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Biomedical subjects

K Sogawa

Publications and source records attributed to K Sogawa.

At least 55 records · Page 3Linked to original sources
PubMed1996

Cooperative interaction between AhR.Arnt and Sp1 for the drug-inducible expression of CYP1A1 gene.

Expression of CYP1A1 gene is regulated in a substrate-inducible manner through at least two kinds of regulatory DNA elements in addition to the TATA sequence, XRE (xenobiotic responsive element), and BTE (basic transcription element), a GC box sequence. The trans-acting factor on the XRE is a heterodimer consisting of arylhydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt), while Sp1 acts as a regulatory factor on the BTE. We have investigated how these factors interact with one another to induce expression of the CYP1A1 gene. Both in vivo transfection assays using Drosophila Schneider line 2 (SL2) cells, which is devoid of endogenous Sp1, AhR, and Arnt, and in vitro transcription assays using baculovirus-expressed AhR, Arnt, and Sp1 proteins revealed that these factors enhanced synergistically expression of the reporter genes driven by a model CYP1A1 promoter, consisting of four repeated XRE sequences and a BTE sequence, in agreement with previous observation (Yanagida, A., Sogawa, K., Yasumoto, K., and Fujii-Kuriyama, Y. (1990) Mol. Cell. Biol. 10, 1470-1475). We have proved by coimmunoprecipitation assays and DNase I footprinting that both AhR and Arnt interact with the zinc finger domain of Sp1 via their basic HLH/PAS domains. When either the AhR.Arnt heterodimer of Sp1 was bound to its cognate DNA element, DNA binding of the second factor was facilitated. Survey of DNA sequences in the promoter region shows that the XRE and GC box elements are commonly found in the genes whose expressions are induced by polycyclic aromatic hydrocarbons, suggesting that the two regulatory DNA elements and their cognate trans-acting factors constitute a common mechanism for induction of a group of drug-metabolizing enzymes.

Animals↗
PubMed1996

Distinct effects of phenobarbital and its N-methylated derivative on liver cytochrome P450 induction.

The relationship between barbiturate structures and their effects on induction of rat cytochrome P450 forms was studied in primary cultured hepatocytes. Treatment of hepatocytes cultured on matrigel with 1 mM barbital, N-methylbarbital, cyclobarbital, hexobarbital, phenobarbital (PB), or mephobarbital (N-methyl-PB) resulted in increased amounts of CYP2B1/2 and CYP2C6 forms. Microsomal CYP3A content was also enhanced by treatment with these barbiturates, except for barbital. Although no relationship was observed between the levels of CYP2B1/2 and CYP3A, ratios of CYP3A/CYP2B1 plus CYP2B2 contents were invariably higher with hepatocytes treated with N-methylated barbiturates than with the nonmethylated analogs. Consistent results were also observed in vivo in rats treated with PB and N-methyl-PB. These results indicate the difference in the structure requirement for induction of CYP2B and CYP3A. In addition, N-methyl-PB was found to suppress PB-mediated induction of CYP2B1. Hepatic levels of CYP2B1 mRNA and protein were increased by treatment with PB or N-methyl-PB alone, but decreased by cotreatment with 1 mM PB and N-methyl-PB. The suppression has been shown to occur at the transcriptional level of the CYP2B1 gene by using a chloramphenicol acetyltransferase reporter-CYP2B1 fused gene system.

Animals↗
PubMed1996

cDNA cloning of a murine homologue of Drosophila single-minded, its mRNA expression in mouse development, and chromosome localization.

