Sex Differences in Inflammation and Muscle Wasting in Aging and Disease.

Only in recent years, thanks to a precision medicine-based approach, have treatments tailored to the sex of each patient emerged in clinical trials. In this regard, both striated muscle tissues present significant differences between the two sexes, which may have important consequences for diagnosis and therapy in aging and chronic illness. In fact, preservation of muscle mass in disease conditions correlates with survival; however, sex should be considered when protocols for the maintenance of muscle mass are designed. One obvious difference is that men have more muscle than women. Moreover, the two sexes differ in inflammation parameters, particularly in response to infection and disease. Therefore, unsurprisingly, men and women respond differently to therapies. In this review, we present an up-to-date overview on what is known about sex differences in skeletal muscle physiology and disfunction, such as disuse atrophy, age-related sarcopenia, and cachexia. In addition, we summarize sex differences in inflammation which may underly the aforementioned conditions because pro-inflammatory cytokines deeply affect muscle homeostasis. The comparison of these three conditions and their sex-related bases is interesting because different forms of muscle atrophy share common mechanisms; for instance, those responsible for protein dismantling are similar although differing in terms of kinetics, severity, and regulatory mechanisms. In pre-clinical research, exploring sexual dimorphism in disease conditions could highlight new efficacious treatments or recommend implementation of an existing one. Any protective factors discovered in one sex could be exploited to achieve lower morbidity, reduce the severity of the disease, or avoid mortality in the opposite sex. Thus, the understanding of sex-dependent responses to different forms of muscle atrophy and inflammation is of pivotal importance to design innovative, tailored, and efficient interventions.

Nano-Immunomodulation: A New Strategy for Skeletal Muscle Diseases and Aging?

The skeletal muscle has a very remarkable ability to regenerate upon injury under physiological conditions; however, this regenerative capacity is strongly diminished in physio-pathological conditions, such as those present in diseased or aged muscles. Many muscular dystrophies (MDs) are characterized by aberrant inflammation due to the deregulation of both the lymphoid and myeloid cell populations and the production of pro-inflammatory cytokines. Pathological inflammation is also observed in old muscles due to a systemic change in the immune system, known as "inflammaging". Immunomodulation represents, therefore, a promising therapeutic opportunity for different skeletal muscle conditions. However, the use of immunomodulatory drugs in the clinics presents several caveats, including their low stability in vivo, the need for high doses to obtain therapeutically relevant effects, and the presence of strong side effects. Within this context, the emerging field of nanomedicine provides the powerful tools needed to control the immune response. Nano-scale materials are currently being explored as biocarriers to release immunomodulatory agents in the damaged tissues, allowing therapeutic doses with limited off-target effects. In addition, the intrinsic immunomodulatory properties of some nanomaterials offer further opportunities for intervention that still need to be systematically explored. Here we exhaustively review the state-of-the-art regarding the use of nano-sized materials to modulate the aberrant immune response that characterizes some physio-pathological muscle conditions, such as MDs or sarcopenia (the age-dependent loss of muscle mass). Based on our learnings from cancer and immune tolerance induction, we also discuss further opportunities, challenges, and limitations of the emerging field of nano-immunomodulation.

Anti­oncogenic and pro­myogenic action of the MKK6/p38/AKT axis induced by targeting MEK/ERK in embryonal rhabdomyosarcoma.

Insights into the molecular and cellular biology of embryonal rhabdomyosarcoma (ERMS), an aggressive paediatric tumour, are required in order to identify new targets for novel treatments that may benefit patients with this disease. The present study examined the functional effects of MKK3 and MKK6, two upstream kinases of p38, and found that the ectopic expression of MKK6 led to rapid p38 activation and the myogenic differentiation of ERMS cells, whereas MKK3 failed to induce differentiation, while maintaining the proliferation state. Myogenin and myosin heavy chain were induced in MKK6­overexpressing ERMS cells and were inhibited by the p38 inhibitor, SB203580. The expression of Myc and ERK­PO4 increased under the effect of SB203580, whereas it decreased in MKK6­overexpressing cells. AKT activation was part of the myogenic program triggered by MKK6 overexpression alone. To the best of our knowledge, the present study demonstrates, for the first time, that the endogenous MKK6 pathway may be recovered by MEK/ERK inhibition (U0126 and trametinib) and that it concomitantly induces the reversal of the oncogenic pattern and the induction of the myogenic differentiation of ERMS cell lines. The effects of MEK/ERK inhibitors markedly increase the potential clinical applications in ERMS, particularly on account of the MEK inhibitor­induced early MKK6/p38 axis activation and of their anti­oncogenic effects. The findings presented herein lend further support to the antitumour effects of MKK6; MKK6 may thus represent a novel target for advanced personalised treatments against ERMS.

