Analysis of muscle protein expression in polyethylene glycol-induced chicken: rat myoblast heterokaryons.

Heterokaryons derived from polyethylene glycol-mediated fusion of myoblasts at different stages of development were used to investigate the transition of cells in the skeletal muscle lineage from the determined to the differentiated state. Heterokaryons were analyzed by immunofluorescence, using rabbit antibodies against the skeletal muscle isoforms of chicken creatine kinase and myosin, and a mouse monoclonal antibody that cross-reacts with chicken and rat skeletal muscle myosin. When cytochalasin B-treated rat L8(E63) myocytes (Konieczny S.F., J. McKay, and J. R. Coleman, 1982, Dev. Biol., 91:11-26) served as the differentiated parental component and chicken limb myoblasts from stage 23-26 or 10-12-d embryos were used as the determined, undifferentiated parental cell, heterokaryons exhibited a progressive extinction of rat skeletal muscle myosin during a 4-6-d culture period, and no precocious expression of chicken differentiated gene products was detected. In the reciprocal experiment, 85-97% of rat myoblast X chicken myocyte heterokaryons ceased expression of chicken skeletal muscle myosin and the M subunit of chicken creatine kinase within 7 d of culture. Extinction was not observed in heterokaryons produced by fusion of differentiated chicken and differentiated rat myocytes and thus is not due to species incompatibility or to the polyethylene glycol treatment itself. The results suggest that, when confronted in a common cytoplasm, the regulatory factors that maintain myoblasts in a proliferating, undifferentiated state are dominant over those that govern expression of differentiated gene products.

The bHLH transcription factor Mist1 is required to maintain exocrine pancreas cell organization and acinar cell identity.

The pancreas is a complex organ that consists of separate endocrine and exocrine cell compartments. Although great strides have been made in identifying regulatory factors responsible for endocrine pancreas formation, the molecular regulatory circuits that control exocrine pancreas properties are just beginning to be elucidated. In an effort to identify genes involved in exocrine pancreas function, we have examined Mist1, a basic helix-loop-helix transcription factor expressed in pancreatic acinar cells. Mist1-null (Mist1(KO)) mice exhibit extensive disorganization of exocrine tissue and intracellular enzyme activation. The exocrine disorganization is accompanied by increases in p8, RegI/PSP, and PAP1/RegIII gene expression, mimicking the molecular changes observed in pancreatic injury. By 12 m, Mist1(KO) mice develop lesions that contain cells coexpressing acinar and duct cell markers. Analysis of the factors involved in cholecystokinin (CCK) signaling reveal inappropriate levels of the CCK receptor A and the inositol-1,4,5-trisphosphate receptor 3, suggesting that a functional defect exists in the regulated exocytosis pathway of Mist1(KO) mice. Based on these observations, we propose that Mist1(KO) mice represent a new genetic model for chronic pancreas injury and that the Mist1 protein serves as a key regulator of acinar cell function, stability, and identity.

Differentiation, not determination, regulates muscle gene activation: transfection of troponin I genes into multipotential and muscle lineages of 10T1/2 cells.

Transcription of quail skeletal muscle troponin I (TnI) genes was examined after stable transfection into multipotential 10T1/2 mouse cells and into determined myoblast lineages derived by 5-azacytidine conversion. Transfected TnI and endogenous mouse muscle genes were inactive both in multipotential 10T1/2 and in proliferating myoblasts but were activated coordinately and to high levels when myoblast lineages differentiated, regardless of whether TnI genes were transfected before or after myoblast lineage determination. We conclude that the TnI gene contains evolutionarily conserved control sequences that activate its transcription in response to differentiation-specific regulatory signals. Myoblast lineage determination, therefore, does not appear to act directly on TnI and other muscle genes but likely establishes a regulatory control system that mediates expression of differentiation-specific transcription signals.

Complex regulation of the muscle-specific contractile protein (troponin I) gene.

