RESUMEN

Collagen VI is a non-fibrillar collagen present in the extracellular matrix (ECM) as a complex polymer; the mainly expressed form is composed of α1, α2 and α3 chains; mutations in genes encoding these chains cause myopathies known as Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and myosclerosis myopathy (MM). The collagen VI α6 chain is a recently identified component of the ECM of the human skeletal muscle. Here we report that the α6 chain was dramatically reduced in skeletal muscle and muscle cell cultures of genetically characterized UCMD, BM and MM patients, independently of the clinical phenotype, the gene involved and the effect of the mutation on the expression of the "classical" α1α2α3 heterotrimer. By contrast, the collagen VI α6 chain was normally expressed or increased in the muscle of patients affected by other forms of muscular dystrophy, the overexpression matching with areas of increased fibrosis. In vitro treatment with TGF-ß1, a potent collagen inducer, promoted the collagen VI α6 chain deposition in the ECM of normal muscle cells, whereas, in cultures derived from collagen VI-related myopathy patients, the collagen VI α6 chain failed to develop a network outside the cells and accumulated in the endoplasmic reticulum. The defect of the α6 chain points to a contribution to the pathogenesis of collagen VI-related disorders.

Asunto(s)

Colágeno Tipo VI/metabolismo , Contractura/metabolismo , Músculo Esquelético/metabolismo , Distrofias Musculares/congénito , Distrofias Musculares/metabolismo , Esclerosis/metabolismo , Adolescente , Adulto , Western Blotting , Células Cultivadas , Niño , Preescolar , Colágeno Tipo VI/genética , Contractura/genética , Contractura/patología , Matriz Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Persona de Mediana Edad , Músculo Esquelético/patología , Distrofias Musculares/genética , Distrofias Musculares/patología , Mutación/genética , Fenotipo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esclerosis/genética , Esclerosis/patología , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Adulto Joven

RESUMEN

BACKGROUND: In biology, it is easy to understand how a damaged functional system may generate wrong signals, but why this should happen when the system is disconnected is less clear. For this reason, among other pain syndromes, neuropathic pain (NP) following spinal cord injury (SCI) leaves most questions unanswered. AIMS AND METHODS: Our purpose is to review current knowledge on NP after SCI, focusing on the mechanisms, assessment and management of the syndrome. RESULTS: The mechanisms responsible for NP following SCI are poorly understood: NP is classically considered a "central pain syndrome" but recent evidence from experimental models reveals a possible "peripheral sensitization". Assessment of NP following SCI is well-established: in addition to clinical evaluation and self-reported scales, many neurophysiological, radiological and microscopic investigations may be performed. The management of NP following SCI is very difficult: evidence of effective drugs is lacking and alternative new treatment approaches yield different outcomes. CONCLUSIONS: Recently clinical and instrumental tools have increased our knowledge on NP, suggesting that the discovery of new treatment agents will depend on an explanation of what changes after SCI: future research must point in this direction.

Asunto(s)

Neuralgia/etiología , Traumatismos de la Médula Espinal/complicaciones , Animales , Humanos , Neuralgia/diagnóstico , Neuralgia/fisiopatología , Neuralgia/terapia , Manejo del Dolor , Dimensión del Dolor , Percepción del Dolor , Umbral del Dolor , Traumatismos de la Médula Espinal/diagnóstico , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/terapia , Resultado del Tratamiento

RESUMEN

Mutations in genes encoding nuclear envelope proteins, particularly LMNA encoding the A-type lamins, cause a broad range of diverse diseases, referred to as laminopathies. The astonishing variety of diseased phenotypes suggests that different mechanisms could be involved in the pathogenesis of laminopathies. In this review we will focus mainly on two of these pathogenic mechanisms: the nuclear damages affecting the chromatin organization, and the oxidative stress causing un-repairable DNA damages. Alteration in the nuclear profile and in chromatin organization, which are particularly impressive in systemic laminopathies whose cells undergo premature senescence, are mainly due to accumulation of unprocessed prelamin A. The toxic effect of these molecular species, which interfere with chromatin-associated proteins, transcription factors, and signaling pathways, could be reduced by drugs which reduce their farnesylation and/or stability. In particular, inhibitors of farnesyl transferase (FTIs), have been proved to be active in rescuing the altered cellular phenotype, and statins, also in association with other drugs, have been included into pilot clinical trials. The identification of a mechanism that accounts for accumulation of un-repairable DNA damage due to reactive oxygen species (ROS) generation in laminopathic cells, similar to that found in other muscular dystrophies (MDs) caused by altered expression of extracellular matrix (ECM) components, suggests that anti-oxidant therapeutic strategies might prove beneficial to laminopathic patients.

