RESUMEN
The Rac1-WAVE-Arp2/3 pathway pushes the plasma membrane by polymerizing branched actin, thereby powering membrane protrusions that mediate cell migration. Here, using knockdown (KD) or knockout (KO), we combine the inactivation of the Arp2/3 inhibitory protein arpin, the Arp2/3 subunit ARPC1A and the WAVE complex subunit CYFIP2, all of which enhance the polymerization of cortical branched actin. Inactivation of the three negative regulators of cortical branched actin increases migration persistence of human breast MCF10A cells and of endodermal cells in the zebrafish embryo, significantly more than any single or double inactivation. In the triple KO cells, but not in triple KD cells, the 'super-migrator' phenotype was associated with a heterogenous downregulation of vimentin (VIM) expression and a lack of coordination in collective behaviors, such as wound healing and acinus morphogenesis. Re-expression of vimentin in triple KO cells largely restored normal persistence of single cell migration, suggesting that vimentin downregulation contributes to the maintenance of the super-migrator phenotype in triple KO cells. Constant excessive production of branched actin at the cell cortex thus commits cells into a motile state through changes in gene expression.
Asunto(s)
Actinas , Pez Cebra , Animales , Humanos , Actinas/metabolismo , Vimentina/genética , Vimentina/metabolismo , Pez Cebra/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Movimiento Celular/fisiología , Proteínas Portadoras/metabolismoRESUMEN
The RAC1-WAVE-Arp2/3 signaling pathway generates branched actin networks that power lamellipodium protrusion of migrating cells. Feedback is thought to control protrusion lifetime and migration persistence, but its molecular circuitry remains elusive. Here, we identify PPP2R1A by proteomics as a protein differentially associated with the WAVE complex subunit ABI1 when RAC1 is activated and downstream generation of branched actin is blocked. PPP2R1A is found to associate at the lamellipodial edge with an alternative form of WAVE complex, the WAVE Shell Complex, that contains NHSL1 instead of the Arp2/3 activating subunit WAVE, as in the canonical WAVE Regulatory Complex. PPP2R1A is required for persistence in random and directed migration assays and for RAC1-dependent actin polymerization in cell extracts. PPP2R1A requirement is abolished by NHSL1 depletion. PPP2R1A mutations found in tumors impair WAVE Shell Complex binding and migration regulation, suggesting that the coupling of PPP2R1A to the WAVE Shell Complex is essential to its function.
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Actinas , Seudópodos , Actinas/metabolismo , Movimiento Celular/fisiología , Seudópodos/metabolismo , Transducción de Señal , Citoplasma/metabolismo , Factores de Transcripción/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismoRESUMEN
Epithelial-mesenchymal transition (EMT) is a critical step in tumor progression that leads to the acquisition by cancer cells the capacity for migration using the mesenchymal motility mode regulated by the RacâWAVEâArp2/3 signaling pathway. Earlier it was shown that proteins interacting with Rac can regulate mesenchymal migration and thus determine the metastatic potential of the cells. The search for new regulators of cell migration is an important theoretical and practical task. The adaptor protein Anks1a is one of the proteins interacting with Rac, whose expression is altered in many types of tumors. The aim of this study was to find whether Anks1a affects the migration of cancer cells and to identify the mechanism underlying this effect. It was suggested that Anks1a can influence cancer cell migration either as a Rac1 effector or by activating human epidermal growth factor receptor 2 (HER2) exchange. We investigated how upregulation and inhibition of Anks1a expression affected migration of breast cancer cells with different HER2 status. Anks1a was shown to interact with the activated form of Rac1. In the MDA-MB-231 cells (triple negative cancer), which lack HER2, Anks1a accumulated at the active cell edge, which is characterized by enrichment with active Rac1, whereas no such accumulation was observed in the HER2-overexpressing SK-BR-3 cells. Downregulation of the ANKS1a expression with esiRNA had almost no effect on the cancer cell motility, except a slight increase in the average migration rate of MDA-MB-231 cells. Among three cell lines tested, overexpression of Anks1a increased the migration rate of HER2-overexpressng SK-BR-3 cells only. We showed that Anks1a is an effector of activated Rac1, but its influence on the cell migration in this capacity was minimal, at least in the studied breast cancer cells. Anks1a affected the motility of breast cancer cells due to its involvement in the EGF receptor exchange.
