Crosstalk between glucocorticoid receptor and obestatin/GPR39 system in skeletal muscle: an avenue for the treatment of muscular atrophy

AUTORIZACIÓN DEL DIRECTOR / TUTOR DE LA TESIS CROSSTALK BETWEEN GLUCOCORTICOID RECEPTOR AND OBESTATIN/GPR39 SYSTEM IN SKELETAL MUSCLE: AN AVENUE FOR THE TREATMENT OF MUSCULAR ATROPHY D. Jesús Pérez Camiña D. Tomás García Caballero Parada INFORMA/N: Que la presente tesis, corresponde con el trabajo realizado por Dña. Tania Cid Díaz , bajo mi dirección, y a utorizo su presentación , considerando que reúne l os r equisitos exigidos en el R eglamento de Estudios de Doctorado de la USC, y que como director de ésta no incurre en las causas de abstención establecidas en Ley 40/2015. En Santiago de Compostela, 10 de Octubre de 2019 Fdo.: Jesús Pérez Camiña Fdo.: Tomás García Caballero Parada Dña.: Tania Cid Díaz DECLARA: No tener ningún conflicto de interés en relación con la tesis En Santiago de Compostela, 10 de outubro de 2019. Fdo.: Tania Cid Díaz INDEX Abstract/Resumen/Resumo 16 sistema Obestatina/GPR39, un sistema autocrino/paracrino con función sobre a mioxénese e con efectos anabólicos no músculo esquelético, podería protexer o músculo fronte a atrofia inducida por glucocorticoides Palabras clave: Señalización Obestatina; Músculo esquelético; Atrofia do músculo esquelético; Atrofia de células do músculo esquelético ABBREVIATIONS Abbreviations 19 4E-BP1: eukaryotic translation initiation factor 4E (eIf4E)- binding protein 1 ALS: autophagy-lysosomal system AMPK: AMP-activated protein kinase BCAA: branched-chain amino acid BCAT: branched-chain amino acid aminotransferase bFGF: basic fibroblast growth factor BSA: bovine serum albumin CAMKII: calmodulin-dependent protein kinase II CDK: cyclin-dependent kinase CK: creatine kinase CP: core particle CSA: cross-sectional area DAPI: 4’,6-diamidino-2-phenylindole Dexa: dexamethasone DM: differentiation medium DMD: Duchenne muscular dystrophy ECL: enhanced chemiluminescence ECM: extracelullar matrix EDL: extensorium digitorium EDTA: ethylenediamine tetra-acetic acid EGF: epidermal growth factor EGFR: epidermal growth factor receptor eIF3-f: eukaryotic translation initiation factor 3 subunit f ER: endoplasmic reticulum ERK1/2: extracellular signal-regulated kinase-1/2 FAPs: muscle like fibroblast-adipogenic progenitors FBS: fetal bovine serum albumin Abbreviations 20 FoxO: forkhead box protein O GAPDH: glyceraldehyde 3-phosphate dehydrogenase GHRL: ghrelin gene GHSR1a: ghrelin receptor GM: growth medium GPCR: G-protein-coupled receptor GPR38: motilin receptor GPR39: G-protein 39-coupled receptor GR: glucocorticoid receptor GREs: glucocorticoid response elements HDACs: histone deacetylases HE: haematoxylin/eosin HEK: human embryonic kidney 293 HS: horse serum Hsc70: heat shock cognate 70 IFN α: interferon α IGF1: insulin-like growth factor-1 IGFR: insulin-like growth factor receptor 1 IL10: interleukin 10 IL4: interleukin 4 IL6: interleukin 6 IRS1: insulin receptor substrate 1 KLF15: Krueppel-like factor 15 LC3: microtubule-associated protein light chain 3 MAFbx: F-box protein 32 MEF2: myocyte enhancer factor-2 MHC: myosin heavy chain Abbreviations 21 MRF4: myogenic regulatory factor 4 MRFs: myogenic regulatory factors mTOR: mammalian target of rapamycin mTORC1: mammalian target of rapamycin complex 1 MuRF1: muscle-Specific RING Finger Protein 1 MuSCs: muscular satellite cells Myf5: myogenic factor 5 MyoD: myoblast determination factor NF-kB: nuclear factor kappa B subunit p65 NFAT: nuclear factor of activated T-cells NLS: nuclear localization sequence NTSR1: neurotensin receptor p62/SQSTM1: sequestosome-1 PAM: peptidyl glycine a-amidating monooxygenase Pax3: paired box protein 3 Pax7: paired box protein 7 PBS: phosphate-buffered saline PGC-1 a : peroxisome proliferator-activated receptor gamma coactivator 1-alpha PI3K: phosphoinositide 3 -kinase PKC: protein kinase C PKD: polycystic kidney disease protein REDD1: protein regulated in development and DNA damage response 1 Rheb: Ras homolog enriched in brain RP: regulatory particle SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEM: standard error of the mean siRNA: small interfering RNA Abbreviations 22 Sirt: sirtuins SIX1: sine oculis homeobox homolog 1 SIX4: sine oculis homeobox homolog 4 SMAD: mothers Against Decapentaplegic Homolog 1 TA: tibialis anterior TNF a : tumor necrosis factor α ULK1: unc-51 like autophagy activating kinase 1 UPS: ubiquitin-proteasome system ZnR: Zn+2-sensing receptor INTRODUCTION Introduction 25 SKELETAL MUSCLE Skeletal muscle is one of the most dynamic and plastic tissues of the human body. In humans, skeletal muscle comprises approximately 40 % of total body weight and contains 50–75 % of all body proteins. Skeletal muscle primary function was described as postural retention as well as locomotion but it is not only a simple scaffold but rather an endocrine organ that produces and releases various types of biological molecules (Frontera WR and Ochala J, 2015; Iizuka K et al., 2014) skeletal muscle produces and releases proteins with autocrine, paracrine, or endocrine functions, termed myokines, in response to contraction, which can influence metabolism. Endocrine functions of myokines are involved in body weight regulation, low-grade inflammation, insulin sensitivity, suppression of tumor growth, and improvement of cognitive function (Hoffmann C and Weigert C, 2017; Iizuka K et al., 2014; Pedersen BK, 2013). Macroscopic organization of muscles show an exceptional level of organization, formed by nerves, blood vessels, conective tissue as well as contractile tissue. Skeletal muscle is attached to bone by tendons, epimysium, within is located the entire muscle composed by bundles of muscle fibers called fascicles surrounded by connective tissue, perimysium. Each fiber is nearby a layer of connective tissue, called endomysium, that includes the basal lamina that is over the sarcomere and where satellite cells are located. These are stem cells like, that play a key role in maintain the pool of myoblasts, the basic muscle cells, and in the repair process following a muscle injury. Fibers are multinucleated cells originated from the fusion of myoblasts and are formed of longitudinally units, myofibrils, surrounded by the sarcomere. The myofibrillar proteins, myosin (the thick filament), actin troponin, tropomyosin (the thin, filament) are enclosed by each sarcomere. These proteins allow the muscle contraction as a reaction to the calcium released from the Introduction 32 MuScs are a heterogenous population, differing in lineage potential, expression profile and proliferation/differentiation potential (Kuang S et al., 2007; Zammit PS, 2008). Most important parameter is that MuSCs are heterogeneus in stemness, it exists two subpopulations based on the expression of the myogenic factor 5 (Myf5). First one, Myf5-, corresponds a low percentage of MuScs, the “true stem cells” and secondly, Myf5+, cells commited to the myogenic program. After transplantation, these two populations diverge in regenerative potential, while Myf5+ cells undergo myogenic differentiation, Myf5cells carry out muscle repair and participate in the renewal of the MuSC compartment (Kuang S et al., 2007). The MuSC remain uniform; however, reduces in population density and efficacy with age (Almada and Wagers, 2016).! Scheme 3. The satellite cells. MuSCs are located over the sarcolemma and under the basal lamina of the myofibers. A, Electron microscopy of the MuSC. B, schematic representation of MuSC in a myofiber. PM, plasma membrane; BL, basal lamina; SC, satellite cell. Figure modified from Fukada S et al. Isolation, characterization, and molecular regulation of muscle stem cells. Front Physiol. 2013;12(4):317 and Pearson Education, Inc, 2009. Is an open-access article Quiescence, activation and differentiation of MuSCs MuSCs resides between basal lamina and sarcolemma of the myofibers. This microenvironment, called niche, limit and orient the migration of the cells during Introduction 33 injury (Sanes JR, 2003). The niche has an important role in maintaining the quiescence and regulating the MuCS fate. It is composed by cellular and noncellular components as, non-myogenic cells that resides in muscle like fibroblastadipogenic progenitors (FAPs), macrophages, extracellular matrix and growth factors (Almada AE, Wagers AJ, 2016). In resting adult muscles MuScs are in dormancy, they are non-mitotic, this state is also known as quiescent, most of them are derived from precursors Pax3+/Pax7+. The expression of Pax7 and the absence of the myoblast determination factor (MyoD) characterize Quiescent MuScs. Quiescent MuSCs become activated following an injury due to several microenvironmental signals, to give rise to myoblast, these myogenic progenitors enter in the differentiation program and fuse to form multinucleated myotubes to fix damaged fibers. The activation process of MuSCs is regulated by basic helix-loop-helix transcription factors called myogenic regulatory factors (MRFs) as Myf5, MyoD, myogenin and myogenic regulatory factor 4 (MRF4). Myf5 and MyoD are decisive during muscle regeneration for the myogenic determination, activated MuSCs express Pax7 and MyoD at the same time, after several proliferation phases, these cells bring out to Pax7-/MyoD+ cells that are committed to proliferation while a few percentage of activated MuSCs derives in Pax7+/MyoD-, these ones return to state of quiescence which is critical to preserve the MuSCs pool (Collins CA et al., 2005; Dumont NA et al., 2015; Zammit PS, 2008). In this sense, the activation of Notch and Wnt signaling is essential for maintaining quiescence in MuSCs by inhibiting MyoD expression and inducing PAX7, respectively (Bjornson CR et al., 2012; Olguin HC and Olwin BB, 2004). Finally, the differentiation phase is marked by the downregulation of MyoD and Myf5 along with the increment in expression of MRF4 and myogenin in early stages (Yin H et al, 2013) whereas the terminal differentiation is marked by the expression of Introduction 34 myofibrillar proteins such as MHC (Scheme 4)(Almada AE and Wagers AJ, 2016; Wang YX et al., 2014). Scheme 4. Hierarchy of transcription factors regulating progression through the myogenic program. Pax3 and Pax7 are the early regulators of myogenic specification. Myf5 and MyoD commited cells to myogenic program. Myogenin and MRF4 are necessary for early and terminal differentiation respectively. Six1 and Six4 govern the regulation of several of named transcription factors during the myogenic program. Self-renewal of MuScs The balance between self-renewal and differentiation of MuScs is crucial for stem cell maintenance and muscle homeostasis as it maintains the stem cell population and supplies the myogenic progenitors needed for muscle regeneration (Kuang S et al., 2007) An error in self-renewal results in a depletion of stem cell population and the consecuent reduce in muscle regeneration. Self-renewal can be performed in two differenten types of cell division: Asymmetric or symmetric. Asymmetric divisions give rise two different doughter cells, one destined to self-renewal, to replenish the stem cell pool and the other, commited to differentiate. On the other Introduction 35 hand, symmetric division generates two