RESUMEN
Functional genomic approaches have facilitated the discovery of rare genetic disorders and improved efforts to decipher their underlying etiology. PPP2R5D-related disorder is an early childhood onset condition characterized by intellectual disability, hypotonia, autism-spectrum disorder, macrocephaly, and dysmorphic features. The disorder is caused by de novo single nucleotide changes in PPP2R5D, which generate heterozygous dominant missense variants. PPP2R5D is known to encode a B'-type (B'56δ) regulatory subunit of a PP2A-serine/threonine phosphatase. To help elucidate the molecular mechanisms altered in PPP2R5D-related disorder, we used a CRISPR-single-base editor to generate HEK-293 cells in which a single transition (c.1258G>A) was introduced into one allele, precisely recapitulating a clinically relevant E420K variant. Unbiased quantitative proteomic and phosphoproteomic analyses of endogenously expressed proteins revealed heterozygous-dominant changes in kinase/phosphatase signaling. These data combined with orthogonal validation studies revealed a previously unrecognized interaction of PPP2R5D with AKT in human cells, leading to constitutively active AKT-mTOR signaling, increased cell size, and uncoordinated cellular growth in E420K-variant cells. Rapamycin reduced cell size and dose-dependently reduced RPS6 phosphorylation in E420K-variant cells, suggesting that inhibition of mTOR1 can suppress both the observed RPS6 hyperphosphorylation and increased cell size. Together, our findings provide a deeper understanding of PPP2R5D and insight into how the E420K-variant alters signaling networks influenced by PPP2R5D. Our comprehensive approach, which combines precise genome editing, isobaric tandem mass tag labeling of peptides generated from endogenously expressed proteins, and concurrent liquid chromatography-mass spectrometry (LC-MS3), also provides a roadmap that can be used to rapidly explore the etiologies of additional genetic disorders.
Asunto(s)
Enfermedades Genéticas Congénitas/genética , Predisposición Genética a la Enfermedad , Proteína Fosfatasa 2/genética , Proteómica , Serina-Treonina Quinasas TOR/genética , Trastorno Autístico/genética , Trastorno Autístico/patología , Sistemas CRISPR-Cas/genética , Enfermedades Genéticas Congénitas/patología , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Megalencefalia/genética , Megalencefalia/patología , Mutación/genética , Polimorfismo de Nucleótido Simple/genética , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
Endoglin and activin receptor-like kinase 1 are specialized transforming growth factor-beta (TGF-ß) superfamily receptors, primarily expressed in endothelial cells. Mutations in the corresponding ENG or ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT1 and HHT2 respectively). To discover proteins interacting with endoglin, ACVRL1 and TGF-ß receptor type 2 and involved in TGF-ß signaling, we applied LUMIER, a high-throughput mammalian interactome mapping technology. Using stringent criteria, we identified 181 novel unique and shared interactions with ACVRL1, TGF-ß receptor type 2, and endoglin, defining potential novel important vascular networks. In particular, the regulatory subunit B-beta of the protein phosphatase PP2A (PPP2R2B) interacted with all three receptors. Interestingly, the PPP2R2B gene lies in an interval in linkage disequilibrium with HHT3, for which the gene remains unidentified. We show that PPP2R2B protein interacts with the ACVRL1/TGFBR2/endoglin complex and recruits PP2A to nitric oxide synthase 3 (NOS3). Endoglin overexpression in endothelial cells inhibits the association of PPP2R2B with NOS3, whereas endoglin-deficient cells show enhanced PP2A-NOS3 interaction and lower levels of endogenous NOS3 Serine 1177 phosphorylation. Our data suggest that endoglin regulates NOS3 activation status by regulating PPP2R2B access to NOS3, and that PPP2R2B might be the HHT3 gene. Furthermore, endoglin and ACVRL1 contribute to several novel networks, including TGF-ß dependent and independent ones, critical for vascular function and potentially defective in HHT.
