Blockade of IGF2R improves muscle regeneration and ameliorates Duchenne muscular dystrophy
2019; Springer Nature; Volume: 12; Issue: 1 Linguagem: Inglês
10.15252/emmm.201911019
ISSN1757-4684
AutoresPamela Bella, Andrea Farini, Stefania Banfi, D. Parolini, Noemi Tonna, Mirella Meregalli, M. Belicchi, Silvia Erratico, Pasqualina D’Ursi, Fabio Bianco, Mariella Legato, Chiara Ruocco, Clementina Sitzia, S. Sangiorgi, Chiara Villa, Giuseppe D’Antona, Luciano Milanesi, Enzo Nisoli, Pierluigi Mauri, Yvan Torrente,
Tópico(s)Children's Physical and Motor Development
ResumoArticle2 December 2019Open Access Source DataTransparent process Blockade of IGF2R improves muscle regeneration and ameliorates Duchenne muscular dystrophy Pamela Bella Pamela Bella Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Andrea Farini Andrea Farini Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Stefania Banfi Stefania Banfi Hematology Department Fondazione IRCCS, Department of Oncology and Hemato-oncology, Istituto Nazionale dei Tumori, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Daniele Parolini Daniele Parolini Thermo Fisher Scientific, Life Technologies Italia, Monza, Italy Search for more papers by this author Noemi Tonna Noemi Tonna Neuro-Zone s.r.l., Open Zone, Milano, Italy Search for more papers by this author Mirella Meregalli Mirella Meregalli Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Marzia Belicchi Marzia Belicchi Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Silvia Erratico Silvia Erratico Novystem Spa, Milan, Italy Search for more papers by this author Pasqualina D'Ursi Pasqualina D'Ursi Institute of Technologies in Biomedicine, National Research Council (ITB-CNR), Milan, Italy Search for more papers by this author Fabio Bianco Fabio Bianco Neuro-Zone s.r.l., Open Zone, Milano, Italy Search for more papers by this author Mariella Legato Mariella Legato Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Chiara Ruocco Chiara Ruocco Department of Medical Biotechnology and Translational Medicine, Center for Study and Research on Obesity, Milan University, Milan, Italy Search for more papers by this author Clementina Sitzia Clementina Sitzia UOC SMEL-1, Scuola di Specializzazione di Patologia Clinica e Biochimica Clinica, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Simone Sangiorgi Simone Sangiorgi Neurosurgery Unit, Department of Surgery, ASST Lariana-S. Anna Hospital, Como, Italy Search for more papers by this author Chiara Villa Chiara Villa Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Giuseppe D'Antona Giuseppe D'Antona Department of Public Health, Experimental and Forensic Medicine, Pavia University, Pavia, Italy Search for more papers by this author Luciano Milanesi Luciano Milanesi Institute of Technologies in Biomedicine, National Research Council (ITB-CNR), Milan, Italy Search for more papers by this author Enzo Nisoli Enzo Nisoli Department of Medical Biotechnology and Translational Medicine, Center for Study and Research on Obesity, Milan University, Milan, Italy Search for more papers by this author PierLuigi Mauri PierLuigi Mauri Institute of Technologies in Biomedicine, National Research Council (ITB-CNR), Milan, Italy Search for more papers by this author Yvan Torrente Corresponding Author Yvan Torrente [email protected] orcid.org/0000-0002-2705-3984 Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Pamela Bella Pamela Bella Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Andrea Farini Andrea Farini Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Stefania Banfi Stefania Banfi Hematology Department Fondazione IRCCS, Department of Oncology and Hemato-oncology, Istituto Nazionale dei Tumori, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Daniele Parolini Daniele Parolini Thermo Fisher Scientific, Life Technologies Italia, Monza, Italy Search for more papers by this author Noemi Tonna Noemi Tonna Neuro-Zone s.r.l., Open Zone, Milano, Italy Search for more papers by this author Mirella Meregalli Mirella Meregalli Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Marzia Belicchi Marzia Belicchi Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Silvia Erratico Silvia Erratico Novystem Spa, Milan, Italy Search for more papers by this author Pasqualina D'Ursi Pasqualina D'Ursi Institute of Technologies in Biomedicine, National Research Council (ITB-CNR), Milan, Italy Search for more papers by this author Fabio Bianco Fabio Bianco Neuro-Zone