Regulation of calcium binding proteins calreticulin and calsequestrin during differentiation in the myogenic cell line L6

In this report we defined the structural and temporal limits within which calreticulin and calsequestrin participate in the muscle cell phenotype, in the L6 model myogenic system. Calreticulin and calsequestrin are two Ca²⁺ binding proteins thought to participate in intracellular Ca²⁺ homeostasis. We show that calsequestrin protein and mRNA were expressed when L6 cells were induced to differentiate, during which time the level of expression of calreticulin protein did not change appreciably. Calreticulin mRNA levels, however, were constant throughout L6 cell differentiation except for slight decline in the mRNA levels at the very late stages of L6 differentiation (day 11–12). We also show that the two Ca²⁺ binding proteins are coexpressed in differentiated L6 cells. Based on its mobility in SDS-PAGE, L6 rat skeletal muscle cells in culture expressed cardiac isoform of calsequestrin. In the mature rat skeletal muscle, calreticulin and calsequestrin were localized to sarcoplasmic reticulum (SR). Calreticulin, but not calsequestrin, staining was also observed in the perinuclear region. These data suggest that expression of calreticulin and calsequestrin may be under different control during myogenesis in rat L6 cells in culture. © 1996 Wiley-Liss, Inc.     

Differentiation markers of mouse C2C12 and rat L6 myogenic cell lines and the effect of the differentiation medium

Summary The differentiation grade of cells in culture is dependent on the composition of the culture medium. Two commonly used myogenic cell lines, mouse C₂C₁₂ and rat L₆, usually differentiate at a low concentration of horse serum. In this study we compared the effect of horse serum with a medium containing a low percentage of Ultroser G and rat brain extract. The maturation grade was evaluated on the basis of various biochemical, (immuno)histochemical and cell-physiological parameters. Substitution of horse serum by Ultroser G and rat brain extract during the differentiation phase resulted in a higher maturation grade of the myotubes of both cell lines, on the basis of creatine kinase activity and the diameter of the myotubes. In addition, the C₂C₁₂ myotubes display cross-striation, contain a higher percentage of creatine kinase muscle-specific isoenzyme MM, show a ninefold increase in acetylcholine receptor (AChR) clusters, form a continuous basement membrane, and have a lower resting cytosolic Ca²⁺ concentration. L₆ myotubes show a fivefold increase in AChR clusters and a twofold increase in the expression of the mRNA of the ɛ-subunit of AChR. C₂C₁₂ cells show spontaneous contraction and response of cytosolic Ca²⁺ to various stimulants in contrast to L₆ cells which do not. These studies established that the Ultroser G/brain extract medium leads to a higher differentiation grade of both cell lines, but parameters appropriate for use as differentiation markers appear to differ between both cell lines.     

Regulation of the p75 neurotrophin receptor in a rat myogenic cell line (L6)

Neurotrophins are expressed in muscle cells both during development and postnatally. Furthermore, during development muscle cells express high levels of the common p75 neurotrophin receptor, which binds all neurotrophins. Only fragmentary and controversial data are available regarding the responsiveness of muscle cells to neurotrophins and the importance of low-affinity p75 receptor in muscle development. The present study investigates in vitro the immunocytochemical expression of p75 in a rat myogenic cell line (L6) at various time points and in response to different coating substrates as a first step in elucidating the regulation of p75 in muscle. We found that in L6 myoblasts, p75 is expressed only at very early stages of maturation and its levels of expression are regulated by the nature of the coating substrates. p75 expression decreases in cells growing on substrates more suitable for myoblast fusion into myotubes. Time course analysis indicates a reverse correlation between myoblast fusion into myotubes and the levels of p75 expression. Myotubes were always p75 negative. Substrates not suitable for the fusion process induced a prolonged presence of p75 in myoblasts with an increase of their apoptosis. We conclude that expression of p75, at least in this in vitro condition, is regulated by the stages of myoblast differentiation and the nature of the coating substrates. According to the observed time- and substrate-related evidences, future studies should investigate in vivo both the regulation of p75 in the myoblast fusion and the effects and the importance of neurotrophins binding during myoblast differentiation.     

