Subtype specific roles of mitogen activated protein kinases in L6E9 skeletal muscle cell differentiation

Role of mitogen activated protein kinases (MAPK) in skeletal muscle differentiation is not fully understood. We investigated subtype-specific functions and their interactions, if any, in the regulation of myogenic differentiation in L6E9 skeletal muscle cells. We show inhibition of extracellular signal-regulated kinase-1 and -2 (ERK-1/-2) and activation of p38 MAP kinase during the differentiation of L6E9 rat skeletal muscle cells under low serum condition. Inhibition of ERK-1/-2 activity dramatically enhanced differentiation as was evident from cellular morphology, expression of muscle differentiation specific marker proteins, suggesting that ERK-1/-2 activation may be inhibitory to initiation and progression of differentiation. In contrast, inhibition of p38 MAP kinase completely prevented differentiation; meaning p38 activation is required from the initiation till terminal differentiation of L6E9 cells. Moreover, inhibition of ERK-1/-2 activities enhanced the activation of p38 MAP kinase that resulted in enhancement of differentiation; whereas inhibition of p38 MAP kinase activity enhanced the ERK-1/-2 activities culminating in abrogation of differentiation. We conclude that ERK-1/-2 and p38 MAP kinase cascades oppositely regulate each other's function(s) thereby regulating L6E9 skeletal muscle differentiation.     

Different roles of PKC and MAP kinases in arteriolar constrictions to pressure and agonists

Protein kinase C (PKC) and mitogen-activated protein (MAP) kinases have been implicated in the modulation of agonist-induced contractions of large vessels. However, their role in pressure- and agonist-induced constrictions of skeletal muscle arterioles, which have a major role in regulating peripheral resistance, is not clearly elucidated. Thus constrictions of isolated rat gracilis muscle arterioles (approximately 80 microm in diameter) to increases in intraluminal pressure and to norepinephrine (NE) or angiotensin II (ANG II) were assessed in the absence or presence of chelerythrine, PD-98058, and SB-203580 (inhibitors of PKC, p42/44 and p38 MAP kinase pathways, respectively). Arteriolar constriction to NE and ANG II were significantly reduced by chelerythrine (by approximately 90%) and unaffected by SB-203580, whereas PD-98058 decreased only ANG II-induced constrictions (by approximately 60%). Pressure-induced increases in wall tension (from 0.1 to 0.7 N/m) resulted in significant arteriolar constrictions (50% maximum) that were abolished by chelerythrine without altering smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)) (fura 2 microfluorimetry). PD-98058 and SB-203580 significantly decreased the magnitude of myogenic tone (by 20% and 60%, respectively) and reduced the sensitivity of the myogenic mechanism to wall tension, causing a significant rightward shift in the wall tension-myogenic tone relationship without affecting smooth muscle [Ca(2+)i]. MAP kinases were demonstrated with Western blotting. Thus in skeletal muscle arterioles 1) PKC is involved in both myogenic and agonist-induced constrictions, 2) PD-98058-sensitive p42/44 MAP kinases modulate both wall tension-dependent and ANG II-induced constrictions, whereas 3) a SB-203580-sensitive p38 MAP kinase pathway seems to be specifically involved in the mechanotransduction of wall tension.     

Involvement of p38 MAPK-mediated signaling in the calpeptin-mediated suppression of myogenic differentiation and fusion in C2C12 cells

Calpeptin inhibits myoblast fusion by inhibiting the activity of calpain. However, the mechanism by which calpeptin inhibits myogenesis is not completely understood. This study examined how calpeptin affects the expression of the myogenic regulatory factors (MRFs) and the phosphorylation of p38 mitogen-activated protein kinase (MAPK) in differentiating C2C12 myoblasts. Consistent with previous reports, calpeptin inhibited the induction of μ-calpain and the formation of myotubes in these cells. In particular, calpeptin inhibited the expression of the early and mid differentiation markers including MyoD, Myf5, myogenin, and MRF4 as well as the expression of the late markers such as troponin T and myosin heavy chain (MyHC). Calpeptin also suppressed the phosphorylation of p38 MAPK in C2C12 cells. SB203580, a specific p38 inhibitor, prevented the expression of the muscle-specific markers and their fusion into myotubes in these cells, which was further accelerated in the presence of calpeptin. These findings suggest that calpeptin inhibits the myogenesis of skeletal muscle cells by down-regulating the MRFs and involving p38 MAPK signaling.     

