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1.
J Am Soc Nephrol ; 29(6): 1706-1719, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29773687

RESUMEN

BackgroundNedd4-2 is an E3 ubiquitin-protein ligase that associates with transport proteins, causing their ubiquitylation, and then internalization and degradation. Previous research has suggested a correlation between Nedd4-2 and BP. In this study, we explored the effect of intercalated cell (IC) Nedd4-2 gene ablation on IC transporter abundance and function and on BP.Methods We generated IC Nedd4-2 knockout mice using Cre-lox technology and produced global pendrin/Nedd4-2 null mice by breeding global Nedd4-2 null (Nedd4-2-/- ) mice with global pendrin null (Slc26a4-/- ) mice. Mice ate a diet with 1%-4% NaCl; BP was measured by tail cuff and radiotelemetry. We measured transepithelial transport of Cl- and total CO2 and transepithelial voltage in cortical collecting ducts perfused in vitro Transporter abundance was detected with immunoblots, immunohistochemistry, and immunogold cytochemistry.Results IC Nedd4-2 gene ablation markedly increased electroneutral Cl-/HCO3- exchange in the cortical collecting duct, although benzamil-, thiazide-, and bafilomycin-sensitive ion flux changed very little. IC Nedd4-2 gene ablation did not increase the abundance of type B IC transporters, such as AE4 (Slc4a9), H+-ATPase, barttin, or the Na+-dependent Cl-/HCO3- exchanger (Slc4a8). However, IC Nedd4-2 gene ablation increased CIC-5 total protein abundance, apical plasma membrane pendrin abundance, and the ratio of pendrin expression on the apical membrane to the cytoplasm. IC Nedd4-2 gene ablation increased BP by approximately 10 mm Hg. Moreover, pendrin gene ablation eliminated the increase in BP observed in global Nedd4-2 knockout mice.Conclusions IC Nedd4-2 regulates Cl-/HCO3- exchange in ICs., Nedd4-2 gene ablation increases BP in part through its action in these cells.


Asunto(s)
Presión Sanguínea/genética , Canales Epiteliales de Sodio/metabolismo , Transporte Iónico/genética , Ubiquitina-Proteína Ligasas Nedd4/genética , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Bicarbonatos/metabolismo , Membrana Celular/metabolismo , Canales de Cloruro/metabolismo , Antiportadores de Cloruro-Bicarbonato/metabolismo , Cloruros/metabolismo , Intercambio Iónico , Túbulos Renales Colectores/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , ATPasas de Translocación de Protón/metabolismo , Protones , Reabsorción Renal/efectos de los fármacos , Simportadores de Sodio-Bicarbonato/metabolismo , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo , Tiazidas/farmacología
2.
Genesis ; 55(7)2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28481036

RESUMEN

Interferon Regulatory Factor 6 (IRF6) is a critical regulator of differentiation, proliferation, and migration of keratinocytes. Mutations in IRF6 cause two autosomal dominant disorders characterized by cleft lip with or without cleft palate. In addition, DNA variation in IRF6 confers significant risk for non-syndromic cleft lip and palate. IRF6 is also implicated in adult onset development and disease processes, including mammary gland development and squamous cell carcinoma. Mice homozygous for a null allele of Irf6 die shortly after birth due to severe skin, limb, and craniofacial defects, thus impeding the study of gene function after birth. To circumvent this, a conditional allele of Irf6 was generated. To validate the functionality of the conditional allele, we used three "deleter" Cre strains: Gdf9-Cre, CAG-Cre, and Ella-Cre. When Cre expression was driven by the Gdf9-Cre or CAG-Cre transgenes, 100% recombination was observed as indicated by DNA genotyping and phenotyping. In contrast, use of the Ella-Cre transgenic line resulted in incomplete recombination, despite expression at the one-cell stage. In sum, we generated a novel tool to delete Irf6 in a tissue specific fashion, allowing for study of gene function past perinatal stages. However, recombination efficiency of this allele was dictated by the Cre-driver used.


