Hisaya Kawate

Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase

Oxygen radicals, which can be produced through normal cellular metabolism, are thought to play an important role in mutagenesis and tumorigenesis. Among various classes of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is most important because of its abundance and mutagenicity. The MTH1 gene encodes an enzyme that hydrolyzes 8-oxo-dGTP to monophosphate in the nucleotide pool, thereby preventing occurrence of transversion mutations. By means of gene targeting, we have established MTH1 gene-knockout cell lines and mice. When examined 18 months after birth, a greater number of tumors were formed in the lungs, livers, and stomachs of MTH1-deficient mice, as compared with wild-type mice. The MTH1-deficient mouse will provide a useful model for investigating the role of the MTH1 protein in normal conditions and under oxidative stress.

The lack of the C-terminal domain of adipose triglyceride lipase causes neutral lipid storage disease through impaired interactions with lipid droplets

CONTEXT The molecular mechanisms by which triglycerides in lipid droplets (LDs) are synthesized, stored, and degraded need to be elucidated. OBJECTIVE The objectives were to report siblings with neutral lipid storage disease with myopathy (NLSDM) with a novel mutation of adipose triglyceride lipase (ATGL) and determine whether the C-terminal part of ATGL containing the hydrophobic region plays a role in the interaction with LDs. DESIGN AND PATIENTS Skin fibroblasts and peripheral blood leukocytes were obtained from NLSDM patients. In vitro experiments were performed with fibroblasts and COS7 cells. MAIN OUTCOME MEASURES Transfection studies were used to assess the effects of various recombinant ATGL proteins on lipase activities and lipid contents. Fluorescence microscopy were used for determination of intracellular distribution of ATGL proteins. RESULTS The direct sequence of ATGL cDNA reveals that a patient is a homozygote for the 4-bp deletion, leading to a premature stop codon and causes the lack of the C terminus of the protein including the hydrophobic domain. Overexpressed control ATGL in NLSDM fibroblasts was found around the rims of LDs and caused significantly reduced cellular lipid accumulation. In contrast, NLSDM ATGL was homogeneously located in the cytoplasm despite the presence of LDs and had almost no effect on LD degradation despite its similar lipase activity. A series of C-terminal truncated ATGLs without the intact hydrophobic domain failed to localize around and degrade LDs. CONCLUSIONS These findings indicate that the domain including the hydrophobic region of ATGL was essential for association with LDs.

Mutational specificity of mice defective in the MTH1 and/or the MSH2 genes.

Oxidative damage of nucleotides within DNA or precursor pools caused by oxygen radicals is thought to play an important role in spontaneous mutagenesis, as well as carcinogenesis and aging. In particular, 8-oxodGTP and 2-OHdATP are potent mutagenic substrate for DNA synthesis. Mammalian MTH1 catalyzes hydrolysis of these mutagenic substrates, suggesting that it functions to prevent mutagenesis caused by these oxidized nucleotides. We have established MTH1(-/-) mice lacking the 8-oxodGTPase activity, which were shown to be susceptible to lung, liver and stomach cancers. To examine in vivo mutation events due to the MTH1-deficiency, a reporter gene, rpsL of Escherichia coli, was introduced into MTH1(-/-) mice. Interestingly, the net frequency of rpsL(-) forward mutants showed no apparent increase in MTH1(-/-) mice as compared to MTH1(+/+) mice. However, we found differences between these two genotypes in the class- and site-distributions of the rpsL(-) mutations recovered from the mice. Unlike MutT-deficient E. coli showing 1000-fold higher frequency of A:T-->C:G transversion than the wild type cells, an increase in frequency of A:T-->C:G transversion was not evident in MTH1 nullizygous mice. Nevertheless, the frequency of single-base frameshifts at mononucleotide runs was 5.7-fold higher in spleens of MTH1(-/-) mice than in those of wild type mice. Since the elevated incidence of single-base frameshifts at mononucleotide runs is a hallmark of the defect in MSH2-dependent mismatch repair system, this weak site-specific mutator effect of MTH1(-/-) mice could be attributed to a partial sequestration of the mismatch repair function that may act to correct mispairs with the oxidized nucleotides. Consistent with this hypothesis, a significant increase in the frequency of G:C-->T:A transversions was observed with MTH1(-/-) MSH2(-/-) mice over MSH2(-/-) mice alone. These results suggest a possible involvement of multiple anti-mutagenic pathways, including the MTH1 protein and other repair system(s), in mutagenesis caused by the oxidized nucleotides.

Inverse associations of serum bilirubin with high sensitivity C-reactive protein, glycated hemoglobin, and prevalence of type 2 diabetes in middle-aged and elderly Japanese men and women.

