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1.
Development ; 150(24)2023 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-37997694

RESUMEN

Identification of signaling events that contribute to innate spinal cord regeneration in zebrafish can uncover new targets for modulating injury responses of the mammalian central nervous system. Using a chemical screen, we identify JNK signaling as a necessary regulator of glial cell cycling and tissue bridging during spinal cord regeneration in larval zebrafish. With a kinase translocation reporter, we visualize and quantify JNK signaling dynamics at single-cell resolution in glial cell populations in developing larvae and during injury-induced regeneration. Glial JNK signaling is patterned in time and space during development and regeneration, decreasing globally as the tissue matures and increasing in the rostral cord stump upon transection injury. Thus, dynamic and regional regulation of JNK signaling help to direct glial cell behaviors during innate spinal cord regeneration.


Asunto(s)
Traumatismos de la Médula Espinal , Regeneración de la Medula Espinal , Animales , Larva , Mamíferos , Regeneración Nerviosa/fisiología , Neuroglía/fisiología , Médula Espinal , Pez Cebra/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos
2.
Nat Methods ; 18(8): 965-974, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34341582

RESUMEN

CRISPR-Cas9 technologies have dramatically increased the ease of targeting DNA sequences in the genomes of living systems. The fusion of chromatin-modifying domains to nuclease-deactivated Cas9 (dCas9) has enabled targeted epigenome editing in both cultured cells and animal models. However, delivering large dCas9 fusion proteins to target cells and tissues is an obstacle to the widespread adoption of these tools for in vivo studies. Here, we describe the generation and characterization of two conditional transgenic mouse lines for epigenome editing, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. By targeting the guide RNAs to transcriptional start sites or distal enhancer elements, we demonstrate regulation of target genes and corresponding changes to epigenetic states and downstream phenotypes in the brain and liver in vivo, and in T cells and fibroblasts ex vivo. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.


Asunto(s)
Sistemas CRISPR-Cas , Epigénesis Genética , Epigenoma , Edición Génica/métodos , Regulación de la Expresión Génica , Animales , Encéfalo/metabolismo , Femenino , Fibroblastos/metabolismo , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Transgénicos , Linfocitos T/metabolismo
3.
Development ; 147(14)2020 07 30.
Artículo en Inglés | MEDLINE | ID: mdl-32665240

RESUMEN

To identify candidate tissue regeneration enhancer elements (TREEs) important for zebrafish fin regeneration, we performed ATAC-seq from bulk tissue or purified fibroblasts of uninjured and regenerating caudal fins. We identified tens of thousands of DNA regions from each sample type with dynamic accessibility during regeneration, and assigned these regions to proximal genes with corresponding expression changes by RNA-seq. To determine whether these profiles reveal bona fide TREEs, we tested the sufficiency and requirements of several sequences in stable transgenic lines and mutant lines with homozygous deletions. These experiments validated new non-coding regulatory sequences near induced and/or essential genes during fin regeneration, including fgf20a, mdka and cx43, identifying distinct domains of directed expression for each confirmed TREE. Whereas deletion of the previously identified LEN enhancer abolished detectable induction of the nearby leptin b gene during regeneration, deletions of enhancers linked to fgf20a, mdka and cx43 had no effect or partially reduced gene expression. Our study generates a new resource for dissecting the regulatory mechanisms of appendage generation and reveals a range of requirements for individual TREEs in control of regeneration programs.


