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 gamma co-activator 1alpha (PGC1alpha) 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 PGC1alpha 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, PGC1alpha-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.

Transgenic mice with dominant negative PKC-theta in skeletal muscle: a new model of insulin resistance and obesity.

Protein kinase C theta (PKC-theta) 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 TGFbeta, nerve-dependent signals and insulin. To study the role of PKC-theta in these mechanisms we generated transgenic mice expressing a "kinase dead" mutant form of PKC-theta (PKC-thetaK/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-theta in skeletal muscle causes obesity associated to insulin resistance, as demonstrated by defective receptor and post-receptorial activation of signaling cascade.

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

Transforming growth factor beta (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-beta autocrine loop in regulating growth and myogenic differentiation in the human rhabdomyosarcoma cell line, RD. We previously reported that the phorbol ester 12-O-tetradecanoylphorbol-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-beta, thus decreasing the concentration of active TGF-beta 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-beta, mediated by the ectopic expression of a soluble type II TGF-beta receptor dominant negative cDNA, induces growth arrest, but does not trigger differentiation. In contrast, a reduction in the TGF-beta concentration, to a range of 0.14-0.20 x 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-beta; furthermore, they show that a 'critical' concentration of TGF-beta is necessary for myogenic differentiation to occur, whereas myogenesis is abolished below and above this concentration. By impairing the TGF-beta 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-beta is required for the differentiation inhibitory effect and TPA inhibits differentiation in these cells probably through a TGF-beta independent mechanism. These data thus clarify the mechanism underlying the multiple roles of TGF-beta in the regulation of both the transformed and differentiated phenotype.

Isolation and characterization of the murine zinc finger coding gene, ZT2: expression in normal and transformed myogenic cells.

In the context of a project aimed at the identification of zinc finger proteins involved in skeletal muscle histogenesis and differentiation, we isolated a murine gene, named ZT2. The 2.44kb partial cDNA clone corresponds to the 3' region of the gene, and contains a 0.54kb open reading frame encoding four C2H2-like zinc finger domains, organized in tandem. This cDNA hybridizes with multiple transcripts (2, 4.5 and 7kb), whose expression levels vary in different tissues and at different developmental stages in the same tissue. At least in skeletal muscle we observed differences in the polyadenylation state of the transcripts at different stages of development. Moreover, ZT2 expression is correlated with cell proliferation and transformation. Sequence analysis and genetic mapping indicate that ZT2 is the homologue of ZNF125, one of the linked zinc finger encoding genes localized on human Chr 11q23. In humans, a high frequency of tumor-associated translocations is found in this chromosome region. As expected, ZT2 maps to the corresponding region on chromosome 9 in the mouse.

Rapid activation and down-regulation of protein kinase C alpha in 12-O-Tetradecanoylphorbol-13-acetate-induced differentiation of human rhabdomyosarcoma cells.

Human rhabdomyosarcoma RD cells express the myogenic regulatory factors MyoD and myogenin but differentiate spontaneously very poorly. Prolonged treatment of RD cells with the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA) induces growth arrest and myogenic differentiation as shown by the accumulation of alpha-actin and myosin light and heavy chains, without affecting the expression of MyoD and myogenin. In this study, we show that short-term phorbol ester treatment of the cultures is sufficient to trigger myogenic differentiation but not growth arrest. Furthermore, PKC inhibitors, such as staurosporine or calphostin C, prevent TPA-induced differentiation but not cell growth arrest. These data suggest that the two events are mediated by different pathways; a possible interpretation is that the activation of one or more PKC isoforms mediates the induction of differentiation, whereas the down-regulation of the same or different isoforms mediates the growth arrest. To address the mechanism whereby TPA affects cell growth and differentiation in RD cells, we first analyzed PKC isoenzyme distribution. We found that RD cells express the alpha, beta 1, gamma, and sigma PKC isoenzymes. Only the alpha isoform is exclusively found in the soluble fraction, but it translocates to the membrane fraction within 5 min of TPA treatment and is completely down-regulated after 6 h. The other isoenzymes are found associated to both the soluble and the particulate fractions and are down-regulated after long-term TPA treatment. By immunofluorescence analysis, we show that the PKC alpha down-regulation is specific for those cells that respond to TPA by activating the muscle phenotype. We propose that TPA-induced differentiation in RD cells is mediated by the transient activation of PKC alpha, which activates some of the intracellular events that are necessary for MyoD and myogenin transacting activity and for the induction of terminal differentiation of RD cells. By contrast, the constitutively active beta 1 and sigma are responsible for the maintenance of cell growth, and their down-regulation is responsible for long-term TPA-induced cell growth arrest.

