RESUMEN
A population of c-kit(+) cardiac stem/progenitor cells (CSPC) has been identified in the heart and shown to contribute to myocardial regeneration after infarction. Previously, we have shown the chemokine, stromal cell derived factor 1α (SDF1) is necessary for the myocardial response to infarction where chronic infusion of the CXCR4 antagonist, AMD3100, exacerbated MI. Notably, AMD3100 increased CSPC proliferation. The effect of SDF1 on CSPC proliferation was further investigated in primary cultures of magnetically sorted c-kit(+) CSPCs. SDF1 facilitated CSPC quiescence by blocking cell cycle progression at the G0 to G1 transition. SDF1 decreased casein kinase 1α (CK1α) consequently attenuating ß-catenin phosphorylation, destabilization, and degradation. Increased levels of ß-catenin with SDF1 were effective, increasing TCF/LEF reporter activity. SDF downregulation of CK1α was dependent on proteasomal degradation and decreased mRNA expression. CK1α siRNA knockdown verified SDF1-dependent CSPC quiescence requires CK1α downregulation and stablilization of ß-catenin. Conversely, ß-catenin knockdown increased CSPC proliferation. SDF1 also increased GSK3ß Y216 phosphorylation responsible for increased activity. SDF1 mediated CK1α downregulation and increase in GSK3ß activity affected cell cycle through Bmi-1 downregulation, increased cyclin D1 phosphorylation, and decreased cyclin D1 levels. In conclusion, SDF1 exerts a quiescent effect on resident c-kit(+) CSPCs by decreasing CK1α levels, increasing GSK3ß activity, stabilizing ß-catenin, and affecting regulation of the cell cycle through Bmi-1 and cyclin D1. SDF1-dependent quiescence is an important factor in stem and progenitor cell preservation under basal conditions, however, with stress or injury in which SDF1 is elevated, quiescence may limit expansion and contribution to myocardial regeneration.
Asunto(s)
Caseína Quinasa Ialfa/genética , Quimiocina CXCL12/genética , Glucógeno Sintasa Quinasa 3/genética , Transducción de Señal/genética , Caseína Quinasa Ialfa/metabolismo , Quimiocina CXCL12/metabolismo , Ciclina D1/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Miocardio/citología , Miocardio/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-kit/genética , Regeneración/genética , Células Madre/metabolismo , Células del Estroma/metabolismoRESUMEN
ecSOD function has prototypically been associated with the extracellular space due to its secretion and localization to the extracellular matrix. A myocyte-specific ecSOD transgenic mouse has shown that it can also be localized to the myocyte intracellular compartment and is capable of attenuating Reactive oxygen species (ROS) formation and increasing NO bioavailability after ischemia reperfusion. Here, the subcellular localization of transgenic ecSOD was further defined by subcellular fractionation, immunofluorescent confocal microscopy, and Western analysis. Its impact on mitochondrial function was assessed by mitochondrial permeability transition (MPT). ecSOD was found to exist in cytosolic and nuclear fractions in addition to membrane. Colocalization of ecSOD with myocardial mitochondria was further demonstrated by confocal microscopy and subcellular fractionation of mitochondria and Western analysis. Isolated ventricular myocytes from cardiac-specific transgenic ecSOD mice were protected from hypoxia reoxygenation injury. Increased ecSOD colocalization to myocardial mitochondria in ecSOD Tg hearts limited MPT in response to Ca(2+) challenge. These results demonstrate that ecSOD is not restricted to the extracellular space and can alter MPT response to Ca(2+) suggesting mitochondrial localization of ecSOD can affect key mitochondrial functions such as MPT which are integral to cell survival.
RESUMEN
Increased levels of extracellular superoxide dismutase (ecSOD) induced by preconditioning or gene therapy protect the heart from ischemia/reperfusion injury. To elucidate the mechanism responsible for this action, we studied the effects of increased superoxide scavenging on nitric oxide (NO) bioavailability in a cardiac myocyte-specific ecSOD transgenic (Tg) mouse. Results indicated that ecSOD overexpression increased cardiac myocyte-specific ecSOD activity 27.5-fold. Transgenic ecSOD was localized to the sarcolemma and, notably, the cytoplasm of cardiac myocytes. Ischemia/reperfusion injury was attenuated in ecSOD Tg hearts, in which infarct size was decreased and LV functional recovery was improved. Using the ROS spin trap, DMPO, electron paramagnetic resonance (EPR) spectroscopy demonstrated a significant decrease in ROS in Tg hearts during the first 20 min of reperfusion. This decrease in ROS was accompanied by an increase in NO production determined by EPR using the NO spin trap, Fe-MGD. Attenuated ROS in ecSOD Tg myocytes was also supported by decreased production of peroxynitrite (ONOO(-)). Increased NO bioavailability was confirmed by attenuated guanylate cyclase-dependent (p-VASP) signaling. In conclusion, attenuation of ROS levels by cardiac-specific ecSOD overexpression increases NO bioavailability in response to ischemia/reperfusion and protects against reperfusion injury. These findings are the first to demonstrate increased NO bioavailability with attenuation of ROS by direct measurement of these reactive species (EPR, reactive fluorescent dyes) with cardiac-specific ecSOD expression. This is also the first indication that the predominantly extracellular SOD isoform is capable of cytosolic localization that affects myocardial intracellular signal transduction and function.