identical daughter cells and this cells can adop different fates in a stocastic way. Some of them, exclusively generate two stem cell daughters, whereas alternaively, it could generate two cells commited to differentiation (Schema 5) (Almada AE and Wagers YX, 2016; Dumont NA et al., 2015; Motohashi N and Asakura A, 2014; Kuang S et al., 2008). The fate of MuSCs in self-renewal is strongly influenced by the stem cell niche. It exists cell polarity within the niche, cell-cell and cell-ECM intractions. The basala lamina side of a satellite cell expreses integrin a7b1 that interacts with laminin, whereas the apical side expresses M-cadherin to dock the satellite cell and transduce signalas from the fiber. In view of asymmetric distribution of apical-basal signals, we could predict that a planar division (symmetric) where the two daughter cells are exposed to both, apical and basal, signals it would be obtained two cells with the same fate. In contrast, an apical-basal-oriented cell division give rise two daughter cells exposed either to apical or basal signals, leading to a different cell fate (Kuang S et al., 2008). Importantly, the daughter thtat remains in contact with the basal lamina adopts a stem cell fate and the daughter that loses the basal lamina contact adopts a commited differentiation fate (Kuang et al., 2007). Introduction 36 Scheme 5. Asymmetric and symmetric division of MuSCs. Asymmetric divisions give rise to one self-renewing cell (Myf5-; blue) and one committed cell (Myf5+; green), it occurs perpendicular to the fiber axis. Symmetric divisions generate two identical (either stem or committed progenitor) daughter cells, it occurs parallel to the muscle fiber axis. From Chang NC et al., 2016. With permission of Elsevier THE OBESTATIN/GPR39 SYSTEM OBESTATIN In 2005, based on bioinformatic predictions, Zhang et al. discovered a new peptide encoded by the ghrelin gene. It was named obestatin, from the Latin “obedere”, that means devour, and statin, indicating suppression. At the first time it was thought to have the opposite physiological effect of ghrelin on food intake, weight and gastric emptying due to it was first isolated in rat stomach (Zhang JV. and Ren PG, 2005). Obestatin is 23-aminoacid hormone derived from the proteolic procesing of preproghrelin and encoded by the ghrelin gene (GHRL). Preproghrelin is composed of 117 amino acids, a 23-amino acid signal peptide and a 94-amino acid peptide called proghrelin which is cleaved in two sites, between arginine 75-phenyalanine 76 and lysine 100-phenyalanine 101 given rise to obestatin precursor. After the cleavage, the lysine residue at the carboxy terminal is cleaved by carboxypeptidase Introduction 37 E, being then amidated in the carboxy-terminal leucine reside by the peptidyl glycine a-amidating monooxygenase (PAM), the presence of a C-terminal amide group is essential for its biological activity (Alén BO et al., 2012; Garg A, 2007; Soares JB and Leite-Moreira AF, 2008 (Scheme 6). Scheme 6. Post-translational processing of preproghrelin to mature ghrelin or obestatin. This polypeptide of 117 amino acids contains a signal peptide of 23 amino acids at the amino terminus, which is the first cleaved site, resulting in proghrelin, formed by 94 amino acids. Blue triangles indicate the sites of proteolytic section. Ghrelin is cleaved between arginine 51alanine 52 and is susceptible to be amidated at serine 3. Obestatin results from proteolytic scission of proghrelin at arginine 75-phenyalanine 76 and lysine 100 and phenyalanine 101, is procesed at the carboyterminal by carbopeptidase E and then in leucine 23. Figure extracted form Garg A., 2007. With permission of Oxford University Press Introduction 38 Moreover, Zhang et al. indicate after a mass spectrometry analysis that a 13 amino acid peptide, obestatin (11-23), with biological active, may be also processed from obestatin (Zhang JV and Ren PG, 2005). Rearding to obestatin distributions, as said, was found to be produced in rat stomach, in rat and human gastric mucosa (Dun SL et al., 2006; Zhang JV and Ren PG, 2005). However its expression was also demonstrated after in endocrine pancreas, adipose tissue, skeletal muscle, liver, lung, thyroid, mammary glands and male reproductive system, proposing an autocrine/paracrine functions in other tissues and organs (Gurriarán-Rodríguez U et al., 2012; Granata R et al., 2008; Granata R et al. 2010; Moretti E et al., 2014; Volante M et al., 2009; Zhao CM et al., 2008). Obestatin receptor: GPR39 The GPR39 receptor was originaly isolated and cloned in 1997 by McKee et al. (McKee KK et al., 1997) and considered an orphan GPCR until Zhang et al. discovered obestatin, their data obtained from radiolabelled ligand binding assay, proposed the obestatin as a ligand of GPR39 (Zhang JV. and Ren PG, 2005). GPR39 receptor gene is located on chromosome 2, band q21-q22, comprising two exons, belongs to the same family as ghrelin (GHSR1a), motilin (GPR38) and neurotensin (NTSR1) receptors (McKee KK et al., 1997) and it is formed by 435 amino acids and seven transmembrane helices. First exon encodes the residues 1 to 285, conforminf the N-terminal region and the first transmembrane helices. Flowing the first exon exists an intron of 200kb that also appears in ghrelin receptor as well as in motilin receptor genes. The residues 286 to 435are encoded by the second exon and correspond to the last two transmembrane helices and the C-terminal region (Egerod KL et al., 2007). Introduction 39 Two splicing variants of the GPCR39 receptor was identified: a full-length receptor called GPR39 1-a, known as canonical form; and a truncated form of the receptor, GPR39 1-b, considered biologically inactive at the first time (Egerod KL et al., 2007). GPR39 expression in the body is mainly circunscribe to the digestive system, but is also found in adipose tissue, liver, spleen, thyroid, lung, heart, reproductive tissues, brain and skeletal muscle (Dong XY et al., 2009; Gurriaran-Rodriguez et al. 2012). This widely expression of the receptor may suggest its role in the regulating diverse physiological functions, as myogenesis, adipogenesis, cell death, hormene secretion ot gastrointestinal motility. Dong XY et al., 2009; Gurriaran-Rodriguez et al. 2011; Gurriaran-Rodriguez et al. 2012) Obestatin bioactivity Despite obestatin was originally describe as a physiological opponent of ghrelin (Zhang JV. and Ren PG, 2005), this action was discarded by several groups (Green BD et al., 2007; Zhang JV et al., 2008). The central effects of obestatin remain unclear, it has been studied its role in peripheral cells and tissues, obestatin acts regulating adipogenesis, pancreatic homeostasis, cardiovascular function and myogenesis (Green BD and Grieve DJ, 2018; Trovato L et al., 2014). Moreover, obestatin regulates metabolic and cell differentiation functions, increasing cell survival and proliferation (Camiña JP et al., 2007; Granata R et al., 2008; Pazos Y et al., 2007) and inhibiting inflammation and apoptosis (Aragno M et al., 2012; Ceranowicz P et al., 2009; Granata R et al., 2012). Obestatin exerts its activity through GPR39 as was demonstrated in several cell lines by knockdown of GPR39 by siRNA experiments producing the inhibition of obestatin signaling (Alén BO et al., 2015; Camiña JP et al., 2007; Gurriarán-Rodríguez U et al., 2011; Gurriarán-Rodríguez U et al., 2012; Santos-Zas I et al., 2016). Moreover, coimmunoprecipitation experiments demonstrated the binding of obestatin to Introduction 40 GPR39 in cultured C2C12 myoblasts (Gurriarán-Rodríguez U et al., 2015). Studies of GPR39 structure and activity related to mouse and human obestatin reveal conformational differences beyond their differences in primary structure. Mouse analogue adopts a different three-dimensional structure that cannot activate human GPR39 (Alén BO et al., 2012). Although there is a proof of the species-specific activity of the obestatin through its receptor, GPR39, it existed a controversy around if GPR39 is the obestatin receptor. First, independent groups perform experiments to elucidate binding and stimulatory function of obestatin on GPR39, but none was able to achieve this aim (Chartrel N et al., 2007; Lauwers E et al., 2006; Tremblay F et al., 2007). This was hardened by the inability to reproduce the binding of obestatin to GPR39 by Zhang’s group as their original experiments(Zhang JV et al, 2007).Following years, it was demonstrate that the quality of obestatin supplied by some companies was deficient (De Spiegeller B et al., 2008), in fact, Zhang et al. pointed that the erratic binding of obestatin to GPR39 could be because of the bioactivity loss of obestatin after its poly-iodination (Zhang JV et al, 2007). Years later was revealed that mono-iodinated obestatin was efficient binding to human embryonic kidney 293 (HEK) cells transiently transfected with plasmids encoding GPR39 (Zhang JV et al., 2008). Other groups propose zinc ions (Zn+2) as the endogenous ligand of GPR39, considering GPR39 as Zn+2-sensing receptor (ZnR) and not the obestatin receptor (Besser L et al., 2009; Holst B et al., 2007). The obestatin signaling an its role in myogenesis Myogenesis is a multistep process. First, myoblast enter in a proliferative phase and then they exit the cell cycle to enter in a differentiation phase, in which they aliniate and fuse forming a multinucleated mature myotubes (Bentzinger CF et al., 2012; Zierath JR and Hawley JA, 2004). Obestatin is expressed in healthy skeletal muscle, in vitro experiments evidence that whether obestatin is up-regulated during early stages of myogenesis and sustained throught terminal differentiation, GPR39 is Introduction 41 expresses in myoblast as well as during differentiation (Scheme 7) (Santos-Zas I et al., 2016). The system is also increase following muscle injury (Gurriarán-Rodríguez U et al., 2012). Schema 7. Obestatin and GPR39 expresion during myogenesis. A. Immunohistochemical detection of obestatin GPR39 in human skeletal muscle tissue and human skeletal muscle cell line. B. Immunoblot analysis of obestatin, GPR39 and myogenic markers in human skeletal muscle cell line. Figure extracted from Santos-Zas I et al. 2016. With permission of Springer Nature After injury, intramuscular injection of obestatin rises regeneration by regulating multiple steps of myogenesis myoblast proliferation, cell cycle exit, differentiation, in order to fuse and form multinucleated myotubes (Gurriarán-Rodríguez U et al.2015). Lately, it was proved that oxidative phenotype of muscle fibre was favor by obestatin through both class II HDAC/MEF2 and PGC-1α mechanisms (SantosZas I et al., 2017). This properties of the obestatin GPR39 system suggest its clinical application in skeletal muscle. Certanly, obestatin demonstrated not only enhance the efficiency of engraftment, but also facilitates the distribution of myoblasts within the host muscle in a cell transplantation therapy experiment, the experiment was based on myoblast- Introduction 48 Dexamethasone in muscle atrophy Dexamethasone (Dexa) is a synthetic glucocorticoid with 20 to 30 times the binding affinity for glucocorticoid receptors of endogenous cortisol. It has anti-inflammatory and immunosuppressant properties being widely used as a potent antiemetic, for