Asunto(s)
Aletas de Animales/metabolismo , Elementos de Facilitación Genéticos/genética , Regeneración/fisiología , Pez Cebra/metabolismo , Aletas de Animales/fisiología , Animales , Animales Modificados Genéticamente/metabolismo , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Conexina 43/genética , Conexina 43/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Leptina/genética , Leptina/metabolismo , Midkina/genética , Midkina/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
4.
BMC Genomics ; 22(1): 585, 2021 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-34340653

RESUMEN

BACKGROUND: Loss of pancreatic insulin-secreting ß-cells due to metabolic or autoimmune damage leads to the development of diabetes. The discovery that α-cells can be efficiently reprogrammed into insulin-secreting cells in mice and humans has opened promising avenues for innovative diabetes therapies. ß-cell loss triggers spontaneous reprogramming of only 1-2% of α-cells, limiting the extent of regeneration. Most α-cells are refractory to conversion and their global transcriptomic response to severe ß-cell loss as well as the mechanisms opposing their reprogramming into insulin producers are largely unknown. Here, we performed RNA-seq on FAC-sorted α-cells to characterize their global transcriptional responses at different time points after massive ß-cell ablation. RESULTS: Our results show that α-cells undergo stage-specific transcriptional changes 5- and 15-days post-diphtheria toxin (DT)-mediated ß-cell ablation. At 5 days, α-cells transiently upregulate various genes associated with interferon signaling and proliferation, including Interferon Induced Protein with Tetratricopeptide Repeats 3 (Ifit3). Subsequently, at 15 days post ß-cell ablation, α-cells undergo a transient downregulation of genes from several pathways including Insulin receptor, mTOR and MET signaling. CONCLUSIONS: The results presented here pinpoint novel markers discriminating α-cells at different stages after acute ß-cell loss, and highlight additional signaling pathways that are modulated in α-cells in this context.


Asunto(s)
Diabetes Mellitus , Células Secretoras de Glucagón , Células Secretoras de Insulina , Animales , Insulina , Ratones , Transcriptoma
5.
Stem Cells ; 38(3): 330-339, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31722129

RESUMEN

To date, most attention on tissue regeneration has focused on the exploration of positive cues promoting or allowing the engagement of natural cellular restoration upon injury. In contrast, the signals fostering cell identity maintenance in the vertebrate body have been poorly investigated; yet they are crucial, for their counteraction could become a powerful method to induce and modulate regeneration. Here we review the mechanisms inhibiting pro-regenerative spontaneous adaptive cell responses in different model organisms and organs. The pharmacological or genetic/epigenetic modulation of such regenerative brakes could release a dormant but innate adaptive competence of certain cell types and therefore boost tissue regeneration in different situations.


Asunto(s)
Medicina Regenerativa/métodos , Cicatrización de Heridas/fisiología , Humanos
6.
Nature ; 514(7523): 503-7, 2014 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-25141178

RESUMEN

Total or near-total loss of insulin-producing ß-cells occurs in type 1 diabetes. Restoration of insulin production in type 1 diabetes is thus a major medical challenge. We previously observed in mice in which ß-cells are completely ablated that the pancreas reconstitutes new insulin-producing cells in the absence of autoimmunity. The process involves the contribution of islet non-ß-cells; specifically, glucagon-producing α-cells begin producing insulin by a process of reprogramming (transdifferentiation) without proliferation. Here we show the influence of age on ß-cell reconstitution from heterologous islet cells after near-total ß-cell loss in mice. We found that senescence does not alter α-cell plasticity: α-cells can reprogram to produce insulin from puberty through to adulthood, and also in aged individuals, even a long time after ß-cell loss. In contrast, before puberty there is no detectable α-cell conversion, although ß-cell reconstitution after injury is more efficient, always leading to diabetes recovery. This process occurs through a newly discovered mechanism: the spontaneous en masse reprogramming of somatostatin-producing δ-cells. The juveniles display 'somatostatin-to-insulin' δ-cell conversion, involving dedifferentiation, proliferation and re-expression of islet developmental regulators. This juvenile adaptability relies, at least in part, upon the combined action of FoxO1 and downstream effectors. Restoration of insulin producing-cells from non-ß-cell origins is thus enabled throughout life via δ- or α-cell spontaneous reprogramming. A landscape with multiple intra-islet cell interconversion events is emerging, offering new perspectives for therapy.