s.r.l., Open Zone, Milano, Italy Search for more papers by this author Mariella Legato Mariella Legato Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Chiara Ruocco Chiara Ruocco Department of Medical Biotechnology and Translational Medicine, Center for Study and Research on Obesity, Milan University, Milan, Italy Search for more papers by this author Clementina Sitzia Clementina Sitzia UOC SMEL-1, Scuola di Specializzazione di Patologia Clinica e Biochimica Clinica, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Simone Sangiorgi Simone Sangiorgi Neurosurgery Unit, Department of Surgery, ASST Lariana-S. Anna Hospital, Como, Italy Search for more papers by this author Chiara Villa Chiara Villa Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Giuseppe D'Antona Giuseppe D'Antona Department of Public Health, Experimental and Forensic Medicine, Pavia University, Pavia, Italy Search for more papers by this author Luciano Milanesi Luciano Milanesi Institute of Technologies in Biomedicine, National Research Council (ITB-CNR), Milan, Italy Search for more papers by this author Enzo Nisoli Enzo Nisoli Department of Medical Biotechnology and Translational Medicine, Center for Study and Research on Obesity, Milan University, Milan, Italy Search for more papers by this author PierLuigi Mauri PierLuigi Mauri Institute of Technologies in Biomedicine, National Research Council (ITB-CNR), Milan, Italy Search for more papers by this author Yvan Torrente Corresponding Author Yvan Torrente [email protected] orcid.org/0000-0002-2705-3984 Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy Search for more papers by this author Author Information Pamela Bella1, Andrea Farini1, Stefania Banfi2, Daniele Parolini3, Noemi Tonna4, Mirella Meregalli1, Marzia Belicchi1, Silvia Erratico5, Pasqualina D'Ursi6, Fabio Bianco4, Mariella Legato1, Chiara Ruocco7, Clementina Sitzia8, Simone Sangiorgi9, Chiara Villa1, Giuseppe D'Antona10, Luciano Milanesi6, Enzo Nisoli7, PierLuigi Mauri6 and Yvan Torrente *,1 1Stem Cell Laboratory, Department of Pathophysiology and Transplantation, Unit of Neurology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Universitá degli Studi di Milano, Milan, Italy 2Hematology Department Fondazione IRCCS, Department of Oncology and Hemato-oncology, Istituto Nazionale dei Tumori, Universitá degli Studi di Milano, Milan, Italy 3Thermo Fisher Scientific, Life Technologies Italia, Monza, Italy 4Neuro-Zone s.r.l., Open Zone, Milano, Italy 5Novystem Spa, Milan, Italy 6Institute of Technologies in Biomedicine, National Research Council (ITB-CNR), Milan, Italy 7Department of Medical Biotechnology and Translational Medicine, Center for Study and Research on Obesity, Milan University, Milan, Italy 8UOC SMEL-1, Scuola di Specializzazione di Patologia Clinica e Biochimica Clinica, Università degli Studi di Milano, Milan, Italy 9Neurosurgery Unit, Department of Surgery, ASST Lariana-S. Anna Hospital, Como, Italy 10Department of Public Health, Experimental and Forensic Medicine, Pavia University, Pavia, Italy *Corresponding author. Tel: +39 0255 033874; E-mail: [email protected] EMBO Mol Med (2020)12:e11019https://doi.org/10.15252/emmm.201911019 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Duchenne muscular dystrophy (DMD) is a debilitating fatal X-linked muscle disorder. Recent findings indicate that IGFs play a central role in skeletal muscle regeneration and development. Among IGFs, insulinlike growth factor 2 (IGF2) is a key regulator of cell growth, survival, migration and differentiation. The type 2 IGF receptor (IGF2R) modulates circulating and tissue levels of IGF2 by targeting it to lysosomes for degradation. We found that IGF2R and the store-operated Ca2+ channel CD20 share a common hydrophobic binding motif that stabilizes their association. Silencing CD20 decreased myoblast differentiation, whereas blockade of IGF2R increased proliferation and differentiation in myoblasts via the calmodulin/calcineurin/NFAT pathway. Remarkably, anti-IGF2R induced CD20 phosphorylation, leading to the activation of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) and removal of intracellular Ca2+. Interestingly, we found that IGF2R expression was increased in dystrophic skeletal muscle of human DMD patients and mdx mice. Blockade of IGF2R by neutralizing antibodies stimulated muscle regeneration, induced force recovery and normalized capillary architecture in dystrophic mdx mice