Asunto(s)
Alelos , Marcación de Gen/métodos , Factores Reguladores del Interferón/genética , Animales , Recombinación Homóloga , Homocigoto , Integrasas/genética , Integrasas/metabolismo , Factores Reguladores del Interferón/metabolismo , Ratones , Fenotipo
3.
Gynecol Oncol ; 139(1): 127-33, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26193427

RESUMEN

OBJECTIVE: The PI3K/Akt pathway is frequently dysregulated in endometrial cancer, the most common gynecologic malignancy. Emerging evidence identifies the ubiquitin ligase NEDD4 as a key regulator of the PI3K/Akt pathway via activation of insulin-like growth factor-1 receptor (IGF-1R). Our objective was to understand the role of NEDD4 in endometrial cancer. METHODS: NEDD4 expression was assessed by immunohistochemistry in a tissue microarray with 77 endometrial lesions ranging from normal benign endometrium to tumor specimens of varying stage and grade. Studies were extended to a panel of eight endometrial cancer cell lines phenotypically representing the most common endometrial patient tumors. RESULTS: Immunohistochemistry demonstrated robust staining of NEDD4 in endometrial tumor specimens, with greater NEDD4 expression in the most aggressive tumors. Expression of NEDD4 was detected in a majority of endometrial cancer cell lines surveyed. Exogenous overexpression of murine Nedd4 in endometrial cancer cell lines with modest endogenous NEDD4 expression resulted in a significant increase in the rate of proliferation. Nedd4 overexpression also promoted an increase in cell surface localization of IGF-1R and activation of Akt. Inhibition of PI3K/Akt signaling reversed the enhanced cell growth in Nedd4-overexpressing endometrial cancer cells. In addition, the expression of NEDD4 in endometrial tumors positively correlated with the Akt downstream effector FoxM1. CONCLUSIONS: This study identifies NEDD4 as a putative oncogene in endometrial cancer that may augment activation of the IGF-1R/PI3K/Akt signaling pathway.


Asunto(s)
Neoplasias Endometriales/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor IGF Tipo 1/metabolismo , Ubiquitina-Proteína Ligasas/genética , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Carcinoma Endometrioide/enzimología , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/patología , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Neoplasias Endometriales/enzimología , Neoplasias Endometriales/patología , Complejos de Clasificación Endosomal Requeridos para el Transporte/biosíntesis , Activación Enzimática , Femenino , Humanos , Inmunohistoquímica , Ubiquitina-Proteína Ligasas Nedd4 , Oncogenes , Receptor IGF Tipo 1/biosíntesis , Análisis de Matrices Tisulares , Ubiquitina-Proteína Ligasas/biosíntesis
4.
J Biol Chem ; 290(19): 11853-64, 2015 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-25787082

RESUMEN

Increased expression of metadherin (MTDH, also known as AEG-1 and 3D3/LYRIC) has been associated with drug resistance, metastasis, and angiogenesis in a variety of cancers. However, the specific mechanisms through which MTDH is involved in these processes remain unclear. To uncover these mechanisms, we generated Mtdh knock-out mice via a targeted disruption of exon 3. Homozygous Mtdh knock-out mice are viable, but males are infertile. The homozygous male mice present with massive loss of spermatozoa as a consequence of meiotic failure. Accumulation of γ-H2AX in spermatocytes of homozygous Mtdh knock-out mice confirms an increase in unrepaired DNA breaks. We also examined expression of the DNA repair protein Rad18, which is regulated by MTDH at the post-transcriptional level. In testes from Mtdh exon 3-deficient mice, Rad18 foci were increased in the lumina of the seminiferous tubules. The Piwi-interacting RNA (piRNA)-interacting protein Mili was expressed at high levels in testes from Mtdh knock-out mice. Accordingly, genome-wide small RNA deep sequencing demonstrated altered expression of piRNAs in the testes of Mtdh knock-out mice as compared with wild type mice. In addition, we observed significantly reduced expression of microRNAs (miRNAs) including miR-16 and miR-19b, which are known to be significantly reduced in the semen of infertile men. In sum, our observations indicate a crucial role for MTDH in male fertility and the DNA repair mechanisms required for normal spermatogenesis.


Asunto(s)
Regulación de la Expresión Génica , Infertilidad Masculina/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , ARN Pequeño no Traducido/metabolismo , Espermatogénesis/genética , Animales , Daño del ADN , Reparación del ADN , Exones , Eliminación de Gen , Genotipo , Homocigoto , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Proteínas de Unión al ARN , Espermatocitos/metabolismo , Espermatozoides/fisiología , Testículo/metabolismo
5.
Physiol Rep ; 3(1)2015 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-25602015

RESUMEN

Preeclampsia is a cardiovascular disorder of late pregnancy that is, commonly characterized by hypertension, renal structural damage and dysfunction, and fetal growth restriction. Prevailing etiologic models of this disorder include T-cell dysfunction as an initiating cause of preeclampsia. Indoleamine 2,3-dioxygenase (IDO), an enzyme that mediates the conversion of tryptophan to kynurenine, has been linked to preeclampsia in humans, and is known to regulate T-cell activity and an endothelial-derived relaxing factor. To test the hypothesis that IDO is causally involved in the pathogenesis of preeclampsia, mice deficient for IDO (IDO-KO) were generated on a C57BL/6 background. IDO-KO and wild-type C57BL/6 mice were bred, and preeclampsia phenotypes were evaluated during pregnancy. Pregnant IDO-KO mice exhibited pathognomonic renal glomerular endotheliosis, proteinuria, pregnancy-specific endothelial dysfunction, intrauterine growth restriction, and mildly elevated blood pressure compared to wild-type mice. Together these findings highlight an important role for IDO in the generation of phenotypes typical of preeclampsia. Loss of IDO function may represent a risk factor for the development of preeclampsia. By extension, increased IDO activity, reductions in IDO reactants, or increases in IDO products may represent novel therapeutic approaches for this disorder.