Asunto(s)
Envejecimiento/fisiología , Transdiferenciación Celular , Diabetes Mellitus Experimental/patología , Células Secretoras de Insulina/citología , Insulina/biosíntesis , Regeneración , Células Secretoras de Somatostatina/citología , Animales , Desdiferenciación Celular , Proliferación Celular , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/terapia , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Células Secretoras de Glucagón/citología , Células Secretoras de Glucagón/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Ratones , Maduración Sexual , Somatostatina/biosíntesis , Somatostatina/metabolismo , Células Secretoras de Somatostatina/metabolismo
7.
Biochim Biophys Acta ; 1818(8): 1919-36, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22001400

RESUMEN

The advent of multicellular organisms was accompanied by the development of short- and long-range chemical signalling systems, including those provided by the nervous and endocrine systems. In turn, the cells of these two systems have developed mechanisms for interacting with both adjacent and distant cells. With evolution, such mechanisms have diversified to become integrated in a complex regulatory network, whereby individual endocrine and neuro-endocrine cells sense the state of activity of their neighbors and, accordingly, regulate their own level of functioning. A consistent feature of this network is the expression of connexin-made channels between the (neuro)hormone-producing cells of all endocrine glands and secretory regions of the central nervous system so far investigated in vertebrates. This review summarizes the distribution of connexins in the mammalian (neuro)endocrine systems, and what we know about the participation of these proteins on hormone secretion, the life of the producing cells, and the action of (neuro)hormones on specific targets. The data gathered since the last reviews on the topic are summarized, with particular emphasis on the roles of Cx36 in the function of the insulin-producing beta cells of the endocrine pancreas, and of Cx40 in that of the renin-producing juxta-glomerular epithelioid cells of the kidney cortex. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.


Asunto(s)
Conexinas/fisiología , Hormonas/metabolismo , Neuronas/metabolismo , Animales , Dopamina/metabolismo , Sistema Endocrino/fisiología , Femenino , Hormona Liberadora de Gonadotropina/metabolismo , Humanos , Insulina/metabolismo , Corteza Renal/metabolismo , Masculino , Modelos Biológicos , Oxitocina/metabolismo , Renina/metabolismo , Transducción de Señal , Vasopresinas/metabolismo
8.
Nat Commun ; 14(1): 4857, 2023 08 11.
Artículo en Inglés | MEDLINE | ID: mdl-37567873

RESUMEN

Unlike adult mammals, zebrafish regenerate spinal cord tissue and recover locomotor ability after a paralyzing injury. Here, we find that ependymal cells in zebrafish spinal cords produce the neurogenic factor Hb-egfa upon transection injury. Animals with hb-egfa mutations display defective swim capacity, axon crossing, and tissue bridging after spinal cord transection, associated with disrupted indicators of neuron production. Local recombinant human HB-EGF delivery alters ependymal cell cycling and tissue bridging, enhancing functional regeneration. Epigenetic profiling reveals a tissue regeneration enhancer element (TREE) linked to hb-egfa that directs gene expression in spinal cord injuries. Systemically delivered recombinant AAVs containing this zebrafish TREE target gene expression to crush injuries of neonatal, but not adult, murine spinal cords. Moreover, enhancer-based HB-EGF delivery by AAV administration improves axon densities after crush injury in neonatal cords. Our results identify Hb-egf as a neurogenic factor necessary for innate spinal cord regeneration and suggest strategies to improve spinal cord repair in mammals.


Asunto(s)
Traumatismos de la Médula Espinal , Regeneración de la Medula Espinal , Animales , Humanos , Ratones , Axones/metabolismo , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Mamíferos , Regeneración Nerviosa/genética , Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/terapia , Traumatismos de la Médula Espinal/metabolismo , Regeneración de la Medula Espinal/fisiología , Pez Cebra/genética
9.
Cell Stem Cell ; 30(1): 96-111.e6, 2023 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-36516837

RESUMEN

The efficacy and safety of gene-therapy strategies for indications like tissue damage hinge on precision; yet, current methods afford little spatial or temporal control of payload delivery. Here, we find that tissue-regeneration enhancer elements (TREEs) isolated from zebrafish can direct targeted, injury-associated gene expression from viral DNA vectors delivered systemically in small and large adult mammalian species. When employed in combination with CRISPR-based epigenome editing tools in mice, zebrafish TREEs stimulated or repressed the expression of endogenous genes after ischemic myocardial infarction. Intravenously delivered recombinant AAV vectors designed with a TREE to direct a constitutively active YAP factor boosted indicators of cardiac regeneration in mice and improved the function of the injured heart. Our findings establish the application of contextual enhancer elements as a potential therapeutic platform for spatiotemporally controlled tissue regeneration in mammals.