Asunto(s)
Receptores de Activinas Tipo II/metabolismo , Antígenos CD/metabolismo , Vasos Sanguíneos/metabolismo , Mapas de Interacción de Proteínas , Receptores de Superficie Celular/metabolismo , Animales , Embrión de Mamíferos , Endoglina , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Unión Proteica , Telangiectasia Hemorrágica Hereditaria/metabolismo , Telangiectasia Hemorrágica Hereditaria/patología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Basic mechanisms by which cellular barriers sense and respond to integrity disruptions remain poorly understood. Despite its tenuous structure and constitutive exposure to disruptive strains, the vascular endothelium exhibits robust barrier function. We show that in response to micrometer-scale disruptions induced by transmigrating leukocytes, endothelial cells generate unique ventral lamellipodia that propagate via integrins toward and across these "micro-wounds" to close them. This novel actin remodeling activity progressively healed multiple micro-wounds in succession and changed direction during this process. Mechanical probe-induced micro-wounding of both endothelia and epithelia suggests that ventral lamellipodia formed as a response to force imbalance and specifically loss of isometric tension. Ventral lamellipodia were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of hydrogen peroxide. Together with Apr2/3, these were functionally required for effective micro-wound healing. We propose that barrier disruptions are detected as local release of isometric tension/force unloading, which is directly coupled to reactive oxygen species-dependent self-restorative actin remodeling dynamics.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/fisiología , Seudópodos/fisiología , Migración Transendotelial y Transepitelial , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Fenómenos Biomecánicos , Adhesión Celular , Células Cultivadas , Técnicas de Cocultivo , Cortactina/metabolismo , Humanos , Linfocitos/fisiología , Microscopía Fluorescente , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico , Imagen de Lapso de Tiempo , Cicatrización de Heridas , Proteína de Unión al GTP rac1/metabolismoRESUMEN
OBJECTIVE: To test the hypothesis that high circulating concentrations of maternal anti-angiogenic factors are associated with increased risk of respiratory distress syndrome (RDS). STUDY DESIGN: This is a nested case-control study of nulliparous women who delivered less than 37 weeks of gestation within the Calcium for Preeclampsia Prevention (CPEP) trial. The study included 116 women with preeclampsia or gestational hypertension and 323 normotensive controls. Soluble fms-like tyrosine kinase 1 (sFlt1), placental growth factor (PlGF) and soluble endoglin (sEng) in maternal serum were measured at 21-32 weeks of gestation. RESULTS: Preterm infants born to hypertensive mothers were more likely to develop RDS (22.5% vs. 20.9%, p = 0.03). After adjustment for gestational age at delivery, the odds ratio for the relationship between hypertension in pregnancy and RDS was 2.18 (95% CI 1.08-4.39). In hypertensive pregnancies women whose infants developed RDS had significantly higher circulating mean sFlt1 levels during midpregnancy (21-32 weeks of gestation) even after adjustment for gestational age at delivery (21,516 pg/mL vs. 7,000 pg/mL, p = 0.01). CONCLUSIONS: Preterm preeclampsia and gestational hypertension, characterized by high circulating levels of sFlt1, are associated with a twofold increased risk of RDS in infants delivered before 37 weeks. Among women with these hypertensive pregnancies circulating sFlt1 concentrations during midpregnancy were substantially higher in women whose infants developed RDS.
