M.I. Senni

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.

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 α‐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.

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.

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.