Asunto(s)
Elementos de Facilitación Genéticos , Terapia Genética , Corazón , Infarto del Miocardio , Miocitos Cardíacos , Regeneración , Animales , Ratones , Proliferación Celular , Corazón/fisiología , Infarto del Miocardio/genética , Infarto del Miocardio/terapia , Miocitos Cardíacos/metabolismo , Pez Cebra/genética , Terapia Genética/métodos , Regeneración/genética
10.
J Membr Biol ; 245(5-6): 263-73, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22729650

RESUMEN

The insulin-producing ß cells of pancreatic islets are coupled by connexin36 (Cx36) channels. To investigate what controls the expression of this connexin, we have investigated its pattern during mouse pancreas development, and the influence of three transcription factors that are critical for ß-cell development and differentiation. We show that (1) the Cx36 gene (Gjd2) is activated early in pancreas development and is markedly induced at the time of the surge of the transcription factors that determine ß-cell differentiation; (2) the cognate protein is detected about a week later and is selectively expressed by ß cells throughout the prenatal development of mouse pancreas; (3) a 2-kbp fragment of the Gjd2 promoter, which contains three E boxes for the binding of the bHLH factor Beta2/NeuroD1, ensures the expression of Cx36 by ß cells; and (4) Beta2/NeuroD1 binds to these E boxes and, in the presence of the E47 ubiquitous cofactor, transactivates the Gjd2 promoter. The data identify Cx36 as a novel early marker of ß cells and as a target of Beta2/NeuroD1, which is essential for ß-cell development and differentiation.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Conexinas/metabolismo , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Inmunoprecipitación de Cromatina , Biología Computacional , Uniones Comunicantes/metabolismo , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Unión Proteica/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína delta-6 de Union Comunicante
11.
Nat Commun ; 12(1): 4458, 2021 07 22.
Artículo en Inglés | MEDLINE | ID: mdl-34294685

RESUMEN

The cellular identity of pancreatic polypeptide (Ppy)-expressing γ-cells, one of the rarest pancreatic islet cell-type, remains elusive. Within islets, glucagon and somatostatin, released respectively from α- and δ-cells, modulate the secretion of insulin by ß-cells. Dysregulation of insulin production raises blood glucose levels, leading to diabetes onset. Here, we present the genetic signature of human and mouse γ-cells. Using different approaches, we identified a set of genes and pathways defining their functional identity. We found that the γ-cell population is heterogeneous, with subsets of cells producing another hormone in addition to Ppy. These bihormonal cells share identity markers typical of the other islet cell-types. In mice, Ppy gene inactivation or conditional γ-cell ablation did not alter glycemia nor body weight. Interestingly, upon ß-cell injury induction, γ-cells exhibited gene expression changes and some of them engaged insulin production, like α- and δ-cells. In conclusion, we provide a comprehensive characterization of γ-cells and highlight their plasticity and therapeutic potential.


