SKELETAL MUSCLE ATROPHY: THE ROLE OF mirnas. ATROFIA DO MÚSCULO ESQUELÉTICO: A FUNÇÃO DOS mirnas - PDF

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SKELETAL MUSCLE ATROPHY: THE ROLE OF mirnas ATROFIA DO MÚSCULO ESQUELÉTICO: A FUNÇÃO DOS mirnas Universidade de Coimbra Faculdade de Medicina SKELETAL MUSCLE ATROPHY: THE ROLE OF mirnas ATROFIA DO

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SKELETAL MUSCLE ATROPHY: THE ROLE OF mirnas ATROFIA DO MÚSCULO ESQUELÉTICO: A FUNÇÃO DOS mirnas Universidade de Coimbra Faculdade de Medicina 2011 SKELETAL MUSCLE ATROPHY: THE ROLE OF mirnas ATROFIA DO MÚSCULO ESQUELÉTICO: A FUNÇÃO DOS mirnas Dissertation presented to the Faculty of Medicine of the University of Coimbra for the fulfillment of the requirements for a Doctoral degree in Health Sciences, branch of Biomedical Sciences. Dissertação apresentada à Faculdade de Medicina da Universidade de Coimbra, para prestação de provas de Doutoramento em Ciências da Saúde, no ramo de Ciências Biomédicas. Universidade de Coimbra Faculdade de Medicina 2011 This work was carried out under the tutorage of the Center for Neurosciences and Cellular Biology of Coimbra, in the context of the PhD program for Experimental Biology and Biomedicine (2005), under the supervision of Doctor Paulo Pereira, Faculty of Medicine, University of Coimbra, Portugal. The practical work was performed under the supervision of Doctor Marco Sandri, Telethon Scientist at the Venetian Institute of Molecular Medicine (VIIM) under the Department of Biomedical sciences of the University of Padova, Italy. This work was supported by the grant SFRH/BD/15890/2005 from Fundação para a Ciência e a Tecnologia (FCT), Lisboa, Portugal and by the grant of the project #14724 from Association Française contre les Myopathies (AFM) Este trabalho foi realizado sob a tutela do Centro de Neurociências e Biologia Celular de Coimbra ao abrigo do Programa Doutoral de Biologia Experimental e Biomedicina (2005), sob a orientação do Doutor Paulo Pereira, Faculdade de Medicina, Universidade de Coimbra, Portugal. O trabalho prático foi realizado sob a orientação do Doutor Marco Sandri, Cientista Telethon do Instituto Veneziano Medicina Molecuar (VIIM) sob o Departamento de Ciencias Biomedicas da Universidade de Padova, Itália. Este trabalho foi financiado pela bolsa SFRH/BD/15890/2005 da Fundação para a Ciência e a Tecnologia (FCT), Lisboa, Portugal e pela bolsa do projecto #14724 da Associação Francesa contra as Myopatias (AFM). Questo lavoro é stato realizzato sotto la tutela del Centro di Neuroscienza e Biologia Cellulare di Coimbra integrato nel Programma Dottorale di Biologia Sperimentale e Biomedicina (2005), sotto la supervisione del Dottor Paulo Pereira, Facoltà di Medicina, Università di Coimbra, Portogallo. Il lavoro pratico é stato realizzato sotto la supervisione del Dottor MarcoSandri, Telethon Scientist presso il Venetian Institute of Molecular Medicine (VIIM) e del Dipartimento di Scienze Biomediche della Università di Padova, Italia. Questo lavoro é stato finanziato con la borsa SFRH/BD/15890/2005 della Fondazione per la Ricerca e per la Tecnologia (FCT), Lisbona, Portogallo e con la borsa del progetto #14724 dell Associazione Francese contro le Miopatie (AFM). I II Acknowledgements / Agradecimentos All journeys have many characters. Those that are always present and those that although not present are always in our thoughts. Like in many journeys, these characters allow us to grow, to become a better person, a better scientist. The journey that ends with this thesis counted with many of these characters to whom I will always be grateful. To Marco Sandri. For giving me the opportunity to do my PhD in your Laboratory. For your supervision and for your help in the most critical moments. For your enthusiastic vision of science. To all the Lab members and company. To Andrea, Anke, Daniela, Dawit, Enrico, Eva, Francesca, Luisa, Giulia, Roberta, Silvia and Vanina. I am glad you were part of my life for these 5 year. Thank you for your friendship. To Cristiano