RESUMO
RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.
Assuntos
Caspase 8/metabolismo , Doenças Hereditárias Autoinflamatórias/metabolismo , Mutação , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Caspase 3/metabolismo , Feminino , Doenças Hereditárias Autoinflamatórias/genética , Doenças Hereditárias Autoinflamatórias/patologia , Humanos , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linhagem , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
In healthy adult skeletal muscle fibers microtubules form a three-dimensional grid-like network. In the mdx mouse, a model of Duchenne muscular dystrophy (DMD), microtubules are mostly disordered, without periodicity. These microtubule defects have been linked to the mdx mouse pathology. We now report that increased expression of the beta 6 class V ß-tubulin (tubb6) contributes to the microtubule changes of mdx muscles. Wild-type muscle fibers overexpressing green fluorescent protein (GFP)-tubb6 (but not GFP-tubb5) have disorganized microtubules whereas mdx muscle fibers depleted of tubb6 (but not of tubb5) normalize their microtubules, suggesting that increasing tubb6 is toxic. However, tubb6 increases spontaneously during differentiation of mouse and human muscle cultures. Furthermore, endogenous tubb6 is not uniformly expressed in mdx muscles but is selectively increased in fiber clusters, which we identify as regenerating. Similarly, mdx-based rescued transgenic mice that retain a higher than expected tubb6 level show focal expression of tubb6 in subsets of fibers. Tubb6 is also upregulated in cardiotoxin-induced mouse muscle regeneration, in human myositis and DMD biopsies, and the tubb6 level correlates with that of embryonic myosin heavy chain, a regeneration marker. In conclusion, modulation of a ß-tubulin isotype plays a role in muscle differentiation and regeneration. Increased tubb6 expression and microtubule reorganization are not pathological per se but reflect a return to an earlier developmental stage. However, chronic elevation of tubb6, as occurs in the mdx mouse, may contribute to the repeated cycles of regeneration and to the pathology of the disease.
Assuntos
Músculo Esquelético/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/fisiologia , Animais , Distrofina/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/fisiologia , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Mioblastos , Cultura Primária de Células , Regeneração , Ativação Transcricional , Regulação para CimaRESUMO
Neutrophils play a key role in host defenses and have recently been implicated in the pathogenesis of autoimmune diseases by various mechanisms, including formation of neutrophil extracellular traps through a recently described distinct form of programmed cell death called NETosis. Techniques to assess and quantitate NETosis in an unbiased, reproducible, and efficient way are lacking, considerably limiting the advancement of research in this field. We optimized and validated, a new method to automatically quantify the percentage of neutrophils undergoing NETosis in real time using the IncuCyte ZOOM imaging platform and the membrane-permeability properties of two DNA dyes. Neutrophils undergoing NETosis induced by various physiological stimuli showed distinct changes, with a loss of multilobulated nuclei, as well as nuclear decondensation followed by membrane compromise, and were accurately counted by applying filters based on fluorescence intensity and nuclear size. Findings were confirmed and validated with the established method of immunofluorescence microscopy. The platform was also validated to rapidly assess and quantify the dose-dependent effect of inhibitors of NETosis. In addition, this method was able to distinguish among neutrophils undergoing NETosis, apoptosis, or necrosis based on distinct changes in nuclear morphology and membrane integrity. The IncuCyte ZOOM platform is a novel real-time assay that quantifies NETosis in a rapid, automated, and reproducible way, significantly optimizing the study of neutrophils. This platform is a powerful tool to assess neutrophil physiology and NETosis, as well as to swiftly develop and test novel neutrophil targets.