representing an encouraging starting point for the development of new biological therapies for DMD. Synopsis IGF2R is over-expressed in Duchenne Muscular Dystrophy (DMD) and mdx muscles. Blockade of IGF2R rescued the dystrophic muscle phenotype, ameliorated vascular architecture defects and improved muscle force. IGF2R expression is increased in dystrophic muscles and binds to CD20. Blockade of IGF2R facilitates IGF2-IGF1R interactions and activates CD20 phosphorylation, promoting the entrance of Ca2+ ions in the sarcoplasm. Increasing levels of Ca2+ ions regulate calcineurin/CaMKII pathway and activates SERCA1 leading to in vitro premature myogenic differentiation and in vivo increased force production and vasculature remodeling. Blockade of IGF2R reestablished the correct oscillating pattern of Ca2+ ion levels in the microenvironment of myofibrils and protects from muscular dystrophy acting on different mechanisms of dystrophic muscles. Despite this variety of mechanisms of action, monoclonal antibodies can be used in DMD to normalize the over-expression of IGF2R and the availability of muscle IGFs. Introduction Duchenne muscular dystrophy (DMD) is a devastating X-linked disease characterized by progressive muscle weakness and caused by a lack of dystrophin protein in the sarcolemma of muscle fibres (Emery, 2002). Impaired muscle regeneration with exhaustion of the satellite cell pool is a major hallmark of DMD. Members of the insulin-like growth factor (IGF) family are secreted during muscle repair and promote muscle regeneration and hypertrophy. Among the IGFs, IGF1 signalling has been extensively characterized for its capacity to promote the proliferation and differentiation of satellite cells, regulate muscle hypertrophy and ameliorate the features of muscular dystrophy (Florini et al, 1996; Barton et al, 2002; Zanou & Gailly, 2013). Nevertheless, little is known about the role of IGF2 in skeletal muscle development and regeneration in vivo. In vitro studies have shown that the IGF2 protein plays a role in a later step of myoblast differentiation (Florini et al, 1991; Wilson et al, 2003; Ge et al, 2011). Interestingly, it was previously shown that there is a link between the Myod and Igf2 genes in myoblast cell culture (Montarras et al, 2005). Further studies suggested that IGF2, by binding to the IGF1 receptor, activates the Akt pathway and downstream targets of Myod, although the exact mechanisms underlying these processes have not been identified (Wilson & Rotwein, 2006, 2007). IGF2 signalling is regulated by IGF-binding proteins, which sequester circulating IGF2; the IGF2 receptor (IGF2R), which reduces IGF2 bioactivity (Brown et al, 2009); and the insulin receptor and IGF1 receptor, both of which can be activated by IGF2 (Livingstone, 2013). The extracytoplasmic region of IGF2R has three binding sites: one for IGF2 in domain 11 and two for Man-6-P in domains 3, 5 and 9 (Dahms et al, 1993; Reddy et al, 2004; Williams et al, 2007). Binding between IGF2 and IGF2R induces the lysosomal degradation of IGF2. Hence, IGF2R serves to clear IGF2 from the circulation or degrade excess circulating IGF2 (Fargeas et al, 2003; Spicer & Aad, 2007). Soluble IGF2R can affect the size of some organs exclusively by reducing the biological activity of IGF2 (Zaina & Squire, 1998). Here, we report that IGF2R and the store-operated Ca2+ channel CD20 share a common hydrophobic binding motif that stabilizes their association. Intracellular Ca2+ regulation is compromised in dystrophic muscle fibres. Mechanisms that affect the influx of Ca2+ into dystrophic muscle fibres include membrane tears (Turner et al, 1988; Straub et al, 1997; Blake et al, 2002), stretch-activated channels (Gervasio et al, 2008), Ca2+ leak channels (Fong et al, 1990) and leaky Ca2+ release channels (Bellinger et al, 2009); it has also been speculated that the function of SERCA, the main protein responsible for Ca2+ reuptake into the sarcoplasmic reticulum (SR), is compromised in mdx mice (Tutdibi et al, 1999; Nicolas-Metral et al, 2001). We found that IGF2R targeting regulated the phosphorylation of CD20 and thereby induced SERCA activation in myoblasts. Interestingly, a delay in muscle differentiation was observed in CD20-silenced (shCD20) myoblasts, whereas the expression levels of early and late differentiation markers were increased after blockade of IGF2R. These features were accompanied by the activation of the calmodulin/calcineurin/NFAT