6.
J Immunol ; 194(5): 2160-7, 2015 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-25632008

RESUMEN

The ubiquitin-ligating enzyme (E3) Itch plays a crucial role in the regulation of inflammation, and Itch deficiency leads to severe airway inflammation. However, the molecular mechanisms by which Itch function is regulated remain elusive. In this study, we found that nontypeable Haemophilus influenzae induces the association of Itch with Ndfip1. Both Itch(-/-) and Ndfip1(-/-) mice exhibited severe airway inflammation in response to nontypeable Haemophilus influenza, which was associated with elevated expression of proinflammatory cytokines. Ndfip1 enhanced Itch ligase activity and facilitated Itch-mediated Tak1 ubiquitination. Mechanistically, Ndfip1 facilitated recruitment of ubiquitin-conjugating enzyme (E2) UbcH7 to Itch. The N-terminal region of Ndfip1 binds to UbcH7, whereas the PY motif binds to Itch. Hence, Ndfip1 acts as an adaptor for UbcH7 and Itch. Reconstitution of full-length Ndfip1 but not the mutants that fail to interact with either UbcH7 or Itch, restored the defect in Tak1 ubiquitination and inhibited elevated proinflammatory cytokine expression by Ndfip1(-/-) cells. These results provide new mechanistic insights into how Itch function is regulated during inflammatory signaling, which could be exploited therapeutically in inflammatory diseases.


Asunto(s)
Proteínas Portadoras/inmunología , Infecciones por Haemophilus/inmunología , Quinasas Quinasa Quinasa PAM/inmunología , Proteínas de la Membrana/inmunología , Sistema Respiratorio/inmunología , Enzimas Ubiquitina-Conjugadoras/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Vectores Genéticos , Células HEK293 , Infecciones por Haemophilus/genética , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/inmunología , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/microbiología , Péptidos y Proteínas de Señalización Intercelular , Lentivirus/genética , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Sistema Respiratorio/metabolismo , Sistema Respiratorio/microbiología , Transducción de Señal , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
7.
Nat Commun ; 5: 4513, 2014 Jul 29.
Artículo en Inglés | MEDLINE | ID: mdl-25072696

RESUMEN

CD40, a member of tumour necrosis factor receptor (TNFR) superfamily, has a pivotal role in B-cell-mediated immunity through various effector pathways including AKT kinase, but the signal transduction of CD40-meidated AKT activation is poorly understood. Here we report that the neural precursor cell expressed developmentally downregulated protein 4 (NEDD4), homologous to E6-AP Carboxyl Terminus family E3 ubiquitin ligase, is a novel component of the CD40 signalling complex. It has a key role in CD40-mediated AKT activation and is involved in modulating immunoglobulin class switch through regulating the expression of activation-induced cytidine deaminase. NEDD4 constitutively interacts with CD40 and mediates K63-linked ubiquitination of TNFR-associated factor3 (TRAF3). The ubiquitination of TRAF3 by NEDD4 is critical for CD40-mediated AKT activation. Thus, NEDD4 is a previously unknown component of the CD40 signalling complex necessary for AKT activation.


Asunto(s)
Antígenos CD40/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Inmunidad Celular/inmunología , Proteína Oncogénica v-akt/metabolismo , Transducción de Señal/inmunología , Factor 3 Asociado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Citometría de Flujo , Humanos , Inmunoprecipitación , Espectrometría de Masas , Ratones , Ratones Noqueados , Ubiquitina-Proteína Ligasas Nedd4 , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ubiquitinación
8.
Circulation ; 128(16): 1748-57, 2013 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-24030498