De Pittà and Matteo Silvestrin. For your help with the microarrays and for all the bioinformatic work. À Manuela Santos. Pelas fundações da minha carreira cientifica. Aos organizadores do Programa Doutoral em Biologia Experimental e Biomedicina do Centro de Neurociências de Coimbra (PDBEB-CNC). Obrigado por terem acreditado em mim e por me terem dado a oportunidade de realizar o meu doutoramento. Obrigado por um incrível e bem organizado primeiro ano. Um obrigado especial ao Dr Paulo Pereira por ter aceitado ser o meu cosupervisor. Aos meus colegas do PDBEB. À Ana Clara, Ana Teles, Carina Santos, Catarina Pimentel, Eduardo Ferreira, Gisela Silva, Helena Sofia Domingues, Joana Lourenço, Mariana Bexiga e Ricardo Marques. Tivemos um primeiro ano bastante intenso mas muito divertido. Como em algumas històrias, existem personagens invisiveis e inominàveis que nos acompanham e ajudam a perceber quem somos. A todas elas um sincero obrigado. Ao Luís, Paulo, Simão e Vítor. Pela vossa amizade e por estarem sempre presente. III Á Lígia. Por teres caminhado sempre a meu lado durante esta aventura. Por toda a tua ajuda. Pelo teu apoio incondicional, amizade e carinho. Ao meu pai. Por sempre me ter incentivado a ir mais alem. Á minha mãe e à minha mana. Por sempre terem acreditado em mim, por sempre me terem apoiado, mesmo nas decisoes mais dificeis. Por me terem ensinado a nunca desistir. IV Acronyms and Abbreviations 19S Regulatory subunit of the proteasome 20S Core particle of the proteasome 3 UTR 3 Untrasnlated Region 40S Ribosomal subunit 4E-BP1 Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 5 UTR 5 Untranslated Region 60S Ribosomal subunit aautp 5-(3-amminoallyl)-UTP ActRIIB Activin receptor IIB Ago Argonaute AKT V-AKT Murine Thymoma Viral Oncogene Homolog ALk4 Activin Receptor-like Kinase 4 ALk5 Activin Receptor-like Kinase 5 ALS Amyotrophic Lateral Sclerosis AMPK AMP-Activated Protein Kinase AP-1 Activator Protein 1 Atg Autophagy-related gene ATP Adenosine triphosphate BaCl 2 Barium Chloride Bcl-2 B-cell lymphoma 2 C2C12 mouse myoblast cell line Ccr4 Chemokine, CC Motif, Receptor4 CDC25A Cell division cycle 25a CDC34 Cell division cycle 34 (ubiquitin conjugating enzyme) CDK6 Cyclin dependent kinase 6 CMA Chaperone-mediated autophagy CMV Cytomegalovirus Col6a1 Collagen VI CreER Cre recombinase fused with the estrogen receptor CSA Cross Section Area Cx43 connexin-43 V CXMD j canine X-linked muscular dystrophy in Japan Cy3 Cyanine Dye 3 Cy5 Cyanine Dye 5 DAPC dystrophin-associated protein complex DAVID Database for Annotation, Visualization and Integrated Discovery DCP2 Deccaping enzyme 2 DGCR8 DiGeorge Syndrome critical region gene 8 DMD Duchene Muscular Dystrophy D-MEM Dulbecco s modified Eagle s medium DNA Dexoxyribonucleic acid DTA Diphtheria toxin A DUB Deubiquitinating enzymes EDL Extensor digitalis longus eif3f Eukaryotic translation initiation factor 3, subunit F eif4e Eukaryotic translation initiation factor 4E eif4g Eukaryotic translation initiation factor 4-gamma eif6 Eukaryotic translation initiation factor 6 Ezh2 Enhancer of Zeste, drosophila, homolog 2 FBS Fetal bovine serum FGF Fibroblast growth factor FGFBP1 Fibroblast growth factor-binding protein FMRP Fragile X Mental Retardation Protein FO Functional Overload FoxO Forkead Box O Fstl1 Follistatin-like 1 Gabarap Gabba receptor-associated proteins GATE16 Golgi associated ATPaseEnhancer 16 KDa) GFP Green Fluorescence protein GSK3 - Glycogen synthase kinase 3 GW182 Trinucleotide repeat-containing gene 6a HDAC4 Histone deacetylase 4 Hox-A11 Homeobox A11 HU Hindlimb unloading HWA448 Torbafylline VI IGF-1 Insulin growth factor-1 IGF-II Insulin-Like growth factor II IKK Inhibitor of Kappa Light chain gene enhancer in B cells, Kinase IL-6 Interleukin 6 IREs Internal Ribosome Entry Site IRS Insulin receptor sunstrate Jumpy myotubularin-related protein JunB V-Jun Avian sarcoma virus 17 oncogene homolog KDa KiloDalton KEGG Kyoto Encyclopedia of Genes and Genomes LAMP-2A Limbic system-associated membrane protein LLN N-acetylleucyl-leucyl-norleucidal