Shinobu Kura

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.

Critical Role of Monocyte Chemoattractant Protein-1 Receptor CCR2 on Monocytes in Hypertension-Induced Vascular Inflammation and Remodeling

Activated monocytes are present in the arterial walls of hypertensive patients and animals. Monocyte chemoattractant protein-1 (MCP-1), which controls monocyte function through its receptor (CCR2), is implicated in hypertensive inflammatory changes in the arterial wall. The role of CCR2 expression on monocytes in hypertension-induced vascular remodeling, however, has not been addressed. We hypothesized that CCR2 on monocytes is critical in hypertension-induced vascular inflammation and remodeling. Hypertension was induced by infusion of angiotensin II (Ang II) into wild-type mice, CCR2-deficient (CCR2−/−) mice, and bone marrow-transferred mice with a leukocyte-selective CCR2 deficiency (BMT-CCR2−/−). In wild-type mice, Ang II increased CCR2 intensity in circulating monocytes, which was prevented by an Ang II type-1 (AT1) receptor blocker or blunted in AT1 receptor–deficient mice. Enhanced CCR2 intensity on monocytes was observed in hypertensive patients and rats, and was reduced by treatment with the Ang II receptor blocker, supporting the clinical relevance of the observation in mice. In CCR2−/− and BMT-CCR2−/− mice, Ang II–induced vascular inflammation and vascular remodeling (aortic wall thickening and fibrosis) were blunted as compared with control mice. In contrast, Ang II–induced left ventricular hypertrophy developed in CCR2−/− and BMT-CCR2−/− mice. The present study suggests that CCR2 expression in monocytes has a critical role in vascular inflammation and remodeling in Ang II–induced hypertension, and possibly in other forms of hypertension.

Bone Marrow–Derived Monocyte Chemoattractant Protein-1 Receptor CCR2 Is Critical in Angiotensin II–Induced Acceleration of Atherosclerosis and Aneurysm Formation in Hypercholesterolemic Mice

Abstract—Angiotensin II (Ang II) is implicated in atherogenesis by activating inflammatory responses in arterial wall cells. Ang II accelerates the atherosclerotic process in hyperlipidemic apoE−/− mice by recruiting and activating monocytes. Monocyte chemoattractant protein-1 (MCP-1) controls monocyte-mediated inflammation through its receptor, CCR2. The roles of leukocyte-derived CCR2 in the Ang II-induced acceleration of the atherosclerotic process, however, are not known. We hypothesized that deficiency of leukocyte-derived CCR2 suppresses Ang II-induced atherosclerosis. Methods and Results—A bone marrow transplantation technique (BMT) was used to develop apoE−/− mice with and without deficiency of CCR2 in leukocytes (BMT-apoE−/−CCR2+/+ and BMT-apoE−/−CCR2−/− mice). Compared with BMT-apoE−/−CCR2+/+ mice, Ang II-induced increases in atherosclerosis plaque size and abdominal aortic aneurysm formation were suppressed in BMT-apoE−/−CCR2−/− mice. This suppression was associated with a marked decrease in monocyte-mediated inflammation and inflammatory cytokine expression. Conclusion—Leukocyte-derived CCR2 is critical in Ang II-induced atherosclerosis and abdominal aneurysm formation. The present data suggest that vascular inflammation mediated by CCR2 in leukocytes is a reasonable target of therapy for treatment of atherosclerosis.

MUTYH-Null Mice Are Susceptible to Spontaneous and Oxidative Stress–Induced Intestinal Tumorigenesis