pathway, suggesting that an IGF post-translational modulatory mechanism regulates muscle differentiation. Notably, IGF2R expression was increased in the dystrophic muscle tissues of mdx mice, while the phosphorylation of IGF2R was significantly decreased. Because IGF2R and CD20 interactions could affect dystrophic muscle tissues, we hypothesized that IGF clearance was faster and its bioavailability lower in dystrophic muscles than in normal muscles and that these changes were accompanied by perturbation of Ca2+ reuptake into the SR. Remarkably, in mdx mice, blockade of IGF2R increased muscle regeneration and significantly recovered muscle force via SERCA activation and Ca2+ reuptake. The IGF2 pathway affects vascular architecture, and the vessel structures of dystrophic skeletal muscles were clearly disorganized in mdx mice; hence, we examined the effect of anti-IGF2R antibodies on blood vessels in the skeletal muscles of mdx mice and found that muscle capillaries were linearized and exhibited normal architecture and maturation. Overall, these data demonstrated that a biological therapy targeting IGF2R leads to improvement of muscle regeneration and suppression of the pathological cascade associated with muscle dystrophic events. Results CD20 phosphorylation is affected by IGF-driven pathway Given the finding that CD20 acts as a mediator/modulator of store-operated calcium entry (SOCE) in skeletal myoblasts (Parolini et al, 2012), we were prompted to evaluate the functional impact of CD20 in C2C12 myoblast differentiation and analyse its possible interactions with the IGF pathway. IGF1 and IGF1R expression were not increased in 10 nM IGF1-treated C2C12 myoblasts (Fig 1A), but IGF1 treatment did induce a significant increase in transcription-dependent IGF2 production (Fig 1A). Immunofluorescence staining revealed that treating C2C12 cells with 10 nM IGF1 increased IGF2 production (Fig 1B). Moreover, over-expressing CD20 in C2C12 cells also resulted in a transcriptional increase in IGF2 expression (Fig 1A). In contrast, IGF2 expression was not detected in untreated C2C12 cells (Fig 1A and B) or CD20 shRNA-transfected C2C12 cells (Fig 1A). To assess the effect of stable CD20 inhibition, C2C12 cells were exposed to lentiviral particles designed to deliver constructs encoding shRNAs that targeted the CD20 mRNA. Knockdown efficiency was assayed after infected cells were selected by WB analysis, which revealed that CD20 protein expression was 60% silenced (Fig EV1A). To assess whether CD20 phosphorylation is affected by an IGF1-driven pathway, C2C12 myoblasts were exposed to 1 nM or 10 nM IGF1 for 2 h or overnight. The impact on CD20 phosphorylation was then detected by specific phosphor-Ser and phosphor-Thr antibodies. After 2 h of exposure to 10 nM IGF1, the level of phosphorylation of CD20 was significantly increased at both Ser and Thr residues (Fig 1C). CD20 serine phosphorylation was also increased after overnight exposure to 1 nM and 10 nM IGF1, although to a lesser extent (Fig 1C). Blockade of IGF2R induced a significant higher level of CD20 phosphorylation than was observed in IGF1-, shCD20- and anti-Flag-treated myoblasts (Fig 1D). Interestingly, CD20 serine phosphorylation was significantly increased in myoblasts co-stimulated with either 10 or 100 nM of IGF2 (Fig 1E). Together, these findings indicate that the activation of CD20-related signalling can be induced by IGFs in skeletal myoblasts. Moreover, IGF2R expression and phosphorylation were reduced in shCD20- and anti-IGF2R-treated myoblasts (Fig 1F). Treatment with anti-IGF2R increased IGF2R-Gαi2 interactions and regulated IGF1R phosphorylation, suggesting a shift in IGF1-IGF1R interactions (Fig 1G and H). Figure 1. IGF2R blockade results in CD20 phosphorylation A. RT–PCR expression and quantification of IGF1, IGF1Rβ and IGF2 levels in untreated C2C12 and shCD20 C2C12 myoblasts and 10 nM IGF1-treated C2C12 and over-expressing CD20 C2C12 myoblasts. Each experiment was replicated independently four times. Two-way ANOVA. ****P < 0.0001. All values are expressed as the mean ± SEM. B. Immunofluorescence for IGF2 (in green) in untreated and 10 nM IGF1-treated C2C12 myoblasts. Scale bars = 75 μm. C–E. Representative CD20 immunoprecipitation using anti-pSer + pThr in (C) C2C12 cells treated with IGF1 for 2 h or overnight (ON), (D) sh-empty (shCTR)- and shCD20-treated C2C12 cells treated with anti-Flag and 10 nM anti-IGF2R, (E) C2C12 cells treated with IGF1 and IGF2, as indicated. Densitometric analysis of data is expressed as the ratio of CD20/vinculin or pSer + pThr/CD20 and is shown normalized to vinculin in arbitrary units in the lower panels. Two-way ANOVA. *P < 0.05; **P < 0.01; ****P < 0.0001. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM. F. Representative WB of IGF2R and phosphorylated IGF2R (pIGF2R) in untreated, shCD20-treated and anti-IGF2R-treated C2C12 cells. Densitometric analysis of data is expressed as the ratio of IGF2R/actin or pIGF2R/IGF2R in arbitrary units in the lower panels. One-way ANOVA. *P < 0.05. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM. G. IGF2R immunoprecipitation products were immunoblotted for Gαi2 and WB expression of IGF2R in untreated, IGF2-treated, anti-IGF2R-treated and IGF2 + anti-IGF2R-treated C2C12 cells. Densitometric analysis of data is expressed as the ratio of Gαi2/IGF2R in arbitrary units in the lower panel. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM. H. Representative WB of IGF1Rβ and IGF1Rβ immunoprecipitation products immunoblotted for pTyr in untreated, IGF2-treated and IGF2 + anti-IGF2R-treated (1:500, 1:1,000 and 1:2,000 dilutions of anti-IGF2R) C2C12 cells (cells were treated for 2 and 24 h). Densitometric analysis of data is expressed as the ratio of pTYR/IGF1Rβ in arbitrary units in the lower panel. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM. Source data are available online for this figure. Source Data for Figure 1 [emmm201911019-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. The effect of CD20 modulation in C2C12 and 3T3 myoblasts A. Knockdown (shCD20) and over-expression (OE) of CD20 was tested by WB analysis. Densitometry analysis of data expressed as the CD20/β-actin ratio in arbitrary units. One-way ANOVA. ****P < 0.0001 (n = 5 independent experiments). All values are expressed as the mean ± SEM. B. Immunofluorescence showing CD20 expression in untreated and CD20 over-expressing C2C12 myoblasts. Scale bars = 75 μm. C–E. 3T3 mouse fibroblasts were labelled for anti-IGF1R tyrosine phosphorylation (green) under basal conditions (C), when co-stimulated with IGF2 and anti-IGF2R (D) and in the presence of IGF2 (E). Scale bars = 75 μm. Source data are available online for this figure. Download figure Download PowerPoint IGF2R binding to CD20 is relevant for myogenic differentiation We next sought to verify whether there are interactions between IGF2R and CD20. Automated protein docking of the transmembrane model of IGF2R with the crystal structure of CD20 predicts the binding pocket and identifies the residue critical for the binding. Structure of IGF2R with IGF2 compared to docking poses of epitope 3 shows that CD20 binds IGF2R domain 11 in the IGF2 binding site. This binding site region consists of a hydrophobic pocket centred on the CD loop, surrounded by polar and charged residues in the AB, EF and HI loops that complement surface charge on IGF2. The helical region (residues 178–184) of CD20 mediates the binding to receptor as well as IGF2 (Fig 2A). Indeed, Tyr18 of CD20 acts as an anchor making contact with the hydrophobic cluster Tyr1542, Phe 1567 and Leu 1629 of IGF2R domain 11 (Fig 2B), while for IGF2 the Phe19 of IGF2 is involved in the hydrophobic interaction (Fig 2C). Site-directed mutagenesis and structural studies have been shown that Phe 19 is important for IGF2R binding (Chuprin et al, 2015). Figure 2. IGF2R binding to CD20 is relevant for myogenic differentiation A–C. Bioinformatic prediction of IGF2R and CD20 cross-reactivity. Cartoon representation of the interaction of IGF2 (yellow) and IGF2R domain 11 complex from X-ray structure (PDB code 2v5p). IGF2R domain 11 AB, CD, EF and HI loops and residues (shown in sticks format) involved in the hydrophobic interactions are shown (A). Cartoon representation of the IGF2R domain 11–CD20 (lime) complex obtained from docking simulations and residues involved in hydrophobic interactions is shown in stick format (B). The structure of IGF2R (in red) and IGF2 (green) is shown compared to docking poses of epitope 3. These data show that the helical region (residues 178–184) of the epitope mediates binding to the receptor as well as IGF2. The C-terminal helix of epitope 3 partially overlaps the first α-helix of IGF2. ClusPro-dock IGF2R-CD20 binding epitope poses corresponding to PDB codes 2v5p-2oslP, 2v5p-2oslQ and 2v5p-3pp4P are coloured in yellow, blue