RESUMEN
During the last decades intermediate filaments (IFs) have emerged as important regulators of cellular signaling events, ascribing IFs with functions beyond the structural support they provide. The organ and developmental stage-specific expression of IFs regulate cell differentiation within developing or remodeling tissues. Lack of IFs causes perturbed stem cell differentiation in vasculature, intestine, nervous system, and mammary gland, in transgenic mouse models. The aberrant cell fate decisions are caused by deregulation of different stem cell signaling pathways, such as Notch, Wnt, YAP/TAZ, and TGFß. Mutations in genes coding for IFs cause an array of different diseases, many related to stem cell dysfunction, but the molecular mechanisms remain unresolved. Here, we provide a comprehensive overview of how IFs interact with and regulate the activity, localization and function of different signaling proteins in stem cells, and how the assembly state and PTM profile of IFs may affect these processes. Identifying when, where and how IFs and cell signaling congregate, will expand our understanding of IF-linked stem cell dysfunction during development and disease.
Asunto(s)
Diferenciación Celular , Proteínas de Filamentos Intermediarios/metabolismo , Filamentos Intermedios/metabolismo , Mutación , Células Madre/metabolismo , Vía de Señalización Wnt , Animales , Humanos , Proteínas de Filamentos Intermediarios/genética , Filamentos Intermedios/genética , Filamentos Intermedios/patología , Células Madre/patologíaRESUMEN
Breaking symmetry in populations of uniform cells, to induce adoption of an alternative cell fate, is an essential developmental mechanism. Similarly, domain and boundary establishment are crucial steps to forming organs during development. Notch signaling is a pathway ideally suited to mediating precise patterning cues, as both receptors and ligands are membrane-bound and can thus act as a precise switch to toggle cell fates on or off. Fine-tuning of signaling by positive or negative feedback mechanisms dictate whether signaling results in lateral induction or lateral inhibition, respectively, allowing Notch to either induce entire regions of cell specification, or dictate binary fate choices. Furthermore, pathway activity is modulated by Fringe modification of receptors or ligands, co-expression of receptors with ligands, mode of ligand presentation, and cell surface area in contact. In this review, we describe how Notch signaling is fine-tuned to mediate lateral induction or lateral inhibition cues, and discuss examples from C.elegans, D. melanogaster and M. musculus. Identifying the cellular machinery dictating the choice between lateral induction and lateral inhibition highlights the versatility of the Notch signaling pathway in development.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Diferenciación Celular/fisiología , Proteínas de Drosophila/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Ratones , Receptores Notch/genéticaRESUMEN
Notch signaling is a key regulator of angiogenesis, in which sprouting is regulated by an equilibrium between inhibitory Dll4-Notch signaling and promoting Jagged-Notch signaling. Whereas Fringe proteins modify Notch receptors and strengthen their activation by Dll4 ligands, other mechanisms balancing Jagged and Dll4 signaling are yet to be described. The intermediate filament protein vimentin, which has been previously shown to affect vascular integrity and regenerative signaling, is here shown to regulate ligand-specific Notch signaling. Vimentin interacts with Jagged, impedes basal recycling endocytosis of ligands, but is required for efficient receptor ligand transendocytosis and Notch activation upon receptor binding. Analyses of Notch signal activation by using chimeric ligands with swapped intracellular domains (ICDs), demonstrated that the Jagged ICD binds to vimentin and contributes to signaling strength. Vimentin also suppresses expression of Fringe proteins, whereas depletion of vimentin enhances Fringe levels to promote Dll4 signaling. In line with these data, the vasculature in vimentin knockout (VimKO) embryos and placental tissue is underdeveloped with reduced branching. Disrupted angiogenesis in aortic rings from VimKO mice and in endothelial 3D sprouting assays can be rescued by reactivating Notch signaling by recombinant Jagged ligands. Taken together, we reveal a function of vimentin and demonstrate that vimentin regulates Notch ligand signaling activities during angiogenesis.