MUTYH is a mammalian DNA glycosylase that initiates base excision repair by excising adenine opposite 8-oxoguanine and 2-hydroxyadenine opposite guanine, thereby preventing G:C to T:A transversion caused by oxidative stress. Recently, biallelic germ-line mutations of MUTYH have been found in patients predisposed to a recessive form of hereditary multiple colorectal adenoma and carcinoma with an increased incidence of G:C to T:A somatic mutations in the APC gene. In the present study, a systematic histologic examination revealed that more spontaneous tumors had developed in MUTYH-null mice (72 of 121; 59.5%) than in the wild type (38 of 109; 34.9%). The increased incidence of intestinal tumors in MUTYH-null mice (11 tumors in 10 of 121 mice) was statistically significant compared with the wild type (no intestinal tumors in 109 mice). Two adenomas and seven adenocarcinomas were observed in the small intestines, and two adenomas but no carcinomas were found in the colons. In MUTYH-null mice treated with KBrO(3), the occurrence of small intestinal tumors dramatically increased. The mean number of polyps induced in the small intestines of these mice was 61.88 (males, 72.75; females, 51.00), whereas it was 0.85 (males, 0.50; females, 1.00) in wild-type mice. The tumors developed predominantly in the duodenum and in the upper region of the (jejunum) small intestines. We conclude that MUTYH suppresses spontaneous tumorigenesis in mammals, thus providing experimental evidence for the association between biallelic germ-line MUTYH mutations and a recessive form of human hereditary colorectal adenoma and carcinoma.

Clearance of apoptotic photoreceptors: Elimination of apoptotic debris into the subretinal space and macrophage-mediated phagocytosis via phosphatidylserine receptor and integrin αvβ3

The effective phagocytotic clearance of apoptotic debris is fundamental to the maintenance of neural tissues during apoptosis. Retinal photoreceptors undergo apoptosis after retinal detachment. Although their induction phase of apoptosis has been well discussed, their phagocytotic process remains quite unclear. We herein demonstrate that apoptotic photoreceptors are selectively eliminated from their physiological localization, the outer nuclear layer, to the subretinal space, and then phagocytosed by monocyte-derived macrophages. This could be shown by an ultrastructural and immunophenotypic analysis. Moreover, in chimera mice expressing transgenic green fluorescent protein in bone marrow-derived cells, the local infiltration of macrophages could be detected after retinal detachment-induced photoreceptor apoptosis. The local injection of an antibody blocking the phosphatidylserine receptor (PSR) or a peptide (GRGDSP)-blocking integrin αvβ3 revealed that phagocytotic clearance involves the PSR as well as integrin αvβ3 in vivo. Importantly, the level of blockade obtained with these reagents was different. Although anti-PSR increased the frequency of apoptotic cells that fail to bind to macrophages, GRGDSP prevented the engulfment (but not the recognition) of apoptotic photoreceptor cells by macrophages. To our knowledge, this is the first report describing the mechanisms through which apoptotic photoreceptors are selectively eliminated via a directional process in the subretinal space.

The characterisation of hyalocytes: the origin, phenotype, and turnover

Aim: To determine the characterisation of hyalocytes: the origin, phenotype, and turnover in the rodent. Methods: To characterise the ultrastructure and distribution of hyalocytes, transmission and scanning electron microscopy was performed in rat eyes. Immunophenotypical analysis was performed by either anti-ED1 or ED2 antibodies. To examine the origin of the hyalocytes, the chimeric mice were created and were used to transplant the bone marrow (BM) cells from enhanced green fluorescent protein (EGFP) transgenic mice. The turnover of hyalocytes was examined at 0, 4, 6, 7, and 12 months after BM transplantation. Results: Hyalocytes were distributed especially in the vitreous cortex and had an irregular shape with a spherical granule. Immunophenotypical studies demonstrated that most of the hyalocytes in rat eyes expressed ED2 but not ED1. In the chimeric mice, the hyalocytes were GFP negative right after BM transplantation. Interestingly, more than 60% of hyalocytes were replaced within 4 months and approximately 90% within 7 months after BM transplantation. Conclusions: The rodent hyalocytes were shown to express tissue macrophage marker, were derived from BM, and totally replaced within 7 months. These data provide the characterisation of hyalocytes in physiological conditions, especially their origin, distribution, and turnover, and may contribute to the better understanding of the pathogenesis of vitreoretinal disease.