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Proteínas Adaptadoras Transductoras de Señales , Neoplasias de la Mama , Femenino , Humanos , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal , Transducción de SeñalRESUMEN
Actin filaments generate mechanical forces that drive membrane movements during trafficking, endocytosis and cell migration. Reciprocally, adaptations of actin networks to forces regulate their assembly and architecture. Yet, a demonstration of forces acting on actin regulators at actin assembly sites in cells is missing. Here we show that local forces arising from actin filament elongation mechanically control WAVE regulatory complex (WRC) dynamics and function, that is, Arp2/3 complex activation in the lamellipodium. Single-protein tracking revealed WRC lateral movements along the lamellipodium tip, driven by elongation of actin filaments and correlating with WRC turnover. The use of optical tweezers to mechanically manipulate functional WRC showed that piconewton forces, as generated by single-filament elongation, dissociated WRC from the lamellipodium tip. WRC activation correlated with its trapping, dwell time and the binding strength at the lamellipodium tip. WRC crosslinking, hindering its mechanical dissociation, increased WRC dwell time and Arp2/3-dependent membrane protrusion. Thus, forces generated by individual actin filaments on their regulators can mechanically tune their turnover and hence activity during cell migration.
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Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Movimiento Celular , Fibroblastos/metabolismo , Mecanotransducción Celular , Seudópodos/metabolismo , Citoesqueleto de Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/genética , Animales , Línea Celular Transformada , Ratones , Microscopía Fluorescente , Pinzas Ópticas , Imagen Individual de Molécula , Estrés Mecánico , Factores de TiempoRESUMEN
BACKGROUND: Functional genomics employs several experimental approaches to investigate gene functions. High-throughput techniques, such as loss-of-function screening and transcriptome profiling, allow to identify lists of genes potentially involved in biological processes of interest (so called hit list). Several computational methods exist to analyze and interpret such lists, the most widespread of which aim either at investigating of significantly enriched biological processes, or at extracting significantly represented subnetworks. RESULTS: Here we propose a novel network analysis method and corresponding computational software that employs the shortest path approach and centrality measure to discover members of molecular pathways leading to the studied phenotype, based on functional genomics screening data. The method works on integrated interactomes that consist of both directed and undirected networks - HIPPIE, SIGNOR, SignaLink, TFactS, KEGG, TransmiR, miRTarBase. The method finds nodes and short simple paths with significant high centrality in subnetworks induced by the hit genes and by so-called final implementers - the genes that are involved in molecular events responsible for final phenotypic realization of the biological processes of interest. We present the application of the method to the data from miRNA loss-of-function screen and transcriptome profiling of terminal human muscle differentiation process and to the gene loss-of-function screen exploring the genes that regulates human oxidative DNA damage recognition. The analysis highlighted the possible role of several known myogenesis regulatory miRNAs (miR-1, miR-125b, miR-216a) and their targets (AR, NR3C1, ARRB1, ITSN1, VAV3, TDGF1), as well as linked two major regulatory molecules of skeletal myogenesis, MYOD and SMAD3, to their previously known muscle-related targets (TGFB1, CDC42, CTCF) and also to a number of proteins such as C-KIT that have not been previously studied in the context of muscle differentiation. The analysis also showed the role of the interaction between H3 and SETDB1 proteins for oxidative DNA damage recognition. CONCLUSION: The current work provides a systematic methodology to discover members of molecular pathways in integrated networks using functional genomics screening data. It also offers a valuable instrument to explain the appearance of a set of genes, previously not associated with the process of interest, in the hit list of each particular functional genomics screening.