TPA-induced differentiation of human rhabdomyosarcoma cells: expression of the myogenic regulatory factors.

RD cells (a cell line derived from a human rhabdomyosarcoma) undergo a very limited myogenic differentiation despite the fact that they express several myogenic determination genes. Since we have previously shown (Aguanno et al., Cancer Res. 50, 3377, 1990) that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces myogenic differentiation in these cells, in this paper we investigate the mechanism by which TPA interferes with the expression and/or function of the myogenic determination genes. Northern blot analysis revealed that RD cells express the myf3 (the human analog of MyoD) and myf4 (the human analog of myogenin) transcripts, but not myf5 or myf6 transcripts. The myf3 and the myf4 gene products are correctly translated and accumulated in the nuclei as shown by immunofluorescence analysis. The tumor promoter (TPA) does not modify the pattern of expression of the myf factors while it induces the accumulation of muscle-specific transcripts, such as alpha-actin and fast myosin light chain 1, and their corresponding proteins. On the other hand, within 1 day of treatment, TPA inhibits the expression of the Id gene, which is a negative regulator of MyoD activity. However, while the TPA-induced inhibition of Id message accumulation correlates with differentiation, cell confluence also causes a reduction in Id message accumulation, without inducing differentiation. Under our experimental conditions, overexpression of any of the myf cDNAs in RD cells does induce spontaneous differentiation but enhances the effect of TPA treatment independently from the level of the expressed message. These data suggest that differentiation of RD cells is likely to depend upon the activity of complexes containing the various members of the MyoD family, which can be regulated by proteins affecting MyoD dimerization such as Id, but also by other mechanisms induced by TPA, such as phosphorylation.

Altered protein phosphorylation in murine muscular dystrophy.

Protein phosphorylation has been studied in the dydy murine muscular dystrophy, both in intact muscle cells and in various membrane fractions derived from them. The results obtained showed that several polypeptides were more heavily phosphorylated in dystrophic myotubes in culture as well as in dystrophic muscle fibers isolated from tibialis anterior. In vitro phosphorylation studies revealed that a large polypeptide of apparent molecular weight of 170,000-150,000 was phosphorylated under basal conditions (3 mM EGTA) in dydy microsomal membranes. The phosphorylation of this polypeptide was not stimulated further by cAMP, calmodulin, cGMP or 12-O-tetradecanoylphorbol 13-acetate (TPA). Under no condition was the corresponding polypeptide phosphorylated at an appreciable rate in normal microsomal membranes. An antibody raised against the voltage-dependent calcium channel reacted, in an immunoblot assay, with a polypeptide, present in both normal and dydy microsomes, which had migration characteristics identical to the phosphorylated 170-150 kDa polypeptide after one- or two-dimensional gel electrophoresis. Additional differences were identified in the phosphorylation of smaller polypeptides of microsomal membranes. When sarcolemmal membranes of normal and dydy muscle were phosphorylated in vitro, no major differences were observed. These results show the existence of an alteration of protein phosphorylation in dystrophic muscle cells in vitro and in vivo, leading to abnormal phosphorylation of the voltage-dependent calcium channel. The possible causes and consequences of this alteration are discussed.

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.

'Early' mammalian myoblasts are resistant to phorbol ester-induced block of differentiation.

