Muscle Creatine Kinase Gene Research Articles

Transgenic and tissue culture analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types.

The muscle creatine kinase (MCK) gene is expressed at high levels only in differentiated skeletal and cardiac muscle. The activity of the cloned enhancer-promoter has previously been shown to be dependent on the Trex element which is specifically bound by a yet unidentified nuclear factor, TrexBF. We have further characterized the function of the Trex site by comparing wild-type and Trex-mutated MCK transgenes in five mouse skeletal muscles: quadriceps, extensor digitorum longus (EDL), soleus, diaphragm, and distal tongue, as well as in heart ventricular muscle. Several types of statistical analysis including analysis of variance (ANOVA) and rank sum tests were used to compare expression between muscle types and between constructs. Upon mutation of the Trex site, median transgene expression levels decreased 3- to 120-fold in the muscles examined, with statistically significant differences in all muscles except the EDL. Expression in the largely slow soleus muscle was more affected than in the EDL, and expression in the distal tongue and diaphragm muscles was affected more than in soleus. Median expression of the transgene in ventricle decreased about 18-fold upon Trex mutation. Transfections into neonatal rat myocardiocytes confirmed the importance of the Trex site for MCK enhancer activity in heart muscle, but the effect is larger in transgenic mice than in cultured cells.

Akt1 and Akt2 differently regulate muscle creatine kinase and myogenin gene transcription in insulin-induced differentiation of C2C12 myoblasts.

Insulin and IGFs are potent inducers of skeletal muscle differentiation. Although PI3K is known to be involved in skeletal muscle differentiation, its downstream targets in this process are not clearly defined. We investigated the roles of Akt and mammalian target of rapamycin (mTOR) in skeletal muscle differentiation. LY294002, a pharmacological inhibitor of PI3K, and the immunosuppressant rapamycin inhibited insulin-induced differentiation of C2C12 myoblasts. LY294002 and rapamycin suppressed myosin heavy chain expression and myotube formation. Transient reporter assays showed that both inhibitors repress muscle creatine kinase (MCK) and myogenin gene transcription. Heterologous expression of Akt1/PKB(alpha) potently suppressed MCK gene transcription without affecting myogenin gene transcription, whereas heterologous expression of Akt2 increased myogenin and MCK gene transcription. Finally, overexpression of myogenin rescued the inhibitory effect of rapamycin on MCK gene transcription, whereas it failed to rescue the inhibitory effect of LY294002 and Akt1. These results suggest that insulin regulates myogenic differentiation chiefly at the level of myogenin gene transcription via PI3K and mTOR. PI3K activity, but not mTOR, may regulate transcriptional activity of myogenin. Our data also suggest that Akt1 and Akt2 play distinct roles in myogenic differentiation.

Akt1 and Akt2 Differently Regulate Muscle Creatine Kinase and Myogenin Gene Transcription in Insulin-Induced Differentiation of C2C12 Myoblasts

Akt1 and Akt2 Differently Regulate Muscle Creatine Kinase and Myogenin Gene Transcription in Insulin-Induced Differentiation of C2C12 Myoblasts

Interaction of Nuclear Proteins with Repeat Sequences in the 5′ Flanking Region of Mouse Muscle Creatine Kinase Gene during Aging

The 5′ flanking region of the mouse muscle creatine kinase (MCK) gene contains two repeat sequences—a mononucleotide repeat, (A)22 (−2694 to −2673), and a tetranucleotide repeat, (GTTT)8 (−2962 to −2931). We show here that these repeats in the mouse MCK gene bind to specific nuclear protein factors. Some of the factors interacting with these sequences are tissue-specific and show age-related decrease in the binding activity. Nonspecific competitor and heterologous DNA probes failed to compete out the complexes showing that the interaction is specific to the repeat sequences. These proteins may have a role in the expression of the gene during aging.

