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1.
Int J Mol Sci ; 22(22)2021 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-34830323

RESUMEN

Sporadic occurrence of inherited eye disorders has been reported in cattle but so far pathogenic variants were found only for rare forms of cataract but not for retinopathies. The aim of this study was to characterize the phenotype and the genetic aetiology of a recessive form of congenital day-blindness observed in several cases of purebred Original Braunvieh cattle. Electroretinography in an affected calf revealed absent cone-mediated function, whereas the rods continue to function normally. Brain areas involved in vision were morphologically normal. When targeting cones by immunofluorescence, a decrease in cone number and an accumulation of beta subunits of cone cyclic-nucleotide gated channel (CNGB3) in the outer plexiform layer of affected animals was obvious. Achromatopsia is a monogenic Mendelian disease characterized by the loss of cone photoreceptor function resulting in day-blindness, total color-blindness, and decreased central visual acuity. After SNP genotyping and subsequent homozygosity mapping with twelve affected cattle, we performed whole-genome sequencing and variant calling of three cases. We identified a single missense variant in the bovine CNGB3 gene situated in a ~2.5 Mb homozygous genome region on chromosome 14 shared between all cases. All affected cattle were homozygous carriers of the p.Asp251Asn mutation that was predicted to be deleterious, affecting an evolutionary conserved residue. In conclusion, we have evidence for the occurrence of a breed-specific novel CNGB3-related form of recessively inherited achromatopsia in Original Braunvieh cattle which we have designated OH1 showing an allele frequency of the deleterious allele of ~8%. The identification of carriers will enable selection against this inherited disorder. The studied cattle might serve as an animal model to further elucidate the function of CNGB3 in mammals.


Asunto(s)
Alelos , Defectos de la Visión Cromática/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Mutación Missense , Subunidades de Proteína/genética , Células Fotorreceptoras Retinianas Conos/metabolismo , Sustitución de Aminoácidos , Animales , Asparagina/metabolismo , Ácido Aspártico/metabolismo , Bovinos , Defectos de la Visión Cromática/diagnóstico por imagen , Defectos de la Visión Cromática/metabolismo , Defectos de la Visión Cromática/patología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/deficiencia , Electrorretinografía , Femenino , Expresión Génica , Frecuencia de los Genes , Homocigoto , Masculino , Fenotipo , Subunidades de Proteína/deficiencia , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Secuenciación Completa del Genoma
2.
J Clin Med ; 10(11)2021 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-34200353

RESUMEN

Glucose is one of the most important metabolic substrates of the retina, and glycaemic imbalances can lead to serious side effects, including retinopathy. We previously showed that hypoglycaemia induces retinal cell death in mice, as well as the implication of glutathione (GSH) in this process. This study aimed to analyse the role of low glucose-induced decrease in GSH levels in endoplasmic reticulum (ER) stress. We cultured 661W photoreceptor-like cells under various glucose conditions and analysed ER stress markers at the mRNA and protein levels. We used the ERAI ("ER stress-activated indicator") mouse model to test ER stress in both ex vivo, on retinal explants, or in vivo, in mice subjected to hypoglycaemia. Moreover, we used buthionine sulfoximine (BSO) and glutamate cysteine ligase (Gclm)-KO mice as models of low GSH to test its effects on ER stress. We show that the unfolded protein response (UPR) is triggered in 661W cells and in ERAI mice under hypoglycaemic conditions. Low GSH levels promote cell death, but have no impact on ER stress. We concluded that low glucose levels induce ER stress independently of GSH levels. Inhibition of ER stress could prevent neurodegeneration, which seems to be an early event in the pathogenesis of diabetic retinopathy.

