Asunto(s)
Hemangioma Cavernoso del Sistema Nervioso Central , Humanos , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Hemangioma Cavernoso del Sistema Nervioso Central/fisiopatología , Animales , Proteína KRIT1/metabolismo , Proteína KRIT1/genética , Proteínas PortadorasRESUMEN
KRIT1 is a 75 kDa scaffolding protein which regulates endothelial cell phenotype by limiting the response to inflammatory stimuli and maintaining a quiescent and stable endothelial barrier. Loss-of-function mutations in KRIT1 lead to the development of cerebral cavernous malformations (CCM), a disease marked by the formation of abnormal blood vessels which exhibit a loss of barrier function, increased endothelial proliferation, and altered gene expression. While many advances have been made in our understanding of how KRIT1, and the functionally related proteins CCM2 and PDCD10, contribute to the regulation of blood vessels and the vascular barrier, some important open questions remain. In addition, KRIT1 is widely expressed and KRIT1 and the other CCM proteins have been shown to play important roles in non-endothelial cell types and tissues, which may or may not be related to their role as pathogenic originators of CCM. In this review, we discuss some of the unsettled questions regarding the role of KRIT1 in vascular physiology and discuss recent advances that suggest this ubiquitously expressed protein may have a role beyond the endothelial cell.
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Células Endoteliales , Hemangioma Cavernoso del Sistema Nervioso Central , Proteína KRIT1 , Humanos , Proteína KRIT1/metabolismo , Proteína KRIT1/genética , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Animales , Células Endoteliales/metabolismo , Células Endoteliales/patología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , MutaciónRESUMEN
Significance: KRIT1 (Krev interaction trapped 1) is a scaffolding protein that plays a critical role in vascular morphogenesis and homeostasis. Its loss-of-function has been unequivocally associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease of genetic origin characterized by defective endothelial cell-cell adhesion and ensuing structural alterations and hyperpermeability in brain capillaries. KRIT1 contributes to the maintenance of endothelial barrier function by stabilizing the integrity of adherens junctions and inhibiting the formation of actin stress fibers. Recent Advances: Among the multiple regulatory mechanisms proposed so far, significant evidence accumulated over the past decade has clearly shown that the role of KRIT1 in the stability of endothelial barriers, including the blood-brain barrier, is largely based on its involvement in the complex machinery governing cellular redox homeostasis and responses to oxidative stress and inflammation. KRIT1 loss-of-function has, indeed, been demonstrated to cause an impairment of major redox-sensitive mechanisms involved in spatiotemporal regulation of cell adhesion and signaling, which ultimately leads to decreased cell-cell junction stability and enhanced sensitivity to oxidative stress and inflammation. Critical Issues: This review explores the redox mechanisms that influence endothelial cell adhesion and barrier function, focusing on the role of KRIT1 in such mechanisms. We propose that this supports a novel model wherein redox signaling forms the common link between the various pathogenetic mechanisms and therapeutic approaches hitherto associated with CCM disease. Future Directions: A comprehensive characterization of the role of KRIT1 in redox control of endothelial barrier physiology and defense against oxy-inflammatory insults will provide valuable insights into the development of precision medicine strategies. Antioxid. Redox Signal. 38, 496-528.