A Pound of Flesh: What Cachexia Is and What It Is Not.

Body weight loss, mostly due to the wasting of skeletal muscle and adipose tissue, is the hallmark of the so-called cachexia syndrome. Cachexia is associated with several acute and chronic disease states such as cancer, chronic obstructive pulmonary disease (COPD), heart and kidney failure, and acquired and autoimmune diseases and also pharmacological treatments such as chemotherapy. The clinical relevance of cachexia and its impact on patients' quality of life has been neglected for decades. Only recently did the international community agree upon a definition of the term cachexia, and we are still awaiting the standardization of markers and tests for the diagnosis and staging of cancer-related cachexia. In this review, we discuss cachexia, considering the evolving use of the term for diagnostic purposes and the implications it has for clinical biomarkers, to provide a comprehensive overview of its biology and clinical management. Advances and tools developed so far for the in vitro testing of cachexia and drug screening will be described. We will also evaluate the nomenclature of different forms of muscle wasting and degeneration and discuss features that distinguish cachexia from other forms of muscle wasting in the context of different conditions.

Lack of PKCθ Promotes Regenerative Ability of Muscle Stem Cells in Chronic Muscle Injury.

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by muscle wasting and chronic inflammation, leading to impaired satellite cells (SCs) function and exhaustion of their regenerative capacity. We previously showed that lack of PKCθ in mdx mice, a mouse model of DMD, reduces muscle wasting and inflammation, and improves muscle regeneration and performance at early stages of the disease. In this study, we show that muscle regeneration is boosted, and fibrosis reduced in mdxθ-/- mice, even at advanced stages of the disease. This phenotype was associated with a higher number of Pax7 positive cells in mdxθ-/- muscle compared with mdx muscle, during the progression of the disease. Moreover, the expression level of Pax7 and Notch1, the pivotal regulators of SCs self-renewal, were upregulated in SCs isolated from mdxθ-/- muscle compared with mdx derived SCs. Likewise, the expression of the Notch ligands Delta1 and Jagged1 was higher in mdxθ-/- muscle compared with mdx. The expression level of Delta1 and Jagged1 was also higher in PKCθ-/- muscle compared with WT muscle following acute injury. In addition, lack of PKCθ prolonged the survival and sustained the differentiation of transplanted myogenic progenitors. Overall, our results suggest that lack of PKCθ promotes muscle repair in dystrophic mice, supporting stem cells survival and maintenance through increased Delta-Notch signaling.

Splenic Ly6Chi monocytes are critical players in dystrophic muscle injury and repair.

Dystrophic muscle is characterized by chronic injury and a steady recruitment of inflammatory Ly6Chi monocytes. Recent studies have identified the spleen as the dominant reservoir of these cells during chronic inflammation. Here, we investigated the contribution of splenic Ly6Chi monocytes to dystrophic muscle pathology. Using the mdx mouse model of muscular dystrophy, we show that Ly6Chi monocytes accumulate in great numbers in the spleen over the course of the disease. The chemokine receptor CCR2 was upregulated on Ly6Chi monocytes in mdx spleen before disease onset, thereby enabling their recruitment to dystrophic muscle. Splenectomy performed before disease onset significantly reduced the number of Ly6Chi monocytes infiltrating dystrophic limb muscle. Moreover, in the absence of splenic Ly6Chi monocytes there was a significant reduction in dystrophic muscle inflammation and necrosis, along with improved regeneration during early disease. However, during late disease, a lack of splenic Ly6Chi monocytes adversely affected muscle fiber repair, due to a delay in the phenotypic shift of proinflammatory F4/80+Ly6ChiCD206lo to antiinflammatory F4/80+Ly6CloCD206+ macrophages. Overall, we show that the spleen is an indispensable source of Ly6Chi monocytes in muscular dystrophy and that splenic monocytes are critical players in both muscle fiber injury and repair.

