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.     

Disruption of the coordinate expression of muscle genes in a transfected BC3H1 myoblast cell line producing a low level of the adenovirus E1A transforming protein.

Mouse BC3H1 myoblasts were stably transfected with the adenovirus 5 E1A gene. One clonal line, BC3E7, was found to differ in some important respects from those previously reported for E1A-transformed myoblasts. In contrast to BC3H1 cells which differentiate when confluent in medium containing 0.5% fetal calf serum (FCS), BC3E7 cells failed to elongate and align, to express acetylcholine receptor and creatine kinase, and to down-regulate expression of beta- and gamma-actins and tropomyosin isoform (TM) 1. However, increased synthesis of TMs 2, 3, and 4, and myosin light chain 1 associated with differentiation in BC3H1 still occurred in BC3E7 cells, and most surprisingly, alpha-actin was produced at a significant level in both proliferating and confluent BC3E7 cells. Interestingly, myogenin was expressed in confluent BC3E7 cells in 0.5% FCS, but not in 20%. The level of E1A expression in BC3E7 cells was found to be very low by analysis of mRNA, by immunoprecipitation of E1A protein, and by the ability of BC3E7 cells to complement the E1A-deficient adenovirus mutant dl312. These results suggest that different levels of E1A may be needed to repress different promoters and that E1A does not block myogenic differentiation by repressing myogenin expression, but represses each muscle gene independently.     

FGF and EGF act synergistically to induce proliferation in BC3H1 myoblasts

BC3H1 muscle cells proliferate when grown in high concentrations of FBS (20%). Lowering the FBS concentration to 0.5% causes the cells to stop proliferating and is permissive for the morphological and biochemical differentiation of BC3H1 cells. Exposure of differentiated BC3H1 myocytes to high concentrations of serum or to the purified growth factors FGF or TGF-b induced a shutdown of this differentiation program but did not induce cell proliferation (Olson et al., J. Cell Biol., 103:1799-1805, 1986; Lathrop et al., J. Cell Biol., 100:1540-1547, 1985, and J. Cell Biol., 101:2194-2198, 1985). We explored the possibility that BC3H1 cells require factors to act synergistically to induce proliferation. We found that EGF and FGF function in a synergistic fashion to stimulate BC3H1 proliferation. Moreover, the temporal requirement for these growth factors suggest that they are functioning as competence and progression factors for BC3H1 cell proliferation.     

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).     

The levels of vascular smooth as well as skeletal muscle actin mRNAs differ substantially among both myoblast and fibroblast lines with different skeletal myogenic potentials.

Little is known about the factors which regulate vascular smooth muscle (vsm) actin gene expression during skeletal myogenesis in culture. We have therefore looked for differences in the levels of accumulation of vsm actin mRNA among six mouse cell lines differing in apparent myogenic potential or in the complement of myogenesis determination genes which they express: NIH 3T3 and 10T1/2 non-myogenic fibroblasts and four myogenic lines--3T3-MyoD1 and 10EMc11s, MyoD/myogenin expressing sublines of the fibroblast lines, derived by transfer into the parent lines of a MyoD cDNA expression construct; C2C12, which expresses all four known myogenesis determination genes; and BC3H1, which expresses myf-5, myogenin, little herculin, and no MyoD. In differentiated cells of all four myogenic lines, vsm actin mRNA was expressed at levels dramatically higher than in growth-arrested NIH 3T3 cells, consistent with expression of vsm actin mRNA as an intrinsic part of the skeletal myogenic program somehow directed by myogenesis determination gene products. Interestingly, however, the level of vsm actin mRNA in growth arrested C3H10T1/2 fibroblasts was also dramatically higher than that in NIH 3T3. In view of these findings, and of the relative ease with which 10T1/2 as opposed to NIH 3T3 cells can be converted to myogenic lines, we hypothesize that factors which can act to regulate vsm actin gene expression in the absence of myogenesis determination gene expression may also influence the skeletal myogenic potential of the cells in which they are found. Among the myogenic lines, the ratio of vsm to skm actin mRNA was highest in BC3H1 cells, raising the possibility that were these cells forced to express MyoD and/or more herculin, as do the other myogenic lines, the ratio would decrease. Thus both fibroblast and myogenic lines will be useful for investigating the mechanisms controlling skeletal myogenesis and vsm and skm actin gene expression during myogenesis.     