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Hemangioma Cavernoso del Sistema Nervioso Central , Humanos , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Células Endoteliales/metabolismo , Transducción de Señal , Oxidación-Reducción , Inflamación , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína KRIT1/metabolismoRESUMEN
Loss of Krev interaction-trapped-1 (KRIT1) expression leads to the development of cerebral cavernous malformations (CCM), a disease in which abnormal blood vessel formation compromises the structure and function of the blood-brain barrier. The role of KRIT1 in regulating endothelial function is well-established. However, several studies have suggested that KRIT1 could also play a role in regulating nonendothelial cell types and, in particular, immune cells. In this study, we generated a mouse model with neutrophil-specific deletion of KRIT1 in order to investigate the effect of KRIT1 deficiency on neutrophil function. Neutrophils isolated from adult Ly6Gtm2621(cre)Arte Krit1flox/flox mice had a reduced ability to attach and spread on the extracellular matrix protein fibronectin and exhibited a subsequent increase in migration. However, adhesion to and migration on ICAM-1 was unchanged. In addition, we used a monomeric, fluorescently-labelled fragment of fibronectin to show that integrin activation is reduced in the absence of KRIT1 expression, though ß1 integrin expression appears unchanged. Finally, neutrophil migration in response to lipopolysaccharide-induced inflammation in the lung was decreased, as shown by reduced cell number and myeloperoxidase activity in lavage samples from Krit1PMNKO mice. Altogether, we show that KRIT1 regulates neutrophil adhesion and migration, likely through regulation of integrin activation, which can lead to altered inflammatory responses in vivo.
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Adhesión Celular , Movimiento Celular , Proteína KRIT1 , Neutrófilos , Animales , Ratones , Adhesión Celular/genética , Adhesión Celular/fisiología , Movimiento Celular/genética , Movimiento Celular/fisiología , Fibronectinas , Integrina beta1/metabolismo , Proteína KRIT1/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neutrófilos/metabolismo , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
The loss function of cerebral cavernous malformation (CCM) genes leads to most CCM lesions characterized by enlarged leaking vascular lesions in the brain. Although we previously showed that NOGOB receptor (NGBR) knockout in endothelial cells (ECs) results in cerebrovascular lesions in the mouse embryo, the molecular mechanism by which NGBR regulates CCM1/2 expression has not been elucidated. Here, we show that genetic depletion of Ngbr in ECs at both postnatal and adult stages results in CCM1/2 expression deficiency and cerebrovascular lesions such as enlarged vessels, blood-brain-barrier hyperpermeability, and cerebral hemorrhage. To reveal the molecular mechanism, we used RNA-sequencing analysis to examine changes in the transcriptome. Surprisingly, we found that the acetyltransferase HBO1 and histone acetylation were downregulated in NGBR-deficient ECs. The mechanistic studies elucidated that NGBR is required for maintaining the expression of CCM1/2 in ECs via HBO1-mediated histone acetylation. ChIP-qPCR data further demonstrated that loss of NGBR impairs the binding of HBO1 and acetylated histone H4K5 and H4K12 on the promotor of the CCM1 and CCM2 genes. Our findings on epigenetic regulation of CCM1 and CCM2 that is modulated by NGBR and HBO1-mediated histone H4 acetylation provide a perspective on the pathogenesis of sporadic CCMs.
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Hemangioma Cavernoso del Sistema Nervioso Central , Histonas , Proteína KRIT1 , Proteínas de Microfilamentos , Receptores de Superficie Celular , Animales , Ratones , Acetilación , Células Endoteliales/metabolismo , Epigénesis Genética , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemorragia , Histonas/genética , Histonas/metabolismo , Proteína KRIT1/metabolismo , Proteínas de Microfilamentos/metabolismo , Permeabilidad , Receptores de Superficie Celular/metabolismoRESUMEN
Cancer-secreted exosomes are critical mediators of cancer-host crosstalk. In the present study, we showed the delivery of miR-21-5p from colorectal cancer (CRC) cells to endothelial cells via exosomes increased the amount of miR-21-5p in recipient cells. MiR-21-5p suppressed Krev interaction trapped protein 1 (KRIT1) in recipient HUVECs and subsequently activated ß-catenin signaling pathway and increased their downstream targets VEGFa and Ccnd1, which consequently promoted angiogenesis and vascular permeability in CRC. A strong inverse correlation between miR-21-5p and KRIT1 expression levels was observed in CRC-adjacent vessels. Furthermore, miR-21-5p expression in circulating exosomes was markedly higher in CRC patients than in healthy donors. Thus, our data suggest that exosomal miR-21-5p is involved in angiogenesis and vascular permeability in CRC and may be used as a potential new therapeutic target.
