Publications (15)45.9 Total impact
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Article: Unlike natural killer (NK) p30, natural cytotoxicity receptor NKp44 binds to multimeric α2,3-NeuNAc-containing N-glycans.
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ABSTRACT: Natural cytotoxicity receptor 2 (NCR2 or natural killer (NK)p44) and NCR3 (NKp30) bind to heparin and heparin sulfate; however, other natural ligands have yet to be identified. We previously reported that NCR1 (NKp46) can bind to multimeric NeuNAc-containing N-glycans and sulfated glycans. In this study, we investigated whether NKp44 and NKp30 can bind to NeuNAc-containing glycans using their common recombinant extracellular domain tagged with 6×His (NKp44-H6 and NKp30-H6). NKp44-H6, but not NKp30-H6, bound multimeric sialyl Lewis X expressing transferrin secreted by HepG2 cells (HepTF) with a K(d) of 420 nM. Competitive and direct binding assays revealed that NKp44-H6 mainly recognizes α2,3-NeuNAc residues on non-reducing ends of N-glycans on HepTF. Moreover, site-directed mutants of NKp44-H6, such as R47Q, R55Q, R92Q, R95Q, K103Q, and R106Q, had reduced binding to α2,3-sialylated N-glycans. These results suggest that NKp44 binds to α2,3-sialylated N-glycans through ionic interactions, and that these binding sites might have some overlap with heparin binding sites.Biological & Pharmaceutical Bulletin 01/2012; 35(4):594-600. · 1.66 Impact Factor -
Article: Hepatocyte nuclear factor 1β induced by chemical stress accelerates cell proliferation and increases genomic instability in mouse liver.
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ABSTRACT: The liver has a considerable capacity of regeneration against the damage. The regulatory factors and molecular mechanism for the capacity are not fully appreciated. In developmental processes, hepatocyte nuclear factor 1β (HNF1β) is a cooperative factor for HNF6, which is a known stimulatory factor for hepatocyte proliferation after partial hepatectomy. We showed that carbon tetrachloride (CCl4)-induced liver injury up-regulated HNF1β, whereas the expression of HNF6 was not affected by the chemical stress, indicating unknown physiological roles of HNF1β against the chemical stress, not in cooperation with HNF6. To determine whether HNF1β has a novel function in the liver regeneration, we overexpressed HNF1β in the mouse liver by adenoviral gene delivery. We revealed that overexpression of HNF1β resulted in accelerated cell proliferation with the protein level up-regulation of plasminogen and plasmin, a converted active form of plasminogen, which play a pivotal role in liver regeneration inducing hepatocyte proliferation. Despite this stimulatory effect for the liver regeneration, HNF1β overexpression significantly increased genomic instability with decreased protein level of mediator of DNA damage checkpoint 1 (MDC1) and dephosphorylation of SP1 transcription factor. The increased expression of HNF1β is associated with several types of hepatocyte carcinomas, indicating possible involvement of the factor in carcinogenesis. Our data extend the current understanding of the mechanism underlying liver regeneration against chemical stress, and identified HNF1β as a novel regulatory factor in this mechanism and as a potential initiator for carcinogenesis.Journal of Receptor and Signal Transduction Research 03/2011; 31(2):132-8. · 1.59 Impact Factor -
Article: Adaptive gene regulation of pyruvate dehydrogenase kinase isoenzyme 4 in hepatotoxic chemical-induced liver injury and its stimulatory potential for DNA repair and cell proliferation.
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ABSTRACT: The processes involved in the adaptation of animals to environmental factors remain unclear. We examined the mechanisms underlying the adaptive potential of the mouse against hepatotoxic chemical-induced injury. Microarray analysis revealed that ethylbenzene, a hepatotoxic chemical, upregulated PDK4 (encoding pyruvate dehydrogenase kinase isoenzyme 4) in mouse livers and that the upregulation was enhanced by previous exposure to the chemical. Although PDK4 is an energy resource regulator induced by starvation, expression of other fasting-inducible genes was unaffected. PDK4 induced by chemical stress developed hepatic accumulation of sirtuin 1 by regulating pyruvate concentration and activated the Nbn and ATM, which are critical for DNA repair and checkpoint activation. PDK4 overexpression on carbon tetrachloride (CCl(4))-induced liver injury resulted in delayed necrotic tissue recovery with cell cycle arrest and decreased γH2AX foci and micronucleus formation. PDK4 silencing on CCl(4)-induced liver injury accelerated necrotic tissue recovery and increased γH2AX foci and micronucleus formation, indicating the essential role of PDK4 in DNA repair and checkpoint activation. PDK4 overexpression induced pancreas-specific transcription factor 1a (Ptf1a) upregulation and transcriptional activation of several pancreatic genes in the liver. Ptf1a overexpression by adenoviral gene delivery resulted in accelerated tissue recovery on CCl(4)-induced liver injury. Our data identified PDK4 as a novel pivotal factor in adaptation to chemical stress.Journal of Receptor and Signal Transduction Research 02/2011; 31(1):85-95. · 1.59 Impact Factor -
Article: Binding of natural cytotoxicity receptor NKp46 to sulfate- and α2,3-NeuAc-containing glycans and its mutagenesis.
