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.     

Protein synthesis and secretion in a myogenic cell line

Myogenesis in a clonal myoblast cell line is accompanied by an increase in the specific activities of creatine phosphokinase and myokinase and in the rates of synthesis and accumulation of myosin heavy chain. Exponentially dividing myoblasts synthesize myosin heavy chain at a rate of about 1% of their rate of total protein synthesis; this rate increases 7-fold during the differentiation process. Both myoblasts and myotubes secrete a minimum of 12-soluble proteins. Although there is a quantitative change in the rates of appearance of five of these proteins during myogenesis, no qualitative changes in the profile of the secreted proteins are detected. Three of the secreted proteins share several properties of soluble collagen molecules. Basal laminae and polymerized collagen fibrils are associated with myotubes, but not with exponentially dividing myoblasts.     

Cell surface changes during muscle differentiation in vitro: a study with the probe 2,4,6-trinitrobenzene sulphonate.

Cell surface changes during muscle differentiation in vitro, were investigated using the non permeant probe 2,4,6-trinitrobenzene sulphonate (TNBS) in order to label the aminogroups of proteins exposed on the outer surface of the plasma membrane. Surface proteins of chick myotubes and 'mature' unfused myoblasts (myoblasts grown for 7 days in a calcium-depleted medium) were found to bind an equal amount of probe, which is twice the amount bound by surface proteins in 'immature' myoblasts (1--2 days of culture) and fibroblasts. This indicates that a 'remodelling' of the plasma membrane outer surface takes place in the course of muscle cell differentiation even in the absence of cell fusion. Moreover, the total amount of TNBS bound to the surface was 4--5 times greater in myotubes than in unfused myoblasts. This appears to result from the surface expansion which occurs in myotubes during the development of the T tubule system.     

Effects of ELF magnetic fields on protein expression profile of human breast cancer cell MCF7

Extremely Low Frequency Magnetic Fields (ELF MF) has been considered as a “possible human carcinogen” by International Agency for Research on Cancer (IARC) while credible mechanisms of its carcinogenicity remain unknown. In this study, a proteomics approach was employed to investigate the changes of protein expression profile induced by ELF MF in human breast cancer cell line MCF7, in order to determine ELF MF-responsive proteins. MCF7 cells were exposed to 50 Hz, 0.4 mT ELF MF for 24 h and the changes of protein profile were examined using two dimensional electrophoresis. Up to 6 spots have been statistically significantly altered (their expression levels were changed at least 5 fold up or down) compared with sham-exposed group. 19 ones were only detected in exposure group while 19 ones were missing. Three proteins were identified by LC-IT Tandem MS as RNA binding protein regulatory subunit Proteasome subunit beta type 7 precursor and Translationally Controlled Tumor Protein. Our finding showed that 50 Hz, 0.4 mT ELF MF alternates the protein profile of MCF7 cell and may affect many physiological functions of normal cell and 2-DE coupled with MS is a promising approach to elucidating cellular effects of electromagnetic fields.     

Comparative proteomes of the proliferating C(2)C(12) myoblasts and fully differentiated myotubes reveal the complexity of the skeletal muscle differentiation program

When cultured in low serum-containing growth medium, the mouse C(2)C(12) cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane- and nuclear-enriched proteins, and phospho-proteins from the proliferating C(2)C(12) cells and the fully differentiated myotubes by the combined methods of two-dimensional PAGE, quantitative PDQuest image analysis, and MS. Quantification of more than 2,000 proteins from C(2)C(12) myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant. In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C(2)C(12) myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes. We found that differential accumulation of 26 phospho-proteins also occurred during conversion of C(2)C(12) myoblasts into myotubes. We identified the differentially expressed proteins by MALDI-TOF-MS and LC-ESI-quadrupole ion trap MS/MS. We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate inter- and intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation.     

Effects of ELF magnetic fields on protein expression profile of human breast cancer cell MCF7

A case-control study in 1979 on electrical wiring configurations and childhood leukemia had stimulated interest in the issue that extremely low frequency magnetic fields (ELF MF) may have harmful biologi-cal effects especially on the incidence of human can-cer[1]. Since then, a large number of studies have been conducted to follow up this important result[2]. The majority of these studies indicate a weak association between exposure to 50―60 Hz ELF MF and the in-cidence of cancer; however…     

Induction of the proliferative phenotype in differentiated myogenic cells by hypoxia

The effect of cellular differentiation on the response of cells to hypoxic stress has been evaluated using the myogenic cell line BC3H1. Aerobic myocytes were predominantly in G0/G1 of the cell cycle and could be induced into S and G2/M of the cell cycle only by replating in high serum-containing medium at subconfluent cell density. In contrast, hypoxic myocytes demonstrated marked progression into S and G2/M upon reoxygenation without replating in the presence of serum. This modulation of myocytes by hypoxia was suggested further by the induction of 100-kDa and 9-kDa proteins (PSP 100 and PSP 9) which were otherwise only detectable in myoblasts. Two-dimensional gel analysis of newly synthesized proteins demonstrated that the five major glucose/oxygen-regulated proteins (GRP/ORP 260, 150, 100, 80, and 33) were induced in hypoxic myogenic cells independent of their state of differentiation. In addition to the GRP/ORPs, synthesis of 20 and 23 other major proteins was influenced in myocytes and myoblasts, respectively. The bulk of these alterations in myoblasts (70%) were inhibitions. In contrast, 75% of the alterations in myocyte protein synthesis were either enhancements or inductions. The data show that hypoxia can modulate the myocyte phenotype and invoke proliferative characteristics. Moreover, the data suggest that ischemia will have a different effect on and prognosis for tissues with a high mitotic index compared with differentiated tissues.     