3.
Front Pharmacol ; 11: 594087, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33447243

RESUMEN

The standard treatment for neovascular age-related macular degeneration (nAMD) consists of intravitreal anti-vascular endothelial growth factors (VEGF). However, for some patients, even maximal anti-VEGF treatment does not entirely suppress exudative activity. The goal of this study was to identify molecular biomarkers in nAMD with incomplete response to anti-VEGF treatment. Aqueous humor (AH) samples were collected from three groups of patients: 17 patients with nAMD responding incompletely to anti-VEGF (18 eyes), 17 patients affected by nAMD with normal treatment response (21 eyes), and 16 control patients without any retinopathy (16 eyes). Proteomic and multiplex analyses were performed on these samples. Proteomic analyses showed that nAMD patients with incomplete anti-VEGF response displayed an increased inflammatory response, complement activation, cytolysis, protein-lipid complex, and vasculature development pathways. Multiplex analyses revealed a significant increase of soluble vascular cell adhesion molecule-1 (sVCAM-1) [ p = 0.001], interleukin-6 (IL-6) [ p = 0.009], bioactive interleukin-12 (IL-12p40) [ p = 0.03], plasminogen activator inhibitor type 1 (PAI-1) [ p = 0.004], and hepatocyte growth factor (HGF) [ p = 0.004] levels in incomplete responders in comparison to normal responders. Interestingly, the same biomarkers showed a high intercorrelation with r2 values between 0.58 and 0.94. In addition, we confirmed by AlphaLISA the increase of sVCAM-1 [ p < 0.0001] and IL-6 [ p = 0.043] in the incomplete responder group. Incomplete responders in nAMD are associated with activated angiogenic and inflammatory pathways. The residual exudative activity of nAMD despite maximal anti-VEGF treatment may be related to both angiogenic and inflammatory responses requiring specific adjuvant therapy. Data are available via ProteomeXchange with identifier PXD02247.

4.
Mol Cell Biol ; 37(24)2017 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-29038159

RESUMEN

Recent work suggested that the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) is increased in the retinal pigment epithelium (RPE) of age-related macular degeneration (ARMD) patients and therefore could be an attractive therapeutic target. Notably, ERK1/2 pathway inhibitors are used in cancer therapy, with severe and noncharacterized ocular side effects. To decipher the role of ERK1/2 in RPE cells, we conditionally disrupted the Erk1 and Erk2 genes in mouse RPE. The loss of ERK1/2 activity resulted in a significant decrease in the level of RPE65 expression, a decrease in ocular retinoid levels concomitant with low visual function, and a rapid disorganization of RPE cells, ultimately leading to retinal degeneration. Our results identify the ERK1/2 pathway as a direct regulator of the visual cycle and a critical component of the viability of RPE and photoreceptor cells. Moreover, our results caution about the need for a very fine adjustment of kinase inhibition in cancer or ARMD treatment in order to avoid ocular side effects.


Asunto(s)
Sistema de Señalización de MAP Quinasas , Degeneración Macular/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , cis-trans-Isomerasas/metabolismo , Animales , Degeneración Macular/terapia , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Modelos Animales , Retina/metabolismo , Retinoides/genética , Retinoides/metabolismo , cis-trans-Isomerasas/genética
5.
Front Aging Neurosci ; 9: 43, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28298893

RESUMEN

Age-related macular degeneration (ARMD) is the leading cause of vision loss in developed countries. Hallmarks of the disease are well known; indeed, this pathology is characterized by lipofuscin accumulation, is principally composed of lipid-containing residues of lysosomal digestion. The N-retinyl-N-retinylidene ethanolamine (A2E) retinoid which is thought to be a cytotoxic component for RPE is the best-characterized component of lipofuscin so far. Even if no direct correlation between A2E spatial distribution and lipofuscin fluorescence has been established in aged human RPE, modified forms or metabolites of A2E could be involved in ARMD pathology. Mitogen-activated protein kinase (MAPK) pathways have been involved in many pathologies, but not in ARMD. Therefore, we wanted to analyze the effects of A2E on MAPKs in polarized ARPE19 and isolated mouse RPE cells. We showed that long-term exposure of polarized ARPE19 cells to low A2E dose induces a strong decrease of the extracellular signal-regulated kinases' (ERK1/2) activity. In addition, we showed that A2E, via ERK1/2 decrease, induces a significant decrease of the retinal pigment epithelium-specific protein 65 kDa (RPE65) expression in ARPE19 cells and isolated mouse RPE. In the meantime, we showed that the decrease of ERK1/2 activity mediates an increase of basic fibroblast growth factor (bFGF) mRNA expression and secretion that induces an increase in phagocytosis via a paracrine effect. We suggest that the accumulation of deposits coming from outer segments (OS) could be explained by both an increase of bFGF-induced phagocytosis and by the decrease of clearance by A2E. The bFGF angiogenic protein may therefore be an attractive target to treat ARMD.