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Neoplasias Colorrectales/irrigación sanguínea , Proteína KRIT1/metabolismo , MicroARNs/metabolismo , Animales , Permeabilidad Capilar , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Embrión de Pollo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Exosomas/genética , Exosomas/metabolismo , Células HCT116 , Células HT29 , Xenoinjertos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteína KRIT1/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/genética , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Microambiente TumoralRESUMEN
Cerebral cavernous malformations (CCMs) are vascular lesions that affect predominantly microvasculature in the brain and spinal cord. CCM can occur either in sporadic or familial form, characterized by autosomal dominant inheritance and development of multiple lesions throughout the patient's life. Three genes associated with CCM are known: CCM1/KRIT1 (krev interaction trapped 1), CCM2/MGC4607 (encoding a protein named malcavernin), and CCM3/PDCD10 (programmed cell death 10). All the mutations identified in these genes cause a loss of function and compromise the protein functions needed for maintaining the vascular barrier integrity. Loss of function of CCM proteins causes molecular disorganization and dysfunction of endothelial adherens junctions. In this review, we provide an overall vision of the CCM pathology, starting with the genetic bases of the disease, describing the role of the proteins, until we reach the cellular level. Thus, we summarize the genetics of CCM, providing a description of CCM genes and mutation features, provided an updated knowledge of the CCM protein structure and function, and discuss the molecular mechanisms through which CCM proteins may act within endothelial cells, particularly in endothelial barrier maintenance/regulation and in cellular signaling.
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Proteínas Reguladoras de la Apoptosis/genética , Encéfalo/irrigación sanguínea , Proteínas Portadoras/genética , Malformaciones Vasculares del Sistema Nervioso Central/genética , Células Endoteliales/metabolismo , Proteína KRIT1/genética , Proteínas de la Membrana/genética , Mutación , Proteínas Proto-Oncogénicas/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Portadoras/metabolismo , Malformaciones Vasculares del Sistema Nervioso Central/metabolismo , Malformaciones Vasculares del Sistema Nervioso Central/patología , Células Endoteliales/patología , Predisposición Genética a la Enfermedad , Humanos , Proteína KRIT1/metabolismo , Proteínas de la Membrana/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de SeñalRESUMEN
KRIT1 is a scaffolding protein that regulates multiple molecular mechanisms, including cell-cell and cell-matrix adhesion, and redox homeostasis and signaling. However, rather little is known about how KRIT1 is itself regulated. KRIT1 is found in both the cytoplasm and the nucleus, yet the upstream signaling proteins and mechanisms that regulate KRIT1 nucleocytoplasmic shuttling are not well understood. Here, we identify a key role for protein kinase C (PKC) in this process. In particular, we found that PKC activation promotes the redox-dependent cytoplasmic localization of KRIT1, whereas inhibition of PKC or treatment with the antioxidant N-acetylcysteine leads to KRIT1 nuclear accumulation. Moreover, we demonstrated that the N-terminal region of KRIT1 is crucial for the ability of PKC to regulate KRIT1 nucleocytoplasmic shuttling, and may be a target for PKC-dependent regulatory phosphorylation events. Finally, we found that silencing of PKCα, but not PKCδ, inhibits phorbol 12-myristate 13-acetate (PMA)-induced cytoplasmic enrichment of KRIT1, suggesting a major role for PKCα in regulating KRIT1 nucleocytoplasmic shuttling. Overall, our findings identify PKCα as a novel regulator of KRIT1 subcellular compartmentalization, thus shedding new light on the physiopathological functions of this protein.
