NLK is required for Ras/ERK/SRF/ELK signaling to tune skeletal muscle development by phosphorylating SRF and antagonizing the SRF/MKL pathway

AbstractSerum response factor (SRF) regulates differentiation and proliferation by binding to RhoA-actin-activated MKL or Ras-MAPK-activated ELK transcriptional coactivators, but the molecular mechanisms responsible for SRF regulation remain unclear. Here, we show that Nemo-like kinase (NLK) is required for the promotion of SRF/ELK signaling in human and mouse cells. NLK was found to interact with and phosphorylate SRF at serine residues 101/103, which in turn enhanced the association between SRF and ELK. The enhanced affinity of SRF/ELK antagonized the SRF/MKL pathway and inhibited mouse myoblast differentiation in vitro. In a skeletal muscle-specific Nlk conditional knockout mouse model, forming muscle myofibers underwent hypertrophic growth, resulting in an increased muscle and body mass phenotype. We propose that both phosphorylation of SRF by NLK and phosphorylation of ELKs by MAPK are required for RAS/ELK signaling, confirming the importance of this ancient pathway and identifying an important role for NLK in modulating muscle development in vivo.

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Long Noncoding RNA Lnc-SEMT Modulates IGF2 Expression by Sponging miR-125b to Promote Sheep Muscle Development and Growth

Cellular Physiology and Biochemistry ◽

10.1159/000492979 ◽

2018 ◽

Vol 49 (2) ◽

pp. 447-462 ◽

Cited By ~ 14

Author(s):

Caihong Wei ◽

Mingming Wu ◽

Chuduan Wang ◽

Ruizao Liu ◽

Huijing Zhao ◽

...

Keyword(s):

Skeletal Muscle ◽

Noncoding Rna ◽

Molecular Mechanisms ◽

Muscle Development ◽

Growth Traits ◽

Noncoding Rnas ◽

Livestock Production ◽

Myoblast Differentiation

Background/Aims: Long noncoding RNAs (lncRNAs) are RNA transcripts that are more than 200 nt long but have little protein-coding potential. Within the last few years, thousands of lncRNAs have been identified and their functions in biological processes have begun to be understood. Although many studies havebegun to examine the functions of many noncoding RNAs, very little is known about the functions of long noncoding (lncRNA) function of livestock production and molecular mechanisms of their functions are still lackingrelated to livestock production. Methods: Expression of sheep enhanced muscularityTranscript lncRNA (lnc-SEMT) and miR-125b were examined in sheep using quantitative reverse-transcription polymerase chain reaction. Expression of Myod (myogenic determination factor), Myog (myoglobin) and Insulin-like growth factor 2 (IGF2)were examined by Western Blot.Luciferase reporter assays were performedto confirm the relationship between lnc-SEMT and miR-125b. Results: Here, we identified a novel lnc-SEMT that promote sheep myoblast differentiation in vitro and enhanced sheep muscularity in vivo. Functional analyses showed that lnc-SEMT accelerates sheep myoblast differentiation in vitro. lnc-SEMT transgenic sheep exhibit a muscle hypertrophy phenotype characterized by increased body weight, and increased the number of muscle fibers indicating that lnc-SEMT play an important role in the regulation of skeletal muscle differentiation in vivo. Our results show that lnc-SEMT acts as a molecular sponge by antagonizing miR-125b to control IGF2 protein labundance in vitro and in vivo. Conclusion: In brief, lnc-SEMT is the first example of a lncRNA could be a useful candidate for improving biological growth traits such as skeletal muscle production in sheep.

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Creating Interactions between Tissue-Engineered Skeletal Muscle and the Peripheral Nervous System

Cells Tissues Organs ◽

10.1159/000443634 ◽

2016 ◽

Vol 202 (3-4) ◽

pp. 143-158 ◽

Cited By ~ 21

Author(s):

Alec S.T. Smith ◽

Samantha L. Passey ◽

Neil R.W. Martin ◽

Darren J. Player ◽

Vivek Mudera ◽

...