Stichopus japonicus arginine kinase: gene structure and unique substrate recognition system

Arginine kinase from the sea cucumber Stichopus japonicus underwent a unique molecular evolution. Unlike the monomeric 40kDa arginine kinases from molluscs and arthropods, Stichopus arginine kinase is dimeric, the same as cytoplasmic isoenzymes of the vertebrate creatine kinases. Its entire amino acid sequence is more similar to creatine kinases than to other arginine kinases, but the guanidino specificity region (GS region) is of the arginine kinase type. To elucidate its unusual evolution, the structure of the Stichopus arginine kinase gene was determined. It consisted of seven exons and six introns, and a part of the exon 2 of the Stichopus gene corresponds to the GS region. Compared with the structure of the human muscle creatine kinase gene (seven exons, six introns), the splice junctions of five introns were conserved exactly between the two genes, suggesting that these introns had been conserved for at least 500 million years. The entire sequence of Stichopus arginine kinase is distinctly included in the creatine kinase cluster in all tree construction methods examined. On the other hand, if the tree is constructed only from sequences corresponding to Stichopus exon 2, it is placed in the arginine kinase cluster. Thus we conclude that Stichopus arginine kinase evolved not from the arginine kinase gene but from the creatine kinase gene, and suggest that its GS region, determining substrate specificity, has been replaced by an arginine kinase type via exon shuffling. In typical arginine kinases four residues, Ser63, Gly64, Val65 and Tyr68 (numbering from the Limulus polyphemus sequence), in the GS region are highly conserved and are associated with substrate binding. Among them, Tyr68 appears to play a crucial role by forming a hydrogen bond with the substrate, and is conserved exactly in all arginine kinases. However, in Stichopus arginine kinase, none of these four conserved residues were present. Nevertheless, the enzyme displays an affinity for the substrate arginine (Km = 0.8mM) comparable with other arginine kinases. This implies that a completely different substrate-binding system has been developed in Stichopus arginine kinase. We propose that the His64 in Stichopus arginine kinase acts as a substitute for the Tyr68 in other arginine kinases, and that the imidazole ring of His64 is hydrogen bonded with the substrate arginine, thus stabilizing it.

Stichopus japonicus arginine kinase: gene structure and unique substrate recognition system

Arginine kinase from the sea cucumber Stichopus japonicus underwent a unique molecular evolution. Unlike the monomeric 40 kDa arginine kinases from molluscs and arthropods, Stichopus arginine kinase is dimeric, the same as cytoplasmic isoenzymes of the vertebrate creatine kinases. Its entire amino acid sequence is more similar to creatine kinases than to other arginine kinases, but the guanidino specificity region (GS region) is of the arginine kinase type. To elucidate its unusual evolution, the structure of the Stichopus arginine kinase gene was determined. It consisted of seven exons and six introns, and a part of the exon 2 of the Stichopus gene corresponds to the GS region. Compared with the structure of the human muscle creatine kinase gene (seven exons, six introns), the splice junctions of five introns were conserved exactly between the two genes, suggesting that these introns had been conserved for at least 500 million years. The entire sequence of Stichopus arginine kinase is distinctly included in the creatine kinase cluster in all tree construction methods examined. On the other hand, if the tree is constructed only from sequences corresponding to Stichopus exon 2, it is placed in the arginine kinase cluster. Thus we conclude that Stichopus arginine kinase evolved not from the arginine kinase gene but from the creatine kinase gene, and suggest that its GS region, determining substrate specificity, has been replaced by an arginine kinase type via exon shuffling. In typical arginine kinases four residues, Ser(63), Gly(64), Val(65) and Tyr(68) (numbering from the Limulus polyphemus sequence), in the GS region are highly conserved and are associated with substrate binding. Among them, Tyr(68) appears to play a crucial role by forming a hydrogen bond with the substrate, and is conserved exactly in all arginine kinases. However, in Stichopus arginine kinase, none of these four conserved residues were present. Nevertheless, the enzyme displays an affinity for the substrate arginine (K(m)=0.8 mM) comparable with other arginine kinases. This implies that a completely different substrate-binding system has been developed in Stichopus arginine kinase. We propose that the His(64) in Stichopus arginine kinase acts as a substitute for the Tyr(68) in other arginine kinases, and that the imidazole ring of His(64) is hydrogen bonded with the substrate arginine, thus stabilizing it.

Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors.

Adenoviral gene transfer holds promise for gene therapy, but effective transduction of a large and distributed tissue such as muscle will almost certainly require systemic delivery. In this context, the use of muscle-specific regulatory elements such as the muscle creatine kinase (MCK) promoter and enhancer will avoid potentially harmful ectopic expression of transgenes. We describe here the development and testing of adenoviral vectors containing small, striated muscle-specific, highly active MCK expression cassettes. One of these regulatory elements (CK6) is less than 600 bp in length and is 12% as active as the CMV promoter/enhancer in muscle. A recombinant adenoviral vector containing this regulatory element retains very high muscle specificity, expressing 600-fold higher levels of transgene in muscle than in liver. Muscle-specific regulatory elements may also increase persistence of transduced muscle cells. Adenoviral transduction of dendritic cells has been shown to stimulate cytotoxic T-lymphocyte (CTL) responses directed against transgene epitopes. We show that human dendritic cells infected in vitro with MCK-containing adenoviruses do not express significant levels of transgene. Furthermore, while adenoviral vectors containing nonspecific promoters are normally cleared from muscle tissue within 1 month, we show that MCK-containing vectors express significant levels of transgene 4 months after intramuscular injection.

