Abstract. The implantation of bone morphogenetic protein (BMP) into muscular tissues induces ectopic bone formation at the site of implantation. To investigate the mechanism underlying this process, we examined whether recombinant bone morphogenetic protein-2 (BMP-2) converts the differentiation pathway of the clonal myoblastic cell line, C2C12, into that of osteoblast lineage. Incubating the cells with 300 ng/ml of BMP-2 for 6 d almost completely inhibited the formation of the multinucleated myotubes expressing troponin T and myosin heavy chain, and induced the appearance of numerous alkaline phosphatase (ALP)-positive cells. BMP-2 dose dependently induced ALP activity, parathyroid hormone (PTH)-dependent 3',5'-cAMP production, and osteocalcin production at concentrations above 100 ng/ml. The concentration of BMP-2 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubating primary muscle cells with 300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas induced ALP activity and osteocalcin production. Incubation with 300 ng/ml of BMP-2 suppressed the expression of mRNA for muscle creatine kinase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-related protein (PTHrP) receptors, and osteocalcin within 24--48 h. BMP-2 completely inhibited the expression of myogenin mRNA by day 3. By day 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was transiently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mRNA was greatly stimulated by BMP-2. When C2C12 cells pretreated with BMP-2 for 6 d were transferred to a colony assay system in the absence of BMP-2, more than 84 % of the colonies generated became troponin T-positive and ALP activity disappeared. TGF-/31 also inhibited myotube formation in C2C12 cells, and suppressed the expression of myogenin and MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteoblastic phenotype was induced by TGF-/31 in C2C12 cells. TGF-/31 potentiated the inhibitory effect of BMP-2 on myotube formation, whereas TGF-/31 reduced ALP activity and osteocalcin production induced by BMP-2 in C2C12 cells. These results indicate that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable.S EVERAL lines of evidence indicate that osteoblasts, chondrocytes, myocytes, and adipocytes are all derived from a common progenitor cell called undifferentiated mesenchymal cells (Taylor and Jones, 1979; Grigoriadis et al., 1988Grigoriadis et al., , 1990 Yamaguchi and Kahn, 1991). During the process of their differentiation, progenitor cells acquire specific phenotypes depending upon the differentiated cell types under the control of respective regulatory factors (for reviews see Rodan and Rodan, 1984;Owen, 1988; Wlodar-Address all correspondence to Dr. Tatsuo Suda, Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Tokyo 142, ski, 1990). The di...

Skeletal muscle development involves the formation of multi-nucleated myotubes. This is thought to proceed by the induction of differentiation (acquisition of fusion competence) of myoblast cells, their aggregation, and union of their plasma membranes. Various membrane proteins including N- and M-cadherins, N- and V-CAMs and integrins participate in myotube formation, but the molecular mechanisms of muscle cell fusion are poorly understood. Here we report the identification of three new, myoblast-expressed gene products, meltrin-alpha, beta and gamma, with homology to both viper haemorrhagic factors and fertilin (PH-30), a membrane protein involved in egg-sperm fusion. Meltrin-alpha, a member of the metalloproteinase/disintegrin protein family, appears to be required for myotube formation. Involvement of a fertilin-related protein in myogenesis suggests that there are common mechanisms in gamete and myoblast fusion.