MAFbx Muscle atrophy F-box MAP1LC3 Microtubule-associated protein 1. Light chain3 MEF2 Mads Box transcription enhancer factor 2 MG132 CBZ-leucyl-leucyl-leucidal MHC Myosin heacy chain Mib1 drosophila homolog of Mindbomb 1 MRF4 Muscle regulatory factor 4 mrna messenger ribonucleic Acid mtor mammalian target of rapamycin Murf-1 muscle-specific ring finger protein Myf5 myogenic factor 5 MyH Myosin Heavy Chain MyoD Myogenic differentiation antigen 1 NF-kB Nuclear factor kappa-b NMJs neuromuscular junctions nptb polypirimidine tract.binding protein N-Ras Neuroblastoma Ras Viral oncogene NUMB Drosophila homolog of Numb ORF Open Reading Frame P27 Cyclin dependent Kinase inhibitor 1B PA200 Proteasome activator, 200 Kda PA28 Proteasome activator 28-alpha VII Pax3 Paired box gene 3 Pax7 Paired box gene 7 PAZ Piwi, Argonaut and Zwille domain PcG Polycomb Group proteins PDCD10 Programmed Cell Death 10 PDH Plant homeo domain PDK1 Phosphoinositide-dependent kinase PDK2 Pyruvate dehydrogenase kinase, isoenzyme 2 PDK4 Pyruvate dehydrogenase kinase, isoenzyme 4 PE Phosphatidylethanolamine PGC1 Peroxisome proliferator-activated receptor-gamma, coactivator 1, alpha PI3K Phosphatidylinositol-3-kinase PI3P Phosphatidyl inositol triphosphate PIP3 Phosphoinositide-3, 4, 5-triphosphate PKB Protein Kinase B PKM2 Muscle piruvate kinase 2 Pola1 DNA polymerase alpha1 PolE4 DNA polymerase epsilon 4 PolK DNA polymerase kappa PS-341 Velcade PTEN Phosphatase and tensin homolog PTX Pentoxyfylline Pur Purine-rich element-binding protein B PYGM Muscle glycogen phosphorylase Ran-GTP Ras-related nuclear protein RISC RNA-induced silencing complex RNAi RNA interference Roc1 Regulator of cullins 1 RT-PCR Real Time Polymerase Chain Reaction Runx1 Runt-Related transcription factor1 S6K1 Ribosomal protein S6 kinase SAM Significance Analysis of micro-arrays SCF Complex composed of Skp1, Cullin and F-box SH-EP1 Neuroblastoma cell line VIII SHIP2 SH2-containing inositol phosphatase 2 shrna Short hairpin RNA Skp1 S-phase kinase-associated protein SMAD Sma- and Mad-related proteins SOD1 Superoxide diemutase Sox6 Sry-box6 Sp3 Transcription factor sp3 SR Sarcoplasmic reticulum SRF Serum response factor STZ Streptozotocin TA Tibialis Anterior TGF- Transforming growth family - THRAP1 Thyroid Hormone Receptor Associated Protein 1 TNF- Tumor necrosis factor - TOR Target of rapamycin TR Thyroid Hormone Receptor TRBP Tar RNA Binding Protein TWEAK TNF-Like weak promoter of apoptosis U2OS Human osteosarcoma cell line U6 Small nuclear RNA U6 UCP2 Uncoupling protein 2 Utrn Utrophin UVRAG Ultraviolet irradiation resistance associated gene VMP1 Vacuole Membrane Protein 1 Vps34 phosphatidylinositol 3-kinase class 3 Xrn exoribonuclease YY1 Yin Yang 1 IX X Thesis Organization This thesis is organized as a scientific paper. It contains seven chapters preceded by an abstract in English and Portuguese. The First Chapter consists of an introduction to the molecular mechanisms implicated in the skeletal muscle atrophy. How is the skeletal muscle organized? What is muscle hypertrophy and atrophy? Which are the degradative mechanisms implicated during muscle atrophy? Which are the molecular regulators of this catabolic process? All these questions are approached in this chapter. Furthermore, there is also a detailed introduction on mirnas. Their origins, their biogenesis, their mechanism of action and their role in muscle cells and adult muscle. In the Second Chapter the major aims of the thesis are presented. A detailed description of the methods used in this thesis is presented in the Third Chapter. In the Fourth and Fifth Chapter, the results obtained are presented. The fourth chapter consist on the identification and functional characterization of the mirnas altered during the atrophic conditions. The fifth chapter describes the identification and functional validation of the target genes of the altered mirnas. this thesis. The Sixth Chapter comprises a general discussion and main conclusions of In the Seventh chapter, the future perspectives of this work are presented. XI