Myogenic differentiation requires signalling through both phosphatidylinositol 3-kinase and p38 MAP kinase

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) or of Akt induces myoblast differentiation. Activation of p38 MAP kinase also triggers myogenic differentiation. The current paper shows that PI 3-kinase and p38 MAP kinase signalling are activated by two separate pathways during myogenic differentiation; both are required for muscle differentiation. p38-induced myogenic differentiation can be inhibited by the PI 3-kinase inhibitor LY294002 without affecting p38 activity. Similarly, a constitutively active form of Akt, myristylated c-Akt (Myr-Akt), induces myogenic differentiation that is inhibited by the p38 inhibitor SB203580. An analysis of the two forms of p38, p38 and p38beta, shows that the activity of both is required for myogenic differentiation. These data suggest that PI 3-kinase and p38 signalling are essential and parallel pathways for myogenic differentiation. They may either affect different downstream targets required for myogenesis or they may converge on shared targets that require input from both signalling pathways.     

Transdifferentiation of esophageal smooth to skeletal muscle is myogenic bHLH factor-dependent

Previously, coexpression of smooth and skeletal differentiation markers, but not myogenic regulatory factors (MRFs), was observed from E16.5 mouse fetuses in a small percentage of diaphragm level esophageal muscle cells, suggesting that MRFs are not involved in the process of initiation of developmentally programmed transdifferentiation in the esophagus. To investigate smooth-to-skeletal esophageal muscle transition, we analyzed Myf5nlacZ knock-in mice, MyoD-lacZ and myogenin-lacZ transgenic embryos with a panel of the antibodies reactive with myogenic regulatory factors (MRFs) and smooth and skeletal muscle markers. We observed that lacZ-expressing myogenic precursors were not detected in the esophagus before E15.5, arguing against the hypothesis that muscle precursor cells populate the esophagus at an earlier stage of development. Rather, the expression of the MRFs initiated in smooth muscle cells in the upper esophagus of E15.5 mouse embryos and was immediately followed by the expression of skeletal muscle markers. Moreover, transdifferentiation was markedly delayed or absent only in the absence of Myf5, suggesting that appropriate initiation and progression of smooth-to-skeletal muscle transdifferentiation is Myf5-dependent. Accordingly, the esophagus of Myf5(-/-):MyoD(-/-)embryos completely failed to undergo skeletal myogenesis and consisted entirely of smooth muscle. Lastly, extensive proliferation of muscularis precursor cells, without programmed cell death, occurred concomitantly with esophageal smooth-to-skeletal muscle transdifferentiation. Taken together, these results indicate that transdifferentiation is the fate of all smooth muscle cells in the upper esophagus and is normally initiated by Myf5.     

Histone deacetylase inhibitor trichostatin A enhances myogenesis by coordinating muscle regulatory factors and myogenic repressors

Histone deacetylase inhibitors (HDACIs) are known to promote skeletal muscle formation. However, their mechanisms that include effects on the expression of major muscle components such as the dystrophin-associated proteins complex (DAPC) or myogenic regulatory factors (MRFs) remain unknown. In this study, we investigated the effects of HDACIs on skeletal muscle formation using the C2C12 cell culture system. C2C12 myoblasts were exposed to trichostatin A (TSA), one of the most potent HDACIs, and differentiation was subsequently induced. We found that TSA enhances the expression of myosin heavy chain without affecting DAPC expression. In addition, TSA increases the expression of the early MRFs, Myf5 and MEF2, whereas it suppresses the expression of the late MRF, myogenin. Interestingly, TSA also enhances the expression of Id1, Id2, and Id3 (Ids). Ids are myogenic repressors that inhibit myogenic differentiation. These findings suggest that TSA promotes gene expression in proliferation and suppresses it in the differentiation stage of muscle formation. Taken together, our data demonstrate that TSA enhances myogenesis by coordinating the expression of MRFs and myogenic repressors.     

Increased MAPK signaling during in vitro muscle wounding

Regeneration of skeletal muscle upon injury is a complex process, involving activation of satellite cells, followed by migration, fusion, and regeneration of damaged myofibers. Previous work concerning the role of the mitogen activated protein (MAP) kinase signaling pathways in muscle injury comes primarily from studies using chemically induced wounding. The purpose of this study was to test the hypothesis that physical injury to skeletal muscle cells in vitro activates the MAP kinase signaling pathways. We demonstrate that extracellular signal regulated kinases (ERKs) 1, 2, and p38 are rapidly and transiently activated in response to injury in C2C12 cells, and are primarily localized to cells adjacent to the wound bed. Culture medium from wounded cells is able to stimulate activation of p38 but not ERK in unwounded cells. These results suggest that both ERK and p38 are involved in the response of muscle cells to physical injury in culture, and reflect what is seen in whole tissues in vivo.     

