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1.
J Cell Sci ; 137(14)2024 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-38904097

RESUMEN

PTPRK is a receptor tyrosine phosphatase that is linked to the regulation of growth factor signalling and tumour suppression. It is stabilized at the plasma membrane by trans homophilic interactions upon cell-cell contact. PTPRK regulates cell-cell adhesion but is also reported to regulate numerous cancer-associated signalling pathways. However, the signalling mechanism of PTPRK remains to be determined. Here, we find that PTPRK regulates cell adhesion signalling, suppresses invasion and promotes collective, directed migration in colorectal cancer cells. In vivo, PTPRK supports recovery from inflammation-induced colitis. In addition, we confirm that PTPRK functions as a tumour suppressor in the mouse colon and in colorectal cancer xenografts. PTPRK regulates growth factor and adhesion signalling, and suppresses epithelial to mesenchymal transition (EMT). Contrary to the prevailing notion that PTPRK directly dephosphorylates EGFR, we find that PTPRK regulation of both EGFR and EMT is independent of its catalytic function. This suggests that additional adaptor and scaffold functions are important features of PTPRK signalling.


Asunto(s)
Transición Epitelial-Mesenquimal , Humanos , Animales , Ratones , Receptores ErbB/metabolismo , Receptores ErbB/genética , Transducción de Señal , Adhesión Celular/genética , Movimiento Celular , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Colitis/patología , Colitis/metabolismo , Colitis/genética , Colitis/inducido químicamente , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Intestinos/patología
2.
Cell Death Dis ; 12(4): 379, 2021 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-33828080

RESUMEN

RIP1 kinase-mediated inflammatory and cell death pathways have been implicated in the pathology of acute and chronic disorders of the nervous system. Here, we describe a novel animal model of RIP1 kinase deficiency, generated by knock-in of the kinase-inactivating RIP1(D138N) mutation in rats. Homozygous RIP1 kinase-dead (KD) rats had normal development, reproduction and did not show any gross phenotypes at baseline. However, cells derived from RIP1 KD rats displayed resistance to necroptotic cell death. In addition, RIP1 KD rats were resistant to TNF-induced systemic shock. We studied the utility of RIP1 KD rats for neurological disorders by testing the efficacy of the genetic inactivation in the transient middle cerebral artery occlusion/reperfusion model of brain injury. RIP1 KD rats were protected in this model in a battery of behavioral, imaging, and histopathological endpoints. In addition, RIP1 KD rats had reduced inflammation and accumulation of neuronal injury biomarkers. Unbiased proteomics in the plasma identified additional changes that were ameliorated by RIP1 genetic inactivation. Together these data highlight the utility of the RIP1 KD rats for target validation and biomarker studies for neurological disorders.


Asunto(s)
Lesiones Encefálicas/genética , Muerte Celular/genética , Isquemia/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Sprague-Dawley , Proteína Serina-Treonina Quinasas de Interacción con Receptores
3.
Immunity ; 52(2): 357-373.e9, 2020 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-32049051

RESUMEN

Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated T cell activation and synergized with anti-PD-1 or anti-PD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Stinggt/gt mice, but not in Cgas-/- mice. Abolishing cGAMP production in Cgas-/- tumor cells, depletion of extracellular ATP, or inactivation of the ATP-gated P2X7R channel also compromised the effects of MerTK blockade. Mechanistically, extracellular ATP acted via P2X7R to enhance the transport of extracellular cGAMP into macrophages and subsequent STING activation. Thus, MerTK blockade increases tumor immunogenicity and potentiates anti-tumor immunity, which has implications for cancer immunotherapy.