6.
PLoS One ; 11(2): e0150266, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26918849

RESUMEN

Glucose is the most important metabolic substrate of the retina and maintenance of normoglycemia is an essential challenge for diabetic patients. Chronic, exaggerated, glycemic excursions could lead to cardiovascular diseases, nephropathy, neuropathy and retinopathy. We recently showed that hypoglycemia induced retinal cell death in mouse via caspase 3 activation and glutathione (GSH) decrease. Ex vivo experiments in 661W photoreceptor cells confirmed the low-glucose induction of death via superoxide production and activation of caspase 3, which was concomitant with a decrease of GSH content. We evaluate herein retinal gene expression 4 h and 48 h after insulin-induced hypoglycemia. Microarray analysis demonstrated clusters of genes whose expression was modified by hypoglycemia and we discuss the potential implication of those genes in retinal cell death. In addition, we identify by gene set enrichment analysis, three important pathways, including lysosomal function, GSH metabolism and apoptotic pathways. Then we tested the effect of recurrent hypoglycemia (three successive 4h periods of hypoglycemia spaced by 48 h recovery) on retinal cell death. Interestingly, exposure to multiple hypoglycemic events prevented GSH decrease and retinal cell death, or adapted the retina to external stress by restoring GSH level comparable to control situation. We hypothesize that scavenger GSH is a key compound in this apoptotic process, and maintaining "normal" GSH level, as well as a strict glycemic control, represents a therapeutic challenge in order to avoid side effects of diabetes, especially diabetic retinopathy.


Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Hipoglucemia/inducido químicamente , Insulina/toxicidad , Familia de Multigenes/genética , Retina/metabolismo , Enfermedad Aguda , Adaptación Fisiológica , Animales , Apoptosis/genética , Fragmentación del ADN , Retinopatía Diabética/prevención & control , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Femenino , Depuradores de Radicales Libres , Regulación de la Expresión Génica/fisiología , Técnica de Clampeo de la Glucosa , Glutatión/metabolismo , Hipoglucemia/genética , Hipoglucemia/metabolismo , Insulina/farmacología , Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Oxidación-Reducción , Distribución Aleatoria , Retina/patología , Factores de Tiempo
7.
Ophthalmic Genet ; 35(1): 12-7, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23373431

RESUMEN

PURPOSE: Age-related macular degeneration (AMD) has been associated with a number of polymorphisms in genes in the complement pathway. We examined the potential genotype-phenotype correlation of complement factor B (CFB) (R32Q) polymorphisms in Caucasian patients with AMD. METHODS: Data from a Central European cohort of 349 patients with early AMD in at least one eye were analyzed for potential associations of the CFB (R32Q/rs641153) polymorphism with phenotypic features of early AMD. Early AMD was classified according to the International Classification and Grading System into predominant drusen size, largest drusen, drusen covered surface, central or ring-like location, peripheral drusen, and pigmentary changes. The potential association with single nucleotide polymorphisms on CFB (R32Q/rs641153) was evaluated for all patients, corrected for age, sex, and the polymorphisms of CFH (Y402H) and ARMS2 (A69S). RESULTS: CFB (R32Q) polymorphisms showed a significant association with smaller drusen size (largest drusen ≤ 250 µm, p = 0.021, predominant drusen ≤ 125 µm, p = 0.016), with smaller surface covered by drusen (≤ 10%; p = 0.02), and with more frequent occurrence of peripheral drusen (p = 0.007). No association was found for pigmentary changes. CONCLUSIONS: The CFB (R32Q) polymorphism was associated with AMD characterized by small drusen only, and appeared to be protective of large drusen (OR 0.48/0.45) and of larger drusen covered area (OR 0.34). Furthermore, peripheral drusen were more frequently found (OR 2.27). This result supports the role of complement components and their polymorphisms in drusen formation and may enable a better understanding of AMD pathogenesis.


Asunto(s)
Factor B del Complemento/genética , Degeneración Macular/genética , Polimorfismo de Nucleótido Simple , Drusas Retinianas/genética , Anciano , Anciano de 80 o más Años , Factor H de Complemento/genética , Femenino , Estudios de Asociación Genética , Técnicas de Genotipaje , Humanos , Masculino , Persona de Mediana Edad , Proteínas/genética
8.
PLoS One ; 8(9): e74162, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24066113