Dedifferentiation of adult human myoblasts induced by ciliary neurotrophic factor in vitro

Ciliary neurotrophic factor (CNTF) is primarily known for its important cellular effects within the nervous system. However, recent studies indicate that its receptor can be highly expressed in denervated skeletal muscle. Here, we investigated the direct effect of CNTF on skeletal myoblasts of adult human. Surprisingly, we found that CNTF induced the myogenic lineage-committed myoblasts at a clonal level to dedifferentiate into multipotent progenitor cells--they not only could proliferate for over 20 passages with the expression absence of myogenic specific factors Myf5 and MyoD, but they were also capable of differentiating into new phenotypes, mainly neurons, glial cells, smooth muscle cells, and adipocytes. These "progenitor cells" retained their myogenic memory and were capable of redifferentiating into myotubes. Furthermore, CNTF could activate the p44/p42 MAPK and down-regulate the expression of myogenic regulatory factors (MRFs). Finally, PD98059, a specific inhibitor of p44/p42 MAPK pathway, was able to abolish the effects of CNTF on both myoblast fate and MRF expression. Our results demonstrate the myogenic lineage-committed human myoblasts can dedifferentiate at a clonal level and CNTF is a novel regulator of skeletal myoblast dedifferentiation via p44/p42 MAPK pathway.     

Transforming Growth Factor-��-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

Satellite cells/myoblasts account for the majority of muscle regenerative potential in response to injury and muscular adaptation to exercise. Although the ability to influence this process would provide valuable benefits for treating a variety of patients suffering from muscle loss, the regulatory mechanisms of myogenesis are not completely understood. We have tested the hypothesis that transforming growth factor-β-activated kinase 1 (TAK1) is an important regulator of skeletal muscle formation. TAK1 is expressed in proliferating C2C12 myoblasts, and its levels are reduced upon differentiation of myoblasts into myotubes. In vivo, TAK1 is predominantly expressed in developing skeletal muscle of young mice. However, the expression of TAK1 was significantly up-regulated in regenerating skeletal muscle of adult mice. Overexpression of a dominant negative mutant of TAK1 or knockdown of TAK1 inhibited the proliferation and differentiation of C2C12 myoblasts. TAK1 was required for the expression of myogenic regulatory factors in differentiating myoblasts. Genetic ablation of TAK1 also inhibited the MyoD-driven transformation of mouse embryonic fibroblasts into myotubes. Inhibition of TAK1 suppressed the differentiation-associated activation of p38 mitogen-activated protein kinase (MAPK) and Akt kinase. Overexpression of a constitutively active mutant of MAPK kinase 6 (MKK6, an upstream activator of p38 MAPK) but not constitutive active Akt restored the myogenic differentiation in TAK1-deficient mouse embryonic fibroblasts. Insulin growth factor 1-induced myogenic differentiation was also found to involve TAK1. Collectively, our results suggest that TAK1 is an important upstream regulator of skeletal muscle cell differentiation.     

Cyclic mechanical stretch stimulates the proliferation of C2C12 myoblasts and inhibits their differentiation via prolonged activation of p38 MAPK

Mitogen-activated protein kinases (MAPKs) play an indispensable role in activation of the myogenic program, which is responsive to mechanical stimulation. Although there is accumulating evidence of mechanical force-mediated cellular responses, the role of MAPK in regulating the myogenic process in myoblasts exposed to cyclic stretch is unclear. Cyclic stretch induced the proliferation of C2C12 myoblasts and inhibited their differentiation into myotubes. In particular, it induced persistent phosphorylation of p38 kinase, and decreased the level of phosphorylation of extracellular-signal regulated kinase (ERK). Partial inhibition of p38 phosphorylation increased cellular levels of MyoD and p-ERK in stretched C2C12 cells, along with increased myotube formation. Treatment with 10 microM PD98059 prevented myogenin expression in response to a low dose of SB203580 (3 microM) in the stretched cells, suggesting that adequate ERK activation is also needed to allow the cells to differentiate into myotubes. These results suggest that cyclic stretch inhibits the myogenic differentiation of C2C12 cells by activating p38-mediated signaling and inhibiting ERK phosphorylation. We conclude that p38 kinase, not ERK, is the upstream signal transducer regulating cellular responses to mechanical stretch in skeletal muscle cells.     

Isoquinoline alkaloids from Coptis japonica stimulate the myoblast differentiation via p38 MAP-kinase and Akt signaling pathway

To overcome the muscle atrophy, such as cachexia and sarcopenia, we tried to find myogenic agents from medicinal plants. From myogenic extract of Coptis japonica, we purified six isoquinoline alkaloids and evaluated their effects on transactivation of myoD and MHC expression in C2C12 cells during differentiation process. Among obtained compounds, magnoflorine most efficiently enhanced the myoblast differentiation by activating the p38 MAP kinase and Akt pathway, and also increased the number of multinucleated and cylinder-shaped myotubes. These results propose that magnoflorine from Coptis japonica might be a promising lead compound for the development of anti-muscle atrophy drug.     