Asunto(s)
Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/enzimología , Óxido Nítrico/metabolismo , Estrés Oxidativo , Superóxido Dismutasa/metabolismo , Animales , Técnicas In Vitro , Ratones , Ratones Transgénicos , Infarto del Miocardio/patología , Miocardio/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Gliosis is a biological process that occurs during injury repair in the central nervous system and is characterized by the overexpression of the intermediate filaments (IFs) glial fibrillary acidic protein (GFAP) and vimentin. A common thread in many retinal diseases is reactive Müller cell gliosis, an untreatable condition that leads to tissue scarring and even blindness. Here, we demonstrate that the vimentin-targeting small molecule withaferin A (WFA) is a novel chemical probe of GFAP. Using molecular modeling studies that build on the x-ray crystal structure of tetrameric vimentin rod 2B domain we reveal that the WFA binding site is conserved in the corresponding domain of tetrameric GFAP. Consequently, we demonstrate that WFA covalently binds soluble recombinant tetrameric human GFAP at cysteine 294. In cultured primary astrocytes, WFA binds to and down-regulates soluble vimentin and GFAP expression to cause cell cycle G(0)/G(1) arrest. Exploiting a chemical injury model that overexpresses vimentin and GFAP in retinal Müller glia, we demonstrate that systemic delivery of WFA down-regulates soluble vimentin and GFAP expression in mouse retinas. This pharmacological knockdown of soluble IFs results in the impairment of GFAP filament assembly and inhibition of cell proliferative response in Müller glia. We further show that a more severe GFAP filament assembly deficit manifests in vimentin-deficient mice, which is partly rescued by WFA. These findings illustrate WFA as a chemical probe of type III IFs and illuminate this class of withanolide as a potential treatment for diverse gliosis-dependent central nervous system traumatic injury conditions and diseases, and for orphan IF-dependent pathologies.
Asunto(s)
Ergosterol/análogos & derivados , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis , Retina , Degeneración Retiniana , Vimentina/metabolismo , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Ciclina D3/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Ergosterol/química , Ergosterol/metabolismo , Ergosterol/farmacología , Proteína Ácida Fibrilar de la Glía/genética , Gliosis/metabolismo , Gliosis/patología , Humanos , Ratones , Ratones Noqueados , Modelos Moleculares , Estructura Secundaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Retina/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Vimentina/química , Vimentina/genética , WitanólidosRESUMEN
PLP1 and DM20, major myelin proteins, are generated by developmentally regulated alternative splicing. In the post-natal brain, PLP1 is the predominant product. Deletion of a splicing enhancer in PLP1 intron 3 causes a mild form of Pelizaeus-Merzbacher disease and reduces PLP1 specific splicing in vitro (Hobson, G. M., Huang, Z., Sperle, K., Stabley, D. L., Marks, H. G., and Cambi, F., 2002. A PLP splicing abnormality is associated with an unusual presentation of PMD. Ann. Neurol. 52, 477-488). We sought to investigate the pathogenic role of the mutation and to determine the consequences on the developmental regulation of PLP1 alternative splicing and myelin stability and function in vivo. We have generated a knockin mouse that carries deletion of the intronic splicing enhancer and have characterized the PLP1/DM20 ratio by Real Time RT-PCR and Western blot analysis in the developing and mature brain and examined the clinical and pathological phenotype by motor testing and electron microscopy. The deletion impairs the increase in the PLP1/DM20 transcript and protein ratio at the time of myelination and in adulthood and results in a PLP1 hypomorph. Electron microscopy shows abnormal myelin wraps with fragmented myelin whorls, which are progressive with age, suggesting a defect in myelin stability. Phenotypic characterization of the knockin mouse shows a defect in motor coordination. The data indicate that the intronic splicing enhancer is necessary for the developmental increase in PLP1/DM20 ratio and that full PLP1 dosage is necessary for myelin stability and brain function. This knockin mouse represents a useful model to investigate the mechanisms of disease in human disorders in which PLP1 expression is reduced.