RESUMEN

BACKGROUND: Atrial fibrillation (AF) is a growing public health problem without adequate therapies. Angiotensin II and reactive oxygen species are validated risk factors for AF in patients, but the molecular pathways connecting reactive oxygen species and AF are unknown. The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a reactive oxygen species-activated proarrhythmic signal, so we hypothesized that oxidized CaMKIIδ could contribute to AF. METHODS AND RESULTS: We found that oxidized CaMKII was increased in atria from AF patients compared with patients in sinus rhythm and from mice infused with angiotensin II compared with mice infused with saline. Angiotensin II-treated mice had increased susceptibility to AF compared with saline-treated wild-type mice, establishing angiotensin II as a risk factor for AF in mice. Knock-in mice lacking critical oxidation sites in CaMKIIδ (MM-VV) and mice with myocardium-restricted transgenic overexpression of methionine sulfoxide reductase A, an enzyme that reduces oxidized CaMKII, were resistant to AF induction after angiotensin II infusion. CONCLUSIONS: Our studies suggest that CaMKII is a molecular signal that couples increased reactive oxygen species with AF and that therapeutic strategies to decrease oxidized CaMKII may prevent or reduce AF.


Asunto(s)
Fibrilación Atrial/etiología , Fibrilación Atrial/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Sistema de Conducción Cardíaco/metabolismo , Anciano , Angiotensina II/metabolismo , Angiotensina II/farmacología , Animales , Fibrilación Atrial/prevención & control , Señalización del Calcio/fisiología , Retroalimentación Fisiológica/efectos de los fármacos , Retroalimentación Fisiológica/fisiología , Femenino , Humanos , Masculino , Metionina Sulfóxido Reductasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Canal Liberador de Calcio Receptor de Rianodina/metabolismo
9.
J Clin Invest ; 123(3): 1262-74, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23426181

RESUMEN

Diabetes increases oxidant stress and doubles the risk of dying after myocardial infarction, but the mechanisms underlying increased mortality are unknown. Mice with streptozotocin-induced diabetes developed profound heart rate slowing and doubled mortality compared with controls after myocardial infarction. Oxidized Ca(2+)/calmodulin-dependent protein kinase II (ox-CaMKII) was significantly increased in pacemaker tissues from diabetic patients compared with that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by myocardial infarction. We developed a knockin mouse model of oxidation-resistant CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease. Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased pacemaker cell survival, maintained normal heart rates, and were resistant to diabetes-attributable mortality after myocardial infarction. Our findings suggest that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden death in diabetic patients after myocardial infarction.


Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Diabetes Mellitus Experimental/enzimología , Infarto del Miocardio/enzimología , Nodo Sinoatrial/enzimología , Animales , Apoptosis , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Gasto Cardíaco , Células Cultivadas , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/mortalidad , Femenino , Fibrosis , Frecuencia Cardíaca , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Cardíacas/metabolismo , Infarto del Miocardio/etiología , Infarto del Miocardio/mortalidad , Miocardio/enzimología , Miocardio/patología , Oxidación-Reducción , Estrés Oxidativo , Péptidos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Nodo Sinoatrial/patología , Nodo Sinoatrial/fisiopatología
10.
J Clin Invest ; 123(2): 657-65, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23348737

RESUMEN

The E3 ubiquitin ligase NEDD4-2 (encoded by the Nedd4L gene) regulates the amiloride-sensitive epithelial Na+ channel (ENaC/SCNN1) to mediate Na+ homeostasis. Mutations in the human ß/γENaC subunits that block NEDD4-2 binding or constitutive ablation of exons 6-8 of Nedd4L in mice both result in salt-sensitive hypertension and elevated ENaC activity (Liddle syndrome). To determine the role of renal tubular NEDD4-2 in adult mice, we generated tetracycline-inducible, nephron-specific Nedd4L KO mice. Under standard and high-Na+ diets, conditional KO mice displayed decreased plasma aldosterone but normal Na+/K+ balance. Under a high-Na+ diet, KO mice exhibited hypercalciuria and increased blood pressure, which were reversed by thiazide treatment. Protein expression of ßENaC, γENaC, the renal outer medullary K+ channel (ROMK), and total and phosphorylated thiazide-sensitive Na+Cl- cotransporter (NCC) levels were increased in KO kidneys. Unexpectedly, Scnn1a mRNA, which encodes the αENaC subunit, was reduced and proteolytic cleavage of αENaC decreased. Taken together, these results demonstrate that loss of NEDD4-2 in adult renal tubules causes a new form of mild, salt-sensitive hypertension without hyperkalemia that is characterized by upregulation of NCC, elevation of ß/γENaC, but not αENaC, and a normal Na+/K+ balance maintained by downregulation of ENaC activity and upregulation of ROMK.


Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/deficiencia , Hipertensión/etiología , Túbulos Renales/fisiopatología , Receptores de Droga/metabolismo , Simportadores/metabolismo , Ubiquitina-Proteína Ligasas/deficiencia , Animales , Presión Sanguínea , Modelos Animales de Enfermedad , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Canales Epiteliales de Sodio/metabolismo , Humanos , Hipertensión/genética , Hipertensión/fisiopatología , Síndrome de Liddle/etiología , Síndrome de Liddle/genética , Síndrome de Liddle/fisiopatología , Ratones , Ratones Noqueados , Ubiquitina-Proteína Ligasas Nedd4 , Potasio/sangre , Potasio/orina , Canales de Potasio de Rectificación Interna/metabolismo , Sodio/sangre , Sodio/orina , Sodio en la Dieta/administración & dosificación , Sodio en la Dieta/efectos adversos , Miembro 3 de la Familia de Transportadores de Soluto 12 , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
11.
J Neurochem ; 124(4): 454-65, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23157328

RESUMEN

Myelin proteolipid protein gene (Plp1) expression is temporally regulated in brain, which peaks during the active myelination period of CNS development. Previous studies with Plp1-lacZ transgenic mice demonstrated that (mouse) Plp1 intron 1 DNA is required for high levels of expression in oligodendrocytes. Deletion-transfection analysis revealed the intron contains a single positive regulatory element operative in the N20.1 oligodendroglial cell line, which was named ASE (antisilencer/enhancer) based on its functional properties in these cells. To investigate the role of the ASE in vivo, the element was deleted from the native gene in mouse using a Cre/lox strategy. Although removal of the ASE from Plp1-lacZ constructs profoundly decreased expression in transfected oligodendroglial cell lines (N20.1 and Oli-neu), the element was dispensable to achieve normal levels of Plp1 gene expression in mouse during development (except perhaps at postnatal day 15) and throughout the remyelination period following cuprizone-induced (acute) demyelination. Thus, it is possible that the ASE is non-functional in vivo, or that loss of the ASE from the native gene in mouse can be compensated for by the presence of other regulatory elements within the Plp1 gene.


Asunto(s)
Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Elementos de Facilitación Genéticos/genética , Intrones/genética , Proteína Proteolipídica de la Mielina/metabolismo , Eliminación de Secuencia/genética , Factores de Edad , Animales , Animales Recién Nacidos , Línea Celular Transformada , Cuprizona/toxicidad , Enfermedades Desmielinizantes/inducido químicamente , Modelos Animales de Enfermedad , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Inhibidores de la Monoaminooxidasa/toxicidad , Proteína Proteolipídica de la Mielina/genética , Oligodendroglía , Transfección
12.
Hypertension ; 60(3): 691-6, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22802227

RESUMEN

Neural precursor cell expressed and developmentally downregulated 4-2 protein (Nedd4-2) facilitates the endocytosis of epithelial Na channels (ENaCs). Both mice and humans with a loss of regulation of ENaC by Nedd4-2 have salt-induced hypertension. ENaC is also expressed in the brain, where it is critical for hypertension on a high-salt diet in salt-sensitive rats. In the present studies we assessed whether Nedd4-2 knockout (-/-) mice have the following: (1) increased brain ENaC; (2) elevated cerebrospinal fluid (CSF) sodium on a high-salt diet; and (3) enhanced pressor responses to CSF sodium and hypertension on a high-salt diet, both mediated by brain ENaC. Prominent choroid plexus and neuronal ENaC staining was present in -/- but not in wild-type mice. In chronically instrumented mice, ICV infusion of Na-rich artificial CSF increased mean arterial pressure 3-fold higher in -/- than in wild-type mice. ICV infusion of the ENaC blocker benzamil abolished this enhancement. In telemetered -/- mice on a high-salt diet (8% NaCl), CSF [Na(+)], mean arterial pressure, and heart rate increased significantly, mean arterial pressure by 30 to 35 mmHg. These mean arterial pressure and heart rate responses were largely prevented by ICV benzamil but only to a minor extent by SC benzamil at the ICV rate. We conclude that increased ENaC expression in the brain of Nedd4-2 -/- mice mediates their hypertensive response to a high-salt diet by causing increased sodium levels in the CSF, as well as hyperresponsiveness to CSF sodium. These findings highlight the possible causative contribution of central nervous system ENaC in the etiology of salt-induced hypertension.