Regulation of glucose transport and glycogen synthesis in L6 muscle cells during oxidative stress. Evidence for cross-talk between the insulin and SAPK2/p38 mitogen-activated protein kinase signaling pathways

We have investigated the cellular mechanisms that participate in reducing insulin sensitivity in response to increased oxidant stress in skeletal muscle. Measurement of glucose transport and glycogen synthesis in L6 myotubes showed that insulin stimulated both processes, by 2- and 5-fold, respectively. Acute (30 min) exposure of muscle cells to hydrogen peroxide (H(2)O(2)) blocked the hormonal activation of both these processes. Immunoblot analyses of cell lysates prepared after an acute oxidant challenge using phospho-specific antibodies against c-Jun N-terminal kinase (JNK), p38, protein kinase B (PKB), and p42 and p44 mitogen-activated protein (MAP) kinases established that H(2)O(2) induced a dose-dependent activation of all five protein kinases. In vitro kinase analyses revealed that 1 mM H(2)O(2) stimulated the activity of JNK by approximately 8-fold, MAPKAP-K2 (the downstream target of p38 MAP kinase) by approximately 12-fold and that of PKB by up to 34-fold. PKB activation was associated with a concomitant inactivation of glycogen synthase kinase-3. Stimulation of the p38 pathway, but not that of JNK, was blocked by SB 202190 or SB203580, while that of p42/p44 MAP kinases and PKB was inhibited by PD 98059 and wortmannin respectively. However, of the kinases assayed, only p38 MAP kinase was activated at H(2)O(2) concentrations (50 microM) that caused an inhibition of insulin-stimulated glucose transport and glycogen synthesis. Strikingly, inhibiting the activation of p38 MAP kinase using either SB 202190 or SB 203580 prevented the loss in insulin-stimulated glucose transport, but not that of glycogen synthesis, by oxidative stress. Our data indicate that activation of the p38 MAP kinase pathway plays a central role in the oxidant-induced inhibition of insulin-regulated glucose transport, and unveils an important biochemical link between the classical stress-activated and insulin signaling pathways in skeletal muscle.     

Abl promotes cadherin-dependent adhesion and signaling in myoblasts

Cell-cell contact promotes myogenic differentiation but the mechanisms that regulate this phenomenon are not well understood. Cdo (also known as Cdon), an Ig superfamily member, functions as a component of cell surface complexes to promote myogenic differentiation through activation of p38a/b MAP kinase. We recently showed that N-cadherin ligation activated p38a/b in a Cdo-dependent manner, whereas N-cadherin ligation-dependent activation of ERK MAP kinase was not affected by loss of Cdo. The non-receptor tyrosine kinase Abl associates with Cdo during myoblast differentiation and is necessary for full activition of p38a/b during this process. The Abl SH3 domain binds to a PxxP motif in the Cdo intracellular domain, and both these motifs are required for their promyogenic activity. Here we show that Abl is necessary for p38a/b activation initiated by N-cadherin ligation, but in contrast to Cdo, Abl is also required for N-cadherin-dependent ERK activation. Moreover, Abl is required for efficient cadherin-mediated myoblast aggregation via modulation of RhoA-ROCK signaling. Therefore, Abl regulates N-cadherin-mediated p38a/b activation by multiple mechanisms, more generally through regulation of cell-cell adhesion and specifically as a component of Cdo-containing complexes. The role of Cdo as a multifunctional coreceptor with roles in several pathways is also discussed.     

Genetic Deficiency of p38α Reveals its Critical Role in Myoblast Cell Cycle Exit:The p38α-JNK Connection

The regulation of skeletal muscle formation (myogenesis) is essential for normal development as well as in pathological conditions such as muscular dystrophies and inflammatory myopathies. Findings published over the past years have established a key role for the p38 MAP kinase signaling pathway in the control of muscle gene expression and myotube formation. However, the relative contribution of the four p38 MAP kinases (p38α, p38β, p38γ and p38δ) to this process was unknown. We have recently demonstrated that myoblasts lacking p38α, but not those lacking p38β or p38δ, were unable to differentiate and form multinucleated myotubes, while p38γ-deficient myoblasts exhibited an attenuated fusion capacity. Defective myogenesis in the absence of p38α was attributed to delayed cell cycle exit and continuous proliferation in differentiation-promoting conditions, caused by enhanced activation of the JNK/cJun pathway. We discuss these findings in the context of the emerging crosstalk of p38 and JNK signaling pathways in controlling cell growth and differentiation.