Regulació del factor de transcripció NFAT5 per les quinases WNK1, SPAK i OSR1 i cerca de quinases activadores de la pròpia WNK1 en resposta a estrés osmòtic

Estudi elaborat a partir d’una estada a la School of Life Sciences de la University of Dundee, Gran Bretanya, entre gener i març del 2007.L'estrès osmòtic causa rà pidament l'activació de la quinasa WNK1, que fosforila i activa a continuació les quinases SPAK i OSR1, que alhora regulen canals i transportadors d’ions preexistents a la membrana cel•lular. El factor de transcripció NFAT5 és el principal regulador de la resposta cel•lular transcripcional secundà ria a hipertonicitat i s’ha descrit que les quinases p38, Fyn, PKA, ERK/MEK i ATM estan involucrades en la seva regulació post-traduccional. No obstant, com que la funció d’aquestes quinases no explica totalment els mecanismes d'activació de NFAT5, s’ha estudiat si l’activitat transcripcional de NFAT5 pot estar regulada per WNK1, SPAK o OSR1. Així doncs, es va observar que l’activitat d’un reporter dependent de NFAT5 no es veu afectada per la presència de cap de les quinases anteriors, en la seva forma wild-type o dominant negatiu. D’altra banda, es va estudiar quin domini de WNK1 és necessari per a que pugui respondre a hipertonicitat i quines quinases poden estar involucrades en la fosforilació de la serina 382 de WNK1. En conclusió, les dades obtingudes apunten que l’activació de WNK1 en resposta a estrès osmòtic requereix la seva fosforilació en la serina 382 per quinases upstream com PAK2 o RSK i que també és necessari un dels seus dominis coiled-coil, almenys els aminoà cids 558 i 561. Aquests processos, però, semblen ser independents de l’activació de NFAT5 en resposta a hipertonicitat.   

Avaluació dels efectes de les procianidines de pinyol de raïm sobre la secreció d'insulina

Les procianidines són un tipus de compostos fenòlics que es troben en aliments com la xocolata, i en begudes com el vi negre. Actualment es coneix que les procianidines exerceixen efectes beneficiosos sobre la salut, especialment sobre el sistema cardiovascular. Estudis realitzats pel grup de recerca “Nutrigenòmica” d’aquesta universitat, mostren indicis que les procianidines afecten la funció secretora d’insulina del pàncrees. En aquest estudi s’han avaluat els efectes que du a terme l’extracte de procianidines de pinyol de raïm (GSPE) sobre la síntesi i la secreció d’insulina en el pàncrees, mitjançant experiments realitzats in vitro, amb la línia cel•lular pancreàtica MIN-6, i in vivo, en models de rata sans i amb síndrome metabòlica. In vitro, s’observa que GSPE realitza una acció inhibidora de la secreció d’insulina en les cèl•lules MIN-6 en condicions basals de glucosa, essent a una concentració de 1 mg/l de GSPE on s’observa un efecte més clar. Els responsables d’aquesta inhibició són els àcids fenòlics i els monòmers presents en l’extracte. En condicions d’estimulació aquest extracte produeix una certa activació de la secreció d’insulina, en la mateixa línia cel•lular, a l’exposar les cèl•lules a una dosi de 10 mg/l. In vivo, l’assaig de l’expressió gènica de la insulina mostra que GSPE redueix la transcripció d’aquest gen, i que aquest efecte depèn tant de la dosi com del període d’administració de l’extracte. Estudis preliminars per determinar el mecanisme d’acció de GSPE, indiquen que aquestes molècules modulen l’expressió del gen de Pdx-1, el qual és un factor de transcripció que estimula l’expressió del gen de la insulina. Amb els resultats obtinguts es pot concloure que GSPE actua frenant tant la síntesi com la secreció d’insulina, tot i que s’ha d’aprofundir més en la descripció de l’efecte observat, i els mecanismes moleculars pels quals te lloc.

