Search

Mice lacking the myristoylated alanine-rich C-kinase substrate, or MARCKS protein, exhibit abnormalities consistent with a defect in the ability of neurons to migrate appropriately during forebrain development. To investigate the possibility that this phenotype could be due to disruption of normal cellular adhesion to extracellular matrix, an assay was developed in which 293 cells co-expressing MARCKS and green fluorescent protein were tested for their adhesion competence on various substrates. Fluorescence-activated cell sorting of adherent and non-adherent green fluorescent protein-expressing cells demonstrated that wild-type MARCKS inhibited adhesion of cells to fibronectin, whereas a non-myristoylated mutant did not inhibit adhesion of cells to a variety of substrates. The fibronectin competitive inhibitor RGD peptide inhibited adhesion of cells expressing all MARCKS variants equally. Cytochalasin D inhibited the adhesion of cells expressing non-myristoylated MARCKS, but did not further decrease the adhesion of cells expressing adhesion-inhibitory proteins. Confocal microscopy demonstrated the presence of inhibitory, myristoylated MARCKS at the plasma membrane, suggesting that localization at this region might be important for MARCKS to inhibit cellular adhesion. These data suggest a possible myristoylation-dependent function of MARCKS to inhibit cellular adhesion to extracellular matrix proteins, indicating a potential mechanism for the cell migration defects seen in the MARCKS-deficient mice.

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.

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.

The importance of regulating the cellular concentrations of the myristoylated alanine-rich C kinase substrate (MARCKS), a major cellular substrate of protein kinase C, is indicated by the fact that mice lacking MARCKS exhibit gross abnormalities of central nervous system development and die shortly after birth. We previously identified a novel means of regulating cellular MARCKS concentrations that involved a specific proteolytic cleavage of the protein and implicated a cysteine protease in this process (Spizz, G., and Blackshear, P. J. (1996) J. Biol. Chem. 271, 553-562). Here we show that p40, the carboxyl-terminal fragment resulting from this cleavage of MARCKS, was associated with the mitochondrial/lysosomal pellet fraction of human diploid fibroblasts and that its generation in cells was sensitive to treatment with NH4Cl. These data suggest the involvement of lysosomes in the generation and/or stability of p40. The MARCKS-cleaving enzyme (MCE) activity was peripherally associated with a 10,000 x g pellet fraction from bovine liver, and it co-purified with the activity and immunoreactivity of a lysosomal protease, cathepsin B. Cathepsin B catalyzed the generation of p40 from MARCKS in a cell-free system and behaved similarly to the MCE with respect to mutants of MARCKS previously shown to be poor substrates for the MCE. Treatment of fibroblasts with a cell-permeable, specific inhibitor of cathepsin B, CA074-Me, resulted in parallel time- and concentration-dependent inhibition of cathepsin B and MCE activity. Incubation of a synthetic MARCKS phosphorylation site domain peptide with purified cathepsin B resulted in cleavage of the peptide at sites consistent with preferred cathepsin B substrate sites. These data provide evidence for the identity of the MCE as cathepsin B and suggest that this cleavage most likely takes place within lysosomes, perhaps as a result of specific lysosomal targeting sequences within the MARCKS primary sequence. The data also suggest a direct interaction between MARCKS and cathepsin B in cells and leave open the possibility that MARCKS may in some way regulate the protease for which it is a substrate.

The myristoylated alanine-rich C kinase substrate (MARCKS) is a major cellular substrate of protein kinase C. Its concentration in cells is important for the normal development of the central nervous system, and perhaps other physiological processes. We found that MARCKS concentrations in cells were regulated in part by a specific proteolytic cleavage; this resulted in two fragments, each representing about half of the intact protein, that co-existed with MARCKS in cells and tissues. These fragments were present in significant concentrations in quiescent fibroblasts; they disappeared, and the amount of intact MARCKS increased, within 15 s of activation of protein kinase C by serum. In vitro experiments demonstrated that phosphorylated MARCKS was a poor substrate for a protease activity present in cell extracts, whereas dephosphorylated MARCKS was a good substrate. Both the protease activity and the specific MARCKS cleavage products were essentially absent in brain, but present in many other cells and tissues. The protease activity, which had the characteristics of a cysteine protease, cleaved MARCKS between Asn147 and Glu148 of the bovine sequence, three amino acids to the amino-terminal side of the MARCKS phosphorylation site domain. These studies demonstrate that MARCKS is subjected to specific cleavage by a cellular protease, in a manner dependent on the phosphorylation state of the substrate. This represents a novel means of regulating cellular MARCKS concentrations; these data also raise the interesting possibility that MARCKS is involved in regulating the activity of this novel cellular protease.

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.

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.

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.

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.

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.

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.

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.