Changes in protein synthesis during myogenesis in a clonal cell line

Methods of quantitative two-dimensional gel electrophoresis have been used to study the changes in protein synthesis that occur during myogenic differentiation in the L6 clonal line of rat skeletal muscle cells. Pure populations of myoblasts were obtained by maintaining the cells at subconfluent densities, and virtually pure populations of fused myotubes have been obtained by sedimentation at 1 × gravity through a serum gradient. The gel analysis reveals major qualitative differences between myoblasts and myotubes, as well as numerous quantitative changes. Both the α and the β forms of tropomyosin and the LC2 myosin light chain were increased in rate of synthesis by at least 1000-fold during myogenesis. Other proteins were detectable in myoblasts but were not synthesized at a detectable rate in myotubes. One of these is a form of tropomyosin which comigrates under several electrophoretic conditions with smooth muscle tropomyosin. Another protein, which is repressed in rate of synthesis by at least 1000-fold during myogenesis, appears to be a major form of collagen. Computer analysis has been used to analyze in detail a particular region containing about 300 spots from the two-dimensional patterns representing protein synthesis in L6 myoblasts, L6 myotubes, and a rat nerve cell line. Quantiative comparisons have shown that, with respect to this set of proteins, the L6 myoblasts and myotubes are no more alike at the level of protein synthesis than are L6 myoblasts and the cells of the nerve line. Therefore, these studies show that L6 differentiation involves not only the qualitative switching on and off of major gene products but also the quantitative alteration of synthetic rates of many of the common proteins.     

c-fos reduces growth factor requirements for mitogenic stimulation of L6 rat myoblasts

Addition of fetal calf serum (FCS) to serum-deprived L6J1 rat myoblasts increases fos-like immunoreactivity. The nuclear immunoreactivity reached a maximum 2 h after serum addition. Effects of the c-fos protein on myoblast proliferation were analyzed in L6J1 rat myoblasts transfected with the murine c-fos gene under control of a metallothionein promoter. L6J1 myoblasts with elevated expression of transfected c-fos reached higher cell densities than neo transfected control myoblasts when approaching a stationary phase in normal culture conditions (5% FCS). The differences in cell densities were even more pronounced at low serum concentrations (0.5% FCS). c-fos transfected cells also had a faster growth rate than did control cells in serum-free medium supplemented with calcium chloride, lithium chloride, sodium selenite, hydrocortisone, and insulin. The cell morphology of c-fos transfected L6J1 myoblasts was not affected compared to control myoblasts. These results suggest that c-fos protein expression in L6J1 myoblasts is activated by serum and that mitogenic stimulation of L6J1 myoblasts is facilitated by the presence of elevated amounts of c-fos protein.     

Indomethacin inhibits the insulin-induced increases in RNA and protein synthesis in L6 skeletal muscle myoblasts

Rates of accretion of RNA and protein and rates of protein synthesis were measured in sub-confluent cultures of L6 myoblasts. Insulin (100 μU/ml) stimulated protein synthesis by 15% within 30 min and by 40% at two and six hours. By six hours insulin also increased the accretion of RNA (+ 15%). The cyclo-oxygenase inhibitor indomethacin did not reduce the basal rate of RNA or protein accretion in L6 cells but reduced the rate of protein synthesis by 16%. When added together with insulin, indomethacin inhibited the hormonally-stimulated rate of protein synthesis and also significantly reduced the accretion of RNA. Indomethacin still reduced the effects of insulin on protein synthesis when added by the incorporation of [³H]-uridine was also stimulated by insulin but was inhibited by indomethacin only when the drug was present throughout the incubation. Inhibition of protein synthesis by cyclo-oxygenase inhibitors may be the result of both a direct action on translational efficiency and an effect on RNA synthesis.     