The levels of vascular smooth as well as skeletal muscle actin mRNAS differ substantially among both myoblast and fibroblast lines with different skeletal myogenic potentials.

Little is known about the factors which regulate vascular smooth muscle (vsm) actin gene expression during skeletal myogenesis in culture. We have therefore looked for differences in the levels of accumulation of vsm actin mRNA among six mouse cell lines differing in apparent myogenic potential or in the complement of myogenesis determination genes which they express: NIH 3T3 and 10T1/2 non-myogenic fibroblasts and four myogenic lines--3T3-MyoD1 and 10EMc11s, MyoD/myogenin expressing sublines of the fibroblast lines, derived by transfer into the parent lines of a MyoD cDNA expression construct; C2C12, which expresses all four known myogenesis determination genes; and BC3H1, which expresses myf-5, myogenin, little herculin, and no MyoD. In differentiated cells of all four myogenic lines, vsm actin mRNA was expressed at levels dramatically higher than in growth-arrested NIH 3T3 cells, consistent with expression of vsm actin mRNA as an intrinsic part of the skeletal myogenic program somehow directed by myogenesis determination gene products. Interestingly, however, the level of vsm actin mRNA in growth arrested C3H10T1/2 fibroblasts was also dramatically higher than that in NIH 3T3. In view of these findings, and of the relative ease with which 10T1/2 as opposed to NIH 3T3 cells can be converted to myogenic lines, we hypothesize that factors which can act to regulate vsm actin gene expression in the absence of myogenesis determination gene expression may also influence the skeletal myogenic potential of the cells in which they are found. Among the myogenic lines, the ratio of vsm to skm actin mRNA was highest in BC3H1 cells, raising the possibility that were these cells forced to express MyoD and/or more herculin, as do the other myogenic lines, the ratio would decrease. Thus both fibroblast and myogenic lines will be useful for investigating the mechanisms controlling skeletal myogenesis and vsm and skm actin gene expression during myogenesis.     

Regulation of differentiation of the BC3H1 muscle cell line through cAMP-dependent and -independent pathways

Serum mitogens, fibroblast growth factor (FGF), and type beta transforming growth factor (TGF-beta) suppress differentiation of the mouse muscle cell line BC3H1; however, the signal transduction pathways whereby these growth factors exert their effects on this system are unknown. The goal of this study was to determine whether the program for differentiation of BC3H1 cells was susceptible to negative regulation by signaling pathways involving cAMP or protein kinase C and whether these intracellular effectors participate in the mechanism by which growth factors prevent establishment of the myogenic phenotype. Exposure of BC3H1 cells to dibutyryl cAMP, 8-bromo-cAMP, or compounds that stimulate adenylate cyclase, i.e. forskolin, prostaglandin E1, and cholera toxin, prevented up-regulation of muscle-specific gene products following growth arrest in mitogen-deficient medium. Conversely, addition of cAMP to differentiated BC3H1 myocytes caused down-regulation of muscle-specific mRNAs. In contrast to the ability of cAMP to block differentiation, chronic exposure to O-tetradecanoylphorbol-13-acetate, the potent activator of protein kinase C, exhibited no apparent effects on expression of muscle-specific gene products. The proto-oncogenes c-myc and c-fos were up-regulated rapidly by cAMP in a manner similar to that observed previously by serum, FGF, and TGF-beta. However, these growth factors failed to increase intracellular cAMP levels, and they did not induce ornithine decarboxylase, which was subject to positive regulation by cAMP and O-tetradecanoyl-13-acetate. Together, these data indicate that differentiation of BC3H1 cells is subject to negative regulation through a cAMP-dependent pathway and that serum mitogens, FGF, and TGF-beta inhibit differentiation through a mechanism independent of cAMP or protein kinase C.     

