津田 英利

Menkes disease is an X-linked disorder of copper metabolism caused by mutations in the ATP7A gene, and female carriers are usually asymptomatic. We describe a 7-month-old female patient with severe intellectual disability, epilepsy, and low levels of serum copper and ceruloplasmin. While heterozygous deletion of exons 16 and 17 of the ATP7A gene was detected in the proband, her mother, and her grandmother, only the proband suffered from Menkes disease clinically. Intriguingly, X chromosome inactivation (XCI) analysis demonstrated that the grandmother and the mother showed skewing of XCI toward the allele with the ATP7A deletion and that the proband had extremely skewed XCI toward the normal allele, resulting in exclusive expression of the pathogenic ATP7A mRNA transcripts. Expression bias analysis and recombination mapping of the X chromosome by the combination of whole genome and RNA sequencing demonstrated that meiotic recombination occurred at Xp21-p22 and Xq26-q28. Assuming that a genetic factor on the X chromosome enhanced or suppressed XCI of its allele, the factor must be on either of the two distal regions derived from her grandfather. Although we were unable to fully uncover the molecular mechanism, we concluded that unfavorable switching of skewed XCI caused Menkes disease in the proband.

Our previous genome-wide association study to explore genetic loci associated with lean nonalcoholic fatty liver disease (NAFLD) in Japan suggested four candidate loci, which were mapped to chr6, chr7, chr12 and chr13. The present study aimed to identify the locus involved functionally in NAFLD around the association signal observed in chr13. Chromosome conformation capture assay and a database survey suggested the intermolecular interaction among DNA fragments in association signals with the adjacent four coding gene promoters. The four genes were further screened by knockdown (KD) in mice using shRNA delivered by an adeno-associated virus vector (AAV8), and KD of G protein-coupled receptor 180 (Gpr180) showed amelioration of hepatic lipid storage. Gpr180 knockout (KO) mice also showed ameliorated hepatic and plasma lipid levels without influencing glucose metabolism after high-fat diet intake. Transcriptome analyses showed downregulation of mTORC1 signaling and cholesterol homeostasis, which was confirmed by weakened phosphorylation of mTOR and decreased activated SREBP1 in Gpr180KO mice and a human hepatoma cell line (Huh7). AAV8-mediated hepatic rescue of GPR180 expression in KO mice showed recovery of plasma and hepatic lipid levels. In conclusion, ablation of GPR180 ameliorated plasma and hepatic lipid levels, which was mediated by downregulation of mTORC1 signaling.

We previously revealed that Kbtbd11 mRNA levels increase during 3T3-L1 differentiation and Kbtbd11 knockdown suppresses whereas its overexpression promotes adipogenesis. However, how Kbtbd11 mRNA is regulated during adipocyte differentiation and how the KBTBD11 protein functions in adipocytes remain elusive. This study aimed to examine the transcriptional regulatory mechanism of Kbtbd11 during adipocyte differentiation, KBTBD11-interacting protein functions, and elucidate the role of KBTBD11 in adipocytes. First, we identified the PPRE consensus sequences in the Kbtbd11 exon 1- and intron 1-containing region and demonstrated that PPARγ acts on this region to regulate Kbtbd11 expression. Next, we purified the KBTBD11 protein complex from 3T3-L1 adipocytes and identified heat shock proteins HSC70 and HSP60 as novel KBTBD11-interacting proteins. HSC70 and HSP60 inhibition increased KBTBD11 protein levels that promoted NFATc1 ubiquitination. These data suggest that HSC70 and HSP60 are involved in KBTBD11 stabilization and are responsible for NFATc1 regulation on the protein level. In summary, this study describes first the protein regulatory mechanism of NFATc1 through the HSC70/HSP60-KBTBD11 interaction that could provide a potential new target for the differentiation and proliferation of various cells, including adipocytes and tumors.

BACKGROUND: Actin, alpha, skeletal muscle 1 (ACTA1) is one of the causative genes of nemaline myopathy (NM) and congenital fiber-type disproportion (CFTD). CFTD is characterized by type 1 fiber atrophy and distinguished from NM in the absence of rods. Eight patients with CFTD, including one patient with dilated cardiomyopathy (DCM), have previously been reported. Herein, we report the case of a 10-year-old boy presenting with CFTD and DCM. METHODS: We performed exome sequencing and analyzed the effect of Met327Lys mutations on cultured C2C12 muscle cells compared with that seen in the wild type (WT, ACTA1) and previously identified Asp294Val mutations associated with a severe phenotype of CFTD without cardiomyopathy. RESULTS: Exome sequencing revealed a de novo mutation, c.980 T > A, p.(Met327Lys), in ACTA1 (NM_001100.4). C2C12 cells transfected with the WT plasmid expressed ACTA1 in the nucleus and cytoplasm. Cells with the Asp294Val mutant showed needle-like structures in the cytoplasm, whereas the expression of the Met327Lys mutant resulted in few aggregations but many apoptotic cells. CONCLUSION: Apoptosis induced in Met327Lys-transfected muscle cells supports the pathogenicity of the mutation and can be implicated as one of the histopathological features associated with CFTD, as in NM.

BACKGROUND: IL-33 is a dual-functional molecule; it acts as a cytokine to enhance type 2 inflammation, and as a nuclear factor. The roles of nuclear IL-33 are not yet fully understood. OBJECTIVE: We aimed to investigate the role of IL-33 in normal human epidermal keratinocytes (NHEKs). METHODS: We utilized RNA interference to knock down cellular IL-33. RESULTS: The IL-33-knockdown (KD) cells showed decreased BrdU incorporation and decreasing tendency in RhoA activity and decreased ECT2 oncogene expression, compared to the controls. Supplementation of IL-33 expression utilizing adenovirus vector recovered the BrdU incorporation in IL-33-KD cells. Increased number of G2/M phase cells and binucleated cells were observed among the KD cells. Overtime observation revealed that IL-33-KD cells could not divide properly, formed binucleated cells, and were less motile than control cells. CONCLUSION: IL-33 KD in NHEKs affected the division and motility, probably by slightly decreasing the RhoA activity by attenuating ECT2 expression.