scholarly journals ROCK2 and Its Alternatively Spliced Isoform ROCK2m Positively Control the Maturation of the Myogenic Program

2007 ◽  
Vol 27 (17) ◽  
pp. 6163-6176 ◽  
Author(s):  
Michele Pelosi ◽  
Francesco Marampon ◽  
Bianca M. Zani ◽  
Sabrina Prudente ◽  
Emerald Perlas ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Intracellular Signaling ◽  
Elongation Factor ◽  
Size Control ◽  
Mitogen Activated Protein Kinase ◽  
Myoblast Differentiation ◽  
Myogenic Cells ◽  
Ribosomal S6 Kinase

ABSTRACT Signal transduction cascades involving Rho-associated kinases (ROCK), the serine/threonine kinases downstream effectors of Rho, have been implicated in the regulation of diverse cellular functions including cytoskeletal organization, cell size control, modulation of gene expression, differentiation, and transformation. Here we show that ROCK2, the predominant ROCK isoform in skeletal muscle, is progressively up-regulated during mouse myoblast differentiation and is highly expressed in the dermomyotome and muscle precursor cells of mouse embryos. We identify a novel and evolutionarily conserved ROCK2 splicing variant, ROCK2m, that is preferentially expressed in skeletal muscle and strongly up-regulated during in vivo and in vitro differentiation processes. The specific knockdown of ROCK2 or ROCK2m expression in C2C12 myogenic cells caused a significant and selective impairment of the expression of desmin and of the myogenic regulatory factors Mrf4 and MyoD. We demonstrate that in myogenic cells, ROCK2 and ROCK2m are positive regulators of the p42 and p44 mitogen-activated protein kinase-p90 ribosomal S6 kinase-eucaryotic elongation factor 2 intracellular signaling pathways and, thereby, positively regulate the hypertrophic effect elicited by insulin-like growth factor 1 and insulin, linking the multifactorial functions of ROCK to an important control of the myogenic maturation.

scholarly journals Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

1997 ◽  
Vol 17 (3) ◽  
pp. 1702-1713 ◽  
Author(s):  
D D Schlaepfer ◽  
M A Broome ◽  
T Hunter
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Intracellular Signaling ◽  
Protein Tyrosine Kinase ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Sh2 Domain ◽  
Mitogen Activated Protein Kinase

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.

scholarly journals In Vivo Activation of STAT3 Signaling in Satellite Cells and Myofibers in Regenerating Rat Skeletal Muscles

2002 ◽  
Vol 50 (12) ◽  
pp. 1579-1589 ◽  
Author(s):  
Katsuya Kami ◽  
Emiko Senba
Keyword(s):  
Skeletal Muscle ◽  
Satellite Cells ◽  
Muscle Regeneration ◽  
Intracellular Signaling ◽  
Skeletal Muscles ◽  
Early Stage ◽  
Skeletal Muscle Regeneration ◽  
Stat3 Signaling

Although growth factors and cytokines play critical roles in skeletal muscle regeneration, intracellular signaling molecules that are activated by these factors in regenerating muscles have been not elucidated. Several lines of evidence suggest that leukemia inhibitory factor (LIF) is an important cytokine for the proliferation and survival of myoblasts in vitro and acceleration of skeletal muscle regeneration. To elucidate the role of LIF signaling in regenerative responses of skeletal muscles, we examined the spatial and temporal activation patterns of an LIF-associated signaling molecule, the signal transducer and activator transcription 3 (STAT3) proteins in regenerating rat skeletal muscles induced by crush injury. At the early stage of regeneration, activated STAT3 proteins were first detected in the nuclei of activated satellite cells and then continued to be activated in proliferating myoblasts expressing both PCNA and MyoD proteins. When muscle regeneration progressed, STAT3 signaling was no longer activated in differentiated myoblasts and myotubes. In addition, activation of STAT3 was also detected in myonuclei within intact sarcolemmas of surviving myofibers that did not show signs of necrosis. These findings suggest that activation of STAT3 signaling is an important molecular event that induces the successful regeneration of injured skeletal muscles.