Asunto(s)
Neovascularización Fisiológica , Receptores Notch/metabolismo , Vimentina/metabolismo , Animales , Aorta/metabolismo , Embrión de Pollo , Endocitosis , Células Endoteliales de la Vena Umbilical Humana , Humanos , Técnicas In Vitro , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Ligandos , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Activación Transcripcional , Vimentina/deficiencia , Vimentina/genéticaRESUMEN
BACKGROUND & AIMS: Alagille syndrome is a genetic disorder characterized by cholestasis, ocular abnormalities, characteristic facial features, heart defects, and vertebral malformations. Most cases are associated with mutations in JAGGED1 (JAG1), which encodes a Notch ligand, although it is not clear how these contribute to disease development. We aimed to develop a mouse model of Alagille syndrome to elucidate these mechanisms. METHODS: Mice with a missense mutation (H268Q) in Jag1 (Jag1+/Ndr mice) were outbred to a C3H/C57bl6 background to generate a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice). Liver tissues were collected at different timepoints during development, analyzed by histology, and liver organoids were cultured and analyzed. We performed transcriptome analysis of Jag1Ndr/Ndr livers and livers from patients with Alagille syndrome, cross-referenced to the Human Protein Atlas, to identify commonly dysregulated pathways and biliary markers. We used species-specific transcriptome separation and ligand-receptor interaction assays to measure Notch signaling and the ability of JAG1Ndr to bind or activate Notch receptors. We studied signaling of JAG1 and JAG1Ndr via NOTCH 1, NOTCH2, and NOTCH3 and resulting gene expression patterns in parental and NOTCH1-expressing C2C12 cell lines. RESULTS: Jag1Ndr/Ndr mice had many features of Alagille syndrome, including eye, heart, and liver defects. Bile duct differentiation, morphogenesis, and function were dysregulated in newborn Jag1Ndr/Ndr mice, with aberrations in cholangiocyte polarity, but these defects improved in adult mice. Jag1Ndr/Ndr liver organoids collapsed in culture, indicating structural instability. Whole-transcriptome sequence analyses of liver tissues from mice and patients with Alagille syndrome identified dysregulated genes encoding proteins enriched at the apical side of cholangiocytes, including CFTR and SLC5A1, as well as reduced expression of IGF1. Exposure of Notch-expressing cells to JAG1Ndr, compared with JAG1, led to hypomorphic Notch signaling, based on transcriptome analysis. JAG1-expressing cells, but not JAG1Ndr-expressing cells, bound soluble Notch1 extracellular domain, quantified by flow cytometry. However, JAG1 and JAG1Ndr cells each bound NOTCH2, and signaling from NOTCH2 signaling was reduced but not completely inhibited, in response to JAG1Ndr compared with JAG1. CONCLUSIONS: In mice, expression of a missense mutant of Jag1 (Jag1Ndr) disrupts bile duct development and recapitulates Alagille syndrome phenotypes in heart, eye, and craniofacial dysmorphology. JAG1Ndr does not bind NOTCH1, but binds NOTCH2, and elicits hypomorphic signaling. This mouse model can be used to study other features of Alagille syndrome and organ development.
