Upregulation of matrix metalloproteinase triggers transdifferentiation of retinal pigmented epithelial cells in Xenopus laevis: A Link between inflammatory response and regeneration.

In adult Xenopus eyes, when the whole retina is removed, retinal pigmented epithelial (RPE) cells become activated to be retinal stem cells and regenerate the whole retina. In the present study, using a tissue culture model, it was examined whether upregulation of matrix metalloproteinases (Mmps) triggers retinal regeneration. Soon after retinal removal, Xmmp9 and Xmmp18 were strongly upregulated in the tissues of the RPE and the choroid.

Meltrin beta (ADAM19) gene: cloning, mapping, and analysis of the regulatory region.

Meltrin beta (ADAM19) is a member of the metalloprotease-disintegrin family. We report here chromosomal mapping of the mouse and rat meltrin beta genes and cloning and analysis of the mouse upstream regulatory regions. The meltrin beta transcript shows a spatially and temporally restricted expression pattern during morphogenesis, indicating that the actions of this membrane-bound protease are regulated, at least in part, at the transcriptional level.

Analysis for transcript expression of meltrin alpha in normal, regenerating, and denervated rat muscle.

Meltrin alpha (a disintegrin and metalloprotease (ADAM) 12) is a recently discovered molecule of the metalloprotease-disintegrin family which has been shown to participate in myotube formation in vitro and in myogenesis in vivo. In this study we investigated meltrin alpha in regenerating rat muscle, which is a condition where satellite cells (SC) contribute to myofiber growth by fusing with one another and with myotubes or muscle fibers. We studied meltrin alpha mRNA expression by RT-PCR and in situ-hybridization in normal adult muscle, in soleus muscle regenerating for 2, 5, or 10 days, and in muscle which had been denervated 1 week, 4 weeks, or 6 months previously.

Roles of Meltrin beta /ADAM19 in the processing of neuregulin.

Meltrin beta/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 beta during mouse development coincided well with that of neuregulin-1 (NRG), a member of the epidermal growth factor family. Then we examined whether meltrin beta participates in the proteolytic processing of membrane-anchored NRGs.

Membrane-anchored metalloprotease MDC9 has an alpha-secretase activity responsible for processing the amyloid precursor protein.

MDC9, also known as meltrin gamma, is a membrane-anchored metalloprotease. MDC9 contains several distinct protein domains: a signal sequence followed by a prodomain and a domain showing sequence similarity to snake venom metalloproteases, a disintegrin-like domain, a cysteine-rich region, an epidermal-growth-factor-like repeat, a transmembrane domain and a cytoplasmic domain. Here we demonstrate that MDC9 expressed in COS cells is cleaved between the prodomain and the metalloprotease domain.

Spatially- and temporally-restricted expression of meltrin alpha (ADAM12) and beta (ADAM19) in mouse embryo.

The cloning of the full-length cDNA encoding meltrin beta (ADAM19), one of the metalloprotease-disintegrins expressed in mouse myogenic cells, revealed that the meltrin beta gene encodes a membrane protein closely related to meltrin alpha (ADAM12) which participates in myotube formation in vitro. To delineate the functions of meltrin alpha and beta, we examined the expression patterns of their transcripts during embryogenesis. The meltrin alpha gene is activated in condensed mesenchymal cells that give rise to skeletal muscle, bones and visceral organs.

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.

Lysophosphatidic acid and bFGF control different modes in proliferating myoblasts.

Myogenic cells provide excellent in vitro models for studying the cell growth and differentiation. In this study we report that lysophosphatidic acid (LPA), a bioactive phospholipid contained in serum, stimulates the growth and inhibits the differentiation of mouse C2C12 myoblast cells, in a distinct manner from basic fibroblast growth factor (bFGF) whose mitotic and anti-differentiation actions have been well investigated. These actions of LPA were both blocked by pertussis toxin, suggesting the involvement of Gi class of G proteins, whereas bFGF acts through receptor tyrosine kinases.

A metalloprotease-disintegrin participating in myoblast fusion.

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.

Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage.

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.

The delta-crystallin enhancer-binding protein delta EF1 is a repressor of E2-box-mediated gene activation.

The repressor delta EF1 was discovered by its action on the DC5 fragment of the lens-specific delta 1-crystallin enhancer. C-proximal zinc fingers of delta EF1 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 delta EF1 accounts for the E2 box repressor activity.

MyoD and myogenin act on the chicken myosin light-chain 1 gene as distinct transcriptional factors.

Expression of MyoD, myogenin, MRF4, and Myf-5 converts nonmuscle cells to muscle cells. In an attempt to analyze the roles of these factors, we have investigated their effects on transcription driven by the promoter of the chicken myosin alkaline light-chain (MLC1) gene. The activation by CMD1 or c-myogenin (chicken MyoD or myogenin, respectively) was dependent on the existence of a muscle-specific regulatory region located from positions -2096 to -1743.

Expression of myogenic factors in denervated chicken breast muscle: isolation of the chicken Myf5 gene.

In this study, we have isolated and characterized the chicken Myf5 gene, and cDNA clones encoding chicken MyoD1 and myogenin. The chicken Myf5 and MRF4 genes are tandemly located on a single genomic DNA fragment, and the chicken Myf5 gene is organized into at least three exons. Using genomic and cDNA probes, we further analyzed the mRNA levels of four myogenic factors during chicken breast muscle development.

