Biomedical subjects
Tokuo Mukai
Publications and source records attributed to Tokuo Mukai.
Inappropriate tall stature and renal ectopy in a male patient with X-linked congenital adrenal hypoplasia due to a novel missense mutation in the DAX-1 gene.
Mutations in DAX-1 gene cause congenital adrenal hypoplasia (AHC). We present a male patient affected by X-linked adrenal hypoplasia congenita due to a novel DAX-1 missense mutation. The mutation V287G affects the C-terminal end of the DAX-1 protein which plays an important role in functioning of the receptor. In addition, our patient presented an inappropriate tall stature and renal ectopy, which have not been described in AHC so far.
A novel isoform of Vinexin, Vinexin gamma, regulates Sox9 gene expression through activation of MAPK cascade in mouse fetal gonad.
Recent loss-of-function and gain-of-function studies have revealed that transcription factor Sox9 is required for testis formation by governing Sertoli cell differentiation, and thereafter regulating transcription of Sertoli marker genes. In the present study, we identified a novel isoform of Vinexin, which is expressed in somatic cells but not germ cells of sexually indifferent stages of fetal gonads. After the sex is determined, the expression continues in testicular Sertoli cells. Immunohistochemical analyses with a specific antibody to Vinexin indicated that Vinexin gamma is localized in the cytoplasm. Functional studies with C3H10T1/2 cells showed that Vinexin gamma acted as a scaffold protein to activate MEK and ERK through interaction with c-Raf and ERK. Ultimately, Sox9 transcription was induced by Vinexin gamma. This up-regulation of Sox9 expression disappeared when the cells were treated with a specific MEK inhibitor, U0126. To determine the role of Vinexin gamma during gonad formation, the gene was disrupted by targeted mutagenesis. The phenotype displayed by the mice indicated that ERK activation was decreased in the Vinexin gamma(-/-) XY gonads, and Sox9 expression was down-regulated. Thus, Vinexin gamma seems to be implicated in regulation of Sox9 gene expression by modulating MAPK cascade in mouse fetal gonads.
Small ubiquitin-like modifier 1 (SUMO-1) modification of the synergy control motif of Ad4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1) regulates synergistic transcription between Ad4BP/SF-1 and Sox9.
An orphan nuclear receptor, Ad4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1), is essential for the development and function of steroidogenic tissues. To examine the transcriptional regulation of Ad4BP/SF-1, two-hybrid screening was performed, and the sumoylation [conjugation of a small ubiqutin-like modifier (SUMO-1)] components Ubc9, protein inhibitor of activated STAT 1 (PIAS1), and protein inhibitor of activated STAT 3 (PIAS3) were isolated. Cultured cell and in vitro studies revealed that Ad4BP/SF-1 is sumoylated at K119 and K194. Because K194 lies within the synergy control (SC) motif defined to repress synergistic transcription from promoters containing multiple binding sites, correlation between the functions of the SC motif and sumoylation was investigated. The K194R mutant of Ad4BP/SF-1, which cannot be sumoylated, showed enhanced synergistic transcription from a promoter containing multiple Ad4/SF-1 sites, suggesting that sumoylation is necessary for repression of transcriptional synergy through the SC motif. It has been established that the Müllerian inhibiting substance gene is transcribed predominantly under the control of Ad4BP/SF-1 and, moreover, its transcription is regulated synergistically with Sox9, Gata4, and Wt1. Interestingly, it was found that all of these factors are sumoylated, and these sumoylation sites occur within SC motifs. Based on the observation that SC motif mutants of Ad4BP/SF-1 and Sox9 resulted in the enhancement of their synergistic transcription, it was concluded that the SC motif regulates synergistic transcription even between distinct types of transcription factors. Considering that both mutants cannot be sumoylated, it is likely that sumoylation is implicated in this regulation. Because it was revealed with an in vitro sumoylated Ad4BP/SF-1 that DNA binding activity and interaction with Sox9 were unaffected, sumoylation may regulate transcription through affecting selective and cooperative interaction among factors constituting transcriptional complexes.
[Congenital adrenal hyperplasia due to 21-hydroxylase deficiency].
21-hydroxylase deficiency occurring in one of 15,000 live births represents the most frequent disorder in female pseudohermaphroditism. Molecular genetic analysis is useful for ascertaining disease condition. Newborn mass-screening has been conducted in Japan. Prenatal diagnosis and treatment is feasible.