Asunto(s)

Membrana Nuclear/patología , Estrés Oxidativo , Humanos , Lamina Tipo A/genética , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Progeria/fisiopatología

RESUMEN

Collagen VI myopathies (Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM), and myosclerosis myopathy) share a common pathogenesis, that is, mitochondrial dysfunction due to deregulation of the permeability transition pore (PTP). This effect was first identified in the Col6a1(-/-) mouse model and then in muscle cell cultures from UCMD and BM patients; the normalizing effect of cyclosporin A (CsA) confirmed the pathogenic role of PTP opening. In order to determine whether mitochondrial performance can be used as a criterion for inclusion in clinical trials and as an outcome measure of the patient response to therapy, it is mandatory to establish whether mitochondrial dysfunction is conserved in primary cell cultures from UCMD and BM patients. In this study we report evidence that mitochondrial dysfunction and the consequent increase of apoptotic rate can be detected not only, as previously reported, in muscle, but also in fibroblast cell cultures established from muscle biopsies of collagen VI-related myopathic patients. However, the mitochondrial phenotype is no longer maintained after nine passages in culture. These data demonstrate that the dire consequences of mitochondrial dysfunction are not limited to myogenic cells, and that this parameter can be used as a suitable diagnostic criterion, provided that the cell culture conditions are carefully established.

Asunto(s)

Ensayos Clínicos como Asunto/métodos , Colágeno Tipo VI/metabolismo , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Adolescente , Adulto , Apoptosis/fisiología , Células Cultivadas , Niño , Contractura/metabolismo , Contractura/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Persona de Mediana Edad , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Distrofias Musculares/congénito , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Evaluación de Resultado en la Atención de Salud , Selección de Paciente , Fenotipo , Cultivo Primario de Células , Esclerosis/metabolismo , Esclerosis/patología

RESUMEN

Lamin A is a nuclear lamina constituent expressed in differentiated cells. Mutations in the LMNA gene cause several diseases, including muscular dystrophy and cardiomyopathy. Among the nuclear envelope partners of lamin A are Sad1 and UNC84 domain-containing protein 1 (SUN1) and Sad1 and UNC84 domain-containing protein 2 (SUN2), which mediate nucleo-cytoskeleton interactions critical to the anchorage of nuclei. In this study, we show that differentiating human myoblasts accumulate farnesylated prelamin A, which elicits upregulation and recruitment of SUN1 to the nuclear envelope and favors SUN2 enrichment at the nuclear poles. Indeed, impairment of prelamin A farnesylation alters SUN1 recruitment and SUN2 localization. Moreover, nuclear positioning in myotubes is severely affected in the absence of farnesylated prelamin A. Importantly, reduced prelamin A and SUN1 levels are observed in Emery-Dreifuss muscular dystrophy (EDMD) myoblasts, concomitant with altered myonuclear positioning. These results demonstrate that the interplay between SUN1 and farnesylated prelamin A contributes to nuclear positioning in human myofibers and may be implicated in pathogenetic mechanisms.

Asunto(s)

Núcleo Celular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Anticolesterolemiantes/farmacología , Diferenciación Celular , Células Cultivadas , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lamina Tipo A , Lovastatina/farmacología , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/patología , Distrofia Muscular de Emery-Dreifuss/metabolismo , Distrofia Muscular de Emery-Dreifuss/patología , Mioblastos/citología , Mioblastos/metabolismo , Prenilación , Células Madre/citología , Células Madre/fisiología