Asunto(s)
Macrófagos/inmunología , Proteínas de la Membrana/metabolismo , Neoplasias/inmunología , Nucleótidos Cíclicos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Tirosina Quinasa c-Mer/inmunología , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Antígeno B7-H1/inmunología , Células Cultivadas , Femenino , Inmunidad Innata , Inmunoterapia , Interferón Tipo I/metabolismo , Macrófagos/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/terapia , Nucleotidiltransferasas/deficiencia , Nucleotidiltransferasas/metabolismo , Fagocitosis , Receptor de Muerte Celular Programada 1/inmunología , Receptores Purinérgicos P2X7/deficiencia , Transducción de Señal/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa c-Mer/genética
4.
Eur J Immunol ; 50(6): 891-902, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32043568

RESUMEN

CD96 is a member of the poliovirus receptor (PVR, CD155)-nectin family that includes T cell Ig and ITIM domain (TIGIT) and CD226. While CD96, TIGIT, and CD226 have important roles in regulating NK cell activity, and TIGIT and CD226 have also been shown to regulate T cell responses, it is unclear whether CD96 has inhibitory or stimulatory function in CD8+ T cells. Here, we demonstrate that CD96 has co-stimulatory function on CD8+ T cells. Crosslinking of CD96 on human or mouse CD8+ T cells induced activation, effector cytokine production, and proliferation. CD96 was found to transduce its activating signal through the MEK-ERK pathway. CD96-mediated signaling led to increased frequencies of NUR77- and T-bet-expressing CD8+ T cells and enhanced cytotoxic effector activity, indicating that CD96 can modulate effector T cell differentiation. Antibody blockade of CD96 or genetic ablation of CD96 expression on CD8+ T cells impaired expression of transcription factors and proinflammatory cytokines associated with CD8+ T cell activation in in vivo models. Taken together, CD96 has a co-stimulatory role in CD8+ T cell activation and effector function.


Asunto(s)
Antígenos CD/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Activación de Linfocitos , Sistema de Señalización de MAP Quinasas/inmunología , Modelos Inmunológicos , Animales , Antígenos CD/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Humanos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Noqueados
5.
PLoS One ; 14(4): e0214110, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30951545

RESUMEN

Peg10 (paternally expressed gene 10) is an imprinted gene that is essential for placental development. It is thought to derive from a Ty3-gyspy LTR (long terminal repeat) retrotransposon and retains Gag and Pol-like domains. Here we show that the Gag domain of PEG10 can promote vesicle budding similar to the HIV p24 Gag protein. Expressed in a subset of mouse endocrine organs in addition to the placenta, PEG10 was identified as a substrate of the deubiquitinating enzyme USP9X. Consistent with PEG10 having a critical role in placental development, PEG10-deficient trophoblast stem cells (TSCs) exhibited impaired differentiation into placental lineages. PEG10 expressed in wild-type, differentiating TSCs was bound to many cellular RNAs including Hbegf (Heparin-binding EGF-like growth factor), which is known to play an important role in placentation. Expression of Hbegf was reduced in PEG10-deficient TSCs suggesting that PEG10 might bind to and stabilize RNAs that are critical for normal placental development.


Asunto(s)
Diferenciación Celular/genética , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Proteínas Nucleares/genética , Placentación/genética , Factores de Transcripción/genética , Animales , Proteínas Reguladoras de la Apoptosis , Linaje de la Célula/genética , Elementos Transponibles de ADN/genética , Proteínas de Unión al ADN , Femenino , Regulación del Desarrollo de la Expresión Génica , Productos del Gen gag/genética , Impresión Genómica/genética , Humanos , Ratones , Placenta/metabolismo , Embarazo , Proteínas de Unión al ARN/genética , Células Madre/citología , Células Madre/metabolismo , Trofoblastos/citología , Trofoblastos/metabolismo
6.
Proc Natl Acad Sci U S A ; 115(50): E11731-E11740, 2018 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-30504141