the treatment of acute exacerbations of multiple sclerosis, allergies, cerebral edema, inflammation, and shocks as well as in patients with conditions such as asthma, atopic and contact dermatitis, and drug hypersensitivity reactions (Johnson DB, Kelley B, 2019; Whelan R and Apfel CC, 2013). Dexa acts through the glucocorticoid receptor and regulates the gene expression binding directly to GREs, known as genomic action (Oakley RH and Cidlowski JA,2009; Patel R et al., 2014; Zhao et al, 2009), or indirectly through direct interactions with cytosolic kinases or specific membrane-bound receptor named non genomic effects (Lowenberg et al, 2008; Stahn et al, 2008). However, is widely use in medicine, high dose of glucocorticoids may lead to muscle wasting, activating the protein degradation in muscle through the UPS and ALS overall (Menconi M et al., 2008; Schakman et al, 2009). The stimulation of the GR by Dexa leads to the activation of these two proteolytic systems increasing the expression of several atrogenes such as FoxO, E3 ubiquitin ligases, MAFbx and MuRF1, and LC3 and Bnip3 (Cid-Diaz T et al., 2017; Mammucari C et al., 2007; Sandri M et al., 2004; Stitt TN et al., 2004). Moreover, is was also described an inhibitory effect of glucocorticoids repressing mTORC1 signaling as a result of enhanced transcription of REDD1 and KLF-15, two genes that are direct targets of GR receptor in muscle. This fact suggests a coordinated interaction between the catabolic signal and the anabolic machinery. REDD1 represses mTORC1 by inhibiting Rheb, which is a positive effector of mTORC1, leading to decreased phosphorylation of both 4E-BP1 and S6K1. On the other hand, KLF15 stimulates the expression of branched-chain amino acid (BCAA) aminotransferase (BCAT), an enzyme which degrades BCAA, and accelerates the Introduction 49 intracellular catabolism of BCAA. Furthermore, KLF-15 cooperates with FOXO1 to upregulate the promoter activity of Atrogin-1 and MuRF1(Shimizu N et al., 2001; Tanaka H et al., 2017) (Scheme 10). Scheme 10. Crosstalk between catabolic process due to glucocorticoids and anabolic process in skeletal muscle. Glucocorticoids exterts its atrophic effect directly promoting the expression of MURF1 or indirectly by the activation of the FOXO family transcription factors and KLF15 which foment MURF1, MAFbx and autophagy protein transcription. They also increase the expression of REDD1 which inhibits the analbolic effect of mTOR. Together these pathways promote muscle atrophy. Figure extracted from Shimizu N et al. 2011. With permission of Elsevier Introduction 50 Ubiquitin proteasome system The UPS is an ATP-dependent proteolytic system that mediates the degradation of target proteins tagged with ubiquitin molecules in the 26S proteasome (Murton AJ et al.,2008). The ubiquitin is a 76 amino acid protein with an 8,5 kDa of molecular weight, it can be added to a lysine un the target protein as a single entity (monoubiquitin) or as a chain of variable length (polyubiquitin) (Kravtsova-Ivantsiv Y and Ciechanover A, 2012). The 26S proteasome is a macromolecular responsible for controlled degradation of ubiquitinated substrates. It is composed for 33 subunits at least, which are assemble in two subcomplexes: the 20S core particle (CP), responsible for proteolytic activities showing highly conserved “barrel”-like structure arranged into four heptameric rings stacked in the order of α7β7β7α7, and the 19S regulatory particle (RP), responsible of substrate recognition, deubiquitination, protein unfolding, and substrate translocation to the 20S CP for degradation (Wang X et al., 2017). The ubiquitylation is regulated by the activity of three enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin protein ligases (E3). First, E1 adenylates the C-terminus of ubiquitin and forms a thioester bond between the ubiquitin C-terminus and a catalytic E1 cysteine residue, in an ATP-dependent process. Then, ubiquitin is transferred to a E2 enzyme, forming another thioester bond between ubiquitin and a cysteine residue of the E2. Finally, the ubiquitin molecule is transferred from the E2 to a lysine residue of the target protein by the action of a E3. E3 enzyme provides with specificity this process as they recognize and bind ubiquitin molecules to a specific target sequence (Bodine SC and Baehr LM, 2014; Rom O and Reznick AZ, 2016). In the context of skeletal muscle, in 2001 two novel E3 ubiquitin-ligases were identified, which expression is significantly increased in response to multiple catabolic conditions characterized by muscle atrophy, MAFbx (FBXO32) and MURF1 (Trim63) (Bodine SC et al., 2001; Gomes MD et al., 2001). MAFbx belongs Introduction 51 to cullinRING E3 ligase family, it has an F-box domain allowing it to bind both skp1 and cullin1, suggesting a function in a SCF ubiquitin ligase complex (Gomes MD et al., 2001). It is found in the cytoplasm but it also possesses a nuclear localization sequence suggesting a possible role in the nucleus. The primary targets of MAFbx in skeletal muscles are the myogenic regulatory factor MyoD and the eukaryotic translation initiation factor 3 subunit f (eIF3-f) (Lagirand-Cantaloube J et al, 2008; Tintignac LA et al., 2005). Further in vitro studies revealed that MAFbx and MyoD are regulated inversely during the differentiation of muscle cells, and overexpression of MAFbx suppressed the differentiation ability due to block the proper myoblast fusion for myotube formation (Tintignac LA et al., 2005). MURF1 is a RING E3 ligase with a zinc-finger domain and a leucine-rich coiled-coiled domain which allows it to form heterodimers with other MURF proteins, and an acidic Cterminus tail (Foletta VC et al., 2011). MURF-2 and MURF-3 are two proteins encoded by a different gene than MUFR-1 that have a high homology with it. They are found in the M-line of the sarcomere. Additionally, MURF1 and MURF-3 are in Z-lines. MURF1 and MURF-2 are also detected in the nucleus. Whereas MURF-2 and MURF-3 have been shown to be important for microtubule stability, just MURF1 have been associated with muscle atrophy (Bodine SC and Baehr LM, 2014). It was demonstrated the interaction of MURF1 with proteins involved in ATP generation and myofibrillar proteins such as nebulin, titin, MLC-2 and cTNI (Witt SH et al., 2005) and in vitro studies reveal that during muscle atrophy MuRF1 participates in the degradation on thick filament proteins following the degradation of MHC (Clarke BA et al., 2007; Cohen S et al., 2009). Knowing the role of this ubiquitin ligases under atrophy conditions, different in vitro studies using mouse cell lines under acute treatment of glucocorticoid demonstrate that both MAFbx and MURF1 undergo a significant upregulation (Cid-Diaz T et al., 2017; Evenson AR et al, 2005; Du J et al, 2000; Hong DH and Forsberg NE, 1995; Marinovic AC et al, 2006; Sacheck JM et Introduction 52 al, 2004; Stitt TN et al, 2004; Thompson MG et al, 1999; Wang L et al, 1998; Yang H et al, 2005). In terms of regulation, both MAFbx and MURF1 are transcriptional regulated by the same transcription factors, the first described were FoxO family which includes FoxO1, FoxO3 and FoxO4 and can bind directly to MAFbx and MURF1 promoter (Waddell DS et al., 2008). One the one hand it was described that FoxO1 was necessary but not sufficient to increase the expression of MAFbx and MURF1 in vitro in response to dexamethasone (Stitt TN et al., 2004), other studies focus in FoxO3 demonstrating its ability to upregulate both ubiquitin ligases during atrophy conditions (Sandri M et al., 2004). However, it have been reported discordances in the regulation of FoxO transcription factors , MAFbx and MURF1 between human and mouse models, suggesting a complex regulation of this system depending on the type of atrophy and between species (Cid-Diaz T et al., 2017; Foletta VC et al., 2009; Gomes MD et al 2001; Gustafsson T et al., 1985; Harber MP et al., 2008; Larsen AE et al., 2006). Moreover, MURF1 has a glucocorticoid response element in the proximal region of the promoter that can bind directly the GR (Waddell DS et al., 2008), whereas MAFbx expression has to be activated indirectly since its promoter does not have direct binding sites to the GR (Shimizu N et al., 2011). Another transcription factor is responsible of the upregulation of both atrogenes, KLF15 is upregulated after dexamethasone treatment, it has the ability to upregulate the expression of FoxO1 and FoxO3, thus, promoting indirectly the expression of MAFbx and MURF1 as well as both atrogenes have KLF15-binding sites, allowing its direct expression by KLF15 (Shimizu N et al., 2011). Introduction 53 Autophagy Lysosomal system Autophagy is a well conserved homeostatic mechanism used for degradation and recycling of bulk cytoplasm, long-lived proteins and organelles (Mizushima N and Komatsu M, 2011). There are three types of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy. Macroautophagy is believed to be the major and the most studied type of autophagy, it needs the formation of a membrane called autophagosome, isolates a small portion of cytoplasm, this autophagosome fuses with the lysosome forming the autolysosome where the invaginated materials are degraded (Mizushima N and Komatsu M, 2011; Mizushima N et al., 2011). Autophagosomes are generated in the proximity to the endoplasmic reticulum (ER) and their formation is carried out by multiple Atg proteins (Nakatogawa H et al., 2009), more specifically, in muscle the autophagic response in atrophy conditions can be initiated via mTOR1 which activation leads to the autophagosome formation by the ULK1 complex (ULK1, ATG13, and FIP200)(Di Rienzo M, et al., 2019). However, autophagy is considered a nonselective degradation pathway, autophagosomes are able to recognize proteins such as p62, which binds target proteins and delivered them as a cargo inside the autophagosome. p62 directly interacts with LC3 on the isolation membrane through the LC3-interacting region being incorporated into the autophagosome to be degraded (Johansen T and Lamark T, 2011; Weidberg H et al., 2011). An impaired autophagy leads to an accumulation of p62 and ubiquitinated aggregates (Komatsu M et al., 2007). It also exists organelle directed autophagy, these include autophagy directed to organelles, as mitophagy, directed to mitochondria, pexophagy to peroxysomes and to intracellular bacteria, called xenophagy (Klionsky DJ et al. 2007). Microautophagy is carried out in the lysosomes itself, the small components of the cytoplasm are invaginated for the lysosomal membrane and then degraded (Li WW Introduction 54 et al., 2012; Mizushima N and Komatsu M, 2011). In muscle is little known about microautophagy, is believe that it can participate in glycogen clearance (Raben N et al., 2008; Takikita S et al., 2010; Lim JA et al 2019) The chaperone-mediated autophagy does not involve membrane reorganization. The chaperone protein heat shock cognate 70 (Hsc70) recognizes specific cytosolic proteins which must contain KFERQ-like pentapeptide, then Lamp-2a acts as a receptor in the lysosome allowing this target proteins to be translocated to the lysosome to be degraded (Mizushima N and Komatsu M, 2011; Mizushima N et al., 2011). This type of autophagy is essential for the