Macrophages fine tune satellite cell fate in dystrophic skeletal muscle of mdx mice.

Satellite cells (SCs) are muscle stem cells that remain quiescent during homeostasis and are activated in response to acute muscle damage or in chronic degenerative conditions such as Duchenne Muscular Dystrophy. The activity of SCs is supported by specialized cells which either reside in the muscle or are recruited in regenerating skeletal muscles, such as for instance macrophages (MΦs). By using a dystrophic mouse model of transient MΦ depletion, we describe a shift in identity of muscle stem cells dependent on the crosstalk between MΦs and SCs. Indeed MΦ depletion determines adipogenic conversion of SCs and exhaustion of the SC pool leading to an exacerbated dystrophic phenotype. The reported data could also provide new insights into therapeutic approaches targeting inflammation in dystrophic muscles.

Disruption of MEK/ERK/c-Myc signaling radiosensitizes prostate cancer cells in vitro and in vivo.

PURPOSE: Prostate cancer (PCa) cell radioresistance causes the failure of radiation therapy (RT) in localized or locally advanced disease. The aberrant accumulation of c-Myc oncoprotein, known to promote PCa onset and progression, may be due to the control of gene transcription and/or MEK/ERK-regulated protein stabilization. Here, we investigated the role of MEK/ERK signaling in PCa. METHODS: LnCAP, 22Rv1, DU145, and PC3 PCa cell lines were used in in vitro and in vivo experiments. U0126, trametinib MEK/ERK inhibitors, and c-Myc shRNAs were used. Radiation was delivered using an x-6 MV photon linear accelerator. U0126 in vivo activity alone or in combination with irradiation was determined in murine xenografts. RESULTS: Inhibition of MEK/ERK signaling down-regulated c-Myc protein in PCa cell lines to varying extents by affecting expression of RNA and protein, which in turn determined radiosensitization in in vitro and in vivo xenograft models of PCa cells. The crucial role played by c-Myc in the MEK/ERK pathways was demonstrated in 22Rv1 cells by the silencing of c-Myc by means of short hairpin mRNA, which yielded effects resembling the targeting of MEK/ERK signaling. The clinically approved compound trametinib used in vitro yielded the same effects as U0126 on growth and C-Myc expression. Notably, U0126 and trametinib induced a drastic down-regulation of BMX, which is known to prevent apoptosis in cancer cells. CONCLUSIONS: The results of our study suggest that signal transduction-based therapy can, by disrupting the MEK/ERK/c-Myc axis, reduce human PCa radioresistance caused by increased c-Myc expression in vivo and in vitro and restores apoptosis signals.

Do neurogenic and cancer-induced muscle atrophy follow common or divergent paths?

Skeletal muscle is a dynamic tissue capable of responding to a large variety of physiological stimuli by adjusting muscle fiber size, metabolism and function. However, in pathological conditions such as cancer and neural disorders, this finely regulated homeostasis is impaired leading to severe muscle wasting, reduced muscle fiber size (atrophy), and impaired function. These disease features develop due to enhanced protein breakdown, which relies on two major degradation systems: the ubiquitin-proteasome and the autophagy-lysosome. These systems are independently regulated by different signalling pathways, which in physiological conditions, determine protein and organelle turnover. However, alterations in one or both systems, as it happens in several disorders, leads to enhanced protein breakdown and muscle atrophy. Although this is a common feature in the different types of muscle atrophy, the relative contribution of each of these systems is still under debate. Here, we will briefly describe the regulation and the activity of the ubiquitin-proteasome and the autophagy-lysosome systems during muscle wasting. We will then discuss what we know regarding how these pathways are involved in cancer induced and in neurogenic muscle atrophy, highlighting common and divergent paths. It is now clear that there is no one unifying common mechanism that can be applied to all models of muscle loss. Detailed understanding of the pathways and proteolysis mechanisms involved in each model will hopefully help the development of drugs to counteract muscle wasting in specific conditions.

Phosphotyrosine phosphatase inhibitor bisperoxovanadium endows myogenic cells with enhanced muscle stem cell functions via epigenetic modulation of Sca-1 and Pw1 promoters.