RESUMEN

Natural killer (NK) cell recognition of tumor cells is mediated through activating receptors such as CD226, with suppression of effector functions often controlled by negative regulatory transcription factors such as FOXO1. Here we show that CD226 regulation of NK cell cytotoxicity is facilitated through inactivation of FOXO1. Gene-expression analysis of NK cells isolated from syngeneic tumors grown in wild-type or CD226-deficient mice revealed dysregulated expression of FOXO1-regulated genes in the absence of CD226. In vitro cytotoxicity and stimulation assays demonstrated that CD226 is required for optimal killing of tumor target cells, with engagement of its ligand CD155 resulting in phosphorylation of FOXO1. CD226 deficiency or anti-CD226 antibody blockade impaired cytotoxicity with concomitant compromised inactivation of FOXO1. Furthermore, inhibitors of FOXO1 phosphorylation abrogated CD226-mediated signaling and effector responses. These results define a pathway by which CD226 exerts control of NK cell responses against tumors.


Asunto(s)
Antígenos de Diferenciación de Linfocitos T/metabolismo , Proteína Forkhead Box O1/antagonistas & inhibidores , Proteína Forkhead Box O1/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Animales , Antígenos de Diferenciación de Linfocitos T/genética , Línea Celular Tumoral , Citotoxicidad Inmunológica , Regulación Neoplásica de la Expresión Génica , Humanos , Ligandos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones , Ratones Noqueados , Nectinas/metabolismo , Fosforilación , Receptores Virales/metabolismo , Transducción de Señal/inmunología
7.
Nat Methods ; 15(7): 512-514, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29786090

RESUMEN

Despite widespread use of CRISPR, comprehensive data on the frequency and impact of Cas9-mediated off-targets in modified rodents are limited. Here we present deep-sequencing data from 81 genome-editing projects on mouse and rat genomes at 1,423 predicted off-target sites, 32 of which were confirmed, and show that high-fidelity Cas9 versions reduced off-target mutation rates in vivo. Using whole-genome sequencing data from ten mouse embryos, treated with a single guide RNA (sgRNA), and from their genetic parents, we found 43 off-targets, 30 of which were predicted by an adapted version of GUIDE-seq.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Ingeniería Genética , Genómica/métodos , Animales , Línea Celular , Femenino , Masculino , Ratones , Reacción en Cadena de la Polimerasa Multiplex/métodos , ARN/genética , Ratas , Secuenciación Completa del Genoma/métodos
8.
PLoS Genet ; 9(2): e1003278, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23408910

RESUMEN

During pancreatic development, transcription factor cascades gradually commit precursor populations to the different endocrine cell fate pathways. Although mutational analyses have defined the functions of many individual pancreatic transcription factors, the integrative transcription factor networks required to regulate lineage specification, as well as their sites of action, are poorly understood. In this study, we investigated where and how the transcription factors Nkx2.2 and Neurod1 genetically interact to differentially regulate endocrine cell specification. In an Nkx2.2 null background, we conditionally deleted Neurod1 in the Pdx1+ pancreatic progenitor cells, the Neurog3+ endocrine progenitor cells, or the glucagon+ alpha cells. These studies determined that, in the absence of Nkx2.2 activity, removal of Neurod1 from the Pdx1+ or Neurog3+ progenitor populations is sufficient to reestablish the specification of the PP and epsilon cell lineages. Alternatively, in the absence of Nkx2.2, removal of Neurod1 from the Pdx1+ pancreatic progenitor population, but not the Neurog3+ endocrine progenitor cells, restores alpha cell specification. Subsequent in vitro reporter assays demonstrated that Nkx2.2 represses Neurod1 in alpha cells. Based on these findings, we conclude that, although Nkx2.2 and Neurod1 are both necessary to promote beta cell differentiation, Nkx2.2 must repress Neurod1 in a Pdx1+ pancreatic progenitor population to appropriately commit a subset of Neurog3+ endocrine progenitor cells to the alpha cell lineage. These results are consistent with the proposed idea that Neurog3+ endocrine progenitor cells represent a heterogeneous population of unipotent cells, each restricted to a particular endocrine lineage.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular , Proteínas de Homeodominio , Páncreas , Factores de Transcripción , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Células Secretoras de Glucagón/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Ratones , Proteínas Nucleares , Páncreas/citología , Páncreas/crecimiento & desarrollo , Páncreas/metabolismo , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra
9.
Development ; 138(15): 3213-24, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21750032