Keyword(s):

Skeletal Muscle ◽

Motor Neurons ◽

Muscle Development ◽

Functional Performance ◽

Cell Types ◽

Original Data ◽

Synaptic Proteins ◽

Myoblast Differentiation

Effective models of mammalian tissues must allow and encourage physiologically (mimetic) correct interactions between co-cultured cell types in order to produce culture microenvironments as similar as possible to those that would normally occur in vivo. In the case of skeletal muscle, the development of such a culture model, integrating multiple relevant cell types within a biomimetic scaffold, would be of significant benefit for investigations into the development, functional performance, and pathophysiology of skeletal muscle tissue. Although some work has been published regarding the behaviour of in vitro muscle models co-cultured with organotypic slices of CNS tissue or with stem cell-derived neurospheres, little investigation has so far been made regarding the potential to maintain isolated motor neurons within a 3D biomimetic skeletal muscle culture platform. Here, we review the current state of the art for engineering neuromuscular contacts in vitro and provide original data detailing the development of a 3D collagen-based model for the co-culture of primary muscle cells and motor neurons. The devised culture system promotes increased myoblast differentiation, forming arrays of parallel, aligned myotubes on which areas of nerve-muscle contact can be detected by immunostaining for pre- and post-synaptic proteins. Quantitative RT-PCR results indicate that motor neuron presence has a positive effect on myotube maturation, suggesting neural incorporation influences muscle development and maturation in vitro. The importance of this work is discussed in relation to other published neuromuscular co-culture platforms along with possible future directions for the field.

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Mammalian target of rapamycin regulates miRNA-1 and follistatin in skeletal myogenesis

The Journal of Cell Biology ◽

10.1083/jcb.200912093 ◽

2010 ◽

Vol 189 (7) ◽

pp. 1157-1169 ◽

Cited By ~ 116

Author(s):

Yuting Sun ◽

Yejing Ge ◽

Jenny Drnevich ◽

Yong Zhao ◽

Mark Band ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Development ◽

Mammalian Target Of Rapamycin ◽

Micro Rna ◽

Skeletal Muscle Regeneration ◽

Target Of Rapamycin ◽

Myoblast Differentiation ◽

Mirna Biogenesis

Mammalian target of rapamycin (mTOR) has emerged as a key regulator of skeletal muscle development by governing distinct stages of myogenesis, but the molecular pathways downstream of mTOR are not fully understood. In this study, we report that expression of the muscle-specific micro-RNA (miRNA) miR-1 is regulated by mTOR both in differentiating myoblasts and in mouse regenerating skeletal muscle. We have found that mTOR controls MyoD-dependent transcription of miR-1 through its upstream enhancer, most likely by regulating MyoD protein stability. Moreover, a functional pathway downstream of mTOR and miR-1 is delineated, in which miR-1 suppression of histone deacetylase 4 (HDAC4) results in production of follistatin and subsequent myocyte fusion. Collective evidence strongly suggests that follistatin is the long-sought mTOR-regulated fusion factor. In summary, our findings unravel for the first time a link between mTOR and miRNA biogenesis and identify an mTOR–miR-1–HDAC4–follistatin pathway that regulates myocyte fusion during myoblast differentiation in vitro and skeletal muscle regeneration in vivo.

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Eph/Ephrin signalling in skeletal muscle development and regeneration

10.32469/10355/46419 ◽

2014 ◽

Author(s):

◽

Danny A. Stark

Keyword(s):