Spontaneous myogenic differentiation of Flk-1-positive cells from adult pancreas and other nonmuscle tissues

2008 ◽  
Vol 294 (2) ◽  
pp. C604-C612 ◽  
Author(s):  
Giuliana Di Rocco ◽  
Alessandra Tritarelli ◽  
Gabriele Toietta ◽  
Ilaria Gatto ◽  
Maria Grazia Iachininoto ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Myogenic Differentiation ◽  
Small Subset ◽  
Myogenic Cells ◽  
Adult Mice ◽  
Primary Myoblasts ◽  
Thymic Medulla ◽  
First Time

At the embryonic or fetal stages, autonomously myogenic cells (AMCs), i.e., cells able to spontaneously differentiate into skeletal myotubes, have been identified from several different sites other than skeletal muscle, including the vascular compartment. However, in the adult animal, AMCs from skeletal muscle-devoid tissues have been described in only two cases. One is represented by thymic myoid cells, a restricted population of committed myogenic progenitors of unknown derivation present in the thymic medulla; the other is represented by a small subset of adipose tissue-associated cells, which we recently identified. In the present study we report, for the first time, the presence of spontaneously differentiating myogenic precursors in the pancreas and in other skeletal muscle-devoid organs such as spleen and stomach, as well as in the periaortic tissue of adult mice. Immunomagnetic selection procedures indicate that AMCs derive from Flk-1+ progenitors. Individual clones of myogenic cells from nonmuscle organs are morphologically and functionally indistinguishable from skeletal muscle-derived primary myoblasts. Moreover, they can be induced to proliferate in vitro and are able to participate in muscle regeneration in vivo. Thus, we provide evidence that fully competent myogenic progenitors can be derived from the Flk-1+ compartment of several adult tissues that are embryologically unrelated to skeletal muscle.

scholarly journals Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase

1993 ◽  
Vol 90 (23) ◽  
pp. 10952-10956 ◽  
Author(s):  
R H Chen ◽  
C Abate ◽  
J Blenis
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Mitogen Activated Protein Kinase ◽  
S6 Kinase ◽  
Downstream Signaling ◽  
C Terminus ◽  
Mitogen Activated Protein ◽  
Ribosomal S6 Kinase

Phosphorylation of the C terminus of c-Fos has been implicated in serum response element-mediated repression of c-fos transcription after its induction by serum growth factors. The growth-regulated enzymes responsible for this phosphorylation in early G1 phase of the cell cycle and the sites of phosphorylation have not been identified. We now provide evidence that two growth-regulated, nucleus- and cytoplasm-localized protein kinases, 90-kDa ribosomal S6 kinase (RSK) and mitogen-activated protein kinase (MAP kinase), contribute to the serum-induced phosphorylation of c-Fos. The major phosphopeptides derived from biosynthetically labeled c-Fos correspond to phosphopeptides generated after phosphorylation of c-Fos in vitro with both RSK and MAP kinase. The phosphorylation sites identified for RSK (Ser-362) and MAP kinase (Ser-374) are in the transrepression domain. Cooperative phosphorylation at these sites by both enzymes was observed in vitro and reflected in vivo by the predominance of the peptide phosphorylated on both sites, as opposed to singly phosphorylated peptides. This study suggests a role for nuclear RSK and MAP kinase in modulating newly synthesized c-Fos phosphorylation and downstream signaling.

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

2016 ◽  
Vol 202 (3-4) ◽  
pp. 143-158 ◽  
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.

scholarly journals Age and prior exercise in vivo determine the subsequent in vitro molecular profile of myoblasts and nonmyogenic cells derived from human skeletal muscle

2019 ◽  
Vol 316 (6) ◽  
pp. C898-C912 ◽  
Author(s):  
Cecilie J. L. Bechshøft ◽  
Simon M. Jensen ◽  
Peter Schjerling ◽  
Jesper L. Andersen ◽  
Rene B. Svensson ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cell ◽  
Human Skeletal Muscle ◽  
Cell Function ◽  
Myogenic Differentiation ◽  
Molecular Profile ◽  
Muscle Stem Cell ◽  
Myogenic Cells

