Masumi Kobayashi

Down-regulation of an ankyrin repeat-containing protein, V-1, during skeletal muscle differentiation and its re-expression in the regenerative process of muscular dystrophy

Using Western blot analysis and immunohistochemical methods, we examined the expression of V-1, a member of the ankyrin repeat-containing protein family, during differentiation and regeneration of skeletal muscle. The expression of V-1 was high in cultured myoblasts and decreased during their differentiation into myotubes, while high expression was maintained when muscle differentiation was inhibited by treatment with basic fibroblast growth factor. Down-regulation of V-1 also occurred during in vivo muscle differentiation from embryonic to postnatal stages, reaching an undetectable level in mature skeletal muscle. In contrast, strong V-1 immunoreactivity was detected again in myoblasts and regenerating muscle fibers with a small diameter, which were observed in Duchenne muscular dystrophy and its animal model, mdx mouse. Thus, it seems that V-1 is a good marker for early stage of muscle regeneration and changes of its expression suggest that V-1 plays a role in prenatal muscle differentiation and postnatal muscle regeneration.

Altered production of immunoregulatory cytokines by invariant Vα19 TCR-bearing cells dependent on the duration and intensity of TCR engagement

Cells bearing invariant Valpha19-Jalpha33 TCR alpha chains are believed to participate in the regulation of inflammatory autoimmune diseases. In this study, the potential to produce immunoregulatory cytokines by these cells was characterized in order to find the mechanism underlying their immunoregulatory functions. Serum levels of IL-4, IL-10, transforming growth factor-beta, IFN-gamma and IL-17 increased in mice over-expressing an invariant Valpha19-Jalpha33 TCR alpha transgene (Valpha19 Tg) in response to anti-CD3 antibody injection. NK1.1(+) Valpha19 Tg(+), but not NK1.1(-) Valpha19 Tg(+) cells, promptly produced immunoregulatory IL-4, IFN-gamma and IL-17 upon invariant TCR engagement with immobilized anti-CD3 antibody in culture. The activation of Valpha19 Tg(+) cells then triggered the production of pro-inflammatory cytokines by bystander cells. Interestingly, the ratio of T(h)2 cytokines such as IL-4, IL-5 and IL-10, but not pro-inflammatory IL-17, to IFN-gamma was increased when the intensity of the stimulation to invariant TCR was attenuated. Collectively, these findings suggest that invariant Valpha19 TCR(+) cells have the potential to participate in the regulation of inflammatory autoimmunity by producing T(h)2-biased cytokines in certain circumstances.

Expression of V-1, a novel catecholamine biosynthesis regulatory protein, is enhanced by hypertension in atrial myocytes of Dahl salt-sensitive rats.

V-1 positively controls catecholamine synthetic gene transcription to promote catecholamine production in PC12D cells. In this study, immunohistochemical analysis revealed that in Wistar rats, V-1 immunoreactivity was localized not only in sympathetic axons but also in the cytoplasm of cardiomyocytes, and that the immunoreactivity in atrial myocytes was more intense than that in ventricular myocytes. Western blot analysis also showed that V-1 expression level in the atrium was higher than that in the ventricle of Wistar rat hearts. When Dahl salt-sensitive (DS) rats were fed an 8% NaCl diet after the age of 6 weeks, blood pressure was raised 230mm Hg at 18 weeks. V-1 expression was shown to be increased in the atrial myocytes of these DS rats, but not in the sympathetic axons, when assayed by immunohistochemistry. These results suggest that in normotensive rats, V-1 is preferentially expressed in the cytoplasm of cardiomyocytes in the atrium rather than in the ventricle. It is also suggested that V-1 expression is increased by hypertension in DS rat atrium.

Generation of Vα14 NKT cells in vitro from hematopoietic precursors residing in bone marrow and peripheral blood

We previously reported the generation of Vα14 invariant TCR+ (Vα14i) NK1.1+ natural killer T (NKT) cells in the cytokine‐activated suspension culture of murine fetal liver cells. In this study, we attempted to apply this finding to the induction of Vα14i NKT cell differentiation in the culture of hematopoietic precursors residing in bone marrow or peripheralblood. Preferential generation of NKT cells was found in the culture of Thy‐1+‐depleted bone marrow cells in the presence of culture supernatant from Con A‐stimulated spleen T cells and a combination of recombinant IL‐3, IL‐4, IL‐7 and GM‐CSF. NKT cell development from peripheral blood hematopoietic precursors was induced when they were cultured on stromal cell monolayers prepared from Thy‐1+‐depleted bone marrow or fetal liver cells, suggesting that certain environments derived from hematopoietic organs are required for the induction of NKT cells from precursors in vitro. A significant fraction of NKT cells generated in the culture were positive for staining with CD1–α‐galactosylceramide tetramer, indicating that Vα14i NKT cells were the major subset among the NKT cells. The present methods for obtaining NKT cells in the culture of bone marrow or peripheral blood cells are applicable to the treatment of patients suffering from diseases with numerical and functional disorders of NKT cells.

Enhancement of Noradrenergic Phenotype Expression in Transgenic Mice Overexpressing V-1, A Cytoplasmic Ankyrin Repeat Protein

Catecholaminergic cells exhibit three types of transmitter phenotypes: dopaminergic, noradrenergic and adrenergic. The catecholaminergic phenotypes are thought to be determined by a phenotype-specific coordinate regulation of gene expression of the four catecholamine synthesizing enzymes, i.e. tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). However, molecular mechanisms that control the coordinate transcriptional regulation of these genes remain to be fully elucidated, although the second messenger systems and transcription factors involved in catecholaminergic cell-specific gene expression for catecholamine synthesizing enzymes have been so far identified and extensively investigated (Zetterstrom et al., 1997; Morin et al., 1997; Lo et al., 1999). Recently, an ankyrin repeat protein designated V-1 has been suggested to act as a possible coordinate regulator for the expression of genes encoding catecholamine synthesizing enzymes, since it has been demonstrated that stable overexpression of V-1 protein in the catecholaminergic cell line, PC12D cell, augments the expression of transcripts encoding TH, AADC and DBH, and as a resuit, catecholamine production is upregulated (Yamakuni et al., 1998). V-1 is a 12 kDa protein containing 2.5 copies of the ankyrin repeat, which has been originally isolated as a developmentally regulated protein from murine cerebellum (Taoka et al., 1992).