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Redes Reguladoras de Genes , Genómica/métodos , Mapas de Interacción de Proteínas , Programas Informáticos , Transcriptoma , Humanos , Mutación con Pérdida de Función , MicroARNs/genética , MicroARNs/metabolismo , Desarrollo de Músculos/genética , FenotipoRESUMEN
International experts in the fields of cellular motility, force generation and mechanosensation met in Baeza, a UNESCO World Heritage city, from the 10th to the 13th of November, 2019. The meeting, part of the 'Current Trends in Biomedicine' series, took place at the 'Sede Antonio Machado', a beautiful 17th century building turned into a conference center of the Universidad Internacional de Andalucía (UNIA), which sponsored the event. The meeting was organized by Alexis Gautreau, Pekka Lappalainen and Miguel Vicente-Manzanares, with the support of the European Molecular Biology Organization (EMBO) and the Spanish-based company IMPETUX. Fifty scientists presented recent results during the talks, poster sessions and thematic discussions. As Baeza itself served as a crossroads of medieval Christian, Moorish and Jewish cultures, the meeting brought together cell biologists, biochemists, biophysicists and engineers from around the world that provided an integrated vision of the role of the actin cytoskeleton, force generation and mechanosensation in diverse physiological processes and pathologies.
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Actinas , Biología MolecularRESUMEN
The actin cytoskeleton generates and senses forces. Here we report that branched actin networks from the cell cortex depend on ARPC1B-containing Arp2/3 complexes and that they are specifically monitored by type I coronins to control cell cycle progression in mammary epithelial cells. Cortical ARPC1B-dependent branched actin networks are regulated by the RAC1/WAVE/ARPIN pathway and drive lamellipodial protrusions. Accordingly, we uncover that the duration of the G1 phase scales with migration persistence in single migrating cells. Moreover, cortical branched actin more generally determines S-phase entry by integrating soluble stimuli such as growth factors and mechanotransduction signals, ensuing from substratum rigidity or stretching of epithelial monolayers. Many tumour cells lose this dependence for cortical branched actin. But the RAC1-transformed tumour cells stop cycling upon Arp2/3 inhibition. Among all genes encoding Arp2/3 subunits, ARPC1B overexpression in tumours is associated with the poorest metastasis-free survival in breast cancer patients. Arp2/3 specificity may thus provide diagnostic and therapeutic opportunities in cancer.
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Actinas/metabolismo , Neoplasias de la Mama/genética , Ciclo Celular , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Quimioterapia Adyuvante , Estudios de Cohortes , Femenino , Humanos , Persona de Mediana Edad , ARN Mensajero/genéticaRESUMEN
IGF-2 mRNA binding protein 3 (IGF2BP3, IMP-3) is a well-known post-transcriptional regulatory factor of gene expression, mainly involved in embryonic development and oncogenesis. We have previously demonstrated that a subset of IMP-3 targets, such as the mRNAs of cyclins D1, D3 and G1, are positively regulated by IMP-3, and that this regulation depends on nuclear localization of IMP-3. In the present study, we show that as a first step following a knock-down of IMP-3, the protein levels of the cyclins rapidly decrease, while their mRNAs remain stable and associated with the polyribosomes, though not translated. We have elucidated the molecular mechanisms of this regulation, demonstrating that IMP-3 and its protein partners ILF3/NF90 and PTBP1 bind to the 3'UTRs of the cyclin mRNAs and protect them from the translational repression induced by miRNA-dependent recruitment of AGO2/GW182 complex in human cancer cells.
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Regiones no Traducidas 3'/genética , Proteínas Argonautas/metabolismo , Autoantígenos/metabolismo , Ciclina D1/genética , Ciclina D3/genética , Biosíntesis de Proteínas/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas Argonautas/genética , Línea Celular Tumoral , Ciclina D1/biosíntesis , Ciclina D3/biosíntesis , Ciclina G1/genética , Proteína 1 Similar a ELAV/genética , Factores Eucarióticos de Iniciación/genética , Humanos , Proteínas del Factor Nuclear 90/genética , Proteínas del Factor Nuclear 90/metabolismo , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genéticaRESUMEN
CNOT6L is a deadenylase subunit belonging to the CCR4-NOT complex, a major deadenylase complex in eukaryotes involved at multiple levels in regulation of gene expression. While CNOT6L is expressed in skeletal muscle cells, its specific functions in this tissue are still largely unknown. Our previous work highlighted the functional of CNOT6L in skeletal muscle cell differentiation. To further explore how CNOT6L regulates myogenesis, we used here gene expression analysis to identify CNOT6L mRNA targets in human myoblasts. Among these novel targets, IL-8 (interleukin 8) mRNA was the most upregulated in CNOT6L knock-down (KD) cells. Biochemical approaches and poly (A) tail length assays showed that IL-8 mRNA is a direct target of CNOT6L, and further investigations by loss- and gain-of-function assays pointed out that IL-8 is an important effector of myogenesis. Therefore, we have characterized CNOT6L-IL-8 as a new signaling axis that regulates myogenesis.