RESUMEN

Glucose is an important metabolic substrate of the retina and diabetic patients have to maintain a strict normoglycemia to avoid diabetes secondary effects, including cardiovascular disease, nephropathy, neuropathy and retinopathy. Others and we recently demonstrated the potential role of hypoglycemia in diabetic retinopathy. We showed acute hypoglycemia to induce retinal cell death both in vivo during an hyperinsulinemic/hypoglycemic clamp and in vitro in 661W photoreceptor cells cultured at low glucose concentration. In the present study, we showed low glucose to induce a decrease of BCL2 and BCL-XL anti-apoptotic proteins expression, leading to an increase of free pro-apoptotic BAX. In parallel, we showed that, in retinal cells, low glucose-induced apoptosis is involved in the process of autophagosomes formation through the AMPK/RAPTOR/mTOR pathway. Moreover, the decrease of LAMP2a expression led to a defect in the autophagosome/lysosome fusion process. Specific inhibition of autophagy, either by 3-methyladenine or by down-regulation of ATG5 or ATG7 proteins expression, increased caspase 3 activation and 661W cell death. We show that low glucose modifies the delicate equilibrium between apoptosis and autophagy. Cells struggled against low nutrient condition-induced apoptosis by starting an autophagic process, which led to cell death when inhibited. We conclude that autophagy defect is associated with low glucose-induced 661W cells death that could play a role in diabetic retinopathy. These results could modify the way of addressing negative effects of hypoglycemia. Short-term modulation of autophagy could be envisioned to treat diabetic patients in order to avoid secondary complications of the disease.


Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Glucosa/farmacología , Células Fotorreceptoras/citología , Células Fotorreceptoras/efectos de los fármacos , Animales , Western Blotting , Células Cultivadas , Inmunohistoquímica , Inmunoprecipitación , Etiquetado Corte-Fin in Situ , Ratones , Células Fotorreceptoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
9.
PLoS One ; 6(6): e21586, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21738719

RESUMEN

BACKGROUND: Glucose is the most important metabolic substrate of the retina and maintenance of normoglycemia is an essential challenge for diabetic patients. Glycemic excursions could lead to cardiovascular disease, nephropathy, neuropathy and retinopathy. A vast body of literature exists on hyperglycemia namely in the field of diabetic retinopathy, but very little is known about the deleterious effect of hypoglycemia. Therefore, we decided to study the role of acute hypoglycemia in mouse retina. METHODOLOGY/PRINCIPAL FINDINGS: To test effects of hypoglycemia, we performed a 5-hour hyperinsulinemic/hypoglycemic clamp; to exclude an effect of insulin, we made a hyperinsulinemic/euglycemic clamp as control. We then isolated retinas from each group at different time-points after the clamp to analyze cells apoptosis and genes regulation. In parallel, we used 661W photoreceptor cells to confirm in vivo results. We showed herein that hypoglycemia induced retinal cell death in mouse via caspase 3 activation. We then tested the mRNA expression of glutathione transferase omega 1 (Gsto1) and glutathione peroxidase 3 (Gpx3), two genes involved in glutathione (GSH) homeostasis. The expression of both genes was up-regulated by low glucose, leading to a decrease of reduced glutathione (GSH). In vitro experiments confirmed the low-glucose induction of 661W cell death via superoxide production and activation of caspase 3, which was concomitant with a decrease of GSH content. Moreover, decrease of GSH content by inhibition with buthionine sulphoximine (BSO) at high glucose induced apoptosis, while complementation with extracellular glutathione ethyl ester (GSHee) at low glucose restored GSH level and reduced apoptosis. CONCLUSIONS/SIGNIFICANCE: We showed, for the first time, that acute insulin-induced hypoglycemia leads to caspase 3-dependant retinal cell death with a predominant role of GSH content.


Asunto(s)
Muerte Celular/fisiología , Hiperglucemia/fisiopatología , Retina/citología , Retina/patología , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/fisiología , Western Blotting , Proteínas Portadoras/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Células Cultivadas , Femenino , Citometría de Flujo , Glutatión/metabolismo , Glutatión Peroxidasa/genética , Glutatión Transferasa/genética , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
10.
PLoS One ; 4(10): e7379, 2009 Oct 12.
Artículo en Inglés | MEDLINE | ID: mdl-19823680

RESUMEN

BACKGROUND: NR2E3 (PNR) is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS) and, more recently, with autosomal dominant retinitis pigmentosa (adRP). NR2E3 acts as a suppressor of the cone generation program in late mitotic retinal progenitor cells. In adult rod photoreceptors, NR2E3 represses cone-specific gene expression and acts in concert with the transcription factors CRX and NRL to activate rod-specific genes. NR2E3 and CRX have been shown to physically interact in vitro through their respective DNA-binding domains (DBD). The DBD also contributes to homo- and heterodimerization of nuclear receptors. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed NR2E3 homodimerization and NR2E3/CRX complex formation in an in vivo situation by Bioluminescence Resonance Energy Transfer (BRET(2)). NR2E3 wild-type protein formed homodimers in transiently transfected HEK293T cells. NR2E3 homodimerization was impaired in presence of disease-causing mutations in the DBD, except for the p.R76Q and p.R104W mutant proteins. Strikingly, the adRP-linked p.G56R mutant protein interacted with CRX with a similar efficiency to that of NR2E3 wild-type and p.R311Q proteins. In contrast, all other NR2E3 DBD-mutant proteins did not interact with CRX. The p.G56R mutant protein was also more effective in abolishing the potentiation of rhodospin gene transactivation by the NR2E3 wild-type protein. In addition, the p.G56R mutant enhanced the transrepression of the M- and S-opsin promoter, while all other NR2E3 DBD-mutants did not. CONCLUSIONS/SIGNIFICANCE: These results suggest different disease mechanisms in adRP- and ESCS-patients carrying NR2E3 mutations. Titration of CRX by the p.G56R mutant protein acting as a repressor in trans may account for the severe clinical phenotype in adRP patients.