Asunto(s)
Inhibidores de la Angiogénesis/sangre , Hipertensión Inducida en el Embarazo/sangre , Síndrome de Dificultad Respiratoria del Recién Nacido/etiología , Adolescente , Adulto , Inhibidores de la Angiogénesis/análisis , Estudios de Casos y Controles , Método Doble Ciego , Femenino , Humanos , Hipertensión Inducida en el Embarazo/epidemiología , Recién Nacido , Recien Nacido Prematuro/fisiología , Embarazo , Ensayos Clínicos Controlados Aleatorios como Asunto , Síndrome de Dificultad Respiratoria del Recién Nacido/sangre , Síndrome de Dificultad Respiratoria del Recién Nacido/epidemiología , Factores de Riesgo , Adulto JovenRESUMEN
OBJECTIVE: Loss-of-function mutations in genes coding for transforming growth factor-beta/bone morphogenetic protein receptors and changes in nitric oxide(*) (NO(*)) bioavailability are associated with hereditary hemorrhagic telangiectasia and some forms of pulmonary arterial hypertension. How these abnormalities lead to seemingly disparate pulmonary pathologies remains unknown. Endoglin (Eng), a transforming growth factor-beta coreceptor, is mutated in hereditary hemorrhagic telangiectasia and involved in regulating endothelial NO(*) synthase (eNOS)-derived NO(*) production and oxidative stress. Because some patients with pulmonary arterial hypertension harbor ENG mutations leading to haplo insufficiency, we investigated the pulmonary vasculature of Eng(+/-) mice and the potential contribution of abnormal eNOS activation to pulmonary arterial hypertension. METHODS AND RESULTS: Hemodynamic, histological, and biochemical assessments and x-ray micro-CT imaging of adult Eng(+/-) mice indicated signs of pulmonary arterial hypertension including increased right ventricular systolic pressure, degeneration of the distal pulmonary vasculature, and muscularization of small arteries. These findings were absent in 3-week-old Eng(+/-) mice and were attributable to constitutively uncoupled eNOS activity in the pulmonary circulation, as evidenced by reduced eNOS/heat shock protein 90 association and increased eNOS-derived superoxide ((*)O(2)(-)) production in a BH(4)-independent manner. These changes render eNOS unresponsive to regulation by transforming growth factor-beta/bone morphogenetic protein and underlie the signs of pulmonary arterial hypertension that were prevented by Tempol. CONCLUSIONS: Adult Eng(+/-) mice acquire signs of pulmonary arterial hypertension that are attributable to uncoupled eNOS activity and increased (*)O(2)(-) production, which can be prevented by antioxidant treatment. Eng links transforming growth factor/bone morphogenetic protein receptors to the eNOS activation complex, and its reduction in the pulmonary vasculature leads to increased oxidative stress and pulmonary arterial hypertension.
Asunto(s)
Endotelio Vascular/fisiopatología , Hipertensión Pulmonar/fisiopatología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Estrés Oxidativo/fisiología , Telangiectasia Hemorrágica Hereditaria/fisiopatología , Animales , Antioxidantes/uso terapéutico , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Óxidos N-Cíclicos/uso terapéutico , Modelos Animales de Enfermedad , Endoglina , Endotelio Vascular/metabolismo , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/prevención & control , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos C57BL , Mutación , Óxido Nítrico Sintasa de Tipo III/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Marcadores de Spin , Telangiectasia Hemorrágica Hereditaria/genética , Telangiectasia Hemorrágica Hereditaria/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Preeclampsia is a pregnancy-specific hypertensive syndrome that causes substantial maternal and fetal morbidity and mortality. Maternal endothelial dysfunction mediated by excess placenta-derived soluble VEGF receptor 1 (sVEGFR1 or sFlt1) is emerging as a prominent component in disease pathogenesis. We report a novel placenta-derived soluble TGF-beta coreceptor, endoglin (sEng), which is elevated in the sera of preeclamptic individuals, correlates with disease severity and falls after delivery. sEng inhibits formation of capillary tubes in vitro and induces vascular permeability and hypertension in vivo. Its effects in pregnant rats are amplified by coadministration of sFlt1, leading to severe preeclampsia including the HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome and restriction of fetal growth. sEng impairs binding of TGF-beta1 to its receptors and downstream signaling including effects on activation of eNOS and vasodilation, suggesting that sEng leads to dysregulated TGF-beta signaling in the vasculature. Our results suggest that sEng may act in concert with sFlt1 to induce severe preeclampsia.
