Studies on the effect of glycoprotein processing inhibitors on fusion of L6 myoblast cell lines

The effect of oligosaccharide processing inhibitors on the fusion of L6 myoblasts was studied. The glucosidase inhibitors, castanospermine, 1-deoxynojirimycin and N-methyl-deoxynojirimycin were potent inhibitors of myoblast fusion, as was the mannosidase II inhibitor, swainsonine. Inhibition of fusion was reversed when inhibitors were removed. However, the mannosidase I inhibitor, 1-deoxymannojirimycin did not inhibit fusion. Changes in cell membrane oligosaccharide structure were followed by monitoring the binding of concanavalin A (conA) and wheat germ agglutinin (WGA) to cell surface membranes in cells treated with processing inhibitors. All the processing inhibitors resulted in increased binding of conA and decreased binding of WGA; this is consistent with the known mechanisms of inhibition of the inhibitors used in the study. Inhibition of fusion by the processing inhibitors also resulted in reduced activities of creatine phosphokinase, an enzyme used as a marker for biochemical differentiation during fusion. Treatment of a non-differentiating conA-resistant cell line with processing inhibitors did not induce fusion, but the cells did show altered lectin-binding properties. The main conclusion drawn from these studies is that cell surface glycoproteins probably containing the mannose (Man)9 structure are important for the fusion reaction.     

Novel evidence that testosterone promotes cell proliferation and differentiation via G protein-coupled receptors in the rat L6 skeletal muscle myoblast cell line

Androgens are known to modulate the skeletal muscle proliferation and differentiation processes. Recent in vitro studies have shown that dihydrotestosterone and anabolic steroids have functions in promoting the proliferation and differentiation of the mouse skeletal muscle myoblast C2C12 cell line through the classical androgen receptor (AR) signaling pathway. But there are contradictory reports that androgen plays its roles through the membrane signaling pathways. In the present study, we show that there is no expression of the classical AR in L6 cells both at gene and protein levels. We then investigated the effects of testosterone (T) on L6 cell proliferation and differentiation. The results show that T promotes L6 cell proliferation after a 24 h treatment, which followed by enhancing L6 cell differentiation, but these effects are not inhibited by flutamide (F), an antagonist of intracellular AR. Further, we tested the effect of testosterone covalently bounding to albumin (T-BSA), which does not cross the plasma membrane. The results demonstrate that T-BSA and free T have similar effects on L6 cell proliferation and differentiation, and that these effects involve G protein-coupled receptors and different downstream pathways. The L6 cell proliferation induced by T involves PKC and ERK1/2 signaling pathways and cell differentiation happens via the PKA signaling pathway. These results suggest that T promotes cell proliferation and differentiation via G protein-coupled receptors and different downstream pathways in the L6 cell line, although the related molecular mechanisms need to be elucidated in future studies.     

Effect of high density lipoproteins on protein expression in myoblast cell lines

We have studied the effects of high-density lipoprotein (HDL) on rat heart (H9c2) and skeletal (L6) myoblasts using two-dimensional gel electrophoresis and tandem mass spectrometry. Gels generated from control cells and cells treated with 250 microg/mL HDL showed significant differences in the 7-10 pI region and the 30-50 kDa mass region. In particular, the membrane binding protein, annexin II, the voltage-dependent anion channel, glyceraldehyde-3-phosphate dehydrogenase and other glycolytic proteins are differentially expressed.     

Proteoglycans of L6J1 myoblast extracellular matrix: Properties and effect on myoblast adhesion

Proteoglycans were isolated from the extracellular matrix (ECM) of L6J1 rat myoblasts; their influence on myoblast adhesion has been studied. Proteoglycan digestion with chondroitinase AC and heparinase III, which degrade polysaccharide moieties, has revealed that chondroitin sulfate proteoglycans are a major class of myoblast extracellular matrix proteoglycans. Electrophoresis of enzymatically processed proteoglycans was used to examine their core proteins. Myoblast adhesion was suppressed by proteoglycans or a mixture of proteoglycans and a fibronectin-extracellular matrix. Myoblast adhesion to a substrate composed of fibronectin and proteoglycans is restored after the substrate was treated with chondroitinase AC. In conclusion, proteoglycans of L6J1 rat myoblast ECMs were isolated and purified. Chondroitin sulfate proteoglycans are a major class of proteoglycans. Isolated proteoglycans suppressed myoblast adhesion; the effect was mediated by polysaccharide moieties of proteoglycans.     