muscle homeostasis in basal conditions, targeting proteins as filamin C which undergoes unfolding and refolding during muscle contraction (Arndt V et al., 2010). During the last years this type of autophagy has raised interest due to its role in aging, neurodegenerative disorders and lysosomal storage diseases (Kon M, Cuervo AM, 2010). In terms of regulation, mTOR is the major metabolic sensor in muscle and coordinates the physiological processes depending on nutritional conditions (Neel BA, 2013). In presence of amino acids, mTOR is activated blocking autophagy via phosphorylation of the trimeric protein complex ULK1/FAK family kinase-interacting protein of 200ka (FIP200)/Atg13 (Neel Ba, 2013; Zachari M and Ganley G, 2017). Upon amino acid deprivation, mTOR located on lysosomal surface is no longer active leading to dephosphorylation of ULK1/FAK/Atg13 complex and concomitant autophagy induction (Zachari M and Ganley G, 2017). However, siRNA and inhibitor studies shown that inhibition of mTOR is not sufficient to alter the autophagic flux (Betzinger CF et al., 2008; Mammucari C et al., 2008; Sandri M, 2010). Moreover, AKT also plays an important role in autophagy regulation through FoxO3 phosphorylation. During catabolic conditions, AKT is not able to inhibit FoxO3 by phosphorylation, thus is translocated to the nucleus where it promotes the Introduction 55 expression of autophagy related genes such as LC3, Bnip3 and cathepsinL (Castets P and Rüegg MA, 2013; Cid-Diaz T et al., 2017; Neel BA, 2013). AMPK is the other well-known regulator of autophagy. Opposing to mTOR activity, during catabolic conditions AMPK can inactivate mTOR blocking the inhibition of ULK1 as well as it can activate ULK1 by phosphorilating a different residue of ULK1 activating the autophagy initiation complex. OBJECTIVES Material&Methods 64 FKHR (H-128) IP 1:50 Santa Cruz Sc-11350 FoxO1 WB 1:1000 Cell Signaling 2880 FoxO3a WB 1:1000 Cell Signaling 12829 FoxO4 WB 1:1000 Cell Signaling 9472 GAPDH WB 1:1000 Abcam ab9485 HDAC4 WB 1:1000 Cell Signaling 7628 LC3A/B WB 1:1000 Cell Signaling 12741 MAFbx WB 1:1000 Santa Cruz sc-166806 Murf1 WB 1:1000 Santa Cruz sc-32920 Myogenin WB 1:1000 Hibridoma Bank F5D Myosin heavy chain WB 1:1000 Hibridoma Bank MF20 Myosin heavy chain IHF 1:200 Hibridoma Bank MF20 p-S6 (S240/244) WB 1:2000 Cell Signaling 2215 p-S6K1(T389) WB 1:1000 Cell Signaling 9234 p4E-BP1 (Thr37/46) WB 1:1000 Cell Signaling 9459 P4E-BP1 (Thr70) WB 1:1000 Cell Signaling 9455 p62 WB 1:1000 Cell Signaling 5114S pAkt(S473) WB 1:1000 Cell Signaling 9275 pFoxO1 (Ser256) WB 1:1000 Cell Signaling 9461 pFoxO1 (Thr24)/FoxO3a (Thr32) WB 1:1000 Cell Signaling 9464 pFoxO1 (Thr24)/FoxO3a (Thr32)/FoxO4 (Thr28) WB 1:1000 Cell Signaling 2599 pFoxO3a (Ser253) WB 1:1000 Cell Signaling 13129 S6 WB 1:1000 Cell Signaling 2217 pHDAC4(S246)/HDAC5(S259)/ HDAC7(S155) WB 1:1000 Cell Signaling 3443 Material&Methods 65 Note: Relation of the primary antibodies used in the different analyses performed in this work. IF, immunofluorescence; WB, western blot. Table 2. Secondary Antibodies Secondary antibody Use Dilution Supplier Reference Goat Anti-Mouse IgG (H&L) Alexa Fluor 488 IF 1:1000 Invitrogen A11029 Goat Anti-Rabbit IgG Fc Alexa Fluor 488 IF 1:1000 Abcam ab150089 Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) WB 1:1000 0 Jackson ImmunoResearch 111-035-003 Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) WB 1:1000 0 Jackson ImmunoResearch 115-035-003 Peroxidase AffiniPure Donkey Anti-Goat IgG (H+L)1 WB 1:1000 0 Jackson ImmunoResearch 705-035-003 Note: Relation of the primary antibodies used in the different analyses performed in this work. IF, immunofluorescence; WB, western blot METHODS Cell culture and differentiation. Mouse C2C12 (ECACC, Whiltshire, UK) myoblasts were cultured as described by the supplier (ECACC, Whiltshire, UK). Briefly, C2C12 myoblasts were maintained in growth medium (GM) containing DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin. For routine differentiation, the cells were grown to ~80% confluence and GM was replaced with differentiation medium (DM) DMEM Material&Methods 66 supplemented with 2% horse serum (HS), 100 U/mL penicillin, and 100 U/mL streptomycin) for 7 days unless otherwise stated. Myogenic primary, C25 cells, and clonal line, KM155C25 Clone 48 (KM155C25 cells), were obtained from the platform for immortalization of human myoblasts of the Center for Myogenic Research in Myology in Paris (Paris, FR) who developed the isolation and immortalization from a biopsy obtained through MYOBANK, a partner in the EU network EuroBioBank (gracilis muscle, donor age 25 years). Primary myogenic cells isolated from biopsies were purified by magnetic activated cell sorting using anti-CD56 (a specific marker of myoblasts) beads (MACS, Miltenyl Biotech). Purity before and after cell sorting was determined by immunolabelling (anti-desmin and anti-mouse IgG1 AlexaFluor 488 antibodies) following the protocols previously described (Mamchaoui K, et al., 2011). Myogenic primary line, C25 cells, were cultured in GM containing Medium 199 (1:4, v/v; Lonza, Pontevedra, ES) supplemented with 20% FBS (v/v), 50 µg/mL gentamicin (Invitrogen, Thermo Fisher Scientific; Massachusetts, US), 25 µg/µL fetuin, 5 ng/mL hEGF, 0.5 ng/mL bFGF, 0.2 µg/mL Dexa (Sigma Chemical, Missouri, US) and 50 µg/mL gentamycin (Invitrogen, Thermo Fisher Scientific; Massachusetts, US) on matrigel-covered cell culture dishes (0.1 mg/mL, 1 mL per 19.5 cm2 dish area, 45 min, 37 °C), incubated at 37 °C, 5% CO2. C25 cells were differentiated in DM, Medium 199: DMEM (1:4, v/v) supplemented with 2% FBS and 1 µg/mL gentamicin on matrigel-covered 12 well multiplates for 7 days unless otherwise stated, incubated at 37 °C, 5% CO2. Stable immortalized cell line from C25, KM155C25 cells was carried out as previously described (Mamchaoui K, et al., 2011). In brief, primary C25 cells were co-transduced with two retroviral vectors expressing hTERT and CDK-4 cDNA (Mamchaoui K, et al., 2011). Co-transduced cells were selected by neomycin and puromycin and then purified using magnetic beads coupled to antibodies directed against the myogenic marker CD56. Following culture at clonal density, individual Material&Methods 67 myogenic clones with extended proliferative lifespans, as compared to the untransduced cells, were isolated from each population. Immortalized human myoblasts, KM155C25 Clone 48 (KM155C25 cells), maintain their capacity to differentiate both in vitro and in vivo after transplantation into the regenerating muscles of immunodeficient mice (Mamchaoui K, et al., 2011; Thorley M et al., 2016). KM155C25 cells were cultured in GM containing Medium 199:DMEM (1:4, v/v; Lonza, Pontevedra, SP) supplemented with 20% FBS (v/v), 50 µg/mL gentamicin (Invitrogen), 25 µg/µL fetuin, 5 ng/mL hEGF, 0.5 ng/mL bFGF, 0.2 µg/mL Dexa (Sigma Chemical, Missouri, US) and 50 µg/mL gentamycin (Invitrogen, Thermo Fisher Scientific; Massachusetts, US) in cell culture dishes incubated at 37 °C, 5% CO2. KM155C25 cells were differentiated in DM Medium 199: DMEM (1:4, v/v) supplemented with 1 µg/mL gentamicin in 12 well multiplates for 7 days unless otherwise stated, incubated at 37 °C, 5% CO2. Murine and human myotubes were treated with Dexa at concentrations from 0.05 to 100 µM for 24 hours in the presence or absence of obestatin from 5 to 100 nM, or with insulin 100 nM as a positive control of atrophy protection. Protein synthesis analysis. We used Click-iT™ Plus OPP Alexa Fluor™ 488 Protein Synthesis Assay Kit (Thermo Fisher Scientific; Massachusetts, US), according to instruction provided by manufacturer. In brief OPP reagent is a puromycin analog containing an alkyne moiety. When added to culture media, OPP is readily taken up by actively growing cells. OPP inhibits protein synthesis by disrupting peptide transfer on ribosomes causing premature chain termination during translation. Addition of the Alexa Fluor® picolyl azide leads to a chemoselective ligation between the picolyl azide dye and the alkyne OPP, allowing the modified proteins to be detected by imaged-based analysis. We treated C2C12 an KM155C25 cells with Dexa 1 µM, Dexa 1 µM + obestatin 10 nM or Dexa 1 µM+ Insulin 100 nM during Material&Methods 68 24 h. After this treatment we added 20 µM of Click-iT® OPP reagent for 30 min. Intact cells were washed once with PBS, fixed with 96% ethanol 15 min at room temperature, washed twice with PBS, permeabilized with 0.5% Triton® X-100 in PBS and incubate for 15 minutes at room temperature. Cells were washed twice with PBS and incubated with Click-iT® Plus OPP reaction cocktail for 30 min, washed twice with PBS incubated and with HCS NuclearMaskTM Blue Stain for 30 min more. Digital images of cell cultures were acquired with a Leica TCS-SP5 spectral confocal microscope (Leica Microsystems, Heidelberg, DE) and then fluorescence intensities were analyzed with Image J. Immunofluorescence. Myoblast cells were cultured on coverslips and differentiated into myotubes under DM for 7 days until a full mature differentiation was visible. Myotubes were then treated with Dexa for 24 h in the presence or absence of obestatin or insulin as indicated above. Intact cells were fixed with 96% ethanol for 15 min at room temperature, washed twice with ice cold PBS, permeabilized with PBST (PBS, 0,3% Triton X-100, 0,1M Glycine) 10 min at room temperature, washed three times with ice cold PBS for 5 min and blocked with 1% BSA in PBST for 30 min at room temperature. Then incubated with primary antibody diluted in 1% BSA in PBST over night at 4°C. After three washes with ice cold PBS for 5 min each, cells were incubated with the secondary antibody (FITC-conjugate goat anti-mouse antibody or FICT-conjugated goat anti-rabbit antibody) in 1% BSA in PBST (1:1000) for 1 h at room temperature, then washed three times in ice cold PBS for 5 min each in dark. Topro or DAPI was used to counterstain the cell nuclei (Invitrogen Thermo Fisher Scientific; Massachusetts, US). Digital images of cell cultures were acquired with a Leica TCS-SP5 spectral confocal microscope (Leica Microsystems, Heidelberg, DE) and Zeiss Axio Vert.A1 (Zeiss, DE). Myotube areas (MHC+ cells, ³ Material&Methods 69 3 nuclei) were quantified by measuring a total of >100 myotube areas from 5 random fields in 3 replicates using ImageJ64 analysis software. Small interfering RNA (siRNA) silencing of gene expression. Chemically synthesized double-stranded siRNA duplexes targeting FoxO4 was selected from ON-TARGETplus SMARTpool siRNA (Dharmacon, Colorado, US). Human FoxO4, AGUCAUGCCUGGAAGCUUU; GAGAAGCGACUGACACUUG; GGAAAUACCAGCUUCAGUC; CAACGAGGCCACCGGCAAA). Chemically synthesized double-stranded siRNA duplexes targeting IGF-1 was a pool of three target sequences (Santa Cruz): 1. CAGACUUUGUACUUCAGAAtt; UUCUGAAGUACAAAGUCUGtt. 2. GCAUGGGUGUUGUAUAGAUtt; AUCUAUACAACACCCAUGCtt. 3.GACUAGAGUUUCAGUUGAAtt; UUCAACUGAAACUCUAGUCtt Chemically synthesized double-stranded siRNA duplexes targeting b-arrestin 1 and b-arrestin 2 was selected from ON-TARGETplus SMARTpool siRNA (Dharmacon, Colorado, US) Human b-arrestin 1, UGGAUAAGGAGAUCUAUUA; AUGGAAAGCUCACCGUCUA; GAACUGCCCUUCACCCUAA; GAACGAGACGCCAGUAGAU; Human b-arrestin 2, CGAACAAGAUGACCAGGUA, CGGCGUAGACUUUGAGAUU, GGGCUUGUCCUUCCGCAAA, UAGAUCACCUGGACAAAGU. Chemically synthesized double-stranded siRNA duplexes targeting KLF15 was a pool of three target sequences (Santa Cruz): 1. CAGCAAGAUGUACACCAAAtt; UUUGGUGUACAUCUUGCUGtt. 