Substrate-associated macromolecules promote cytodifferentiation of BC3H1 myogenic cells.

Differentiated mouse BC3H1 myogenic cells secrete substrate-associated macro-molecules (SAM) which restrict the proliferation of undifferentiated cells and promote both cell shape changes and expression of predominantly the vascular smooth muscle (VSM)-specific isoform of the contractile protein alpha-actin. While we previously reported that high cell density was required for stimulating maximal expression of VSM alpha-actin in BC3H1 cells (Strauch and Reeser: Journal of Biological Chemistry 264:8345-8355, 1989), the permissive effect of SAM on myoblast cytodifferentiation was not at all dependent on the formation of cell to cell contacts. This observation suggests that biogenesis of an extracellular matrix rather than the formation of physical contacts between cells may be the rate-limiting step for induction of VSM alpha-actin expression at high cell density. The biologically active moieties in SAM that promote cytodifferentiation also are expressed by mouse embryonic fibroblast cell lines and are distinctly different from a class of adheron-like macromolecules released by differentiated BC3H1 myocytes directly into the culture medium. While SAM was cell growth restrictive, reconstituted particulate material (RPM) prepared from myocyte-conditioned medium promoted the adhesion and proliferation of growth-arrested myoblasts. SAM and RPM are composed of different polypeptide subunits which collectively may establish microenvironmental conditions that are permissive for BC3H1 myogenic cell differentiation.     

Myristic acid utilization and processing in BC3H1 muscle cells.

Because myristic acid (14:0) is important in regulating cell function, we have studied its utilization in BC3H1 muscle cells. Phosphatidylcholine contained 70-80% of the [9,10-3H]14:0 radioactivity incorporated into the cell phospholipids. In both myoblasts and myocytes, however, large amounts of radioactivity also accumulated in a labile neutral lipid pool consisting mostly of triacylglycerol. Therefore, radioactive lipid products formed when BC3H1 cells labeled with 14:0 are stimulated are not necessarily derived only from phosphatidylcholine. Elongation of [9,10-3H]14:0 occurred rapidly in the myoblasts and myocytes, and extensive desaturation also occurred in the myoblasts. Thus, even after short periods of labeling, substantial amounts of radioactivity are contained in fatty acids other than 14:0. The labeling of proteins with [9,10-3H]myristic acid was generally similar in the myoblasts and myocytes. A number of lipid-soluble, polar radioactive metabolites were released into the medium during incubation of [9,10-3H]14:0 with the cells. [1-14C] 14:0 was not converted to these compounds, indicating that they are chain-shortened 14:0 derivatives. Based on chemical analysis, two of the major products appear to be hydroxylated fatty acids. This oxidation process shows some specificity for 14:0 because similar compounds were not produced from palmitic, oleic, or linoleic acids. The myocytes formed larger amounts of the metabolites than the myoblasts, suggesting that differentiation may increase the activity of this 14:0 oxidative pathway.     

Induction of vascular smooth muscle alpha-isoactin expression in BC3H1 cells

An isoactin analysis was performed on L-[35S]cysteine labeled BC3H1 cells to determine if these smooth muscle-like cells synthesize vascular smooth muscle actin. Three different NH2-terminal peptides were identified on thin layer electrophoretograms of DNase I-purified and trypsin-digested BC3H1 cell actin. Results obtained from secondary digestion with thermolysin or Staphylococcus aureus V8 protease showed that the most acidic NH2-terminal peptide was derived from vascular smooth muscle alpha-isoactin. Treatment of cell monolayers with serum-free medium caused a 3-fold increase in the level of alpha-isoactin expression and a concomitant decrease in the level of non-muscle beta- and gamma-isoactin. Cell-cell contact was required for induction of alpha-isoactin, and the effects of serum depletion on isoactin expression and cell growth were reversible. The intensity of about 11 out of 500 polypeptide spots on two-dimensional gels of BC3H1 cell polypeptides also was influenced by the culture conditions. The finding that smooth muscle isoactin expression was coupled to cell growth conditions indicate the potential usefulness of BC3H1 cells in studies of isoactin expression and utilization during vascular smooth muscle development.     

