Marina Bouché

In vitro differentiation of satellite cells isolated from normal and dystrophic mammalian muscles. A comparison with embryonic myogenic cells

Satellite cells were isolated from skeletal muscles of adult normal and dystrophic mice (C57/6J/dy strain) by sequential digestion of tissue fragments with collagenase, hyaluronidase and trypsin. These cells exhibit in culture similar behaviour to that of embryonic myoblasts, undergoing an initial duplicative period lasting about 2--3 days, followed by a shorter phase (1--2 days) of rapid cell fusion. During the duplicative phase most of the satellite cells appear round-shaped, whereas embryonic myoblasts appear typically spindle-shaped: both cell types actively incorporate [3H]thymidine. During the subsequent days of culture an increasing number of satellite cells becomes spindle-shaped; afterwards the cells contact each other and fuse into multinucleated myotubes. The majority of spindle-shaped satellite cells is unable to incorporate [3H]thymidine, thus behaving as post-mitotic cells. Concomitantly with satellite cell fusion, an increase of about 80-fold of creatine phosphokinase (CPK) specific activity is observed. Satellite cells are able to recognize co-cultured embryonic myoblasts ([3H]thymidine-labelled): hybrid myotubes containing labelled and unlabelled nuclei are formed in these experimental conditions. Satellite cells from dystrophic animals are able to differentiate in culture and do not show appreciable differences as compared to their normal counterparts. In dystrophic myotubes, however, CPK specific activity is almost twice that observed in normal myotubes. Human dystrophic satellite cells from biopsies of adult muscle cultured in similar conditions grow and fuse into multinucleated myotubes showing a behaviour identical to normal controls.

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

We have previously suggested that PKCα 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 PKCα 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 PKCα, 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.

Transgenic mice with dominant negative PKC‐theta in skeletal muscle: A new model of insulin resistance and obesity

Protein kinase C θ (PKC‐θ) is the PKC isoform predominantly expressed in skeletal muscle, and it is supposed to mediate many signals necessary for muscle histogenesis and homeostasis, such as TGFβ, nerve‐dependent signals and insulin. To study the role of PKC‐θ in these mechanisms we generated transgenic mice expressing a “kinase dead” mutant form of PKC‐θ (PKC‐θK/R), working as “dominant negative,” specifically in skeletal muscle. These mice are viable and fertile, however, by the 6–7 months of age, they gain weight, mainly due to visceral fat deposition. Before the onset of obesity (4 months of age), they already show increased fasting and fed insulin levels and reduced insulin‐sensitivity, as measured by ipITT, but normal glucose tolerance, as measured by ipGTT. After the 6–7 months of age, transgenic mice develop hyperinsulinemia in the fasting and fed state. The ipGTT revealed in the transgenic mice both hyperglycemia and hyperinsulinemia. At the molecular level, impaired activation of the IR/IRS/PI3K pathway and a significant decrease both in the levels and in insulin‐stimulated activation of the serine/threonine kinase Akt were observed. Taken together these data demonstrate that over‐expression of dominant negative PKC‐θ in skeletal muscle causes obesity associated to insulin resistance, as demonstrated by defective receptor and post‐receptorial activation of signaling cascade.

TGF-β autocrine loop regulates cell growth and myogenic differentiation in human rhabdomyosarcoma cells

Transforming growth factor β (TGF) is a well‐known inhibitor of myogenic differentiation as well as an autocrine product of rhabdomyosarcoma cells. We studied the role of the TGF‐β autocrine loop in regulating growth and myogenic differentiation in the human rhabdomyosarcoma cell line, RD. We previously reported that the phorbol ester 12‐O‐tetradeca‐noylphorbol‐13‐acetate (TPA) induces growth arrest and myogenic differentiation in these cells, which constitutively express muscle regulatory factors. We show that TPA inhibits the activation of secreted latent TGF‐β, thus decreasing the concentration of active TGF‐β to which the cells are exposed. This event is mediated by the TPA‐induced alteration of the uPA/ PAI serine‐protease system. Complete removal of TGF‐β, mediated by the ectopic expression of a soluble type II TGF‐β receptor dominant negative cDNA, induces growth arrest, but does not trigger differentiation. In contrast, a reduction in the TGF‐β concentration, to a range of 0.14–0.20 × 10−2 ng/ml (which is similar to that measured in TPA‐treated cells), mimics TPA‐induced differentiation. Taken together, these data demonstrate that cell growth and suppression of differentiation in rhabdomyosarcoma cells require overproduction of active TGF‐β; furthermore, they show that a ‘critical’ concentration of TGF‐β is necessary for myogenic differentiation to occur, whereas myogenesis is abolished below and above this concentration. By impairing the TGF‐β autocrine loop, TPA stabilizes the factor concentration within the range compatible for differentiation to occur. In contrast, in human primary muscle cells a much higher concentration of exogenous TGF‐β is required for the differentiation inhibitory effect and TPA inhibits differentiation in these cells probably through a TGF‐β independent mechanism. These data thus clarify the mechanism underlying the multiple roles of TGF‐β in the regulation of both the transformed and differentiated phenotype.—Bouche, M., Canipari, R., Mel‐chionna, R., Willems, D., Senni, M. I., Molinaro, M. TGF‐β autocrine loop regulates cell growth and myo‐genic differentiation in human rhabdomyosarcoma cells. FASEB J. 14, 1147–1158 (2000)