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Transporte Activo de Núcleo Celular , Proteína KRIT1/metabolismo , Proteína Quinasa C-alfa , Células HeLa , Humanos , Fosforilación , Proteína Quinasa C-alfa/genética , Acetato de TetradecanoilforbolRESUMEN
Krev interaction trapped protein 1 (KRIT1) is a scaffold protein known to form functional complexes with distinct proteins, including Malcavernin, PDCD10, Rap1 and others. It appears involved in several cellular signaling pathways and exerts a protective role against inflammation and oxidative stress. KRIT1 has been studied as a regulator of endothelial cell functions and represents a determinant in the pathogenesis of Cerebral Cavernous Malformation (CCM), a cerebrovascular disease characterized by the formation of clusters of abnormally dilated and leaky blood capillaries, which predispose to seizures, neurological deficits and intracerebral hemorrhage. Although KRIT1 is ubiquitously expressed, few studies have described its involvement in pathologies other than CCM including cancer. Cutaneous melanoma represents the most fatal skin cancer due to its high metastatic propensity. Despite the numerous efforts made to define the signaling pathways activated during melanoma progression, the molecular mechanisms at the basis of melanoma growth, phenotype plasticity and resistance to therapies are still under investigation.
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Proteína KRIT1/metabolismo , Melanoma/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Movimiento Celular/fisiología , Núcleo Celular/metabolismo , Proliferación Celular/genética , Regulación hacia Abajo , Femenino , Técnicas de Silenciamiento del Gen/métodos , Humanos , Proteína KRIT1/genética , Masculino , Melanocitos/metabolismo , Melanoma/patología , Persona de Mediana Edad , beta Catenina/metabolismoRESUMEN
Cerebral cavernous malformations (CCMs) is a disorder of endothelial cells predominantly localized in the brain. Although a complete inactivation of each CCM protein has been found in the affected endothelium of diseased patients and a necessary and additional role of microenvironment has been demonstrated to determine in vivo the occurrence of vascular lesions, a microvascular endothelial model based on knockdown of a CCM gene represents today a convenient method to study some of critical signaling events regulating pathogenesis of CCM. For these reasons, in our laboratory we developed a microvascular cerebral endothelial model of Krit1 deficiency performing silencing experiments of CCM1 gene (Krit1) with siRNA procedure.
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Endotelio Vascular/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/diagnóstico , Fenotipo , Biomarcadores , Técnicas de Cultivo de Célula , Susceptibilidad a Enfermedades , Células Endoteliales/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Hemangioma Cavernoso del Sistema Nervioso Central/etiología , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Humanos , Proteína KRIT1/genética , Proteína KRIT1/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
Cerebral cavernous malformation (CCM) is a vascular malformation of the central nervous system that is associated with leaky capillaries, and a predisposition to serious clinical conditions including intracerebral hemorrhage and seizures. Germline or sporadic mutations in the CCM1/KRIT1 gene are responsible for the majority of cases of CCM. In this article, we describe the original characterization of the CCM1/KRIT1 gene. This cloning was done through the use of a variant of the yeast two-hybrid screen known as the interaction trap, using the RAS-family GTPase KREV1/RAP1A as a bait. The partial clone of KRIT1 (Krev1 Interaction Trapped) initially identified was extended through 5'RACE and computational analysis to obtain a full-length cDNA, then used in a sequential screen to define the integrin-associated ICAP1 protein as a KRIT1 partner protein. We discuss how these interactions are relevant to the current understanding of KRIT1/CCM1 biology, and provide a protocol for library screening with the Interaction Trap.
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Estudios de Asociación Genética , Proteína KRIT1/genética , Técnicas del Sistema de Dos Híbridos , Estudios de Asociación Genética/métodos , Genotipo , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Humanos , Proteína KRIT1/metabolismo , Mutación , Biblioteca de Péptidos , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Transformación Genética , Flujo de TrabajoRESUMEN
Cerebral cavernous malformation (CCM) proteins play critical roles for endothelial cell functions, including cytoskeletal remodeling, cell-cell interactions, cell polarity, tube formation, and angiogenesis. It has been shown that the mutation of even one of the CCM genes involved in CCMs can determine an alteration in the angiogenesis process, but the precise mechanism is yet to be clarified.Here using a model of cerebral microvascular endothelial cells (hBMEC) transiently silenced by CCM1, we tried to mimic the physiological conditions that occur in the presence of CCM1 gene know-down evaluating their ability to form tube structures through an in vitro angiogenesis assay.