Muscle Differentiation Is Antagonized by SOX15, a New Member of the SOX Protein Family

SOX proteins belong to a multigenic family characterized by a unique DNA binding domain, known as the high mobility group box, that is related to that of the testis determining gene SRY. cDNA sequences for more than 30 SOX genes have been identified, and some are known to have diverse roles in vertebrate differentiation and development. Here, we report the isolation and characterization of mouse Sox15 that was uncovered during a screen for high mobility group box containing transcription factors that are expressed at different levels during skeletal muscle differentiation. Sox15 cDNAs were found at a much higher frequency in myoblasts prior to their differentiation into myotubes. Electrophoretic mobility shift assays indicated that recombinant SOX15 protein was capable of binding to a consensus DNA binding site for SOX proteins. When overexpressed in C2C12 myoblasts, wild type SOX15, but not a C-terminal truncated form or the related protein SOX11, specifically inhibited activation of muscle-specific genes and expression of the basic helix-loop-helix myogenic factors myogenin and MyoD, resulting in a failure of the cells to differentiate into myotubes. These results suggest a specific and repressive role for SOX15, requiring the C-terminal domain, during myogenesis.

DNA immunization utilizing a herpes simplex virus type 2 myogenic DNA vaccine protects mice from mortality and prevents genital herpes

A gene transfer vector for DNA immunization was developed in which the promoter was derived from the murine muscle creatine kinase (MCK) gene; a gene expressed only in differentiated skeletal muscle. In vitro, we observed high-level, but unrestricted, gene expression from the cytomegalovirus (CMV) promoter unlike expression from the MCK promoter which was weak but restricted to myofibers. A myogenic DNA vaccine (MDV) that encoded the glycoprotein D gene from herpes simplex virus type-2 (HSV-2) was used to DNA immunize mice. MDV immunization resulted in virus specific immunity that protected HSV-2 infected mice from mortality and prevented the development of genital herpes. Therefore, we conclude that high-level gene expression or the use of a strong transcription unit was not a prerequisite for an efficacious DNA vaccine and the use of a nonviral tissue specific promoter could suffice.

Helix-Loop-Helix Proteins: Regulators of Transcription in Eucaryotic Organisms

Helix-Loop-Helix Proteins: Regulators of Transcription in Eucaryotic Organisms

Muscle-specific Overexpression of FAT/CD36 Enhances Fatty Acid Oxidation by Contracting Muscle, Reduces Plasma Triglycerides and Fatty Acids, and Increases Plasma Glucose and Insulin

Increasing evidence has implicated the membrane protein CD36 (FAT) in binding and transport of long chain fatty acids (FA). To determine the physiological role of CD36, we examined effects of its overexpression in muscle, a tissue that depends on FA for its energy needs and is responsible for clearing a major fraction of circulating FA. Mice with CD36 overexpression in muscle were generated using the promoter of the muscle creatine kinase gene (MCK). Transgenic (MCK-CD36) mice had a slightly lower body weight than control litter mates. This reflected a leaner body mass with less overall adipose tissue, as evidenced by magnetic resonance spectroscopy. Soleus muscles from transgenic animals exhibited a greatly enhanced ability to oxidize fatty acids in response to stimulation/contraction. This increased oxidative ability was not associated with significant alterations in histological appearance of muscle fibers. Transgenic mice had lower blood levels of triglycerides and fatty acids and a reduced triglyceride content of very low density lipoproteins. Blood cholesterol levels were slightly lower, but no significant decrease in the cholesterol content of major lipoprotein fractions was measured. Blood glucose was significantly increased, while insulin levels were similar in the fed state and higher in the fasted state. However, glucose tolerance curves, determined at 20 weeks of age, were similar in control and transgenic mice. In summary, the study documented, in vivo, the role of CD36 to facilitate cellular FA uptake. It also illustrated importance of the uptake process in muscle to overall FA metabolism and glucose utilization.