2. CCAAGGCCAGAACUUUAGUtt; ACUAAAGUUCUGGCCUUGGtt. 3. CAUAGGUCCAUCCACAUAAtt; UUAUGUGGAUGGACCUAUGtt. An ON-TARGETplus nontargeting siRNA (Dharmacon, Colorado, US) was used as a control for all siRNA experiments. Human KM155C25 myotubes were transfected Material&Methods 70 at day-6 post-differentiation with Lipofectamine 2000 (Invitrogen Thermo Fisher Scientific; Massachusetts, US), following manufacturer’s instructions with 50nM of SiRNA targeting FoxO4, IGF-1, b-arrestin 1 or b-arrestin 2 for 24 h, after block the protein of interest cells were stimulated with Dexa, obestatin or a combination of both as was indicated above. SDS-PAGE and western blot analysis. The cell samples were directly lysed in icecold RIPA buffer [50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP40, 0.25% (w/v) Na-deoxycholate, protease inhibitor cocktail, phosphatase inhibitor cocktail (Sigma Chemical, Missouri, US)]. The lysates were clarified by centrifugation (14,000xg for 15 min at 4°C) and the protein concentration was quantified using the QuantiProTM BCA assay kit (Sigma Chemical, Missouri, US). For immunoblotting, equal amounts of protein were fractionated by SDS-PAGE and transferred onto nitrocellulose membranes. Immunoreactive bands were detected by enhanced chemiluminescence (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific; Massachusetts, US). Co-immunoprecipitation assays. Following treatment [DM, DM+Dexa (1 µM, 24 h), DM+Dexa (1 µM, 24 h)+obestatin (10 nM, 24 h)], KM155C25 myotubes cells were washed twice with ice-cold PBS and lysed in co-immunoprecipitation lysis buffer (50 mM Tris, 100 mM NaCl, 5 mM EDTA, 50 mM NaF, 1% Triton X-100, 10 mM glycerol phosphate, 200 µM, sodium orthovanadate, 2.5 mM sodium pyrophosphate plus protease and phosphatase inhibitors). FoxO1 was immunoprecipitated using rabbit anti-FoxO1 antibody coupled to protein ADynabeads according to instruction provided by manufacturer (Thermo Fisher Scientific; Massachusetts, US). The washed immunoprecipitates were subjected to western blot analysis using the indicated antibodies. Material&Methods 71 Data analysis. All values are presented as mean ± standard error of the mean (SEM). Student t test were performed to assess the statistical significance of 2-way analysis. For multiple comparisons, ANOVA was employed. P <0.05 was considered as statistically significant (*). RESULTS Results 80 Figure 1.3. Obestatin restores myotube area in Dexa atrophied myotubes. (a) Immunofluorescence detection of MHC and Topro on differentiated C2C12 mouse myotubes under DM, DM+Dexa 1 µM, DM+Dexa 1 µM+obestatin 5nM, DM+Dexa 1 µM+obestatin 25nM, DM+Dexa 1 µM+obestatin 100nM DM+Dexa 1 µM+insulin 100nM during 24h. (b) Evaluation of myotube area and distribution. Data were expressed as mean±SEM (n=3; P< 0.05 versus *DM or # DM + Dexa1µM values. Student t-test) Results 81 Furthermore, when we analyzed the muscle-specific transcription factor myogenin. Myogenin was upregulated following Dexa treatment as well as after obestatin and insulin co-treatments by ~24% compared with control cells (Fig 1.4), this result was consistent with the dual role of myogenin in skeletal muscle, not only promoting the expression of the MuRF1 and MAFbx under catabolic conditions, but also counteracting the atrophy phenotype favoring muscle growth (Moresi, et al., 2010). We studied autophagy as one of the mayor degradative pathways in catabolic conditions. The levels of microtubule-associated protein 1 light chain 3 isoform I (LC3-I) did not change in Dexa treated myotubes, whereas the lipidated form (LC3II), a reliable marker of autophagosome formation, was significantly elevated (Fig. 1.4c), evidenced by an increase in the LC3II/LC3I ratio observed in Dexa treated C2C12 myotubes. This increase was reversed by 18-34% upon obestatin treatment consistent with decreased LC3-II levels (Fig. 1.4). Furthermore, significant accumulation of p62 protein was seen in obestatin-stimulated cells as compared to Dexa treated C2C12 myotubes (144-169%) indicating a block in autophagy (Fig. 1.4c). By contrast, obestatin stimulation was associated with a reduction in the expression of the lysosomal enzyme Cathepsin L by 31-29% as compared to Dexa treated myotubes, further supporting, at least, a partial inactivation of autophagy. Insulin treatment decreased LC3-II/LC3-I ratio and increased p62 accumulation, although it failed to modify Cathepsin L levels (Fig. 1.4c). Results 82 Figure 1.4. Obestatin counteract the atrophy pathways activated during Dexa treatment. Immunoblot analysis of MuRF1, MAFbx, myogenin, MHC, LC3I/II, p62 and cathepsinL on differentiated C2C12 mouse myotubes under DM, DM+Dexa1µM, DM+Dexa1µM+obestatin 5nM and DM+Dexa1µM+insulin 100nM during 24h. Data were expressed as mean±SEM obtained from intensity scans (n=3; P< 0.05 versus *DM or # DM + Dexa1µM values. Student t-test) Results 83 The obestatin/GPR39 system restored protein synthesis in dexamethasone treated C2C12 mouse myotubes Given that we saw a restored level of MHC we decided to study whether or not protein synthesis pathway could be altered after Dexa treatment and if the obestatin/GPR39, as an anabolic signal, could be restoring this pathway. By immunoblot analysis we found a 1.8to 2.9-fold increase in S6K1 phosphorylation at S371 [pS6K1(S371) related to Dexa treated cells; Fig. 1.5a] and consequently a 2.5to 2.6-fold increase in phosphorylation of its downstream target, the ribosomal protein S6 at S240/244 [pS6(S240/244); Fig. 1.4]. In insulin-treated cells, pS6K1(S371) and pS6(S240/244) were also significantly increased 1.9and 2.5-fold, respectively (Fig. 1.5a). Furthermore, obestatin markedly promoted 4E-BP1 hyperphosphorylation at T70 [p4E-BP1(T70)], especially concerning the g form (2.0to 2.9-fold), altering the basal phosphorylation of β and g forms at T37/T46 [p4E-BP1(T37/46); 1.3to 1.6fold and 2.0to 2.9-fold, respectively] in Dexa treated cells (Fig. 1.5a). The p4EBP1(T70) g form was also increased by insulin (1.9-fold related to Dexa treated cells). Insulin did not appreciably alter basal T37/T46 phosphorylation (β or g forms). To further assess the protein synthesis status, we performed a protein synthesis assay in presence or not of cycloheximide, a protein synthesis inhibitor (Bonifacio A et al., 2017). As shown in Fig 1.5b, a significant increase in protein synthesis was observed after obestatin treatment related to Dexa-treated cells (Fig 1.5b). Results 84 Figure 1.5. Obestatin restores protein synthesis after Dexa treatment. (a) Immunoblot analysis of S6K1, S6 and 4EBP1 on differentiated C2C12 mouse myotubes under DM, DM+Dexa1µM, DM+Dexa1µM+obestatin5nM and DM+Dexa1µM+insulin 100nM during 24h. (b) Evaluation of protein synthesis on differentiated C2C12 mouse myotubes under DM, DM+Dexa1µM, DM+Dexa1µM+obestatin5nM and DM+Dexa1µM+insulin 100nM during 24h using Click-iT™ Plus OPP Alexa Fluor™ 488 Protein Synthesis. Data were expressed as mean±SEM obtained from intensity scans (n=3; P< 0.05 versus *DM or # DM + Dexa1µM values. Student t-test) Results 85 The obestatin/GPR39 system regulates the interplay between AKT and FoxO signaling in dexamethasone-induced atrophy in C2C12 mouse myotubes The down-regulation of ubiquitin E3-ligases and autophagy and up-regulation of protein synthesis seen after obestatin treatment suggested the implication obestatin/AKT-mTOR signaling pathway. Indeed, the regulatory residue S473 [pAKT(S473)] of AKT was analyzed showing an increase of 2.5to 3.8-fold in obestatin-stimulated cells as compared to Dexa treated C2C12 myotubes. This upregulation in AKT activity was concomitant with a phosphorylation at S2448 residue [pmTOR(S2448)] of mTOR, 2.6to 2.8-fold increase in obestatin treated cells related to Dexa treated cells (Fig 1.6). Figure 1.6. Obestatin restores anabolic signaling after Dexa treatment. Immunoblot analysis of AKT and mTOR on differentiated C2C12 mouse myotubes under DM, DM+Dexa1µM, DM+Dexa1µM+obestatin5nM and DM+Dexa1µM+insulin 100nM during 24h. Data were expressed as mean±SEM obtained from intensity scans (n=3; P< 0.05 versus *DM or # DM + Dexa1µM values. Student t-test) Results 86 One mechanism by which AKT reduces the expression of the ubiquitin E3-ligases is the phosphorylation and subsequent nuclear exclusion of FoxO family members. Since mTOR is a suppressor of the autophagy-lysosome system, while FoxO is an inducer of autophagy-dependent degradation, we decided to study the FoxO family status under the obestatin/GPR39 system activation. As shown in Figure 1.7 obestatin markedly increased FoxO4 phosphorylation at T28 [pFoxO4(T28)] by 1.9to 3.4-fold related to Dexa treated C2C12 myotubes but did not change FoxO3a and FoxO1 phosphorylation (Fig. 1.7). In contrast, insulin treatment did appreciably increase phosphorylation of FoxO1 at T24 [pFoxO1(T24) 3.8-fold related to Dexa treated C2C12 myotubes] but had no effect at S256 [pFoxO1(S256)] (Fig. 1.7). Furthermore, insulin increased FoxO3a phosphorylation at T32 and S253 [pFoxO3a(T32) (S253) 2.2-and 3.2-fold, respectively] with minor effect on FoxO4 phosphorylation (1.4-fold; Fig. 1.7). Taken together these results, our data support a model where the interplay between AKT, mTOR and FoxO4 regulates two of the main proteolytic systems, the ubiquitinproteasome and the autophagy-lysosome systems, and the signaling associated with protein translation in response to the obestatin/GPR39 system. Effect of dexamethasone treatment in KM155C25 human myotubes In order to validate whether the obestatin/GPR39 signaling is conserved between human and mouse, we used an in vitro cell culture model of human skeletal muscle: the human muscle stem cell line immortalized from a control individual, KM155C25 cells (Mamchaoui K, et al., 2011; Thorley M et al., 2016). Treatment of this cell line with Dexa resulted in dose-dependent increases in the MuRF1 and MAFbx protein content with a maximal effect at 1 µM of Dexa [5.4–5.9-fold, respectively (Fig1.8)]. Results 87 Figure 1.7. Obestatin controls through FOXO4 inhibition. (a) Inmunoblot analysis of FOXO1, FOXO3a and FOXO4 on differentiated C2C12 mouse myotubes under DM, DM+Dexa1µM, DM+Dexa1µM+obestatin5nM and DM+Dexa1µM+insulin 100nM during 24h. Data were expressed as mean±SEM obtained from intensity scans (n=3; P< 0.05 versus *DM or # DM + Dexa1µM values. Student t-test) Up-regulation of ubiquitin E3-ligases was concomitant with a decrease in AKT activity and MHC expression (Fig1.8). Intriguingly, myogenin expression was down- Results 88 regulated following Dexa treatment, contrary to what was observed in C2C12 myotubes, possibly related to the fact that myogenin is up-regulated during the initial phases of differentiation, and the kinetics of differentiation may differ between C2C12 and KM155C25 (Fig1.8). Figure 1.8. Dose response of Dexa activates atrophy program in humans. Immunoblot analysis of MuRF1, MAFbx, MHC, myogenin and AKT on differentiated KM155C25 human myotubes under Dexa treatment (0,05-100 µM) during 24h. Data were expressed as mean±SEM obtained from intensity scans (n=3; P< 0.05 versus *DM values. Student t-test). Results 