Growth factors, signaling pathways, and the regulation of proliferation and differentiation in BC3H1 muscle cells. I. A pertussis toxin- sensitive pathway is involved

Cells of the nonfusing muscle cell line BC3H1 stop proliferating and express a family of muscle-specific proteins when the FBS concentration is reduced from 20 to 0.5% (Munson, R., K.L. Caldwell, and L. Glaser. 1982. J. Cell Biol. 92:350-356). Several growth factors have been shown to block differentiation in this cell line. To begin to investigate the potential role of G proteins in signal transducing pathways from these receptors, we have examined the effects of cholera toxin (CT) and pertussis toxin (PT) on proliferation and differentiation in BC3H1 cells. PT specifically ADP ribosylates a protein with an apparent molecular mass of 40 kD in BC3H1 cell membranes, whereas CT specifically ADP ribosylates three proteins of 35-43 kD. When added to exponentially growing cells in 20% FBS, CT and PT inhibited [3H]thymidine incorporation by up to 75% in a dose-dependent fashion. We found the synthesis of creatine kinase (CK) and skeletal muscle myosin light chain was reversibly induced in cells in 20% FBS treated with PT, but no increased synthesis was seen in cells treated with CT or in control cells; Northern analysis indicated this induction was at the level of mRNA. In cells shifted to 0.5% FBS, CT inhibited the normally induced synthesis of CK whereas PT potentiated it by approximately 50%. Forskolin also inhibited growth in 20% FBS and differentiation in 0.5% FBS medium in a dose-dependent fashion. both forskolin and CT elevated cAMP levels compared with control or PT-treated cells, suggesting that CT is blocking proliferation and differentiation by elevating cAMP levels. These results establish that a PT-sensitive pathway is involved in regulating proliferation and differentiation in BC3H1 cells, and we postulate that PT functions by ADP ribosylating a G protein that transduces signals from growth factor receptors in these cells.     

The expression of sarcomeric muscle-specific contractile protein genes in BC3H1 cells: BC3H1 cells resemble skeletal myoblasts that are defective for commitment to terminal differentiation

The BC3H1 cell line has been used widely as a model for studying regulation of muscle-related proteins, such as the acetylcholine receptor, myokinase, creatine kinase, and actin. These cells, derived from a nitrosourea-induced mouse brain neoplasm, have some of the morphological characteristics of smooth muscle and have been shown to express the vascular smooth muscle isoform of alpha-actin. To provide further information about the contractile protein phenotype of BC3H1 and to gain additional insights into the possible tissue of origin of these cells, we have examined the expression of a battery of contractile protein genes. During rapid growth, subconfluent BC3H1 cells express the nonmuscle isoform of alpha-tropomyosin (alpha-Tm) and the nonsarcomeric isoforms of myosin heavy and light chains (MHCs and MLCs, respectively), but do not express troponin T(TnT). However, when BC3H1 cells differentiate in response to incubation in serum-deprived medium or upon approaching confluence, they express TnT as well as sarcomeric muscle isoforms of MHC, MLC 2 and 3, alpha-Tm, and alpha-actin. These results suggest that BC3H1 is a skeletal muscle cell line of ectodermal origin that is defective for commitment to terminal differentiation.     

Multiple controls for the synthesis of muscle-specific proteins in BC3H1 cells

The regulation of the synthesis of muscle-specific proteins has been examined in BC3H1 cells, a smooth muscle-like cell line isolated by Schubert et al. (J. Cell Biol., 1974, 61: 398-413.). The synthesis of both creatine kinase and the acetylcholine receptor appear to be under dual control, a positive control due to cell-cell contact which increases the rate of synthesis of this protein, and a negative signal, elicited by serum components, that decreases the rate of synthesis of these proteins. Induction of muscle-specific proteins in BC3H1 cells is a reversible process and can be arrested after partial induction has taken place by the addition of serum or high-molecular-weight protein fraction from serum to these cells. The high-molecular-weight protein fraction from serum is not by itself mitogenic for Bc3H1 cells and cannot be replaced by a variety of known hormones (mitogenic factors).     