Upstream region of the myogenin gene confers transcriptional activation in muscle cell lineages during mouse embryogenesis.

Myogenin, one of the MyoD-related factors containing basic-helix-loop-helix motifs, is transcriptionally activated in skeletal muscle lineages in somites and limb buds during embryogenesis. In an attempt to understand regulatory mechanisms which govern transcriptional activation of the myogenin gene, transgenic mice bearing the lacZ gene driven by the upstream region of the myogenin gene were generated. Stereoscopic visualization of LacZ-positive cells of these transgenic mouse embryos revealed that the upstream region of the myogenin gene conferred its transcriptional activation in cells of the skeletal muscle lineages in somites, limb buds, and visceral arches.

Differential trans-activation of muscle-specific regulatory elements including the mysosin light chain box by chicken MyoD, myogenin, and MRF4.

We have isolated cDNAs encoding a chicken homologue of MRF4 (cMRF4) in addition to chicken MyoD (CMD1) and myogenin (c-myogenin) described previously. In an attempt to understand the roles that cMRF4, CMD1, and c-myogenin play in chicken myogenesis, the effects of these factors on muscle-specific cis-elements identified in regulatory regions of myosin alkali light chain (MLC) genes were examined. The promoter analysis of some of MLC genes has revealed two sorts of muscle-specific positive regulatory elements to date, an enhancer located upstream of the adult type LC1 gene and a cis-element, termed an MLC box, conserved among promoters of various MLC genes.

Localization of mRNAs coding for CMD1, myogenin and the alpha-subunit of the acetylcholine receptor during skeletal muscle development in the chicken.

Myogenin and CMD1, the chicken homologue of MyoD, transactivate the promoter of the alpha-subunit of the acetylcholine receptor (AChR) in chicken fibroblasts. The expression of these three genes was followed by in situ hybridization. In two-day-old embryos the CMD1 gene is expressed shortly before the AChR alpha-subunit and the myogenin genes.

Positive and negative gene regulation in muscle.

By the analysis of cis and trans-acting element involved in transcriptional regulation of chicken myosin alkali light chain genes, we have identified the MLC box, muscle specific enhancer element and negative regulatory element. The MLC box is an essential element for the expression of MLC genes located at approximately 100 bp upstream from mRNA start sites. The core sequence of MLC box is similar to the consensus of actin gene CArG box and SRE of c-fos oncogene.

A stable cellular marker for the analysis of mouse chimeras: the bacterial chloramphenicol acetyltransferase gene driven by the human elongation factor 1 alpha promoter.

We have developed a method of marking of mouse cells by means of transfection of a foreign gene. The transgene chosen here was the plasmid pEF321CAT which contains the bacterial chloramphenicol acetyl transferase (CAT) gene linked to the promoter region of the human polypeptide chain elongation factor 1 alpha (hEF1 alpha) gene. Evaluation of the plasmid pEF321CAT as a cellular marker for mouse cells involved intensive examination of a transgenic mouse carrying pEF321CAT.

Myogenin contains two domains conserved among myogenic factors.

Previously, three structurally related proteins, MyoD, myogenin, and Myf5, have been identified, and each of them was found to convert C3H10T1/2 fibroblasts to myoblasts when their respective cDNAs were expressed under the control of a viral promoter. Here, we describe the cloning and DNA sequencing of myogenin cDNAs from chicken and mouse. They encode polypeptides highly homologous to each other, but the polypeptide sequences we have obtained are not homologous in the carboxyl-terminal 70 amino acids with those previously reported for mouse, rat, and human because of a single base deletion in the previously reported cDNAs.

Regulation of the chicken embryonic myosin light-chain (L23) gene: existence of a common regulatory element shared by myosin alkali light-chain genes.

The transcriptional regulation of the chicken myosin alkali light-chain (MLC) L23 gene was analyzed. Two different types of cis-regulatory regions were identified: one was a silencerlike region located between 3.7 and 2.

Transcriptional regulation of 3-methylcholanthrene-inducible P-450 gene responsible for metabolic activation of aromatic carcinogenes.

We have investigated the transcriptional regulation of 3-methylcholanthrene (3MC)-inducible P-450c gene which is involved in the metabolic activation of polycyclic aromatic carcinogens. Reverse genetic study using the fusion gene composed of the 5' upstream sequence of P-450c gene and the structure gene for bacterial chloramphenicol acetyltransferase (CAT) and a cultured cell line of Hepa-1 cells localized two kinds of cis-acting regulatory DNA elements. One is designated XRE or xenobiotic responsive element which is responsible for the inducibility of the gene and is distributed 5 times in the region from -3.

Repression of cytochrome P-450c gene expression by cotransfection with adenovirus E1a DNA.

Gene expression of rat cytochrome P-450c (P-450c) depends upon inducible enhancers scattered in the 5'-upstream region of the gene. We show that expression of the P-450c gene is repressed by contransfection with adenovirus E1a DNA, regardless of the presence or absence of inducers, in a transient expression system of HeLa cells. Since cotransfection of either 13S or 12S E1a cDNA was effective in the repression, the region necessary for repression could be separated from that of transactivation of other adenovirus early genes.