Mesenchymal cells were isolated from somites and limbs of mouse embryos at different developmental stages. When grown in tissue culture, some of the cells underwent muscle differentiation as indicated by synthesis of sarcomeric myosin, acetylcholine receptor and, in the case of limb cells, fusion into multinucleated myotubes. When the tumour promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) was added to these cultures, it caused differential effects, depending upon the age of the embryo from which cells were isolated. In cultures of somites or limb bud from embryos up to 12 days post coitum, TPA did not interfere with the appearance of differentiated muscle cells. When TPA was added to cultures from older embryos, it inhibited muscle differentiation with an efficiency which increased with the age of the embryo, reaching about 90% inhibition at 15 days. After this period, a new population of myogenic cells appeared in the limb, which were able to differentiate in the presence of TPA and represented the great majority of myoblasts after day 18 of embryonic development. The simplest interpretation of these data can be based on the existence of three major classes of myogenic cell precursors, which appear sequentially during muscle histogenesis: 'early' myoblasts, which appear resistant to tumour promoters; 'late' myoblasts, whose differentiation is inhibited by tumour promoters and 'satellite' cells which, like early myoblasts, show no sensitivity to TPA.

Expression of adult fast pattern of acetylcholinesterase molecular forms by mouse satellite cells in culture.

The pattern of acetylcholinesterase (AChE) molecular forms, obtained by sucrose gradient sedimentation, was studied at different in vitro developmental stages of myogenic cells isolated from adult mouse skeletal muscle. Only the globular forms were present in rapidly dividing satellite cells during the first days in culture. After myotube formation, a pattern similar to that described in mammalian fast-twitch skeletal muscle was observed. This pattern did not change during the following period in culture (up to 1 month) nor could it be modified by co-culturing with spinal cord motoneurons or by addition of brain-derived extracts. The internal-external localization of AChE molecular forms has been determined by the use of echothiophate iodide, a membrane-impermeant irreversible inhibitor of AChE. Echothiophate-treated cultures showed about 40% of both asymmetric and globular forms localized on the sarcolemma, with their active sites oriented outward. Analysis of culture medium from untreated cultures revealed the presence of both asymmetric and globular forms. When the same analysis was repeated on cultures of myoblasts derived from 16-day-old mouse embryos, the pattern of AChE forms was different. The myotubes derived from these cells exhibit a very small proportion of asymmetric form, which was not released into the medium. This pattern was not further modified during the following days of culture, nor by co-cultures with spinal cord motoneurons or by incubations with brain-derived extracts. Thus, the myotubes derived from myoblasts express in culture a clear phenotypic difference when compared to the corresponding myotubes from satellite cells, supporting the view that these two myogenic cells are endowed with different developmental programs.

Effect of phorbol esters and liposome-delivered phospholipids on the differentiation program of normal and dystrophic satellite cells.

Satellite cells, isolated from hind limb of normal C57BL/6J mice, differentiate in culture in the presence of concentrations of phorbol esters which inhibit differentiation of embryonic myoblasts. However, if phosphatidylserine containing liposomes were added to the culture medium together with TPA, differentiation of satellite cells was reversibly inhibited. Under these conditions, the withdrawal of these cells from the cell cycle still occurred as in untreated cells. Phosphatidylserine liposomes alone or liposomes containing phosphatidylcholine (either alone or in combination with TPA) had no effect on satellite cell differentiation. In the case of satellite cells from dystrophic C57BL/6J/dydy mice, TPA addition (0.1 microM) to the culture medium partially (about 70%) inhibited morphological and biochemical differentiation. This effect could be prevented by preincubating dystrophic satellite cells with liposomes containing phosphatidylcholine but not other phospholipids. These data indicate that it is possible to change the sensitivity to TPA of satellite cells by modifying the phospholipid composition of their plasma membrane. Possible relationships of these phenomena with activation of protein kinase C or phosphatidylinositol breakdown have been investigated. The results obtained are discussed with regard to possible modulation of the intracellular response to agonist binding.

Altered distribution of protein kinase C in dystrophic muscle cells and its modulation by liposome-delivered phospholipids.