A combination of the RT-PCR method and subsequent screening of the cDNA library of mouse skeletal muscle with the cDNA isolated by RT-PCR used as a probe led to isolation of cDNAs encoding a polypeptide (mSim) with bHLH and PAS domains which show high similarity to the corresponding regions of Drosophila Sim, a master regulator in neurogenesis. Experiments using a GST-fusion protein demonstrated that mSim heterodimerizes with Arnt (Ah receptor nuclear translocator), even more efficiently than AhR (Ah receptor) does with Arnt. RNA blot analysis using RNAs from various tissues of mice indicated that mSim transcript is expressed in several limited tissues such as muscle, kidney and lung of adult animals. Distribution of mSim mRNA was always accompanied with that of Arnt. All the results suggest a regulatory role of mSim in partnership with Arnt. Chromosomal location of the mSim gene was determined by fluorescent in situ hybridization to be localized on the C3.3-C4 band of mouse chromosome 16 which is syntenic with the human chromosome 21q22 carrying the Down syndrome critical region, where a gene highly homologous to the Drosophila sim was localized. Whole mount in situ hybridization using a unique part of mSim cDNA sequence showed that mSim mRNA was expressed in the ventral diencephalon, branchial arches and limbs. These findings will provide an approach to the cause of the Down syndrome as well as the elucidation of the functional roles of mSim in animal development.

Age Factors↗
PubMed1996

Enhanced expression of the protein kinase substrate annexin in human hepatocellular carcinoma.

Annexin (AX) constitutes a new family of Ca2+-dependent membrane-binding proteins; 13 of them have been described. Among these, annexin-1 (AX-I) has displayed many biological functions in vitro. Its actual role in vivo, however, remains unknown. We already reported that AX-I was expressed in proliferating (regenerating) hepatocytes at both protein and messenger RNA (mRNA) levels. The role of AX-I in human hepatocellular carcinoma (HCC) remains obscure. In this study, the amounts of AX-I at protein and mRNA levels, as well as its localization, have been determined in the normal human liver, chronic hepatitis liver, and nontumorous and tumorous portions of HCC. AX-I was rarely found in normal and chronic liver tissues, whereas it is overexpressed at both the transcriptional and translational levels in tumorous and nontumorous regions of HCC. In addition, more AX-I was expressed in the tumorous portion than the nontumorous portion of HCC. AX-I was present in the hepatocytes and HCC cells, localized mainly in the cytoplasm. AX-I was expressed in poorly differentiated cancer cells. Furthermore, AX-I was tyrosine-phosphorylated in HCC. We also found that some of the AX-I- positive hepatocytes in the nontumorous tissues were derived from a particular subset of parenchymal cells (stem or oval cells). These results indicate that AX-I plays an important role in the malignant transformation process leading to HCC and that it is closely related to the histological grade of HCC. HCC would offer a novel tool with which to study the function of AX-I in malignant transformation.

Annexin A1↗
PubMed1996

Purification and characterization of the DNA-binding domain of BTEB, a GC box-binding transcription factor, expressed in Escherichia coli.

BTEB is a GC-binding protein that regulates the transcription of genes with a single GC-box or tandemly repeated GC-boxes in the promoter. The DNA-binding domain of BTEB consists of three contiguous Cys2-His2 zinc finger motifs and short segments adjacent to their N- and C-terminal sides [Kobayashi et al. (1995) J. Biochem. 117, 91-95]. The truncated BTEB (residues 120 to 244) containing the DNA-binding domain was expressed in Escherichia coli and purified to homogeneity under denaturing conditions. DNA-binding activity of the BTEB was regenerated by refolding in the presence of Zn2+. The efficiency in regeneration was 70 +/- 10%, and the dissociation constant (Kd) of the DNA-complex was 4 +/- 2 nM. Co2+ also regenerated the DNA-binding affinity of BTEB, albeit with less efficiency than Zn2+. Co-BTEB showed a slightly lower affinity to the specific DNA than Zn-BTEB. Refolding in the presence of Cd2+ resulted in an extremely low efficiency in regeneration of the DNA-binding activity. Zn-BTEB is in a monomer state at concentrations lower than 0.5 microM, and forms a dimer in the concentration range of about 10 to 200 microM.

Amino Acid Sequence↗
PubMed1996

cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt).