Epidermal growth factor controls smooth muscle alpha-isoactin expression in BC3H1 cells

We have examined the effects of epidermal growth factor (EGF), platelet-derived growth factor, and insulin on the differentiation of a mouse vascular smooth muscle-like cell line, the BC3H1 cells. On the basis of cell morphology and smooth muscle alpha-isoactin synthesis, we demonstrate that EGF at physiological concentrations prevents the differentiation of these cells, whereas platelet-derived growth factor has no apparent effect. The induction of alpha-isoactin synthesis by serum deprivation is inhibited by EGF in a dose-dependent manner with a half-maximal effect at 3-5 ng/ml and a maximal inhibition at approximately 30 ng/ml. Northern analysis also shows that EGF blocks the accumulation of alpha-isoactin mRNA normally observed during cell differentiation. Addition of EGF to differentiated cells results in a repression of alpha-isoactin synthesis, a stimulation of beta- and gamma-isoactin synthesis, and the stabilization of the nonmuscle isoactins. The synthesis of creatine phosphokinase, a muscle-specific noncontractile protein, is also regulated by EGF in a similar fashion. Modulation by EGF of alpha-isoactin expression is not affected by aphidicolin and is therefore independent of its mitogenic effect on these cells. Insulin is not required for observation of the EGF-dependent effects but instead seems to promote differentiation. Our results show that EGF can replace serum in controlling the differentiation of BC3H1 cells.     

Control of expression of the 1,4-dihydropyridine receptor in BC3H1 cells

To determine whether expression of the 1,4-dihydropyridine receptor of skeletal muscle Ca2+ channels is regulated by signals that impinge on muscle-specific gene expression, BC3H1 muscle cells were analyzed using (+)[3H]PN200-110 as a probe for the receptor. No dihydropyridine binding sites were detected in proliferating cells. Binding site density increased following serum withdrawal, peaking at day six, with little or no change in Kd (approximately equal to 250 pM, similar to that seen in skeletal muscle). No DHP binding sites were detected in BC3H1 cells bearing an activated c-H-ras oncogene. Induction of the dihydropyridine receptor was reversibly blocked by 200 pM transforming growth factor beta. The results indicate that formation of dihydropyridine-sensitive Ca2+ channels may require up-regulation of the dihydropyridine receptor itself, and that transforming growth factor beta is a potent, reversible inhibitor of this receptor in BC3H1 muscle cells.     

Regulation of creatine phosphokinase expression during differentiation of BC3H1 cells

The intracellular mechanisms involved in the regulation of creatine phosphokinase expression in the BC3H1 muscle-like cell line have been examined under conditions of enzyme induction and repression. In the presence of low serum concentrations, BC3H1 cells cease to grow and synthesize high levels of creatine phosphokinase. When differentiated BC3H1 cultures are exposed to media containing high serum concentrations, cell division is reinitiated and further induction of creatine phosphokinase is inhibited. Accumulation of creatine phosphokinase-mRNA appears to be intimately coupled to the state of growth of BC3H1 cells. Log phase cells do not contain detectable levels of translatable creatine phosphokinase-mRNA; however, following cessation of growth, creatine phosphokinase-mRNA accumulates in approximate proportion to the increase in creatine phosphokinase activity. Reinitiation of cell division in quiescent differentiated cultures results in the arrest of further accumulation of creatine phosphokinase-mRNA but does not inhibit the translation of pre-existing creatine phosphokinase-mRNA. Under conditions of enzyme repression, however, the newly synthesized creatine phosphokinase appears to be enzymatically inactive. These results indicate that the expression of the muscle phenotype in BC3H1 cells is regulated by components present in serum and that myogenic differentiation is at least partially reversible following re-entry of quiescent cells into the cell cycle.     