The decline in skeletal muscle regenerative capacity with age is partly attributed to muscle stem cell (satellite cell) dysfunction. Recent evidence has pointed to a strong interaction between myoblasts and fibroblasts, but the influence of age on this interaction is unknown. Additionally, while the native tissue environment is known to determine the properties of myogenic cells in vitro, how the aging process alters this cell memory has not been established at the molecular level. We recruited 12 young and 12 elderly women, who performed a single bout of heavy resistance exercise with the knee extensor muscles of one leg. Five days later, muscle biopsies were collected from both legs, and myogenic cells and nonmyogenic cells were isolated for in vitro experiments with mixed or separated cells and analyzed by immunostaining and RT-PCR. A lower myogenic fusion index was detected in the cells from the old versus young women, in association with differences in gene expression levels of key myogenic regulatory factors and senescence, which were further altered by performing exercise before tissue sampling. Coculture with nonmyogenic cells from the elderly led to a higher myogenic differentiation index compared with nonmyogenic cells from the young. These findings show that the in vitro phenotype and molecular profile of human skeletal muscle myoblasts and fibroblasts is determined by the age and exercise state of the original in vivo environment and help explain how exercise can enhance muscle stem cell function in old age.

scholarly journals Nur77 is phosphorylated in cells by RSK in response to mitogenic stimulation

2006 ◽  
Vol 393 (3) ◽  
pp. 715-724 ◽  
Author(s):  
Andrew D. Wingate ◽  
David G. Campbell ◽  
Mark Peggie ◽  
J. Simon C. Arthur
Keyword(s):  
Protein Kinase ◽  
Orphan Receptor ◽  
Mitogen Activated Protein Kinase ◽  
Mitogenic Stimulation ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Ribosomal S6 Kinase ◽  
In Cells

Nur77 is a nuclear orphan receptor that is able to activate transcription independently of exogenous ligand, and has also been shown to promote apoptosis on its localization to mitochondria. Phosphorylation of Nur77 on Ser354 has been suggested to reduce ability of Nur77 to bind DNA; however, the kinase responsible for this phosphorylation in cells has not been clearly established. In the present study, we show that Nur77 is phosphorylated on this site by RSK (ribosomal S6 kinase) and MSK (mitogen- and stress-activated kinase), but not by PKB (protein kinase B) or PKA (protein kinase A), in vitro. In cells, phosphorylation of Nur77 in vivo is catalysed by RSK, which is activated downstream of the classical MAPK (mitogen-activated protein kinase) cascade. Phosphorylation of Nur77 by RSK is able to promote the binding of Nur77 to 14-3-3 proteins in vitro, however, no evidence could be seen for this interaction in cells. We have established that two related proteins, Nurr1 and Nor1, are also phosphorylated on the equivalent site by RSK in cells in response to mitogenic stimulation.

scholarly journals Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts

2020 ◽  
Vol 21 (3) ◽  
pp. 745
Author(s):  
Orly Lacham-Kaplan ◽  
Donny M. Camera ◽  
John A. Hawley
Keyword(s):  
Synergistic Effect ◽  
Striated Muscle ◽  
Morphological Changes ◽  
Estrogen Receptor Α ◽  
Mitogen Activated Protein Kinase ◽  
Myoblast Differentiation ◽  
C2c12 Myoblasts ◽  
Myotube Formation

Estrogen (E2) and polyunsaturated fatty acids (n-3PUFA) supplements independently support general wellbeing and enhance muscle regeneration in-vivo and myotube formation in-vitro. However, the combined effect of E2 and n-3PUFA on myoblast differentiation is not known. The purpose of the study was to identify whether E2 and n-3PUFA possess a synergistic effect on in-vitro myogenesis. Mouse C2C12 myoblasts, a reliable model to reiterate myogenic events in-vitro, were treated with 10nM E2 and 50μM eicosapentaenoic acid (EPA) independently or combined, for 0–24 h or 0–120 h during differentiation. Immunofluorescence, targeted qPCR and next generation sequencing (NGS) were used to characterize morphological changes and differential expression of key genes involved in the regulation of myogenesis and muscle function pathways. E2 increased estrogen receptor α (Erα) and the expression of the mitogen-activated protein kinase 11 (Mapk11) within 1 h of treatment and improved myoblast differentiation and myotube formation. A significant reduction (p < 0.001) in myotube formation and in the expression of myogenic regulatory factors Mrfs (MyoD, Myog and Myh1) and the myoblast fusion related gene, Tmem8c, was observed in the presence of EPA and the combined E2/EPA treatment. Additionally, EPA treatment at 48 h of differentiation inhibited the majority of genes associated with the myogenic and striated muscle contraction pathways. In conclusion, EPA and E2 had no synergistic effect on myotube formation in-vitro. Independently, EPA inhibited myoblast differentiation and overrides the stimulatory effect of E2 when used in combination with E2.

