Varga et al.: Macrophage PPARγ, a Lipid Activated Transcription Factor Controls the Growth Factor GDF3 and Skeletal Muscle Regeneration Immunity. 2016 Nov 15;45(5):1038-1051. doi: 10.1016/j.immuni.2016.10.016.

Tissue regeneration requires inflammatory and reparatory activity of macrophages. Macrophages detect and eliminate the damaged tissue and subsequently promote regeneration. This dichotomy requires the switch of effector functions of macrophages coordinated with other cell types inside the injured tissue. The gene regulatory events supporting the sensory and effector functions of macrophages involved in tissue repair are not well understood. Here we show that the lipid activated transcription factor, PPARγ, is required for proper skeletal muscle regeneration, acting in repair macrophages. PPARγ controls the expression of the transforming growth factor-β (TGF-β) family member, GDF3, which in turn regulates the restoration of skeletal muscle integrity by promoting muscle progenitor cell fusion. This work establishes PPARγ as a required metabolic sensor and transcriptional regulator of repair macrophages. Moreover, this work also establishes GDF3 as a secreted extrinsic effector protein acting on myoblasts and serving as an exclusively macrophage-derived regeneration factor in tissue repair

TGF-β promotes microtube formation in glioblastoma through Thrombospondin 1

International audienceAbstract Background Microtubes (MTs), cytoplasmic extensions of glioma cells, are important cell communication structures promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular, glioblastomas (GBMs), while they are uncommon in chemosensitive IDH-mutant and 1p/19q co-deleted oligodendrogliomas. The aim of this study was to identify potential signaling pathways involved in MT formation. Methods Bioinformatics analysis of TCGA was performed to analyze differences between GBM and oligodendroglioma. Patient-derived GBM stem cell lines were used to investigate MT formation under transforming growth factor-beta (TGF-β) stimulation and inhibition in vitro and in vivo in an orthotopic xenograft model. RNA sequencing and proteomics were performed to detect commonalities and differences between GBM cell lines stimulated with TGF-β. Results Analysis of TCGA data showed that the TGF-β pathway is highly activated in GBMs compared to oligodendroglial tumors. We demonstrated that TGF-β1 stimulation of GBM cell lines promotes enhanced MT formation and communication via calcium signaling. Inhibition of the TGF-β pathway significantly reduced MT formation and its associated invasion in vitro and in vivo. Downstream of TGF-β, we identified thrombospondin 1 (TSP1) as a potential mediator of MT formation in GBM through SMAD activation. TSP1 was upregulated upon TGF-β stimulation and enhanced MT formation, which was inhibited by TSP1 shRNAs in vitro and in vivo. Conclusion TGF-β and its downstream mediator TSP1 are important mediators of the MT network in GBM and blocking this pathway could potentially help to break the complex MT-driven invasion/resistance network

Rôle de la protéine liant l'ARN, IMP-2 (Insulin-like Growth factor II mRNA-binding-Protein 2) au cours de la myogenèse normale

Le maintien de l’équilibre entre prolifération et différenciation est assuré par larégulation de l’expression de gènes qui sont impliqués dans la différenciation ou laprolifération cellulaire, ou qui gouvernent le passage d’une voie à l’autre. Unealtération de cet équilibre peut ainsi conduire à des situations pathologiquesmajeures comme la formation de tumeurs. Ainsi les cellules cancéreuses prolifèrentde manières incontrôlées et échappent au programme de différenciation cellulaire.IMP-2 est une protéine retrouvée surexprimée dans les rhabdomyosarcomes,cellules cancéreuses issues de précurseurs myogéniques, et exprimée au cours dudéveloppement embryonnaire. Pourtant, aucune étude ne s’était encore penchée surson rôle au cours la myogénèse normale, par opposition aux rhabdomyosarcomes.Nous pensons que la compréhension des mécanismes liés à la voie IMP-2 normale,pourrait aider à mieux comprendre pourquoi ces protéines sont retrouvéessurexprimées dans les rhadbomyosarcomes.Nous avons démontré qu’IMP-2 interagit avec un grand nombre d’ARNm, et laplupart de ces interactions ont été infirmées ou confirmées par la présence dans lecomplexe protéique d’IMP-2 de ces ARNm. Nous avons aussi pu démontrer qu’IMP-2 fait partie de complexes protéiques impliqués dans l’épissage, la traduction, ouencore le transport des ARN.Même si l’inhibition d’IMP-2 semble provoquer des remaniements morphologiquesvisibles sur les lignées de myoblastes de souris (C2C12), son rôle au sein de lacellule n’est pas encore clair. Il paraît évident qu’IMP-2 a un rôle au cours de lamyogenèse, même si nous n’avons pas pu déterminer un rôle précis. Ses différentspartenaires interagissent avec IMP-2 dans différents sous complexes ayantcertainement des rôles distincts et agissant chacun sur des ARN différents

Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis by increasing translation efficiency