RESUMEN

The homeodomain transcription factor Nkx2.2 is essential for pancreatic development and islet cell type differentiation. We have identified Tm4sf4, an L6 domain tetraspanin family member, as a transcriptional target of Nkx2.2 that is greatly upregulated during pancreas development in Nkx2.2(-/-) mice. Tetraspanins and L6 domain proteins recruit other membrane receptors to form active signaling centers that coordinate processes such as cell adhesion, migration and differentiation. In this study, we determined that Tm4sf4 is localized to the ductal epithelial compartment and is prominent in the Ngn3(+) islet progenitor cells. We also established that pancreatic tm4sf4 expression and regulation by Nkx2.2 is conserved during zebrafish development. Loss-of-function studies in zebrafish revealed that tm4sf4 inhibits α and ß cell specification, but is necessary for ε cell fates. Thus, Tm4sf4 functional output opposes that of Nkx2.2. Further investigation of how Tm4sf4 functions at the cellular level in vitro showed that Tm4sf4 inhibits Rho-activated cell migration and actin organization in a ROCK-independent fashion. We propose that the primary role of Nkx2.2 is to inhibit Tm4sf4 in endocrine progenitor cells, allowing for delamination, migration and/or appropriate cell fate decisions. Identification of a role for Tm4sf4 during endocrine differentiation provides insight into islet progenitor cell behaviors and potential targetable regenerative mechanisms.


Asunto(s)
Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Islotes Pancreáticos/embriología , Islotes Pancreáticos/crecimiento & desarrollo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular , Ghrelina/genética , Ghrelina/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Humanos , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares , Alineación de Secuencia , Células Madre/citología , Células Madre/fisiología , Factores de Transcripción/genética , Pez Cebra/anatomía & histología , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo
10.
BMC Bioinformatics ; 12: 62, 2011 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-21352540

RESUMEN

BACKGROUND: The creation of a complete genome-wide map of transcription factor binding sites is essential for understanding gene regulatory networks in vivo. However, current prediction methods generally rely on statistical models that imperfectly model transcription factor binding. Generation of new prediction methods that are based on protein binding data, but do not rely on these models may improve prediction sensitivity and specificity. RESULTS: We propose a method for predicting transcription factor binding sites in the genome by directly mapping data generated from protein binding microarrays (PBM) to the genome and calculating a moving average of several overlapping octamers. Using this unique algorithm, we predicted binding sites for the essential pancreatic islet transcription factor Nkx2.2 in the mouse genome and confirmed >90% of the tested sites by EMSA and ChIP. Scores generated from this method more accurately predicted relative binding affinity than PWM based methods. We have also identified an alternative core sequence recognized by the Nkx2.2 homeodomain. Furthermore, we have shown that this method correctly identified binding sites in the promoters of two critical pancreatic islet ß-cell genes, NeuroD1 and insulin2, that were not predicted by traditional methods. Finally, we show evidence that the algorithm can also be applied to predict binding sites for the nuclear receptor Hnf4α. CONCLUSIONS: PBM-mapping is an accurate method for predicting Nkx2.2 binding sites and may be widely applicable for the creation of genome-wide maps of transcription factor binding sites.