Proteoglycans of L6J1 myoblast extracellular matrix. Characteristics and effect on myoblast adhesion

Proteoglycans were isolated from extracellular matrix of L6J1 rat myoblasts and their influence on myoblast adhesion was studied. Proteoglycan digestion with chondroitinase AC and heparinase III degrading the polysaccharide moieties revealed that chondroitin sulfate proteoglycans are the main class of myoblast extracellular matrix proteoglycans. Electrophoresis of enzymatically processed proteoglycans was used to examine their core proteins. Myoblast adhesion was suppressed by proteoglycans or the mixture of proteoglycans and fibronectin/extracellular matrix. When being processed with chondroitinase AC the combined substrate of fibronectin and proteoglycans lost the capability of myoblast adhesion suppression. Thus, as a result of presented work the proteoglycans of L6J1 rat myoblast extracellular matrix were isolated and purified. The main class of proteoglycans was chondroitin sulphate proteoglycans. Isolated proteoglycans suppressed myoblast adhesion and this effect was mediated by polysaccharide moieties of proteoglycans.     

Glycolipids and glycosyltransferases in permanent cell lines established from human medulloblastomas.

Medulloblastoma biopsies are heterogenous and might contain normal brain tissue, which limits the usefulness of such tumor material for biochemical analyses. We have, therefore, examined the gangliosides and their metabolism using the medulloblastoma cell lines. Daoy and D341 Med, cultured both in vitro and as xenografts in nude mice. The ganglioside patterns in the Daoy showed a switch from a high GM2, 70% (mol% of total ganglioside sialic acid) and low lactoseries gangliosides (2%) content in monolayer cultures, to a high proportion of lactoseries gangliosides (50%) and virtually no GM2 (1%) in xenografts, but an increased proportion of other a-series gangliosides. The D341 Med showed a similar change regarding the lacto-series gangliosides from 1% in suspension culture to 10% in xenografts. The activity of five glycosyltransferases, GM3, GD3, GM2, GM1 and LA2 synthases, did not parallel the ganglioside patterns and could not account for the noted variations therein. In the Daoy cell line the LA2 synthase as well as the GM2 synthase activity was relatively high in both culture systems, despite the marked difference in the expression of GM2 and the lactoseries gangliosides. These results suggest that environmental factors play a crucial role for the in vivo activity of the glycosyltransferases.     

A unique subset of developmentally regulated surface proteins turns over rapidly during fusion of the L6 rat myoblast cell line

We have previously identified several developmentally regulated surface polypeptides in the L6 rat myoblast cell line, on the basis of their susceptibility to lactoperoxidase catalyzed iodination. An analysis of the turnover rates of these polypeptides now indicates that while the bulk of the iodinated polypeptides have a half-life of 20-30 h, four low-molecular-weight polypeptides have half lives of 2-7 h. The half-lives of all of the rapid turnover class surface polypeptides were greatly increased in cultures where fusion was inhibited by chloroquine and in nonfusing variants of the L6 cell line. In contrast, inhibition of fusion by the metalloendoprotease inhibitor 1, 10-phenanthroline did not alter the turnover of any iodinatable surface proteins. We propose that some or all of the rapid turnover class of polypeptides may be surface receptors which control cell surface alterations involved in the acquisition of fusion competence or in fusion itself.     

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.     

Polyamine depletion inhibits the differentiation of L6 myoblast cells

Exposure to alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, inhibited the insulin induced differentiation of L6 myoblast cells. Differentiation was assessed by measuring creatine kinase activity and by determining the percentage of nuclei in myotubes. The levels of putrescine and spermidine increased in stimulated cultures prior to their differentiation and these increases were blocked by alpha-difluoromethylornithine. Provision of exogenous putrescine was able to reverse the inhibitory effect of the drug. The anti-differentiative effect is observed only if alpha-difluoromethylornithine is added within twenty-four hours of insulin stimulation. In the experimental protocol used, alpha-difluoromethylornithine was added as the cultures approached confluence and had no effect on their ultimate DNA content. Therefore, the effect of alpha-difluoromethylornithine on myoblast differentiation is not secondary to an effect on cellular proliferation. These results indicate that polyamines may be involved in the mediation of muscle cell differentiation.     