Assuntos
Morte Celular , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Ensaios de Triagem em Larga Escala , Imagem Molecular , Neutrófilos/imunologia , Neutrófilos/metabolismo , Descoberta de Drogas , Armadilhas Extracelulares/efeitos dos fármacos , Humanos , Processamento de Imagem Assistida por Computador , Lúpus Eritematoso Sistêmico , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Neutrófilos/efeitos dos fármacosRESUMO
Studies in skeletal muscle cell cultures suggest that the cortical actin cytoskeleton is a major requirement for insulin-stimulated glucose transport, implicating the ß-actin isoform, which in many cell types is the main actin isoform. However, it is not clear that ß-actin plays such a role in mature skeletal muscle. Neither dependency of glucose transport on ß-actin nor actin reorganization upon glucose transport have been tested in mature muscle. To investigate the role of ß-actin in fully differentiated muscle, we performed a detailed characterization of wild type and muscle-specific ß-actin knockout (KO) mice. The effects of the ß-actin KO were subtle; however, we confirmed the previously reported decline in running performance of ß-actin KO mice compared with wild type during repeated maximal running tests. We also found insulin-stimulated glucose transport into incubated muscles reduced in soleus but not in extensor digitorum longus muscle of young adult mice. Contraction-stimulated glucose transport trended toward the same pattern, but the glucose transport phenotype disappeared in soleus muscles from mature adult mice. No genotype-related differences were found in body composition or glucose tolerance or by indirect calorimetry measurements. To evaluate ß-actin mobility in mature muscle, we electroporated green fluorescent protein (GFP)-ß-actin into flexor digitorum brevis muscle fibers and measured fluorescence recovery after photobleaching. GFP-ß-actin showed limited unstimulated mobility and no changes after insulin stimulation. In conclusion, ß-actin is not required for glucose transport regulation in mature mouse muscle under the majority of the tested conditions. Thus, our work reveals fundamental differences in the role of the cortical ß-actin cytoskeleton in mature muscle compared with cell culture.
Assuntos
Actinas/metabolismo , Actinas/fisiologia , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Feminino , Teste de Tolerância a Glucose , Técnicas In Vitro , Masculino , Camundongos , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Ribonucleotídeos/farmacologia , Corrida/fisiologiaRESUMO
OBJECTIVE: We and others previously demonstrated that sirtuin 1 (SIRT-1) regulates apoptosis and cartilage-specific gene expression in human chondrocytes and mouse models. This study was undertaken to determine if SIRT-1 enzymatic activity plays a protective role in cartilage homeostasis in vivo, by investigating mice with SIRT-1 mutations to characterize their cartilage. METHODS: Articular cartilage was harvested from the paws and knees of 5- and 6-month-old wild-type (WT) mice and mice homozygous for SIRT-1tm2.1Mcby (SIRT-1y/y), an allele carrying a point mutation that encodes a SIRT-1 protein with no enzymatic activity (y/y mice). Mice ages 2 days old and 6-7 days old were also examined. Mouse joint cartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures. RESULTS: We found that articular cartilage tissue sections from y/y mice of up to 6 months of age contained reduced levels of type II collagen, aggrecan, and glycosaminoglycan compared to sections from WT mice. In contrast, protein levels of matrix metalloproteinase 8 (MMP-8), MMP-9, and MMP-13 were elevated in the cartilage of y/y mice. In addition, chondrocyte apoptosis was elevated in SIRT-1 mutant mice as compared to their WT littermates. Consistent with these observations, protein tyrosine phosphatase 1b was elevated in the y/y mice. CONCLUSION: Our in vivo findings in this animal model demonstrate that mice with defective SIRT-1 also have defective cartilage, with elevated rates of cartilage degradation with age. Hence, normal cartilage homeostasis requires enzymatically active SIRT-1 protein.
Assuntos
Cartilagem/enzimologia , Condrócitos/enzimologia , Homeostase/fisiologia , Osteoartrite/fisiopatologia , Sirtuína 1/metabolismo , Agrecanas , Animais , Técnicas de Cultura de Células , Colágeno Tipo II , Modelos Animais de Doenças , Proteínas da Matriz Extracelular , Feminino , Immunoblotting , Imuno-Histoquímica , Camundongos , Mutação Puntual , Sirtuína 1/genéticaAssuntos
Artrite/genética , Diferenciação Celular/genética , Mutação com Ganho de Função/genética , Células Germinativas/patologia , Mutação/genética , Fator 88 de Diferenciação Mieloide/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Osso e Ossos/patologia , Proteína C-Reativa/genética , Linhagem Celular , Exoma/genética , Feminino , Humanos , Inflamação/genética , Monócitos/patologiaRESUMO
Pompe disease is a lysosomal storage disorder caused by the deficiency of acid alpha-glucosidase, the enzyme that degrades glycogen in the lysosomes. The disease manifests as a fatal cardiomyopathy and skeletal muscle myopathy in infants; in milder late-onset forms skeletal muscle is the major tissue affected. We have previously demonstrated that autophagic inclusions in muscle are prominent in adult patients and the mouse model. In this study we have evaluated the contribution of the autophagic pathology in infants before and 6 months after enzyme replacement therapy. Single muscle fibers, isolated from muscle biopsies, were stained for autophagosomal and lysosomal markers and analyzed by confocal microscopy. In addition, unstained bundles of fixed muscles were analyzed by second harmonic imaging. Unexpectedly, the autophagic component which is so prominent in juvenile and adult patients was negligible in infants; instead, the overwhelming characteristic was the presence of hugely expanded lysosomes. After 6 months on therapy, however, the autophagic buildup becomes visible as if unmasked by the clearance of glycogen. In most fibers, the two pathologies did not seem to coexist. These data point to the possibility of differences in the pathogenesis of Pompe disease in infants and adults.