Asunto(s)
Proteínas de Homeodominio/química , Mutación , Receptores Nucleares Huérfanos/química , Transactivadores/química , Línea Celular , Dimerización , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/química , Humanos , Mitosis , Modelos Moleculares , Estructura Terciaria de Proteína , Retina/citología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Retinitis Pigmentosa/metabolismo , Células Madre/citología
11.
J Diabetes ; 1(4): 263-72, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20923527

RESUMEN

BACKGROUND: Peroxisome proliferator-activated receptor α (PPARα) regulates the expression of fatty acid metabolism genes and is thought to play a role in the regulation of insulin secretion and lipid detoxification. We have examined the mechanism whereby glucose decreases PPARα gene expression in the pancreatic ß-cell. METHODS: INS832/13 ß-cell and isolated rat islets were incubated at 3 and 20 mM glucose for 18 h in the absence or presence of adenosine monophosphate (AMP)-activated protein kinase (AMPK) activators and inhibitors, as well as p38 mitogen-activated protein kinase (p38 MAPK) inhibitors. In another set of experiments, INS832/13 were infected with an adenovirus expressing a dominant-negative form of AMPK. PPARα expression levels were measured by reverse transcription polymerase chain reaction and Western blot. RESULTS: Elevated glucose reduced the abundance of the PPARα transcript and protein, and its target genes acyl-coenzyme A (CoA) oxidase (ACO) and uncoupling protein 2 (UCP-2) in INS832/13 ß-cell and isolated rat islets. Glucose reduced AMPK activity, while the AMPK activators 5-amino-4-imidazolecarboxamide riboside and metformin increased PPARα expression and suppressed the action of glucose. By contrast, the AMPK inhibitor compound C mimicked the glucose effect. A dominant negative form of AMPKα reduced the PPARα, ACO and UCP-2 transcripts to the same extent as elevated glucose. Pharmacological evidence indicated that glucose-regulated PPARα expression does not involve p38 MAPK, a target of AMPK in several cell types. CONCLUSIONS: The results indicate that glucose represses PPARα gene expression via AMPK, but not via p38 MAPK in the ß-cell.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Islotes Pancreáticos/fisiología , PPAR alfa/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Cartilla de ADN , Regulación hacia Abajo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/enzimología , PPAR alfa/efectos de los fármacos , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos
12.
Hum Mutat ; 30(3): 342-51, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19006237

RESUMEN

NR2E3, a photoreceptor-specific nuclear receptor (PNR), represses cone-specific genes and activates several rod-specific genes. In humans, mutations in NR2E3 have been associated with the recessively-inherited enhanced short-wavelength sensitive S-cone syndrome (ESCS) and, recently, with autosomal dominant (ad) retinitis pigmentosa (RP) (adRP). In the present work, we describe two additional families affected by adRP that carry a heterozygous c.166G>A (p.G56R) mutation in the NR2E3 gene. Functional analysis determined the dominant negative activity of the p.G56R mutant protein as the molecular mechanism of adRP. Interestingly, in one pedigree, the most common causal variant for ESCS (p.R311Q) cosegregated with the adRP-linked p.G56R mutation, and the compound heterozygotes exhibited an ESCS-like phenotype, which in 1 of the 2 cases was strikingly "milder" than the patients carrying the p.G56R mutation alone. Impaired repression of cone-specific genes by the corepressors atrophin-1 (dentatorubral-pallidoluysian atrophy [DRPLA] gene product) and atrophin-2 (arginine-glutamic acid dipeptide repeat [RERE] protein) appeared to be a molecular mechanism mediating the beneficial effect of the p.R311Q mutation. Finally, the functional dominance of the p.R311Q variant to the p.G56R mutation is discussed.


