modèles d angiogenèse in vivo et in vitro nous avons montré que LOXL2 est impliquée dans la formation de capillaires. La perte d expression de LOXL2 entraine une diminution importante de l incorporation du collagène IV dans la MEC aussi bien chez l embryon de poisson zèbre que dans les HUVEC in vitro. De plus, la perte d expression de la chaine 1 du collagène IV dans les HUVEC inhibe la tubulogenèse de façon similaire à la perte d expression de LOXL2, suggérant que l interaction de LOXL2 avec le collagène de type IV est à même de médier les effets décrits. Alors que la perte d expression de LOXL2 altère fortement l angiogenèse et l assemblage du collagène IV les effets de l inhibition pharmacologique de son activité catalytique sont très limités. Par une approche structure-fonction menée in vivo et in vitro nous avons démontré que l activité catalytique de LOXL2 n est impliquée ni dans la formation de capillaires ni dans l assemblage du collagène IV. LOXL2 apparaît donc comme un régulateur majeur de l angiogenèse. L induction de son expression par des facteurs pro-angiogéniques initie l assemblage du collagène IV dans la lame basale constituant ainsi un microenvironnement optimal pour la formation d un vaisseau fonctionnelSprouting angiogenesis is associated with extensive extracellular matrix (ECM) remodeling. The molecular mechanisms involved in building the vascular microenvironment and its impact on capillary formation remain elusive. We identified lysyl oxidase like protein 2 (LOXL2) which accumulates in the endothelial hypoxic ECM. We showed that LOXL2 is expressed in capillaries during developmental and pathological angiogenesis. Knocking-down LOXL2 proper organization of endothelial cells and formation of capillaries, resulting in non functional intersegmental vessels (ISV) in zebrafish embryos. Surprisingly, pharmacological inhibition of lysyl oxidase activity did not affect ISV formation. Further investigation in a 3D culture model confirmed that LOXL2 expression was required for capillary formation. Pharmacological inhibition of LOXL2 enzymatic activity only slightly affected lumen formation, suggesting that mechanisms independent of LOXL2 enzymatic activity were responsible for defective capillary morphogenesis. We hypothesized that LOXL2 could regulates organization of the vascular basement membrane. Whereas knocking-down LOXL2 expression led to inhibition of collagen IV assembly, inhibition of LOXL2 enzymatic activity only affected collagen IV crosslinking. In conclusion, we show that LOXL2 regulates neovessel formation through assembly of the vascular basal lamina and collagen IV organization and provide further novel evidence that LOXL2 regulates sprouting angiogenesis independently of its lysyl oxidase activityPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

    Lymphatic vascular specification and its modulation during embryonic development

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    Despite its essential roles in development and disease, the lymphatic vascular system has been less studied than the blood vascular network. In recent years, significant advances have been made in understanding the mechanisms that regulate lymphatic vessel formation, both during development and in pathological conditions. Remarkably, lymphatic endothelial cells are specified as a subpopulation of pre-existing venous endothelial cells. Here, we summarize the current knowledge of the transcription factor pathways responsible for lymphatic specification and we also focus on the factors that promote or restrict this event

    Vegfc Regulates Bipotential Precursor Division and Prox1 Expression to Promote Lymphatic Identity in Zebrafish

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    Lymphatic vessels arise chiefly from preexisting embryonic veins. Genetic regulators of lymphatic fate are known, but how dynamic cellular changes contribute during the acquisition of lymphatic identity is not understood. We report the visualization of zebrafish lymphatic precursor cell dynamics during fate restriction. In the cardinal vein, cellular commitment is linked with the division of bipotential Prox1-positive precursor cells, which occurs immediately prior to sprouting angiogenesis. Following precursor division, identities are established asymmetrically in daughter cells; one daughter cell becomes lymphatic and progressively upregulates Prox1, and the other downregulates Prox1 and remains in the vein. Vegfc drives cell division and Prox1 expression in lymphatic daughter cells, coupling signaling dynamics with daughter cell fate restriction and precursor division

    Dominant-negative Sox18 function inhibits dermal papilla maturation and differentiation in all murine hair types

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    SOX family proteins SOX2 and SOX18 have been reported as being essential in determining hair follicle type; however, the role they play during development remains unclear. Here, we demonstrate that Sox18 regulates the normal differentiation of the dermal papilla of all hair types. In guard (primary) hair dermal condensate (DC) cells, we identified transient Sox18 in addition to SOX2 expression at E14.5, which allowed fate tracing of primary DC cells until birth. Similarly, expression of Sox18 was detected in the DC cells of secondary hairs at E16.5 and in tertiary hair at E18.5. Dominant-negative Sox18 mutation (opposum) did not prevent DC formation in any hair type. However, it affected dermal papilla differentiation, restricting hair formation especially in secondary and tertiary hairs. This Sox18 mutation also prevented neonatal dermal cells or dermal papilla spheres from inducing hair in regeneration assays. Microarray expression studies identified WNT5A and TNC as potential downstream effectors of SOX18 that are important for epidermal WNT signalling. In conclusion, SOX18 acts as a mesenchymal molecular switch necessary for the formation and function of the dermal papilla in all hair types

    Vegfc/d-dependent regulation of the lymphatic vasculature during cardiac regeneration is influenced by injury context

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    The lymphatic vasculature mediates essential physiological functions including fluid homeostasis, lipid and hormone transport, and immune cell trafficking. Recent studies have suggested that promoting lymphangiogenesis enhances cardiac repair following injury, but it is unknown whether lymphangiogenesis is required for cardiac regeneration. Here, we describe the anatomical distribution, regulation, and function of the cardiac lymphatic network in a highly regenerative zebrafish model system using transgenic reporter lines and loss-of-function approaches. We show that zebrafish lacking functional vegfc and vegfd signaling are devoid of a cardiac lymphatic network and display cardiac hypertrophy in the absence of injury, suggesting a role for these vessels in cardiac tissue homeostasis. Using two different cardiac injury models, we report a robust lymphangiogenic response following cryoinjury, but not following apical resection injury. Although the majority of mutants lacking functional vegfc and vegfd signaling were able to mount a full regenerative response even in the complete absence of a cardiac lymphatic vasculature, cardiac regeneration was severely impaired in a subset of mutants, which was associated with heightened pro-inflammatory cytokine signaling. These findings reveal a context-dependent requirement for the lymphatic vasculature during cardiac growth and regeneration
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