Assuntos
Autofagia/fisiologia , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo II/patologia , Lisossomos/patologia , Adulto , Criança , Pré-Escolar , Terapia de Reposição de Enzimas , Doença de Depósito de Glicogênio Tipo II/enzimologia , Humanos , Lactente , Recém-Nascido , Lisossomos/enzimologia , Fibras Musculares Esqueléticas/patologia , alfa-Glucosidases/deficiência , alfa-Glucosidases/metabolismo , alfa-Glucosidases/uso terapêuticoRESUMO
N-RAP is a striated muscle-specific scaffolding protein that organizes alpha-actinin and actin into symmetrical I-Z-I structures in developing myofibrils. Here we determined the order of events during myofibril assembly through time-lapse confocal microscopy of cultured embryonic chick cardiomyocytes coexpressing fluorescently tagged N-RAP and either alpha-actinin or actin. During de novo myofibril assembly, N-RAP assembled in fibrillar structures within the cell, with dots of alpha-actinin subsequently organizing along these structures. The initial fibrillar structures were reminiscent of actin fibrils, and coassembly of N-RAP and actin into newly formed fibrils supported this. The alpha-actinin dots subsequently broadened to Z-lines that were wider than the underlying N-RAP fibril, and N-RAP fluorescence intensity decreased. FRAP experiments showed that most of the alpha-actinin dynamically exchanged during all stages of myofibril assembly. In contrast, less than 20% of the N-RAP in premyofibrils was exchanged during 10-20 min after photobleaching, but this value increased to 70% during myofibril maturation. The results show that N-RAP assembles into an actin containing scaffold before alpha-actinin recruitment; that the N-RAP scaffold is much more stable than the assembling structural components; that N-RAP dynamics increase as assembly progresses; and that N-RAP leaves the structure after assembly is complete.
Assuntos
Actinina/metabolismo , Actinas/metabolismo , Proteínas Musculares/metabolismo , Miócitos Cardíacos/fisiologia , Miofibrilas/fisiologia , Animais , Células Cultivadas , Embrião de Galinha , Humanos , Miócitos Cardíacos/ultraestrutura , Ligação ProteicaRESUMO
Differentiated mammalian cells and tissues, such as skeletal muscle fibers, acquire an organization of Golgi complex and microtubules profoundly different from that in proliferating cells and still poorly understood. In adult rodent skeletal muscle, the multinucleated muscle fibers have hundreds of Golgi elements (GE), small stacks of cisternae that serve as microtubule-organizing centers. We are interested in the role of the GE in organizing a peculiar grid of microtubules located in the fiber cortex, against the sarcolemma. Modifications of this grid in the mdx mouse model of Duchenne muscular dystrophy have led to identifying dystrophin, the protein missing in both human disease and mouse model, as a microtubule guide. Compared to wild-type (WT), mdx microtubules are disordered and more dense and they have been linked to the dystrophic pathology. GE themselves are disordered in mdx. Here, to identify the causes of GE and microtubule alterations in the mdx muscle, we follow GFP-tagged microtubule markers in live mdx fibers and investigate the recovery of GE and microtubules after treatment with nocodazole. We find that mdx microtubules grow 10% faster but in 30% shorter bouts and that they begin to form a tangled network, rather than an orthogonal grid, right after nucleation from GE. Strikingly, a large fraction of microtubules in mdx muscle fibers seem to dissociate from GE after nucleation. Moreover, we report that mdx GE are mispositioned and increased in number and size. These results were replicated in WT fibers overexpressing the beta-tubulin tubb6, which is elevated in Duchenne muscular dystrophy, in mdx and in regenerating muscle. Finally, we examine the association of GE with ER exit sites and ER-to-Golgi intermediate compartment, which starts during muscle differentiation, and find it persisting in mdx and tubb6 overexpressing fibers. We conclude that GE are full, small, Golgi complexes anchored, and positioned through ER Exit Sites. We propose a model in which GE mispositioning, together with the absence of microtubule guidance due to the lack of dystrophin, determines the differences in GE and microtubule organization between WT and mdx muscle fibers.