AIM The aim of this study was to examine the association of serum bilirubin, an endogenous antioxidant, with serum high sensitivity C-reactive protein (hs-CRP) level, HbA(1c), and the prevalence of type 2 diabetes in middle-aged and elderly Japanese men and women (n=12,400). METHODS Analysis of covariance and logistic regression analysis were used to estimate geometric means of hs-CRP and HbA(1c) and odds ratios of prevalent diabetes according to bilirubin concentrations, respectively, with statistical adjustment for behavioral factors and liver enzymes. RESULTS Geometric means of hs-CRP and HbA(1c) were progressively lower with increasing concentrations of serum bilirubin in men and women each. An inverse association between serum bilirubin and HbA(1c) was slightly attenuated after adjustment for hs-CRP, but still remained highly significant (trend P=0.0004 in men and trend P=10(-5) in women). Multivariate-adjusted odds ratios of prevalent diabetes for the lowest to highest quintiles of serum total bilirubin were 1.00, 1.00, 0.73, 0.80, and 0.73 (trend P=0.002), without adjustment for hs-CRP, and 1.00, 1.04, 0.76, 0.86, and 0.79 (trend P=0.01), with adjustment for hs-CRP. CONCLUSIONS Higher concentrations of serum bilirubin probably confer protection against the development of type 2 diabetes.

Nuclear Compartmentalization of N-CoR and Its Interactions with Steroid Receptors

ABSTRACT The repression mechanisms by the nuclear receptor corepressor (N-CoR) of steroid hormone receptor (SHR)-mediated transactivation were examined. Yellow fluorescent protein (YFP)-N-CoR was distributed as intranuclear discrete dots, while coexpression of androgen receptor (AR), glucocorticoid receptor α, and estrogen receptor α ligand-dependently triggered redistribution of YFP-N-CoR. In fluorescence recovery after photobleaching analysis, mobility of the N-CoR was reduced by 5α-dihydrotestosterone (DHT)-bound AR. The middle region of N-CoR mostly contributed to the interaction with agonist-bound SHRs and the suppression of their transactivation function. N-CoR impaired the DHT-induced N-C interaction of AR, and the impaired interaction was dose-dependently recovered by coexpression of SRC-1 and CBP. N-CoR also impaired the intranuclear complete (distinct) focus formation of SHRs. Coexpression of SRC-1 or CBP released YFP-N-CoR or endogenous N-CoR from incomplete foci and simultaneously recovered complete foci of AR-green fluorescent protein. These results indicate that the relative ratio of coactivators and corepressors determines the conformational equilibrium between transcriptionally active and inactive SHRs in the presence of agonists. The intranuclear foci formed by agonist-bound SHRs were completely destroyed by actinomycin D and α-amanitin, indicating that the focus formation does not precede the transcriptional activation. The focus formation may reflect the accumulation of SHR/coactivator complexes released from the transcriptionally active sites and thus be a mirror of transcriptionally active complex formation.

Intracellular localization and function of DNA repair methyltransferase in human cells

An antibody preparation specific for human O6-methylguanine-DNA methyltransferase (EC 2.1.1.63) was obtained by immunoaffinity purification on two types of affinity columns with the purified human and mouse methyltransferase proteins as ligands. The antibodies were used in Western blotting analysis of fractionated cell extracts. More than 90% of the methyltransferase protein was recovered in the cytoplasmic fractions with both human HeLa S3 cells and MR-M cells, the latter overproducing the enzyme 36 times as much as the former. Cytoplasmic localization of the methyltransferase in HeLa S3 cells was further confirmed by in situ immunostaining. By Western blotting analysis of fractionated cell extracts from HeLa S3 cells treated with alkylating agents, we found that amounts of the enzyme decreased more rapidly in the nuclear fraction than in the cytoplasmic fraction, and recovery of the enzyme level in the cytoplasmic fraction was slower than that in the other. These results suggest that the methyltransferase protein is degraded in the nucleus after it commits the repair reaction and that the cytoplasmic enzyme is transported into the nucleus as the nuclear methyltransferase is used up in this manner.