Akt fosforila FoxG1 en cèl·lules de glioma

En un treball recent s’ha descrit l’amplificació del gen del factor de transcripció FoxG1, homòleg de l'oncogen víric aviar Qin, en mostres de meduloblastoma, un tipus de tumor cerebral que representa el 20% dels tumors cerebrals infantils malignes (Adesina et al.¸2007). El tumor cerebral més freqüent i agressiu en l’adult és el glioma, especialment la seva forma més maligna: el glioblastoma multiforme (glioma de grau IV segons la classificació de l'OMS). En aquest treball hem estudiat l'expressió proteica del factor de transcripció FoxG1, homòleg de l'oncogen víric aviar Qin, en mostres de glioma. Vam analitzar 15 mostres de glioma, detectant FoxG1 en 9 d’elles, i amb diferents nivells d’expressió. Intentant aprofundir en el coneixement de la funció i la regulació de FoxG1, vam estudiar si FoxG1 podia ser fosforilat. Vam detectar, tant per assaig cinasa com per espectrometria de masses, que FoxG1 és un substracte directe de la cinasa Akt, el principal efector de la via de PI3K (phosphoinositide 3-kinase). En la línia cel•lular de glioblastoma U373MG, vam observar que Akt endogen fosforila FoxG1 en un pèptid situat a l’extrem C-terminal del domini forkhead. Aquesta fosforilació és contrarestada per un inhibidor farmacològic de PI3K. Al contrari del que passa en FoxO on la fosforilació per Akt inhibeix l’activitat de FoxO promovent la seva exportació del nucli, la fosforilació de FoxG1 per Akt no promou cap canvi en la seva localització subcel•lular, i FoxG1 es manté nuclear. Actualment estem estudiant els efectes biològics de la fosforilació de FoxG1 per Akt.

PGC-1α regula la respuesta celular frente a estrés oxidativo en el miocardio tras síndrome coronario agudo

PGC-1α es un factor de transcripción maestro en la regulación mitocondrial de genes de protección frente a estrés oxidativo. Decidimos analizar el papel de la molécula en la regulación celular miocárdica tras infarto agudo. Evaluamos 38 pacientes con diagnóstico de SCACEST sometidos a estrategia de reperfusión. Encontramos que los pacientes con nivel basal de expresión reducido y mayor inducción de PGC-1α tras el evento presentaban infartos más extensos estimados por resonancia cardiaca. Concluimos que PGC-1α participa en la regulación de la respuesta celular frente a isquemia, en base a la activación de enzimas de protección mitocondrial.

Progresión tumoral y búsqueda de nuevas dianas terapéuticas en la familia de tumores del sarcoma de Ewing: función biológica de la caveolina-1

El sarcoma de Ewing es el segundo tumor óseo infantil más frecuente y presenta una alta incidencia de enfermedad metastática. Este tipo de tumores presentan una traslocación génica característica que da origen a una proteína de fusión, normalmente EWS/FLI1. Esta proteína de fusión actúa como factor de transcripción aberrante regulando la expresión de diferentes genes implicados en la iniciación, mantenimiento y progresión del tumor. Nuestro grupo describió como uno de estos genes diana a la caveolina 1 (CAV1) describiendo además su papel determinante en el fenotipo maligno del sarcoma de Ewing, en la tumorigénesis y en la resistencia a apoptosis inducida por quimioterapia. Para investigar el papel concreto de CAV1 en el proceso metastático de este sarcoma, creamos un modelo de baja expresión de CAV1 en líneas celulares de sarcoma de Ewing y determinamos cambios en su capacidad migratoria, invasiva y metastática. En los ensayos in vitro hallamos una menor capacidad migratoria de las células knockdown de CAV1 y una reducción en la expresión de MMP9 y en la actividad de MMP2. La regulación de la actividad de MMP2 parece estar relacionada con la posible regulación que ejerce CAV1 en la función de MT1-MMP, proteína fundamental para la activación de MMP2. Por otro lado, en este estudio proponemos que CAV1 promueve la expresión de MMP9 tanto transcripcionalmente, regulando la vía de señalización ERK1/2, como a nivel post-transcripcional regulando la vía RSK1/rpS6. Además, en los ensayos de metástasis experimental in vivo las células knockdown de CAV1 presentaron una menor incidencia de metástasis pulmonar, hecho que correlacionó con una disminución en la expresión de SPARC, una proteína de adhesión importante en procesos metastáticos. En resumen, nuestros resultados evidencian la importancia de CAV1 en el proceso metastático del sarcoma de Ewing.