RESUMO
Cancer-related fatigue (CRF) is a common burden in cancer patients and little is known about its underlying mechanism. The primary aim of this study was to identify gene signatures predictive of post-radiotherapy fatigue in prostate cancer patients. We employed Fisher Linear Discriminant Analysis (LDA) to identify predictive genes using whole genome microarray data from 36 men with prostate cancer. Ingenuity Pathway Analysis was used to determine functional networks of the predictive genes. Functional validation was performed using a T lymphocyte cell line, Jurkat E6.1. Cells were pretreated with metabotropic glutamate receptor 5 (mGluR5) agonist (DHPG), antagonist (MPEP), or control (PBS) for 20 min before irradiation at 8 Gy in a Mark-1 γ-irradiator. NF-κB activation was assessed using a NF-κB/Jurkat/GFP Transcriptional Reporter Cell Line. LDA achieved 83.3% accuracy in predicting post-radiotherapy fatigue. "Glutamate receptor signaling" was the most significant (p = 0.0002) pathway among the predictive genes. Functional validation using Jurkat cells revealed clustering of mGluR5 receptors as well as increased regulated on activation, normal T cell expressed and secreted (RANTES) production post irradiation in cells pretreated with DHPG, whereas inhibition of mGluR5 activity with MPEP decreased RANTES concentration after irradiation. DHPG pretreatment amplified irradiation-induced NF-κB activation suggesting a role of mGluR5 in modulating T cell activation after irradiation. These results suggest that mGluR5 signaling in T cells may play a key role in the development of chronic inflammation resulting in fatigue and contribute to individual differences in immune responses to radiation. Moreover, modulating mGluR5 provides a novel therapeutic option to treat CRF.
Assuntos
Fadiga/etiologia , NF-kappa B/metabolismo , Neoplasias da Próstata/radioterapia , Radioterapia/efeitos adversos , Receptor de Glutamato Metabotrópico 5/agonistas , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Idoso , Estudo de Associação Genômica Ampla , Humanos , Células Jurkat , Aprendizado de Máquina , Masculino , Metoxi-Hidroxifenilglicol/análogos & derivados , Metoxi-Hidroxifenilglicol/farmacologia , Pessoa de Meia-Idade , Piridinas/farmacologia , Dosagem Radioterapêutica , Linfócitos T/metabolismo , TranscriptomaRESUMO
Inflammasomes are innate immune sensors that respond to pathogen- and damage-associated signals with caspase-1 activation, interleukin (IL)-1ß and IL-18 secretion, and macrophage pyroptosis. The discovery that dominant gain-of-function mutations in NLRP3 cause the cryopyrin-associated periodic syndromes (CAPS) and trigger spontaneous inflammasome activation and IL-1ß oversecretion led to successful treatment with IL-1-blocking agents. Herein we report a de novo missense mutation (c.1009A > T, encoding p.Thr337Ser) affecting the nucleotide-binding domain of the inflammasome component NLRC4 that causes early-onset recurrent fever flares and macrophage activation syndrome (MAS). Functional analyses demonstrated spontaneous inflammasome formation and production of the inflammasome-dependent cytokines IL-1ß and IL-18, with the latter exceeding the levels seen in CAPS. The NLRC4 mutation caused constitutive caspase-1 cleavage in cells transduced with mutant NLRC4 and increased production of IL-18 in both patient-derived and mutant NLRC4-transduced macrophages. Thus, we describe a new monoallelic inflammasome defect that expands the monogenic autoinflammatory disease spectrum to include MAS and suggests new targets for therapy.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas de Ligação ao Cálcio/genética , Inflamassomos/genética , Inflamação/genética , Síndrome de Ativação Macrofágica/genética , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Criança , Exoma/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação/sangue , Interleucina-18/sangue , Interleucina-18/metabolismo , Síndrome de Ativação Macrofágica/sangue , Macrófagos/metabolismo , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Skeletal muscle microtubules (MTs) form a nonclassic grid-like network, which has so far been documented in static images only. We have now observed and analyzed dynamics of GFP constructs of MT and Golgi markers in single live fibers and in the whole mouse muscle in vivo. Using confocal, intravital, and superresolution microscopy, we find that muscle MTs are dynamic, growing at the typical speed of â¼9 µm/min, and forming small bundles that build a durable network. We also show that static Golgi elements, associated with the MT-organizing center proteins γ-tubulin and pericentrin, are major sites of muscle MT nucleation, in addition to the previously identified sites (i.e., nuclear membranes). These data give us a framework for understanding how muscle MTs organize and how they contribute to the pathology of muscle diseases such as Duchenne muscular dystrophy.
