Biomedical subjects

G Spizz

Publications and source records attributed to G Spizz.

11 recordsLinked to original sources

PubMed1992

Growth factors promote inositol uptake in BC3H1 cells.

BC3H1 cells induced to differentiate by serum withdrawal were found to incorporate substantially less [3H]inositol into their phosphoinositides than cells induced to differentiate by growth in the presence of high serum. This decrease was found to be due to a decline in the rate of [3H]inositol uptake by the serum-starved cells. Addition of purified growth factors such as TGF-beta, EGF and FGF to these cells promoted inositol uptake and lead to an increase in the incorporation of [3H]inositol into phosphoinositides. Stimulation of inositol uptake by TGF-beta required at least a 24 hr exposure to the growth factor. These data indicate that growth factors regulate phosphoinositide metabolism at many different levels including at the level of inositol uptake.

Animals↗

PubMed1989

A ras-dependent pathway abolishes activity of a muscle-specific enhancer upstream from the muscle creatine kinase gene.

Differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific genes whose products are required for the specialized functions of the mature muscle fiber. The program for myogenic differentiation is subject to negative control by several peptide growth factors and by the products of mutationally activated ras oncogenes, which persistently activate intracellular cascades normally triggered by specific growth factors. Previously, we reported that induction of the muscle creatine kinase (mck) gene during myogenesis was dependent on a distal upstream enhancer that cooperated with a proximal promoter to direct high levels of expression in developing muscle cells (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, T. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909). To investigate the mechanisms whereby ras blocks the induction of muscle-specific genes, we have examined the ability of mck 5' regulatory elements to direct expression of the linked reporter gene for chloramphenicol acetyltransferase (cat) in C2 myoblasts bearing mutant N-ras and H-ras oncogenes. In this paper we report that expression of activated ras alleles abolishes activity of the mck upstream enhancer but does not affect the activity of the mck promoter. The ability of ras to repress the expression of mck-cat fusion genes that have been transfected either transiently or stably into myoblasts suggests that ras may exert its effects on muscle-specific genes through mechanisms independent of chromatin configurations or DNA methylation. These results also suggest that ras blocks establishment of the myogenic phenotype by preventing the accumulation of regulatory factors required for transcriptional induction of muscle-specific genes.

Animals↗

PubMed1988

Decrease in transforming growth factor-beta binding and action during differentiation in muscle cells.

We report here the effects of differentiation on the binding and action of transforming growth factor-beta (TGF-beta) in three lines of myogenic cells. In two lines (L6-A1 and C2) which irreversibly differentiate by fusing to form postmitotic myotubes, there is a virtual disappearance of TGF-beta binding sites as differentiation occurs. Analyses of the binding curves by the method of Scatchard indicates that there is little or no change in affinity but a substantial decrease in the number of binding sites. In L6-A1 cells, responsiveness to TGF-beta decreases in parallel to the loss of receptors. The decreases in TGF-beta binding and activity with differentiation are not paralleled by similar changes in another growth factor, insulin-like growth factor-I, which exhibits little change in binding and only a modest decrease in activity as L6-A1 myoblasts differentiate to form myotubes. In a third cell line (BC3H1), which exhibits reversible differentiation without fusion, there is little or no change in TGF-beta binding as the cells differentiate. Comparisons with reported decreases in binding of fibroblast and epidermal growth factors indicates that there are substantial differences in growth factor binding and actions as muscle cells differentiate, but it is not possible to make the simple generalization that differentiation is accompanied by a decrease in binding of all growth factors.

Animals↗

PubMed1988

Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene.

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

Animals↗

PubMed1987

Inhibition of myogenic differentiation by fibroblast growth factor or type beta transforming growth factor does not require persistent c-myc expression.

Skeletal muscle differentiation is accompanied by accumulation of the mRNA encoding the muscle isoenzyme of creatine kinase (MCK) and can be suppressed by serum components, fibroblast growth factor (FGF), or type beta transforming growth factor (TGF beta). Using the nonfusing myogenic cell line, BC3H1, the potential involvement of c-myc in growth factor-dependent inhibition of myogenesis was examined. Withdrawal of undifferentiated myoblasts from the cell cycle in medium with 0.5% serum was associated with a precipitous decline in expression of c-myc mRNA followed by induction of MCK mRNA. In 0.5% serum containing TGF beta, c-myc mRNA declined to a level identical to that in differentiated cells; however, MCK mRNA was not expressed. Exposure of quiescent differentiated cells to FGF or TGF beta caused disappearance of muscle-specific gene products and was accompanied by only transient low level induction of c-myc mRNA. These data indicate that persistent c-myc expression is not required for growth factor-mediated inhibition of myogenic differentiation.