Protein turnover in senescent cultured chick embryo fibroblasts

The over-all rates of protein synthesis, degradation and net accumulation were estimated in rapidly growing young and slowly doubling old cultures of chick fibroblasts. We find that not only the rate of protein synthesis is reduced in senescent cultures, but the average rate of protein degradation is also slowed down considerably. This decrease in the rate of protein breakdown in aging cells stands in contrast with the previously observed acceleration of this process by other conditions (such as serum deprivation or overcrowding) that lead to the cessation of cellular growth. Though the retarded protein degradation may contribute to the acculation of abnormal proteins in senescent cells we find that the breakdown of grossly abnormal puromycin peptides proceeds equally rapidly in young and old cultures. The protein content of senescent cells increases by 1.8-fold as compared to young cells, while the average cell volume is increased even more (almost 5-fold). By contrast, consideration of the over-all balance of protein metabolism in these cells indicates that the average concentration of metabolically turning-over proteins is somewhat higher in senescent than in young fibroblasts.     

Indomethacin inhibits the insulin-induced increases in RNA and protein synthesis in L6 skeletal muscle myoblasts

Rates of accretion of RNA and protein and rates of protein synthesis were measured in sub-confluent cultures of L6 myoblasts. Insulin (100 microU/ml) stimulated protein synthesis by 15% within 30 min and by 40% at two and six hours. By six hours insulin also increased the accretion of RNA (+15%). The cyclo-oxygenase inhibitor indomethacin did not reduce the basal rate of RNA or protein accretion in L6 cells but reduced the rate of protein synthesis by 16%. When added together with insulin, indomethacin inhibited the hormonally-stimulated rate of protein synthesis and also significantly reduced the accretion of RNA. Indomethacin still reduced the effects of insulin on protein synthesis when added to the cells two hours after the hormone. Synthesis of RNA measured by the incorporation of [3H]-uridine was also stimulated by insulin but was inhibited by indomethacin only when the drug was present throughout the incubation. Inhibition of protein synthesis by cyclo-oxygenase inhibitors may be the result of both a direct action on translational efficiency and an effect on RNA synthesis.     

Prostaglandin production in myotube cultures. Influence on protein turnover.

The production of prostaglandins (PG) E2 and F2 alpha and their possible role in regulation of protein turnover in cultured skeletal-muscle cells were examined. Primary chick myoblasts and myotubes, and L8 myotubes, produced PGE2 and PGF2 alpha from endogenous arachidonic acid. PG production by all three cell types was increased manyfold by the addition of exogenous arachidonic acid. Arachidonate-stimulated PG production was inhibited by the addition of indomethacin (0.1 mM). When L8 and chick myotubes were treated with PGE2, PGF2 alpha, arachidonic acid (0.01 mM) or indomethacin (0.1 mM), no significant alterations in rates of protein synthesis or degradation were observed. Rates of protein synthesis and degradation in these cells were responsive to the addition of 10% fetal-bovine serum under identical experimental conditions. Thus, in contrast with incubated adult skeletal muscle, it appears that the production of prostaglandin metabolites from arachidonic acid is unrelated to regulation of protein turnover in cultured muscle cells.     

Effect of beta agonists on protein turnover in adipose tissue.

Chronic administration (21 days) of the beta agonist cimaterol to rats decreased epididymal fat by 27%, and inhibited in vitro rates of protein synthesis by 34% and net protein breakdown by 71% in adipose tissue. Likewise, incubation of rat adipose tissue with cimaterol and isoproterenol stimulated lipolysis, and inhibited protein synthesis and degradation. Thus, in addition to affecting muscle mass and lipid metabolism, beta agonists appear to slow rates of protein turnover in adipose tissue.     

Inactivation of protein synthesis stimulating activity in serum by cells

When Ehrlich ascites cells were cultivated in serum-free media their cellular protein synthetic rate declined to a new steady-state level and the cells stopped multiplying. On addition of serum the cellular protein synthetic rate increased to the level before serum starvation and cells resumed multiplication. The activity in serum stimulating protein synthesis was inactivated on incubation with cells. At cell concentrations of the usual culture conditions this inactivation took several hours; at very high cell concentrations it was complete in ten minutes. Serum-starved cells inactivated low serum (2%–6%) media in the same length of time. Studies of inactivation of high serum media demonstrated that cells had a limted capacity to inactivate. Cells grown in 10% serum were unable to inactivate. Inactivation was not due to accumulation in the medium of either low molecular or macromolecular cell products. Inactivation was strongly inhibited at 4° or by treatment of cells with fluoride or cycloheximide (long exposure): less inhibited by treatment with 2-deoxyglucose or glutaraldehyde; and slightly inhibited by treatment with cyanide or cycloheximide (short exposure). Inactivating ability was unaffected by trypsinization. These findings are best explained by the hypothesis that cells take up the serum activity by endocytosis.     