Asunto(s)
Encéfalo/metabolismo , Canales Epiteliales de Sodio/metabolismo , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Síndrome de Liddle/metabolismo , Cloruro de Sodio Dietético/efectos adversos , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Modelos Animales de Enfermedad , Complejos de Clasificación Endosomal Requeridos para el Transporte/deficiencia , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Canales Epiteliales de Sodio/efectos de los fármacos , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Ubiquitina-Proteína Ligasas Nedd4 , Sodio/líquido cefalorraquídeo , Cloruro de Sodio Dietético/farmacología , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
13.
Acta Neuropathol ; 123(6): 807-23, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22539017

RESUMEN

Ubiquitin-immunoreactive neuronal inclusions composed of TAR DNA binding protein of 43 kDa (TDP-43) are a major pathological feature of frontotemporal lobar degeneration (FTLD-TDP). In vivo studies with TDP-43 knockout mice have suggested that TDP-43 plays a critical, although undefined role in development. In the current report, we generated transgenic mice that conditionally express wild-type human TDP-43 (hTDP-43) in the forebrain and established a paradigm to examine the sensitivity of neurons to TDP-43 overexpression at different developmental stages. Continuous TDP-43 expression during early neuronal development produced a complex phenotype, including aggregation of phospho-TDP-43, increased ubiquitin immunoreactivity, mitochondrial abnormalities, neurodegeneration and early lethality. In contrast, later induction of hTDP-43 in the forebrain of weaned mice prevented early death and mitochondrial abnormalities while yielding salient features of FTLD-TDP, including progressive neurodegeneration and ubiquitinated, phospho-TDP-43 neuronal cytoplasmic inclusions. These results suggest that neurons in the developing forebrain are extremely sensitive to TDP-43 overexpression and that timing of TDP-43 overexpression in transgenic mice must be considered when distinguishing normal roles of TDP-43, particularly as they relate to development, from its pathogenic role in FTLD-TDP and other TDP-43 proteinopathies. Finally, our adult induction of hTDP-43 strategy provides a mouse model that develops critical pathological features that are directly relevant for human TDP-43 proteinopathies.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Neuronas/metabolismo , Proteinopatías TDP-43/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/metabolismo , Neuronas/citología , Proteinopatías TDP-43/genética , Factores de Tiempo , Ubiquitina/metabolismo
14.
J Cell Biol ; 196(1): 29-36, 2012 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-22213801

RESUMEN

PTEN (phosphatase and tensin homologue deleted on chromosome TEN) is the major negative regulator of phosphatidylinositol 3-kinase signaling and has cell-specific functions including tumor suppression. Nuclear localization of PTEN is vital for tumor suppression; however, outside of cancer, the molecular and physiological events driving PTEN nuclear entry are unknown. In this paper, we demonstrate that cytoplasmic Pten was translocated into the nuclei of neurons after cerebral ischemia in mice. Critically, this transport event was dependent on a surge in the Nedd4 family-interacting protein 1 (Ndfip1), as neurons in Ndfip1-deficient mice failed to import Pten. Ndfip1 binds to Pten, resulting in enhanced ubiquitination by Nedd4 E3 ubiquitin ligases. In vitro, Ndfip1 overexpression increased the rate of Pten nuclear import detected by photobleaching experiments, whereas Ndfip1(-/-) fibroblasts showed negligible transport rates. In vivo, Ndfip1 mutant mice suffered larger infarct sizes associated with suppressed phosphorylated Akt activation. Our findings provide the first physiological example of when and why transient shuttling of nuclear Pten occurs and how this process is critical for neuron survival.


Asunto(s)
Isquemia Encefálica/metabolismo , Proteínas Portadoras/fisiología , Proteínas de la Membrana/fisiología , Neuronas/fisiología , Fosfohidrolasa PTEN/metabolismo , Animales , Isquemia Encefálica/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Supervivencia Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte/fisiología , Péptidos y Proteínas de Señalización Intercelular , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ubiquitina-Proteína Ligasas Nedd4 , Fotoblanqueo , Transporte de Proteínas , Ubiquitina-Proteína Ligasas/fisiología , Ubiquitinación
15.
Dev Dyn ; 241(2): 340-9, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22113860

RESUMEN

BACKGROUND: DNA variation in Interferon Regulatory Factor 6 (IRF6) contributes risk for orofacial clefting, including a common DNA variant rs642961. This DNA variant is located in a multi-species conserved sequence that is 9.7 kb upstream from the IRF6 transcriptional start site (MCS9.7). The MCS9.7 element was shown to possess enhancer activity that mimicked the expression of endogenous Irf6 at embryonic day 11.5 in transient transgenic embryos, and also contains a p63 binding site that transactivates IRF6 expression. To analyze whether the MCS9.7 enhancer is sufficient to drive IRF6 expression, we generated stable transgenic murine lines that carry a MCS9.7-lacZ transgene. We hypothesized that MCS9.7 was sufficient to recapitulate the endogenous expression of Irf6 at other time-points during embryonic development. RESULTS: We observed that MCS9.7 activity recapitulated endogenous Irf6 expression in most tissues, but not in the medial edge epithelium (MEE) at E14.5, when Irf6 expression was high during secondary palatal fusion. Also, while MCS9.7 activity and Irf6 expression were associated with p63 expression, we observed MCS9.7 activity and Irf6 expression in periderm, although p63 was absent. CONCLUSION: These data suggest that MCS9.7 enhancer activity is not sufficient to recapitulate IRF6 expression, and that p63 expression is not always necessary nor sufficient for transactivation of IRF6.