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ABSTRACT: Natural cytotoxicity receptor 1 (NCR1, NKp46) binds to heparin and heparan sulfate; however, other natural ligands for NKp46 have yet to be elucidated. Using the recombinant extracellular region (coding for AA 22-258) of NKp46 tagged with 6× His (NKp46-H6), and mutants K136Q, R139Q, H142Q, R145Q, and K149Q, we determined their binding affinities to sulfate- and NeuAc-containing glycans-coated plates. NKp46-H6 directly bound to plates coated with heparin- and heparan sulfate-conjugated bovine serum albumin with K(d) values of 770 and 850 nM, respectively. The binding of NKp46-H6 to heparin-BSA was suppressed by soluble heparin, herparan sulfate, fucoidan, λ-carrageenan, and dextran sulfate, but not by 2-O-, 6-O-, and N-desulfated heparin. NKp46-H6 also bound to multimeric sialyl Lewis X expressing transferrin secreted by human hepatoma HepG2 cells (HepTF) with a K(d) value of 530 nM, but not to desialylated HepTF, commercially available TF, or 1-acid glycoprotein. Moreover, mutants R139Q, R145Q, and K149Q had significantly reduced binding to these sulfate-containing glycans, and K136Q and K149Q to HepTF, indicating that NKp46 binds to sulfate- and 2,3-NeuAc-containing glycans mainly via ionic interactions. However, the binding sites of NKp46 were different.Biochemical and Biophysical Research Communications 02/2011; 406(3):377-82. · 2.48 Impact Factor -
Article: A single nucleotide mutation in the mouse renin promoter disrupts blood pressure regulation.
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ABSTRACT: Renin, a major regulatory component of the renin-angiotensin system, plays a pivotal role in regulating blood pressure and electrolyte homeostasis and is predominantly expressed in the kidney. Several cAMP-responsive elements have been identified within renin gene promoters. Here, we study how 2 such elements, renin proximal promoter element-2 (RP-2) and overlapping cAMP and negative regulatory elements (CNRE), affect the transcriptional regulation of renin. We generated Tg mice (TgM) bearing BACs containing either WT or mutant RP-2 or CNRE, integrated at single chromosomal loci. Analysis of the TgM revealed that RP-2 was essential to basal promoter activity in the kidney, while renin mRNA levels did not significantly change in any tissues tested in the CNRE mutant TgM. To evaluate the physiological significance of these mutations, we used the BAC Tg to rescue hypotensive Renin-null mutant mice. As predicted, no renin expression was observed in the kidneys of RP-2 mutant/Renin-null compound mice, whereas renin expression in CNRE mutant compound mice was indistinguishable from that in control mice. Consistent with this, RP-2 mutant animals were hypotensive, while CNRE mutants had normal blood pressure. Thus, transcriptional regulation of renin expression via RP-2 but not CNRE is critical for blood pressure regulation by this gene.Journal of Clinical Investigation 04/2008; 118(3):1006-16. · 15.39 Impact Factor -
Article: Structural organization of the mouse neurochondrin gene.
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ABSTRACT: Neurochondrin is a brain and bone specific leucine-rich protein. We previously cloned the two types of mRNAs (neurochondrin-1; 729 amino acids and neurochondrin-2; 712 amino acids) from mouse and human species. As a first step, to better understand the mechanism of the bone and brain specific and developmentally regulated expression of the neruochondrin gene, the genomic organization of murine neurochondrin was determined. It consists of 7 exons and spans about 10 kb; all splice junctions conform to the GT/AG rule. It codes for two alternatively spliced messenger RNAs, neurochondrin-1 containing all 7 exons and neurochondrin-2 lacking exon 1b but containing the other exons. Cap site analysis showed that the major transcription initiation occurs at 765 bp upstream of the ATG start codon of neurochondrin-1. The promoter region has no TATA and CAAT box-like sequence but contains potential AP-1 and SP-1 binding sites. The neurochondrin gene is localized to mouse chromosome 4D1 and rat chromosome 5q36.11.International Journal of Molecular Medicine 10/2004; 14(3):361-6. · 1.98 Impact Factor -
Article: Finb, a multiple zinc finger protein, represses transcription of the human angiotensinogen gene.