Skeletal Muscle ◽

Satellite Cells ◽

Tyrosine Kinases ◽

Muscle Development ◽

Receptor Tyrosine Kinases ◽

Repulsive Interaction ◽

Type I ◽

Ephrin Ligands

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Skeletal muscle can be isolated into 642 individual muscles and makes up to one third to one half of the mass of the human body. Each of these muscles is specified and patterned prenatally and after birth they will increase in size and take on characteristics suited to each muscle's unique function. To make the muscles functional, each muscle cell must be innervated by a motor neuron, which will also affect the characteristics of the mature muscle. In a healthy adult, muscles will maintain their specialized pattern and function during physiological homeostasis, and will also recapitulate them if the integrity or health of the muscle is disrupted. This repair and regeneration is dependent satellite cells, the skeletal muscle stem cells. In this dissertation, we study a family of receptor tyrosine kinases, Ephs, and their juxtacrine ephrin ligands in the context of skeletal muscle specification and regeneration. First, using a classical ephrin 'stripe' assay to test for contact-mediated repulsion, we found that satellite cells respond to a subset of ephrins with repulsive motility in vitro and that these forward signals through Ephs also promote patterning of differentiating myotubes parallel to ephrin stripes. This pattering can be replicated in a heterologous in vivo system (the hindbrain of the developing quail, where neural crest cells migrate in streams to the branchial arches, and in the forelimb of the developing quail, where presumptive limb myoblasts emigrate from the somite). Second, we present evidence that specific pairwise interactions between Eph receptor tyrosine kinases and ephrin ligands are required to ensure appropriate muscle innervation when it is originally set during postnatal development and when it is recapitulated after muscle or nerve trauma during adulthood. We show expression of a single ephrin, ephrin-A3, exclusively on type I (slow) myofibers shortly after birth, while its receptor EphA8 is only localized to fast motor endplates, suggesting a functional repulsive interaction for motor axon guidance and/or synaptogenesis. Adult EFNA3-/- mutant mice show a significant loss of slow myofibers, while misexpression of ephrin-A3 on fast myofibers results in a switch from a fast fiber type to slow in the context of sciatic nerve injury and regrowth. Third, we show that EphA7 is expressed on satellite cell derived myocytes in vitro, and marks both myocytes and regenerating myofibers in vivo. In the EPHA7 knockout mouse, we find a regeneration defect in a barium chloride injury model starting 3 days post injection in vivo, and that cultured mutant satellite cells are slow to differentiate and divide. Finally, we present other potential Ephs and ephrins that may affect skeletal muscle, such as EphB1 that is expressed on all MyHC-IIb fibers and a subset of MyHC-IIx fibers, and we show a multitude of Ephs and ephrins at the neuromuscular junction that appear to localize on specific myofibers and at different areas of the synapse. We propose that Eph/ephrin signaling, though well studied in development, continues to be important in regulating post natal development, regeneration, and homeostasis of skeletal muscle.

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Overlapping functions of the myogenic bHLH genes MRF4 and MyoD revealed in double mutant mice

Development ◽

10.1242/dev.125.13.2349 ◽

1998 ◽

Vol 125 (13) ◽

pp. 2349-2358 ◽

Cited By ~ 7

Author(s):

A. Rawls ◽

M.R. Valdez ◽

W. Zhang ◽

J. Richardson ◽

W.H. Klein ◽

...

Keyword(s):

Skeletal Muscle ◽

Double Mutant ◽

Muscle Development ◽

Expression Patterns ◽

Muscle Fibers ◽

Mutant Mice ◽

Myoblast Differentiation ◽

Bhlh Genes ◽

Bhlh Factors

The myogenic basic helix-loop-helix (bHLH) genes - MyoD, Myf5, myogenin and MRF4 - exhibit distinct, but overlapping expression patterns during development of the skeletal muscle lineage and loss-of-function mutations in these genes result in different effects on muscle development. MyoD and Myf5 have been shown to act early in the myogenic lineage to establish myoblast identity, whereas myogenin acts later to control myoblast differentiation. In mice lacking myogenin, there is a severe deficiency of skeletal muscle, but some residual muscle fibers are present in mutant mice at birth. Mice lacking MRF4 are viable and have skeletal muscle, but they upregulate myogenin expression, which could potentially compensate for the absence of MRF4. Previous studies in which Myf5 and MRF4 null mutations were combined suggested that these genes do not share overlapping myogenic functions in vivo. To determine whether the functions of MRF4 might overlap with those of myogenin or MyoD, we generated double mutant mice lacking MRF4 and either myogenin or MyoD. MRF4/myogenin double mutant mice contained a comparable number of residual muscle fibers to mice lacking myogenin alone and myoblasts from those double mutant mice formed differentiated multinucleated myotubes in vitro as efficiently as wild-type myoblasts, indicating that neither myogenin nor MRF4 is absolutely essential for myoblast differentiation. Whereas mice lacking either MRF4 or MyoD were viable and did not show defects in muscle development, MRF4/MyoD double mutants displayed a severe muscle deficiency similar to that in myogenin mutants. Myogenin was expressed in MRF4/MyoD double mutants, indicating that myogenin is insufficient to support normal myogenesis in vivo. These results reveal unanticipated compensatory roles for MRF4 and MyoD in the muscle differentiation pathway and suggest that a threshold level of myogenic bHLH factors is required to activate muscle structural genes, with this level normally being achieved by combinations of multiple myogenic bHLH factors.