Asunto(s)
Síndrome de Alagille/genética , Proteína Jagged-1/genética , Mutación Missense , Síndrome de Alagille/metabolismo , Síndrome de Alagille/patología , Animales , Conductos Biliares Intrahepáticos/metabolismo , Conductos Biliares Intrahepáticos/patología , Diferenciación Celular , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Proteína Jagged-1/metabolismo , Masculino , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfogénesis , Organoides , Fenotipo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Transducción de Señal , TransfecciónRESUMEN
The Notch pathway is a conserved signaling pathway and a form of direct cell-cell communication related to many biological processes during development and adulthood. Deregulation of the Notch pathway is involved in many diseases, including cancer. Almost 20% of all cancer cases have an infectious etiology, with viruses responsible for at least 1.5 million new cancer cases per year. Seven groups of viruses have been classified as oncogenic: hepatitis B and C viruses (HBV and HCV respectively), Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV), human T lymphotropic virus (HTLV-1), human papillomavirus (HPV), and Merkel cell polyomavirus (MCPyV). These viruses share the ability to manipulate a variety of cell pathways that are critical in proliferation and differentiation, leading to malignant transformation. Viral proteins interact directly or indirectly with different members of the Notch pathway, altering their normal function. This review focuses exclusively on the direct interactions of viral oncoproteins with Notch elements, providing a deeper understanding of the dual behavior of the Notch pathway as activator or suppressor of neoplasia in virus-related cancers.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Transformación Celular Viral , Virus Oncogénicos/fisiología , Receptores Notch/metabolismo , Transducción de Señal , Animales , Biomarcadores , Susceptibilidad a Enfermedades , Humanos , Neoplasias/etiología , Neoplasias/metabolismo , Neoplasias/patología , Unión Proteica , Receptores Notch/químicaRESUMEN
Adult neurogenesis is regulated by a number of cellular players within the neurogenic niche. Astrocytes participate actively in brain development, regulation of the mature central nervous system (CNS), and brain plasticity. They are important regulators of the local environment in adult neurogenic niches through the secretion of diffusible morphogenic factors, such as Wnts. Astrocytes control the neurogenic niche also through membrane-associated factors, however, the identity of these factors and the mechanisms involved are largely unknown. In this study, we sought to determine the mechanisms underlying our earlier finding of increased neuronal differentiation of neural progenitor cells when cocultured with astrocytes lacking glial fibrillary acidic protein (GFAP) and vimentin (GFAP(-/-) Vim(-/-) ). We used primary astrocyte and neurosphere cocultures to demonstrate that astrocytes inhibit neuronal differentiation through a cell-cell contact. GFAP(-/-) Vim(-/-) astrocytes showed reduced endocytosis of Notch ligand Jagged1, reduced Notch signaling, and increased neuronal differentiation of neurosphere cultures. This effect of GFAP(-/-) Vim(-/-) astrocytes was abrogated in the presence of immobilized Jagged1 in a manner dependent on the activity of γ-secretase. Finally, we used GFAP(-/-) Vim(-/-) mice to show that in the absence of GFAP and vimentin, hippocampal neurogenesis under basal conditions as well as after injury is increased. We conclude that astrocytes negatively regulate neurogenesis through the Notch pathway, and endocytosis of Notch ligand Jagged1 in astrocytes and Notch signaling from astrocytes to neural stem/progenitor cells depends on the intermediate filament proteins GFAP and vimentin.
Asunto(s)
Astrocitos/metabolismo , Proteínas de Unión al Calcio/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Neurogénesis/genética , Receptores Notch/genética , Vimentina/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Astrocitos/citología , Proteínas de Unión al Calcio/metabolismo , Comunicación Celular/genética , Diferenciación Celular , Técnicas de Cocultivo , Endocitosis , Regulación del Desarrollo de la Expresión Génica , Proteína Ácida Fibrilar de la Glía , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Cultivo Primario de Células , Receptores Notch/metabolismo , Proteínas Serrate-Jagged , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo , Vimentina/deficiencia , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Spontaneous bleeds are a leading cause of death in the pediatric JAG1-related liver disease Alagille syndrome (ALGS). We asked whether there are sex differences in bleeding events in patients, whether Jag1Ndr/Ndr mice display bleeds or vascular defects, and whether discovered vascular pathology can be confirmed in patients non-invasively. We performed a systematic review of patients with ALGS and vascular events following PRISMA guidelines, in the context of patient sex, and found significantly more girls than boys reported with spontaneous intracranial hemorrhage. We investigated vascular development, homeostasis, and bleeding in Jag1Ndr/Ndr mice, using retina as a model. Jag1Ndr/Ndr mice displayed sporadic brain bleeds, a thin skull, tortuous blood vessels, sparse arterial smooth muscle cell coverage in multiple organs, which could be aggravated by hypertension, and sex-specific venous defects. Importantly, we demonstrated that retinographs from patients display similar characteristics with significantly increased vascular tortuosity. In conclusion, there are clinically important sex differences in vascular disease in ALGS, and retinography allows non-invasive vascular analysis in patients. Finally, Jag1Ndr/Ndr mice represent a new model for vascular compromise in ALGS.