Retinoic acid induces myogenin synthesis and myogenic differentiation in the rat rhabdomyosarcoma cell line BA-Han-1C

The F3 molecule is a member of the immunoglobulin superfamily anchored to membranes by a glycane-phosphatidylinositol, and is predominantly expressed on subsets of axons of the central and peripheral nervous system. In a previous paper (Gennarini, G., P. Durbec, A. Boned, G. Rougon, and C. Goridis. 1991. Neuron. 6:595-606), we have established that F3 fulfills the operational definition of a cell adhesion molecule and that it stimulates neurite outgrowth when presented to sensory neurons as a surface component of transfected CHO cells. In the present study the question as to whether soluble forms of F3 would be functionally active was addressed in vitro on cultures of mouse dorsal root ganglion neurons. We observed that preparations enriched in soluble F3 had no effect on neuron attachment but enhanced neurite initiation and neurite outgrowth in a dose-dependent manner. By contrast, soluble NCAM-120 does not have any measurable effect on these phenomena. Addition of anti-F3 monovalent antibodies reduced the number of process-bearing neurons and the neuritic output per neuron to control values. Addition of cerebrospinal fluid, a natural source of soluble F3, also stimulated neurite extension, and this effect was partially blocked by anti-F3 antibodies. Our results suggest that the soluble forms of adhesive proteins with neurite outgrowth-promoting properties could act at a distance from their site of release in a way reminiscent of growth and trophic factors.     

Reduced DNA repair during differentiation of a myogenic cell line

Repair synthesis induced by 4-nitroquinoline-1-oxide (4NQO) in L6 myoblasts before and after cellular fusion was measured by [3H] thymidine incorporation into unreplicated DNA. The level of repair synthesis was reuced after the cells had fused into myotubes. The terminal addition of radioactive nucleotides into DNA strands occurred only to a minor extent, and the dilution of [3H] thymidine by intracellular nucleotide pools was shown not to be responsible for the observed difference in repair synthesis, Both the initial rate and the overall incorporation of [3H] thymidine were found to be 50% lower in the myotubes. 4NQO treatment of myoblasts and myotubes induced modifications in the DNA which were observed as single-strand breaks during alkaline sucrose sedimentation. After the myoblasts were allowed a post-treatment incubation, most of the single-strand breaks were not longer apparent. In contrast, a post-treatment incubation of myotubes did not change the extent of single-strand breakage seen. Both myoblasts and myotubes were equally effective in repairing single- strand breaks induced by X radiation. It would appear that when myoblasts fuse, a repair enzyme activity is lost, probably an endonuclease that recognizes one of the 4 NQO modifications of DNA. The result observed is a partial loss of repair synthetic ability and a complete loss of ability to remove the modification that appears as a single-strand break in alkali.     

A myogenic cell line with altered serum requirements for differentiation

Dfferentiation properties of a cell line, L84, which originated from a non-fusing clone isolated from the myogenic line L8, are described. In nutritional medium supplemented with 10% serum used routinely with L8 cells, L84 cells continue to proliferate to very high densities and fail to form multinucleated fibres. When grown in medium supplemented with 2% horse serum of 2% horse serum plus 0.1% microng/ml insulin, L84 cells behave very similarly to L8 cells grown in medium supplemented with 10% horse serum: when the cultures reach confluency, proliferation decreases and cells start to fuse and form a dense network of fibres. Large increases in creatine kinase activity and synthesis of myosin are associated with cell fusion. Under conditions in which L84 cells do not fuse the increase in these synthetic activities is not observed, even after extremely high cell densities are reached. The data show that L84 cells retain the programme for their differentiation into muscle fibres. The difference between L84 and its progenitor line L8 lies in the sensitivity to the environmental conditions which trigger the expression of this programme.     

Expression of transformation-associated traits in the myogenic cell lines L6 and L8

The presence of cellular alterations, usually associated with transformation, has been studied in two permanent myogenic cell lines, L6 and L8, that retain the ability to differentiate in vitro. We present evidence that, beside being immortal, both cell lines are anchorage-independent for proliferation, a feature not found in primary muscle cells. L6 secretes constitutively high levels of plasminogen activator. L8 is able to undergo multinucleation in the presence of cytochalasin B (cytB) and is tumorigenic in vivo. Single anchorage-independent clones were shown to possess differentiative potentials similar to those of the parental line. Moreover, cell fusion could be directly observed in L8 while still growing as colonies in soft agar. We discuss our data with respect to (i) the reported differences in the regulation of differentiation between primary myogenic cells and continuous cell lines; (ii) the relationship between transformation and differentiation in muscle cells.     