Asunto(s)
Insulina/biosíntesis , Células Secretoras de Polipéptido Pancreático/metabolismo , Polipéptido Pancreático/metabolismo , Precursores de Proteínas/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal , Linaje de la Célula/genética , Femenino , Técnicas de Sustitución del Gen , Humanos , Células Secretoras de Insulina/clasificación , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Transgénicos , Páncreas/citología , Páncreas/embriología , Páncreas/crecimiento & desarrollo , Polipéptido Pancreático/deficiencia , Polipéptido Pancreático/genética , Células Secretoras de Polipéptido Pancreático/clasificación , Células Secretoras de Polipéptido Pancreático/citología , Embarazo , RNA-Seq
12.
Dis Model Mech ; 13(5)2020 05 27.
Artículo en Inglés | MEDLINE | ID: mdl-32461216

RESUMEN

Spinal cord injury is a devastating condition in which massive cell death and disruption of neural circuitry lead to long-term chronic functional impairment and paralysis. In mammals, spinal cord tissue has minimal capacity to regenerate after injury. In stark contrast, the regeneration of a completely transected spinal cord and accompanying reversal of paralysis in adult zebrafish is arguably one of the most spectacular biological phenomena in nature. Here, we review reports from the last decade that dissect the mechanisms of spinal cord regeneration in zebrafish. We highlight recent progress as well as areas requiring emphasis in a line of study that has great potential to uncover strategies for human spinal cord repair.


Asunto(s)
Regeneración de la Medula Espinal/fisiología , Pez Cebra/fisiología , Animales , Neovascularización Fisiológica , Células-Madre Neurales/citología , Neuronas/citología , Transducción de Señal
13.
Cell Rep ; 32(9): 108089, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-32877671

RESUMEN

Zebrafish regenerate heart muscle through division of pre-existing cardiomyocytes. To discover underlying regulation, we assess transcriptome datasets for dynamic gene networks during heart regeneration and identify suppression of genes associated with the transcription factor Tp53. Cardiac damage leads to fluctuation of Tp53 protein levels, concomitant with induced expression of its central negative regulator, mdm2, in regenerating cardiomyocytes. Zebrafish lacking functional Tp53 display increased indicators of cardiomyocyte proliferation during regeneration, whereas transgenic Mdm2 blockade inhibits injury-induced cardiomyocyte proliferation. Induced myocardial overexpression of the mitogenic factors Nrg1 or Vegfaa in the absence of injury also upregulates mdm2 and suppresses Tp53 levels, and tp53 mutations augment the mitogenic effects of Nrg1. mdm2 induction is spatiotemporally associated with markers of de-differentiation in injury and growth contexts, suggesting a broad role in cardiogenesis. Our findings reveal myocardial Tp53 suppression by mitogen-induced Mdm2 as a regulatory component of innate cardiac regeneration.


Asunto(s)
Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Regeneración/fisiología , Proteína p53 Supresora de Tumor/genética , Proteínas de Pez Cebra/genética , Animales , Proliferación Celular/fisiología , Genes p53 , Miocardio/citología , Miocitos Cardíacos/citología , Pez Cebra
14.
Dev Cell ; 48(6): 853-863.e5, 2019 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-30713073

RESUMEN

Attaining proper organ size during development and regeneration hinges on the activity of mitogenic factors. Here, we performed a large-scale chemical screen in embryonic zebrafish to identify cardiomyocyte mitogens. Although commonly considered anti-proliferative, vitamin D analogs like alfacalcidol had rapid, potent mitogenic effects on embryonic and adult cardiomyocytes in vivo. Moreover, pharmacologic or genetic manipulation of vitamin D signaling controlled proliferation in multiple adult cell types and dictated growth rates in embryonic and juvenile zebrafish. Tissue-specific modulation of vitamin D receptor (VDR) signaling had organ-restricted effects, with cardiac VDR activation causing cardiomegaly. Alfacalcidol enhanced the regenerative response of injured zebrafish hearts, whereas VDR blockade inhibited regeneration. Alfacalcidol activated cardiac expression of genes associated with ErbB2 signaling, while ErbB2 inhibition blunted its effects on cell proliferation. Our findings identify vitamin D as mitogenic for cardiomyocytes and other cell types in zebrafish and indicate a mechanism to regulate organ size and regeneration.