Animals↗

PubMed1987

Autonomous expression of c-myc in BC3H1 cells partially inhibits but does not prevent myogenic differentiation.

Myogenic differentiation is obligatorily coupled to withdrawal of myoblasts from the cell cycle and is inhibited by specific polypeptide growth factors. To investigate the potential involvement of c-myc in the control of myogenesis, the BC3H1 muscle cell line was stably transfected with a simian virus 40 promoter:c-myc chimeric gene. In quiescent cells in 0.5% serum, the exogenous c-myc gene was expressed at a level more than threefold greater than the level of endogenous c-myc in undifferentiated, proliferating cells of the parental line in 20% serum. The transfected myc gene partially inhibited the expression of both muscle creatine kinase and the nicotinic acetylcholine receptor, but was not sufficient to prevent the induction of these muscle differentiation products upon mitogen withdrawal.

Animals↗

PubMed1987

The oncogenic forms of N-ras or H-ras prevent skeletal myoblast differentiation.

Differentiation of skeletal muscle involves withdrawal of myoblasts from the cell cycle, fusion to form myotubes, and the coordinate expression of a variety of muscle-specific gene products. Fibroblast growth factor and type beta transforming growth factor specifically inhibit myogenesis; however, the transmembrane signaling pathways responsible for suppression of differentiation by these growth factors remain elusive. Because ras proteins have been implicated in the transduction of growth factor signals across the plasma membrane, we used DNA-mediated gene transfer to investigate the potential involvement of this family of regulatory proteins in the control of myogenesis. Transfection of the mouse skeletal muscle cell line C2 with the oncogenic forms of H-ras or N-ras completely suppressed both myoblast fusion and induction of the muscle-specific gene products nicotinic acetylcholine receptor and creatine kinase. Inhibition of differentiation by activated ras genes occurred at the level of muscle-specific mRNA accumulation. In contrast, proto-oncogenic forms of N-ras or H-ras had no apparent effects on the ability of C2 cells to differentiate. Myoblasts transfected with activated ras genes exhibited normal growth properties and ceased proliferating in the absence of mitogens, indicating that ras inhibited differentiation through a mechanism independent of cell proliferation. These results demonstrate that activated ras gene products mimic the inhibitory effects of fibroblast growth factor and type beta transforming growth factor on myogenic differentiation and suggest that each of these regulators of myogenesis may operate through a common intracellular pathway.

Animals↗

PubMed1986

Serum and fibroblast growth factor inhibit myogenic differentiation through a mechanism dependent on protein synthesis and independent of cell proliferation.

Myogenesis is accompanied by the withdrawal of proliferating myoblasts from the cell cycle, their fusion to form myotubes, and the coordinate expression of a variety of muscle-specific gene products, such as the muscle isoenzyme of creatine kinase (MCK). In the present study we used the nonfusing muscle cell line, BC3H1, to examine the mechanisms involved in regulation of MCK mRNA expression. Proliferating BC3H1 cells, in media with 20% fetal calf serum, had undetectable levels of MCK mRNA. Exposure of undifferentiated cells to media containing 0.5% serum resulted in withdrawal of cells from the cell cycle and in a several hundred-fold increase in the steady state level of MCK mRNA. Induction of this muscle-specific mRNA could be rapidly reversed by exposure of quiescent differentiated cells to media containing either 20% serum or pituitary fibroblast growth factor. The decline in the steady state level of MCK mRNA following mitogenic stimulation was not dependent upon reentry of cells into the cell cycle, but it did require protein synthesis. Together, these data indicate that fibroblast growth factor can specifically inhibit muscle-specific gene expression through a mechanism independent of cell proliferation. The finding that MCK mRNA was down-regulated by a mechanism that required protein synthesis suggests that mitogen-inducible early gene products may be involved in regulation of muscle gene expression.

Animals↗

PubMed1986

Fatty acylation of cellular proteins. Temporal and subcellular differences between palmitate and myristate acylation.