Differential expression of myogenic determination genes in muscle cells: possible autoactivation by the Myf gene products.

The development of muscle cells involves the action of myogenic determination factors. In this report, we show that human skeletal muscle tissue contains, besides the previously described Myf-5, two additional factors Myf-3 and Myf-4 which represent the human homologues of the rodent proteins MyoD1 and myogenin. The genes encoding Myf-3, Myf-4 and Myf-5 are located on human chromosomes 11, 1, and 12 respectively. Constitutive expression of a single factor is sufficient to convert mouse C3H 10T1/2 fibroblasts to phenotypically normal muscle cells. The myogenic conversion of 10T1/2 fibroblasts results in the activation of the endogenous MyoD1 and Myf-4 (myogenin) genes. This observation suggests that the expression of Myf proteins leads to positive autoregulation of the members of the Myf gene family. Individual myogenic colonies derived from MCA C115 cells (10T1/2 fibroblast transformed by methylcholanthrene) express various levels of endogenous MyoD1 mRNA ranging from nearly zero to high levels. The Myf-5 gene was generally not activated in 10T1/2 derived myogenic cell lines but was expressed in some MCA myoblasts. In primary human muscle cells Myf-3 and Myf-4 mRNA but very little Myf-5 mRNA is expressed. In mouse C2 and P2 muscle cell lines MyoD1 is abundantly synthesized together with myogenin. In contrast, the rat muscle lines L8 and L6 and the mouse BC3H1 cells express primarily myogenin and low levels of Myf-5 but no MyoD1. Myf-4 (myogenin) mRNA is present in all muscle cell lines at the onset of differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of MRF4, a myogenic helix-loop-helix protein, produces multiple changes in the myogenic program of BC3H-1 cells.

Expression of MRF4, a myogenic regulatory factor of the basic helix-loop-helix type, produced multiple changes in the myogenic program of the BC3H-1 cell line. BC3H-1 cells that stably expressed exogenous MRF4 were prepared and termed BR cell lines. Upon differentiation, the BR cells were found to have three muscle-specific properties (endogenous MyoD expression, myoblast fusion, and fast myosin light-chain 1 expression) that the parent BC3H-1 cells did not have. Of the four known myogenic regulatory factors (MyoD, myogenin, Myf-5, and MRF4), only MRF4 was capable of activating expression of the endogenous BC3H-1 myoD gene. In addition, the pattern of Myf-5 expression in BR cells was the opposite of that in BC3H-1 cells. Myf-5 expression was low in BR myoblasts and showed a small increase upon myotube formation, whereas Myf-5 expression was high in BC3H-1 myoblasts and decreased upon differentiation. Though the MRF4-transfected BR cells fused to form large myotubes and expressed fast myosin light-chain 1, the pattern of myosin heavy-chain isoform expression was the same in the BR and the nonfusing parent BC3H-1 cells, suggesting that factors in addition to the MyoD family members regulate myosin heavy-chain isoform expression patterns in BC3H-1 cells. In contrast to the changes produced by MRF4 expression, overexpression of Myf-5 did not alter BC3H-1 myogenesis. The results suggest that differential expression of the myogenic regulatory factors of the MyoD family may be one mechanism for generating cells with diverse myogenic phenotypes.     

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.     