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Diferenciación Celular/genética , Interleucina-8/genética , Músculo Esquelético/metabolismo , Ribonucleasas/genética , Adulto , Animales , Western Blotting , Línea Celular , Células Cultivadas , Perfilación de la Expresión Génica , Humanos , Interleucina-8/metabolismo , Microscopía Fluorescente , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citología , Mioblastos/citología , Mioblastos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasas/metabolismo , Transducción de Señal/genética , Transcripción GenéticaRESUMEN
BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disorder and is one of the most common forms of muscular dystrophy. We have recently shown that some hallmarks of FSHD are already expressed in fetal FSHD biopsies, thus opening a new field of investigation for mechanisms leading to FSHD. As microRNAs (miRNAs) play an important role in myogenesis and muscle disorders, in this study we compared miRNAs expression levels during normal and FSHD muscle development. METHODS: Muscle biopsies were obtained from quadriceps of both healthy control and FSHD1 fetuses with ages ranging from 14 to 33 weeks of development. miRNA expression profiles were analyzed using TaqMan Human MicroRNA Arrays. RESULTS: During human skeletal muscle development, in control muscle biopsies we observed changes for 4 miRNAs potentially involved in secondary muscle fiber formation and 5 miRNAs potentially involved in fiber maturation. When we compared the miRNA profiles obtained from control and FSHD biopsies, we did not observe any differences in the muscle specific miRNAs. However, we identified 8 miRNAs exclusively expressed in FSHD1 samples (miR-330, miR-331-5p, miR-34a, miR-380-3p, miR-516b, miR-582-5p, miR-517* and miR-625) which could represent new biomarkers for this disease. Their putative targets are mainly involved in muscle development and morphogenesis. Interestingly, these FSHD1 specific miRNAs do not target the genes previously described to be involved in FSHD. CONCLUSIONS: This work provides new candidate mechanisms potentially involved in the onset of FSHD pathology. Whether these FSHD specific miRNAs cause deregulations during fetal development, or protect against the appearance of the FSHD phenotype until the second decade of life still needs to be investigated.
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Feto/metabolismo , MicroARNs/genética , Músculo Esquelético/embriología , Músculo Esquelético/patología , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/patología , Transcriptoma , Biopsia , Estudios de Casos y Controles , Biología Computacional , Femenino , Feto/embriología , Feto/patología , Humanos , Masculino , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapulohumeral/embriologíaRESUMEN
BACKGROUND: Elevated levels of the inflammatory cytokine TNF-α are common in chronic diseases or inherited or degenerative muscle disorders and can lead to muscle wasting. By contrast, IGF1 has a growth promoting effect on skeletal muscle. The molecular mechanisms mediating the effect of TNF-α and IGF1 on muscle cell differentiation are not completely understood. Muscle cell proliferation and differentiation are regulated by microRNAs (miRNAs) which play a dominant role in this process. This study aims at elucidating how TNF-α or IGF1 regulate microRNA expression to affect myoblast differentiation and myotube formation. RESULTS: In this study, we analyzed the impact of TNF-α or IGF1 treatment on miRNA expression in myogenic cells. Results reveal that i) TNF-α and IGF1 regulate miRNA expression during skeletal muscle cell differentiation in vitro, ii) microRNA targets can mediate the negative effect of TNF-α on fusion capacity of skeletal myoblasts by targeting genes associated with axon guidance, MAPK signalling, focal adhesion, and neurotrophin signalling pathway, iii) inhibition of miR-155 in combination with overexpression of miR-503 partially abrogates the inhibitory effect of TNF-α on myotube formation, and iv) MAPK/ERK inhibition might participate in modulating the effect of TNF-α and IGF1 on miRNA abundance. CONCLUSIONS: The inhibitory effects of TNF-α or the growth promoting effects of IGF1 on skeletal muscle differentiation include the deregulation of known muscle-regulatory miRNAs as well as miRNAs which have not yet been associated with skeletal muscle differentiation or response to TNF-α or IGF1. This study indicates that miRNAs are mediators of the inhibitory effect of TNF-α on myoblast differentiation. We show that intervention at the miRNA level can ameliorate the negative effect of TNF-α by promoting myoblast differentiation. Moreover, we cautiously suggest that TNF-α or IGF1 modulate the miRNA biogenesis of some miRNAs via MAPK/ERK signalling. Finally, this study identifies indicative biomarkers of myoblast differentiation and cytokine influence and points to novel RNA targets.