We isolated mouse cDNA clones (Arnt2) that are highly similar to but distinct from the aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt). The composite cDNA covered a 2,443-bp sequence consisting of a putative 2,136-bp open reading frame encoding a polypeptide of 712 amino acids. The predicted Arnt2 polypeptide carries a characteristic basic helix-loop-helix (bHLH)/PAS motif in its N-terminal region with close similarity (81% identity) to that of mouse Arnt and has an overall sequence identity of 57% with Arnt. Biochemical properties and interaction of Arnt2 with other bHLH/PAS proteins were investigated by coimmunoprecipitation assays, gel mobility shift assays, and the yeast two-hybrid system. Arnt2 interacted with AhR and mouse Sim as efficiently as Arnt, and the Arnt2-AhR complex recognized and bound specifically the xenobiotic responsive element (XRE) sequence. Expression of Arnt2 successfully rescued XRE-driven reporter gene activity in the Arnt-defective c4 mutant of Hepa-1 cells. RNA blot analysis revealed that expression of Arnt2 mRNA was restricted to the brains and kidneys of adult mice, while Arnt mRNA was expressed ubiquitously. In addition, whole-mount in situ hybridization of 9.5-day mouse embryos showed that Arnt2 mRNA was expressed in the dorsal neural tube and branchial arch 1, while Arnt transcripts were detected broadly in various tissues of mesodermal and endodermal origins. These results suggest that Arnt2 may play different roles from Arnt both in adult mice and in developing embryos. Finally, sequence comparison of the currently known bHLH/PAS proteins indicates a division into two phylogenetic groups: the Arnt group, containing Arnt, Arnt2, and Per, and the AhR group, consisting of AhR, Sim, and Hif-1alpha.

Amino Acid Sequence↗
PubMed1996

Role of protein phosphatase in malignant osteogenic and soft tissue tumors.

The expression of the three catalytic subunits of protein phosphatase (PP) type 1 and 2A, PP1 alpha, PP1 gamma 1, and PP2AC, was examined in osteogenic tumors and soft tissue tumors by immunohistochemical analysis. The percentage of cells stained positively with antiserum against PP1 catalytic subunit isoform PP1 gamma 1, was significantly higher in malignant osteogenic tumors (chondrosarcoma, osteosarcoma, and Ewing's sarcoma) and in malignant soft tissue tumors (liposarcoma and malignant fibrous histiocytoma [M.F.H.]) than in benign tumors (osteochondroma, osteoblastoma, ossifying fibroma, enchondroma and lipoma). Furthermore, the malignant tumor lesions showed a markedly high number of cells in the S-phase fraction of the cell cycle, as compared to benign tumors. These results suggest that PP1 gamma 1 is involved in the accelerated growth of malignant tumor cells.

Bone Neoplasms↗
PubMed1995

Possible function of Ah receptor nuclear translocator (Arnt) homodimer in transcriptional regulation.

Arnt (Ah receptor nuclear translocator) is a member of a transcription factor family having characteristic motifs designated bHLH (basic helix-loop-helix) and PAS and was originally found as a factor forming a complex with Ah receptor (AhR) to bind the specific xenobiotic responsive element (XRE) sequence for induction of drug-metabolizing P4501A1. We have examined interaction of Arnt with other PAS proteins--Drosophila Per, Sim, and AhR--by the coimmunoprecipitation method. Arnt formed a homodimer with itself as well as heterodimers with the others by means of the PAS and HLH domains in a cooperative way. The Arnt homodimer binds the sequence of adenovirus major late promoter (MLP) with the E box core sequence CACGTG, suggesting that the CAC half of the XRE, CACGCN(A/T), recognized by the AhR-Arnt heterodimer is a target for Arnt. Cotransfection experiments using CV-1 cells with an Arnt expression plasmid and a MLP chloramphenicol acetyltransferase (CAT) reporter plasmid revealed that Arnt markedly activated CAT expression, indicative of a newly discovered regulatory role of Arnt.