Lin-28 is a highly conserved, RNA-binding, microRNA-regulated protein that is involved in regulation of developmental timing in Caenorhabditis elegans. In mammals, Lin-28 is stage-specifically expressed in embryonic muscle, neurons, and epithelia, as well as in embryonic carcinoma cells, but is suppressed in most adult tissues, with the notable exception of skeletal and cardiac muscle. The specific function and mechanism of action of Lin-28 are not well understood. Here we used loss-of-function and gain-of-function assays in cultured myoblasts to show that expression of Lin-28 is essential for skeletal muscle differentiation in mice. In order to elucidate the specific function of Lin-28, we used a combination of biochemical and functional assays, which revealed that, in differentiating myoblasts, Lin-28 binds to the polysomes and increases the efficiency of protein synthesis. An important target of Lin-28 is IGF-2, a crucial growth and differentiation factor for muscle tissue. Interaction of Lin-28 with translation initiation complexes in skeletal myoblasts and in the embryonic carcinoma cell line P19 was confirmed by localization of Lin-28 to the stress granules, temporary structures that contain stalled mRNA–protein translation complexes. Our results unravel novel mechanisms of translational regulation in skeletal muscle and suggest that Lin-28 performs the role of “translational enhancer” in embryonic and adult cells and tissues

Acetylation is important for MyoD function in adult mouse

Acetylation is a post-translational modification that influences the activity of numerous proteins in vitro. Among them, the myogenic transcription factor MyoD displays an increased transcriptional activity in vitro when acetylated on two lysines, lysine 99 and 102. Here, we have investigated the biological relevance of this acetylation in vivo. Using specific antibodies, we demonstrate that endogenous MyoD is acetylated on lysine 99 and 102 in myoblasts. Moreover, we show the functional importance of acetylation in live animals, using a mutant of MyoD in which lysines 99 and 102 were replaced by arginines. Knock-in (KI) embryos homozygous for the MyoD R 99,102 allele expressed slightly reduced levels of MyoD, but they developed normally. However, the KI homozygous adult mice showed a phenotype close to that of MyoD knock-out animals, including delayed muscle regeneration in vivo, and increased numbers of myoblasts but with reduced differentiation potential in vitro. Taken together, these results demonstrate the importance of MyoD acetylation for adult myogenesis

Derivation and Characterization of Immortalized Human Muscle Satellite Cell Clones from Muscular Dystrophy Patients and Healthy Individuals

In Duchenne muscular dystrophy (DMD) patients, absence of dystrophin causes muscle wasting by impacting both the myofiber integrity and the properties of muscle stem cells (MuSCs). Investigation of DMD encompasses the use of MuSCs issued from human skeletal muscle. However, DMD-derived MuSC usage is restricted by the limited number of divisions that human MuSCs can undertake in vitro before losing their myogenic characteristics and by the scarcity of human material available from DMD muscle. To overcome these limitations, immortalization of MuSCs appears as a strategy. Here, we used CDK4/hTERT expression in primary MuSCs and we derived MuSC clones from a series of clinically and genetically characterized patients, including eight DMD patients with various mutations, four congenital muscular dystrophies and three age-matched control muscles. Immortalized cultures were sorted into single cells and expanded as clones into homogeneous populations. Myogenic characteristics and differentiation potential were tested for each clone. Finally, we screened various promoters to identify the preferred gene regulatory unit that should be used to ensure stable expression in the human MuSC clones. The 38 clonal immortalized myogenic cell clones provide a large collection of controls and DMD clones with various genetic defects and are available to the academic community

The microRNA miR-181 targets the homeobox protein Hox-A11 during mammalian myoblast differentiation.

Deciphering the mechanisms underlying skeletal muscle-cell differentiation in mammals is an important challenge. Cell differentiation involves complex pathways regulated at both transcriptional and post-transcriptional levels. Recent observations have revealed the importance of small (20-25 base pair) non-coding RNAs (microRNAs or miRNAs) that are expressed in both lower organisms and in mammals. miRNAs modulate gene expression by affecting mRNA translation or stability. In lower organisms, miRNAs are essential for cell differentiation during development; some miRNAs are involved in maintenance of the differentiated state. Here, we show that miR-181, a microRNA that is strongly upregulated during differentiation, participates in establishing the muscle phenotype. Moreover, our results suggest that miR-181 downregulates the homeobox protein Hox-A11 (a repressor of the differentiation process), thus establishing a functional link between miR-181 and the complex process of mammalian skeletal-muscle differentiation. Therefore, miRNAs can be involved in the establishment of a differentiated phenotype - even when they are not expressed in the corresponding fully differentiated tissue

Acetylation is important for MyoD function in adult mice

Acetylation is a post-translational modification that influences the activity of numerous proteins in vitro. Among them, the myogenic transcription factor MyoD shows an increased transcriptional activity in vitro when acetylated on two lysines (K): lysines 99 and 102. Here, we have investigated the biological relevance of this acetylation in vivo. Using specific antibodies, we show that endogenous MyoD is acetylated on lysines 99 and 102 in myoblasts. Moreover, we show the functional importance of acetylation in live animals by using a mutant of MyoD in which lysines 99 and 102 were replaced by arginines (R). Knock-in embryos homozygous for the MyoD(R99,102) allele expressed slightly reduced levels of MyoD but developed normally. However, the knock-in homozygous adult mice showed a phenotype that was almost identical to that of MyoD-knockout animals, including delayed muscle regeneration in vivo and an increased number of myoblasts but with reduced differentiation potential in vitro. Together, these results show the importance of MyoD acetylation for adult myogenesis