The activity and subcellular distribution of the calcium-phospholipid dependent protein kinase (protein kinase C) were studied in normal and dystrophic muscle cells in vitro. Clonal strains of satellite cells, isolated from normal and dystrophic (C57BL/6J/dydy) mice, differentiate in vitro at a comparable level (over 80% of fusion). Differentiated myotubes were homogenized and separated into a soluble and a particulate fraction. The activity of protein kinase C was assayed in both fractions, and was found to be mainly in the cytosol of normal cells, whereas it was mainly associated to the membrane fraction of dystrophic cells. This altered distribution of the enzyme was likely consequent to alterations in the phospholipid composition of the dystrophic cell membrane, since it was possible to partially revert the situation by modifying the membranes with liposome-delivered phospholipids. Splenic lymphocytes from dystrophic mice showed an altered distribution of protein kinase C similar to that observed in muscle cells. The possible biochemical basis and the functional consequences of this altered distribution of the enzyme in the dystrophic cells are discussed.

A muscle cell line from dystrophic mice expressing an altered phenotype in vitro.

The isolation and characterization of a myogenic cell line from C57BL/6J/dydy mice is described. This line (DyA4) maintains the morphological, biochemical and electrophysiological characteristics of the primary cultured cells, at least for 20 passages. The cells actively divide as long as they are subcultured in media supplemented with horse serum and embryo extract. If the cells are not subcultured for a few days, they fuse into multinucleated contracting myotubes, which readily synthesize specific muscle products such as acetylcholinesterase and acetylcholine receptor. This dystrophic cell line expresses in vitro the same altered phenotype that is characteristic of dystrophic muscle cells in primary cultures, namely reduced acetylcholine sensitivity and reduced acetylcholine receptor expression. Because they can be grown in large amounts, and represent a pure muscle cell population which express an altered phenotype in an in vitro aneural avascular environment, DyA4 cells provide a very useful model system for investigating the pathogenesis of murine muscular dystrophy.

Acetylcholine stimulates phosphatidylinositol turnover at nicotinic receptors of cultured myotubes.

Acetylcholine treatment of [3H]inositol pre-labelled cultured chick embryo myotubes results in the stimulation of phosphatidylinositol breakdown, as shown by the measurement of inositol-1-phosphate accumulating in the presence of lithium. The described effect is dependent on agonist concentration and incubation time, and is inhibited by tubocurarine and alpha-bungarotoxin. The activation of phosphatidylinositol breakdown by acetylcholine at extrajunctional nicotinic receptors is likely to be involved in the modulation of the functional activity of the receptor.

Increased endocytosis of acetylcholine receptors by dystrophic mouse myotubes in vitro.

Multinucleated myotubes, grown in vitro from satellite cells of dystrophic mice (C57BL/6J/dydy) exhibit a reduced sensitivity to ACh. This reduction correlates with a reduced density of 125I-alpha-bungarotoxin (125I-BTX) binding sites on the surface of dystrophic myotubes. Denervated adult muscle fibers from dystrophic mice respond to Ach similarly to denervated normal muscle fibers. Furthermore, cultured dystrophic myotubes, treated with a brain extract which induces AChR clusterization, still show an impaired response to ACh and reduced 125I-BTX binding. Thus AChR function appears altered in dystrophic muscle cells in culture while it appears normal in dystrophic adult muscle, regardless of whether the receptors are dispersed on the membrane or clustered at the junctional site. Metabolic studies on the reduced AChR level in dystrophic myotubes revealed a dramatically reduced half-life (2 vs 10 hr) while the rate of synthesis was unchanged. An increased rate of internalization of AChR was observed in dystrophic myotubes with a corresponding relative increase of the "hidden AChR pool," which could be partially reduced by agents which disrupt the cytoskeleton. No structural alterations could be detected on the AChR molecule as its sedimentation coefficient and subunit composition appeared identical between normal and dystrophic myotubes. Thus the increased turnover of AChR in dystrophic myotubes either reflects subtle alterations of the molecule or a more generalized increase of endocytosis in this form of myopathy.

Membrane acetylcholinesterase in murine muscular dystrophy In vivo and in cultured myotubes.