Assuntos
Complexo de Golgi/fisiologia , Microtúbulos/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Animais , Antígenos/metabolismo , Técnicas de Transferência de Genes , Complexo de Golgi/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia de Fluorescência , Microscopia de Vídeo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Imagem com Lapso de Tempo , Tubulina (Proteína)/metabolismoRESUMO
OBJECTIVE: We previously demonstrated that Sirt1 regulates apoptosis in cartilage in vitro. Here we attempt to examine in vivo cartilage homeostasis, using Sirt1 total body knockout (KO) mice. METHOD: Articular cartilage was harvested from hind paws of 1-week and 3-week-old mice carrying wild type (WT) or null Sirt1 gene. Knees of Sirt1 haploinsufficient mice also were examined, at 6 months. Joint cartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures. RESULTS: We found that articular cartilage tissue sections from Sirt1 KO mice up to 3 weeks of age exhibited low levels of type 2 collagen, aggrecan, and glycosaminoglycan content. In contrast, protein levels of MMP-13 were elevated in the Sirt1 KO mice, leading to a potential increase of cartilage breakdown, already shown in the heterozygous mice. Additional results showed elevated chondrocyte apoptosis in Sirt1 KO mice, as compared to WT controls. In addition to these observations, PTP1b (protein tyrosine phosphatase b) was elevated in the Sirt1 KO mice, in line with previous reports. CONCLUSION: The findings from this animal model demonstrated that Sirt1 KO mice presented an altered cartilage phenotype, with an elevated apoptotic process and a potential degradative cartilage process.
Assuntos
Cartilagem Articular/química , Osteoartrite/genética , Animais , Apoptose , Doenças das Cartilagens/genética , Doenças das Cartilagens/metabolismo , Doenças das Cartilagens/patologia , Cartilagem Articular/patologia , Condrócitos/fisiologia , Modelos Animais de Doenças , Predisposição Genética para Doença , Homeostase , Camundongos , Camundongos Knockout , Osteoartrite/metabolismo , Osteoartrite/patologia , FenótipoRESUMO
A wave of structural reorganization involving centrosomes, microtubules, Golgi complex and ER exit sites takes place early during skeletal muscle differentiation and completely remodels the secretory pathway. The mechanism of these changes and their functional implications are still poorly understood, in large part because all changes occur seemingly simultaneously. In an effort to uncouple the reorganizations, we have used taxol, nocodazole, and the specific GSK3-ß inhibitor DW12, to disrupt the dynamic microtubule network of differentiating cultures of the mouse skeletal muscle cell line C2. Despite strong effects on microtubules, cell shape and cell fusion, none of the treatments prevented early differentiation. Redistribution of centrosomal proteins, conditional on differentiation, was in fact increased by taxol and nocodazole and normal in DW12. Redistributions of Golgi complex and ER exit sites were incomplete but remained tightly linked under all circumstances, and conditional on centrosomal reorganization. We were therefore able to uncouple microtubule reorganization from the other events and to determine that centrosomal proteins lead the reorganization hierarchy. In addition, we have gained new insight into structural and functional aspects of the reorganization of microtubule nucleation during myogenesis.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Microtúbulos/fisiologia , Animais , Transporte Biológico , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Camundongos , Microtúbulos/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Nocodazol/farmacologia , Paclitaxel/farmacologiaRESUMO
OBJECTIVE: Fractures can initiate an immune response that disturbs osteoblastic and osteoclastic cellular homeostasis through cytokine production and release. The aim of our study was to investigate gamma/delta T cells, innate lymphocytes known to be involved in tissue repair, as potential cellular components of the osteoimmune system's response to an in vivo model of bone injury. The absence of such cells or their effector cytokines influences the fate of other responder cells in proliferation, differentiation, matrix production, and ultimate callus formation. METHODS: Tibia fractures were created in 60 gamma/delta T cell-deficient mice (also called delta T cell receptor [TCR]-knockout mice) and 60 control C57BL/6 mice. Analysis included radiographs, basic histology, mechanical testing, flow cytometry, and immunohistochemical localization of gamma/delta TCR-positive subsets from control animals and of CD44 expression from both groups, as well as enzyme-linked immunosorbent assay for the effector