RESUMEN

Farnesylated prelamin A is a processing intermediate produced in the lamin A maturation pathway. Accumulation of a truncated farnesylated prelamin A form, called progerin, is a hallmark of the severe premature ageing syndrome, Hutchinson-Gilford progeria. Progerin elicits toxic effects in cells, leading to chromatin damage and cellular senescence and ultimately causes skin and endothelial defects, bone resorption, lipodystrophy and accelerated ageing. Knowledge of the mechanism underlying prelamin A turnover is critical for the development of clinically effective protein inhibitors that can avoid accumulation to toxic levels without impairing lamin A/C expression, which is essential for normal biological functions. Little is known about specific molecules that may target farnesylated prelamin A to elicit protein degradation. Here, we report the discovery of rapamycin as a novel inhibitor of progerin, which dramatically and selectively decreases protein levels through a mechanism involving autophagic degradation. Rapamycin treatment of progeria cells lowers progerin, as well as wild-type prelamin A levels, and rescues the chromatin phenotype of cultured fibroblasts, including histone methylation status and BAF and LAP2alpha distribution patterns. Importantly, rapamycin treatment does not affect lamin C protein levels, but increases the relative expression of the prelamin A endoprotease ZMPSTE24. Thus, rapamycin, an antibiotic belonging to the class of macrolides, previously found to increase longevity in mouse models, can serve as a therapeutic tool, to eliminate progerin, avoid farnesylated prelamin A accumulation, and restore chromatin dynamics in progeroid laminopathies.

Asunto(s)

Autofagia/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Proteínas Nucleares/metabolismo , Progeria/patología , Precursores de Proteínas/metabolismo , Sirolimus/farmacología , Antibacterianos/farmacología , Western Blotting , Células Cultivadas , Niño , Cromatina/metabolismo , Humanos , Lamina Tipo A , Membrana Nuclear/efectos de los fármacos , Prenilación

RESUMEN

Potentially viable therapeutic approaches for Duchenne muscular dystrophy (DMD) are now within reach. Indeed, clinical trials are currently under way. Two crucial aspects still need to be addressed: maximizing therapeutic efficacy and identifying appropriate and sensible outcome measures. Nevertheless, the end point of these trials remains painful muscle biopsy to show and quantify protein restoration in treated boys. In this study we show that PMMA/N-isopropil-acrylamide+ (NIPAM) nanoparticles (ZM2) bind and convey antisense oligoribonucleotides (AONs) very efficiently. Systemic injection of the ZM2-AON complex restored dystrophin protein synthesis in both skeletal and cardiac muscles of mdx mice, allowing protein localization in up to 40% of muscle fibers. The mdx exon 23 skipping level was up to 20%, as measured by the RealTime assay, and dystrophin restoration was confirmed by both reverse transcription-PCR and western blotting. Furthermore, we verified that dystrophin restoration also occurs in the smooth muscle cells of the dorsal skin arrector pili, an easily accessible histological structure, in ZM2-AON-treated mdx mice, with respect to untreated animals. This finding reveals arrector pili smooth muscle to be an appealing biomarker candidate and a novel low-invasive treatment end point. Furthermore, this marker would also be suitable for subsequent monitoring of the therapeutic effects in DMD patients. In addition, we demonstrate herein the expression of other sarcolemma proteins such as alpha-, beta-, gamma- and delta-sarcoglycans in the human skin arrector pili smooth muscle, thereby showing the potential of this muscle as a biomarker for other muscular dystrophies currently or soon to be the object of clinical trials.

Asunto(s)

Distrofina/biosíntesis , Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Nanopartículas/administración & dosificación , Oligorribonucleótidos Antisentido/administración & dosificación , Acrilamidas/administración & dosificación , Acrilamidas/química , Animales , Distrofina/genética , Exones , Corazón , Humanos , Masculino , Ratones , Ratones Endogámicos mdx , Músculo Liso/metabolismo , Nanopartículas/química , Oligorribonucleótidos Antisentido/química , Oligorribonucleótidos Antisentido/genética , Polimetil Metacrilato/administración & dosificación , Polimetil Metacrilato/química , Sarcoglicanos/genética , Piel/metabolismo

RESUMEN

Lamin A is a component of the nuclear lamina mutated in a group of human inherited disorders known as laminopathies. Among laminopathies, progeroid syndromes and lipodystrophies feature accumulation of prelamin A, the precursor protein which, in normal cells, undergoes a multi-step processing to yield mature lamin A. It is of utmost importance to characterize the prelamin A form accumulated in each laminopathy, since existing evidence shows that drugs acting on protein processing can improve some pathological aspects.We report that two antibodies raised against differently modified prelamin A peptides show a clear specificity to full-length prelamin A or carboxymethylated farnesylated prelamin A, respectively. Using these antibodies, we demonstrated that inhibition of the prelamin A endoprotease ZMPSTE24 mostly elicits accumulation of full-length prelamin A in its farnesylated form, while loss of the prelamin A cleavage site causes accumulation of carboxymethylated prelamin A in progeria cells. These results suggest a major role of ZMPSTE24 in the first prelamin A cleavage step.

