Michio Kawasuji

Rad18 guides polη to replication stalling sites through physical interaction and PCNA monoubiquitination

The DNA replication machinery stalls at damaged sites on templates, but normally restarts by switching to a specialized DNA polymerase(s) that carries out translesion DNA synthesis (TLS). In human cells, DNA polymerase η (polη) accumulates at stalling sites as nuclear foci, and is involved in ultraviolet (UV)‐induced TLS. Here we show that polη does not form nuclear foci in RAD18−/− cells after UV irradiation. Both Rad18 and Rad6 are required for polη focus formation. In wild‐type cells, UV irradiation induces relocalization of Rad18 in the nucleus, thereby stimulating colocalization with proliferating cell nuclear antigen (PCNA), and Rad18/Rad6‐dependent PCNA monoubiquitination. Purified Rad18 and Rad6B monoubiquitinate PCNA in vitro. Rad18 associates with polη constitutively through domains on their C‐terminal regions, and this complex accumulates at the foci after UV irradiation. Furthermore, polη interacts preferentially with monoubiquitinated PCNA, but polδ does not. These results suggest that Rad18 is crucial for recruitment of polη to the damaged site through protein–protein interaction and PCNA monoubiquitination.

C-C chemokine receptor 2 (CCR2) deficiency improves bleomycin-induced pulmonary fibrosis by attenuation of both macrophage infiltration and production of macrophage-derived matrix metalloproteinases

Macrophage infiltration is implicated in various types of pulmonary fibrosis. One important pathogenetic process associated with pulmonary fibrosis is injury to basement membranes by matrix metalloproteinases (MMPs) that are produced mainly by macrophages. In this study, C‐C chemokine receptor 2‐deficient (CCR2−/−) mice were used to explore the relationship between macrophage infiltration and MMP activity in the pathogenesis of pulmonary fibrosis, using the bleomycin‐induced model of this disease process. CCR2 is the main (if not only) receptor for monocyte chemoattractant protein‐1/C‐C chemokine ligand 2 (MCP‐1/CCL2), which is a critical mediator of macrophage trafficking, and CCR2 −/− mice demonstrate defective macrophage migration. Pulmonary fibrosis was induced in CCR2−/− and wild‐type (CCR2+/+) mice by intratracheal instillation of bleomycin. No significant differences in the total protein concentration in bronchoalveolar lavage (BAL) fluid, or in the degree of histological lung inflammation, were observed in the two groups until day 7. Between days 3 and 21, however, BAL fluid from CCR2−/− mice contained fewer macrophages than BAL fluid from CCR2+/+ mice. Gelatin zymography of BAL fluid and in situ zymography revealed reduced gelatinolytic activity in CCR2−/− mice. Immunocytochemical staining showed weaker expression of MMP‐2 and MMP‐9 in macrophages in BAL fluid from CCR2−/− mice at day 3. Gelatin zymography of protein extracted from alveolar macrophages showed reduced gelatinolytic activity of MMP‐2 and MMP‐9 in CCR2−/− mice. At days 14 and 21, lung remodelling and the hydroxyproline content of lung tissues were significantly reduced in CCR2−/− mice. These results suggest that the CCL2/CCR2 functional pathway is involved in the pathogenesis of bleomycin‐induced pulmonary fibrosis and that CCR2 deficiency may improve the outcome of this disease by regulating macrophage infiltration and macrophage‐derived MMP‐2 and MMP‐9 production. Copyright

Tumor suppressor WARTS ensures genomic integrity by regulating both mitotic progression and G1 tetraploidy checkpoint function.

Defects in chromosomes or mitotic spindles activate the spindle checkpoint, resulting in cell cycle arrest at prometaphase. The prolonged activation of spindle checkpoint generally leads to mitotic exit without segregation after a transient mitotic arrest and the consequent formation of tetraploid G1 cells. These tetraploid cells are usually blocked to enter the subsequent S phase by the activation of p53/pRb pathway, which is referred to as the G1 tetraploidy checkpoint. A human homologue of the Drosophila warts tumor suppressor, WARTS, is an evolutionarily conserved serine–threonine kinase and implicated in development of human tumors. We previously showed that WARTS plays a crucial role in controlling mitotic progression by forming a regulatory complex with zyxin, a regulator of actin filament assembly, on mitotic apparatus. However, when WARTS is activated during cell cycle and how the loss of WARTS function leads to tumorigenesis have not been elucidated. Here we show that WARTS is activated during mitosis in mammalian cells, and that overexpression of a kinase-inactive WARTS in Rat1 fibroblasts significantly induced mitotic delay. This delay resulted from prolonged activation of the spindle assembly checkpoint and was frequently followed by mitotic slippage and the development of tetraploidy. The resulting tetraploid cells then abrogated the G1 tetraploidy checkpoint and entered S phase to achieve a DNA content of 8N. This impairment of G1 tetraploidy checkpoint was caused as a consequence of failure to induce p53 expression by expressing a kinase-inactive WARTS. WARTS thus plays a critical role in maintenance of ploidy through its actions in both mitotic progression and the G1 tetraploidy checkpoint.