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Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Microvasos/metabolismo , Neovascularización Patológica/metabolismo , Fenotipo , Técnicas de Cultivo de Célula , Células Cultivadas , Hemangioma Cavernoso del Sistema Nervioso Central/etiología , Humanos , Proteína KRIT1/genética , Proteína KRIT1/metabolismoRESUMEN
Cerebral cavernous malformations (CCM) or cavernomas are slow-flow capillary vascular malformations with a mulberry-like appearance, which are predominantly located in the central nervous system. CCM can occur in a sporadic or a familial form. The latter is inherited in an autosomal dominant manner, and in the majority of the fragile lesions, mutations in the genes CCM1 (KRIT1), CCM2 (OSM), or CCM3 (PDCD10) can be detected. Loss of these genes leads to numerous alterations in endothelial cell signaling resulting in a disturbed vessel architecture and function. Lower activity of Notch signaling occurs upon loss of CCM1, CCM3, or the CCM1-interacting protein ICAP1 in cell culture and animal models. Notch signaling in endothelial cells is an essential regulator of angiogenesis, arterial-venous differentiation, vascular permeability and stability, mural cell recruitment, and flux of metabolites across the vessel wall. The purpose of this chapter is to briefly summarize the current understanding of Notch signaling in familial CCM and to provide a protocol for detecting cleaved Notch1 receptor proteins on paraformaldehyde-fixed paraffin-embedded mouse tissue.
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Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Dominios y Motivos de Interacción de Proteínas , Receptor Notch1/metabolismo , Transducción de Señal , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Biomarcadores , Células Endoteliales/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/etiología , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Humanos , Inmunohistoquímica , Proteína KRIT1/genética , Proteína KRIT1/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Neovascularización Patológica/metabolismo , Pericitos/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptor Notch1/químicaRESUMEN
Glyoxalase 1 (Glo1) is a glutathione (GSH)-dependent enzyme that catalyzes the isomerization of the hemithioacetal formed non-enzymatically from methylglyoxal (MG) and GSH to S-D-lactoylglutathione (SLG). The activity of Glo1 is measured spectrophotometrically by following the increase of absorbance at 240 nm and 25 °C, attributable to the formation of SLG. The hemithioacetal is preformed by incubation of 2 mM MG and 1 mM GSH in 0.1 M sodium phosphate buffer (PBS) pH 7.2, at 25 °C for 10 min. The cell extract is then added, and the A240 is monitored over 5-min incubation against correction for blank. Glo1 activity is given in units per mg of protein where one unit activity is defined as 1 µmole of SLG produced per min under assay conditions. Here, we describe measurement of Glo1 activity in established cellular models of cerebral cavernous malformation (CCM) disease, including KRIT1-knockout mouse embryonic fibroblast (MEF) and KRIT1-silenced human brain microvascular endothelial (hBMEC) cells.