Asunto(s)
Sitios de Unión , Biología Computacional/métodos , Proteínas de Homeodominio/genética , Análisis por Matrices de Proteínas , Factores de Transcripción/genética , Algoritmos , Animales , Genoma , Proteína Homeobox Nkx-2.2 , Ratones , Páncreas , Regiones Promotoras Genéticas , Unión Proteica , Análisis de Regresión , Proteínas de Pez Cebra
11.
Mol Endocrinol ; 24(2): 381-90, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19965928

RESUMEN

Nkx2.2 is an essential regulator of pancreatic endocrine differentiation. Nkx2.2-null mice are completely devoid of beta-ells and have a large reduction of alpha- and PP cells. In the place of these islet populations, there is a corresponding increase in the ghrelin-positive epsilon-cells. Molecular studies have indicated that Nkx2.2 functions as an activator and repressor to regulate islet cell fate decisions. To determine whether Nkx2.2 is solely important for islet cell fate decisions or also has the capability to control ghrelin at the promoter level, we studied the transcriptional regulation of the ghrelin promoter within the pancreas, in vitro and in vivo. These studies demonstrate that both of the previously identified transcriptional start sites in the ghrelin promoter are active within the embryonic pancreas; however, the long transcript is preferentially up-regulated in the Nkx2.2-null pancreas. We also show that the promoter region between -619 and -488 bp upstream of the translational start site is necessary for repression of ghrelin in alphaTC1 and betaTC6 cells. Surprisingly, we also show that Nkx2.2 is able to bind to and activate the ghrelin promoter in several cell lines that do or do not express endogenous ghrelin. Together, these results suggest that the up-regulation of ghrelin expression in the Nkx2.2-null mice is not due to loss of repression of the ghrelin promoter in the nonghrelin islet populations. Furthermore, Nkx2.2 may contribute to the activation of ghrelin in mature islet epsilon-cells.


Asunto(s)
Regulación de la Expresión Génica , Ghrelina/genética , Proteínas de Homeodominio/metabolismo , Islotes Pancreáticos/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Inmunoprecipitación de Cromatina , Secuencia de Consenso , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Embrión de Mamíferos , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Humanos , Ratones , Ratones Transgénicos , Células 3T3 NIH , Proteínas Nucleares , Páncreas/metabolismo , ARN Mensajero/metabolismo , Alineación de Secuencia , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Proteínas de Pez Cebra
12.
BMC Dev Biol ; 9: 65, 2009 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-20003319

RESUMEN

BACKGROUND: The homeodomain containing transcription factor Nkx2.2 is essential for the differentiation of pancreatic endocrine cells. Deletion of Nkx2.2 in mice leads to misspecification of islet cell types; insulin-expressing beta cells and glucagon-expressing alpha cells are replaced by ghrelin-expressing cells. Additional studies have suggested that Nkx2.2 functions both as a transcriptional repressor and activator to regulate islet cell formation and function. To identify genes that are potentially regulated by Nkx2.2 during the major wave of endocrine and exocrine cell differentiation, we assessed gene expression changes that occur in the absence of Nkx2.2 at the onset of the secondary transition in the developing pancreas. RESULTS: Microarray analysis identified 80 genes that were differentially expressed in e12.5 and/or e13.5 Nkx2.2-/- embryos. Some of these genes encode transcription factors that have been previously identified in the pancreas, clarifying the position of Nkx2.2 within the islet transcriptional regulatory pathway. We also identified signaling factors and transmembrane proteins that function downstream of Nkx2.2, including several that have not previously been described in the pancreas. Interestingly, a number of known exocrine genes are also misexpressed in the Nkx2.2-/- pancreas. CONCLUSIONS: Expression profiling of Nkx2.2-/- mice during embryogenesis has allowed us to identify known and novel pancreatic genes that function downstream of Nkx2.2 to regulate pancreas development. Several of the newly identified signaling factors and transmembrane proteins may function to influence islet cell fate decisions. These studies have also revealed a novel function for Nkx2.2 in maintaining appropriate exocrine gene expression. Most importantly, Nkx2.2 appears to function within a complex regulatory loop with Ngn3 at a key endocrine differentiation step.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Páncreas/embriología , Factores de Transcripción/metabolismo , Animales , Femenino , Proteína Homeobox Nkx-2.2 , Masculino , Ratones , Páncreas/citología , Proteínas de Pez Cebra
13.
J Biol Chem ; 284(45): 31236-48, 2009 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-19759004