89 The obestatin/GPR39 system attenuates atrophy-related gene expression by targeting FoxO4 in dexamethasone-induced atrophy in human KM155C25 Having evaluated the effect of Dexa under basal conditions, the action of obestatin/GPR39 signaling was determined on Dexa!related myotubes at 1 µM concentration, based on the maximal effect on the MuRF1 and MAFbx expression at that dose. Consistent with the up-regulation of the studied atrogenes after Dexa treatment, myotubes showed a decrease in area of 63 ± 3% in response to Dexa (Fig1.9). These changes were reversed by obestatin (10 nM, 24 h) as revealed by a marked increase in myotube area (~100 ± 12% over Dexa-treated myotubes, Fig1.9). Furthermore, the myotube areas were ~40% larger in the obestatin treated cells than insulin treated cells (60 ± 8% increase over Dexa-treated myotubes; Fig1.9), used as positive control. Figure 1.9. Obestatin restores myotube area in Dexa atrophied myotubes. Immunofluorescence detection of MHC and DAPI on differentiated KM155C25 human myotubes under DM, DM+Dexa 1 µM, DM+Dexa 1 µM+obestatin 10nM, DM+Dexa 1 µM+insulin 100nM during 24h. Data were expressed as mean±SEM (n=3; P< 0.05 versus *DM or # DM + Dexa1µM values. Student t-test) Results 96 As KM155C25 cells are a no commercial immortalized cell line, we decided to investigate whether the pathways activated after Dexa treatment and the signaling of obestatin/GPR39 system was also maintained on the primary human cells. As shown in Fig1.15, in a immunofluorescence assay for MHC, Dexa treatment in C25 myotubes was able to reproduce muscle atrophy phenotype by reducing the myotube area (62,7±2% referred to control cells, (Fig1.15), whether obestatin treatment restored the myotube area by 185,5±2% over Dexa treated cells. In addition, by a immunoblot analysis we confirmed that obestatin treatment not only reversed Dexa-induced myotube atrophy decreasing MuRF1 and MAFbx expression (Fig1.16) but also increased the amount of phosphorylated FoxO4 at T28 which correlated with decreased in the atrogenes expression. Moreover, FoxO3 and FoxO1 remain inhibited and unaltered respectively after Dexa or obestatin treatments (Fig1.16). Figure 1.15. Obestatin counteract Dexa atrophy in C25 primary cells. Immunofluorescence detection of MHC and DAPI on differentiated C25 primary human myotubes under DM, DM+Dexa 1 µM, DM+Dexa 1 µM+obestatin 10nM during 24h. Data were expressed as mean±SEM (n=3; P< 0.05 versus *DM or # DM + Dexa1µM values. Student t-test) Results 97 Figure 1.16. Obestatin counteract the atrophy pathways activated during Dexa treatment in C25 primary cells. Immunoblot analysis of MURF1, MAFbx, myogenin and MHC on KM155C25 human myotubes under DM, DM+Dexa 1 µM, DM+Dexa 1 µM+obestatin 10nM, DM+Dexa 1 µM+insulin 100nM during 24h. Data were expressed as mean±SEM obtained from intensity scans (n=3; P< 0.05 versus *DM or # DM + Dexa1µM values. Student t-test) Results 98 We further tested by siRNA experiments the role of FoxO4 governing the activation of muscle atrophy genes. siRNA targeting FoxO4 (81 ± 4% reduction relative to sicontrol) significantly decreased the MuRF1 and MAFbx expression in Dexa stimulated cells by 55 ± 1 and 61 ± 1% relative to si-control, respectively (Fig1.17). Under these conditions, si-FoxO4 experiments increased the MuRF1 and MAFbx expression in obestatin!stimulated cells by 88 ± 12 and 82 ± 6% relative to sicontrol, respectively. Furthermore, the FoxO4 knockdown showed deregulations of autophagy!related proteins. The acute FoxO4 deficiency on Dexa treated cells increased the expression levels of p62 by 69 ± 3% relative to si-control (Fig1.17). In contrast, the Cathepsin L levels and the LC3II/LC3I ratio were decreased by 52 ± 6 and 57 ± 5%, respectively. Silencing of FoxO4 increased the p62 and Cathepsin L levels as well as the LC3II/LC3I ratio in obestatin!treated myotubes by 94 ± 6, 564 ± 15 and 86 ± 3% relative to si-control, respectively (Fig1.17). These results demonstrate not only the role of FoxO4 in triggering the atrophy program, but also that its activity is tightly controlled by the obestatin/GPR39 system. Results 99 Figure 1.17. Obestatin counteracts the atrophy effect of Dexa treatment through FoxO4. Immunoblot analysis for MAFbx, MuRF1, p62, cathepsinL and LC3 in KM155C25 human myotubes under DM, DM+Dexa 1 µM, DM+Dexa 1 µM+obestatin 10nM during 24h. Data were expressed as mean±SEM obtained from intensity scans (n=3; *, #, € P < 0.05 vs. control. Student t-test) Results 100 We further investigated the role of obestatin on FoxO family regulation by KLF15. siRNA targeting KLF15 (44±6% reduction relative to si-control) significantly decreased the expression of FoxO3, FoxO1 and FoxO4 in Dexa-treated cells related to si-control (34±1, 31±2, and 26±1 %, respectively). MAFbx and MuRF1 expression was decreased by 33±3 and 55±2 %, respectively (Fig 1.18). Under obestatin treatment, FoxO4 was clearly downregulated related to Dexa treatment in both sicontrol and si-KLF15 treated cells (Fig1.18). By contrast, neither FoxO3 nor FoxO1 expression showed differences related to Dexa treatment neither si-control nor siKLF15 treated cells (Fig1.18). These results suggested the involvement of alternative signaling nodes in the regulation of FoxO4 expression. Indeed, MAFbx and MURF1 expression was affected to a greater extent by obestatin (Fig1.18). Figure 1.18. Obestatin regulates FoxO4 independent of KLF15. Immunoblot analysis for MAFbx, MURF1, FoxO1, FoxO3 and FoxO4 in KM155C25 human myotubes under DM, DM+Dexa 1 µM, DM+Dexa 1 µM+obestatin 10nM during 24h. Data were expressed as mean±SEM obtained from intensity scans (n=3; P< 0.05 versus *si control values. Student t-test). Results 101 Post-translational regulation of FoxO1 by the obestatin/GPR39 system in dexamethasone induced atrophy in human KM155C25 myotubes While the obestatin/GPR39 signaling clearly controlled the FoxO4 activity by phosphorylation, altering its subcellular location, its role in regulating FoxO1 was not so clear. Nuclear/cytoplasmic shuttling of FoxO1 suggested the implication of posttranslational modifications beyond protein phosphorylation, i.e. acetylation and ubiquitination. By immunoblot analysis we saw that in response to Dexa, FoxO1 was acetylated (Ac-FoxO1), and the Ac-FoxO1 level was significantly decreased by 59– 76% in response to obestatin (Fig 1.19). The acetylation status of FoxO transcription factors is generally balanced by histone acetylases and histone deacetylase (HDAC), including the NAD+-dependent sirtuins. As shown in Figure FoxO1 acetylation was accompanied by Dexa induced phosphorylation of HDAC4 [pHDAC4(S246), 1.7-fold], but this phosphorylation was clearly decreased 29–61% in response to obestatin. In addition, Dexa treatment increased Sirt1 expression by 1.7-fold, and this expression was decreased in obestatin-treated cells (36–60% inhibition related to Dexa treated cells; Fig 1.19). Sirt2 was also increased 1.6-fold in response to Dexa and this expression was decreased in obestatin-treated cells (36–42% inhibition related to Dexa treated cells; Fig 1.19). No significant modification of Sirt3 expression was observed in any condition (Fig 1.19). Depending on the cell/tissue type and upstream stimulus, multiple kinase families including the Ca+2/CaM-dependent protein kinases (CAMKs), protein kinase D (PKDs) and AMPK can phosphorylate and regulate the localization of the class IIa HDACs (Mihaylova & Shaw, 2013) The activation of PKD [pPKD/PKCμ(S916)] was up-regulated 1.7-fold in the presence of Dexa, but this action was reduced in response to obestatin (35–50% Results 102 inhibition related to Dexa treated cells; Fig 1.19). Dexa treatment increased 2.0-fold the activation of CAMKII, estimated by the phosphorylation of CAMKII at T286 [pCAMKII(T286)], being decreased by obestatin stimulation (33–45% inhibition related to Dexa treated cells; Fig 1.19). Finally, activation of AMPK, estimated by the phosphorylation of AMPK at T172 [pAMPK(T172)], was increased by 2.4-fold in response to Dexa but decreased by 34–73% under obestatin stimulation (Fig1.19). Therefore, HDAC4 activity was regulated by interplay among PKD, CAMKII and AMPK, which suggests some mechanisms for the modulation of FoxO1 acetylation. Next, we looked for evidence of potential FoxO1-interacting proteins in control, Dexa (1 µM) and Dexa (1 µM)/obestatin (10 nM) treated cells. Proteins of myotube cells were immunoprecipitated with anti-FoxO1 and probed with anti-Ac-FoxO1, antiSirt1, anti-Sirt2, anti-Sirt3, anti-HDAC4 antibodies or ant-Ubiquitin. Figure shows that Sirt1 interacted with FoxO1, but this interaction was clearly decreased by 59 ± 1% in response to Dexa. In addition, the interaction between Sirt1 and FoxO1 was clearly increased by obestatin stimulation by 105 ± 2% as compared with Dexa treated cells. In contrast, a significant decrease in Sirt2 was detected in Dexa treated or obestatin treated cells (Fig 1.20), excluding the direct involvement of this NAD+-dependent histone deacetylase in regulating FoxO1 acetylation through obestatin. No detectable changes in Sirt3 were observed after the treatments. However, we found that HDAC4 interacted with FoxO1 and this interaction was decreased by 20 ± 3% in response to Dexa but increased by 57 ± 10% in obestatin treated cells. We also investigated whether Dexa or obestatin signaling could modify the ubiquitination of FoxO1. Dexa treatment decreased by 54 ± 2% the ubiquitination of FoxO1 related to control levels (Fig 1.20). This effect was partially counteracted by obestatin resulting in 47 ± 3% increase in the level of ubiquitination when compared with Dexa treated cells. Thus, obestatin/GPR39 signaling controls Results 103 FoxO1 activity by altering an intricate combination of post-translational modifications, such as phosphorylation, acetylation and ubiquitination. Figure 1.19. Obestatin counteracts the atrophy effect of Dexa treatment through FoxO1 posttranslational modifications. Immunoblot analysis of Ac-FoxO1, CAMKII, HDAC, PKD, AMPK, Sirt1, Sirt2 and Sirt3 in KM155C25 human myotubes under DM, DM+Dexa 1 µM, DM+Dexa 1 µM+obestatin 10nM during 24h. Data were expressed as mean±SEM obtained from intensity scans (n=3; P< 0.05 versus *DM or # DM + Dexa1µM values. Student t-test). Results 104 Figure 1.20. Obestatin promotes FoxO1 acetylation and ubiquitination during Dexa atrophy. Immunoblot analysis of immunoprecipitation assay of Ac-FoxO1, Sirt1, Sirt2, Sirt3 and Ubiquitin in KM155C25 human myotubes under DM, DM+Dexa 1 µM, DM+Dexa 1 µM+obestatin 10nM during 24h. Data were expressed as mean±SEM obtained from intensity scans (n=3; P< 0.05 versus *DM or # DM + Dexa1µM values. Student t-test). Obestatin/GPR39 system induce negative regulation of FoxO transcription factors through b -arrestin signal complex and it is independent of IGF signaling Having shown that obestatin induced negative regulation of FoxO transcription factors, we investigated the involvement of the b-arrestin signal complex (SantosZas I, et al., 2016) The effect of down-regulation of b-arrestin 1 and b-arrestin 2 by specific siRNA was evaluated in KM155C25 myotubes. The siRNAs decreased b-arrestin 1 and barrestin 2 expressions by 58 ± 3 and 61 ± 4%, respectively (Fig 1.21). In these conditions, obestatin