Ryanodine receptor protein is expressed during differentiation in the muscle cell lines BC3H1 and C2C12

BC3H1 and C2C12, murine cell lines, were assessed as model systems for the expression of ryanodine receptor protein during myogenesis. The ryanodine receptor is a calcium release channel of the sarcoplasmic reticulum and a component of the triad junction, a structure which is essential to excitation-contraction coupling in mature striated muscle. BC3H1 and C2C12 cells do not express the ryanodine receptor at detectable levels in a proliferative, nondifferentiated state. The ryanodine receptor protein is expressed during differentiation in BC3H1 and C2C12 cells, becoming detectable within 24 hr of the onset of differentiation. In both cell lines the ryanodine receptor is assembled in oligomeric form and binds [3H]ryanodine with high affinity. Fusion is not required for expression of the ryanodine receptor in either BC3H1 or nonfusing C2C12 cells. The level of expression of the ryanodine receptor protein is modulated by incubation with the growth factors TGF-beta and bFGF in a manner similar to that of other muscle-specific proteins. These initial observations suggest that the BC3H1 and C2C12 cell lines provide a model system for further investigations of the expression and function of the ryanodine receptor during myogenic differentiation.     

Glucose promotes epithelial‐mesenchymal transitions in bladder cancer by regulating the functions of YAP1 and TAZ

Glucose levels and type 2 diabetes (T2D) are both associated with tumorigenesis and epithelial‐mesenchymal transitions (EMTs). EMTs facilitate bladder cancer (BC) metastasis development, but the mechanism by which high‐glucose levels promote these EMTs in BC remains unclear. Therefore, we sought to elucidate the mechanism underlying EMT promotion due to increased glucose levels. T24 and UMUC‐3 cells were cultured in media containing different glucose concentrations. YAP1, TAZ, GLUT1 and EMT‐associated marker expression was analysed via Western blotting and qPCR. BC cell proliferation and invasion were assessed using MTT and Transwell assays, respectively. A xenograft nude mouse model of diabetes was used to evaluate tumour growth and metastasis in vivo. T2D was positively associated with pathologic grade (P = .016) and TNM stage (P < .001) in BC. High glucose triggered BC cell proliferation and invasion in both in vitro and in vivo conditions. High‐glucose levels also promoted EMTs in BC cells and increased YAP1 and TAZ expression. YAP1 or TAZ knockdown altered EMT marker expression and decreased GLUT1 expression. Overall, our results suggest that high‐glucose levels promote EMTs in BC cells via YAP1 and TAZ regulation. These effector molecules may be promising therapeutic targets for BC cases comorbid with T2D.     

Inositol trisphosphate receptor calcium release is required for cerebral artery smooth muscle cell proliferation

Vascular damage signals smooth muscle cells to proliferate, often exacerbating existing pathologies. Although the role of changes in "global" Ca2+ in vascular smooth muscle (VSM) cell dedifferentiation has been studied, the role of specific Ca2+ signals in determining VSM phenotype remains relatively unexplored. Earlier work with cultured VSM cells suggests that inositol 1,4,5-trisphosphate receptor (IP3R) expression and sarcoplasmic reticulum (SR) Ca2+ release may be linked to VSM cell proliferation in native tissue. Thus we hypothesized that SR Ca2+ release through IP3Rs in the form of discrete transient signals is necessary for VSM cell proliferation. To investigate this hypothesis, we used mouse cerebral arteries to design an organ culture system that permitted examination of Ca2+ dynamics in native tissue. Explanted arteries were cultured in normal medium with 10% FBS, and appearance of individual VSM cells migrating from explanted arteries (outgrowth cells) was tracked daily. Initial exposure to 10% FBS increased Ca2+ waves in myocytes in the arteries that were blocked by the IP3R antagonist 2-aminoethoxydiphenylborate (2-APB). Inhibition of IP3R opening (via 100 microM 2-APB, 10 microM xestospongin C, or 25 microM U-73122) dramatically reduced outgrowth cell number compared with untreated or ryanodine-treated (10 microM) arteries. Consistent with this finding, 2-APB inhibited cell proliferation, as measured by reduced proliferating cell nuclear antigen immunostaining within 48 h of culture but did not inhibit cell migration. These results indicate that activation of IP3R Ca2+ release is required for VSM cell proliferation in these arteries.