Asunto(s)

Proteínas de la Membrana/fisiología , Metaloendopeptidasas/fisiología , Proteínas Nucleares/metabolismo , Progeria/metabolismo , Precursores de Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Endopeptidasas/metabolismo , Fibroblastos/metabolismo , Humanos , Lamina Tipo A , Proteínas de la Membrana/antagonistas & inhibidores , Metaloendopeptidasas/antagonistas & inhibidores , Progeria/patología , Prenilación de Proteína , Conejos/inmunología

RESUMEN

Lamin A is a component of the nuclear lamina mutated in a group of human inherited disorders known as laminopathies. Among laminopathies, progeroid syndromes and lipodystrophies feature accumulation of prelamin A, the precursor protein which, in normal cells, undergoes a multi-step processing to yield mature lamin A. It is of utmost importance to characterize the prelamin A form accumulated in each laminopathy, since existing evidence shows that drugs acting on protein processing can improve some pathological aspects. We report that two antibodies raised against differently modified prelamin A peptides show a clear specificity to full-length prelamin A or carboxymethylated farnesylated prelamin A, respectively. Using these antibodies, we demonstrated that inhibition of the prelamin A endoprotease ZMPSTE24 mostly elicits accumulation of full-length prelamin A in its farnesylated form, while loss of the prelamin A cleavage site causes accumulation of carboxymethylated prelamin A in progeria cells. These results suggest a major role of ZMPSTE24 in the first prelamin A cleavage step.

RESUMEN

Of various proposed alternatives to autogenous bone, a synthetic, degradable copolymer of PLA-GLA and dextrane seems to be a promising biomaterial for maxillary sinus lift. Consecutive partially edentulous patients showing severe monolateral posterior maxillary atrophy were treated via sinus lift using PLA-GLA-dextrane copolymer as the sole filler. Delayed implant positioning was performed and cores of regenerated tissues and native bone controls were retrieved and evaluated by light and electron microscopy, histomorphometry, microhardness and qualitative X-ray analysis. Seven sinuses in 7 patients were augmented with PLA-GLA-dextrane copolymer. Six to nine months after the copolymer 'graft', 17 bone cores were retrieved: all histological sections contained newly synthesized, mineralized material and new bone in various stages of development. Histomorphometry revealed average Trabecular Bone Volume (TBV) values ranging from 51% (6 months) to 77% (9 months). Backscattered scanning electron microscopy (BSE) in experimental and control samples confirmed histology findings. Microhardness values suggested newly formed bone at nine months was not as hard as native bone. Ca and P content was similar in 9-month regenerated and native bone. Seventeen implants were inserted in the second stage of surgery: resulting Implant Success (SR) and Cumulative Success (CSR) up to 3 years were 100% following Albrektssons criteria. Sinus lift augmentation using PLA-GLA-dextrane copolymer as the sole filler resulted in uneventful surgeries. New bone formation was evident histologically and its maturation was still in progress after 9 months. Successful, staged implant positioning was achieved in regenerated tissue.

Asunto(s)

Dextranos/química , Ácido Láctico/química , Seno Maxilar/cirugía , Ácido Poliglicólico/química , Adulto , Microanálisis por Sonda Electrónica , Femenino , Humanos , Masculino , Seno Maxilar/patología , Persona de Mediana Edad , Osteogénesis , Proyectos Piloto , Copolímero de Ácido Poliláctico-Ácido Poliglicólico

RESUMEN

The precursor protein of the nuclear lamina constituent lamin A is a 74-kDa protein called prelamin A which undergoes subsequent steps of posttranslational modification at its C-terminal CaaX residue. The unexpected finding that accumulation of unprocessable prelamin A is the molecular basis of the most severe laminopathies so far identified, including Hutchinson-Gilford progeria and restrictive dermopathy, has opened new perspectives in the study of the pathogenic mechanisms causing all lamin A/C-linked disorders, as well as new interest in the analysis of molecular mechanisms regulating prelamin A processing. However, complete knowledge of the cellular pathways affected downstream of prelamin A accumulation is still lacking, but it could give new insights both in normal and pathogenic mechanisms regulated by lamins. In this article, we review the involvement of defects of prelamin A processing in the pathogenesis of a group of laminopathies. In particular, we discuss the possibility that mutations leading to accumulation of particular forms of prelamin A result in specific nuclear abnormalities and impairment of nuclear functions leading to cell senescence or altered metabolism.