Proteostasis of aging and stem cells

Estudi realitzat a partir d’una estada a la the Salk Institute, Estats Units, entre 2010 i 2012. L'estabilitat del genoma és essencial per a la supervivència de les cèl • lules mare, però, l'estabilitat del proteoma pot tenir un paper igualment important en la identitat de cèl • lules mare i la seva funció. La nostra hipòtesi és que les cèl • lules mare tenen la capacitat de proteostasis augmentada en comparació amb els seus homòlegs diferenciats i ens varem preguntar si l'activitat del proteasoma és diferent a les cèl • lules mare embrionàries humanes (hESCs). En particular, els nostres resultats mostren que les poblacions de cèl• lules mare presenten una activitat del proteasoma que es correlaciona amb majors nivells de la subunitat 19S del proteasoma PSMD11/RPN-6 i un corresponent augment del ensamblatge del 26S/30S proteasoma. L'expressió ectòpica de PSMD11 és suficient per augmentar l'activitat del proteasoma. Sorprenentment, varem trobar que la llarga vida del GLP-1 C. elegans mutant té també un augment dramàtic en l'activitat del proteasoma associat a nivells augmentats en l'expressió de RPN-6. El factor de transcripció DAF-16 és essencial per l'augment de la longevitat de GLP-1 i els cucs mutants que trobem DAF-16 necessari per a l'augment d'expressió de RPN-6 i, per tant, per l'activació de l'activitat del proteasoma en GLP-1 mutant animals. Una possibilitat interessant és que els gens que regulen la vida i la resistència a l'estrès en C. elegans poden també regular la funció hESCs de mamífer, cèl • lules que son considerades immortals. Aquests resultats ens van portar a la conclusió de que FOXO4, un factor de transcripció sensible a la insulina/IGF-1, regula l'activitat del proteasoma en hESCs, el que suggereix un paper per FOXO4 en la funció d’aquestes cèl • lules. En efecte, FOXO4 es necessari per a la diferenciació en llinatges neuronals de les hESCs. Els nostres resultats estableixen una nova regulació de laproteostasis en hESCs que uneix la longevitat i la resistència a l'estrès en invertebrats amb la funció i identitat de les hESCs.

Avoiding transcription factor competition at promoter level increases the chances of obtaining oscillations

Background: The ultimate goal of synthetic biology is the conception and construction of genetic circuits that are reliable with respect to their designed function (e.g. oscillators, switches). This task remains still to be attained due to the inherent synergy of the biological building blocks and to an insufficient feedback between experiments and mathematical models. Nevertheless, the progress in these directions has been substantial. Results: It has been emphasized in the literature that the architecture of a genetic oscillator must include positive (activating) and negative (inhibiting) genetic interactions in order to yield robust oscillations. Our results point out that the oscillatory capacity is not only affected by the interaction polarity but by how it is implemented at promoter level. For a chosen oscillator architecture, we show by means of numerical simulations that the existence or lack of competition between activator and inhibitor at promoter level affects the probability of producing oscillations and also leaves characteristic fingerprints on the associated period/amplitude features. Conclusions: In comparison with non-competitive binding at promoters, competition drastically reduces the region of the parameters space characterized by oscillatory solutions. Moreover, while competition leads to pulse-like oscillations with long-tail distribution in period and amplitude for various parameters or noisy conditions, the non-competitive scenario shows a characteristic frequency and confined amplitude values. Our study also situates the competition mechanism in the context of existing genetic oscillators, with emphasis on the Atkinson oscillator.