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Accelerated Response of the myogenin Gene to Denervation in Mutant Mice Lacking Phosphorylation of Myogenin at Threonine 87

Molecular and Cellular Biology ◽

10.1128/mcb.24.5.1983-1989.2004 ◽

2004 ◽

Vol 24 (5) ◽

pp. 1983-1989 ◽

Cited By ~ 11

Author(s):

Chris S. Blagden ◽

Larry Fromm ◽

Steven J. Burden

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Electrical Activity ◽

Muscle Development ◽

Mutant Mice ◽

Threonine Residue ◽

E Boxes ◽

Bhlh Proteins

ABSTRACT Gene expression in skeletal muscle is regulated by a family of myogenic basic helix-loop-helix (bHLH) proteins. The binding of these bHLH proteins, notably MyoD and myogenin, to E-boxes in their own regulatory regions is blocked by protein kinase C (PKC)-mediated phosphorylation of a single threonine residue in their basic region. Because electrical stimulation increases PKC activity in skeletal muscle, these data have led to an attractive model suggesting that electrical activity suppresses gene expression by stimulating phosphorylation of this critical threonine residue in myogenic bHLH proteins. We show that electrical activity stimulates phosphorylation of myogenin at threonine 87 (T87) in vivo and that calmodulin-dependent kinase II (CaMKII), as well as PKC, catalyzes this reaction in vitro. We find that phosphorylation of myogenin at T87 is dispensable for skeletal muscle development. We show, however, that the decrease in myogenin (myg) expression following innervation is delayed and that the increase in expression following denervation is accelerated in mutant mice lacking phosphorylation of myogenin at T87. These data indicate that two distinct innervation-dependent mechanisms restrain myogenin activity: an inactivation mechanism mediated by phosphorylation of myogenin at T87, and a second, novel regulatory mechanism that regulates myg gene activity independently of T87 phosphorylation.

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Regulation of myogenic progenitor proliferation in human fetal skeletal muscle by BMP4 and its antagonist Gremlin

The Journal of Cell Biology ◽

10.1083/jcb.200511036 ◽

2006 ◽

Vol 175 (1) ◽

pp. 99-110 ◽

Cited By ~ 50

Author(s):

Natasha Y. Frank ◽

Alvin T. Kho ◽

Tobias Schatton ◽

George F. Murphy ◽

Michael J. Molloy ◽

...

Keyword(s):

Skeletal Muscle ◽

Molecular Mechanisms ◽

Side Population ◽

Functional Studies ◽

Morphogenetic Protein ◽

Study Gene Expression ◽

Main Population ◽

Myogenic Progenitor

Skeletal muscle side population (SP) cells are thought to be “stem”-like cells. Despite reports confirming the ability of muscle SP cells to give rise to differentiated progeny in vitro and in vivo, the molecular mechanisms defining their phenotype remain unclear. In this study, gene expression analyses of human fetal skeletal muscle demonstrate that bone morphogenetic protein 4 (BMP4) is highly expressed in SP cells but not in main population (MP) mononuclear muscle-derived cells. Functional studies revealed that BMP4 specifically induces proliferation of BMP receptor 1a–positive MP cells but has no effect on SP cells, which are BMPR1a-negative. In contrast, the BMP4 antagonist Gremlin, specifically up-regulated in MP cells, counteracts the stimulatory effects of BMP4 and inhibits proliferation of BMPR1a-positive muscle cells. In vivo, BMP4-positive cells can be found in the proximity of BMPR1a-positive cells in the interstitial spaces between myofibers. Gremlin is expressed by mature myofibers and interstitial cells, which are separate from BMP4-expressing cells. Together, these studies propose that BMP4 and Gremlin, which are highly expressed by human fetal skeletal muscle SP and MP cells, respectively, are regulators of myogenic progenitor proliferation.

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Endogenous Galectin-3 is Required for Skeletal Muscle Repair

10.1101/2020.10.01.322461 ◽

2020 ◽

Author(s):

Daniel Giuliano Cerri ◽

Lilian Cataldi Rodrigues ◽

Vani Maria Alves ◽

Juliano Machado ◽

Víctor Alexandre Félix Bastos ◽

...

Keyword(s):