Actin isoform utilization during differentiation and remodeling of BC3H1 myogenic cells

Mouse BC3H1 myogenic cells and a bi-functional chemical cross linking reagent were utilized to investigate the polymerization of newly-synthesized vascular smooth muscle (α-actin) and non-muscle (β- and γ-actin) actin monomers into native F-actin filament structures during myogenesis. Two actin dimer species were identified by SDS-PAGE analysis of phenylenebismaleimide-cross linked fractions of BC3H1 myoblasts and myocytes. P-dimer was derived from the F-actin-enriched, detergent-insoluble cytoskeleton. Pulse-chase analysis revealed that D-dimer initially was associated with the cytoskeleton but then accumulated in the soluble fraction of lysed muscle cells that contained a non-filamentous or aggregated actin pool. Immunoblot analysis indicated that non-muscle and smooth muscle actins were capable of forming both types of dimer. However, induction of smooth muscle α-actin in developing myoblasts coincided with an increase in D-dimer level which may facilitate actin stress fiber assembly. Smooth muscle α-actin was rapidly utilized in differentiating myoblasts to assemble extraction-resistant F-actin filaments in the cytoskeleton whereas non-muscle β- and γ-actin filaments were more readily dissociated from the cytoskeleton by an extraction buffer containing ATP and EGTA. The data indicate that cytoarchitectural remodeling in developing BC3H1 myogenic cells is accompanied by selective actin isoform utilization that effectively segregates multiple isoactins into different sub-cellular domains and/or supramolecular entities. J. Cell. Biochem. 67:514–527, 1997. © 1997 Wiley-Liss, Inc.     

High-affinity [3H]inositol uptake by dissociated brain cells and cultured fibroblasts from fetal mice

The accumulation of [³H]inositol by mechanically dissociated brain cells and cultured skin fibroblasts from fetal mice was examined. Uptake by both tissues was strongly dependent on temperature and the presence of sodium ions. Brain and fibroblast uptake also responded similarly to inhibition by inositol isomers and phloridzin. At lower concentrations of inositol, both tissues exhibited high-affinity uptake kinetics with apparent K_m values near 30 M, similar to values observed previously in human fibroblasts and other cultured cells. The activity of brain high-affinity uptake was nearly an order of magnitude lower than that of fibroblasts, however, and was in part confounded by the presence of a low-affinity or simple diffusion system operating at inositol concentrations above 100M. Brain preparation from adult mice also showed evidence of high-affinity, Na⁺ dependent uptake, but its activity was significantly diminished relative to that of fetal brain preparations. Our results demonstrate that a high-affinity inositol transport system closely resembling that found in cultured cells is expressed in the developing mouse brain.     

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.     

Control of muscle differentiation in BC3H1 cells by fibroblast growth factor and vanadate

We have examined the control of actin isoform synthesis by pituitary-derived fibroblast growth factor and serum in BC3H1 cells, a tumor-derived nonfusing muscle cell line. Under differentiating conditions in BC3H1 cells, the synthesis of beta- and gamma-actin ceases, and the rate of alpha-actin synthesis is increased concomitant with cessation of cell growth. Addition of fetal calf serum to differentiated cells reverses the process, whereas the addition of pituitary-derived fibroblast growth factor inhibits synthesis of alpha-actin but fails to induce the synthesis of beta- and gamma-actin. Analysis of RNA from differentiated BC3H1 cells after the addition of fetal calf serum indicated that the serum-induced increase in beta- and gamma-actin synthesis reflected an increase in their mRNA levels. In contrast, the repression of alpha-actin synthesis by fetal calf serum or fibroblast growth factor appears to reflect the translation efficiency of alpha-actin mRNA. Fibroblast growth factor is a competence factor for BC3H1 cells which allows them to progress from G0 4 h into the G1 phase of the cell cycle. In order to understand the nature of the intracellular signals responsible for the effect of fibroblast growth factor, we treated cells with vanadate, a known inhibitor of tyrosine-specific protein phosphatases. Vanadate fully mimics the action of fibroblast growth on actin synthesis and creatine phosphokinase synthesis and causes BC3H1 cells to exit the G0 portion of the cell cycle, as demonstrated by the induction of the c-fos proto-oncogene following addition of serum, vanadate, or bovine pituitary-derived fibroblast growth factor to these cells. We conclude that repression of alpha-actin synthesis and induction of the synthesis of beta- and gamma-actin are under independent control and that the induction of beta- and gamma-nonmuscle actin synthesis following serum addition is independent from movement into the cell cycle, and dependent on as yet unidentified serum components. The rate of synthesis of alpha-actin can be controlled by a defined mitogenic polypeptide fibroblast growth factor, which in short term experiments primarily affects the rate of translation of alpha-actin mRNA. The repression by fibroblast growth factor is most likely due to activation of a tyrosine specific protein kinase(s).     