Transcription factor map alignment of promoter regions

We address the problem of comparing and characterizing the promoter regions of genes with similar expression patterns. This remains a challenging problem in sequence analysis, because often the promoter regions of co-expressed genes do not show discernible sequence conservation. In our approach, thus, we have not directly compared the nucleotide sequence of promoters. Instead, we have obtained predictions of transcription factor binding sites, annotated the predicted sites with the labels of the corresponding binding factors, and aligned the resulting sequences of labels—to which we refer here as transcription factor maps (TF-maps). To obtain the global pairwise alignment of two TF-maps, we have adapted an algorithm initially developed to align restriction enzyme maps. We have optimized the parameters of the algorithm in a small, but well-curated, collection of human–mouse orthologous gene pairs. Results in this dataset, as well as in an independent much larger dataset from the CISRED database, indicate that TF-map alignments are able to uncover conserved regulatory elements, which cannot be detected by the typical sequence alignments.

Hepatocyte nuclear factor-4 alpha regulates the human apolipoprotein AV gene: Identification of a novel response element and involvement in the control by peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, AMP-activated protein kinase, and mitogen-activated protein kinase pathway

The recently discovered apolipoprotein AV (apoAV) gene has been reported to be a key player in modulating plasma triglyceride levels. Here we identify the hepatocyte nuclear factor-4 (HNF-4 ) as a novel regulator of human apoAV gene. Inhibition of HNF-4 expression by small interfering RNA resulted in down-regulation of apoAV. Deletion, mutagenesis, and binding assays revealed that HNF-4 directly regulates human apoAV promoter through DR1 [a direct repeat separated by one nucleotide (nt)], and via a novel element for HNF-4 consisting of an inverted repeat separated by 8 nt (IR8). In addition, we show that the coactivator peroxisome proliferator-activated receptor- coactivator-1 was capable of stimulating the HNF-4 -dependent transactivation of apoAV promoter. Furthermore, analyses in human hepatic cells demonstrated that AMP-activated protein kinase (AMPK) and the MAPK signaling pathway regulate human apoAV expression and suggested that this regulation may be mediated, at least in part, by changes in HNF-4 . Intriguingly, EMSAs and mice with a liver-specific disruption of the HNF-4 gene revealed a species-distinct regulation of apoAV by HNF-4 , which resembles that of a subset of HNF-4 target genes. Taken together, our data provide new insights into the binding properties and the modulation of HNF-4 and underscore the role of HNF-4 in regulating triglyceride metabolism.

Growth Factor Stimulation Improves The Structure And Properties Of Scaffold-free Engineered Auricular Cartilage Constructs.

The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.

Macrophage Cell Activation With Acute Apical Abscess Contents Determined By Interleukin-1 Beta And Tumor Necrosis Factor Alpha Production.