Asunto(s)

Laminina/fisiología , Proteínas Nucleares/fisiología , Precursores de Proteínas/fisiología , Animales , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Lamina Tipo A , Mutación , Proteínas Nucleares/genética , Precursores de Proteínas/genética , Procesamiento Proteico-Postraduccional

RESUMEN

Here we present an overview of the experimental evidence and of the conceptual basis for the involvement of lamins and nuclear envelope proteins in a group of genetic diseases collectively referred to as laminopathies. Some of these diseases affect a specific tissue (skeletal and/or cardiac muscles, subcutaneous fat, peripheral nerves), while others affect a variety of tissues; this suggests that the pathogenic mechanism of laminopathies could reside in the alteration of basic mechanisms affecting gene expression. On the other hand, a common feature of cells from laminopathic patients is represented by nuclear shape alterations and heterochromatin rearrangements. The definition of the role of lamins in the fine regulation of heterochromatin organization may help understanding not only the pathogenic mechanism of laminopathies but also the molecular basis of cell differentiation and ageng.

Asunto(s)

Envejecimiento/fisiología , Enfermedades Genéticas Congénitas , Membrana Nuclear/metabolismo , Animales , Núcleo Celular/metabolismo , Humanos , Laminas/metabolismo , Proteínas Nucleares/metabolismo

RESUMEN

The involvement of the nuclear envelope in the modulation of chromatin organization is strongly suggested by the increasing number of human diseases due to mutations of nuclear envelope proteins. A common feature of these diseases, named laminopathies, is the occurrence of major chromatin defects. We previously reported that cells from laminopathic patients show an altered nuclear profile, and loss or detachment of heterochromatin from the nuclear envelope. Recent evidence indicates that processing of the lamin A precursor is altered in laminopathies featuring pre-mature aging and/or lipodystrophy phenotype. In these cases, pre-lamin A is accumulated in the nucleus and heterochromatin is severely disorganized. Here we report evidence indicating that pre-lamin A is mis-localized in the nuclei of Emery-Dreifuss muscular dystrophy fibroblasts, either bearing lamin A/C or emerin mutations. Abnormal pre-lamin A-containing structures are formed following treatment with a farnesyl-transferase inhibitor, a drug that causes accumulation of pre-lamin A. Pre-lamin A-labeled structures co-localize with heterochromatin clumps. These data indicate that in almost all laminopathies the expression of the mutant lamin A precursor disrupts the organization of heterochromatin domains. Our results further show that the absence of emerin expression alters the distribution of pre-lamin A and of heterochromatin areas, suggesting a major involvement of emerin in pre-lamin A-mediated mechanisms of chromatin remodeling.

Asunto(s)

Ensamble y Desensamble de Cromatina , Heterocromatina , Lamina Tipo A , Membrana Nuclear/metabolismo , Envejecimiento Prematuro/genética , Animales , Ensamble y Desensamble de Cromatina/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lipodistrofia/genética , Lipodistrofia/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/metabolismo , Mutación , Proteínas Nucleares , Timopoyetinas/genética , Timopoyetinas/metabolismo

RESUMEN

The fate of emerin during skeletal muscle regeneration was investigated in an animal model by means of crush injury. Immunofluorescence, immunoblotting and mRNA analysis demonstrated that emerin level is increased in regenerating rat muscle fibers with respect to normal mature myofibers. This finding suggests an involvement of emerin during the muscle fiber regeneration process, in analogy with its reported involvement in muscle cell differentiation in vitro. The impairment of skeletal muscle physiological regeneration or reorganization could be a possible pathogenetic mechanism for Emery Dreifuss muscular dystrophy.