A cloned quail troponin I contractile protein gene, stably transfected into a mouse myogenic cell line, exhibits appropriate developmental activation and quantitative expression during myoblast differentiation. Deletion mutagenesis analyses reveal that the troponin I gene has two distinct cis regulatory elements required for its developmental expression, as measured by mRNA accumulation and nuclear runoff transcription assays. One element in the 5' flanking region is required for maximum quantitative expression, and a second larger regulatory element (1.5 kilobases) within the first intron is responsible for differentiation-specific transcription. The upstream region is highly sensitive to negative repression by interaction with pBR322 sequences. The larger intragenic region retains some activity when moved to the 5' and 3' flanking regions and when inverted but is maximally active in its native intragenic site. The concerted activities of these two regulatory regions produce a 100- to 200-fold transcriptional activation during myoblast differentiation. The conserved 5' exon-intron organization of troponin I and other contractile protein genes suggests a possible mechanism by which intragenic control elements coordinate contractile protein gene regulation during skeletal myogenesis.

Fibroblast growth factor inhibits MRF4 activity independently of the phosphorylation status of a conserved threonine residue within the DNA-binding domain.

MRF4 is a member of the muscle-specific basic helix-loop-helix transcription factor family that also includes MyoD, myogenin, and Myf-5. Each of these proteins, when overexpressed in fibroblasts, converts the cells to differentiated muscle fibers that express several skeletal muscle genes, such as those for alpha-actin, muscle creatine kinase, and troponin I. Despite the fact that MRF4 functions as a positive transcriptional regulator, the MRF4 protein is subject to negative regulation by a variety of agents, most notably by exposure of cells to purified growth factors, such as basic fibroblast growth factor (bFGF). In an effort to establish whether bFGF inhibits MRF4 activity through specific posttranslational modifications, we examined whether MRF4 exists in vivo as a phosphoprotein and whether the phosphorylation status of the protein regulates its activity. Our results indicate that MRF4 is phosphorylated predominantly on serine residues, with weak phosphorylation occurring on threonine residues. Both cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) phosphorylate MRF4 in vitro as well as in vivo, and the overexpression of each kinase inhibits MRF4 activity and thus blocks terminal differentiation. PKC-directed phosphorylation of a conserved threonine residue (T-99) situated within the DNA-binding domain inhibits MRF4 from binding in vitro to specific DNA targets. However, although T-99 itself is essential for myogenic activity, our studies demonstrate that the phosphorylation status of T-99 does not play a major role in regulating MRF4 activity in vivo, since PKA, PKC, and bFGF inhibit the activity of MRF4 proteins in which the identified PKA and PKC sites have been mutated. We suggest that the negative regulation of MRF4 imposed by bFGF does not involve a direct modification of the protein at the identified PKA and PKC sites but instead may involve the modification of specific coregulators that interact with this muscle regulatory factor.

Muscle-specific expression of the troponin I gene requires interactions between helix-loop-helix muscle regulatory factors and ubiquitous transcription factors.

The quail fast skeletal troponin I (TnI) gene is a member of the contractile protein gene set and is expressed exclusively in differentiated skeletal muscle cells. TnI gene transcription is controlled by an internal regulatory element (IRE), located within the first intron, that functions as a muscle-specific enhancer. Recent studies have shown that the TnI IRE may interact directly with the muscle regulatory factors MyoD, myogenin, and Myf-5 to produce a muscle-specific expression pattern, since these factors trans-activate cotransfected TnI gene constructs in C3H10T1/2 fibroblasts. In this study, we have examined the protein-IRE interactions that are responsible for transcriptionally activating the TnI gene during skeletal muscle development. We demonstrate that the helix-loop-helix muscle regulatory factors MyoD, myogenin, Myf-5, and MRF4, when complexed with the immunoglobulin enhancer-binding protein E12, interact with identical nucleotides within a muscle regulatory factor-binding site (MRF site) located in the TnI IRE. The nuclear proteins that bind to the MRF site are restricted to skeletal muscle cells, since protein extracts from HeLa, L, and C3H10T1/2 fibroblasts do not contain similar binding activities. Importantly, the TnI MRF site alone is not sufficient to elicit the full enhancer activity associated with the IRE. Instead, two additional regions (site I and site II) are required. The proteins that interact with site I and site II are expressed in both muscle and nonmuscle cell types and by themselves are ineffective in activating TnI gene expression. However, when the MRF site is positioned upstream or downstream of site I and site II, full enhancer activity is restored. We conclude that helix-loop-helix muscle regulatory factors must interact with ubiquitously expressed proteins to generate the active TnI transcription complex that is present in differentiated muscle fibers.