RESUMEN

Nkx2.2 and NeuroD1 are two critical regulators of pancreatic beta cell development. Nkx2.2 is a homeodomain transcription factor that is essential for islet cell type specification and mature beta cell function. NeuroD1 is a basic helix-loop-helix transcription factor that is critical for islet beta cell maturation and maintenance. Although both proteins influence beta cell development directly downstream of the endocrine progenitor factor, neurogenin3 (Ngn3), a connection between the two proteins in the regulation of beta cell fate and function has yet to be established. In this study, we demonstrate that Nkx2.2 transcriptional activity is required to facilitate the activation of NeuroD1 by Ngn3. Furthermore, Nkx2.2 is necessary to maintain high levels of NeuroD1 expression in developing mouse and zebrafish islets and in mature beta cells. Interestingly, Nkx2.2 regulates NeuroD1 through two independent promoter elements, one that is bound and activated directly by Nkx2.2 and one that appears to be regulated by Nkx2.2 through an indirect mechanism. Together, these findings suggest that Nkx2.2 coordinately activates NeuroD1 with Ngn3 within the endocrine progenitor cell and also plays a role in the maintenance of NeuroD1 expression to regulate beta cell function in the mature islet. Collectively, these findings further define the conserved regulatory networks involved in islet beta cell formation and function.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/crecimiento & desarrollo , Ratones , Ratones Noqueados , Ratones Transgénicos , Factores de Transcripción/genética , Transcripción Genética , Pez Cebra , Proteínas de Pez Cebra/genética
14.
Regul Pept ; 157(1-3): 51-6, 2009 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-19268691

RESUMEN

Ghrelin is a peptide hormone that has been implicated in the regulation of food intake and energy homeostasis. Ghrelin is predominantly produced in the stomach, but is also expressed in many other tissues where its functions are not well characterized. In the rodent and human pancreas, ghrelin levels peak at late gestation and gradually decline postnatally. Several studies have suggested that ghrelin regulates beta cell function during embryonic development and in the adult. In addition, in a number of mouse models, ghrelin cells appear to replace insulin- and glucagon-producing cells in the islet. In this analysis, we investigated whether the absence or overexpression of ghrelin influenced the development and differentiation of the pancreatic islet during embryonic development. These studies revealed that ghrelin is dispensable for normal pancreas development during gestation. Conversely, we demonstrated that elevated ghrelin in the Nkx2.2 null islets is not responsible for the absence of insulin- and glucagon-producing cells. Finally, we have also determined that in the absence of insulin, ghrelin cells form in their normal numbers and ghrelin is expressed at wild type levels.


Asunto(s)
Ghrelina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/crecimiento & desarrollo , Animales , Diferenciación Celular , Ghrelina/deficiencia , Ghrelina/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/metabolismo , Islotes Pancreáticos/embriología , Ratones , Ratones Noqueados , Proteínas Nucleares , ARN Mensajero/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra
15.
Proc Natl Acad Sci U S A ; 101(10): 3697-702, 2004 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-14990799