Analysis of the expression of chicken and rat gene products in myoblast x myoblast cell hybrids

Hybrid cells were isolated by fusing primary chicken myoblasts to HPRT-deficient rat L6 myoblasts and incubating the cells in medium containing HAT and ouabain. All hybrid clones contained both rat and chicken chromosomes and expressed a number of gene products characteristic of both species. Although all clones were capable of fusing spontaneously to form myofibers, immunofluorescence and isoenzyme analysis revealed only the rat forms of skeletal muscle myosin and MM-creatine kinase. No differentiated gene products of chicken origin were detected. Analysis of the expression of chicken HPRT revealed that some hybrid clones were capable of modulating this enzyme activity when switched from HAT medium into thioguanine medium and back into HAT, even though HPRT is normally a constitutively expressed enzyme. Parental control cells were incapable of this modulation phenomenon.     

A study of metabolic cooperation with established myoblast cell lines.

The present study was undertaken to test the action of ConA on the distribution of intramembranous particles (IMPs) and on the reassembly of junctional contacts in isolated and reaggregated embryonic neuronal and glial cells. The lectin ConA causes all embryonic cells to aggregate in unorganized cell patterns. ConA does not alter the distribution of IMPs but it inhibits the formation of the zonula occludens (ZO) by preventing the alignment and fusion of IMPs or by inducing them to become arranged in bizarre arrays. The possible relationship between ConA receptor sites and the IMPs is discussed. From a morphological viewpoint the aggregation of embryonic cells influenced by lectin is distinctly different from the normal processes of cell adhesion, cell sorting and establishment of intercellular contacts.     

Phenotypic expression in chick erythrocyte X rat myoblast hybrids and in chick myoblast X rat myoblast hybrids

Attempts were made to reprogram chick erythrocyte nuclei to specify the synthesis of chick myosin. Chick erythrocytes were fused with rat myogenic cells with the aid of UV-inactivated Sendai virus. In the heterokaryons and hybrid myotubes which resulted from this fusion, the erythrocyte nuclei resumed RNA synthesis and formed nucleoli. Although some new chick antigens developed in those myotubes which contained fully reactivated chick erythrocyte nuclei, accumulation of chick myosin could not be detected by immunological methods. Neither small heterokaryons nor large hybrid myotubes which were actively synthesizing rat myosin reacted with antibodies directed against chick myosin. A small number of mononucleated cells, believed to be synkaryons formed by mitotic division of heterokaryons, did, however, react strongly with antibodies directed against chick myosin and showed a cross striation typical of skeletal muscle. The frequency of such cells was too low, however, to permit karyological analysis or further characterization of the antigen. Hybrids between chick myoblasts and rat myoblasts produced both chick and rat myosin thus indicating that simultaneous translation of chick and rat mRNA for myosin in a common cytoplasm was possible. In summary the evidence obtained suggested that reprogramming of chick erythrocyte nuclei, if it did occur in the present system, was a rare phenomenon.The possibility that hybrids between chick erythrocytes and rat myoblasts expressed markers typical of an erythroid phenotype was examined by immune staining with antibodies directed against chick haemoglobin. The results suggested that haemoglobin was introduced into hybrid cells by erythrocytes which failed to lyse before fusion. The intensity of this immune fluorescence decreased with increasing time after fusion. The rate at which this decrease occurred was not affected by inhibition of RNA synthesis. Thus, there was no evidence for the accumulation of haemoglobin in the hybrid cells.     