An internal regulatory element controls troponin I gene expression.

During skeletal myogenesis, approximately 20 contractile proteins and related gene products temporally accumulate as the cells fuse to form multinucleated muscle fibers. In most instances, the contractile protein genes are regulated transcriptionally, which suggests that a common molecular mechanism may coordinate the expression of this diverse and evolutionarily unrelated gene set. Recent studies have examined the muscle-specific cis-acting elements associated with numerous contractile protein genes. All of the identified regulatory elements are positioned in the 5'-flanking regions, usually within 1,500 base pairs of the transcription start site. Surprisingly, a DNA consensus sequence that is common to each contractile protein gene has not been identified. In contrast to the results of these earlier studies, we have found that the 5'-flanking region of the quail troponin I (TnI) gene is not sufficient to permit the normal myofiber transcriptional activation of the gene. Instead, the TnI gene utilizes a unique internal regulatory element that is responsible for the correct myofiber-specific expression pattern associated with the TnI gene. This is the first example in which a contractile protein gene has been shown to rely primarily on an internal regulatory element to elicit transcriptional activation during myogenesis. The diversity of regulatory elements associated with the contractile protein genes suggests that the temporal expression of the genes may involve individual cis-trans regulatory components specific for each gene.

Differential trans activation associated with the muscle regulatory factors MyoD1, myogenin, and MRF4.

Expression of the mammalian muscle regulatory factors MyoD1, myogenin, and MRF4 will convert C3H10T1/2 fibroblasts to stable muscle cell lineages. Recent studies have shown that MyoD1 and myogenin also trans-activate expression of a number of cotransfected contractile protein genes, suggesting that these muscle regulatory factors are involved in controlling terminal differentiation events. The extent and specificity of trans activation by the muscle regulatory factors, however, have not been compared directly. In this study, we found that MyoD1, myogenin, and MRF4 exhibited different trans-activation capacities. In contrast to MyoD1 and myogenin, MRF4 was inefficient in trans-activating most of the genes tested, although conversion of C3H10T1/2 fibroblasts to a myogenic lineage was observed at similar frequencies with all three factors. Addition of basic fibroblast growth factor to cells expressing exogenous muscle regulatory factors inhibited the transcriptional activation of cotransfected genes, demonstrating that MyoD1, myogenin, or MRF4 proteins alone are not sufficient to produce a terminally differentiated phenotype. In all cases, trans activation was dependent on signal transduction pathways that are regulated by fibroblast growth factor. Our observations, coupled with previous studies showing differences in the temporal expression and protein structure of MyoD1, myogenin, and MRF4, suggest that the individual members of the muscle regulatory factor family have distinct biological roles in controlling skeletal muscle development.

Expression of the troponin complex genes: transcriptional coactivation during myoblast differentiation and independent control in heart and skeletal muscles.

We compared the developmental regulation of the three troponin genes that encode the proteins of the Ca2+ regulatory complex in striated muscles of the Japanese quail. Nuclear run-on transcription and RNA protection analyses showed that the fast skeletal troponin I, the fast skeletal troponin T, and the slow skeletal-cardiac troponin C genes were transcriptionally coactivated and that transcripts rapidly accumulated within 6 to 12 h after the initiation of myoblast differentiation. The fast-isoform mRNAs of troponin I and troponin T were coexpressed at similar levels in different skeletal muscles, whereas the slow-cardiac troponin C mRNA varied independently and was the only one of these genes expressed in embryonic and adult heart. We conclude that these troponin genes are transcriptionally coactivated during skeletal myoblast differentiation, indicating that their transcription is under precise temporal control. However, this troponin C gene is regulated independently is specialized striated muscles.