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ABSTRACT: We previously identified a regulatory element at the 3'-downstream region of the human angiotensinogen (hANG) gene. Using this element as a probe by the Southwestern screening, we isolated a cDNA clone, encoding Finb, a transcriptional activator with multiple zinc finger domains. The N-terminal zinc finger domain of Finb bound to the GGATGG sequence within the regulatory element. Unexpectedly, Finb repressed transcription dependent on the regulatory element. Inspection of the 5'-flanking region in the hANG promoter identified the GGATGG-like elements, which prompted us to examine the effect of Finb on the hANG promoter activity. We also found the two Finb binding elements in the 5'-flanking region of the hANG gene by the gel shift assay, both of which were necessary for transcriptional repression of the hANG promoter. These findings suggest that Finb functions as a sequence-specific transcriptional repressor of the hANG gene.International Journal of Molecular Medicine 06/2004; 13(5):637-42. · 1.98 Impact Factor -
Article: Identification of the mouse neurochondrin promoter region and the responsible region for cell type specific gene regulation.
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ABSTRACT: Neurochondrin is a cytoplasmic protein possibly involved in neurite outgrowth and chondrocyte differentiation. In the present study, we have identified 202 bp of the mouse neurochondrin minimal promoter sequences encompassing the transcriptional initiation site, and both of the activating and repressing regions in the first exon. These two regulatory regions in the first exon had a cell type dependent effect on the identified minimal promoter. In the regulatory region, the duplication of potential binding sites for GATA family transcriptional factors was observed. Prospective binding sites for sex determining region Y and c-Ets1 were also found in the minimal promoter region. These factors could be potential regulators for the mouse neurochondrin gene.Neuroscience Letters 03/2004; 356(2):107-10. · 2.11 Impact Factor -
Article: Targeted disruption of the neurochondrin/norbin gene results in embryonic lethality.
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ABSTRACT: Neurochondrin/norbin is a cytoplasmic protein involved in dendrite outgrowth. The expression of the gene has been restricted to neural, bone, and chondral tissues. To identify the functions of the gene in vivo, we have generated mice with a disrupted mutation in the neurochondrin/norbin gene. Histological analysis of heterozygous mutant mice indicates the possibility of specific functions of neurochondrin/norbin in chondrocyte differentiation. We defined the expression patterns of neurochondrin/norbin-lacZ fusion protein in the central nervous system. In the developing olfactory bulb, beta-galactosidase activity was detected in the mantle layer at 12.5 dpc and the strongest activity was detected in the presumptive mitral or tufted cell layer at 15.5 dpc. beta-Galactosidase activity was also detected in the lateral choroid plexus. In homozygous (-/-) mutant mice, the disruption of the neurochondrin/norbin gene leads to early embryonic death between 3.5 and 6.5 dpc. This result indicates that neurochondrin/norbin gene function is essential for the early embryogenesis.Biochemical and Biophysical Research Communications 11/2003; 310(4):1219-26. · 2.48 Impact Factor -
Article: Cloning and characterization of a novel splicing isoform of USF1.
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ABSTRACT: The ubiquitous basic helix-loop-helix transcription factor USFs encoded by two distinct genes (USF1 and USF2) recognize a core motif, CACGTG, termed E box and regulate the expression of a variety of genes. USF1 and USF2 proteins form homo- and heterodimers to bind the target core motif DNA. Here, we report the molecular cloning and functional characterization of a novel alternative splicing variant of human USF1 (hUSF1), termed USF1/BD. Compared with USF1 wild-type (wt), USF1/BD lacks the N-terminal transactivation domain. Cloning and characterization of the hUSF1 genomic region revealed that USF1/BD is generated by excising the sequence corresponding to a part of exon 4. In transiently transfected cells, USF1/BD was localized in the nucleus and repressed the promoter activity of the human angiotensinogen gene. In vitro translated USF1/BD possessed DNA binding activity as a homodimer and a heterodimer with USF1 (wt). These results suggest that USF1/BD plays a role as a modulator of USF1 to control the expression of target genes.International Journal of Molecular Medicine 09/2003; 12(2):161-7. · 1.98 Impact Factor -
Article: Renin-dependent Cardiovascular Functions and Renin-independent Blood-Brain Barrier Functions Revealed by Renin-deficient Mice
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ABSTRACT: Renin plays a key role in controlling blood pressure through its specific cleavage of angiotensinogen to generate angiotensin I (AI). Although possible existence of the other angiotensin forming enzymes has been discussed to date, its in vivo function remains to be elucidated. To address the contribution of renin, we generated renin knockout mice. Homozygous mutant mice show neither detectable levels of plasma renin activity nor plasma AI, lowered blood pressure 20–30 mm Hg less than normal, increased urine and drinking volume, and altered renal morphology as those observed in angiotensinogen-deficient mice. We recently found the decreased density in granular layer cells of hippocampus and the impaired blood-brain barrier function in angiotensinogen-deficient mice. Surprisingly, however, such brain phenotypes were not observed in renin-deficient mice. Our results demonstrate an indispensable role for renin in the circulating angiotensin generation and in the maintenance of blood pressure, but suggest a dispensable role for renin in the blood-brain barrier function.Journal of Biological Chemistry 01/2000; 275(1):5-8. · 4.77 Impact Factor -