Asunto(s)
Elementos de Facilitación Genéticos , Epidermis/embriología , Regulación del Desarrollo de la Expresión Génica , Factores Reguladores del Interferón/genética , Hueso Paladar/embriología , Fosfoproteínas/genética , Transactivadores/genética , Activación Transcripcional , Animales , Labio Leporino/genética , Fisura del Paladar/genética , Epitelio/embriología , Ratones , Ratones Transgénicos , Hueso Paladar/metabolismo , Sitio de Iniciación de la Transcripción , beta-Galactosidasa/genética
16.
Cell Metab ; 14(5): 635-46, 2011 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-22019085

RESUMEN

The iron exporter ferroportin (Fpn) is essential to transfer iron from cells to plasma. Systemic iron homeostasis in vertebrates is regulated by the hepcidin-mediated internalization of Fpn. Here, we demonstrate a second route for Fpn internalization; when cytosolic iron levels are low, Fpn is internalized in a hepcidin-independent manner dependent upon the E3 ubiquitin ligase Nedd4-2 and the Nedd4-2 binding protein Nfdip-1. Retention of cell-surface Fpn through reductions in Nedd4-2 results in cell death through depletion of cytosolic iron. Nedd4-2 is also required for internalization of Fpn in the absence of ferroxidase activity as well as for the entry of hepcidin-induced Fpn into the multivesicular body. C. elegans lacks hepcidin genes, and C. elegans Fpn expressed in mammalian cells is not internalized by hepcidin but is internalized in response to iron deprivation in a Nedd4-2-dependent manner, supporting the hypothesis that Nedd4-2-induced internalization of Fpn is evolutionarily conserved.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Hierro/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/deficiencia , Péptidos Catiónicos Antimicrobianos/genética , Evolución Biológica , Caenorhabditis elegans , Proteínas Portadoras/genética , Proteínas de Transporte de Catión/genética , Células Cultivadas , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Células HEK293 , Hepcidinas , Homeostasis/fisiología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Macrófagos/citología , Macrófagos/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Ubiquitina-Proteína Ligasas Nedd4 , Plásmidos , ARN Interferente Pequeño , Proteínas Recombinantes de Fusión/genética , Transfección , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación
17.
J Am Soc Nephrol ; 22(9): 1707-19, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21852580

RESUMEN

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.


Asunto(s)
Aldosterona/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Túbulos Renales Distales/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Simportadores del Cloruro de Sodio/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Animales , Regulación hacia Abajo , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Ubiquitina-Proteína Ligasas Nedd4 , Fosforilación , Transducción de Señal , Ubiquitinación , Proteínas de Xenopus , Xenopus laevis
18.
Pancreas ; 40(6): 861-71, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21697765

RESUMEN

OBJECTIVES: These studies examined the effect of homozygous deletion of vasoactive intestinal peptide receptor type 1 (VPAC1) on development and function of intestines and pancreas. METHODS: Genetically engineered VPAC1-null mutant mice were monitored for growth, development, and glucose homeostasis. Expression of VPAC1 was examined during embryonic development using VPAC1 promoter-driven ß-galactosidase transgenic mice. RESULTS: Homozygous deletion of VPAC1 resulted in fetal, neonatal, and postweaning death owing to failure to thrive, intestinal obstruction, and hypoglycemia. Histological findings demonstrated disorganized hyperproliferation of intestinal epithelial cells with mucus deposition and bowel wall thickening. The pancreas demonstrated small dysmorphic islets of Langerhans containing α, ß, and δ cells. Expression of a VPAC1 promoter-driven transgene was observed in E12.5 and E14.5 intestinal epithelial and pancreatic endocrine cells. Vasoactive intestinal peptide receptor type 1-null mutant animals had lower baseline blood glucose levels compared to both heterozygous and wild-type littermates. Vasoactive intestinal peptide receptor type 1-deficient mice responded to oral glucose challenge with normal rise in blood glucose followed by rapid hypoglycemia and failure to restore baseline glucose levels. Insulin challenge resulted in profound hypoglycemia and inadequate glucose homeostasis in VPAC1-null mutant animals. CONCLUSIONS: These observations support a role for VPAC1 during embryonic and neonatal development of intestines and endocrine pancreas.