Casein kinase II increases the transcriptional activities of MRF4 and MyoD independently of their direct phosphorylation.

The myogenic regulatory factors (MRFs) are a subclass of a much larger group of basic helix-loop-helix transcription factors which includes members of the E protein such as E47, E2-2, and HEB. Although the MRFs are unique in their ability to confer a myogenic phenotype on nonmuscle cells, they require E protein partners to form a MRF-E protein heterodimer, which represents the functional myogenesis-inducing complex. The mechanisms controlling homodimer and heterodimer formation in vivo remain largely unknown, although it is likely that posttranslational modification of one or both basic helix-loop-helix partners is critical to this regulatory event. In this respect, MyoD and MRF4, both members of the MRF family, exist in vivo as phosphoproteins and contains multiple consensus phosphorylation sites, including sites for casein kinase II (CKII) phosphorylation. In this study, we demonstrate that overexpression of CKII increases the transcriptional activities of MRF4 and MyoD in vivo. Interestingly, mutation of the individual CKII sites within MRF4 and MyoF does not alter the ability of CKII to enhance MRF transcriptional activity, suggesting that the effect of CKII expression on the MRFs is indirect. Given that the MRFs require dimerization with E protein partners to activate muscle-specific transcription, the effects of CKII expression on E protein function also were examined. Our studies show that E47 serves as an in vitro substrate for CKII and that CKII-phosphorylated E-47 proteins no longer bind to DNA. These observations were confirmed by in vivo experiments showing that overexpressing of CKII produces a dramatic reduction in E47 homodimer-directed transcription. We conclude from these studies that CKII may act as a positive regulator of myogenesis by preventing E protein homodimers from binding to muscle gene regulatory elements.

Muscle LIM protein promotes myogenesis by enhancing the activity of MyoD.

The muscle LIM protein (MLP) is a muscle-specific LIM-only factor that exhibits a dual subcellular localization, being present in both the nucleus and in the cytoplasm. Overexpression of MLP in C2C12 myoblasts enhances skeletal myogenesis, whereas inhibition of MLP activity blocks terminal differentiation. Thus, MLP functions as a positive developmental regulator, although the mechanism through which MLP promotes terminal differentiation events remains unknown. While examining the distinct roles associated with the nuclear and cytoplasmic forms of MLP, we found that nuclear MLP functions through a physical interaction with the muscle basic helix-loop-helix (bHLH) transcription factors MyoD, MRF4, and myogenin. This interaction is highly specific since MLP does not associate with nonmuscle bHLH proteins E12 or E47 or with the myocyte enhancer factor-2 (MEF2) protein, which acts cooperatively with the myogenic bHLH proteins to promote myogenesis. The first LIM motif in MLP and the highly conserved bHLH region of MyoD are responsible for mediating the association between these muscle-specific factors. MLP also interacts with MyoD-E47 heterodimers, leading to an increase in the DNA-binding activity associated with this active bHLH complex. Although MLP lacks a functional transcription activation domain, we propose that it serves as a cofactor for the myogenic bHLH proteins by increasing their interaction with specific DNA regulatory elements. Thus, the functional complex of MLP-MyoD-E protein reveals a novel mechanism for both initiating and maintaining the myogenic program and suggests a global strategy for how LIM-only proteins may control a variety of developmental pathways.

Ras p21Val inhibits myogenesis without altering the DNA binding or transcriptional activities of the myogenic basic helix-loop-helix factors.