Understanding the regulation of the stem cell fate is fundamental for designing novel regenerative medicine strategies. Previous studies have suggested that pharmacological treatments with small molecules provide a robust and reversible regulation of the stem cell program. Previously, we showed that treatment with a vanadium compound influences muscle cell fatein vitro In this study, we demonstrate that treatment with the phosphotyrosine phosphatase inhibitor bisperoxovanadium (BpV) drives primary muscle cells to a poised stem cell stage, with enhanced function in muscle regenerationin vivofollowing transplantation into injured muscles. Importantly, BpV-treated cells displayed increased self-renewal potentialin vivoand replenished the niche in both satellite and interstitial cell compartments. Moreover, we found that BpV treatment induces specific activating chromatin modifications at the promoter regions of genes associated with stem cell fate, includingSca-1andPw1 Thus, our findings indicate that BpV resets the cell fate program by specific epigenetic regulations, such that the committed myogenic cell fate is redirected to an earlier progenitor cell fate stage, which leads to an enhanced regenerative stem cell potential.-Smeriglio, P., Alonso-Martin, S., Masciarelli, S., Madaro, L., Iosue, I., Marrocco, V., Relaix, F., Fazi, F., Marazzi, G., Sassoon, D. A., Bouché, M. Phosphotyrosine phosphatase inhibitor bisperoxovanadium endows myogenic cells with enhanced muscle stem cell functionsviaepigenetic modulation of Sca-1 and Pw1 promoters.

Cavin-1 and Caveolin-1 are both required to support cell proliferation, migration and anchorage-independent cell growth in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a childhood soft tissue tumor with broad expression of markers that are typically found in skeletal muscle. Cavin-1 is a recently discovered protein actively cooperating with Caveolin-1 (Cav-1) in the morphogenesis of caveolae and whose role in cancer is drawing increasing attention. Using a combined in silico and in vitro analysis here we show that Cavin-1 is expressed in myogenic RMS tumors as well as in human and primary mouse RMS cultures, exhibiting a broad subcellular localization, ranging from nuclei and cytosol to plasma membrane. In particular, the coexpression and plasma membrane interaction between Cavin-1 and Cav-1 characterized the proliferation of human and mouse RMS cell cultures, while a downregulation of their expression levels was observed during the myogenic differentiation. Knockdown of Cavin-1 or Cav-1 in the human RD and RH30 cells led to impairment of cell proliferation and migration. Moreover, loss of Cavin-1 in RD cells impaired the anchorage-independent cell growth in soft agar. While the loss of Cavin-1 did not affect the Cav-1 protein levels in RMS cells, Cav-1 overexpression and knockdown triggered a rise or depletion of Cavin-1 protein levels in RD cells, respectively, in turn reflecting on increased or decreased cell proliferation, migration and anchorage-independent cell growth. Collectively, these data indicate that the interaction between Cavin-1 and Cav-1 underlies the cell growth and migration in myogenic tumors.

From innate to adaptive immune response in muscular dystrophies and skeletal muscle regeneration: the role of lymphocytes.

Skeletal muscle is able to restore contractile functionality after injury thanks to its ability to regenerate. Following muscle necrosis, debris is removed by macrophages, and muscle satellite cells (MuSCs), the muscle stem cells, are activated and subsequently proliferate, migrate, and form muscle fibers restoring muscle functionality. In most muscle dystrophies (MDs), MuSCs fail to properly proliferate, differentiate, or replenish the stem cell compartment, leading to fibrotic deposition. However, besides MuSCs, interstitial nonmyogenic cells and inflammatory cells also play a key role in orchestrating muscle repair. A complete understanding of the complexity of these mechanisms should allow the design of interventions to attenuate MDs pathology without disrupting regenerative processes. In this review we will focus on the contribution of immune cells in the onset and progression of MDs, with particular emphasis on Duchenne muscular dystrophy (DMD). We will briefly summarize the current knowledge and recent advances made in our understanding of the involvement of different innate immune cells in MDs and will move on to critically evaluate the possible role of cell populations within the acquired immune response. Revisiting previous observations in the light of recent evidence will likely change our current view of the onset and progression of the disease.

Knock down of caveolin-1 affects morphological and functional hallmarks of human endothelial cells.