Requirement for protein synthesis in the regulation of protein breakdown in cultured hepatoma cells.

The modes of action of insulin and of inhibitors of protein synthesis on the degradation of labeled cellular proteins have been studied in cultured hepatoma (HTC) cells. Protein breakdown is accelerated upon the deprivation of serum (normally present in the culture medium), and this enhancement is inhibited by either insulin or cycloheximide. An exception is a limited class of rapidly turning over cellular proteins, the degradation of which is not influenced by insulin or cycloheximide. Alternative hypotheses to explain the relationship of protein synthesis to the regulation of protein breakdown, viz., control by the levels of precursors of protein synthesis, regulation by the state of the ribosome cycle, or requirement for a product of protein synthesis, have been examined. Protein breakdown was not influenced by amino acid deprivation, and measurements of valyl-tRNA levels in HTC cells subjected to various experimental conditions showed no correlation between the levels of charged tRNAVal and the rates of protein degradation. Three different inhibitors of protein synthesis (puromycin, pactamycin, and cycloheximide) suppressed enhanced protein breakdown in a similar fashion. A direct relationship was found between the respective potencies of these drugs to inhibit protein synthesis and to block enhanced protein breakdown. When cycloheximide and insulin were added following a prior incubation of HTC cells in a serum-free medium, protein breakdown was maximally suppressed within 15-30 min. Actinomycin D inhibited protein breakdown only after a time lag of about 90 min. It is suggested that the regulation of protein breakdown in hepatoma cells requires the continuous formation of a product of protein synthesis, in a manner analogous to the mode of the control of this process in bacteria.     

Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis.     

Inhibition of N-linked glycosylation induces early apoptosis in human promyelocytic HL-60 cells

Inhibition of protein N-glycosylation by tunicamycin induced morphological changes characteristic of apoptosis in human promyelocytic HL-60 cells. Internu-cleosomal DMA fragmentation could be detected after short-time incubation (between 6 and 9 h) of HL-60 cells with low doses of tunicamycin (0.05 μg/ml). Under these conditions the synthesis of glycoproteins was reduced to 17% of control values, while no significant changes in the rates of total protein synthesis could be observed. Tunicamycin ability to induce DNA fragmentation was in good correlation with its potency as glycosylation inhibitor in several myeloid cell lines. Tunicamycin-induced apoptosis was potentiated by activation of protein kinease C (PKC) by phorbol esters and partially prevented by the PKC inhibitor staurosporine. Inhibitors of RNA and protein synthesis displayed a protective effect. Treatment of HL-60 cells with tunicamycin did not elicit the expression of cell surface differentiation antigens or their ability to generate superoxide anion. In contrast, tunicamycin significantly inhibited these processes during dimethyl sulfoxide (DMSO)-induced myeloid differentiation. These observations indicate that the main effect of tunicamycin in HL-60 cells is the induction of apoptosis. © 1995 Wiley-Liss, Inc.     

Effects of anabolic agents on protein breakdown in L6 myoblasts.

1. Protein degradation in rat L6 myoblasts is inhibited by high concentrations of insulin as well as by foetal bovine serum and bovine colostrum, mixtures rich in growth-factor activity. 2. Growth factors achieve maximal effects within 2 h after addition to the cell cultures, but these diminish with time. Indeed, during incubations greater than 12 h, foetal calf serum actually stimulates protein breakdown. The changed response, however, is not due to the depletion of growth factors from serum. 3. Protein breakdown is stimulated by dexamethasone by a process that takes several hours to be expressed, but is more pronounced over a 4 h measurement period than over 18h. The glucocorticoid response is prevented by insulin or by cycloheximide. 4. Anabolic agents such as trenbolone, diethylstilboestrol and testosterone do not alter rates of intracellular protein breakdown and do not interfere with the glucocorticoid-induced catabolic response. 5. The results are consistent with anabolic steroids and related agents acting indirectly on muscle, perhaps via altering concentrations of growth factors of the somatomedin type.     