Amino Acid Sequence↗
PubMed1995

Enhanced expression of catalytic subunit isoform PP1 gamma 1 of protein phosphatase type 1 associated with malignancy of osteogenic tumor.

The expressions of the three catalytic subunits of protein phosphatase (PP) type 1 and 2A, PP1 alpha, PP1 gamma 1, and PP2AC, were examined in 14 cases of three types of osteogenic tumor using immunohistochemical analysis. The percentage of tumor cells stained positively with antiserum against PP1 catalytic subunit-isoform PP1 gamma 1 was significantly higher in malignant osteogenic tumors than in benign osteogenic tumors. Furthermore, malignant osteogenic tumor showed markedly high S-phase fraction in the cell cycle of tumor cells, as compared to benign osteogenic tumors. These results suggest that PP1 gamma 1 is involved in the accelerated growth of malignant cells in osteogenic tumors.

Adolescent↗
PubMed1995

Transcriptional activation domains of the Ah receptor and Ah receptor nuclear translocator.

The Ah receptor (AhR) and Ah receptor nuclear translocator (Arnt) heterodimer bind the xenobiotic-responsive element (XRE) sequence in the upstream region of the genes for some drug-metabolizing enzymes, such as P4501A1 and glutathione S-transferase Ya, to activate their transcription. This paper describes transcriptional activation domains of the AhR and Arnt as examined in vivo by DNA transfection experiments using GAL4-AhR or GAL4-Arnt chimeric plasmids and a reporter plasmid containing five GAL4 DNA binding sites. The major activation domain of Arnt was localized in a short segment of the C-terminal 34 amino acids, while the glutamine-rich domain of Arnt showed no transcriptional activity. This activation domain of Arnt could be further divided into two subdomains with some sequence similarity. Point mutation analysis of one of the subdomains revealed that bulky hydrophobic amino acids and neighboring acidic amino acids were necessary for the transcription-enhancing activity of Arnt. The C-terminal half of the AhR showed a strong transcription-stimulating activity, apparently five times as strong as that of Arnt. Further analysis of the activity revealed that the C-terminal transcriptional activity was distributed in several activation domains, one of which is rich in glutamine residues. These results indicate that the glutamine-rich domains of the AhR and Arnt function differently in the heterodimer regulatory complex. Previously, we showed that the enhancer activity of XRE was repressed by E1A proteins, especially the 12S form of E1A. Cotransfection experiments using an E1A12S expression plasmid and a GAL4-AhR or GAL4-Arnt expression plasmid demonstrated that E1A protein rather predominantly inhibited the transcriptional activity of Arnt.

Adenovirus E1A Proteins↗
PubMed1995

Analysis of functional domains of a GC box-binding protein, BTEB.

BTEB is a small GC box-binding protein containing three contiguous zinc finger structures in the C-terminal region of the molecule, and activates or represses the transcription of genes with the GC box sequence in the promoter, depending on the repetitiveness of the GC box sequence [Imataka et al. (1992) EMBO J. 11, 3663-3671]. We have analyzed functional domains of BTEB in a transient expression system using Y-1 cells (a mouse adrenal cortex cell line). BTEB contained two regions responsible for the transcriptional activation. These regions showed a sequence similarity to each other and appeared to enhance the transcription independently. The two sequences were rich in hydrophobic amino acids and showed no similarity to the sequences of transactivation domains so far elucidated. The DNA-binding properties of BTEB were also analyzed by means of the gel mobility shift assay. Three contiguous zinc finger motifs were needed for the binding activity. Furthermore, it was revealed that a short basic region immediately N-terminal to the zinc finger motifs was required for the DNA binding.

Amino Acid Sequence↗
PubMed1995

Polymorphic forms of the Ah receptor and induction of the CYP1A1 gene.