This clinical study has investigated the antigenic activity of bacterial contents from exudates of acute apical abscesses (AAAs) and their paired root canal contents regarding the stimulation capacity by levels of interleukin (IL)-1 beta and tumor necrosis factor alpha (TNF-α) throughout the root canal treatment against macrophage cells. Paired samples of infected root canals and exudates of AAAs were collected from 10 subjects. Endodontic contents were sampled before (root canal sample [RCS] 1) and after chemomechanical preparation (RCS2) and after 30 days of intracanal medication with calcium hydroxide + chlorhexidine gel (Ca[OH]2 + CHX gel) (RCS3). Polymerase chain reaction (16S rDNA) was used for detection of the target bacteria, whereas limulus amebocyte lysate was used to measure endotoxin levels. Raw 264.7 macrophages were stimulated with AAA exudates from endodontic contents sampled in different moments of root canal treatment. Enzyme-linked immunosorbent assays were used to measure the levels of TNF-α and IL-1 beta. Parvimonas micra, Porphyromonas endodontalis, Dialister pneumosintes, and Prevotella nigrescens were the most frequently detected species. Higher levels of endotoxins were found in samples from periapical exudates at RCS1 (P < .005). In fact, samples collected from periapical exudates showed a higher stimulation capacity at RCS1 (P < .05). A positive correlation was found between endotoxins from exudates with IL-1 beta (r = 0.97) and TNF-α (r = 0.88) production (P < .01). The significant reduction of endotoxins and bacterial species achieved by chemomechanical procedures (RCS2) resulted in a lower capacity of root canal contents to stimulate the cells compared with that at RCS1 (P < .05). The use of Ca(OH)2 + CHX gel as an intracanal medication (RCS3) improved the removal of endotoxins and bacteria from infected root canals (P < .05) whose contents induced a lower stimulation capacity against macrophages cells at RCS1, RCS2, and RCS3 (P < .05). AAA exudates showed higher levels of endotoxins and showed a greater capacity of macrophage stimulation than the paired root canal samples. Moreover, the use of intracanal medication improved the removal of bacteria and endotoxins from infected root canals, which may have resulted in the reduction of the inflammatory potential of the root canal content.

Vascular Endothelial Growth Factor Increases During Blood-brain Barrier-enhanced Permeability Caused By Phoneutria Nigriventer Spider Venom.

Phoneutria nigriventer spider accidental envenomation provokes neurotoxic manifestations, which when critical, results in epileptic-like episodes. In rats, P. nigriventer venom (PNV) causes blood-brain barrier breakdown (BBBb). The PNV-induced excitotoxicity results from disturbances on Na(+), K(+) and Ca(2+) channels and glutamate handling. The vascular endothelial growth factor (VEGF), beyond its angiogenic effect, also, interferes on synaptic physiology by affecting the same ion channels and protects neurons from excitotoxicity. However, it is unknown whether VEGF expression is altered following PNV envenomation. We found that adult and neonates rats injected with PNV showed immediate neurotoxic manifestations which paralleled with endothelial occludin, β-catenin, and laminin downregulation indicative of BBBb. In neonate rats, VEGF, VEGF mRNA, and Flt-1 receptors, glutamate decarboxylase, and calbindin-D28k increased in Purkinje neurons, while, in adult rats, the BBBb paralleled with VEGF mRNA, Flk-1, and calbindin-D28k increases and Flt-1 decreases. Statistically, the variable age had a role in such differences, which might be due to age-related unequal maturation of blood-brain barrier (BBB) and thus differential cross-signaling among components of the glial neurovascular unit. The concurrent increases in the VEGF/Flt-1/Flk-1 system in the cerebellar neuron cells and the BBBb following PNV exposure might imply a cytokine modulation of neuronal excitability consequent to homeostatic perturbations induced by ion channels-acting PNV neuropeptides. Whether such modulation represents neuroprotection needs further investigation.

Granulocyte Colony-stimulating Factor (g-csf) Positive Effects On Muscle Fiber Degeneration And Gait Recovery After Nerve Lesion In Mdx Mice.