stimulated phosphorylation of AKT in S437, FoxO3a in T32, S318 and S321, FoxO4 in T32 and FoxO1 S256 but not in T24. This effect were reduced by depletion of b-arrestin 1 or 2 [pAKT(S473): 66±1 or 72±2%; pFoxO3a(T32): 68±1 or 67±3%; pFoxO4(T28): 78±2 or 67±2%; pFoxO1(T256): 47±2 Results 105 or 53 ±3%; pFoxO3a(S318/321): 66±2% or 63±3%, respectively Fig1.21]. This finding provides a functional activity for b-arrestin dependent signalplex being specific signaling arm to activate AKT/FoxO signaling in myotubes. Figure 1.21. The obestatin/GPR39 system controls the atrophy program through b-arrestin signaling. Immunoblot analysis of b-arrestin 1, b-arrestin 2, AKT, FoxO1, FoxO3 and FoxO4 in KM155C25 human myotubes under DM, DM+Dexa 1 µM, DM+Dexa 1 µM+obestatin 10nM during 24h. Data were expressed as mean±SEM obtained from intensity scans (n=3; P< 0.05 versus *DM or # DM + Dexa1µM values. Student t-test). Discussion 112 sites for the 14-3-3 chaperone protein leading to nuclear export and inactivation of HDACs (McKinsey TA et al., 2000; McKinsey TA et al., 2002). The increase HDAC4 activity occurred together with significant changes in the activation of PKD/PKCµ and CAMKII, enzymes known to regulate class II HDAC activation via phosphorylation (Backs J et al., 2006; Vega RB et al., 2006). This is further supported by results of immunoprecipitation assays that demonstrate that HDAC4 interacts with FoxO1, and that this interaction is clearly decreased in response to Dexa. We also observed an increased interaction between FoxO1 and Sirt1, and not Sirt2, under obestatin stimulation. Taking into account the role of acetylation on FoxO1 activity on autophagy, (Brunet A et al., 2004; Zhao Y et al., 2010)FoxO1 could function in eliciting autophagy in response to Dexa by directly binding to the promoter region of autophagic genes (Qiang L et al., 2010; Liu J et al., 2015) or by specific interaction with Atg7, an E1-following protein, to influence the autophagic process (Zhao Y et al., 2010). On the other hand, the lack of AKT specific phosphorylation of FoxO1 at S256 prevents its degradation (Huang H et al., 2005). Thus, we propose that the obestatin/GPR39 signaling has exerted its specific function by reducing FoxO1 activity by deacetylation and phosphorylation. Deacetylation of FoxO1 occurs as a result of its association to Sirt1 and HDAC4, and the deacetylated FoxO1 fails to induce the autophagic process. Obestatin related AKT signaling promotes the ubiquitin dependent degradation via FoxO1 phosphorylation, thereby inhibiting its transcriptional function. This is further supported by our results showing that obestatin can increase the ubiquitination status of FoxO1, and thus accelerate its degradation. Third, the Dexa-induced expression pattern of myogenin is different between mouse and human myotubes. Myogenin plays a dual role as both a regulator of muscle development and an inducer of neurogenic atrophy by directly activating the expression of MuRF1 and MAFbx in mice (Moressi V et al., 2010; Tang H and Goldman D, 2006). These Discussion 113 contrasting activities reflect differential modulation by signaling pathways that enable myogenin to regulate distinct sets of target genes (Tang H and Goldman D, 2006). Although myogenin was upregulated in C2C12 myotubes following Dexa treatment, at no time, this transcription factor did show an increase in human myotubes. This might be ascribed to differences in kinetics of myogenin expression between cellular models. The precise mechanisms underlying this phenomenon are unclear; however, the fact that MuRF1 and MAFbx were downregulated under obestatin treatment in the absence of myogenin upregulation appears to exclude its implication as inducer of the E3 ubiquitin ligases in glucocorticoid-induced atrophy. In this regard, myogenin is not induced in response to other forms of atrophy such as cancer, fasting or diabetes (Lecker SH et al., 2004; Sacheck JM et al., 2007). Together, these findings highlight the existence of fundamental differences between the regulatory circuitry in human and mouse muscle wasting and their modulation. Further experimentation will be required to fully elucidate the functional consequences of these differences. The obestatin/GPR39 system arises as one of the autocrine systems for coordinating muscle growth, enhancing muscle repair and increasing muscle mass through regulatory role on proliferation, differentiation and hypertrophy of muscle cells (Gurriaran-Rodriguéz U et al., 2012; Santos-Zas I et al., 2016). If one adds to this a role in regulating muscle atrophy through regulation of ubiquitin E3-ligases expression and autophagy, this system displays clear functional similarities with the IGF and/or insulin signaling, but acts using distinct receptors and signaling pathways. The major has been the identification of key anti-atrophic signaling nodes in humans, in particular FoxO4 and FoxO1, in response to obestatin. It is worth underlining that, siRNA targeting FoxO4 suppress the ability of obestatin to regulate E3 ubiquitin ligases and autophagy during glucocorticoid treatment. Furthermore, FoxO1 is regulated by post-translational modifications, for example Discussion 114 acetylation/deacetylation and ubiquitination. Interestingly, the obestatin action was not due to an effect on the FoxO3, the most critical factor for the atrophy program (Mammucari C et al., 2007a; Milan G et al., 2015; Zhao J et al., 2007). Indeed, we provide functional evidence for a FoxO3 phosphorylation switch that explains how glucocorticoids, per se, regulate transcriptional activation of the gene but subsequently inactivate the corresponding protein by site-specific phosphorylation and nuclear export. Therefore, FoxO4 and FoxO1 are both required for the optimal regulation of the proteostasis in response to the obestatin/GPR39 system, at least during glucocorticoid-induced atrophy. In skeletal muscle, GR activates a transcription network driven by KLF15 that induces the expression of atrogenes such as MAFbx and Murf1 as well as regulate the expression of FoxOs (Shimizu N et al., 2011). In this case in particular, we demonstrated the implication of KLF15 in the expression of FoxOs and atrogenes in human myoblasts. The functional cooperativity of GR, FoxOs, and KLF15 in the expression of the atrogenes denote the molecular basis for the involvement of GR in muscle atrophy. Intriguingly, GR-mediated transcription through KLF15 of FoxO3 and FoxO1 was not repressed by obestatin but did so for FoxO4. This support that obestatin signaling regulates FoxO3 and FoxO1 activities via post-transcriptional modification. However, obestatin modulates FoxO4 activity by both transcriptional and post-transcriptional modification. Although it is well stablished the functional cooperativity of GR and KLF15 in the expression of FoxOs, obestatin appears to modulate FoxO4 expression independently to KLF15 axis. Indeed, cotreatment of Dexa and obestatin downregulated to a greater extent the FoxO4 expression under siRNA against KLF15 conditions related to Dexa treatment. Since this action represses to a further extent the expression of atrogenes, it would be of particular interest to identify the molecular mechanisms involved for the development of Discussion 115 treatments for glucocorticoid-induced and wasting disorder-related skeletal muscle atrophy. Further studies are clearly needed to clarify this issue. In summary, these results demonstrate that the activation of obestatin/GPR39 system impaired proteolysis via specific inactivation of FoxO4 and FoxO1 and efficiently counteracts the catabolic processes provoked by glucocorticoids. Notably, AKT/mTOR activation combined with PKD/CAMKII/AMPK inactivation by the obestatin signaling is required to inhibit FoxO dependent atrogenes and autophagy in human muscle cells. This crosstalk between glucocorticoid receptor and the obestatin/GPR39 signaling is a coordinated interaction between an anabolic signal and the catabolic machinery. Modulation of the activity of this system may represent a new strategy to ameliorate the debilitating effects of the muscle atrophic response to glucocorticoids. CONCLUSIONS Conclusions 119 1. The obestatin/GPR39 system regulates protein synthesis, ubiquitinproteasome and autophagy systems through a coordinated regulation involving Akt-, PKD/PKCµ-, CAMKII-, AMPKand p38-dependent mechanisms with different sets of effector proteins that eventually affect FoxO transcription factors. 2. Some key regulators in glucocorticoid induced muscle atrophy process differ between human and mouse. Although the final effect is the same, in order to design pharmacological treatment, the targets must be assessed. 3. A specific pattern of FoxO post-translational modifications, including FoxO4 phosphorylation and FoxO1 deacetylation is critical in the regulation of autophagy and the ubiquitin-proteasome system. 4. These results demonstrate that the obestatin/GPR39 signaling pathways not only promote myogenesis but is able to counteract deregulations in the proteostasis, e.g. those associated to glucocorticoid-induced myotube atrophy, and to restore efficient basal homeostasis. RESUMEN Resumen 128 el tratamiento con glucocorticoides. 2) Estudiar la relación entre la degradación y síntesis proteica y mostrar evidencias de que el sistema obestatina/GPR39 actua a través del eje AKT/FOXO controlando los sistemas ubiquitin-proteosoma y autofagia-lisosomal. 3) Dilucidar las posibles diferencias en la regulación de las vías de señalización catabólicas entre humanos y ratón. Para el desarrollo de este trabajo empleamos dos modelos in vitro de células musculares. La línea celular de mioblastos de ratón C2C12, la línea celular de mioblastos humanos KM155C25 y el cultivo primario de mioblastos humanos C25. Sometimos a los mioblastos a un proceso de diferenciación durante 7 días, una vez obtuvimos miotubos maduros, procedimos al tratamiento de los mismos con dexametasona o con dexametasona más obestatina, con la finalidad de dilucidar si la activación del sistema obestatina/GPR39 podría contrarrestar el efecto catabólico de los glucocorticoides en ambos modelos y establecer las posibles diferencias en cuanto a regulación de estos. Los resultados de este trabajo demostraron que, en miotubos de ratón C2C12 tanto la regulación de la maquinaria proteolítica como síntesis proteica, era llevada a cabo por la activación de la vía de señalización de AKT/mTOR a través del sistema obestatina/GPR39. Pese a que la activación de AKT es capaz de regular la familia de factores de transcripción FOXO, en este caso bajo el tratamiento con obestatina, no se observó una inhibición de FOXO3 ni FOXO1 por fosforilación, al contrario que FOXO4, sugiriendo que la obestatina regula de manera específica esta isoforma. De esta manera, bajo el tratamiento con obestatina se produjo una inhibición del sistema ubiquitin-proteosoma, tal y como pudimos determinar por la inhibición de las ubiquitin-ligasas MAFbx y MuRF1, a la par que el flujo autofágico se vio frenado tal y como demostró la redución en la forma lipidada de LC3, la disminución en la expresión de catepsina L y la acumulación de p62. En cuanto a la síntesis proteica la activación de la vía mTOR fue demostrada a través de la activación de