Induction of cytochrome P4501A1 (CYP1A1) by certain xenobiotics is mediated by the Ah receptor/Arnt complex. The present knowledge about the molecular process of induction is summarized with special attention to our recent work on characterization of the polymorphic forms of the Ah receptor.

Cloning, Molecular↗
PubMed1994

Dioxin binding activities of polymorphic forms of mouse and human arylhydrocarbon receptors.

The genetic difference in the susceptibility of mice to environmental toxicities induced by dioxin and related chemicals is governed by polymorphism of the arylhydrocarbon receptor (AhR) (Poland, A., and Knutson, C. (1982) Annu. Rev. Pharmacol. Toxicol. 22, 517-554). cDNA cloning of AhR cDNA from responder (C57BL/6) and non-responder (DBA/2J) mice allowed us to analyze the structure and function of these AhRs. Both of the AhRs, which were expressed in COS-7 cells transfected with their expression plasmids, showed a clear 9 S complex with 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) in a linear glycerol gradient centrifugation, consistent with the result of the endogenously expressed AhR in Hepa-1 cells. This result provides the first direct evidence that the cDNA-encoded protein binds the ligand specifically. Scatchard plot analysis revealed that the dissociation constant (Kd) of C57BL AhR for TCDD is 0.27 nM, while that of DBA AhR is elevated up to six times that high. Chimeric plasmids between the two cDNAs and site-directed mutagenesis revealed two critical alterations responsible for the reduced ligand binding activity: an Ala375 to Val alteration and an elongated carboxyl-terminal sequence due to a T to C mutation at the first letter of the termination codon of C57BL AhR. Two variants with reduced and intermediate ligand binding activity were also found in human AhRs with amino acid alterations equivalent to those of the DBA AhR. Importance of the amino acid at position 381 of human AhR (equivalent to position 375 of mouse AhR) in the ligand binding was confirmed by the fact that the mutation of Val381 to Asp completely abolished the ligand binding activity of human AhR.

Amino Acid Sequence↗
PubMed1994

Human arylhydrocarbon receptor: functional expression and chromosomal assignment to 7p21.

We isolated the human arylhydrocarbon receptor (AhR) cDNA from a human lung cDNA library, by using mouse AhR cDNA as a labeled probe. The nucleotide sequence of cloned human AhR cDNA is identical to the previously reported human AhR sequence [Dolwick et al. (1993), Mol. Pharmacol. 44, 911-917] from cell line HepG2. The overall amino acid identity with mouse AhR from cell line Hepa-1 is 72.5%. The human AhR expressed either in COS-7 cells or in a reticulocyte lysate in vitro translation system showed specific dioxin-binding activity and Arnt-dependent DNA-binding activity. Chromosomal localization of the AhR gene was determined to be chromosome 7p21 by fluorescent in situ hybridization and DNA blot hybridization using 23 human x mouse or Chinese hamster hybrid cell DNAs.

Amino Acid Sequence↗
PubMed1994

A complete structure of the mouse Ah receptor gene.

A complete sequence of the Ah receptor gene was cloned from a mouse genomic library by using the Ah receptor cDNA as a probe. The Ah receptor gene is 37.5 kb long and is split into 11 exons by 10 introns. Sequence analysis of the 5' flanking region of the Ah receptor gene reveals that there is neither a TATA box nor a CAAT box in the promoter region. Instead, this gene has a few GC boxes and other enhancer elements in the 5' upstream flanking region. Southern blot analysis indicated that the Ah receptor gene is a unique gene.

Animals↗
PubMed1994

Ah receptor: a novel ligand-activated transcription factor.

Two years have passed since the Ah receptor (AhR) and the AhR nuclear translocator (Arnt) were revealed to constitute a novel ligand-activated transcription factor by molecular biological studies on cloned cDNAs. Many interesting properties of the AhR/Arnt system have begun to be elucidated. We present here a brief summary of the molecular biology of AhR and Arnt.

Amino Acid Sequence↗