Caveolin-1 (CAV1) is the principal structural component of caveolae which functions as scaffolding protein for the integration of a variety of signaling pathways. In this study, we investigated the involvement of CAV1 in endothelial cell (EC) functions and show that siRNA-induced CAV1 silencing in the human EC line EA.hy926 induces distinctive morphological changes, such as a marked increase in cell size and formation of stress fibers. Design-based stereology was employed in this work to make unbiased quantification of morphometric properties such as volume, length, and surface of CAV1 silenced versus control cells. In addition, we showed that downregulation of CAV1 affects cell cycle progression at G1/S phase transition most likely by perturbation of AKT signaling. With the aim to assess the contribution of CAV1 to typical biological processes of EC, we report here that CAV1 targeting affects cell migration and matrix metalloproteinases (MMPs) activity, and reduces angiogenesis in response to VEGF, in vitro. Taken together our data suggest that the proper expression of CAV1 is important not only for maintaining the appropriate morphology and size of ECs but it might represent a prospective molecular target for studying key biological mechanisms such as senescence and tumorigenesis.

Intracellular signaling in ER stress-induced autophagy in skeletal muscle cells.

Skeletal muscle remodeling in response to muscle disuse and unloading is known to be associated with so-called ER stress, which, in turn, activates autophagy and contributes to muscle atrophy. Different molecules are involved in ER stress-induced autophagy, among which PKCθ has recently been described. In this study, we dissected both in vitro and in vivo ER stress-induced autophagy pathways in muscle. Using C2C12 muscle cells in culture, we demonstrated that PKC activation induced autophagy in the absence of ER stress. We further demonstrated that PKCθ was strongly activated in cultured myoblasts and myotubes during ER stress induced by different stimuli, such as TG or TN treatment, and that it localized into Lc3-positive autophagic dots upon TG treatment. Neither Akt dephosphorylation nor Foxo or GSK3ß activation was observed in these conditions. Moreover, PKCθ inhibition in myoblasts and myotubes prevented ER stress-induced Lc3 activation and autophagic dot formation, but not ER stress. In vivo, lack of PKCθ prevented both food deprivation- and immobilization-induced autophagy and muscle atrophy, irrespective of Akt pathway inhibition. Taken together, these results demonstrate that PKCθ functions as an ER stress sensor in skeletal muscle, required for ER-stress-dependent autophagy activation, and can be proposed as a novel molecular target to maintain muscle homeostasis in response to external stimuli, such as disuse and unloading, still allowing intracellular clearance.

PKC theta ablation improves healing in a mouse model of muscular dystrophy.

Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, limiting muscle regeneration and resulting in fibrotic and fatty tissue replacement of muscle, which exacerbates the wasting process in dystrophic muscles. Limiting immune response is thus one of the therapeutic options to improve healing, as well as to improve the efficacy of gene- or cell-mediated strategies to restore dystrophin expression. Protein kinase C θ (PKCθ) is a member of the PKCs family highly expressed in both immune cells and skeletal muscle; given its crucial role in adaptive, but also innate, immunity, it is being proposed as a valuable pharmacological target for immune disorders. In our study we asked whether targeting PKCθ could represent a valuable approach to efficiently prevent inflammatory response and disease progression in a mouse model of muscular dystrophy. We generated the bi-genetic mouse model mdx/θ(-/-), where PKCθ expression is lacking in mdx mice, the mouse model of Duchenne muscular dystrophy. We found that muscle wasting in mdx/θ(-/-) mice was greatly prevented, while muscle regeneration, maintenance and performance was significantly improved, as compared to mdx mice. This phenotype was associated to reduction in inflammatory infiltrate, pro-inflammatory gene expression and pro-fibrotic markers activity, as compared to mdx mice. Moreover, BM transplantation experiments demonstrated that the phenotype observed was primarily dependent on lack of PKCθ expression in hematopoietic cells.These results demonstrate a hitherto unrecognized role of immune-cell intrinsic PKCθ activity in the development of DMD. Although the immune cell population(s) involved remain unidentified, our findings reveal that PKCθ can be proposed as a new pharmacological target to counteract the disease, as well as to improve the efficacy of gene- or cell- therapy approaches.

Thyroid hormone T3 counteracts STZ induced diabetes in mouse.