Insertion of two independent enhancers in the long terminal repeat of a self-inactivating vector results in high-titer retroviral vectors with tissue-specific expression.

The use of retroviral vectors (RVs) derived from the murine oncoretroviruses for gene therapy is associated with the risk of malignant transformation of infected cells and ectopic expression of the proteins of interest. Targeting retroviral vectors to specific tissues would increase their safety and clinical applicability. To explore the potential of targeting vector expression to skeletal muscle, the murine leukemia virus broad transcriptional tropism was modified by substituting the viral promoter and/or enhancer with a transcriptional cassette containing the human T cell leukemia virus type I Tax-responsive element and the minimal muscle creatine kinase enhancer and promoter. The resulting retroviral vectors could be transcriptionally trans-activated by tax. In the absence of Tax, however, the viruses showed muscle-specific expression. Trans-complementing packaging and indicator cells stably expressing Tax were used to isolate high-titer producer cell clones (10(6) CFU/ml). In vitro, the levels of expression of these RVs in Tax-expressing fibroblasts were 10,000-fold higher than in normal fibroblasts and 1000-fold higher in C2C12 myotubes than in C2C12 myoblasts. Expression of the vectors and the endogenous muscle creatine kinase gene was similarly dependent on the maturity of the muscle cultures. One vector with modified LTRs was also tested in vivo in regenerating muscle and showed a delayed pattern of expression in myofibers compared with the vector containing the wild-type LTRs. These vectors can be easily modified to contain different tissue-specific enhancer and promoter elements and the availability of complementing packaging and indicator cells expressing Tax should allow their application in a variety of gene therapy settings.

Collaborative interactions between MEF‐2 and Sp1 in muscle‐specific gene regulation

Previous investigations have demonstrated synergistic interactions in vivo between CCAC and A/T-rich nucleotide sequence motifs as functional components of muscle-specific transcriptional enhancers. Using CCAC and A/T-rich elements from the myoglobin and muscle creatine kinase (MCK) gene enhancers, Sp1 and myocyte-specific enhancer factor-2 (MEF-2) were identified as cognate binding proteins that recognize these sites. Physical interactions between Sp1 and MEF-2 were demonstrated by immunological detection of both proteins in DNA binding complexes formed in vitro by nuclear extracts in the presence of only the A/T sequence motif, by coprecipitation of recombinant MEF-2 in the presence of a glutathione-S-transferase–Sp1 fusion protein bound to glutathione beads, and by a two-hybrid assay in Saccharomyces cerevisiae. The interaction with Sp1 in vitro and in vivo is specific for MEF-2 and was not observed with serum response factor, a related MADS domain protein. Forced expression of Sp1 and MEF-2 in insect cells otherwise lacking these factors promotes synergistic transcriptional activation of a promoter containing binding sites for both proteins. These data expand the repertoire of functional and physical interactions between lineage-restricted (MEF-2) and ubiquitous (Sp1) transcription factors that may be important for myogenic differentiation. J. Cell. Biochem. 70:366–375, 1998. © 1998 Wiley-Liss, Inc.

Collaborative interactions between MEF-2 and Sp1 in muscle-specific gene regulation

Previous investigations have demonstrated synergistic interactions in vivo between CCAC and A/T-rich nucleotide sequence motifs as functional components of muscle-specific transcriptional enhancers. Using CCAC and A/T-rich elements from the myoglobin and muscle creatine kinase (MCK) gene enhancers, Sp1 and myocyte-specific enhancer factor-2 (MEF-2) were identified as cognate binding proteins that recognize these sites. Physical interactions between Sp1 and MEF-2 were demonstrated by immunological detection of both proteins in DNA binding complexes formed in vitro by nuclear extracts in the presence of only the A/T sequence motif, by coprecipitation of recombinant MEF-2 in the presence of a glutathione-S-transferase-Sp1 fusion protein bound to glutathione beads, and by a two-hybrid assay in Saccharomyces cerevisiae. The interaction with Sp1 in vitro and in vivo is specific for MEF-2 and was not observed with serum response factor, a related MADS domain protein. Forced expression of Sp1 and MEF-2 in insect cells otherwise lacking these factors promotes synergistic transcriptional activation of a promoter containing binding sites for both proteins. These data expand the repertoire of functional and physical interactions between lineage-restricted (MEF-2) and ubiquitous (Sp1) transcription factors that may be important for myogenic differentiation.