Asunto(s)
Antígenos CD/metabolismo , Preeclampsia/metabolismo , Preñez , Receptores de Superficie Celular/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Endoglina , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Edad Gestacional , Hemodinámica , Humanos , Riñón/metabolismo , Riñón/patología , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados , Persona de Mediana Edad , Datos de Secuencia Molecular , Óxido Nítrico Sintasa de Tipo III/metabolismo , Placenta/metabolismo , Placenta/patología , Preeclampsia/etiología , Preeclampsia/fisiopatología , Embarazo , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/genética , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta1 , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
OBJECTIVE: To determine if angiogenesis is altered in adult Endoglin heterozygous (Eng(+/-)) mice, the animal model for the vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1). METHODS: Primary cultures of endothelial cells were generated from Eng(+/-) and Eng(+/+) mice and analyzed for proliferation, migration, and ability to form capillary-like tubes. Endothelial cells derived from umbilical veins of newborns (HUVEC) with an HHT1 genotype were also tested for capillary formation. Two in vivo models of angiogenesis were tested in the Eng(+/-) and Eng(+/+) mice: Matrigel implant-dependent angiogenesis and reperfusion following hindlimb ischemia. RESULTS: The Eng(+/-) endothelial cells displayed significantly reduced proliferation and migration, increased collagen production, and decreased NO synthase expression and vascular endothelial growth factor (VEGF) secretion. They also showed impaired capillary tube formation in vitro, as did the HHT1 HUVEC. These endothelial cell-specific abnormalities were associated with impaired Matrigel-dependent capillary tube formation in vivo and delayed reperfusion following hindlimb ischemia. CONCLUSIONS: Although vascular development is normal in Eng(+/-) mice, angiogenic abnormalities were observed in the adult mice and their isolated endothelial cells. These results suggest that a normal level of endoglin is required for full angiogenic activity.
Asunto(s)
Células Endoteliales/fisiología , Endotelio Vascular/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Telangiectasia Hemorrágica Hereditaria/metabolismo , Animales , Northern Blotting/métodos , Western Blotting/métodos , Movimiento Celular , Proliferación Celular , Colágeno/metabolismo , Endoglina , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Heterocigoto , Miembro Posterior/irrigación sanguínea , Péptidos y Proteínas de Señalización Intracelular/genética , Isquemia/metabolismo , Isquemia/patología , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Neovascularización Fisiológica , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We tested the hypothesis that induction of neuronal NO synthase (nNOS) impairs vascular smooth muscle contractility after hypoxia. nNOS protein was increased in aorta, mesenteric arterioles, pulmonary arteries, brain, and diaphragm from rats exposed to 8% O2 for 48 hours and in human aortic SMCs after hypoxic incubation (1% O2). Ca-dependent NO synthase activity was increased in endothelium-denuded aortic segments from hypoxia-exposed rats. N-nitro-L-arginine methyl ester enhanced the contractile responses of endothelium-denuded aortic rings and mesenteric arterioles from hypoxia-exposed but not normoxic rats (P < 0.05). The hypoxia-inducible mRNA transcript expressed by human cells was found to contain a novel 5'-untranslated region, consistent with activation of transcription in the genomic region contiguous with exon 2. Translational efficiency of this transcript is markedly increased compared with previously described human nNOS mRNAs. Transgenic mice possessing a lacZ reporter construct under control of these genomic sequences demonstrated expression of the construct after exposure to hypoxia (8% O2, 48 hours) in the aorta, mesenteric arterioles, renal papilla, and brain. These results reveal a novel human nNOS promoter that confers the ability to rapidly upregulate nNOS expression in response to hypoxia with a functionally significant effect on vascular smooth muscle contraction.