Murine muscular dystrophy is characterized by a reduction of the 10S molecular form of acetylcholinesterase (AChE); this reduction occurs in both strains of dystrophic mice and at the time of the phenotypic appearance of the disease. In the present study we have analyzed the biochemical features, the cellular distribution and the developmental appearance of the AChE alteration. Sequential extractions with low salt, detergent and high salt revealed that this alteration affects only membrane-bound forms (those requiring Triton X-100 for solubilization), while both the low salt soluble and the high salt soluble forms appeared almost identical in normal and dystrophic muscles. Specific activity, sensitivity to different ions, pH dependence and Km were found to be identical in the enzymes from normal and dystrophic muscles, suggesting that the catalytic site of the 10S form is probably not altered. Further analysis, by non-denaturing gel electrophoresis, of the detergent soluble forms separated by sedimentation, revealed a single band for the 4S, a doublet for the 6S and three bands for the 10S peaks, indicating the existence of charge heterogeneity in AChE molecular forms. The corresponding molecular forms from dystrophic muscles behaved identically upon electrophoresis: the residual activity in the detergent soluble 10S form could still be separated into three bands, comigrating with their normal counterparts. Neuraminidase treatment resulted in a reduction of migration of both the 6S and 10S derived bands, but not of the 4S species, showing that sialic acid is added only to polymeric forms. Interestingly, the reduction of the 10S form appears to be linked to a developmental stage not reached in cell cultures, as cultured myotubes from muscles of dystrophic mice contained normal amounts of membrane-bound AChE forms. The molecular mechanism underlying the reduction of the tetrameric membrane bound AChE form in dystrophic muscle and the possible functional consequences are discussed.

Acetylcholinesterase activity and molecular forms during denervation and reinnervation in extensor digitorum longus muscle of the rat.

The four principal molecular forms of acetylcholinesterase characteristic of the mammalian muscle (16.1 S., 12.5 S, 10.2 S, and 3.6. S) were identified by sucrose gradient sedimentation as the four activity peaks H, H1, M and L. After denervation obtained by crushing the sciatic nerve five stages of the denervation-reinnervation process were examined. Days 7, 14, 22, 30, and 60 were chosen on the basis of previous electrophysiological and histochemical studies. The AChE activity showed an initial drop followed by recovery after nerve arrival at the muscle which was completed by day 60. Marked changes in the relative proportions of the four molecular forms were observed. The 16.1 S almost disappeared during the denervation period, reappeared after nerve arrival and was completely restored at day 60. Changes were also observed in the intermediate and lower forms and were tentatively related to processes of degradation, reaggregation and de novo synthesis. A comparison of the present data with those from parallel electrophysiological and histochemical studies suggests the presence and the functional role of molecular forms other than 16S in the neuromuscular junction.

Black widow spider toxin-induced calcium fluxes and transmitter release in a neurosecretory cell line.

Several polypeptide neurotoxins affect presynaptic functions by interfering with chemical neurotransmission. This group of toxins includes botulinum toxin, tetanus toxin, beta-bungaro-toxin and black widow spider toxin (BWSTx). While the effect of the first three toxins is mainly a rapid and severe block of neurotransmitter release, BWSTx affects transmission by a massive stimulation of mediator release. Despite various hypotheses put forward to explain the action of BWSTx at the level of nerve terminals, there is still a considerable degree of uncertainty as to the cation dependence of venom action. Study of the toxin mode of action at the biochamical level has been hampered by the complexity and cellular heterogeneity of the preparations used, neuromuscular junction or synaptosomes. PC12 cell line, derived from a rat phaeochromocytoma, seems to be an excellent model in view of its property of synthesising and storing noradrenaline, dopamine and acetylcholine, and releasing them in depolarising conditions. We have recently shown that highly purified BWSTx stimulates secretion from PC12 cells of previously taken up radioactive dopamine (DA) and noradrenaline (NA) (ref. 14 and manuscript in preparation). We report here that the earliest detectable event after toxin treatment of such cells is a massive increase of cytosolic calcium.