Asunto(s)

Proteínas de la Membrana/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/fisiología , Regeneración/fisiología , Timopoyetinas/metabolismo , Animales , Diferenciación Celular/fisiología , Núcleo Celular/metabolismo , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Immunoblotting , Inmunohistoquímica , Masculino , Modelos Animales , Fibras Musculares Esqueléticas/citología , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , Proteínas Nucleares , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Regulación hacia Arriba

RESUMEN

Hutchinson-Gilford progeria (HGPS) is a premature aging syndrome associated with LMNA mutations. Progeria cells bearing the G608G LMNA mutation are characterized by accumulation of a mutated lamin A precursor (progerin), nuclear dysmorphism and chromatin disorganization. In cultured HGPS fibroblasts, we found worsening of the cellular phenotype with patient age, mainly consisting of increased nuclear-shape abnormalities, progerin accumulation and heterochromatin loss. Moreover, transcript distribution was altered in HGPS nuclei, as determined by different techniques. In the attempt to improve the cellular phenotype, we applied treatment with drugs either affecting protein farnesylation or chromatin arrangement. Our results show that the combined treatment with mevinolin and the histone deacetylase inhibitor trichostatin A dramatically lowers progerin levels, leading to rescue of heterochromatin organization and reorganization of transcripts in HGPS fibroblasts. These results suggest that morpho-functional defects of HGPS nuclei are directly related to progerin accumulation and can be rectified by drug treatment.

Asunto(s)

Heterocromatina/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Progeria/tratamiento farmacológico , Progeria/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Células Cultivadas , Niño , Metilación de ADN , Heterocromatina/ultraestructura , Histonas/metabolismo , Humanos , Lamina Tipo A/deficiencia , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lovastatina/farmacología , Progeria/metabolismo , Progeria/patología , Precursores de Proteínas/metabolismo , Ribonucleoproteínas/metabolismo

RESUMEN

BACKGROUND: Skeletal muscle disorders associated with mutations of lamin A/C gene include autosomal Emery-Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B. The pathogenic mechanism underlying these diseases is unknown. Recent data suggest an impairment of signalling mechanisms as a possible cause of muscle malfunction. A molecular complex in muscle cells formed by lamin A/C, emerin, and nuclear actin has been identified. The stability of this protein complex appears to be related to phosphorylation mechanisms. OBJECTIVE: To analyse lamin A/C phosphorylation in control and laminopathic muscle cells. METHODS: Lamin A/C N-terminal phosphorylation was determined in cultured mouse myoblasts using a specific antibody. Insulin treatment of serum starved myoblast cultures was carried out to evaluate involvement of insulin signalling in the phosphorylation pathway. Screening of four Emery-Dreifuss and one limb girdle muscular dystrophy 1B cases was undertaken to investigate lamin A/C phosphorylation in both cultured myoblasts and mature muscle fibres. RESULTS: Phosphorylation of lamin A was observed during myoblast differentiation or proliferation, along with reduced lamin A/C phosphorylation in quiescent myoblasts. Lamin A N-terminus phosphorylation was induced by an insulin stimulus, which conversely did not affect lamin C phosphorylation. Lamin A/C was also hyperphosphorylated in mature muscle, mostly in regenerating fibres. Lamin A/C phosphorylation was strikingly reduced in laminopathic myoblasts and muscle fibres, while it was preserved in interstitial fibroblasts. CONCLUSIONS: Altered lamin A/C interplay with a muscle specific phosphorylation partner might be involved in the pathogenic mechanism of Emery-Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B.

Asunto(s)

Lamina Tipo A/metabolismo , Distrofia Muscular de Emery-Dreifuss/metabolismo , Mioblastos/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Diferenciación Celular , Línea Celular , Humanos , Insulina/metabolismo , Lamina Tipo A/genética , Ratones , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Cinturas/genética , Distrofia Muscular de Cinturas/metabolismo , Distrofia Muscular de Cinturas/patología , Distrofia Muscular de Emery-Dreifuss/genética , Fosforilación , Transducción de Señal

RESUMEN

The existence of a nuclear polyphosphoinositol metabolism independent from that at the plasma membrane is now widely recognized. Specific changes in the nuclear phosphatidylinositol (Ptdlns) metabolism have been implicated in cell growth, differentiation, and neoplastic transformation. Here we shall review the main features of nuclear inositol lipid signaling through type I IGF receptor, focusing the attention on the role of inositide-specific phospholipase C (PI-PLC) beta1 in cell proliferation and differentiation, given its peculiar localization in the nuclear compartment.