Adrenocorticotropin is a specific mitogen for mammalian myogenic cells.

Peptides derived from proopiomelanocortin (POMC) have been found to stimulate the proliferation of murine myogenic cells. Among these peptides, adrenocorticotropin (ACTH) and alpha-, beta-, and gamma-melanocyte-stimulating hormones (MSH) were found to be active, whereas the opioid peptides were not. At clonal density, both ACTH and MSH caused a three- to fourfold increase in the average number of cells per clone in myogenic but not in fibroblast colonies. At high cell density, ACTH and MSH caused a three- to fourfold increase in proliferation of myogenic cells, reflected by an increased accumulation of skeletal myosin. On the other hand mouse embryo skin or muscle fibroblasts or vertebral chondroblasts did not increase proliferation in response to POMC-derived peptides. The half-maximal dose at which ACTH stimulated myoblast proliferation was around 5 nM, and the mitogenic effect was doubled by suboptimal doses of fibroblast growth factor. The possible physiological significance of the mitogenic effect of ACTH on myogenic cells is discussed.

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.

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 GSK3β 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.—Madaro, L., Marrocco, V., Carnio, S., Sandri, M., Bouché, M. Intracellular signaling in ER stress‐induced autophagy in skeletal muscle cells. FASEB J. 27, 1990–2000 (2013). www.fasebj.org

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 sacrified. 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.

PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase

Using both in vivo and in vitro protein kinase C (PKC) θ mutant models, we found that PKCθ, the PKC isoform predominantly expressed in skeletal muscle, is required for myoblast fusion and myofiber growth, by regulating focal adhesion kinase activity and, in turn, the expression of the pro-fusion genes caveolin-3 and β1D-integrin.

Protein kinase C theta co-operates with calcineurin in the activation of slow muscle genes in cultured myogenic cells.

Adult skeletal muscle fibers can be divided into fast and slow twitch subtypes on the basis of specific contractile and metabolic properties, and on distinctive patterns of muscle gene expression. The calcium, calmodulin‐dependent protein phosphatase, calcineurin, stimulates slow fiber‐specific genes (myoglobin (Mb), troponin I slow) in cultured skeletal muscle cells, as well as in transgenic mice, through the co‐operation of peroxisome‐proliferation‐activator receptor γ co‐activator 1α (PGC1α) myocyte enhancer factor 2 (MEF2), and nuclear factor of activated T cells (NFAT) transcription factors. Specific protein kinase C isoforms have been shown to functionally co‐operate with calcineurin in different cellular models. We investigated whether specific protein kinase C isoforms are involved in calcineurin‐induced slow skeletal muscle gene expression. By pharmacological inhibition or exogenous expression of mutant forms, we show that protein kinase C theta (the protein kinase C isoform predominantly expressed in skeletal muscle) is required and co‐operates with calcineurin in the activation of the Mb promoter, as well as in the induction of slow isoforms of myosin and troponin I expression, in cultured muscle cells. This co‐operation acts primarily regulating MEF2 activity, as shown by using reporter gene expression driven by the Mb promoter mutated in the specific binding sites. MEF2 activity on the Mb promoter is known to be dependent on both PGC1α and inactivation of histone deacetylases (HDACs) activity. We show in this study that protein kinase C theta is required for, even though it does not co‐operate in, PGC1α‐dependent Mb activation. Importantly, protein kinase C theta regulates the HDAC5 nucleus/cytoplasm location. We conclude that protein kinase C theta ensures maximal activation of MEF2, by regulating both MEF2 transcriptional complex formation and HDACs nuclear export. J. Cell. Physiol. 207: 379–388, 2006.