Fragments of bovine insulin-like growth factors I and II stimulate proliferation of rat L6 myoblast cells

The active sites of bovine insulin-like growth factor (IGF) I and II fragments were studied. Overlapping fragments of IGF I (residues 1-25, 11-35, 21-45, 31-55, and 41-70) and of IGF II (residues 1-24, 10-34, 20-44, 30-54, and 40-67) were chemically synthesized. The activity of the fragments was measured by stimulating the proliferation of rat L6 myoblast cells. Two fragments of IGF I (residues 21-45 and 31-55) and two fragments of IGF II (residues 20-44 and 30-54) were active while the other fragments were inactive in stimulating cell proliferation. Although the activity of these fragments was observed only at a high concentration of 0.1 mM, the results imply that the active site is located around residues 31-45 for IGF I fragments and residues 30-44 for IGF II fragments. Consequently, an IGF I fragment (residues 26-50) having a five-residue extension to both the N- and C-terminal sites of residues 31-45 also stimulated the proliferation of L6 myoblast cells. Furthermore, the substitution of Ile-35 in two IGF II fragments (residues 21-45 and 31-55) by Ser inactivated these fragments. This suggests that Ile-35 is an essential residue for IGF II fragment activity. Ser-35, which was reported in the original sequencing of bovine IGF II, is incorrect in the sequence and furthermore has been consistently found to be an Ile-35 in our hands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of an L6 myoblast in vitro model of moniliformin toxicosis

L6 myoblasts were used as an in vitro model to investigate the role of moniliformin and its interaction with monensin in turkey knockdown syndrome and sudden death syndromes in poultry. Cell viability and microscopic and ultrastructural alterations noted in L6 myoblasts cultured in the presence of moniliformin (0.0–0.3 g/l) were compared to those observed in parallel cultures also containing one of the following compounds: selenium (0–0.004 ng/l), thiamine (0–0.3 g/l), or pyruvate (0–0.46 g/l). Marked dilation of the RER, membranous whorls, glycogen deposition, membrane-bound cytoplasmic inclusions and necrosis were observed in myoblasts exposed to 0.03/2-0.30 g moniliformin/l medium. Supplementation of medium with thiamine and pyruvate, or selenium, provided significant protection to cells exposed to 0.0–0.3 g/l or 0.0–0.15 g moniliformin/l, respectively. Dose-dependent differences in protein and ATP production were not detected. Myoblasts grown in medium containing 0–0.15 g moniliformin/l and 7.5–50.0 M A23187, beauvericin or monensin had degrees of cytotoxicity similar to parallel cultures receiving only an ionophore. L6 myoblasts were a useful model of moniliformin toxicosis. The findings of this study suggest cytotoxicity due to moniliformin in L6 myoblasts may be due in part to oxidative damage and altered pyruvate metabolism, and that moniliformin does not predispose myoblasts to ionophore toxicosis. This study supports the results of in vivo investigations in poultry that moniliformin and monensin do not act synergistically to induce knockdown or monensin toxicosis.     

Major chondroitin sulfate proteoglycans identified in L6J1 myoblast culture

The major proteoglycans from L6J1 rat myoblast culture were identified. The proteoglycans were isolated from different constituents of cell culture: culture medium, extracellular matrix (ECM), and myoblasts. To identify their core proteins, the proteoglycans were treated with enzymes specifically digesting chondroitin/dermatan sulfates or chondroitin sulfates. Subsequent electrophoresis and mass spectrometry revealed versican, collagen XII, and inter-α-trypsin inhibitor classified as chondroitin sulfate proteoglycans and biglycan known to be chondroitin/dermatan sulfate proteoglycan. Versican was identified in ECM and the other proteoglycans in the culture medium. Such difference in localization is likely to be a consequence of different biological functions. Versican, collagen XII, and biglycan are synthesized by myoblasts and inter-α-trypsin inhibitor originates from fetal bovine serum (a culture medium component).     

Effects of inhibitors of ornithine and S-adenosylmethionine decarboxylases on L6 myoblast proliferation