Modulation of DNA synthesis in cultured muscle cells by 1,25-dihydroxyvitamin D-3

Biphasic effects of 1,25-dihydroxyvitamin D-3 on DNA synthesis were shown in primary cultured (24 h) chick embryo myoblasts exposed to physiological concentrations of the hormone. The sterol stimulated [3H]thymidine incorporation into DNA in proliferating myoblasts, e.g., at early stages of culture prior to cell fusion or in high serum-treated cells. The opposite effects were observed during the subsequent stage of myoblast differentiation in low-serum media. The mitogenic effect of 1,25-dihydroxyvitamin D-3 was correlated with an increase in c-myc mRNA and a decrease in c-fos mRNA levels, whereas its inhibitory action on DNA synthesis was accompanied by increased myofibrillar and microsomal protein synthesis and an elevation of creatine kinase activity, the latter suggesting a stimulation of muscle cell differentiation by the sterol. These data are in agreement with the results of previous morphological studies. Treatment of myoblasts with the calcium ionophore X-537 A or the phorbol ester TPA caused only a transient stimulation of [3H]thymidine incorporation into DNA, which occurred earlier than the response elicited by 1,25-dihydroxyvitamin D-3, suggesting that changes in intracellular Ca2+ and kinase C activity are not major mediators of the hormone effects. A similar temporal profile of changes in calmodulin mRNA levels as that of [3H]thymidine incorporation into DNA was observed after treatment of myoblasts with the sterol, in accordance with the role of calmodulin in the regulation of cell proliferation. 1,25-dihydroxyvitamin D-3 may play a function in embryonic muscle growth and differentiation.     

A collagen-binding protein involved in the differentiation of myoblasts recognizes the Arg-Gly-Asp sequence

We had earlier demonstrated that a 46-kDa glycoprotein is involved in the differentiation of rat skeletal myoblasts. We now show that the binding of this glycoprotein to collagen and gelatin is disrupted by Arg-Gly-Asp (RGD) containing peptide but not by Arg-Gly-Glu (RGE). The former peptide also selectively elutes the 46-kDa glycoprotein bound to gelatin-Sepharose. Since all other proteins which bind RGD sequences have been found at the cell surface, we attempted to localize the 46-kDa glycoprotein by means of immuno fluorescent staining and radioiodine labeling. Surprisingly, the majority of the protein was found to be localized in the endoplasmic reticulum. Protease treatment of a microsomal fraction revealed that the protein is in the interior of the reticulum. Immunoprecipitation experiments, using a polyclonal antibody against the 46-kDa protein, demonstrated that no closely related proteins exist in myoblasts and also confirmed that the protein was not a fragment of a cell-surface localized protein. These findings suggest that the RGD sequence is also used in protein recognition within the cell.     

2-DE proteome analysis of a proliferating and differentiating human neuronal stem cell line (ReNcell VM)

The proteome of a proliferating human stem cell line was analyzed and then utilized to detect stem cell differentiation-associated changes in the protein profile. The analysis was conducted with a stable human fetal midbrain stem cell line (ReNcell VM) that displays the properties of a neural stem cell. Therefore, acquisition of proteomic data should be representative of cultured human neural stem cells (hNSCs) in general. Here we present a 2-DE protein-map of this cell line with annotations of 402 spots representing 318 unique proteins identified by MS. The subsequent proteome profiling of differentiating cells of this stem cell line at days 0, 4 and 7 of differentiation revealed changes in the expression of 49 identified spots that could be annotated to 45 distinct proteins. This differentiation-associated expression pattern was validated by Western blot analysis for transgelin-2, proliferating cell nuclear antigen, as well as peroxiredoxin 1 and 4. The group of regulated proteins also included NudC, ubiquilin-1, STRAP, stress-70 protein, creatine kinase B, glial fibrillary acidic protein and vimentin. Our results reflect the large rearrangement of the proteome during the differentiation process of the stem cells to terminally differentiated neurons and offer the possibility for further characterization of specific targets driving the stem cell differentiation.     