MRF4, MyoD, myogenin, and Myf-5 are muscle-specific basic helix-loop-helix transcription factors that share the ability to activate the expression of skeletal muscle genes such as those encoding alpha-actin, myosin heavy chain, and the acetylcholine receptor subunits. The muscle regulatory factors (MRFs) also exhibit the unique capacity to initiate the myogenic program when ectopically expressed in a variety of nonmuscle cell types, most notably C3H10T1/2 fibroblasts (10T1/2 cells). The commitment of myoblasts to terminal differentiation, although positively regulated by the MRFs, also is controlled negatively by a variety of agents, including several growth factors and oncoproteins such as fibroblast growth factor (FGF-2), transforming growth factor beta 1 (TGF-beta 1), and Ras p21Val. The molecular mechanisms by which these varied agents alter myogenic terminal differentiation events remain unclear. In an effort to establish whether Ras p21Val represses MRF activity by directly targeting the MRF proteins, we examined the DNA binding and transcription activation potentials of MRF4 and MyoD when expressed in 10T1/2 cells or in 10T1/2 cells expressing Ras p21Val. Our results demonstrate that Ras p21Val inhibits terminal differentiation events by targeting the basic domain of the MRFs, and yet the mechanism underlying this inhibition does not involve altering the DNA binding or the inherent transcriptional activity of these regulatory factors. In contrast, FGF-2 and TGF-beta 1 block terminal differentiation by repressing the transcriptional activity of the MRFs. We conclude that the Ras p21Val block in differentiation operates via an intracellular signaling pathway that is distinct from the FGF-2 and TGF-beta 1 pathways.

The MRF4 activation domain is required to induce muscle-specific gene expression.

MRF4 is a member of the basic helix-loop-helix muscle regulatory factor family that also includes MyoD, myogenin, and Myf-5. Overexpression of MRF4 or the other muscle regulatory factors in fibroblasts converts the cells to differentiated muscle fibers and transcriptionally activates expression of endogenous and cotransfected muscle genes. Although these factors induce a similar phenotype, they also exhibit some distinct biological activities. For example, MyoD trans activates alpha-actin and troponin I reporter genes to very high levels, whereas MRF4 efficiently activates only alpha-actin expression. Since these proteins have a common basic helix-loop-helix domain, it is likely that portions of the proteins outside of this region impart some specificity to the activity of each muscle regulatory factor. As an initial step in determining the mechanism by which MRF4 and MyoD activate gene transcription, the transcriptional activation domain of MRF4 has been characterized. Experiments utilizing chimeric proteins containing the yeast GAL4 DNA-binding domain and portions of the MRF4 protein indicate that the MRF4 activation domain is located within amino acids 10 to 30. This amino terminus is both necessary and sufficient to elicit a transcriptional response in transfected cells. The MRF4 activation domain and the related amino-terminal MyoD activation domain are capable of substituting for one another in converting fibroblasts to a myogenic phenotype and in activating expression of an alpha-actin reporter gene, although the MRF4 and MyoD activation domains on these chimeric proteins also dictate the specificity of transcriptional activation. The different primary amino acid sequences of these regions leave open the possibility that different coregulator proteins interact with the muscle regulatory factors to elicit their correct transcriptional activity during skeletal muscle development.

Fibroblast growth factor and transforming growth factor beta repress transcription of the myogenic regulatory gene MyoD1.

In this report, we demonstrate that myogenic cultures inhibited from differentiating by treatment with fibroblast growth factor or transforming growth factor beta show reduced levels of MyoD1 mRNA. Although this repression may contribute to the inhibition of myogenesis by growth factors, additional regulatory pathways must be affected, since inhibition still occurs in cultures engineered to constitutively express MyoD1 mRNA.

Signaling through mitogen-activated protein kinase and Rac/Rho does not duplicate the effects of activated Ras on skeletal myogenesis.