Skeletal Muscle ◽

Cell Culture ◽

Molecular Mechanisms ◽

Repair Process ◽

Muscle Repair ◽

Galectin 3 ◽

Self Repair ◽

Myoblast Cell

ABSTRACTSkeletal muscle has the intrinsic ability to self-repair through a multifactorial process, but many aspects of its cellular and molecular mechanisms are not fully understood. There is increasing evidence that some members of the mammalian β-galactoside-binding protein family (galectins) are involved in the muscular repair process (MRP), including galectin-3 (Gal-3). However, there are many questions about the role of this protein on muscle self-repair. Here, we demonstrate that endogenous Gal-3 is required for: i) muscle repair in vivo using a chloride-barium myolesion mouse model, and ii) mouse primary myoblasts myogenic programming. Injured muscle from Gal-3 knockout mice (GAL3KO) showed persistent inflammation associated with compromised muscle repair and the formation of fibrotic tissue on the lesion site. In GAL3KO mice, osteopontin expression remained high even after 7 and 14 days of the myolesion, while MyoD and myogenin had decreased their expression. In GAL3KO mouse primary myoblast cell culture, Pax7 detection seems to sustain even when cells are stimulated to differentiation and MyoD expression is drastically reduced. These findings suggest that the detection and temporal expression levels of these transcriptional factors appear to be altered in Gal-3-deficient myoblast cell culture compared to Wild Type (WT) cells. We observed Gal-3 expression in WT states, both in vivo and in vitro, in sarcoplasm/cytoplasm and myonuclei; as differentiation proceeds, Gal-3 expression is drastically reduced, and its location is confined to the sarcolemma/plasma cell membrane. We also observed a change in the temporal-spatial profile of Gal-3 expression and muscle transcription factors levels during the myolesion. Overall, these results demonstrate that endogenous Gal-3 is required for the skeletal muscle repair process.

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Histone H3K36me2-specific methyltransferase ASH1L is required for the MLL-AF9-induced leukemogenesis

10.1101/2021.08.05.455280 ◽

2021 ◽

Author(s):

Mohammad B. Aljazi ◽

Yuen Gao ◽

Yan Wu ◽

George I. Mias ◽

Jin He

Keyword(s):

Enzymatic Activity ◽

Transcriptional Activation ◽

Gene Knockout ◽

Conditional Knockout ◽

Leukemic Cells ◽

Gene Promoters ◽

Leukemic Transformation ◽

Knockout Mouse Model

ASH1L and MLL1 are two histone methyltransferases that facilitate transcriptional activation during normal development. However, the roles of ASH1L and its enzymatic activity in the development of MLL-rearranged leukemias are not fully elucidated in the Ash1L gene knockout animal models. In this study, we used an Ash1L conditional knockout mouse model to show that loss of ASH1L in hematopoietic progenitor cells impaired the initiation of MLL-AF9-induced leukemic transformation in vitro. Furthermore, genetic deletion of ASH1L in the MLL-AF9-transformed cells impaired the maintenance of leukemic cells in vitro and largely blocked the leukemia progression in vivo. Importantly, the loss of ASH1L function in the Ash1L-deleted cells could be rescued by wild-type but not the catalytic-dead mutant ASH1L, suggesting the enzymatic activity of ASH1L was required for its function in promoting MLL-AF9-induced leukemic transformation. At the molecular level, ASH1L enhanced the MLL-AF9 target gene expression by directly binding to the gene promoters and modifying the local histone H3K36me2 levels. Thus, our study revealed the critical functions of ASH1L in promoting the MLL-AF9-induced leukemogenesis, which provides a molecular basis for targeting ASH1L and its enzymatic activity to treat MLL-arranged leukemias.

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Srf controls satellite cell fusion through the maintenance of actin architecture

The Journal of Cell Biology ◽

10.1083/jcb.201705130 ◽

2017 ◽

Vol 217 (2) ◽

pp. 685-700 ◽

Cited By ~ 20

Author(s):

Voahangy Randrianarison-Huetz ◽

Aikaterini Papaefthymiou ◽

Gaëlle Herledan ◽

Chiara Noviello ◽

Ulduz Faradova ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Serum Response Factor ◽

Control Cell ◽

Muscle Growth ◽

Muscle Stem Cells ◽

Hypertrophic Growth ◽

Proliferation And Differentiation ◽

Muscle Homeostasis

Satellite cells (SCs) are adult muscle stem cells that are mobilized when muscle homeostasis is perturbed. Here, we show that serum response factor (Srf) is needed for optimal SC-mediated hypertrophic growth. We identified Srf as a master regulator of SC fusion required in both fusion partners, whereas it was dispensable for SC proliferation and differentiation. We show that SC-specific Srf deletion leads to impaired actin cytoskeleton and report the existence of finger-like actin–based protrusions at fusion sites in vertebrates that were notoriously absent in fusion-defective myoblasts lacking Srf. Restoration of a polymerized actin network by overexpression of an α-actin isoform in Srf mutant SCs rescued their fusion with a control cell in vitro and in vivo and reestablished overload-induced muscle growth. These findings demonstrate the importance of Srf in controlling the organization of actin cytoskeleton and actin-based protrusions for myoblast fusion in mammals and its requirement to achieve efficient hypertrophic myofiber growth.

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