Asunto(s)
Corazón/anatomía & histología , Corazón/fisiología , Miocitos Cardíacos/citología , Regeneración/efectos de los fármacos , Vitamina D/farmacología , Pez Cebra/anatomía & histología , Pez Cebra/fisiología , Animales , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Corazón/efectos de los fármacos , Mitógenos/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Especificidad de Órganos , Transducción de Señal/efectos de los fármacos , Pez Cebra/embriología , Proteínas de Pez Cebra/metabolismo
15.
Nat Cell Biol ; 20(11): 1267-1277, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30361701

RESUMEN

The mechanisms that restrict regeneration and maintain cell identity following injury are poorly characterized in higher vertebrates. Following ß-cell loss, 1-2% of the glucagon-producing α-cells spontaneously engage in insulin production in mice. Here we explore the mechanisms inhibiting α-cell plasticity. We show that adaptive α-cell identity changes are constrained by intra-islet insulin- and Smoothened-mediated signalling, among others. The combination of ß-cell loss or insulin-signalling inhibition, with Smoothened inactivation in α- or δ-cells, stimulates insulin production in more α-cells. These findings suggest that the removal of constitutive 'brake signals' is crucial to neutralize the refractoriness to adaptive cell-fate changes. It appears that the maintenance of cell identity is an active process mediated by repressive signals, which are released by neighbouring cells and curb an intrinsic trend of differentiated cells to change.


Asunto(s)
Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Transducción de Señal , Receptor Smoothened/metabolismo , Animales , Diferenciación Celular , Plasticidad de la Célula , Proliferación Celular , Femenino , Células Secretoras de Glucagón/citología , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Receptor Smoothened/genética
16.
Mol Cell Endocrinol ; 448: 108-121, 2017 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-28390953

RESUMEN

Pannexins (Panx's) are membrane proteins involved in a variety of biological processes, including cell death signaling and immune functions. The role and functions of Panx's in pancreatic ß-cells remain to be clarified. Here, we show Panx1 and Panx2 expression in isolated islets, primary ß-cells, and ß-cell lines. The expression of Panx2, but not Panx1, was downregulated by interleukin-1ß (IL-1ß) plus interferon-γ (IFNγ), two pro-inflammatory cytokines suggested to contribute to ß-cell demise in type 1 diabetes (T1D). siRNA-mediated knockdown (KD) of Panx2 aggravated cytokine-induced apoptosis in rat INS-1E cells and primary rat ß-cells, suggesting anti-apoptotic properties of Panx2. An anti-apoptotic function of Panx2 was confirmed in isolated islets from Panx2-/- mice and in human EndoC-ßH1 cells. Panx2 KD was associated with increased cytokine-induced activation of STAT3 and higher expression of inducible nitric oxide synthase (iNOS). Glucose-stimulated insulin release was impaired in Panx2-/- islets, and Panx2-/- mice subjected to multiple low-dose Streptozotocin (MLDS) treatment, a model of T1D, developed more severe diabetes compared to wild type mice. These data suggest that Panx2 is an important regulator of the insulin secretory capacity and apoptosis in pancreatic ß-cells.


Asunto(s)
Apoptosis/efectos de los fármacos , Conexinas/deficiencia , Citocinas/farmacología , Intolerancia a la Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Animales , Conexinas/metabolismo , Técnicas de Silenciamiento del Gen , Intolerancia a la Glucosa/patología , Humanos , Hiperglucemia/patología , Inflamación/patología , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Factor de Transcripción STAT3/metabolismo , Estreptozocina
17.
PLoS One ; 11(3): e0150880, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26959991