cytokines interleukin-2 (IL-2), interferon-gamma (IFNgamma), and IL-6. RESULTS: Animals deficient in gamma/delta T cells demonstrated more mature histologic elements and quantitative increases in the expression of major bone (bone sialoprotein) and cartilage (type II collagen) matrix proteins and in the expression of bone morphogenetic protein 2 at a critical reparative phase. Moreover, only gamma/delta T cell-deficient animals had a decrease in the osteoprogenitor antiproliferative cytokines IL-6 and IFNgamma at the reparative phase. The result was improved stability at the repair site and an overall superior biomechanical strength in gamma/delta T cell-deficient mice compared with controls. CONCLUSION: The evidence for a role of gamma/delta T cells in the context of skeletal injury demonstrates the importance of the immune system's effect on bone biology, which is relevant to the field of osteoimmunology, and offers a potential molecular platform from which to develop essential therapeutic strategies.
Assuntos
Consolidação da Fratura/fisiologia , Imunidade Inata/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Linfócitos T/fisiologia , Animais , Matriz Óssea/metabolismo , Cartilagem/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Citocinas/metabolismo , Receptores de Hialuronatos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Osteoblastos/metabolismo , Osteoblastos/patologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T/patologiaRESUMO
During muscle differentiation, microtubule stability, nucleation and orientation all undergo profound changes, which are simultaneous with and possibly necessary for the elongation and fusion of muscle cells. We do not yet understand these events, but they present similarities with the polarized migration of fibroblasts, in which EB1 is necessary for microtubule stabilization. However, it was recently reported that EB3, not EB1, is involved in muscle cell elongation and fusion, and that neither of these two proteins influences microtubule stabilization. To re-examine the role of EB1, we have generated C2 cell lines permanently expressing EB1-targeted shRNAs. In these lines, EB1 is specifically knocked down by more than 90% before any differentiation-related changes can take place. We find that differentiation (assessed by myogenin expression), elongation and fusion are prevented. In addition, two early events that normally precede differentiation - microtubule stabilization and the accumulation of cadherin and beta-catenin on the plasma membrane - are inhibited. Re-expression of EB1 as EB1-GFP restores all aspects of normal differentiation, whereas overexpression of EB3-GFP restores elongation but not fusion. We conclude that EB1 is necessary for the early stages of muscle differentiation.
Assuntos
Diferenciação Celular/fisiologia , Fusão Celular , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Músculo Esquelético , Mioblastos Esqueléticos , Animais , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Forma Celular , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/fisiologia , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Skeletal muscle differentiation involves a complete reorganization of the microtubule network. Nearly 20 years ago, Tassin et al. [1985: J Cell Biol 100:35-46] suggested a mechanism for this reorganization by showing a redistribution of the microtubule organizing center from the centrosome to the nuclear membrane. Little progress has been made since. It is still not clear whether centrosomal proteins are redistributed together, whether microtubules are nucleated at the nuclear membrane or transported there post-nucleation, and whether gamma-tubulin (gammatub) remains necessary for nucleation in myotubes. To investigate these questions, we have examined the redistribution of the centrosomal proteins pericentrin (PC), gammatub, and ninein in the C2 muscle cell line. Immunofluorescence of differentiated myotubes shows PC along the nuclear membrane whereas gammatub is only detected there after pre-fixation detergent extraction. After expression of a GFP-tagged gammatub, we observe a weak fluorescence along the nuclear membrane, confirming the presence of gammatub at a low concentration relative to PC. Microinjection of anti-gammatub antibodies into myotubes blocks microtubule growth from both nuclear membranes and centrosomal sites. The centrosomal microtubule-anchoring protein, ninein, is found at the nuclear membrane as well and its distribution appears independent of microtubule integrity. We conclude that centrosomal proteins are redistributed independently during muscle differentiation, to sites that nucleate microtubules both along the nuclear membranes and through the cytoplasm.