IGF-I-induced differentiation of L6 myogenic cells requires the activity of cAMP-phosphodiesterase

Inhibition of type 4 cAMP-specific phosphodiesterase (PDE4) activity in L6-C5 and L6-E9 abolished myogenic differentiation induced by low-serum medium and IGF-I. L6-C5 cells cultured in low-serum medium displayed a PDE4 activity higher than cells cultured in serum-free medium, a condition not sufficient to induce differentiation. In the presence of serum, PDE4D3, the major isoform natively expressed in L6-C5 cells, translocated to a Triton-insoluble fraction, which increased the PDE specific activity of the fraction, and exhibited a Mr shift typical of phosphorylation of this isoform. Furthermore, serum promoted the localization of PDE4D3 to a vesicular subcellular compartment. In L6-C5 cells, IGF-I is a stronger inducer of myogenic differentiation in the presence than in absence of serum. Its ability to trigger differentiation in the absence of serum was restored by overexpressing wild-type PDE4D3, but not a phosphorylation-insensitive mutant. This finding was confirmed in single cells overexpressing a GFP-PDE4D3 fusion protein by assessing nuclear accumulation of myogenin in both L6-C5 and L6-E9. Overexpression of other PDE isoforms was less efficient, confirming that PDE4D3 is the physiologically relevant phosphodiesterase isoform in the control of myogenesis. These results show that downregulation of cAMP signaling through cAMP-phosphodiesterase stimulation is a prerequisite for induction of myogenesis.     

Retargeting of viral glycoproteins into a non-exporting compartment during the myogenic differentiation of rat L6 cells

We have analysed protein trafficking during the differentiation of rat L6 myoblasts into myotubes. Different proteins were found to lose different amounts of their processing by the Golgi apparatus during the myogenic differentiation, indicating that they were transported to this organelle with differing efficiencies. In order to investigate the destination of the nonprocessed glycoproteins we analysed the behaviour of vesicular stomatitis virus (VSV) and Semliki Forest virus glycoproteins in the presence of Brefeldin A, which returns the enzymes of the Golgi apparatus to the ER. Such experiments indicated that during myogenesis a fraction of both glycoproteins was shunted into a compartment that did not participate recycling with the Golgi apparatus. Immunofluorescence studies with the mutant VSV tsO45 G protein suggested that this compartment was diffusively distributed. We investigated whether the cytoplasmic tail had a role in the myogenic transport modulation by analysing the behaviour of recombinant VSV G proteins. Exchanging the cytoplasmic tail or the tail plus the membrane anchor had no effect, suggesting that the luminal portion was responsible for the diverted transport. Taken together, the results suggest that during the myogenesis of L6 myoblasts, varying fractions of different viral glycoproteins were sorted from the ER into a specific compartment that did not recycle with the Golgi apparatus.     

Phorbol ester-induced differentiation of L6 myogenic cells involves phospholipase D activation

TPA, a potent PKC activator, inhibits myogenic differentiation and activates phospholipase D (PLD). We evaluated the involvement of PLD in the TPA effects on L6 myoblasts differentiation. TPA, at concentrations inhibiting differentiation of L6 cells, induced a strong, though transient, PLD activation. Surprisingly, at nanomolar concentration, TPA induced both myogenic differentiation and sustained activation of PLD. Differential effect of TPA can be ascribed to PKC downregulation induced by highest TPA concentrations. TPA-induced differentiation was inhibited by 1-butanol, confirming the involvement of PLD in this effect. These data suggest that prolonged elevation of PLD activity is required for myogenic differentiation.     

Commitment,fusion and biochemical differentiation of a myogenic cell line in the absence of DNA synthesis

L₆E₉ rat myoblasts derived from the L₆ cell line can be induced to differentiate to a very high percentage by manipulating the culture conditions. Under standard differentiating conditions, L₆E₉ cells divide an average of 2.5 times before differentiating and >99% of them incorporate ³H-TdR before fusing. By inhibiting DNA replication by a variety of means, data have been obtained which demonstrate that this DNa synthesis is not required to switch from growth to differentiation. After every cell division, L₆E₉ cells have the option either to fuse or to proliferate without intervening DNA synthesis.Cell cloning and DNA labeling experiments show a direct correlation between the time of culture in differentiating medium and a progressive loss of proliferative capacity of mononucleated L₆E₉ cells, demonstrating that these cells become irreversibly committed to differentiation and withdraw from the cell cycle prior to and not as a consequence of cell fusion. The commitment step occurs during the G1 phase prior to fusion. This G1 phase has a latent period during which no irreversible step toward differentiation occurs and the cells remain ambivalent toward growth or differentiation. Under proper conditions, this period is followed by an irreversible commitment toward differentiation and a loss of proliferative capacity. The kinetics of this commitment step strongly suggest that L₆E₉ cells become irreversibly committed in a stochastic manner. Once the cells have become committed, with or without DNA synthesis, they will fuse to form myotubes and biochemically differentiate in a deterministic fashion.The data presented are consistent with a stochastic model of differentiation for L₆E₉ cells and demonstrate that the switch from a proliferating to a differentiating genetic program can occur in the absence of DNA synthesis.     