XII Table of Contents Acknowledgements / Agradecimentos... III Acronyms and Abbreviations... V Thesis Organization... XI Table of Contents... XIII Abstract... XV Resumo... XVII Chapter 1 - Introduction Skeletal Muscle: the main player Skeletal Muscle Architecture Skeletal muscle fiber type Skeletal Muscle Hypertrophy and Atrophy Skeletal Muscle Hypertrophy Skeletal Muscle Atrophy The Ubiquitin/Proteasome system The Proteasome Ubiquitin and the ubiquitin conjugating cascade The Ubiquitin-Proteasome System in skeletal muscle The Autophagy/Lysosome Pathway Autophagic machinery Autophagy Regulation Autophagy in skeletal muscle The main orchestrators of skeletal muscle atrophy mirnas: from their origins to their functions in muscle atrophy Genomic distribution and transcription regulation Overview of mirnas Biogenesis Prediction of mirnas Targets Mechanisms of mrna posttranscriptional repression by mirnas Post-transcriptional Repression by mirnas Degradation of the target mrnas P-Bodies and the compartmentalization of mirna-mediated repression Functions of mirnas mirnas in skeletal muscle mirnas and the differentiation of muscle cells Role of mirnas in adult skeletal muscle Role of mirnas in fiber type switch Role of mirnas in muscle regeneration Role of mirnas in sarcopenia Role of mirnas in muscle hypertrophy and atrophy mirnas in muscle pathology Chapter 2 Aims Chapter 3 - Methods XIII 3.1- Animal models and surgical procedures Cell Culture RNA and mirna purification Micro Array of mirnas and mrnas Integration of the data obtained with the mrna expression profile with those of the mirna expression profile Validation of the microarray results Cross-sectional area measurements Luciferase assays Cloning and Plasmids Protein extraction and Western blotting Chapter 4 mirna expression signature in different atrophic condition Overview of the mirna expression profiles in different atrophic conditions Validation of the microarray results mirna-21 and mirna-206 expression is independent of its host genes mirna-21 and mirna-206 expression levels are affected by FoxO3 and NF mirna-206 and mirna-21 cooperate synergistically to induce skeletal muscle atrophy mirna-206 activates the promoter of Atrogin Inhibition of mirna-206 and mirna-21 partially protects from denervation-induced atrophy Chapter5 Identification of target genes regulated by mirna-206 and mirna Skeletal muscle atrophy is a transcriptionally regulated process Skeletal muscle atrophy is a transcriptionally regulated process fine tuned by mirnas PolK, YY1, eif4e3 and PDCD10 are possible targets of mirna-206 and mirna The 3 UTR of PolK, YY1, eif4e3 and PDCD10 are modulated during denervation The 3 UTR of PolK, YY1, eif4e3 and PDCD10 are modulated by mirna-206 and mirna Over-expression of mirna-206 and mirna-21 down-regulate eif4e3, PDCD10 and YY1 in C2C12 myoblasts Chapter 6 General Discussion and Conclusions Different atrophic conditions activate specific mirna programs mirna-206 induces atrophy in adult skeletal muscle Atrophy related mirnas regulate gene expression mainly by inducing degradation of target mrnas Chapter 7 Future Perspectives References XIV Abstract Skeletal muscle atrophy is a condition associated to loss of muscle mass in many diseases. Atrophy is a characteristic response to starvation, aging and disuse conditions (such as immobilization, denervation or unloading) but it also occurs as a complication in several chronic diseases such as cancer, diabetes, sepsis, AIDS, renal and heart failure and others. Independently of the cause, the main feature of muscle wasting is the enhancement of protein degradation that overcomes protein synthesis. Skeletal muscle atrophy is a transcriptionally regulated process (Lecker et al., 2004; Sandri et al., 2004a; Stitt et al., 2004). FoxOs are critical transcription factors involved in the regulation of critical rate-limiting enzymes belonging to the two most important degradative pathways: the ubiquititn/proteasome (Gomes et al., 2001; Sandri et al., 2004a) and the autophagy/lysosome (Mammucari et