Activation of mitogen-activated protein kinase in BC3H1 myocytes by fluoroaluminate.

Treatment of BC3H1 myocytes or 3T3-L1 fibroblasts with fluoroaluminate (AlF4-), a direct activator of G proteins, increased the tyrosine phosphorylation of a 42-kDa cytosolic protein. AlF4- induced a parallel increase in protein kinase activity toward myelin basic protein (MBP) in partially purified cell extracts. To test whether AlF4- was activating the 42-kDa MAP (mitogen-activated protein) kinase, extracts from AlF4--treated cells were taken through the chromatographic steps routinely used to purify MAP kinase from growth factor-stimulated cells. Following phenyl-Superose chromatography, a peak of MBP kinase activity eluted at a position characteristic of MAP kinase. Immunoblotting of the active fractions with anti-phosphotyrosine antibodies revealed a single reactive protein band of Mr 42,000. Stimulation of MAP kinase by AlF4- was rapid, peaking within 15 min and persisting for at least 1 h. In contrast, the activation of MAP kinase by insulin was transient, characteristic of its activation by growth factors in other cell types. Although concentrations of sodium fluoride greater than 1 mM also activated MAP kinase, this effect was shown to be dependent upon the simultaneous presence of aluminum ions in the medium. Activation of MAP kinase by AlF4- was not affected by either cellular depletion of protein kinase C or pretreatment of cells with pertussis toxin. Potential sites of action of AlF4- are discussed. These findings suggest that activation of a G protein(s) in intact cells can initiate events that result in tyrosine phosphorylation and activation of MAP kinase.     

The oligosaccharide component of alpha 1-adrenergic receptors from BC3H1 and DDT1 muscle cells. Studies with glycosidases and photoaffinity labelling of intact cells.

In this study, we clarify the structural aspects of the oligosaccharides associated with the alpha 1-adrenergic receptor in two muscle cell lines. Photoaffinity labelling of intact BC3H1 or DDT1 muscle cells with 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline ([125I]azidoprazosin) followed by SDS/polyacrylamide-gel electrophoresis (PAGE) and autoradiography revealed specifically labelled proteins of molecular mass = 87,000 and 81,000, respectively. Treatment of photoaffinity-labelled receptors in DDT1 cells with 33 u. of endoglycosidase F/ml for 24 h resulted in the loss of the 81 kDa receptor and the appearance of a 52.5 kDa protein. When lower concentrations of glycosidase or shorter incubation times were used, the 81 kDa receptor was converted to a 66 kDa protein. Treatment of the photoaffinity-labelled BC3H1 receptor with endoglycosidase F resulted in the appearance of a 50.5 kDa protein. Neither alpha-mannosidase nor endoglycosidase H had an effect on the photoaffinity labelling patterns of the receptor from the two cell types. alpha 1-Adrenergic receptors, solubilized from membranes prepared from BC3H1 and DDT1 cells, bound to wheat germ agglutinin-Sepharose and were displaced by N-acetylglucosamine. Taken together, these results indicate that alpha 1-adrenergic receptors in BC3H1 and DDT1 cells contain complex, but not high, mannose oligosaccharide chains; differences in the composition or number of chains partially accounts for the different molecular mass of the receptor in the two cell lines. The results further indicate that the oligosaccharide chains contribute substantially to the apparent molecular mass of alpha 1-adrenergic receptors, as detected by SDS/PAGE, and that the protein backbone of these receptors is likely to be approximately 50 kDa.     