Previous studies demonstrated that palmitate and myristate are covalently linked to distinct sets of cellular proteins and that the linkages through which these fatty acids are attached to the polypeptide chains are different (Olson, E. N., Towler, D. A., and Glaser, L. (1985) J. Biol. Chem. 260, 3784-3790). In the present study, the kinetics and subcellular sites of acylation of proteins with palmitate and myristate were examined in the BC3H1 muscle cell line. Acylation with myristate was an extremely early modification that appeared to take place cotranslationally or shortly thereafter for a variety of soluble and membrane-bound proteins. In contrast, acylation of proteins with palmitate was a post-translational event that occurred exclusively on membrane proteins. To begin to understand the intracellular pathways that acyl proteins follow during their maturation, the degree of glycosylation, and the nature of the interaction of these proteins with membranes were examined. The majority of acyl proteins were tightly associated with membranes and could not be removed by conditions that release peripheral proteins from membranes. However, only a minor fraction of acylated proteins were N-glycosylated. These data suggest that the acyltransferases that attach palmitate and myristate to proteins are present in different subcellular locations and demonstrate that these fatty acids are attached to newly synthesized acyl proteins at different times during their maturation. The lack of carbohydrate on the majority of integral membrane acyl proteins suggests that these proteins may follow intracellular pathways that are different from those followed by cell surface glycoproteins.

Acyltransferases↗

PubMed1986

Regulation of myogenic differentiation by type beta transforming growth factor.

Type beta transforming growth factor (TGF beta) has been shown to be both a positive and negative regulator of cellular proliferation and differentiation. The effects of TGF beta also are cell-type specific and appear to be modulated by other growth factors. In the present study, we examined the potential of TGF beta for control of myogenic differentiation. In mouse C-2 myoblasts, TGF beta inhibited fusion and prevented expression of the muscle-specific gene products, creatine kinase and acetylcholine receptor. Differentiation of the nonfusing muscle cell line, BC2Hl, was also inhibited by TGF beta in a dose-dependent manner (ID50 approximately 0.5 ng/ml). TGF beta was not mitogenic for either muscle cell line, indicating that its inhibitory effects do not require cell proliferation. Inhibition of differentiation required the continual presence of TGF beta in the culture media. Removal of TGF beta led to rapid appearance of muscle proteins, which indicates that intracellular signals generated by TGF beta are highly transient and require continuous occupancy of the TGF beta receptor. Northern blot hybridization analysis using a muscle creatine kinase cDNA probe indicated that TGF beta inhibited differentiation at the level of muscle-specific mRNA accumulation. These results provide the first demonstration that TGF beta is a potent regulator of myogenic differentiation and suggest that TGF beta may play an important role in the control of tissue-specific gene expression during development.

Animals↗

PubMed1986

Mitogens and protein synthesis inhibitors induce ornithine decarboxylase gene transcription through separate mechanisms in the BC3H1 muscle cell line.

Ornithine decarboxylase (ODCase), the rate-limiting enzyme in polyamine biosynthesis, exhibits dramatic fluctuations in activity in response to a variety of hormones and growth factors and has been shown to be down-regulated during myogenesis. In the present study, the molecular mechanisms involved in expression of ODCase mRNA were examined in cells of the BC3H1 muscle line. Proliferating, undifferentiated cells in medium with 20% fetal calf serum displayed high levels of ODCase mRNA and enzyme activity. The transfer of proliferating cells to medium containing 0.5% serum resulted in their withdrawal from the cell cycle and a 20- to 50-fold reduction in the steady-state level of ODCase mRNA within 24 h. Down-regulation of ODCase mRNA was paralleled by a decrease in ODCase enzyme activity and ODCase gene transcription. ODCase mRNA was rapidly reinduced by exposure of quiescent, differentiated cells to medium with 20% serum or by inhibition of protein synthesis with cycloheximide. The accumulation of ODCase mRNA after mitogenic stimulation or protein synthesis inhibition was accompanied by an increase in ODCase gene transcription. The mechanisms whereby mitogens and protein synthesis inhibitors induced ODCase transcription appeared to be different since cycloheximide potentiated the effects of mitogens, resulting in superinduction of ODCase transcription to a level significantly greater than in the presence of mitogens alone. These results indicate that ODCase down-regulation during myogenesis is controlled primarily at the level of ODCase gene transcription. These data also demonstrate that ODCase expression is regulated by antagonistic signals, positive signals for transcription elicited by mitogens and negative signals from endogenous protein repressors that influence ODCase transcription.

Animals↗