Atsuko Fujisawa-Sehara

Roles of meltrin beta/ADAM19 in the processing of neuregulin

Meltrin β/ADAM19 is a member of ADAMs (a d isintegrin andmetalloproteases), 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 ofmeltrin β during mouse development coincided well with that of neuregulin-1 (NRG), a member of the epidermal growth factor family. Then we examined whether meltrin β participates in the proteolytic processing of membrane-anchored NRGs. When NRG-β1 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 β, which lead to the significant decrease in the cell surface exposure of extracellular domains of NRG-β1. Furthermore, expression of protease-deficient mutants of meltrin β exerted dominant negative effects on the basal processing of NRG-β1. These results indicate that meltrin β participates in the processing of NRG-β1. Since meltrin β affected the processing of NRG-β4 but not that of NRG-α2, meltrin β was considered to have a preference for β-type NRGs as substrate. Furthermore, the effects of the secretory pathway inhibitors suggested that meltrin β participates in the intracellular processing of NRGs rather than the cleavage on the cell surface.

Spatially- and temporally-restricted expression of meltrin α (ADAM12) and β (ADAM19) in mouse embryo

Abstract The cloning of the full-length cDNA encoding meltrin β (ADAM19), one of the metalloprotease-disintegrins expressed in mouse myogenic cells, revealed that the meltrin β gene encodes a membrane protein closely related to meltrin α (ADAM12) which participates in myotube formation in vitro. To delineate the functions of meltrin α and β, we examined the expression patterns of their transcripts during embryogenesis. The meltrin α gene is activated in condensed mesenchymal cells that give rise to skeletal muscle, bones and visceral organs. Meltrin β mRNA, in contrast, is markedly expressed in craniofacial and dorsal root ganglia and ventral horns of the spinal cord, where peripheral neuronal cell lineages differentiate. Heart, skeletal muscle, intestine and lung also express meltrin β mRNA transiently. Although the meltrin α and β transcripts exhibit distinct expression patterns during embryogenesis, both genes are mainly activated in mesenchymal cells that are derived from both mesoderm and ectoderm.

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. Its distal half, containing a pair of E boxes (CANNTG), had been previously characterized as an enhancer responsive to CMD1 but not to c-myogenin. In this study, we report the identification of another enhancer in the muscle-specific regulatory region which is preferentially responsive to c-myogenin. Deletion and mutation analyses indicated that this enhancer requires a single E box and its flanking sequences. Furthermore, analysis of chimeric proteins of CMD1 and c-myogenin indicated that regions outside the basic helix-loop-helix domain of c-myogenin are involved in the specificity of the enhancer. These results show that CMD1 and c-myogenin act on the MLC1 gene by recognizing different upstream DNA sequences and that direct or indirect interactions between the regions outside the basic helix-loop-helix domain and flanking sequences of E boxes are involved in the target sequence specificity.

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 2.7 kilobases upstream of the mRNA initiation site, and the other was essential for the expression of L23 in skeletal muscle cells and was located between 106 and 91 base pairs upstream of the cap site. This 16-base-pair cis-acting element was designated as the MLC box since it is well conserved in various muscle-specific MLC promoter regions. The activity of the MLC box showed tissue specificity. To analyze the relationship between the nucleotide sequence and the activity of the MLC box precisely, mutation analysis was performed. The 16-base-pair sequence was indispensable for the active transcription of L23 gene, and the MLC box could function in either orientation. The inverted sequence of the MLC box was similar to the sequence of the alpha-actin CArG box. By using a gel mobility retardation assay, the nuclear protein(s) that binds to both MLC box and CArG box was detected with nuclear extract prepared from chicken embryonic breast muscle. These observations imply that a common factor regulates the coordinate expression of these contractile proteins in muscle differentiation.

Upregulation of matrix metalloproteinase triggers transdifferentiation of retinal pigmented epithelial cells in Xenopus laevis: A Link between inflammatory response and regeneration: MMP Triggers Retinal 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. In the culture, Mmp expression in the RPE cells corresponded with their migration from the choroid. A potent MMP inhibitor, 1,10‐PNTL, suppressed RPE cell migration, proliferation, and formation of an epithelial structure in vitro. The mechanism involved in upregulation of Mmps was further investigated. After retinal removal, inflammatory cytokine genes, IL‐1β and TNF‐α, were upregulated both in vivo and in vitro. When the inflammation inhibitors dexamethasone or Withaferin A were applied in vitro, RPE cell migration was severely affected, suppressing transdifferentiation. These results demonstrate that Mmps play a pivotal role in retinal regeneration, and suggest that inflammatory cytokines trigger Mmp upregulation, indicating a direct link between the inflammatory reaction and retinal regeneration.