P53 protein is activated during muscle differentiation and participates with MyoD in the transcription of muscle creatine kinase gene.

The p53 protein is a transcription factor involved in processes of cell growth and differentiation. The muscle creatine kinase (MCK) gene whose transcription is induced during muscle differentiation contains p53-binding sites. In this study we tested the involvement of p53 in the activation of MCK transcription during muscle differentiation of C2 cells. We have shown that the p53 protein is stabilized and its DNA binding and transcriptional activities are induced during muscle differentiation. At the stage of muscle-differentiation, p53 protein can induce the accurate transcription of a minimal p53-dependent MCK reporter gene. Moreover, p53 cooperates with MyoD in the induction of MCK transcription. The expression of a dominant negative p53 protein in muscle cells reduced the expression of endogenous MCK gene. The dominant negative p53 protein abolished the cooperativity of wild type p53 with MyoD. Amino and carboxy terminal residues of MyoD required for the cooperation with p53 in transcription were identified. The cooperativity between the two proteins occurs also at the stage of DNA binding. We suggest that p53 protein is activated during myoblast differentiation and participates with MyoD in the induction of MCK transcription.

Synergistic transcriptional activation of the MCK promoter by p53: tetramers link separated DNA response elements by DNA looping.

The WAF1, Cyclin G and muscle creatine kinase (MCK) genes, all contain multiple copies of the consensus p53-binding element within their regulatory regions. We examined the role of these elements in transactivation of the muscle creatine kinase (MCK) gene by p53. The MCK promoter possesses distal (-3182 to -3133) and proximal (-177 to -81) p53-binding elements within which residues -3182 to -3151 (distal) and -176 to -149 (proximal) show homology to the consensus p53-binding site. Using promoter deletion studies, we find that both proximal and distal elements are required for high level, synergistic transcriptional activation in vivo. Electron microscopy indicates that p53-p53 interactions link proximal and distal p53-binding elements and cause looping out of intervening DNA, suggesting that this DNA sequence may be dispensable for synergy. This idea was confirmed by progressive deletion of the DNA between p53-binding elements. Synergism persisted with spacing reduced to only 150 bp. Tetramerization-deficient p53 mutants were defective for transcriptional activation but still capable of synergy. Our results provide evidence for a model by which high level transcriptional activation of promoters with multiple p53 response elements is achieved.

Molecular characterization of the creatine kinases and some historical perspectives.

Over the last 15 years, molecular characterization of the creatine kinase (CK) gene family has paralleled the molecular revolution of understanding gene structure, function, and regulation. In this review, we present a summary of advances in molecular analysis of the CK gene family with a few vignettes of historical interest. We describe how the muscle CK gene provided an essential model system to examine myogenic regulatory mechanisms, leading to the discovery of the binding site for the MyoD family of basic helix-loop-helix transcription factors essential in skeletal myogenesis and the characterization of the MEF2 family of factors with an A/T rich consensus binding site essential in skeletal myogenesis and cardiogenesis. Cloning and characterization of the four mRNAs and nuclear genes encoding the cytosolic CKs, muscle and brain CKs, and the mitochondrial (Mt) CKs, sarcomeric MtCK and ubiquitous MtCK, has allowed intriguing study of tissue-specific and cell-specific expression of the different CKs and analysis of structural, functional, regulatory, and evolutionary relationships among both the four CK proteins and genes. Current and future studies focus on understanding both cellular energetics facilitated by the CK enzymes, especially energy channelling from the site of production, the mitochondrial matrix and inner membrane, to various cytosolic foci of utilization, and regulation of MtCK gene expression at the cell and tissue-specific level as models of regulation of energy producing genes.

Multiple muscle-specific regulatory elements are associated with a DNase I hypersensitive site of the cardiac beta-myosin heavy-chain gene.

Using nuclei isolated from neonatal cardiomyocytes, we have mapped the DNase I hypersensitive sites (DHSs) residing within the 5'-upstream regions of the hamster cardiac myosin heavy-chain (MyHC) gene. Two cardiac-specific DHSs within the 5 kb upstream region of the cardiac MyHC gene were identified. One of the DHSs was mapped to the -2.3 kb (beta-2.3 kb) region and the other to the proximal promoter region. We further localized the beta-2.3 kb site to a range of 250 bp. Multiple, conserved, muscle regulatory motifs were found within the beta-2.3 kb site, consisting of three E-boxes, one AP-2 site, one CArG motif, one CT/ACCC box and one myocyte-specific enhancer factor-2 site. This cluster of regulatory elements is strikingly similar to a cluster found in the enhancer of the mouse muscle creatine kinase gene (-1256 to -1050). The specific interaction of the motifs within the beta-2.3 kb site and the cardiac nuclear proteins was demonstrated using gel mobility-shift assays and footprinting analysis. In addition, transfection analysis revealed a significant increase in chloramphenicol acetyltransferase activity when the beta-2.3 kb site was linked to a heterologous promoter. These results suggest that previously undefined regulatory elements of the beta-MyHC gene may be associated with the beta-2.3 kb site.