Article: Regulated Expression of Human Angiotensinogen Gene by Hepatocyte Nuclear Factor 4 and Chicken Ovalbumin Upstream Promoter-Transcription Factor
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ABSTRACT: We previously identified various upstream and downstream regulatory elements and factors important for hepatic expression of the human angiotensinogen (ANG) gene, the precursor of vasoactive octapeptide angiotensin II. In the present study, to further investigate the molecular mechanism of human ANG transcriptional regulation, we generated transgenic mice carrying the fusion gene composed of the 1.3-kilobase promoter of the human ANG gene, its downstream enhancer, and the chloramphenicol acetyltransferase reporter gene. Because expression of the chloramphenicol acetyltransferase gene was observed strongly in the liver and weakly in the kidney, we suspected that hepatocyte nuclear factor (HNF) 4 with a tissue expression pattern similar to that of the reporter gene would regulate ANG transcription. In vitro assays indicated that HNF4 bound to the promoter elements and strongly activated the ANG transcription, but that chicken ovalbumin upstream promoter transcription factor (COUP-TF), a transcriptional repressor, dramatically repressed human ANG transcription through the promoter elements and the downstream enhancer core elements. Furthermore, COUP-TF dramatically decreased the human ANG transcription in the mouse liver by the Helios Gene Gun system in vivo. These results suggest that an interplay between HNF4 and COUP-TF could be important in hepatic human ANG transcription.Journal of Biological Chemistry 12/1999; 274(49):34605-34612. · 4.77 Impact Factor -
Article: Identification of two distinct Sp1- and RBF-1-like nuclear factors that bind to the upstream region of the human angiotensinogen promoter
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ABSTRACT: Angiotensionogen, the protein precursor of angiotensin II that is a crucial regulator of blood pressure and electrolyte balance, is constitutively produced by the liver. In the present study, we identified two nuclear factors that are possibly involved in maintaining the constitutive promoter activity of the human angiotensinogen gene. The 32 bp DNA region between −344 and −313 located in the 1.3 kb angiotensinogen upstream region (−1222 to +44) partially contributed to the maintenance of the efficient promoter activity in HepG2 cells. This segment was able to form the complexes with HepG2 nuclear extracts, which could be dissociated by competing recognition sequences that contain those of either Sp1 or RBF-1. Anin vivo competition experiment demonstrated that the parental promoter activity is reduced about 65% by an RBF-1 competitor more effectively than by an Sp1 competitor. These results suggested that Sp1- and RBF-1-like factors play roles in maintaining the constitutively active angiotensinogen promoter.Endocrine 04/1995; 3(7):543-547. · 1.42 Impact Factor -
Article: Molecular cloning and expression of human neurochondrin-1 and -2
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ABSTRACT: Human neurochondrins have been cloned from a brain cDNA library. The human neurochondrin-1 and -2 predict leucine-rich (15.8 and 15.9%) proteins of 729 and 712 amino acid residues, with molecular weights of 78.9 and 77.2 kDa, respectively. The deduced amino acid sequence indicates 98% identity among human, mouse and rat species. Northern analysis indicates that about 4 kb human neurochondrin mRNAs are abundant in the fetal and the adult brain.Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression 1446(3):397-402. · 1.70 Impact Factor -
Article: ATF-like Element Contributes to Hepatic Activation of Human Angiotensinogen Promoter
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ABSTRACT: Angiotensinogen is the precursor protein of angiotensin II that is involved in regulating blood pressure and electrolyte homeostasis, and it is mainly synthesized in the liver. In the present study, we analyzed the human angiotensinogen proximal promoter region by means of Chloramphenicol acetyltransferase assays, and suggested that the region from −106 to +44 is sufficient for hepatoma cell line (HepG2)-specific expression. Electrophoretic mobility shift assays using ALE (TF-ikelement, −102 to −87) fragment identified CREB/ATF family nuclear factors and novel ones, ALF (E-bindingactor). The deletion andin vivocompetition of ALE decreased the human angiotensinogen promoter activity. Furthermore, the heterologous promoter analysis demonstrated that ALE acts as a HepG2-dependent activating element. These results indicate that ALE plays an important role in hepatic expression of human angiotensinogen gene.Biochemical and Biophysical Research Communications.
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Institutions
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1995–2008
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University of Tsukuba
- Institute of Applied Biochemistry
Tsukuba, Ibaraki-ken, Japan
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