scholarly journals Androgens induce growth of the limb skeletal muscles in a rapamycin-insensitive manner

2018 ◽  
Vol 315 (4) ◽  
pp. R721-R729 ◽  
Author(s):  
Michael L. Rossetti ◽  
David H. Fukuda ◽  
Bradley S. Gordon
Keyword(s):  
Skeletal Muscle ◽  
Fiber Type ◽  
Muscle Growth ◽  
Total Protein Content ◽  
Cross Sectional ◽  
S6 Kinase ◽  
Mechanistic Target Of Rapamycin ◽  
Ribosomal S6 Kinase

Signaling through the mechanistic target of rapamycin complex 1 (mTORC1) has been well defined as an androgen-sensitive transducer mediating skeletal muscle growth in vitro; however, this has yet to be tested in vivo. As such, male mice were subjected to either sham or castration surgery and allowed to recover for 7 wk to induce atrophy of skeletal muscle. Then, castrated mice were implanted with either a control pellet or a pellet that administered rapamycin (~2.5 mg·kg−1·day−1). Seven days postimplant, a subset of castrated mice with control pellets and all castrated mice with rapamycin pellets were given once weekly injections of nandrolone decanoate (ND) to induce muscle growth over a six-week period. Effective blockade of mTORC1 by rapamycin was noted in the skeletal muscle by the inability of insulin to induce phosphorylation of ribosomal S6 kinase 1 70 kDa (Thr389) and uncoordinated-like kinase 1 (Ser757). While castration reduced tibialis anterior (TA) mass, muscle fiber cross-sectional area, and total protein content, ND administration restored these measures to sham levels in a rapamycin-insensitive manner. Similar findings were also observed in the plantaris and soleus, suggesting this rapamycin-insensitive effect was not specific to the TA or fiber type. Androgen-mediated growth was not due to changes in translational capacity. Despite these findings in the limb skeletal muscle, rapamycin completely prevented the ND-mediated growth of the heart. In all, these data indicate that mTORC1 has a limited role in the androgen-mediated growth of the limb skeletal muscle; however, mTORC1 was necessary for androgen-mediated growth of heart muscle.

Mitogen-activated protein kinase signaling is necessary for the maintenance of skeletal muscle mass

2009 ◽  
Vol 296 (5) ◽  
pp. C1040-C1048 ◽  
Author(s):  
Hao Shi ◽  
Jason M. Scheffler ◽  
Caiyun Zeng ◽  
Jonathan M. Pleitner ◽  
Kevin M. Hannon ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Mass ◽  
Skeletal Muscle Mass ◽  
Fluorescent Protein ◽  
Mapk Signaling ◽  
26S Proteasome ◽  
Mitogen Activated Protein Kinase ◽  
Reporter Activity

The signal transduction cascades that maintain muscle mass remain to be fully defined. Herein, we report that inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in vitro decreases myotube size and protein content after 3-day treatment with a MEK inhibitor. Neither p38 nor JNK inhibitors had any effect on myotube size or morphology. ERK1/2 inhibition also upregulated gene transcription of atrogin-1 and muscle-specific RING finger protein 1 and downregulated the phosphorylation of Akt and its downstream kinases. Forced expression of enhanced green fluorescent protein-tagged MAPK phosphatase 1 (MKP-1) in soleus and gastrocnemius muscles decreased both fiber size and reporter activity. This atrophic effect of MKP-1 was time dependent. Analysis of the reporter activity in vivo revealed that the activities of nuclear factor-κB and 26S proteasome were differentially activated in slow and fast muscles, suggesting muscle type-specific mechanisms may be utilized. Together, these findings suggest that MAPK signaling is necessary for the maintenance of skeletal muscle mass because inhibition of these signaling cascades elicits muscle atrophy in vitro and in vivo.

Sign in / Sign up

Export Citation Format

Share Document