Protein kinase C theta (PKCθ) modulates the ClC-1 chloride channel activity and skeletal muscle phenotype: a biophysical and gene expression study in mouse models lacking the PKCθ

In skeletal muscle, the resting chloride conductance (gCl), due to the ClC-1 chloride channel, controls the sarcolemma electrical stability. Indeed, loss-of-function mutations in ClC-1 gene are responsible of myotonia congenita. The ClC-1 channel can be phosphorylated and inactivated by protein kinases C (PKC), but the relative contribution of each PKC isoforms is unknown. Here, we investigated on the role of PKCθ in the regulation of ClC-1 channel expression and activity in fast- and slow-twitch muscles of mouse models lacking PKCθ. Electrophysiological studies showed an increase of gCl in the PKCθ-null mice with respect to wild type. Muscle excitability was reduced accordingly. However, the expression of the ClC-1 channel, evaluated by qRT-PCR, was not modified in PKCθ-null muscles suggesting that PKCθ affects the ClC-1 activity. Pharmacological studies demonstrated that although PKCθ appreciably modulates gCl, other isoforms are still active and concur to this role. The modification of gCl in PKCθ-null muscles has caused adaptation of the expression of phenotype-specific genes, such as calcineurin and myocyte enhancer factor-2, supporting the role of PKCθ also in the settings of muscle phenotype. Importantly, the lack of PKCθ has prevented the aging-related reduction of gCl, suggesting that its modulation may represent a new strategy to contrast the aging process

Rapid Activation and Down-Regulation of Protein Kinase C a in 12-O-Tetradecanoyl phorbol-13-acetate-induced Differentiation of Human Rhabdomyosarcoma Cells.

Human rhabdomyosarcoma RD cells express the myogenic regulatory fadors MyoD and myogenin but differentiate spontaneously very poorly. Prolonged treatment of RD cells with the protein kinase C (PKC) adivator 1 2-O-tetradecanoylphorbol-1 3-acetate (TPA) induces growth arrest and myogenic differentiation as shown by the accumulation of a-adin and myosin light and heavy chains, without affeding the expression of MyoD and myogenin. In this study, we show that shortterm 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 adivation of one or more PKC isoforms mediates the indudion of differentiation, whereas the down-regulation of the same or different isoforms mediates the growth arrest. To address the mechanism whereby TPA affeds cell growth and differentiation in RD cells, we first analyzed PKC isoenzyme distribution. We found that RD cells express the a, ıi1, 7, and ı PKC isoenzymes. Only the a isoform is exclusively found in the soluble fradion, but it translocates to the membrane fradion within 5 mm of TPA treatment and is completely down-regulated after 6 h. The other isoenzymes are found associated to both the soluble and the particulate fradions and are downregulated after long-term TPA treatment. By immunofluorescence analysis, we show that the PKC a down-regulation is specific for those cells that respond to TPA by adivating the muscle phenotype. We propose that TPA-induced differentiation in RD cells is mediated by the transient adivation of PKC a, which adivates some of the intracellular events that are necessary for MyoD and myogenin transading adivity and for the indudion of terminal differentiation of RD cells. By contrast, the constitutively adive ı and ı are responsible for the maintenance of cell growth, and their down-regulation is responsible for long-term TPA-induced cell growth arrest

Differential response of embryonic and fetal myoblasts to TGF-beta: a possible regulatory mechanism of skeletal muscle histogenesis

Embryonic and fetal skeletal myoblasts were grown in culture in the presence of TGF beta. Under the conditions employed, TGF beta inhibited differentiation of fetal but not of embryonic myoblasts, To investigate the possible relevance of these data to skeletal muscle histogenesis in vivo, we studied the proliferation/differentiation state of mesodermal cells in the proximal region of the limb bud at the time of primary fiber formation. BrdU labeling and immunostaining for myosin heavy chains revealed that very few mesodermal cells enter the S phase of the cycle when differentiated primary fibers fist appear. However, a few hours later, many cells in S phase surround newly formed muscle fibers, suggesting that the latter may be a source of mitogens for undifferentiated myoblasts. Coculture experiments supported this hypothesis, showing that medium conditioned by fiber-containing explants can stimulate myoblast proliferation. Taken together these data suggested a possible mechanism for the regulation of muscle fiber formation. The model assumes that fibers form in the proximal region of the limb bud, where TGF beta is known to be present, and BrdU labeling experiments did not reveal cells in S phase. It is conceivable that non-dividing embryonic myoblasts (which do not respond to TGF beta) can undergo differentiation, while fetal myoblasts are inhibited by TGF beta. Once formed, primary fibers may stimulate a new wave of proliferation in fetal myoblasts, in order to expand the pool of cells needed to form secondary fibers. o test this model we developed an organ culture for limb buds which resulted in the production of myotubes with a phenotype similar to embryonic (primary) and fetal (secondary) fibers, roughly at the time when primary and secondary fibers form in vivo. When these cultures were treated with TGF beta, embryonic myotubes did form (as expected), but fetal myotubes never appeared. Conversely, when these cultures were treated with anti-TGF beta neutralizing antibodies, fetal myotubes developed earlier than in control cultures, suggesting that endogenously produced TGF beta may repress differentiation of fetal cells in vitro and, possibly, in vivo