The epidermal growth factor-mediated inhibition of growth in A431 cells is accompanied by an atypical stimulation of protein breakdown

Addition of epidermal growth factor (EGF) to serum-free or serum-supplemented cultures of A431 cells stimulates protein breakdown without affecting rates of protein synthesis. These effects are atypical because in other cell lines, including AG2804-transformed human fibroblasts examined for comparison, EGF inhibits protein breakdown and stimulates protein synthesis. The response to EGF in A431 cells does not reflect a general post-receptor modification in growth factor action, since addition of insulin to the cells leads to the normal inhibition of protein breakdown. These findings indicate that the unusual growth inhibition produced by EGF in A431 cells can be explained by an increased rate of intracellular protein breakdown.     

Triacylglycerol lipase activities of cultured rat L6 myoblasts

The utilization of exogenous triacylglycerol by fusing and non-fusing rat L6 myoblasts grown in culture was investigated. Although small quantities of triacylglycerol were accumulated by both cell lines during an incubation of 2 h, no evidence could be found for the presence of lipoprotein lipase, either in the cells or released into the medium. Cell homogenate studies confirmed the absence of lipoprotein lipase but revealed the presence of an acid lipase having a pH optimum at 4.6. Acid lipase activity was mainly associated with a 15 000 g pellet and was capable of hydrolysing triolein at maximum velocity in the millimolar range. Unlike lipoprotein lipase, acid lipase was strongly inhibited by serum and preliminary investigations suggest that the inhibitory component of serum is located amongst the higher density lipoproteins. It is likely that the acid lipase is of lysosomal origin and is responsible for the hydrolysis of internalized triacylglycerol for subsequent utilization by the cell.     

Lack of differentiation following mouse serum treatment of cultured chick limb myoblasts

This investigation was conducted to assess the effects of mouse serum on chick skeletal muscle cell differentiation. In light of earlier findings of altered membrane phospholipid metabolism following mouse serum treatment of Friend erythroleukemic and chick chondrogenic cells, it was of interest to determine whether similar changes would modulate the fusion of mononucleated myoblasts, which is necessary for the formation of multinucleated skeletal muscle fibers. When mouse serum is added to low density cultures of enriched chick myoblasts shortly following cell attachment to the substratum, fusion is inhibited and neutral lipid accumulation ensues. There is an early inhibitory effect on DNA synthesis but not on protein synthesis. There is no increase in the uptake of 2-deoxyglucose following insulin stimulation of the cells, which suggests that while the cells are accumulating large amounts of lipid, they are not being converted into typical adipocytes. Finally, even in cultures of mouse serum-treated cells that undergo significant fusion, one observes thinner myotubes that do not spontaneously contract as do those of control cultures, as well as a disorganization of fluorescently stained actin and myosin myofilaments. These findings demonstrate that mouse serum acts in a dose-dependent manner, is not cytotoxic to the cells, but is capable of modulating normal developmental events of myoblasts as reported for other cell and tissue types.     

Endoglin modulation of TGF-beta1-induced collagen synthesis is dependent on ERK1/2 MAPK activation.