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Hemangioma Cavernoso del Sistema Nervioso Central/enzimología , Lactoilglutatión Liasa/metabolismo , Espectrofotometría , Animales , Activación Enzimática , Fibroblastos/enzimología , Hemangioma Cavernoso del Sistema Nervioso Central/etiología , Humanos , Proteína KRIT1/genética , Proteína KRIT1/metabolismo , Lactoilglutatión Liasa/química , Ratones , Estrés Oxidativo , Espectrofotometría/métodosRESUMEN
Cerebral cavernous malformations (CCMs) are microvascular anomalies in the brain that result in increased susceptibility to stroke. Three genes have been identified as causes of CCMs: cerebral cavernous malformations 1 [(CCM1) also termed Krev interaction trapped 1 (KRIT1)], cerebral cavernous malformation 2 [(CCM2) also termed MGC4607] and cerebral cavernous malformation 3 [(CCM3) also termed programmed cell death 10 (PDCD10)]. It has been demonstrated that both CCM1 and CCM3 bind to CCM2 to form a CCM signaling complex (CSC) with which to modulate multiple signaling cascades. CCM proteins have been reported to play major roles in microvascular angiogenesis in human and animal models. However, CCM proteins are ubiquitously expressed in all major tissues, suggesting an unseen broader role of the CSC in biogenesis. Recent evidence suggests the possible involvement of the CSC complex during tumorigenesis; however, studies concerning this aspect are limited. This is the first report to systematically investigate the expression patterns of CCM proteins in major human tumors using realtime quantitative PCR, RNAfluorescence in situ hybridization, immunohistochemistry and multicolor immunofluorescence imaging. Our data demonstrated that differential expression patterns of the CSC complex are correlated with certain types and grades of major human cancers, indicating the potential application of CCM genes as molecular biomarkers for clinical oncology. Our data strongly suggest that more efforts are needed to elucidate the role of the CSC complex in tumorigenesis, which may have enormous clinical potential for cancer diagnostic, prognostic and therapeutic applications.
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Proteínas Reguladoras de la Apoptosis/genética , Proteínas Portadoras/genética , Proteína KRIT1/genética , Proteínas de la Membrana/genética , Neoplasias/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proteínas Portadoras/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Proteína KRIT1/metabolismo , Proteínas de la Membrana/metabolismo , Clasificación del Tumor , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
Nuclear accumulation of the small phosphoprotein integrin cytoplasmic domain-associated protein-1 (ICAP1) results in recruitment of its binding partner, Krev/Rap1 interaction trapped-1 (KRIT1), to the nucleus. KRIT1 loss is the most common cause of cerebral cavernous malformation, a neurovascular dysplasia resulting in dilated, thin-walled vessels that tend to rupture, increasing the risk for hemorrhagic stroke. KRIT1's nuclear roles are unknown, but it is known to function as a scaffolding or adaptor protein at cell-cell junctions and in the cytosol, supporting normal blood vessel integrity and development. As ICAP1 controls KRIT1 subcellular localization, presumably influencing KRIT1 function, in this work, we investigated the signals that regulate ICAP1 and, hence, KRIT1 nuclear localization. ICAP1 contains a nuclear localization signal within an unstructured, N-terminal region that is rich in serine and threonine residues, several of which are reportedly phosphorylated. Using quantitative microscopy, we revealed that phosphorylation-mimicking substitutions at Ser-10, or to a lesser extent at Ser-25, within this N-terminal region inhibit ICAP1 nuclear accumulation. Conversely, phosphorylation-blocking substitutions at these sites enhanced ICAP1 nuclear accumulation. We further demonstrate that p21-activated kinase 4 (PAK4) can phosphorylate ICAP1 at Ser-10 both in vitro and in cultured cells and that active PAK4 inhibits ICAP1 nuclear accumulation in a Ser-10-dependent manner. Finally, we show that ICAP1 phosphorylation controls nuclear localization of the ICAP1-KRIT1 complex. We conclude that serine phosphorylation within the ICAP1 N-terminal region can prevent nuclear ICAP1 accumulation, providing a mechanism that regulates KRIT1 localization and signaling, potentially influencing vascular development.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Núcleo Celular/metabolismo , Serina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Células CHO , Dominio Catalítico , Cricetinae , Cricetulus , Humanos , Proteína KRIT1/metabolismo , Mutagénesis Sitio-Dirigida , Fosforilación , Quinasas p21 Activadas/química , Quinasas p21 Activadas/metabolismoRESUMEN