Asunto(s)
Proteína Proteolipídica de la Mielina/genética , Vaina de Mielina/fisiología , Enfermedad de Pelizaeus-Merzbacher/genética , Enfermedad de Pelizaeus-Merzbacher/fisiopatología , Empalme Alternativo , Animales , Conducta Animal , Encéfalo/crecimiento & desarrollo , Encéfalo/patología , Modelos Animales de Enfermedad , Elementos de Facilitación Genéticos , Eliminación de Gen , Técnicas de Sustitución del Gen , Intrones/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Electrónica , Actividad Motora , Proteínas de la Mielina/genética , Proteínas de la Mielina/metabolismo , Proteína Proteolipídica de la Mielina/metabolismo , Vaina de Mielina/patología , Vaina de Mielina/ultraestructura , Nervio Óptico/crecimiento & desarrollo , Nervio Óptico/patología , Nervio Óptico/ultraestructura , Enfermedad de Pelizaeus-Merzbacher/patologíaRESUMEN
Alternative splicing of competing 5' splice sites is regulated by enhancers and silencers in the spliced exon. We have characterized sequences and splicing factors that regulate alternative splicing of PLP and DM20, myelin proteins produced by oligodendrocytes (OLs) by selection of 5' splice sites in exon 3. We identify a G-rich enhancer (M2) of DM20 5' splice site in exon 3B and show that individual G triplets forming M2 are functionally distinct and the distal group plays a dominant role. G-rich M2 and a G-rich splicing enhancer (ISE) in intron 3 share similarities in function and protein binding. The G-rich sequences are necessary for binding of hnRNPs to both enhancers. Reduction in hnRNPH and F expression in differentiated OLs correlates temporally with increased PLP/DM20 ratio. Knock down of hnRNPH increased PLP/DM20 ratio, while hnRNPF did not. Silencing hnRNPH and F increased the PLP/DM20 ratio more than hnRNPH alone, demonstrating a novel synergistic effect. Mutation of M2, but not ISE reduced the synergistic effect. Replacement of M2 and all G runs in exon 3B abolished it almost completely. We conclude that developmental changes in hnRNPH/F associated with OLs differentiation synergistically regulate PLP alternative splicing and a G-rich enhancer participates in the regulation.
Asunto(s)
Empalme Alternativo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Proteínas de la Membrana/genética , Proteína Proteolipídica de la Mielina/genética , Oligodendroglía/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico , Secuencia de Bases , Sitios de Unión , Western Blotting , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Exones , Guanina/análisis , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/antagonistas & inhibidores , Humanos , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteína Proteolipídica de la Mielina/metabolismo , Interferencia de ARN , Sitios de Empalme de ARNRESUMEN
Proteolipid protein (PLP) and DM20 are generated by alternative splicing of exon 3B of PLP1 transcript in differentiating oligodendrocytes. We investigated the role of exonic splicing enhancers (ESE) in the selection of PLP 5' donor site, focusing on putative ASF/SF2, and SC35 binding motifs in exon 3B on the basis of mutations that cause disease in humans. Mutations in a putative ASF/SF2 binding motif (nucleotides 406-412) reduced PLP 5' donor site selection, whereas a mutation in a putative SC35 binding motif (nucleotides 382-389) had no effect. UV crosslinking and immunoprecipitation (IP) assays using an antibody to ASF/SF2 showed that the ASF/SF2 protein specifically binds to the ESE (nucleotides 406-412). The single nucleotide mutations that reduced PLP splice site selection greatly diminished ASF/SF2 protein binding to this motif. We next tested the effect of overexpressed ASF/SF2 on PLP 5'splice selection in differentiating oligodendrocytes. ASF/SF2 positively regulates PLP splice site selection in a concentration-dependent manner. Disruption of the putative ASF/SF2 binding site in exon 3B reduced the positive effect of ASF/SF2 on PLP splicing. We conclude that an ESE in exon3B regulates PLP 5' donor site selection and that ASF/SF2 protein participates in the regulation of PLP alternative splicing in oligodendrocytes.
Asunto(s)
Empalme Alternativo , Elementos de Facilitación Genéticos , Exones , Proteína Proteolipídica de la Mielina/genética , Proteínas del Tejido Nervioso/genética , Oligodendroglía/fisiología , Secuencias de Aminoácidos , Animales , Sitios de Unión , Diferenciación Celular , Linaje de la Célula/fisiología , Células Cultivadas , Humanos , Mutación , Regiones Promotoras Genéticas , Sitios de Empalme de ARN , Ratas , Ratas Sprague-Dawley , TransfecciónRESUMEN
Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily that play important roles in bone formation, embryonic patterning, and epidermal-neural cell fate decisions. BMPs signal through pathway specific mediators such as Smads1 and 5, but the upstream regulation of BMP-specific Smads has not been fully characterized. Here we report the identification of SANE (Smad1 Antagonistic Effector), a novel protein with significant sequence similarity to nuclear envelop proteins such as MAN1. SANE binds to Smad1/5 and to BMP type I receptors and regulates BMP signaling. SANE specifically blocks BMP-dependent signaling in Xenopus embryos and in a mammalian model of bone formation but does not inhibit the TGF-beta/Smad2 pathway. Inhibition of BMP signaling by SANE requires interaction between SANE and Smad1, because a SANE mutant that does not bind Smad1 does not inhibit BMP signaling. Furthermore, inhibition appears to be mediated by inhibition of BMP-induced Smad1 phosphorylation, blocking ligand-dependent nuclear translocation of Smad1. These studies define a new mode of regulation for intracellular BMP/Smad1 signaling.