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Diferenciación Celular/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , MicroARNs/biosíntesis , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Adulto , Células Cultivadas , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Músculo Esquelético/citología , Mioblastos Esqueléticos/citologíaRESUMEN
A genome-wide screen had previously shown that knocking down miR-98 and let-7g, two miRNAs of the let-7 family, leads to a dramatic increase in terminal myogenic differentiation. In the present paper, we report that a transcriptomic analysis of human myoblasts, where miR-98 was knocked down, revealed that approximately 240 genes were sensitive to miR-98 depletion. Among these potential targets of miR-98, we identified the transcriptional repressor E2F5 and showed that it is a direct target of miR-98. Knocking down simultaneously E2F5 and miR-98 almost fully restored normal differentiation, indicating that E2F5 is involved in the regulation of skeletal muscle differentiation. We subsequently show that E2F5 can bind to the promoters of two inhibitors of terminal muscle differentiation, ID1 (inhibitor of DNA binding 1) and HMOX1 (heme oxygenase 1), which decreases their expression in skeletal myoblasts. We conclude that miR-98 regulates muscle differentiation by altering the expression of the transcription factor E2F5 and, in turn, of multiple E2F5 targets.
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Diferenciación Celular/genética , Factor de Transcripción E2F5/genética , Regulación del Desarrollo de la Expresión Génica , MicroARNs/genética , Mioblastos Esqueléticos/metabolismo , Factor de Transcripción E2F5/antagonistas & inhibidores , Factor de Transcripción E2F5/metabolismo , Perfilación de la Expresión Génica , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Proteína 1 Inhibidora de la Diferenciación/genética , Proteína 1 Inhibidora de la Diferenciación/metabolismo , MicroARNs/antagonistas & inhibidores , MicroARNs/metabolismo , Desarrollo de Músculos/genética , Mioblastos Esqueléticos/citología , Regiones Promotoras Genéticas , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , TranscriptomaRESUMEN
The messenger RNA 3'-untranslated region (3'UTR) plays an important role in regulation of gene expression on the posttranscriptional level. The 3'UTR controls gene expression via orchestrated interaction between the structural components of mRNAs (cis-element) and the specific trans-acting factors (RNA binding proteins and non-coding RNAs). The crosstalk of these factors is based on the binding sequences and/or direct protein-protein interaction, or just functional interaction. Much new evidence that has accumulated supports the idea that several RNA binding factors can bind to common mRNA targets: to the non-overlapping binding sites or to common sites in a competitive fashion. Various factors capable of binding to the same RNA can cooperate or be antagonistic in their actions. The outcome of the collective function of all factors bound to the same mRNA 3'UTR depends on many circumstances, such as their expression levels, affinity to the binding sites, and localization in the cell, which can be controlled by various physiological conditions. Moreover, the functional and/or physical interactions of the factors binding to 3'UTR can change the character of their actions. These interactions vary during the cell cycle and in response to changing physiological conditions. Abnormal functioning of the factors can lead to disease. In this review we will discuss how alterations of these factors or their interaction can affect cancer development and promote or enhance the malignant phenotype of cancer cells. Understanding these alterations and their impact on 3'UTR-directed posttranscriptional gene regulation will uncover promising new targets for therapeutic intervention and diagnostics. We will also discuss emerging new tools in cancer diagnostics and therapy based on 3'UTR binding factors and approaches to improve them.