RESUMEN

Signalling through gap junctions contributes to control insulin secretion and, thus, blood glucose levels. Gap junctions of the insulin-producing ß-cells are made of connexin 36 (Cx36), which is encoded by the GJD2 gene. Cx36-null mice feature alterations mimicking those observed in type 2 diabetes (T2D). GJD2 is also expressed in neurons, which share a number of common features with pancreatic ß-cells. Given that a synonymous exonic single nucleotide polymorphism of human Cx36 (SNP rs3743123) associates with altered function of central neurons in a subset of epileptic patients, we investigated whether this SNP also caused alterations of ß-cell function. Transfection of rs3743123 cDNA in connexin-lacking HeLa cells resulted in altered formation of gap junction plaques and cell coupling, as compared to those induced by wild type (WT) GJD2 cDNA. Transgenic mice expressing the very same cDNAs under an insulin promoter revealed that SNP rs3743123 expression consistently lead to a post-natal reduction of islet Cx36 levels and ß-cell survival, resulting in hyperglycemia in selected lines. These changes were not observed in sex- and age-matched controls expressing WT hCx36. The variant GJD2 only marginally associated to heterogeneous populations of diabetic patients. The data document that a silent polymorphism of GJD2 is associated with altered ß-cell function, presumably contributing to T2D pathogenesis.


Asunto(s)
Conexinas/metabolismo , Células Secretoras de Insulina/metabolismo , ARN Mensajero/genética , Animales , Western Blotting , Membrana Celular/metabolismo , Conexinas/genética , Femenino , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/química , Proteína delta-6 de Union Comunicante
19.
Pancreas ; 44(8): 1234-44, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26465951

RESUMEN

The pancreas produces enzymes with a digestive function and hormones with a metabolic function, which are produced by distinct cell types of acini and islets, respectively. Within these units, secretory cells coordinate their functioning by exchanging information via signals that flow in the intercellular spaces and are generated either at distance (several neural and hormonal inputs) or nearby the pancreatic cells themselves (inputs mediated by membrane ionic-specific channels and by ionic- and metabolite-permeant pannexin channels and connexin "hemichannels"). Pancreatic secretory cells further interact via the extracellular matrix of the pancreas (inputs mediated by integrins) and directly with neighboring cells, by mechanisms that do not require extracellular mediators (inputs mediated by gap and tight junction channels). Here, we review the expression and function of the connexins and pannexins that are expressed by the main secretory cells of the exocrine and endocrine pancreatic cells. Available data show that the patterns of expression of these proteins differ in acini and islets, supporting distinct functions in the physiological secretion of pancreatic enzymes and hormones. Circumstantial evidence further suggests that alterations in the signaling provided by these proteins are involved in pancreatic diseases.


Asunto(s)
Conexinas/fisiología , Islotes Pancreáticos/metabolismo , Páncreas Exocrino/metabolismo , Jugo Pancreático/metabolismo , Animales , Conexinas/metabolismo , Humanos , Islotes Pancreáticos/citología , Islotes Pancreáticos/fisiología , Modelos Biológicos , Páncreas Exocrino/citología , Páncreas Exocrino/fisiología , Enfermedades Pancreáticas/metabolismo , Enfermedades Pancreáticas/patología , Enfermedades Pancreáticas/fisiopatología , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Transducción de Señal
20.
Diabetes Res Clin Pract ; 99(3): 250-9, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23176806

RESUMEN

Proper functioning of pancreatic islets requires that numerous ß-cells are properly coordinated. With evolution, many mechanisms have converged, which now allow individual ß-cells to sense the state of activity of their neighbors as well as the changes taking place in the extracellular medium, and to regulate accordingly their own function. Here, we review one such mechanism for intercellular coordination, which depends on connexins. These integral membrane proteins accumulate at sites of close apposition between adjacent islet cell membranes, referred to as gap junctions. Recent evidence demonstrates that connexin-dependent signaling is relevant for the in vivo control of insulin biosynthesis and release, as well as for the survival of ß-cells under stressing conditions. The data suggest that alterations of this signaling may be implicated in the ß-cell alterations which characterize most forms of diabetes, raising the tantalizing possibility that targeting of the direct intercellular communications ß-cells establish within each pancreatic islet may provide a novel, therapeutically useful strategy.


Asunto(s)
Comunicación Celular/fisiología , Conexinas/fisiología , Células Secretoras de Insulina/fisiología , Animales , Apoptosis/fisiología , Uniones Comunicantes/fisiología , Humanos , Insulina/biosíntesis , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/fisiología , Proteína delta-6 de Union Comunicante
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