This study intended to demonstrate that the thyroid hormone T3 counteracts the onset of a Streptozotocin (STZ) induced diabetes in wild type mice. To test our hypothesis diabetes has been induced in Balb/c male mice by multiple low dose Streptozotocin injection; and a group of mice was contemporaneously injected with T3. After 48 h mice were tested for glucose tolerance test, insulin serum levels and then sacrificed. Whole pancreata were utilized for morphological and biochemical analyses, while protein extracts and RNA were utilized for expression analyses of specific molecules. The results showed that islets from T3 treated mice were comparable to age- and sex-matched control, untreated mice in number, shape, dimension, consistency, ultrastructure, insulin and glucagon levels, Tunel positivity and caspases activation, while all the cited parameters and molecules were altered by STZ alone. The T3-induced pro survival effect was associated with a strong increase in phosphorylated Akt. Moreover, T3 administration prevented the STZ-dependent alterations in glucose blood level, both during fasting and after glucose challenge, as well as in insulin serum level. In conclusion we demonstrated that T3 could act as a protective factor against STZ induced diabetes.

Synthetic sulfonyl-hydrazone-1 positively regulates cardiomyogenic microRNA expression and cardiomyocyte differentiation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are obtained from adult cells through overexpression of pluripotency factors. iPSCs share many features with embryonic stem cells (ESCs), circumventing ethical issues, and, noteworthy, match donor's genotype. iPSCs represent therefore a valuable tool for regenerative medicine. Cardiac differentiation of ESCs can be enhanced via microRNAs (miRNAs) and small chemical compounds, which probably act as chromatin remodelers. Cardiomyogenic potential of iPSCs is currently intensely investigated for cell therapy or in vitro drug screening and disease modeling. However, influences of small compounds on iPSC-related cardiomyogenesis have not yet been investigated in details. Here, we compared the effects of two small molecules, bis-peroxo-vanadium (bpV) and sulfonyl-hydrazone-1 (SHZ) at varying concentrations, during cardiac differentiation of murine iPSCs. SHZ (5 µM) enhanced specific marker expression and cardiomyocyte yield, without loss of cell viability. In contrast, bpV showed negligible effects on cardiac differentiation rate and appeared to induce Casp3-dependent apoptosis in differentiating iPSCs. Furthermore, SHZ-treated iPSCs were able to increase beating foci rate and upregulate early and late cardiomyogenic miRNA expression (miR-1, miR-133a, and miR-208a). Thus, our results demonstrate that small chemical compounds, such as SHZ, can constitute a novel and clinically feasible strategy to improve iPSC-derived cardiac differentiation.

PKCalpha-mediated ERK, JNK and p38 activation regulates the myogenic program in human rhabdomyosarcoma cells.

We have previously suggested that PKCalpha has a role in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediated growth arrest and myogenic differentiation in human embryonal rhabdomyosarcoma cells (RD). Here, by monitoring the signalling pathways triggered by TPA, we demonstrate that PKCalpha mediates these effects by inducing transient activation of c-Jun N-terminal protein kinases (JNKs) and sustained activation of both p38 kinase and extracellular signal-regulated kinases (ERKs) (all referred to as MAPKs). Activation of MAPKs following ectopic expression of constitutively active PKCalpha, but not its dominant-negative form, is also demonstrated. We investigated the selective contribution of MAPKs to growth arrest and myogenic differentiation by monitoring the activation of MAPK pathways, as well as by dissecting MAPK pathways using MEK1/2 inhibitor (UO126), p38 inhibitor (SB203580) and JNK and p38 agonist (anisomycin) treatments. Growth-arresting signals are triggered either by transient and sustained JNK activation (by TPA and anisomycin, respectively) or by preventing both ERK and JNK activation (UO126) and are maintained, rather than induced, by p38. We therefore suggest a key role for JNK in controlling ERK-mediated mitogenic activity. Notably, sarcomeric myosin expression is induced by both TPA and UO126 but is abrogated by the p38 inhibitor. This finding indicates a pivotal role for p38 in controlling the myogenic program. Anisomycin persistently activates p38 and JNKs but prevents myosin expression induced by TPA. In accordance with this negative role, reactivation of JNKs by anisomycin, in UO126-pre-treated cells, also prevents myosin expression. This indicates that, unlike the transient JNK activation that occurs in the TPA-mediated myogenic process, long-lasting JNK activation supports the growth-arrest state but antagonises p38-mediated myosin expression. Lastly, our results with the MEK inhibitor suggest a key role of the ERK pathway in regulating myogenic-related morphology in differentiated RD cells.