Dax-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1) gene transcription is regulated by wnt4 in the female developing gonad.
Dax-1 [dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (NR0B1)] is an orphan nuclear receptor acting as a suppressor of Ad4 binding protein/steroidogenic factor 1 [Ad4BP/SF-1 (NR5A1)] and as an anti-Sry factor in the process of gonadal sex differentiation. The roles of these nuclear receptors in the differentiation of the gonads and the adrenal cortex have been established through studies of the mutant phenotype in both mice and humans. However, the mechanisms underlying transcriptional regulation of these genes remain largely unknown. Here, we examined the relationship between Dax-1 gene transcription and the Wnt4 pathway. Reporter gene analysis revealed that Dax-1 gene transcription was activated by beta-catenin, a key signal-transducing protein in the Wnt pathway, acting in synergy with Ad4BP/SF-1. Interaction between beta-catenin and Ad4BP/SF-1 was observed using yeast two-hybrid and in vitro pull-down assays. The region of Ad4BP/SF-1 essential for this interaction consists of an acidic amino acid cluster, which resides in the first helix of the ligand-binding domain. Mutation of the amino acid cluster impaired transcriptional activation of Dax-1 as well as interaction of Ad4BP/SF-1 with beta-catenin. These results were supported by in vivo observations using Wnt4 gene-disrupted mice, in which Dax-1 gene expression was decreased significantly in sexually differentiating female gonads. We thus conclude that Wnt4 signaling mediates the increased expression of Dax-1 as the ovary becomes sexually differentiated.
Molecular pathogenesis of lipoid adrenal hyperplasia and adrenal hypoplasia congenita.
Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH in which the synthesis of all gonadal and adrenal cortical steroids is markedly impaired. Lipoid CAH may be caused by the defect in either the steroidogenic acute regulatory (StAR) protein or the P450scc. More than 34 different mutations in StAR gene have been identified. Clinically, most of the patients manifest adrenal insufficiency from 1 day to 2 months of age, but some patient show delayed onset of adrenal insufficiency. Affected 46, XY subjects do not show pubertal development, whereas affected 46, XX subjects undergo spontaneous feminization, breast development and cyclical vaginal bleeding at the usual age of puberty. X-linked adrenal hypoplasia congenital (AHC) is a rare congenital adrenal disorder characterized by severe adrenal insufficiency and hypogonadotropic hypogonadism. More than 80 different several intragenic mutations of DAX-1 have been identified. The failure of pubertal development may be caused by either abnormal hypothalamic or pituitary regulation of gonadotropin secretion. In addition, although the testicular steroidogenesis is largely intact, the functional maturity of Sertoli cells and also spermatogenesis are impaired. The type of mutation does not predict clinical phenotype. Thus, unified mechanism how DAX-1 gene defect gives rise to adrenal insufficiency, hypothalamic/pituitary hypogonadism and impaired spermatogenesis remains established.
Sexually dimorphic expression of Dax-1 in the adrenal cortex.
BACKGROUND: The DAX-1 (also known as AHC) gene encodes an unusual member of the nuclear receptor superfamily, and the gene product plays a pivotal role during gonadal and adrenal differentiation. Mutations of the human DAX-1 gene cause X-linked adrenal hypoplasia congenita associated with hypogonadotropic hypogonadism. However, the molecular mechanisms underlying the transcriptional regulation of the gene are not well understood. RESULTS: Sexually dimorphic expression of Dax-1 (NR0B1) in the adrenal cortex was observed by RT-PCR, Western blotting and immunohistochemistry. The differential expression was abolished by gonadectomy and was restored again by sex steroid replacement. Our results suggested that the Dax-1 gene transcription is suppressed by androgens and androgen receptor (AR/NR3C4). Dax-1 gene transcription is regulated by Ad4BP/SF-1 (NR5A1), therefore we investigated the functional correlation between A4BP/SF-1 and AR. Interestingly, AR down-regulated the Dax-1 gene transcription mediated by Ad4BP/SF-1 in the presence of the ligand. DNA binding by AR was not essential for the suppressive action, however the suppression seemed to be dependent on the promoter contexts. An interaction between Ad4BP/SF-1 and AR was detected in the presence of the ligand for AR. CONCLUSION: The present study revealed that the expression of Dax-1 in the adrenal cortex is regulated by androgen and the receptor. Interestingly, AR acts as a suppressor in the presence of the ligand through interaction with Ad4BP/SF-1.