RESUMEN
MiRNAs impact on the control of cell fate by regulating gene expression at the post-transcriptional level. Here, using mammalian muscle differentiation as a model and a phenotypic loss-of-function screen, we explored the function of miRNAs at the genome-wide level. We found that the depletion of a high number of miRNAs (63) impacted on differentiation of human muscle precursors, underscoring the importance of this post-transcriptional mechanism of gene regulation. Interestingly, a comparison with miRNA expression profiles revealed that most of the hit miRNAs did not show any significant variations of expression during differentiation. These constitutively expressed miRNAs might be required for basic and/or essential cell function, or else might be regulated at the post-transcriptional level. MiRNA inhibition yielded a variety of phenotypes, reflecting the widespread miRNA involvement in differentiation. Using a functional screen (the STarS--Suppressor Target Screen--approach, i. e. concomitant knockdown of miRNAs and of candidate target proteins), we discovered miRNA protein targets that are previously uncharacterized controllers of muscle-cell terminal differentiation. Our results provide a strategy for functional annotation of the human miRnome.
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Diferenciación Celular/genética , Genoma Humano/genética , MicroARNs/genética , Mioblastos/metabolismo , Animales , Western Blotting , Línea Celular , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mioblastos/citología , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Skeletal muscle regeneration after injury follows a remarkable sequence of synchronized events. However, the mechanisms regulating the typical organization of the regenerating muscle at different stages remain largely unknown. Here we show that muscle regeneration in mice lacking either CD9 or CD81 is abnormal and characterized by the formation of discrete giant dystrophic myofibres, which form more quickly in the absence of both tetraspanins. We also show that, in myoblasts, these two tetraspanins associate with the immunoglobulin domain molecule CD9P-1 (EWI-F/FPRP), and that grafting of CD9P-1-depleted myoblasts in regenerating muscles also leads to abnormal regeneration. In vitro myotubes lacking CD9P-1 or both CD9 and CD81 fuse with a higher frequency than normal myotubes. Our study unveils a mechanism preventing inappropriate fusion of myotubes that has an important role in the restitution of normal muscle architecture during muscle regeneration.
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Fusión Celular , Músculo Esquelético/fisiología , Regeneración , Tetraspanina 28/fisiología , Tetraspanina 29/fisiología , Animales , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/citología , Regulación hacia ArribaRESUMEN
BACKGROUND: miRNA profiling performed in myogenic cells and biopsies from skeletal muscles has previously identified miRNAs involved in myogenesis. RESULTS: Here, we have performed miRNA transcriptome profiling in human affinity-purified CD56+ myoblasts induced to differentiate in vitro. In total, we have identified 60 miRNAs differentially expressed during myogenic differentiation. Many were not known for being differentially expressed during myogenic differentiation. Of these, 14 (miR-23b, miR-28, miR-98, miR-103, miR-107, miR-193a, miR-210, miR-324-5p, miR-324-3p, miR-331, miR-374, miR-432, miR-502, and miR-660) were upregulated and 6 (miR-31, miR-451, miR-452, miR-565, miR-594 and miR-659) were downregulated. mRNA transcriptome profiling performed in parallel resulted in identification of 6,616 genes differentially expressed during myogenic differentiation. CONCLUSIONS: This simultaneous miRNA/mRNA transcriptome profiling allowed us to predict with high accuracy target genes of myogenesis-related microRNAs and to deduce their functions.