Asunto(s)
Intestinos/embriología , Intestinos/fisiopatología , Páncreas/embriología , Páncreas/fisiopatología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/deficiencia , Animales , Secuencia de Bases , Glucemia/metabolismo , Cartilla de ADN/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Prueba de Tolerancia a la Glucosa , Heterocigoto , Homocigoto , Intestinos/patología , Islotes Pancreáticos/embriología , Islotes Pancreáticos/patología , Islotes Pancreáticos/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Páncreas/patología , Embarazo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/fisiología
19.
Fertil Steril ; 96(2): 422-427.e2, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21676393

RESUMEN

OBJECTIVE: To identify factors secreted by the human embryo and correlate levels with embryo morphology and pregnancy outcome. DESIGN: A laboratory-based study of human embryo protein synthesis and secretion and a retrospective analysis of spent embryo culture media as it relates to pregnancy outcome. SETTING: University-based academic IVF program. PATIENT(S): IVF patients who had donated cryopreserved human pronuclear-stage embryos. Patients undergoing fresh IVF cycles resulting in a blastocyst transfer who donated spent media drops. INTERVENTION(S): In vitro embryo culture and collection of spent media. MAIN OUTCOME MEASURE(S): Protein analysis and identification by two-dimensional gel electrophoresis and mass spectrometry, ApoA1 quantification by ELISA, and mRNA analysis by quantitative reverse transcriptase-polymerase chain reaction. RESULT(S): By protein gel electrophoresis, apolipoprotein A1 (ApoA1) was increased in the culture media from good-quality blastocysts (n = 6 embryos) compared to either cleavage-arrested embryos (n = 6 embryos) or poor-quality blastocysts (n = 6 embryos) using spent media from culture days 4 and 5, respectively. Apolipoprotein A1 concentrations were 23.1% greater in day 5 spent culture media from good-grade blastocysts (n = 30) when compared to poor-grade embryos (n = 30). However, in a group of patients (n = 20) with transfer of two good-quality blastocysts, ApoA1 levels from day 5 spent media did not correlate with embryo implantation and pregnancy. Quantitative reverse transcriptase-polymerase chain reaction confirmed the presence of ApoA1 mRNA transcripts in human blastocysts. CONCLUSION(S): Apolipoprotein A1 is produced by human preimplantation embryos, and increased levels are present in spent culture media containing blastocysts of higher morphologic grade. These results suggest a role for lipoproteins in early embryologic development.


Asunto(s)
Apolipoproteína A-I/metabolismo , Blastocisto/metabolismo , Apolipoproteína A-I/genética , Medios de Cultivo/metabolismo , Electroforesis en Gel Bidimensional , Técnicas de Cultivo de Embriones , Implantación del Embrión , Transferencia de Embrión , Ensayo de Inmunoadsorción Enzimática , Femenino , Fertilización In Vitro , Regulación del Desarrollo de la Expresión Génica , Humanos , Embarazo , Índice de Embarazo , Proteómica/métodos , ARN Mensajero/metabolismo , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masas en Tándem , Factores de Tiempo , Regulación hacia Arriba
20.
Blood ; 117(2): 638-46, 2011 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-20959604

RESUMEN

The divalent metal ion transporter DMT1 is critical for nonheme iron import. We have previously shown that DMT1 is regulated in vitro by ubiquitination that is facilitated by the adaptor proteins Ndfip1 and Ndfip2. Here we report that in Ndfip1(-/-) mice fed a low- iron diet, DMT1 expression and activity in duodenal enterocytes are significant higher than in the wild-type animals. This correlates with an increase in serum iron levels and transferrin saturation. Liver and spleen iron stores were also increased in Ndfip1(-/-) mice fed a normal diet. Counterintuitive to the increase in iron uptake, Ndfip1(-/-) mice fed a low iron diet develop severe microcytic, hypochromic anemia. We demonstrate that this is due to a combination of iron deficiency and inflammatory disease in Ndfip1(-/-) mice, because Ndfip1(-/-)/Rag1(-/-) immunodeficient mice fed a low iron diet did not develop anemia and showed an iron overload phenotype. These data demonstrate that Ndfip1 is a critical mediator of DMT1 regulation in vivo, particularly under iron restricted conditions.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión/metabolismo , Homeostasis/fisiología , Hierro/metabolismo , Proteínas de la Membrana/metabolismo , Anemia Ferropénica/etiología , Anemia Ferropénica/metabolismo , Animales , Immunoblotting , Inmunohistoquímica , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Hierro de la Dieta/metabolismo , Espectrometría de Masas , Ratones , Ratones Noqueados , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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