The role of polyamines in myoblast proliferation was studied by treating cells of Yaffe's L6 line of rat myoblasts with inhibitors of polyamine synthesis. Both an irreversible inhibitor of ornithine decarboxylase--difluoromethyl-ornithine (DFMO)--and a competitive inhibitor of S-adenosyl-methionine decarboxylase--methylglyoxal-bis(guanylhydrazone) (MGBG)--depressed spermidine levels and inhibited myoblast proliferation. Spermine levels were not significantly depressed by either inhibitor and putrescine levels were decreased only by DFMO. Putrescine and spermidine, but not magnesium, prevented inhibition of myoblast proliferation by DFMO and MGBG; determination of 14C-DFMO uptake in the presence and absence of these compounds demonstrated that they did not reduce the rate or extent of inhibitor uptake and thus prevent its inhibition of ornithine decarboxylase. Thus it seems likely that these inhibitors reduce cell proliferation by inhibiting polyamine formation. Addition of spermidine to the cells led to a substantial reduction in the activity of S-adenosyl-methionine-decarboxylase, suggesting that the enzyme is subject to negative regulation by the products of the polyamine biosynthetic pathway. Unexpectedly, addition of spermidine also increased intracellular putrescine levels; this apparently resulted from conversion of spermidine to putrescine. Addition of putrescine or spermidine in the absence of serum did not increase the rate of myoblast proliferation although it did elevate intracellular polyamine levels as expected. We conclude that some threshold level of one or more polyamines (probably spermidine) is necessary but not sufficient for initiation and maintenance of myoblast proliferation in culture.     

Integrin-linked kinase is a positive mediator of L6 myoblast differentiation

Overexpression of ILK in L6 myoblasts results in increased ILK kinase activity, stimulating myotube formation and induction of biochemical differentiation markers. Expression of a dominant negative ILK mutant, ILK(E359K), inhibits endogenous ILK activation and L6 differentiation. Cell cycle analysis of ILK(E359K) cells cultured in serum-free conditions indicates significant apoptosis (11-19% sub-diploid peak) which is not seen in insulin treated cells. Expression of ILK variants does not have significant effects on S-phase transit, however. Known targets of ILK, PKB/Akt or glycogen synthase kinase 3beta are not obviously involved in ILK-induced L6 differentiation. Insulin-stimulated phosphorylation of PKB at Ser473 is unimpaired in the ILK(E359K) cells, suggesting that PKB is not a myogenic target of ILK. Inhibition of GSK3beta by LiCl blocks L6 myogenesis, indicating that ILK-mediated inhibition of GSK3beta is not sufficient for differentiation. Our data do suggest that a LiCl-sensitive interaction of ILK is important in L6 myoblast differentiation.     

Selective inhibition of cathepsin B with cell-permeable CA074Me negatively affects L6 rat myoblast differentiation.

Active cathepsin B, in concert with other cellular proteases, has been implicated in the catabolic restructuring associated with myotube formation during skeletal myoblast cell differentiation (i.e., myogenesis). We have examined this role in differentiating myoblasts using the cell-permeable, cathepsin B selective inhibitor CA074Me. Cathepsin B activity levels in differentiating L6 rat myoblasts treated with CA074Me were significantly lower than levels in control myoblasts. Inhibition of cathepsin B activity by CA074Me occurred at each stage of differentiation and was dose related. Myotube size and number and induced levels of fusion-related creatine phosphokinase activity and myosin heavy-chain protein were reduced from 30 to 50% in CA074Me-treated myoblasts. These reductions were also dose related. In contrast, CA074Me did not affect levels of myogenin, an early marker of myogenesis, or levels of cathepsin L type and myokinase activities, two nonspecific enzymes. The negative effects associated with CA074Me were reversed when the drug was removed. Collectively, these data suggest that active cathepsin B plays a role in myoblast-myoblast fusion and consequently may be necessary for the complete expression of those genes associated with the fusion process.     

Analysis of the expression of chicken and rat gene products in myoblast × myoblast cell hybrids

Hybrid cells were isolated by fusing primary chicken myoblasts to HPRT-deficient rat L6 myoblasts and incubating the cells in medium containing HAT and ouabain. All hybrid clones contained both rat and chicken chromosomes and expressed a number of gene products characteristic of both species. Although all clones were capable of fusing spontaneously to form myofibers, immunofluorescence and isoenzyme analysis revealed only the rat forms of skeletal muscle myosin and MM-creatine kinase. No differentiated gene products of chicken origin were detected. Analysis of the expression of chicken HPRT revealed that some hybrid clones were capable of modulating this enzyme activity when switched from HAT medium into thioguanine medium and back into HAT, even though HPRT is normally a constitutively expressed enzyme. Parental control cells were incapable of this modulation phenomenon.