sus dianas Resumen 129 S6K1, S6 y 4EBP1, de manera concomitante observamos un efecto antiatrófico tanto a nivel fenotípico, con una recuperación del área de los miotubos tratados con obestatina, a la vez que observamos un aumento en el contenido proteico de los mismos con una recuperación de la expresión de MHC. Estos resultados demostraron que la obestatina controla tanto la síntesis como la degradación proteica, regulando de manera especifica tanto el anabolismo como la actividad proteolítica en miotubos de ratón. Pudimos comprobar que, de la misma manera, la obestatina, fue capaz de revertir el fenotipo atrófico en miotubos humanos. Tanto la actividad proteasomal, analizada a través de la expresión de las ubiquitin-ligasas MAFbx y MuRF1, como el flujo autofágico, a través del análisis de LC3II, catepsina L y p62, se vieron reducidos bajo el tratamiento con obestatina, a la par que era capaz de restaurar la síntesis de MHC y recuperar el área de los miotubos. Sin embargo, es sabido que, entre humanos y ratón, ciertos genes clave y vías de señalización pueden diferir sustancialmente en el proceso miogénico. Nuestros resultados demostraron que el modelo celular de ratón C2C12, reflejaba únicamente de manera parcial los mecanismos involucrados en la señalización en células humanas. En primer lugar, observamos diferencias en la regulación de los factores de transcripción FOXO. FOXO3 fue activado de manera transcripcional tras el tratamiento con dexametasona a la vez que se vio inhibido por fosforilación en el residuo T32 y S318/321 bajo el mismo tratamiento, en este sentido los glucocorticoides parecen ejercer un papel dual en la regulación de FOXO3 en humanos, combinando una regulación transcripcional y post-traduccional, haciendo esta isoforma inactiva en el proceso atrófico por corticoides. FOXO1, no solo se vio regulado por fosforilación en el residuo S256 a través de la activación de AKT tras el estímulo con obestatina, si no que también se vio regulado por acetilación en los miotubos humanos. Los resultados obtenidos parecen demostrar que, la diminución en la acetilación de Resumen 130 FOXO1 tras el tratamiento con obestatina, fue llevado a cabo a través de la activación de HDAC4 por defosforilación. A la par de el aumento de actividad de HDAC4, también se observó una disminución de la actividad de PKF/PKCµ y CAMKII, todas ellas enzimas conocidas por regular la actividad de histonas deacetilasas de clase II. Estos resultados fueron apoyados además por los ensayos de inmunoprecipitación de FOXO1, donde se pudo observar una interacción directa entre FOXO1 y HDAC4 además de una interacción con SIRT1 en las células control, que se vio claramente disminuida tras el tratamiento con dexametasona; tras el tratamiento con obestatina se observó una recuperación de la interacción entre FOXO1 HDAC4 y SIRT1. Además, pudimos observar un aumento del estado de ubiquitinación de FOXO1 tras el tratamiento con obestatina, evidenciando su marcaje para ser degradado en el sistema ubiquitin-proteasomal. Tanto en el modelo humano como en el de ratón, FOXO4 parece ser la isoforma regulada de manera específica por fosforilación tras el tratamiento con obestatina. De esta manera, en ensayos de siRNA contra FOXO4, observamos que, el bloqueo de este factor de transcripción era suficiente para regular de manera negativa la expresión de las ubiquitin-ligasas MAFbx y MURF1, además de reducir el flujo autofágico. Más allá de la regulación post-traduccional de los factores de transcripción FOXO por fosforilación o acetilación en miotubos humanos, observamos tras un ensayo de siRNA contra KLF15, un regulador clave de la expresión de los factores de transcripción FOXO, que la obestatina no era capaz de regular la expresión de FOXO1 ni FOXO3, si embargo si fomentaba la disminución de la expresión de FOXO4 independientemente de KLF15. Los resultados demostraron que FOXO1 y FOXO4 son necesarios para la regulación de la proteostasis en respuesta al estímulo del sistema obestatina/GPR39, en el modelo de atrofia muscular inducida por glucocorticoides, mientras que FOXO3, a pesar de ser descrito como un factor clave en el desarrollo de este tipo de programa atrófico, fue regulado Resumen 131 transcripcionalmente de manera positiva, al mismo tiempo que se inhibia por fosforilación tras el tratamiento con corticoides, excluyendo su implicación promoviendo la expresión de atrogenes. La estimulación de miotubos humanos con obestatina, demostró ser capaz de frenar el programa atrófico a través de la señalización mediada por b-arrestinas e independientemente de la señalización de IGF1, así fue demostrado en dos ensayos de siRNA, de manera que, bloqueando la señalización de b-arrestinas, la obesatina no fue capaz de promover la fosforilación de FOXO3, FOXO1 ni FOXO4, además de no fomentar la activación de AKT por fosforilación en su residuo S473. En cuanto al bloqueo de IGF1, la ausencia de está proteína no tuvo ningún efecto, ni en el desarrollo del programa atrófico mediado por glucocorticoides, ni en la capacidad de la obestatina de contrarrestar los efectos catabólicos de los mismos. Otra de las diferencias que encontramos entre la señalización de humanos y ratón fue el patrón de expresión de el factor de transcripción miogenina, ha sido descrito en la bibliografía que ejerce un papel dual en el músculo, regulando el proceso miogénico a la par que fomenta la expresión de atrogenes en ratones. En el modelo de ratón observamos que la miogenina no se encontraba activada, mientras que, si lo estaba en el modelo humano, este hecho, junto a la disminución de la expresión de MAFbx y MURF1 tras el tratamiento con obestatina, nos hacen pensar que se podría tratar, simplemente, de diferencias en la cinética de expresión de esta proteína entre humanos y ratón. El sistema obestatina/GPR39 demostró ser un sistema autocrino, regulando el crecimiento muscular y promoviendo la regeneración. En el proceso atrófico inducido por glucocorticoides, la obestatina fue capaz de regular la expresión de atrogenes tales como las ubiquitin-ligasas MAFbx y MURF1, además del flujo autofágico, mostrando claras similitudes con el sistema IGF1 o insulina pero actuando a través de otras vías de señalización, implicando la regulación de Resumen 132 FOXO4 y FOXO1 bajo una regulación post-traducional específica de fosforilación, acetilación y ubiquitinación, contrarrestando de manera eficiente el proceso catabólico. De manera notable, la activación de la vía de señalización a través de la obestatina, de AKT/mTOR, combinada con la inhibición de PKD/CAMKII/AMPK, es necesaria para la inhibición de la actividad de los factores de transcripción FOXO y la expresión de atrogenes dependiente de los mismos. Cabe destacar que existen diferencias en algunos reguladores clave del programa atrófico entre humanos y ratón, que hace que, a pesar de que el resultado final sea el mismo, a la hora de diseñar fármacos destinados al tratamiento de la atrofia, las dianas moleculares han de ser comprobadas. Finalmente, con este trabajo se sugiere que la señalización entre el receptor de glucocorticoides y el sistema obestatina/GPR39 se trata de una interacción entre la señalización anabólica y la maquinaria catabólica. La capacidad de modular la actividad de este sistema hace que represente una buena estrategia para paliar los efectos de la atrofia muscular inducida por glucocorticoides. AGRADECIMIENTOS Agradecimientos Como en cada fin de etapa, te pones a reflexionar sobre todo el tiempo que ha pasado y lo corto que parece. Han pasado 6 años desde que llegué a mi laboratorio y la verdad se han pasado volando, si bien es cierto que el viaje no ha sido fácil en algunos momentos, ahora que se acaba, se que ha merecido la pena. En primer lugar, me gustaría agradecer a las instituciones que han hecho posible el desarrollo de mi trabajo. El Instituto de Salud Carlos III por financiar la investigación a través de los proyectos PI15/01537 y PI18/00760, a la Fundación Ramón Domínguez por concederme una beca pre-doctoral durante los dos primeros años de mi tesis y a la Organización Europea de Biología Molecular (EMBO) por darme la oportunidad de realizar una estancia internacional en Liverpool. Además, agradecer a mis directores de tesis, el Dr. Jesús Pérez Camiña y el Dr. Tomás García-Caballero por guiarme y asesorarme a lo largo de esta tesis. Me gustaría agradecer de manera especial al Dr. Jesús Pérez Camiña por abrirme las puertas de su laboratorio que además de un lugar de trabajo, se ha vuelto una pequeña familia. Gracias por ayudarme todos estos años en mi formación y por haberme hecho crecer como investigadora. Has tenido mucha paciencia conmigo, animándome y enseñándome a centrarme, por que el que mucho abarca poco aprieta. Muchas gracias por entender que las situaciones personales son prioritarias, me llevo tanto enseñanzas científicas como personales que sé que me ayudaran en un futuro. A la Dra. Yolanda Pazos por ser una fuente de energía positiva incansable, gracias por esas charlas matutinas que alivian los días duros y por todo el apoyo y consejos que me has dado. A mis compañeros del laboratorio 4 y 12, en especial a Saúl, por todas las conversaciones de marujas y las comilonas con nuestra italiana favorita Giulia, ya sabes que después de estos años nos hemos convertido en los hermanos que nunca tuvimos. A Carlos, tu ayuda a sido esencial, tanto laboral como personal, tus chistes “buenos” son los mejores. También me gustaría agradecer a las nuevas incorporaciones, María, mi gitana, eres alegría y la representación de superación, no tengo duda de que tu trayectoria será brillante, por que todo esfuerzo tiene su Agradecimientos 136 recompensa. A Fátima, me has alegrado los días en esta última etapa, las conversaciones, las gominolas, galletas, té…eres la compañera de laboratorio que todo el mundo quisiera tener, siempre dispuesta a ayudar. Además, no tengo duda que llegarás a Japón a cumplir tu sueño neuro-asiático, buena suerte. A mi “host laboratory” en Liverpool. Gracias a la Dra. Ainhoa Mielgo, por darme la oportunidad de trabajar en su laboratorio y enseñarme como trabajar en un nuevo campo. A mis compañeros ingleses, muchas gracias por la paciencia con mi inglés macarrónico y por haberme hecho sentir como en casa. A mis padres, por apoyarme durante toda mi carrera científica, a pesar de muchas veces no entender bien que es lo que estaba haciendo. Me habéis hecho la vida muy fácil y eso se ve reflejado en todos mis éxitos, muchas gracias, os quiero. A Mila y Ángel, por ser mi segunda familia y tratarme como una hija, muchas gracias. A mis amigas Nuria y Laura, que desde Ourense siempre he sentido vuestro cariño y apoyo, se que no nos podemos ver mucho, pero la distancia no es un problema para nosotras, sé que siempre estaréis ahí y los cafés de los viernes también. A mis amigos de la carrera, Las Leyendas de Biología, gracias por todos estos años de risas y apoyo, juntos hemos superado el primer curso de Bolonia y con orgullo. Aunque cada uno hemos tomado nuestro camino, esas cenas de Navidad anuales nunca faltaran y seguiremos riéndonos y quejándonos de lo viejos que nos hacemos. Finalmente agradecer a Anxo, una constante en mi vida, un apoyo incondicional. 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