The ability of basic helix-loop-helix muscle regulatory factors (MRFs), such as MyoD, to convert nonmuscle cells to a myogenic lineage is regulated by numerous growth factor and oncoprotein signaling pathways. Previous studies have shown that H-Ras 12V inhibits differentiation to a skeletal muscle lineage by disrupting MRF function via a mechanism that is independent of the dimerization, DNA binding, and inherent transcriptional activation properties of the proteins. To investigate the intracellular signaling pathway(s) that mediates the inhibition of MRF-induced myogenesis by oncogenic Ras, we tested two transformation-defective H-Ras 12V effector domain variants for their ability to alter terminal differentiation. H-Ras 12V,35S retains the ability to activate the Raf/MEK/mitogen-activated protein (MAP) kinase cascade, whereas H-Ras 12V,40C is unable to interact directly with Raf-1 yet still influences other signaling intermediates, including Rac and Rho. Expression of each H-Ras 12V variant in C3H10T1/2 cells abrogates MyoD-induced activation of the complete myogenic program, suggesting that MAP kinase-dependent and -independent Ras signaling pathways individually block myogenesis in this model system. However, additional studies with constitutively activated Rac1 and RhoA proteins revealed no negative effects on MyoD-induced myogenesis. Similarly, treatment of Ras-inhibited myoblasts with the MEK1 inhibitor PD98059 revealed that elevated MAP kinase activity is not a significant contributor to the H-Ras 12V effect. These data suggest that an additional Ras pathway, distinct from the well-characterized MAP kinase and Rac/Rho pathways known to be important for the transforming function of activated Ras, is primarily responsible for the inhibition of myogenesis by H-Ras 12V.

Myogenin and MEF2 function synergistically to activate the MRF4 promoter during myogenesis.

The basic helix-loop-helix muscle regulatory factor (MRF) gene family encodes four distinct muscle-specific transcription factors known as MyoD, myogenin, Myf-5, and MRF4. These proteins represent key regulatory factors that control many aspects of skeletal myogenesis. Although the MRFs often exhibit overlapping functional activities, their distinct expression patterns during embryogenesis suggest that each protein plays a unique role in controlling aspects of muscle development. As a first step in determining how MRF4 gene expression is developmentally regulated, we examined the ability of the MRF4 gene to be expressed in a muscle-specific fashion in vitro. Our studies show that the proximal MRF4 promoter contains sufficient information to direct muscle-specific expression. Located within the proximal promoter are a single MEF2 site and E box that are required for maximum MRF4 expression. Mutation of the MEF2 site or E box severely impairs the ability of this promoter to produce a muscle-specific response. In addition, the MEF2 site and E box function in concert to synergistically activate the MRF4 gene in nonmuscle cells coexpressing MEF2 and myogenin proteins. Thus, the MRF4 promoter is regulated by the MEF2 and basic helix-loop-helix MRF protein family through a cross-regulatory circuitry. Surprisingly, the MRF4 promoter itself is not transactivated by MRF4, suggesting that this MRF gene is not subject to an autoregulatory pathway as previously implied by other studies. Understanding the molecular mechanisms regulating expression of each MRF gene is central to fully understanding how these factors control developmental events.

Abrogating cholesterol esterification suppresses growth and metastasis of pancreatic cancer.

Cancer cells are known to execute reprogramed metabolism of glucose, amino acids and lipids. Here, we report a significant role of cholesterol metabolism in cancer metastasis. By using label-free Raman spectromicroscopy, we found an aberrant accumulation of cholesteryl ester in human pancreatic cancer specimens and cell lines, mediated by acyl-CoA cholesterol acyltransferase-1 (ACAT-1) enzyme. Expression of ACAT-1 showed a correlation with poor patient survival. Abrogation of cholesterol esterification, either by an ACAT-1 inhibitor or by shRNA knockdown, significantly suppressed tumor growth and metastasis in an orthotopic mouse model of pancreatic cancer. Mechanically, ACAT-1 inhibition increased intracellular free cholesterol level, which was associated with elevated endoplasmic reticulum stress and caused apoptosis. Collectively, our results demonstrate a new strategy for treating metastatic pancreatic cancer by inhibiting cholesterol esterification.

Inhibition of myogenic differentiation by the H-ras oncogene is associated with the down regulation of the MyoD1 gene.