G-CSF has been shown to decrease inflammatory processes and to act positively on the process of peripheral nerve regeneration during the course of muscular dystrophy. The aims of this study were to investigate the effects of treatment of G-CSF during sciatic nerve regeneration and histological analysis in the soleus muscle in MDX mice. Six-week-old male MDX mice underwent left sciatic nerve crush and were G-CSF treated at 7 days prior to and 21 days after crush. Ten and twenty-one days after surgery, the mice were euthanized, and the sciatic nerves were processed for immunohistochemistry (anti-p75(NTR) and anti-neurofilament) and transmission electron microscopy. The soleus muscles were dissected out and processed for H&E staining and subsequent morphologic analysis. Motor function analyses were performed at 7 days prior to and 21 days after sciatic crush using the CatWalk system and the sciatic nerve index. Both groups treated with G-CSF showed increased p75(NTR) and neurofilament expression after sciatic crush. G-CSF treatment decreased the number of degenerated and regenerated muscle fibers, thereby increasing the number of normal muscle fibers. The reduction in p75(NTR) and neurofilament indicates a decreased regenerative capacity in MDX mice following a lesion to a peripheral nerve. The reduction in motor function in the crushed group compared with the control groups may reflect the cycles of muscle degeneration/regeneration that occur postnatally. Thus, G-CSF treatment increases motor function in MDX mice. Nevertheless, the decrease in baseline motor function in these mice is not reversed completely by G-CSF.

Inhibition Of Tumor Necrosis Factor-alpha And Cyclooxigenase-2 By Isatin: A Molecular Mechanism Of Protection Against Tnbs-induced Colitis In Rats.

Isatin, an indole alkaloid has been shown to have anti-microbial, anti-tumor and anti-inflammatory effects. Due to its findings, we evaluated whether this alkaloid would have any effect on TNBS-induced colitis. Animals (male Unib:WH rats, aged 8 weeks old) were induced colitis through a rectal administration of 2,4,6-trinitrobenzene sulphonic acid using a catheter inserted 8 cm into the rectum of the animals. The rats were divided into two major groups: non-colitic and colitic. The colitic group was sub-divided into 6 groups (10 animals per group): colitic non-treated, Isatin 3; 6; 12.5; 18.75 and 25 mg/kg. Our main results showed that the oral treatment with Isatin 6 and 25 mg/kg were capable of avoiding the increase in TNF-α, COX-2 and PGE₂ levels when compared to the colitic non-treated group. Interestingly, the same doses (6 and 25 mg/kg) were also capable of preventing the decrease in IL-10 levels comparing with the colitic non-treated group. The levels of MPO, (an indirect indicator of neutrophil presence), were also maintained lower than those of the colitic non-treated group. Isatin also prevented the decrease of SOD activity and increase of GSH-Px and GSH-Rd activity as well as the depletion of GSH levels. In conclusion, both pre-treatments (6 and 25 mg/kg) were capable of protecting the gut mucosa against the injury caused by TNBS, through the combination of antioxidant and anti-inflammatory properties, which, together, showed a protective activity of the indole alkaloid Isatin.

Frequency Of The Allelic Variant C.1150t > C In Exon 10 Of The Fibroblast Growth Factor Receptor 3 (fgfr3) Gene Is Not Increased In Patients With Pathogenic Mutations And Related Chondrodysplasia Phenotypes.

Mutations in the FGFR3 gene cause the phenotypic spectrum of FGFR3 chondrodysplasias ranging from lethal forms to the milder phenotype seen in hypochondroplasia (Hch). The p.N540K mutation in the FGFR3 gene occurs in ∼70% of individuals with Hch, and nearly 30% of individuals with the Hch phenotype have no mutations in the FGFR3, which suggests genetic heterogeneity. The identification of a severe case of Hch associated with the typical mutation c.1620C > A and the occurrence of a c.1150T > C change that resulted in a p.F384L in exon 10, together with the suspicion that this second change could be a modulator of the phenotype, prompted us to investigate this hypothesis in a cohort of patients. An analysis of 48 patients with FGFR3 chondrodysplasia phenotypes and 330 healthy (control) individuals revealed no significant difference in the frequency of the C allele at the c.1150 position (p = 0.34). One patient carrying the combination `pathogenic mutation plus the allelic variant c.1150T > C' had a typical achondroplasia (Ach) phenotype. In addition, three other patients with atypical phenotypes showed no association with the allelic variant. Together, these results do not support the hypothesis of a modulatory role for the c.1150T > C change in the FGFR3 gene.