Asunto(s)
Mutación , Receptores Citoplasmáticos y Nucleares/genética , Degeneración Retiniana/genética , Factores de Transcripción/genética , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Animales , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Salud de la Familia , Femenino , Genes Dominantes , Genes Recesivos , Genotipo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Receptores Nucleares Huérfanos , Linaje , Unión Proteica , Receptores Citoplasmáticos y Nucleares/metabolismo , Degeneración Retiniana/patología , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/patología , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismo , Adulto Joven
13.
FASEB J ; 22(6): 1905-13, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18263705

RESUMEN

c-Jun N-terminal kinases (SAPK/JNKs) are activated by inflammatory cytokines, and JNK signaling is involved in insulin resistance and beta-cell secretory function and survival. Chronic high glucose concentrations and leptin induce interleukin-1beta (IL-1beta) secretion from pancreatic islets, an event that is possibly causal in promoting beta-cell dysfunction and death. The present study provides evidence that chronically elevated concentrations of leptin and glucose induce beta-cell apoptosis through activation of the JNK pathway in human islets and in insulinoma (INS 832/13) cells. JNK inhibition by the dominant inhibitor JNK-binding domain of IB1/JIP-1 (JNKi) reduced JNK activity and apoptosis induced by leptin and glucose. Exposure of human islets to leptin and high glucose concentrations leads to a decrease of glucose-induced insulin secretion, which was partly restored by JNKi. We detected an interplay between the JNK cascade and the caspase 1/IL-1beta-converting enzyme in human islets. The caspase 1 gene, which contains a potential activating protein-1 binding site, was up-regulated in pancreatic sections and in isolated islets from type 2 diabetic patients. Similarly, cultured human islets exposed to high glucose- and leptin-induced caspase 1 and JNK inhibition prevented this up-regulation. Therefore, JNK inhibition may protect beta-cells from the deleterious effects of high glucose and leptin in diabetes.


Asunto(s)
Apoptosis/efectos de los fármacos , Glucosa/farmacología , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Leptina/farmacología , Caspasa 1/genética , Células Cultivadas , Diabetes Mellitus Tipo 2/patología , Humanos , Secreción de Insulina , Islotes Pancreáticos/citología , Regulación hacia Arriba/efectos de los fármacos
14.
Apoptosis ; 13(3): 343-53, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18253836

RESUMEN

The retinal pigment epithelium (RPE) is constantly exposed to external injuries which lead to degeneration, dysfunction or loss of RPE cells. The balance between RPE cells death and proliferation may be responsible for several diseases of the underlying retina, including age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Signaling pathways able to control cells proliferation or death usually involve the MAPK (mitogen-activated protein kinases) pathways, which modulate the activity of transcription factors by phosphorylation. UV exposure induces DNA breakdown and causes cellular damage through the production of reactive oxygen species (ROS) leading to programmed cell death. In this study, human retinal pigment epithelial cells ARPE19 were exposed to 100 J/m(2) of UV-C and MAPK pathways were studied. We first showed the expression of the three major MAPK pathways. Then we showed that activator protein-1 (AP-1) was activated through phosphorylation of cJun and cFos, induced by JNK and p38, respectively. Specific inhibitors of both kinases decreased their respective activities and phosphorylation of their nuclear targets (cJun and cFos) and reduced UV-induced cell death. The use of specific kinases inhibitors may provide excellent tools to prevent RPE apoptosis specifically in RPE diseases involving ROS and other stress-related compounds such as in AMD.


Asunto(s)
Apoptosis/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Epitelio Pigmentado Ocular/fisiopatología , Apoptosis/efectos de la radiación , Línea Celular , Humanos , MAP Quinasa Quinasa 4/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/efectos de la radiación , Proteína Quinasa 9 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/efectos de la radiación , Epitelio Pigmentado Ocular/citología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Retina/enzimología , Retina/efectos de la radiación , Factor de Transcripción AP-1/metabolismo , Rayos Ultravioleta , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de la radiación
15.
J Biomol Screen ; 11(8): 1015-26, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17092917

RESUMEN

Protein kinases are directly implicated in many human diseases; therefore, kinase inhibitors show great promises as new therapeutic drugs. In an effort to facilitate the screening and the characterization of kinase inhibitors, a novel application of the AlphaScreen technology was developed to monitor JNK activity from (1) purified kinase preparations and (2) endogenous kinase from cell lysates preactivated with different cytokines. The authors confirmed that both adenosine triphosphate (ATP) competitive as well as peptide-based JNK inhibitors were able to block the activity of both recombinant and HepG2 endogenous JNK activity. Using the same luminescence technique adapted for binding studies, the authors characterized peptide inhibitor mechanisms by measuring the binding affinity of the inhibitors for JNK. Because of the versatility of the technology, this cell-based JNK kinase assay could be adapted to other kinases and would represent a powerful tool to evaluate endogenous kinase activity and test a large number of potential inhibitors in a more physiologically relevant environment.