Measles Virus Infection of SLAM (CD150) Knockin Mice Reproduces Tropism and Immunosuppression in Human Infection

ABSTRACT The human signaling lymphocyte activation molecule (SLAM, also called CD150), a regulator of antigen-driven T-cell responses and macrophage functions, acts as a cellular receptor for measles virus (MV), and its V domain is necessary and sufficient for receptor function. We report here the generation of SLAM knockin mice in which the V domain of mouse SLAM was replaced by that of human SLAM. The chimeric SLAM had an expected distribution and normal function in the knockin mice. Splenocytes from the SLAM knockin mice permitted the in vitro growth of a virulent MV strain but not that of the Edmonston vaccine strain. Unlike in vitro infection, MV could grow only in SLAM knockin mice that also lacked the type I interferon receptor (IFNAR). After intraperitoneal or intranasal inoculation, MV was detected in the spleen and lymph nodes throughout the body but not in the thymus. Notably, the virus appeared first in the mediastinal lymph node after intranasal inoculation. Splenocytes from MV-infected IFNAR−/− SLAM knockin mice showed suppression of proliferative responses to concanavalin A. Thus, MV infection of SLAM knockin mice reproduces lymphotropism and immunosuppression in human infection, serving as a useful small animal model for measles.

Targeted disruption of one allele of the Y‐box binding protein‐1 (YB‐1) gene in mouse embryonic stem cells and increased sensitivity to cisplatin and mitomycin C

The eukaryotic Y‐box binding protein‐1 (YB‐1) functions in various biological processes, including transcriptional and translational control, DNA repair, drug resistance, and cell proliferation. To elucidate the physiological role of the YB‐1 protein, we disrupted one allele of mouse YB‐1 in embryonic stem (ES) cells. Northern blot analysis revealed that YB‐1+/‐ ES cells with one intact allele contain approximately one‐half the amount of mRNA detected in wild‐type (YB‐1+/+) cells. We further found that the protein level of YB‐1+/‐ cells was reduced to approximately 50–60% compared with that of YB‐1+/+ cells. However, no apparent growth difference was found between YB‐1+/‐ and YB‐1+/+ cells. YB‐1+/‐ cells showed increased sensitivity to cisplatin and mitomycin C, but not to etoposide, X‐ray or UV irradiation, as compared to YB‐1+/+ cells. YB‐1 may have the capacity to exert a protective role against cytotoxic effects of DNA damaging agents, and may be involved in certain aspects of drug resistance.

UV-induced mutations in epidermal cells of mice defective in DNA polymerase η and/or ι

Xeroderma pigmentosum variant (XP-V) is a human rare inherited recessive disease, predisposed to sunlight-induced skin cancer, which is caused by deficiency in DNA polymerase η (Polη). Polη catalyzes accurate translesion synthesis (TLS) past pyrimidine dimers, the most prominent UV-induced lesions. DNA polymerase ι (Polι) is a paralog of Polη that has been suggested to participate in TLS past UV-induced lesions, but its function in vivo remains uncertain. We have previously reported that Polη-deficient and Polη/Polι double-deficient mice showed increased susceptibility to UV-induced carcinogenesis. Here, we investigated UV-induced mutation frequencies and spectra in the epidermal cells of Polη- and/or Polι-deficient mice. While Polη-deficient mice showed significantly higher UV-induced mutation frequencies than wild-type mice, Polι deficiency did not influence the frequencies in the presence of Polη. Interestingly, the frequencies in Polη/Polι double-deficient mice were statistically lower than those in Polη-deficient mice, although they were still higher than those of wild-type mice. Sequence analysis revealed that most of the UV-induced mutations in Polη-deficient and Polη/Polι double-deficient mice were base substitutions at dipyrimidine sites. An increase in UV-induced mutations at both G:C and A:T pairs associated with Polη deficiency suggests that Polη contributes to accurate TLS past both thymine- and cytosine-containing dimers in vivo. A significant decrease in G:C to A:T transition in Polη/Polι double-deficient mice when compared with Polη-deficient mice suggests that Polι is involved in error-prone TLS past cytosine-containing dimers when Polη is inactivated.