Asunto(s)
Variación Genética , Hipoxia/enzimología , Óxido Nítrico Sintasa de Tipo I/genética , ARN Mensajero/biosíntesis , Animales , Aorta Torácica/metabolismo , Western Blotting , Genes Reporteros , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Óxido Nítrico Sintasa de Tipo I/biosíntesis , Regiones Promotoras Genéticas , Biosíntesis de Proteínas/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Decreased endothelial NO synthase (eNOS)-derived NO bioavailability and impaired vasomotor control are crucial factors in cardiovascular disease pathogenesis. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a vascular disorder associated with ENDOGLIN (ENG) haploinsufficiency and characterized by venous dilatations, focal loss of capillaries, and arteriovenous malformations (AVMs). We report that resistance arteries from Eng+/- mice display an eNOS-dependent enhancement in endothelium-dependent dilatation and impairment in the myogenic response, despite reduced eNOS levels. We have found that eNOS is significantly reduced in endoglin-deficient endothelial cells because of decreased eNOS protein half-life. We demonstrate that endoglin can reside in caveolae and associate with eNOS, suggesting a stabilizing function of endoglin for eNOS. After Ca2+-induced activation, endoglin-deficient endothelial cells have reduced eNOS/Hsp90 association, produce less NO, and generate more eNOS-derived superoxide (O2-), indicating that endoglin also facilitates eNOS/Hsp90 interactions and is an important regulator in the coupling of eNOS activity. Treatment with an O2- scavenger reverses the vasomotor abnormalities in Eng(+/-) arteries, suggesting that uncoupled eNOS and resulting impaired myogenic response represent early events in HHT1 pathogenesis and that the use of antioxidants may provide a novel therapeutic modality.
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Péptidos y Proteínas de Señalización Intracelular/fisiología , Óxido Nítrico Sintasa/fisiología , Resistencia Vascular/fisiología , Acetilcolina/farmacología , Animales , Antígenos CD , Presión Sanguínea/efectos de los fármacos , Caveolina 1 , Caveolinas/análisis , Células Cultivadas/efectos de los fármacos , Regulación hacia Abajo , Endoglina , Endotelio Vascular/fisiología , Activación Enzimática/fisiología , Proteínas HSP90 de Choque Térmico/análisis , Proteínas HSP90 de Choque Térmico/fisiología , Heterocigoto , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Hígado/enzimología , Microdominios de Membrana/química , Microdominios de Membrana/enzimología , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/enzimología , Arterias Mesentéricas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/fisiología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Nitroprusiato/farmacología , Fenilefrina/farmacología , Receptores de Superficie Celular , Transducción de Señal/fisiología , Superóxido Dismutasa/farmacología , Telangiectasia Hemorrágica Hereditaria/enzimología , Telangiectasia Hemorrágica Hereditaria/genética , Venas Umbilicales/citología , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/fisiología , Resistencia Vascular/genética , Vasodilatación/efectos de los fármacosRESUMEN
BACKGROUND AND PURPOSE: Hereditary hemorrhagic telangiectasia type 1 (HHT1) is an autosomal dominant vascular dysplasia caused by mutations in the endoglin gene and characterized by dilated vessels and arteriovenous malformations (AVMs). To understand the etiology of this disorder, we evaluated the cerebral vasculature of endoglin heterozygous (Eng+/-) mice, which represent the only animal model of HHT1. METHODS: The cerebral vasculature of Eng+/- and Eng+/+ mice from C57BL/6 (B6) and 129/Ola (129) strains with a differential susceptibility to HHT1 was studied with corrosion casting. Casts were observed by scanning electron microscopy to detect malformations and evaluate arterial diameters and orientation of endothelial nuclei. Measurements were taken to assess relative constriction at arteriolar branching points and downstream relative dilatation. RESULTS: Three of 10 Eng+/- mice demonstrated abnormal vascular findings including AVMs, while none of 15 Eng+/+ mice did. The incidence of relative constriction at arteriolar branching points was significantly less in both Eng+/- groups than in their Eng+/+ counterparts. The occurrence of relative dilatation was significantly greater in B6-Eng+/- than in B6-Eng+/+ mice. Endothelial nuclei were significantly rounder and deviated more from the direction of blood flow in Eng+/- than in Eng+/+ mice. CONCLUSIONS: Eng+/- mice showed significant structural alterations in cerebral blood vessels, indicating that the level of endoglin on endothelium is critical for maintenance of normal vasculature. Since endoglin haploinsufficiency is associated with HHT1, such changes in arteriolar structures might occur in HHT1 patients and predispose them to AVMs and their sequelae.