al., 2007; Zhao et al., 2007). Also NF-kB is involved in transcriptional regulation during muscle wasting (Cai et al., 2004; Hunter and Kandarian, 2004). Furthermore, a restricted group of genes, called atrophy-related genes or atrogenes, are commonly up- or down-regulated to all the atrophic conditions (Lecker et al., 2004). These findings suggest the presence of a shared molecular mechanism that controls muscle atrophy. Among the atrogenes there are genes involved in several fundamental biological processes that may require an additional regulation to fine-tune their action during muscle wasting. This action might be accomplished by a new class of regulatory molecules, the mirnas. mirnas are predicted to regulate several genes from the same pathway. Their role in adult skeletal muscle is largely unknown. mirnas are small non-coding RNAs with approximately 22 nucleotides that regulate posttranscriptionally gene expression. They are highly conserved among species and they are predicted to regulate the expression of approximately 60% of protein coding genes. Conventionally, mirnas are known to regulate gene expression by binding to the 3 -Untranslated Regions (3 UTRs) of the mrnas and, therefore, blocking translation or inducing mrna degradation. Each mirna has the potential to target hundreds of different mrnas. On the other hand each mrna can be targeted by different mirnas creating in this way complex regulatory networks. One of the hallmarks of mirnas is their specificity. In fact, several mirnas are involved in developmental and physiological processes that require tissue- and stage-specific expression. The tight regulation of mirnas expression is crucial and alterations are correlated with pathological conditions. The essential role of these regulators in skeletal muscle was clearly demonstrated in several animal models in which the mirna pathway was blocked leading to a compromised myogenic XV development (Kwon et al., 2005; O'Rourke et al., 2007; Sokol and Ambros, 2005). Furthermore, several mirna show a muscle-specific expression and are called myomirs. This group is composed by mirna-1, mirna-133, mirna-206, mirna-208a, mirna-208b and mirna-499. Muscle specific mirnas are involved in several processes of the muscle physiology including myogenesis, fiber type establishment and muscle regeneration. Besides myomirs, other mirnas were shown to be involved in the regulation of these processes. The involvement of several mirnas in the regulation of several aspect of muscle biology creates a complex regulatory network increasing the complexity of muscle biology. Moreover mirna deregulation is associated with muscle disease (De, V et al., 2010; Eisenberg et al., 2007; McCarthy and Esser, 2007a; Williams et al., 2009; Yuasa et al., 2008a). However, regardless of the growing evidences on mirnas function few studies have addressed their biological role in vivo. In this thesis, we studied the role that mirnas play in skeletal muscle atrophy. We have used an in vivo approach supported by bioinformatic analyses to identify some of the mechanisms controlled by mirnas. In the first part of the thesis we have established the mirna expression signature of several atrophic conditions by microarray analysis. According to our results, each atrophic condition has a specific mirna expression profile. Only middle-to-late atrophic conditions showed a significant alteration of the mirnas expression levels. Although no common mirna was found between the different conditions, two highly up-regulated mirnas were found in denervation, mirna-206 and mirna-21. Thus, we decided to address their biological role in vivo. Our studies showed, for the first time, that in vivo over-expression of these two mirnas leads to an atrophic phenotype, while inhibition of these mirnas induced hypertrophy. In the second part of this thesis, we performed mrna expression profile by using the same samples used for mirna profile. A bioinformatic approach based on gene expression data allowed us
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