Transgene expression in the QM myogenic cell line

We have isolated an avian muscle cell line (QM) which has the essential features of established mammalian muscle cell lines. The experiments reported here were undertaken to determine the suitability of QM cells for the introduction and analysis of cloned transgenes. The promoter of the cardiac troponin T (cTNT) gene has been previously shown to contain sequence elements which govern muscle-specific expression of the chloramphenicol acetyltransferase (CAT) gene in transiently transfected primary cell cultures. We show here that QM cells stably harboring cTNT promoter-CAT fusion genes up-regulate CAT expression in concert with myogenic differentiation, and that as few as 110 upstream nucleotides are sufficient for such differentiation-dependent regulation. In addition, both transient and stable transfection experiments demonstrate that differentiated QM cells possess trans-acting factors necessary for the expression of the skeletal alpha-actin promoter, despite the absence of mRNA or protein product from the endogenous sarcomeric actin genes in these cells. Finally, to follow the developmental potential of QM cells in vivo, we created a clone, QM2ADH, which constitutively expresses the histochemical marker transgene encoding Drosophila alcohol dehydrogenase. When surgically inserted into the limb buds of developing chick embryos, QM2ADH cells are incorporated into endogenous developing muscles, indicating that QM cells are capable of recognizing and responding to host cues governing muscle morphogenesis. Thus, QM cells are versatile as recipients of transgenes for the in vitro and in vivo analysis of molecular events in muscle development.     

Changes in adenyl cyclase specific activity during differentiation on an established myogenic cell line

The variation of specific activity of adenyl cyclase has been studied during differentiation of an established line of myoblast, strain L₆D and of a temperature sensitive developmental variant strain, H₆, derived from it. The specific activities of both basal and NaF stimulated adenyl cyclase were found to decrease 2 to 3 folds after fusion of myoblasts into myotubes in cultures of L₆D. Cultures of strain H₆ displayed the same decrease in specific activity of adenyl cyclase when grown at temperature which allows differentiation, while no decrease was observed at the temperature which does not allow cell fusion. These results indicare that the decrease in specific activity of adenyl cyclase is associated with cell fusion and reflects membrane changes ocurring during differentiation of myogenic cells.     

Effect of sodium butyrate on gene expression in a rat myogenic cell line

Sodium butyrate, when added in millimolar concentration to a culture of myoblasts of the L6 cell line, inhibits reversibly cell proliferation and differentiation. In the present work, we have studied the effect of Na butyrate on the translational efficiency of the overall poly (A)+ RNA. The mRNA from treated cells was translated in vitro as efficiently as proliferating myoblasts mRNA, while a decrease of translation efficiency was observed with myotubes mRNA. In addition this RNA directs the synthesis of several new polypeptides. on the switch on of alpha actin and myosin heavy chains (MHC), muscle specific genes by the dot blot and Northern blot techniques using cloned probes. Na butyrate prevented the expression of MHC and allowed the switch on of alpha actin gene but at a lesser extent than in normal myotubes. In addition the drug prevented the translocation of alpha actin mRNA into the cytoplasm.     

Insulin and glucose-dependent regulation of the glucose transport system in the rat L6 skeletal muscle cell line

Differentiated rat L6 skeletal muscle cell cultures maintained in glucose-deficient medium containing 25 mM xylose displayed a rapid, reversible, time- and concentration-dependent 3-5-fold increase in glucose transport activity. Glucose deprivation in the continuous presence of insulin (24 h) resulted in an overall 9-10-fold stimulation of glucose transport activity. In contrast, acute (30 min) and chronic (24 h) insulin treatment of L6 cells maintained in high glucose (25 mM)-containing medium resulted in a 1.5- and 4-fold induction of glucose transport activity, respectively. Acute glucose deprivation and/or insulin treatment had no significant effect on the total amount of glucose transporter protein, whereas the long-term insulin- and glucose-dependent regulation of glucose transport activity directly correlated with an increase in the cellular expression of the glucose transporter protein. In situ hybridization of the L6 cells demonstrated a 3-, 4-, and 6-fold increase in glucose transporter mRNA induced by glucose deprivation, insulin, and glucose deprivation plus insulin treatments, respectively. Similarly, Northern blot analysis of total RNA isolated from glucose-deprived, insulin, and glucose-deprived plus insulin-treated cells resulted in a 4-, 3-, and 9-fold induction of glucose transporter mRNA, respectively. The continuous presence of insulin in the medium, either in the presence or absence of glucose, resulted in a transient alteration of the glucose transporter mRNA. The relative amount of the glucose transporter mRNA was maximally increased at 6-12 h which subsequently returned to the basal steady-state level within 48 h. These data demonstrate a role for insulin and glucose in the overall regulation of glucose transporter gene expression which may account for the alteration of glucose transporter activity of muscle tissue observed in pathophysiological states such as type II diabetes mellitus.