An analysis of myo-[3H]inositol trisphosphates found in myo-[3H]inositol prelabelled avian erythrocytes.

Evidence is presented to show that acid extracts of avian erythrocytes prelabelled for 24-48 h with myo-[3H]inositol contain the following myo-[3H]inositol trisphosphates (expressed as a percentage of total myo-[3H]inositol trisphosphates extracted): 36% myo-[3H]inositol 1,4,5-trisphosphate; 33.7% myo-[3H]inositol 1,3,4-trisphosphate; 13% myo-[3H]inositol 3,4,5-trisphosphate; 9.7% myo-[3H]inositol 3,4,6-trisphosphate; 4.4% myo-[3H]inositol 1,4,6-trisphosphate and 3.3% myo-[3H]inositol 1,3,6-trisphosphate. The only phosphatidyl-myo-[3H]inositol bisphosphate that could be detected in [3H]Ins-prelabelled avian erythrocytes was phosphatidyl-myo-[3H]inositol 4,5-bisphosphate. Cellular myo-[3H]inositol 3,4,5-trisphosphate may be synthesized by dephosphorylation of myo-[3H]inositol 3,4,5,6-tetrakisphosphate. D- and L-myo-[3H]inositol 1,4,6-trisphosphate and D- and L-myo-[3H]inositol 1,3,6-trisphosphate may be dephosphorylation products of myo-[3H]inositol 1,3,4,6-tetrakisphosphate.     

Retinoic acid treatment of fibroblasts causes a rapid decrease in [3H]inositol uptake

NIH 3T3 fibroblasts treated with all-trans-retinoic acid (RA) showed a dramatic decrease in the uptake of [3H]inositol compared to solvent-treated controls. The onset of RA-induced inhibition of [3H]inositol uptake was rapid with a 10-15% decrease occurring after 2-3 h of RA exposure and 60-70% reduction after 16 h of RA treatment. A progressive dose-dependent decrease in inositol uptake was found as the concentration of RA increased from 10(-8) to 10(-5) M and the effect was fully reversible within 48 h after RA removal. The Vmax and Kt for the controls were 10 nmol/2.5 x 10(6) cells/2 h and 51 microM; and for RA-treated cells the values were 4 nmol/2.5 x 10(6) cells/2 h and 52 microM. The decreased [3H]inositol uptake was not due to a change in the affinity (Kt) of the transporter for the inositol but to a decrease in the Vmax. The maximal effect on inositol uptake was dependent on RA treatment of the cells after they reached saturation density or if made quiescent by serum starvation. RA was the most active of the different retinoids examined in the order RA greater than 13-cis-RA = retinyl acetate greater than all-trans-retinol greater than 5,6-dihydroxyretinoic acid methyl ester greater than N-4-hydroxyphenyl retinamide. In contrast to this effect on inositol, the uptake of fucose, mannose, galactose, and glucose was either not affected or enhanced (for mannose and fucose) by RA treatment. RA inhibition of inositol uptake was also observed in 3T3-Swiss and Balb/3T3 cells but not in two virally transformed 3T3 cell lines. Phlorizin, amiloride, and monensin inhibited inositol uptake by 66, 74, and 58%, respectively, and this inhibition was additive when the cells were treated with RA as well as these inhibitors. A decreased incorporation of [3H]inositol into polyphosphoinositides was also observed in RA-treated cells but not to the same extent as for [3H]inositol uptake. In conclusion, RA treatment of 3T3 fibroblasts decreases the uptake of [3H]inositol by up to 70% within 8 to 10 h at near physiological concentrations in a reversible and specific manner.