Role of the p66Shc isoform in insulin-like growth factor I receptor signaling through MEK/Erk and regulation of actin cytoskeleton in rat myoblasts

To investigate the role of Shc in IGF action and signaling in skeletal muscle cells, Shc protein levels were reduced in rat L6 myoblasts by stably overexpressing a Shc cDNA fragment in antisense orientation (L6/Shcas). L6/Shcas myoblasts showed marked reduction of the p66Shc protein isoform and no change in p52Shc or p46Shc proteins compared with control myoblasts transfected with the empty vector (L6/Neo). When compared with control, L6/Shcas myoblasts demonstrated 3-fold increase in Erk-1/2 phosphorylation under basal conditions and blunted Erk-1/2 stimulation by insulin-like growth factor I (IGF-I), in the absence of changes in total Erk-1/2 protein levels. Increased basal Erk-1/2 activation was paralleled by a greater proportion of phosphorylated Erk-1/2 in the nucleus of L6/Shcas myoblasts in the absence of IGF-I stimulation. The reduction of p66Shc in L6/Shcas myoblasts resulted in marked phenotypic abnormalities, such as rounded cell shape and clustering in islets or finger-like structures, and was associated with impaired DNA synthesis in response to IGF-I and lack of terminal differentiation into myotubes. In addition, L6/Shcas myoblasts were characterized by complete disruption of actin filaments and cell cytoskeleton. Treatment of L6/Shcas myoblasts with the MEK inhibitor PD98059 reduced the abnormal increase in Erk-1/2 activation to control levels and restored the actin cytoskeleton, re-establishing the normal cell morphology. Thus, the p66Shc isoform exerts an inhibitory effect on the mitogen-activated protein kinase signaling pathway in rodent myoblasts, which is necessary for maintenance of IGF responsiveness of the MEK/Erk pathway and normal cell phenotype.     

Coordinate accumulation of contractile protein mRNAs during myoblast differentiation.

The synthesis of contractile protein mRNAs has been studied during the differentiation of quail skeletal muscle myoblasts in culture. Eight contractile protein mRNAs were identified by translation of total cellular RNA isolated from differentiated myofibers in wheat germ and reticulocyte lysates. Products of the translation systems were fractionated by two-dimensional gel electrophoresis, and incorporation of [³⁵S]methionine into individual contractile proteins was quantitated by computerized densitometry of autoradiograms. These translation assay systems were used to quantitate levels of contractile protein mRNAs in cultures of myoblasts undergoing highly synchronous differentiation. Our results show that dividing myoblasts contain very little, if any, translatable contractile protein mRNA. The mRNAs coding for myosin heavy chain, the musclespecific actin, three myosin light chains, two tropomyosin subunits, and one troponin subunit begin to coordinately accumulate at fusion, when contractile protein synthesis is activated. Their levels increase 50- to 200-fold during the next 30 hr, paralleling increases in the rates of contractile protein synthesis. These results indicate that the contractile protein mRNAs accumulate coordinately during myoblast differentiation and that contractile protein synthesis is regulated by changes in the levels of these mRNAs.     

Proteomic analysis of plasma membrane lipid rafts of HL-60 cells

Neutrophils acquire phagocytic activity as they differentiate. Recently, plasma membrane lipid rafts have been shown to play important roles in the process of phagocytosis in neutrophils. To characterize the proteins involved in phagocytosis and to elucidate the process by which they acquire phagocytic activity, we investigated by nano-LC-MS/MS analysis the changes in protein composition of plasma membrane lipid rafts during DMSO-induced differentiation of the human leukemia cell line HL-60 cells into neutrophilic lineage. Based on the spectrum counts of 147 proteins identified, 25 proteins were upregulated and 49 were downregulated by DMSO treatment. CD11b/CD18 subunits of beta2-integrin Mac-1, CD35, and GPI-80, which are known to be upregulated during differentiation, were dominantly detected in the lipid rafts of DMSO-treated cells. Many known membrane proteins, G proteins, and cytoskeletal proteins were also detected and they showed characteristic distributions. Absolute quantification of nine proteins in the lipid rafts using internal standard peptides labeled with stable isotopes showed that the amount of protein almost corresponded to the results obtained by spectrum count. Identified proteins, expression of which was altered by DMSO treatment, are expected to be candidate proteins involved in differentiation and functions of neutrophils.     

CELF and PTB proteins modulate the inclusion of the β-tropomyosin exon 6B during myogenic differentiation

Exon 6B from the chicken β-tropomyosin pre-mRNA is alternatively spliced during myogenic differentiation. Exon 6B is excluded in mRNA from myoblasts and included in mRNA from myotubes. We investigated the regulation of exon 6B inclusion ex vivo in a quail myogenic cell line, which behaves as myoblasts in undifferentiated state and as myotubes after differentiation. We show that the β-tropomyosin exon 6B is a novel target of CUG-BP and ETR-3-like factor (CELF). Overexpression of CELF proteins in myoblasts activates splicing of exon 6B. Using a dominant-negative form of CELF4, we demonstrate that CELF proteins are involved in switching splicing from exon 6A towards exon 6B inclusion during myogenic differentiation. We also found that polypyrimidine tract binding protein (PTB) is required for splicing repression of exon 6B in myoblasts. CELF and PTB proteins exhibit antagonistic properties toward inclusion of exon 6B during myogenic differentiation. Our results suggest that a change in the protein level of CUGBP1 and PTB proteins, associated with a distinct pattern of PTB during the transition from myoblasts to myotubes is one of the parameters involved in regulating splicing of exon 6B during myogenesis.