BACKGROUND/AIMS: Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in the extracellular matrix accumulation observed in fibrotic diseases. Endoglin is an important component of the TGF-beta receptor complex highly expressed in tissues undergoing fibrotic processes. Endoglin expression regulates the effect of TGF-beta on extracellular matrix synthesis. The purpose of our study has been to understand the molecular mechanism by which endoglin exerts its effects on fibrosis and the possible role of MAP kinases in these effects. METHODS: We have assessed in mock and in endoglin-transfected L6E9 myoblasts the effect of TGF-beta1 on collagen mRNA by Northern blot and effect of TGF-beta1 on collagen content in the cultured medium by [(3)H]-Proline incorporation into collagen proteins. Total and activated MAPK and their role on collagen synthesis were assessed by Western blot. RESULTS: TGF-beta1 induced an increase on alpha(2) (I) collagen mRNA expression and collagen accumulation in mock-transfected myoblasts, whereas the response was much lower in endoglintransfected cells. TGF-beta1 activated the ERK1/2 and p38 MAPK pathways but not the JNK pathway in L6E9 myoblasts. TGF-beta1-induced alpha(2) (I) collagen mRNA expression and collagen accumulation were completely inhibited by SB203580, in either mock or endoglintransfected myoblasts. PD98059 increased TGF-beta1 induced-collagen synthesis and accumulation in endoglin-transfected myoblasts but not in mock cells. CONCLUSION: Our studies demonstrate that TGF-beta1- induced collagen synthesis is mediated by p38 MAPK activation in L6E9 myoblasts. Furthermore, endoglin expression reduces basal and TGF-beta1 induced collagen synthesis when ERK1/2 pathway is operating.     

Endoglin expression regulates basal and TGF-beta1-induced extracellular matrix synthesis in cultured L6E9 myoblasts.

BACKGROUND/AIMS: TGF-beta1 plays a major role in extracellular matrix (ECM) accumulation in tissue fibrosis. Connective tissue growth factor appears to play a critical role in this effect. Endoglin is a component of the transforming growth factor b (TGF-beta) receptor complex. Endoglin is upregulated by TGF-beta1, but its functional role in ECM regulation is unknown. Using rat myoblasts as a model system, we have assessed the role of endoglin on regulating CTGF expression and ECM synthesis and accumulation in the presence or absence of TGF-beta1. METHODS: L6E9 myoblast cell line was transfected with human endoglin, and collagen, fibronectin and CTGF production was assessed by Western blot and by proline incorporation to collagen proteins. RESULTS: Northern blot analysis revealed that parental rat myoblasts L6E9 do not express endogenous endoglin. Upon endoglin transfection, endoglin-expressing cells displayed a decreased CTGF expression and decreased collagen and fibronectin accumulation respect to mock transfectants. Northern blot analysis also revealed a decreased alpha2 (I) procollagen mRNA expression in endoglin transfectants. TGF-beta1 treatment induced an increase in CTGF expression and collagen synthesis and accumulation in L6E9 myoblasts. This effect was significantly lower in endoglin-transfected than in mock-transfected cells. CONCLUSION: These results demonstrate that endoglin expression negatively regulates basal and TGF-beta1-induced CTGF and collagen expression and synthesis.     

Conditions necessary for inhibition of protein synthesis and production of cytopathic effect in Aedes albopictus cells infected with vesicular stomatitis virus.

The relationship between the development of cytopathic effect (CPE) and the inhibition of host macromolecular synthesis was examined in a CPE-susceptible cloned line of Aedes albopictus cells after infection with vesicular stomatitis virus. To induce rapid and maximal CPE, two conditions were required: (i) presence of serum in the medium and (ii) incubation at 34 degrees C rather than at 28 degrees C. In the absence of serum, incubation of infected cultures at 34 degrees C resulted in a significant increase in viral protein and RNA synthesis compared with that observed at 28 degrees C. However, when serum was present in the medium, by 6 h after infection protein synthesis (both host and viral) was markedly inhibited when infected cells were maintained at 34 degrees C. RNA synthesis (host and viral) was also inhibited in vesicular stomatitis virus-infected cells maintained at 34 degrees C with serum, but somewhat more slowly than protein synthesis. Examination of polysome patterns indicated that when infected cultures were maintained under conditions which predispose to CPE, more than half of the ribosomes existed as monosomes, suggesting that protein synthesis was being inhibited at the level of initiation. In addition, the phosphorylation of one (or two) polysome-associated proteins was reduced when protein synthesis was inhibited. Our findings indicate a strong correlation between virus-induced CPE in the LT-C7 clone of A. albopictus cells and the inhibition of protein synthesis. Although the mechanism of the serum effect is not understood, incubation at 34 degrees C probably predisposes to CPE and inhibition of protein synthesis by increasing the amount of viral gene products made.