Cerebral cavernous malformation (CCM) is a cerebrovascular disorder of proven genetic origin characterized by abnormally dilated and leaky capillaries occurring mainly in the central nervous system, with a prevalence of 0.3-0.5% in the general population. Genetic studies have identified causative mutations in three genes, CCM1/KRIT1, CCM2 and CCM3, which are involved in the maintenance of vascular homeostasis. However, distinct studies in animal models have clearly shown that CCM gene mutations alone are not sufficient to cause CCM disease, but require additional contributing factors, including stochastic events of increased oxidative stress and inflammation. Consistently, previous studies have shown that up-regulation of NADPH oxidase-mediated production of reactive oxygen species (ROS) in KRIT1 deficient endothelium contributes to the loss of microvessel barrier function. In this study, we demonstrate that KRIT1 loss-of-function in stromal cells, such as fibroblasts, causes the up-regulation of NADPH oxidase isoform 1 (NOX1) and the activation of inflammatory pathways, which in turn promote an enhanced production of proangiogenic factors, including vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2). Furthermore and importantly, we show that conditioned media from KRIT1 null fibroblasts induce proliferation, migration, matrix metalloproteinase 2 (MMP2) activation and VE-cadherin redistribution in wild type human endothelial cells. Taken together, our results demonstrate that KRIT1 loss-of-function in stromal cells affects the surrounding microenvironment through a NOX1-mediated induction and release of angiogenic factors that are able to promote paracrine proangiogenic responses in human endothelial cells, thus pointing to a novel role for endothelial cell-nonautonomous effects of KRIT1 mutations in CCM pathogenesis, and opening new perspectives for disease prevention and treatment.
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Proteína KRIT1/metabolismo , NADPH Oxidasa 1/metabolismo , Neovascularización Fisiológica , Comunicación Paracrina , Regulación hacia Arriba , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Ciclooxigenasa 2/metabolismo , Dinoprostona/biosíntesis , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones Noqueados , Neovascularización Fisiológica/efectos de los fármacos , Comunicación Paracrina/efectos de los fármacos , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Cerebral cavernous malformation (CCM) is a genetic, cerebrovascular disease. Familial CCM is caused by genetic mutations in KRIT1, CCM2, or PDCD10 Disease onset is earlier and more severe in individuals with PDCD10 mutations. Recent studies have shown that lesions arise from excess mitogen-activated protein kinase kinase kinase 3 (MEKK3) signaling downstream of Toll-like receptor 4 (TLR4) stimulation by lipopolysaccharide derived from the gut microbiome. These findings suggest a gut-brain CCM disease axis but fail to define it or explain the poor prognosis of patients with PDCD10 mutations. Here, we demonstrate that the gut barrier is a primary determinant of CCM disease course, independent of microbiome configuration, that explains the increased severity of CCM disease associated with PDCD10 deficiency. Chemical disruption of the gut barrier with dextran sulfate sodium augments CCM formation in a mouse model, as does genetic loss of Pdcd10, but not Krit1, in gut epithelial cells. Loss of gut epithelial Pdcd10 results in disruption of the colonic mucosal barrier. Accordingly, loss of Mucin-2 or exposure to dietary emulsifiers that reduce the mucus barrier increases CCM burden analogous to loss of Pdcd10 in the gut epithelium. Last, we show that treatment with dexamethasone potently inhibits CCM formation in mice because of the combined effect of action at both brain endothelial cells and gut epithelial cells. These studies define a gut-brain disease axis in an experimental model of CCM in which a single gene is required for two critical components: gut epithelial function and brain endothelial signaling.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Encéfalo/metabolismo , Tracto Gastrointestinal/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Encéfalo/patología , Proteínas Portadoras/metabolismo , Colitis/complicaciones , Dexametasona/farmacología , Dexametasona/uso terapéutico , Sulfato de Dextran , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Microbioma Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/patología , Hemangioma Cavernoso del Sistema Nervioso Central/tratamiento farmacológico , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Proteína KRIT1/metabolismo , Ligandos , Ratones , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismoRESUMEN
Loss-of-function mutations of the gene encoding Krev interaction trapped protein 1 (KRIT1) are associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries and affecting 0.5% of the human population. However, growing evidence demonstrates that KRIT1 is implicated in the modulation of major redox-sensitive signaling pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, suggesting that its loss-of-function mutations may have pathological effects not limited to CCM disease. The aim of this study was to address whether KRIT1 loss-of-function predisposes to the development of pathological conditions associated with enhanced endothelial cell susceptibility to oxidative stress and inflammation, such as arterial endothelial dysfunction (ED) and atherosclerosis. Silencing of KRIT1 in human aortic endothelial cells (HAECs), coronary artery endothelial cells (HCAECs), and umbilical vein endothelial cells (HUVECs) resulted in increased expression of endothelial proinflammatory adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) and in enhanced susceptibility to tumor necrosis factor alpha (TNF-α)-induced apoptosis. These effects were associated with a downregulation of Notch1 activation that could be rescued by antioxidant treatment, suggesting that they are consequent to altered intracellular redox homeostasis induced by KRIT1 loss-of-function. Furthermore, analysis of the aorta of heterozygous KRIT1+/- mice fed a high-fructose diet to induce systemic oxidative stress and inflammation demonstrated a 1.6-fold increased expression of VCAM-1 and an approximately 2-fold enhanced fat accumulation (7.5% vs 3.6%) in atherosclerosis-prone regions, including the aortic arch and aortic root, as compared to corresponding wild-type littermates. In conclusion, we found that KRIT1 deficiency promotes ED, suggesting that, besides CCM, KRIT1 may be implicated in genetic susceptibility to the development of atherosclerotic lesions.
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Aorta/metabolismo , Aterosclerosis/genética , Endotelio Vascular/metabolismo , Proteína KRIT1/genética , Mutación con Pérdida de Función , Animales , Aorta/patología , Apoptosis , Aterosclerosis/metabolismo , Endotelio Vascular/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Proteína KRIT1/deficiencia , Proteína KRIT1/metabolismo , Metabolismo de los Lípidos , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Receptor Notch1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
BACKGROUND: The CRISPR/Cas9 system has opened new perspectives to study the molecular basis of cerebral cavernous malformations (CCMs) in personalized disease models. However, precise genome editing in endothelial and other hard-to-transfect cells remains challenging. METHODS: In a proof-of-principle study, we first isolated blood outgrowth endothelial cells (BOECs) from a CCM1 mutation carrier with multiple CCMs. In a CRISPR/Cas9 gene correction approach, a high-fidelity Cas9 variant was then transfected into patient-derived BOECs using a ribonucleoprotein complex and a single-strand DNA oligonucleotide. In addition, patient-specific CCM1 knockout clones were expanded after CRISPR/Cas9 gene inactivation. RESULTS: Deep sequencing demonstrated correction of the mutant allele in nearly 33% of all cells whereas no CRISPR/Cas9-induced mutations in predicted off-target loci were identified. Corrected BOECs could be cultured in cell mixtures but demonstrated impaired clonal survival. In contrast, CCM1-deficient BOECs displayed increased resistance to stress-induced apoptotic cell death and could be clonally expanded to high passages. When cultured together, CCM1-deficient BOECs largely replaced corrected as well as heterozygous BOECs. CONCLUSION: We here demonstrate that a non-viral CRISPR/Cas9 approach can not only be used for gene knockout but also for precise gene correction in hard-to-transfect endothelial cells (ECs). Comparing patient-derived isogenic CCM1+/+ , CCM1+/- , and CCM1-/- ECs, we show that the inactivation of the second allele results in clonal evolution of ECs lacking CCM1 which likely reflects the initiation phase of CCM genesis.