Asunto(s)
Técnicas Químicas Combinatorias/métodos , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/química , Proteínas Quinasas/metabolismo , Sitios de Unión , Unión Competitiva , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , MAP Quinasa Quinasa 4/metabolismo
16.
EMBO J ; 25(4): 785-97, 2006 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-16456539

RESUMEN

Islet-brain 1 (IB1 or JIP-1) is a scaffold protein that interacts with components of the c-Jun N-terminal kinase (JNK) signal-transduction pathway. IB1 is expressed at high levels in neurons and in pancreatic beta-cells, where it controls expression of several insulin-secretory components and secretion. IB1 has been shown to homodimerize, but neither the molecular mechanisms nor the function of dimerization have yet been characterized. Here, we show that IB1 homodimerizes through a novel and unique set of Src homology 3 (SH3)-SH3 interactions. X-ray crystallography studies show that the dimer interface covers a region usually engaged in PxxP-mediated ligand recognition, even though the IB1 SH3 domain lacks this motif. The highly stable IB1 homodimer can be significantly destabilized in vitro by three individual point mutations directed against key residues involved in dimerization. Each mutation reduces IB1-dependent basal JNK activity in 293T cells. Impaired dimerization also results in a reduction in glucose transporter type 2 expression and in glucose-dependent insulin secretion in pancreatic beta-cells. Taken together, these results indicate that IB1 homodimerization through its SH3 domain has pleiotropic effects including regulation of the insulin secretion process.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Regulación de la Expresión Génica/fisiología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Sustitución de Aminoácidos , Línea Celular , Cristalografía por Rayos X , Dimerización , Transportador de Glucosa de Tipo 2/metabolismo , Humanos , Secreción de Insulina , MAP Quinasa Quinasa 4/química , Neuronas/metabolismo , Mutación Puntual , Dominios Homologos src/genética
17.
FEBS Lett ; 579(29): 6581-6, 2005 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-16298369

RESUMEN

A role for cytosolic malonyl-CoA decarboxylase (MCD) as a regulator of fatty acid oxidation has been postulated. However, there is no direct evidence that MCD is present in the cytosol. To address this issue, we performed cell fractionation and electron microscopic colloidal gold studies of rat liver to determine the location and activity of MCD. By both methods, substantial amounts of MCD protein and activity were found in the cytosol, mitochondria and peroxisomes, the latter with the highest specific activity. MCD species with different electrophoretic mobility were observed in the three fractions. The data demonstrate that active MCD is present in the cytosol, mitochondria and peroxisomes of rat liver, consistent with the view that MCD participates in the regulation of cytosolic malonyl-CoA levels and of hepatic fatty acid oxidation.


Asunto(s)
Carboxiliasas/análisis , Citosol/enzimología , Hepatocitos/citología , Animales , Carboxiliasas/fisiología , Fraccionamiento Celular , Masculino , Microscopía Electrónica , Mitocondrias Hepáticas/enzimología , Peroxisomas/enzimología , Ratas , Ratas Sprague-Dawley
18.
Endocrinology ; 146(1): 375-82, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15459119

RESUMEN

The cellular response to fasting and starvation in tissues such as heart, skeletal muscle, and liver requires peroxisome proliferator-activated receptor-alpha (PPARalpha)-dependent up-regulation of energy metabolism toward fatty acid oxidation (FAO). PPARalpha null (PPARalphaKO) mice develop hyperinsulinemic hypoglycemia in the fasting state, and we previously showed that PPARalpha expression is increased in islets at low glucose. On this basis, we hypothesized that enhanced PPARalpha expression and FAO, via depletion of lipid-signaling molecule(s) for insulin exocytosis, are also involved in the normal adaptive response of the islet to fasting. Fasted PPARalphaKO mice compared with wild-type mice had supranormal ip glucose tolerance due to increased plasma insulin levels. Isolated islets from the PPARalpha null mice had a 44% reduction in FAO, normal glucose use and oxidation, and enhanced glucose-induced insulin secretion. In normal rats, fasting for 24 h increased islet PPARalpha, carnitine palmitoyltransferase 1, and uncoupling protein-2 mRNA expression by 60%, 62%, and 82%, respectively. The data are consistent with the view that PPARalpha, via transcriptionally up-regulating islet FAO, can reduce insulin secretion, and that this mechanism is involved in the normal physiological response of the pancreatic islet to fasting such that hypoglycemia is avoided.