Asunto(s)

Núcleo Celular/fisiología , Receptor IGF Tipo 1/fisiología , Transducción de Señal/fisiología , Fosfolipasas de Tipo C/fisiología , Animales , Núcleo Celular/enzimología , Humanos , Fosfoinositido Fosfolipasa C , Hidrolasas Diéster Fosfóricas/fisiología

RESUMEN

Among two-chain ribosome-inactivating proteins (RIPs), volkensin is the most toxic to cells and animals, and is retrogradely axonally transported in the rat central nervous system, being an effective suicide transport agent. Here we studied the binding, endocytosis, intracellular routeing, degradation and exocytosis of this RIP. The interaction of volkensin with HeLa cells was compared to that of nigrin b, as an example of a type 2 RIP with low toxicity, and of ricin, as a reference toxin. Nigrin b and volkensin bound to cells with comparable affinity (approx. 10(-10) M) and had a similar number of binding sites (2 x 10(5)/cell), two-log lower than that reported for ricin. The cellular uptake of volkensin was lower than that reported for nigrin b and ricin. Confocal microscopy showed the rapid localization of volkensin in the Golgi stacks with a perinuclear localization similar to that of ricin, while nigrin b was distributed between cytoplasmic dots and the Golgi compartment. Consistently, brefeldin A, which disrupts the Golgi apparatus, protected cells from the inhibition of protein synthesis by volkensin or ricin, whereas it was ineffective in the case of nigrin b. Of the cell-released RIPs, 57% of volkensin and only 5% of ricin were active, whilst exocytosed nigrin b was totally inactive. Despite the low binding to, and uptake by, cells, the high cytotoxicity of volkensin may depend on (i) routeing to the Golgi apparatus, (ii) the low level of degradation, (iii) rapid recycling and (iv) the high percentage of active toxin remaining after exocytosis.

Asunto(s)

Exocitosis/fisiología , Glicoproteínas/metabolismo , Lectinas de Plantas/metabolismo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , N-Glicosil Hidrolasas/metabolismo , Proteínas de Plantas/metabolismo , Inhibidores de la Síntesis de la Proteína/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 2 , Ricina/metabolismo

RESUMEN

Elastofibroma dorsi is a pseudotumoral fibroproliferative lesion characterized by polymorphic fiber-like deposits of elastinophilic material. Several theories have been reported explaining the pathogenesis of elastofibroma. Recent cytogenetic studies have demonstrated chromosomal instability in elastofibromas, not normally observed in non-neoplastic tissues. These chromosomal defects are commonly observed in aggressive fibromatosis too. Such clinical observations suggest a multistage pathogenetic mechanism for the onset of elastofibroma. This study, using histochemical, immunohistochemical staining techniques, and ultrastructural examination, describes the detection of an otherwise typical elastofibroma contextual to a high grade sarcoma. Hence, the coexistence of elastofibroma and high-grade sarcoma may suggest a causal link between the two pathological entities. The results obtained suggest that the coexistence of the two pathological entities is conceivably coincidental.

Asunto(s)

Fibroma/ultraestructura , Leiomiosarcoma/ultraestructura , Recurrencia Local de Neoplasia/ultraestructura , Neoplasias de los Tejidos Blandos/ultraestructura , Femenino , Fibroma/diagnóstico , Fibroma/cirugía , Humanos , Inmunohistoquímica/métodos , Leiomiosarcoma/diagnóstico , Leiomiosarcoma/cirugía , Persona de Mediana Edad , Recurrencia Local de Neoplasia/diagnóstico , Recurrencia Local de Neoplasia/cirugía , Neoplasias de los Tejidos Blandos/diagnóstico , Neoplasias de los Tejidos Blandos/cirugía

RESUMEN

Strong evidence has been obtained during the last 16 years suggesting that phosphoinositides, which are involved in the regulation of a large variety of cellular processes in the cytoplasm and in the plasma membrane, are present within the nucleus. A number of advances has resulted in the discovery that nuclear phosphoinositides and their metabolizing enzymes are deeply involved in cell growth and differentiation. Remarkably, the nuclear inositide metabolism is regulated independently from that present elsewhere in the cell. Even though nuclear inositol lipids generate second messengers such as diacyglycerol and inositol 1,4,5-trisphosphate, it is becoming increasingly clear that in the nucleus polyphosphoinositides may act by themselves to influence functions such as pre-mRNA splicing and chromatin structure. This review aims at highlighting the most significant and up-dated findings about inositol lipid metabolism in the nucleus.

Asunto(s)

Núcleo Celular/metabolismo , Fosfatidilinositoles/metabolismo , Transducción de Señal/fisiología , Fosfolipasas de Tipo C/metabolismo , Animales , Diferenciación Celular/fisiología , Humanos , Isoenzimas/metabolismo , Fosfolipasa C beta