Asunto(s)
Síndrome de Alagille , Femenino , Masculino , Animales , Ratones , Síndrome de Alagille/complicaciones , Caracteres Sexuales , Retina , Factores de RiesgoRESUMEN
Notch is an evolutionary, conserved, cell-cell signaling pathway that is central to several biological processes, from tissue morphogenesis to homeostasis. It is therefore not surprising that several genetic mutations of Notch components cause inherited human diseases, especially cardiovascular disorders. Despite numerous efforts, current in vivo models are still insufficient to unravel the underlying mechanisms of these pathologies, hindering the development of utmost needed medical therapies. In this perspective review, we discuss the limitations of current murine models and outline how the combination of microphysiological systems (MPSs) and targeted computational models can lead to breakthroughs in this field. In particular, while MPSs enable the experimentation on human cells in controlled and physiological environments, in silico models can provide a versatile tool to translate the in vitro findings to the more complex in vivo setting. As a showcase example, we focus on Notch-related cardiovascular diseases, such as Alagille syndrome, Adams-Oliver syndrome, and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Impact statement In this review, a comprehensive overview of the limitations of current in vivo models of genetic Notch cardiovascular diseases is provided, followed by a discussion over the potential of microphysiological systems and computational models in overcoming these limitations and in potentiating drug testing and modeling of these pathologies.
Asunto(s)
Enfermedades Cardiovasculares , Displasia Ectodérmica , Deformidades Congénitas de las Extremidades , Animales , Enfermedades Cardiovasculares/genética , Humanos , Ratones , Receptores Notch/genética , Transducción de SeñalRESUMEN
The intermediate filament (IF) cytoskeleton has been proposed to regulate morphogenic processes by integrating the cell fate signaling machinery with mechanical cues. Signaling between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) through the Notch pathway regulates arterial remodeling in response to changes in blood flow. Here we show that the IF-protein vimentin regulates Notch signaling strength and arterial remodeling in response to hemodynamic forces. Vimentin is important for Notch transactivation by ECs and vimentin knockout mice (VimKO) display disrupted VSMC differentiation and adverse remodeling in aortic explants and in vivo. Shear stress increases Jagged1 levels and Notch activation in a vimentin-dependent manner. Shear stress induces phosphorylation of vimentin at serine 38 and phosphorylated vimentin interacts with Jagged1 and increases Notch activation potential. Reduced Jagged1-Notch transactivation strength disrupts lateral signal induction through the arterial wall leading to adverse remodeling. Taken together we demonstrate that vimentin forms a central part of a mechanochemical transduction pathway that regulates multilayer communication and structural homeostasis of the arterial wall.
Asunto(s)
Aorta/metabolismo , Hemodinámica , Receptores Notch/metabolismo , Transducción de Señal , Estrés Fisiológico , Remodelación Vascular , Vimentina/metabolismo , Animales , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Ratones , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptores Notch/genética , Activación Transcripcional , Vimentina/genéticaRESUMEN
Activation of Notch signaling requires intracellular routing of the receptor, but the mechanisms controlling the distinct steps in the routing process is poorly understood. We identify PKCζ as a key regulator of Notch receptor intracellular routing. When PKCζ was inhibited in the developing chick central nervous system and in cultured myoblasts, Notch-stimulated cells were allowed to undergo differentiation. PKCζ phosphorylates membrane-tethered forms of Notch and regulates two distinct routing steps, depending on the Notch activation state. When Notch is activated, PKCζ promotes re-localization of Notch from late endosomes to the nucleus and enhances production of the Notch intracellular domain, which leads to increased Notch activity. In the non-activated state, PKCζ instead facilitates Notch receptor internalization, accompanied with increased ubiquitylation and interaction with the endosomal sorting protein Hrs. Collectively, these data identify PKCζ as a key regulator of Notch trafficking and demonstrate that distinct steps in intracellular routing are differentially modulated depending on Notch signaling status.