Skeletal muscle development is regulated by a complex series of genetic and environmental cues that control the establishment of the myogenic lineage and the differentiation of determined myoblasts. Numerous agents, including growth factors and oncogene products, have been shown to inhibit skeletal muscle development, possibly by affecting the pattern of signal transduction that is required for myogenesis. Among the eukaryotic G proteins that have been implicated as mediators of signal transduction are the protein products of the mammalian ras genes (p21s). In this study, we demonstrate that expression of a transfected, oncogenic, human H-ras gene in C3H10T1/2-derived myoblasts has dramatic, yet varied, effects on skeletal myogenesis. While some H-ras transformed myoblast clones are differentiation-defective, other clones are inhibited from morphologically differentiating but retain a limited ability to biochemically differentiate. The H-ras induced inhibition of differentiation usually is associated with a decreased expression of the myogenic determination gene, MyoD1. Introduction of a MyoD1 cDNA expression vector into differentiation-defective H-ras expressing myoblasts partially restores the myogenic potential in these cells. Our results suggest that activated H-ras p21 inhibits the terminal differentiation of myoblasts by producing a general reduction in the differentiation competence of cells which, in the most extreme case, is a consequence of the down-regulation of the MyoD1 determination gene.

Differential effects of Ras signaling through NFkappaB on skeletal myogenesis.

Oncogenic Ras (H-Ras G12V) inhibits skeletal myogenesis through multiple signaling pathways. Previously, we demonstrated that the major downstream effectors of Ras (i.e., MEK/MAPK, RalGDS and Rac/Rho) play a minor, if any, role in the differentiation-defective phenotype of Ras myoblasts. Recently, NFkappaB, another Ras signaling target, has been shown to inhibit myogenesis presumably by stimulating cyclin D1 accumulation and cell cycle progression. In this study, we address the involvement of NFkappaB activation in the Ras-induced inhibition of myogenesis. Using H-Ras G12V and three G12V effector-loop variants, we detect high levels of NFkappaB transcriptional activity in C3H10T1/2-MyoD cells treated with differentiation medium. Myogenesis is blocked by all Ras proteins tested, yet only in the case of H-Ras G12V are cyclin D1 levels increased and cell cycle progression maintained. Expression of IkappaBalpha SR, an inhibitor of NFkappaB, does not reverse the differentiation-defective phenotype of Ras expressing cultures, but does induce differentiation in cultures treated with tumor necrosis factor (TNFalpha) or in cultures expressing the RelA/p65 subunit of NFkappaB. These data confirm that NFkappaB is a target of Ras and suggest that the cellular actions of NFkappaB require additional signals that are discriminated by the Ras effector-loop variants. Results with IkappaBalpha SR convincingly demonstrate that H-Ras G12V does not rely on NFkappaB activity to block myogenesis, an observation that continues to implicate another unidentified signaling pathway(s) in the inhibition of skeletal myogenesis by Ras.

Structure and myofiber-specific expression of the rat muscle regulatory gene MRF4.

We have cloned an 11.3-kb rat genomic DNA fragment encompassing the muscle regulatory factor 4 (MRF4) protein-coding sequence, 8.5 kb of 5'-flanking sequence, and 1.0 kb of 3'-flanking sequence. In order to study MRF4 gene expression, the rat myogenic cell line, L6J1-C, which expresses the endogenous MRF4 gene only in differentiated myofibers, was transfected stably with the full-length genomic clone and various 5' deletions. RNase protection assays demonstrated that MRF4 genes containing as little as 430 bp of 5'-flanking sequence exhibited an increase in expression as the cells differentiated into myofibers, indicating that elements responsible for fiber-specific expression are contained within this cloned DNA fragment. Similar up-regulation was observed with genes containing 1.5 kb of 5'-flanking sequence. Interestingly, MRF4 genes containing 5.0 kb and 8.5 kb of 5'-flanking sequence were up-regulated to even higher levels, suggesting that additional myofiber-specific regulatory elements located between 1.5 and 5.0 kb upstream from the coding region play a role in regulating the expression of this muscle-specific gene.