and cyan, respectively (C). D. Over-expressing CD20 in C2C12 myoblasts that co-expressed CD20 (in green) and IGF2R (in red). DAPI-labelled nuclei are shown in blue. Scale bars = 75 μm. E. Representative CD20 and IGF2R immunoprecipitation products immunoblotted for IGF2R and CD20, respectively, in untreated and shCD20-treated C2C12 cell membranes and whole lysates of proteins. The immunoprecipitation output is shown as IP neg. F. Myotube immunofluorescence of cells in proliferation medium (P.M.) and after 2, 4 and 6 days of myogenic differentiation in serum-free medium. Control (untreated), shCD20-treated C2C12 cells, C2C12 and HSkM myoblasts pre-treated with anti-IGF2R for 24 h were stained. Desmin-positive myotubes are shown in green. Scale bars = 75 μm. G. Fusion index quantification after 6 days of differentiation. One-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Each experiment was performed in triplicate wells. All values are expressed as mean ± SEM. H. Representative WB of anti-myogenin, anti-Myf5, anti-MyHC and anti-β-actin in total protein lysates obtained from untreated, shCD20-treated C2C12 cells, and C2C12 and HSkM myoblasts pre-treated with anti-IGF2R for 24 h; cells were collected under P.M. and after 2, 4 and 6 days of myogenic differentiation. Densitometric analysis of WB data expressed as the ratio of the indicated antibody/β-actin in arbitrary units. Two-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 in comparison with the results obtained in untreated cells at the corresponding time point. ##P < 0.01; ###P < 0.001; ####P < 0.0001 in comparison with the results obtained in shCD20-treated C2C12 cells at the corresponding time point. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM. Source data are available online for this figure. Source Data for Figure 2 [emmm201911019-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint CD20 (Swiss-Prot code: P11836) is an integral membrane protein with intracellular N- and C-termini, four transmembrane spans (TM1–4) and an extracellular domain located between TM3 and TM4. Its only extracellular portions are two short loops located at positions 72–80 and 142–182. Mutagenesis and epitope mapping studies identified two CD20 epitopes in these extracellular domains. Epitope 2 spans residues 146–160, while epitope 3 spans residues 168–175. The X-ray structures of the Fab fragment in the complex with epitope 3 mimicked the large extracellular loop of CD20. The complex structure of human IGF2R domains 11–13, which bind to IGF2 (PDB code 2v5p), is also available. Using a protein–protein docking procedure, we evaluated the IGF2R-CD20 epitope 3 interaction using the ClusPro server. Four initial CD20 epitope 3 conformations (obtained with the PDB codes 2osl, 3bky and 3pp4) and one IGF2R conformation were used as the starting structures for the docking simulations. Computational steps were performed as follows: (i) rigid-body docking by sampling billions of conformations, (ii) RMSD-based clustering of the 1,000 structures with the lowest energy to find highly populated clusters that will represent the most likely models of the complex and (iii) refinement of the selected structures using energy minimization. For each simulation, ClusPro returned ten clusters with low energy, and a representative pose was chosen from the most populated cluster with the lowest energy on visual inspection (Fig 2C). In co-immunoprecipitation experiments, the IGF2R protein formed a stable complex with CD20 (Fig 2E), and shCD20 abolished or decreased the ability of IGF2R to interact with CD20 (Fig 2E). These findings suggest that IGF2R and CD20 share a common hydrophobic binding motif that stabilizes their association. Moreover, over-expression of CD20 increased the expression of IGF2R in C2C12 myoblasts (Fig 2D). These results verify that functional interactions occur between CD20 and IGF2R during myoblast differentiation. The expression of early and late differentiation myogenic markers demonstrated that myoblasts started to express myogenin after 2 days in differentiation medium (DM) and that its expression peaked after 4 days before decreasing after 6 days of differentiation (Fig 2F–H). Accordingly, the expression of MyHC was first observed on day 4 and persisted at day 6. Myoblasts exposed to blockade of IGF2R showed a significant increase in the expression of early and late differen
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