Muscle-specific overexpression of human lipoprotein lipase in mice causes increased intracellular free fatty acids and induction of peroxisomal enzymes

A transgenic mouse model for peroxisomal and mitochondrial induction caused by increased uptake of fatty acids in muscle was established. Transgenic mouse lines were generated using a human lipoprotein lipase (LPL) mini gene (3–20 copies) driven by the promoter of the muscle creatine kinase gene. Expression of human LPL was only observed in skeletal and cardiac muscle. In proportion to the level of LPL overexpression increased LPL activity in skeletal (up to 24-fold) and cardiac (up to three-fold) muscle, decreased plasma triglyceride levels, elevated free fatty acid (FFA) uptake by muscle tissue, weight loss (due to a reduction in muscle mass as well as adipose tissue mass) and premature death were observed. A remarkable increase in the number of mitochondria and peroxisomes was detected using oxide-electron microscopy. Proliferation of mitochondria and peroxisomes was confirmed by a dose-dependent increase of marker enzyme activity (succinate-dehydrogenase and catalase). In addition, peroxisomal acyl-CoAse enzyme protein was markedly elevated whereas mRNA was increased only up to two-fold. No changes in peroxisomal proliferator activated receptor α mRNA was found. This degree of proliferation and enzyme activity of mitochondria and peroxisomes suggests that FFA play an important role in the induction of these organelles. In addition, myopathy characterized by excessive glycogen storage, muscle fiber degeneration, and fiber atrophy with centralization of nuclei, mimicking several forms of human myopathies was noted. Our results imply that improper regulation of muscle LPL leading to increased fatty acid levels in muscle can cause severe pathological changes. This effect may be important in the pathogenesis of human myopathies. We conclude that these transgenic mouse lines could serve as a useful animal model for the investigation of myopathies and the effects of fatty acids on the induction of mitochondria and peroxisomes.

Bone Morphogenetic Protein-2 Inhibits Terminal Differentiation of Myogenic Cells by Suppressing the Transcriptional Activity of MyoD and Myogenin

Bone morphogenetic protein (BMP) is a family of cytokines that induce ectopic bone formation when implanted into muscular tissues. We reported that BMP-2 inhibits the terminal differentiation of C2C12 myoblasts and converts them into osteoblast lineage cells (Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Takahashi, N., Ikeda, T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A., and Suda, T. (1994) J. Cell Biol.127, 1755–1766). In the present study, we examined the molecular mechanism of the inhibitory effect of BMP-2 on terminal differentiation of myogenic cells. When either MyoD or myogenin cDNA was introduced into C3H10T1/2 (10T1/2) cells with a muscle-specific CAT reporter containing four copies of the right E-box of muscle creatine kinase (MCK) enhancer, the CAT activity was dose-dependently suppressed by BMP-2. Furthermore, BMP-2 inhibited the terminal differentiation of these subclonal 10T1/2 cells that stably expressed MyoD or myogenin into mature myotubes that expressed myosin heavy chain and troponin T. The differentiation of a subclone of the MyoD-transfected NIH3T3 cells into mature muscle cells was also inhibited by BMP-2. BMP-2 induced alkaline phosphatase activity in 10T1/2-derived, but not in NIH3T3-derived MyoD-transfected cells. These cells constitutively expressed exogenous MyoD and myogenin, which were localized exclusively in the nuclei irrespective of the presence and the absence of BMP-2. However, these cells failed to express the mRNAs of endogenous myogenic factors and MCK when cultured with BMP-2. In the electrophoresis mobility shift assay using nuclear extracts of the myogenic cells, MyoD and myogenin bound to the right E-box in the enhancer region of the MCK gene even in the presence of BMP-2. These results suggest that BMP-2 inhibits the terminal differentiation of myogenic cells by suppressing the transcriptional activity of the myogenic factors.