RESUMEN

Corticotropin-releasing factor (CRF) and its paralogues urocortin (Ucn)I, -II, and -III signal by activating their receptors, CRF receptors (CRFR)1 and -2, to maintain homeostasis through endocrine, autonomic, and behavioral responses. CRFR2 is found in cardiomyocytes and in endothelial and smooth muscle cells of the systemic vasculature. Echocardiography and cardiac catheterization were used in mice to assess the physiologic effects of i.v. UcnII and CRFR2 deficiency on left ventricular function and the systemic vasculature. UcnII treatment augmented heart rate, exhibited potent inotropic and lusitropic actions on the left ventricle, and induced a downward shift of the diastolic pressure-volume relation. UcnII also reduced systemic arterial pressure, associated with a lowering of systemic arterial elastance (end-systolic pressure/stroke volume) and systemic vascular resistance. CRFR2-deficient mice showed no alteration in cardiac contractility or blood pressure in response to UcnII administration, suggesting that the effects of UcnII are specific to CRFR2 function. Pretreatment with a beta-adrenergic receptor antagonist, esmalol, had no effect on the inotropic or lusitropic effects of UcnII in vivo, indicating that its actions are independent of beta-adrenergic receptors. Single i.v. bolus administration of UcnII to a heart failure model (muscle-specific LIM protein-deficient mice) produced significant enhancement of inotropic and lusitropic effects on left ventricular function and improved cardiac output. These results demonstrate the potent cardiovascular physiologic actions of UcnII in both wild-type and cardiomyopathic mice and support a potential beneficial use of this peptide in therapy of congestive heart failure.


Asunto(s)
Fenómenos Fisiológicos Cardiovasculares/efectos de los fármacos , Hormona Liberadora de Corticotropina/farmacología , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/fisiopatología , Animales , Presión Sanguínea/efectos de los fármacos , Hormona Liberadora de Corticotropina/administración & dosificación , Insuficiencia Cardíaca/genética , Proteínas con Dominio LIM , Ratones , Ratones Noqueados , Proteínas Musculares/deficiencia , Proteínas Musculares/genética , Proteínas Musculares/fisiología , Contracción Miocárdica/efectos de los fármacos , Receptores de Hormona Liberadora de Corticotropina/deficiencia , Receptores de Hormona Liberadora de Corticotropina/genética , Receptores de Hormona Liberadora de Corticotropina/fisiología , Urocortinas , Resistencia Vascular/efectos de los fármacos , Función Ventricular Izquierda/efectos de los fármacos
16.
Endocrinology ; 144(6): 2580-7, 2003 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12746321

RESUMEN

Corticotropin-releasing factor (CRF) and its family of ligands are key regulators of energy balance. These ligands function via activation of their two receptors, CRFR1 and CRFR2. CRFR1 has been shown to be the dominant receptor in activation of the hypothalamic-pituitary-adrenal axis in response to stress as well as a key mediator of anxiety in the limbic system. To specifically examine the role of CRFR2 in energy balance, mice deficient for CRFR2 were exposed to physiological perturbations of homeostasis, including high-fat diet, repeated cold stress, and glucose and insulin challenges, and their responses measured. While on a high-fat diet, CRFR2-mutant mice consumed substantially more food and maintaining the same weight but had significantly lower body fat and lower plasma lipids than their wild-type littermates. These mice were also less inclined to develop diet-induced insulin resistance and more sensitive to changes in plasma glucose, indicating increased insulin sensitivity. Following repeated cold stress, mutant mice had significantly lower body fat and a transient reduction in feed efficiency, despite similar body weights, suggesting a possible preference for fat as an energy substrate. Elevated levels of uncoupling protein-1 in brown adipose tissue as well as smaller white and brown adipocytes from CRFR2-mutant mice were indications of possible increased sympathetic tone. These results demonstrate that CRFR2 plays a critical role in regulation of energy expenditure and is important for responses to homeostatic challenges.


Asunto(s)
Frío , Grasas de la Dieta/farmacología , Homeostasis/fisiología , Receptores de Hormona Liberadora de Corticotropina/genética , Estrés Fisiológico/fisiopatología , Adipocitos/metabolismo , Adipocitos/patología , Tejido Adiposo/patología , Tejido Adiposo/fisiología , Animales , Glucemia , Proteínas Portadoras/metabolismo , Ingestión de Alimentos/fisiología , Metabolismo Energético/fisiología , Glucosa/farmacología , Hipoglucemiantes/sangre , Hipoglucemiantes/farmacología , Insulina/sangre , Insulina/farmacología , Canales Iónicos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Mutantes , Proteínas Mitocondriales , Actividad Motora/fisiología , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Proteína Desacopladora 1
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