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Diferenciación Celular/genética , MicroARNs/genética , Desarrollo de Músculos/genética , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , ARN Mensajero/metabolismo , Antígeno CD56/genética , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , MicroARNs/fisiología , ARN Mensajero/genética , Regulación hacia ArribaRESUMEN
PURPOSE: The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats. METHODS: Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis. RESULTS: We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b. CONCLUSIONS: Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.
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Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/metabolismo , Proteínas de Homeodominio/metabolismo , MicroARNs/metabolismo , Células Ganglionares de la Retina/metabolismo , Células Horizontales de la Retina/metabolismo , Transducción de Señal/genética , eIF-2 Quinasa/metabolismo , Animales , Apoptosis/genética , Western Blotting , Diabetes Mellitus Experimental/genética , Retinopatía Diabética/genética , Regulación de la Expresión Génica , Genes Reporteros , Proteínas de Homeodominio/genética , Hibridación in Situ , Luciferasas/análisis , Masculino , MicroARNs/genética , Ratas , Ratas Wistar , Células Ganglionares de la Retina/citología , Células Horizontales de la Retina/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , eIF-2 Quinasa/genéticaRESUMEN
The discovery of regulatory small non-coding RNAs represents a revolution in our understanding of gene regulation. These small non-coding RNAs are powerful tools for exploring cellular pathways and for artificially controlling gene expression. Natural small RNAs also represent potential therapeutic targets.
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Epigénesis Genética , MicroARNs/genética , ARN Interferente Pequeño/genética , HumanosRESUMEN
Insulin-like growth factor 2 (IGF-2) mRNA-binding proteins (IMPs) are a family of posttranscriptional regulatory factors with well-understood roles in embryonic development and cancer but with poorly characterized functions in normal adult cells and tissues. We now show that IMP-2, the most ubiquitously expressed member of the family, is abundant in human and mouse adult skeletal myoblasts, where it is indispensable for cell motility and for stabilization of microtubules. To explore the functions of IMP-2, we analyzed the transcripts that were differentially regulated in IMP-2-depleted myoblasts and bound to IMP-2 in normal myoblasts. Among them were the mRNAs of PINCH-2, an important mediator of cell adhesion and motility, and MURF-3, a microtubule-stabilizing protein. By gain- and loss-of-function assays and gel shift experiments, we show that IMP-2 regulates the expression of PINCH-2 and MURF-3 proteins via direct binding to their mRNAs. Upregulation of PINCH-2 in IMP-2-depleted myoblasts is the key event responsible for their decreased motility. Our data reveal how the posttranscriptional regulation of gene expression by IMP-2 contributes to the control of adhesion structures and stable microtubules and demonstrate an important function for IMP-2 in cellular motility.
Asunto(s)
Movimiento Celular/fisiología , Células Musculares/fisiología , Mioblastos/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas con Dominio LIM , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Células Musculares/citología , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/citología , Mioblastos/citología , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMEN
Mechanisms of transcriptional repression are important during cell differentiation. Mammalian heterochromatin protein 1 isoforms HP1alpha, HP1beta, and HP1gamma play important roles in the regulation of chromatin structure and function. We explored the possibility of different roles for the three HP1 isoforms in an integrated system, skeletal muscle terminal differentiation. In this system, terminal differentiation is initiated by the transcription factor MyoD, whose target genes remain mainly silent until myoblasts are induced to differentiate. Here we show that HP1alpha and HP1beta isoforms, but not HP1gamma, interact with MyoD in myoblasts. This interaction is direct, as shown using recombinant proteins in vitro. A gene reporter assay revealed that HP1alpha and HP1beta, but not HP1gamma, inhibit MyoD transcriptional activity, suggesting a model in which MyoD could serve as a bridge between nucleosomes and chromatin-binding proteins such as HDACs and HP1. Chromatin immunoprecipitation assays show a preferential recruitment of HP1 proteins on MyoD target genes in proliferating myoblasts. Finally, modulation of HP1 protein level impairs MyoD target gene expression and muscle terminal differentiation. Together, our data show a nonconventional interaction between HP1 and a tissue-specific transcription factor, MyoD. In addition, they strongly suggest that HP1 isoforms play important roles during muscle terminal differentiation in an isoform-dependent manner.