Asunto(s)
Adaptación Fisiológica , Ayuno/fisiología , Ácidos Grasos/metabolismo , Islotes Pancreáticos/fisiología , PPAR alfa/fisiología , Transcripción Genética , Regulación hacia Arriba , Animales , Expresión Génica/fisiología , Glucosa/metabolismo , Glucosa/fisiología , Prueba de Tolerancia a la Glucosa , Hormonas/metabolismo , Insulina/sangre , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Ratones , Ratones Noqueados , Oxidación-Reducción , PPAR alfa/deficiencia , PPAR alfa/metabolismo , Ratas , Ratas Wistar
19.
Diabetes ; 53(11): 2815-23, 2004 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15504961

RESUMEN

Pancreatic islet transplantation may successfully restore normoglycemia in type 1 diabetic patients. However, successful grafting requires transplantation of a sufficient number of islets, usually requiring two or more donors. During the isolation process and following clinical transplantation, islets are subjected to severe adverse conditions that impair survival and ultimately contribute to graft failure. Here, we have mapped the major intracellular stress-signaling pathways that may mediate human islet loss during isolation and following cytokine attack. We found that the isolation procedure potently recruits two pathways consisting of |mitogen-activated protein kinase kinase (MKK)7 --> Jun NH(2)-terminal kinase (JNK)/p38 --> c-fos| and the |nuclear factor-kappaB (NF-kappaB) --> iNOS| module. Cytokines activate the |NF-kappaB --> iNOS| and |MKK4/MKK3/6 --> JNK/p38| pathways without recruitment of c-fos. Culturing the islets for 48 h after isolation allows for the activated pathways to return to background levels, with expression of MKK7 becoming undetectable. These data indicate that isolation and cytokines recruit different death pathways. Therefore, strategies might be rationally developed to avoid possible synergistic activation of these pathways in mediating islet loss during isolation and following grafting.


Asunto(s)
Citocinas/farmacología , Islotes Pancreáticos/fisiología , Apoptosis/efectos de los fármacos , Secuencia de Bases , Cartilla de ADN , Regulación de la Expresión Génica/efectos de los fármacos , Genes fos/genética , Humanos , Interleucina-1/farmacología , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 4 , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
20.
Diabetes ; 53(4): 1007-19, 2004 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15047616

RESUMEN

The malonyl-CoA/long-chain acyl-CoA (LC-CoA) model of glucose-induced insulin secretion (GIIS) predicts that malonyl-CoA derived from glucose metabolism inhibits fatty acid oxidation, thereby increasing the availability of LC-CoA for lipid signaling to cellular processes involved in exocytosis. For directly testing the model, INSr3 cell clones overexpressing malonyl-CoA decarboxylase in the cytosol (MCDc) in a tetracycline regulatable manner were generated, and INS(832/13) and rat islets were infected with MCDc-expressing adenoviruses. MCD activity was increased more than fivefold, and the malonyl-CoA content was markedly diminished. This was associated with enhanced fat oxidation at high glucose, a suppression of the glucose-induced increase in cellular free fatty acid (FFA) content, and reduced partitioning at elevated glucose of exogenous palmitate into lipid esterification products. MCDc overexpression, in the presence of exogenous FFAs but not in their absence, reduced GIIS in all beta-cell lines and in rat islets. It also markedly curtailed the stimulation of insulin secretion by other fuel and nonfuel secretagogues. In the absence of MCDc overexpression, the secretory responses to all types of secretagogues were amplified by the provision of exogenous fatty acids. In the presence of exogenous FFAs, the fatty acyl-CoA synthetase inhibitor triacsin C reduced secretion in response to glucose and nonfuel stimuli. The data show the existence of important links between the metabolic coupling factor malonyl-CoA, the partitioning of fatty acids, and the stimulation of insulin secretion to both fuel and nonfuel stimuli.


Asunto(s)
Acilcoenzima A/fisiología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Lípidos/fisiología , Malonil Coenzima A/metabolismo , Transducción de Señal/fisiología , Animales , Carboxiliasas/genética , Carboxiliasas/metabolismo , Células Clonales , Glucosa/farmacología , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Modelos Biológicos , Proteínas Recombinantes/metabolismo , Transfección
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