The repressor 8EF1 was discovered by its action on the DC5 fragment of the lens-specific 81-crystallin enhancer. C-proximal zinc fingers of 8EF1 were found responsible for binding to the DC5 fragment and had specificity to CACCT as revealed by selection of high-affinity binding sequences from a random oligonucleotide pool. CACCT is present not only in DC5 but also in the E2 box (CACCTG) elements which are the binding sites of various basic helix-loop-helix activators and also the target of an unidentified repressor, raising the possibility that 8EF1 accounts for the E2 box repressor activity. 8EF1 competed with E47 for binding to an E2 box sequence in vitro. In lymphoid cells, endogenous 8EF1 activity as a repressor was detectable, and exogenous 8EF1 repressed immunoglobulin K enhancer by binding to the KE2 site. Moreover, bEF1 repressed MyoD-dependent activation of the muscle creatine kinase enhancer and MyoD-induced myogenesis of 1OT1/2 cells. Thus, 8lEF1 counteracts basic helix-loop-helix activators through binding site competition and fulfills the conditions of the E2 box repressor. In embryonic tissues, the most prominent site of &;EF1 expression is the myotome. Myotomal expression as well as the above results argues for a significant contribution of 6EF1 in regulation of embryonic myogenesis through the modulation of the actions of MyoD family proteins.Cumulative evidence has indicated that repressors interacting with activators play crucial roles in developmental gene regulation. In the best-studied cases of transcriptional regulatory elements generating developmental specificities, it is found that a binding site of a repressor overlaps with a binding site of an activator so that the regulators of the opposite effects compete for occupancy of the same site (16,28,29,32). In general, the repressor is more widely distributed in spatial and temporal terms than the activators. Thus, in the majority of cell types the repressor occupies the element and shuts off expression of the gene, and only under conditions in which binding of the activator to the element dominates over that of the repressor is expression of the gene turned on. This seems one of the basic mechanisms to elicit stage-specific or cell-typespecific gene expression.An example is found in immunoglobulin enhancers in which E2 box activator binding sites with the core sequence of CACCTG have been identified. A group of basic helix-loophelix (bHLH) activator proteins (25) which are encoded by E2A and E2-2 genes (3) and bind to the E2 site of the immunoglobulin K enhancer (and the ,uE5 site of the immunoglobulin heavy-chain enhancer as well) are in competition with a repressor, and only in differentiated lymphoid cells is the action of the activator effectuated and immunoglobulin K enhancer activated (29). The same scenario holds true for regulatory elements of myocyte-specific genes such as AChRb in which activators containing myogenic MyoD family proteins as their component bind to an E2-box-activating element (32). In these cases, action of the r...

The DNA element governing the inducible expression of drug-metabolizing P-450c gene by xenobiotic treatments was investigated by gene transfer methods. A variety of dissected fragments from -844 to -1140bp region which was essential for the inducibility of P-450c gene were placed on the heterologous SV40 promoter for testing the inducibility. Mapping studies in combination with gel retardation assay defined the presence of the two xenobiotic responsive elements (XRE, XRE1, -1007 - -1021bp; XRE2, -1088 - -1092bp) composed of about 15 nucleotides which expressed the enhancer activity in response to xenobiotic inducers. The two XREs share 10 nucleotides in common out of 15 as expressed in the sequence CG/CTG/CC/TTG/CTCACGCT/AA and are arranged in the inverse orientation. They are different from DREs (drug responsive element) proposed previously (Sogawa, K. et al. Proc. Natl. Acad. Sci. 83, 8044-8048 (1986] and expressed a strong enhancer activity in response to 3-methylcholanthrene. The XRE shows a significant homology with glucocorticoid regulatory elements and apparently needs normal functions of a putative xenobiotic receptor for the inducible enhancer activity.

Meltrin ␤/ADAM19 is a member of ADAMs (a disintegrin and metalloproteases), which are a family of membrane-anchored glycoproteins that play important roles in fertilization, myoblast fusion, neurogenesis, and proteolytic processing of several membrane-anchored proteins. The expression pattern of meltrin ␤ during mouse development coincided well with that of neuregulin-1 (NRG), a member of the epidermal growth factor family. Then we examined whether meltrin ␤ participates in the proteolytic processing of membrane-anchored NRGs. When NRG-␤1 was expressed in mouse L929 cells, its extracellular domain was constitutively processed and released into the culture medium. This basal processing activity was remarkably potentiated by overexpression of wild-type meltrin ␤, which lead to the significant decrease in the cell surface exposure of extracellular domains of NRG-␤1. Furthermore, expression of protease-deficient mutants of meltrin ␤ exerted dominant negative effects on the basal processing of NRG-␤1. These results indicate that meltrin ␤ participates in the processing of NRG-␤1. Since meltrin ␤ affected the processing of NRG-␤4 but not that of NRG-␣2, meltrin ␤ was considered to have a preference for ␤-type NRGs as substrate. Furthermore, the effects of the secretory pathway inhibitors suggested that meltrin ␤ participates in the intracellular processing of NRGs rather than the cleavage on the cell surface.