Requirement of the Pro-Cys-His-Arg sequence for O6-methylguanine-DNA methyltransferase activity revealed by saturation mutagenesis with negative and positive screening

O6-Methylguanine-DNA methyltransferase catalyzes transfer of a methyl group from O6-methylguanine and O4-methylthymine of DNA to a cysteine residue of the enzyme protein, thereby repairing the mutagenic and carcinogenic lesions in a single-step reaction. There are highly conserved amino acid sequences around the methyl-accepting cysteine site in eleven molecular species of methyltransferases. To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site-directed mutagenesis of the cloned DNA for Escherichia coli Ogt methyltransferase, and the activity and stability of mutant forms of the enzyme were examined. When cysteine-139, to which methyl transfer occurs, was replaced by other amino acids, all of the mutants showed the methyltransferase-negative phenotype. Methyltransferase-positive revertants, isolated from one of the negative mutants, had restored codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and is not replaceable by other amino acids. Using this negative and positive selection procedure, the analysis was extended to other residues near the acceptor site. At the histidine-140 and arginine-141 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild-type protein. At proline-138, five substitutions (serine, glutamine, threonine, histidine, and alanine) exhibited the positive phenotype but levels of methyltransferase activity in extracts of cells harboring these mutant forms were very low. This suggests that the proline residue at this site is important for maintaining the proper conformation of the protein. With valine-142 substitutions there were seven types of positive revertants, among which mutants carrying isoleucine, cysteine, leucine, and alanine showed relatively high levels of methyltransferase activity. These results indicate that the sequence Pro-Cys-His-Arg is a sine qua non for methyltransferase to exert its function.

Relation of coffee consumption and serum liver enzymes in Japanese men and women with reference to effect modification of alcohol use and body mass index.

Abstract Objective. Previous studies have shown that coffee consumption is inversely related to serum levels of liver enzymes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT), but few have addressed the relation in women and effect modifications of alcohol use and obesity. We examined the association of coffee and green tea consumption with serum activities of liver enzymes in free-living Japanese men and women, focusing on sex difference and effect modifications of alcohol and obesity. Material and methods. The data were derived from the baseline survey of the Kyushu University Fukuoka Cohort Study, and included 12,020 Japanese men and women aged 49–76 years who were free of chronic liver diseases. Results. There was an inverse association between coffee consumption and elevated ALT in men, and the association between the two was weaker in women. In the analyses stratified by aminotransferases category, inverse associations of coffee consumption with serum activities of liver enzymes were observed in both men and women within the whole range and among those with aminotransferases within the reference range (ALT/AST ≤40 IU/L for men and ALT/AST ≤30 IU/L for women). Inverse associations of coffee with liver enzymes were more evident in those with high alcohol consumption and in those with low body mass index. Conclusions. Coffee drinking probably confers protection against alcohol-related increase in liver enzymes.

Tob proteins suppress steroid hormone receptor-mediated transcriptional activation

Although sex steroid hormones have significant effects on bone metabolism, the molecular mechanisms of these actions have not been fully elucidated yet. We examined the functional relationship between steroid hormone receptors and Tob, a member of an anti-proliferative protein family and a negative regulator of osteoblast proliferation and differentiation. Luciferase assay using promoters carrying hormone-responsive elements revealed that both Tob1 and Tob2 proteins but not PC3 suppressed steroid hormone receptor-dependent transcriptional activation in MC3T3-E1 osteoblastic cells. Mutated Tob proteins carrying amino acid substitutions at an LXXLL motif also showed the same degree of inhibition of the transcriptional activation as the wild type. By observation of androgen receptor (AR)-tagged with green fluorescent protein under a confocal laser scanning microscope, we found that Tob1 inhibits the nuclear foci formation of dihydrotestosterone-bound AR. These results indicate that Tob family proteins may negatively regulate sex steroid hormone action in bone formation.

Mutual transactivational repression of Runx2 and the androgen receptor by an impairment of their normal compartmentalization

Steroid hormones play important roles not only in the reproductive system but also in bone metabolism. We examined the functional relationship between steroid hormone receptors and the Runx2 transcription factor that is essential for osteoblast differentiation and proliferation. A functional reporter assay using promoters carrying steroid hormone-responsive elements revealed that Runx2 suppressed ligand-dependent transcriptional activation mediated by receptors. To examine intracellular localization of these proteins, a three-dimensional imaging study was performed by laser scanning confocal microscopy of green fluorescent protein (GFP)-fused proteins. As previously reported, ligand-bound human androgen receptor (AR) was translocated from the cytoplasm to the nucleus and formed subnuclear fine foci. Coexpression of human Runx2 disrupted the AR subnuclear fine foci formation, and the intranuclear fluorescent pattern of AR became similar to that of Runx2. On the other hand, ligand-bound ARs repressed the Runx2-mediated transactivation function. Runx2 was also extracted from its original compartment by ligand-bound ARs. These results suggest that both Runx2 and ARs repress the transactivation function of the other protein by extracting it from its original compartment. The AR and Runx2 may play a mutual role in transcriptional activation in osteoblasts.