Macrophage-Derived Angiopoietin-Like Protein 2 Accelerates Development of Abdominal Aortic Aneurysm

Objective—Recently, we reported that angiopoietin-like protein 2 (Angptl2) functions in various chronic inflammatory diseases. In the present study, we asked whether Angptl2 and its associated chronic inflammation contribute to abdominal aortic aneurysm (AAA). Methods and Results—Immunohistochemistry revealed that Angptl2 is abundantly expressed in infiltrating macrophages within the vessel wall of patients with AAA and in a CaCl2-induced AAA mouse model. When Angptl2-deficient mice were used in the mouse model, they showed decreased AAA development compared with wild-type mice, as evidenced by reduction in aneurysmal size, less severe destruction of vessel structure, and lower expression of proinflammatory cytokines and matrix metalloproteinase-9. However, no difference in the number of infiltrating macrophages within the aortic aneurysmal vessel wall was observed between genotypes. AAA development was also significantly suppressed in wild-type mice that underwent Angptl2-deficient bone marrow transplantation. Expression levels of proinflammatory cytokines and metalloproteinase-9 in Angptl2-deficient macrophages were significantly decreased, and those decreases were rescued by treatment of Angptl2 deficient macrophages with exogenous Angptl2. Conclusion—Macrophage-derived Angptl2 contributes to AAA development by inducing inflammation and degradation of extracellular matrix in the vessel wall, suggesting that targeting the Angptl2-induced inflammatory axis in macrophages could represent a new strategy for AAA therapy.

Methylated DNA-binding domain 1 and methylpurine-DNA glycosylase link transcriptional repression and DNA repair in chromatin

The methyl–CpG dinucleotide containing a symmetrical 5-methylcytosine (mC) is involved in gene regulation and genome stability. We report here that methylation-mediated transcriptional repressor methylated DNA-binding domain 1 (MBD1) interacts with methylpurine–DNA glycosylase (MPG), which excises damaged bases from substrate DNA. MPG itself actively represses transcription and has a synergistic effect on gene silencing together with MBD1. Chromatin immunoprecipitation analysis reveals the molecular movement of MBD1 and MPG in vivo: (i) The MBD1–MPG complex normally exists on the methylated gene promoter; (ii) treatment of cells with alkylating agent methylmethanesulfonate (MMS) induces the dissociation of MBD1 from the methylated promoter, and MPG is located on both methylated and unmethylated promoters; and (iii) after completion of the repair, the MBD1–MPG complex is restored on the methylated promoter. Mobility-shift and structural analyses show that the MBD of MBD1 binds a methyl–CpG pair (mCpG × mCpG) but not the methyl–CpG pair containing a single 7-methylguanine (N) (mCpG × mCpN) that is known as one of the major lesions caused by MMS. We further demonstrate that knockdown of MBD1 by specific small interfering RNAs significantly increases cell sensitivity to MMS. These data suggest that MBD1 cooperates with MPG for transcriptional repression and DNA repair. We hypothesize that MBD1 functions as a reservoir for MPG and senses the base damage in chromatin.

Perivascular adipose tissue-secreted angiopoietin-like protein 2 (Angptl2) accelerates neointimal hyperplasia after endovascular injury

Much attention is currently focused on the role of perivascular adipose tissue in development of cardiovascular disease (CVD). Some researchers view it as promoting CVD through secretion of cytokines and growth factors called adipokines, while recent reports reveal that perivascular adipose tissue can exert a protective effect on CVD development. Furthermore, adiponectin, an anti-inflammatory adipokine, reportedly suppresses neointimal hyperplasia after endovascular injury, whereas such vascular remodeling is enhanced by pro-inflammatory adipokines secreted by perivascular adipose, such as tumor necrosis factor-α (TNF-α). These findings suggest that extent of vascular remodeling, a pathological process associated with CVD development, depends on the balance between pro- and anti-inflammatory adipokines secreted from perivascular adipose tissue. We previously demonstrated that angiopoietin-like protein 2 (Angptl2), a pro-inflammatory factor secreted by adipose tissue, promotes adipose tissue inflammation and subsequent systemic insulin resistance in obesity. Here, we examined whether Angptl2 secreted by perivascular adipose tissue contributes to vascular remodeling after endovascular injury in studies of transgenic mice expressing Angptl2 in adipose tissue (aP2-Angptl2 transgenic mice) and Angptl2 knockout mice (Angptl2(-/-) mice). To assess the role of Angptl2 secreted by perivascular adipose tissue on vascular remodeling after endovascular injury, we performed adipose tissue transplantation experiments using these mice. Wild-type mice with perivascular adipose tissue derived from aP2-Angptl2 mice exhibited accelerated neointimal hyperplasia after endovascular injury compared to wild-type mice transplanted with wild-type tissue. Conversely, vascular inflammation and neointimal hyperplasia after endovascular injury were significantly attenuated in wild-type mice transplanted with Angptl2(-/-) mouse-derived perivascular adipose tissue compared to wild-type mice transplanted with wild-type tissue. RT-PCR analysis revealed that mouse Angptl2 expression in perivascular adipose tissue was significantly increased by aging, hypercholesterolemia, and endovascular injury, all risk factors for coronary heart disease (CHD). Immunohistochemical and RT-PCR analysis of tissues from patients with CHD and from non-CHD patients indicated that ANGPTL2 expression in epicardial adipose tissue was unchanged. Interestingly, that analysis also revealed a positive correlation in ANGPTL2 and ADIPONECTIN expression in epicardial adipose tissue of non-CHD patients, a correlation not seen in CHD patients. However, in epicardial adipose tissue from CHD patients, ANGPTL2 expression was positively correlated with that of TNF-α, a correlation was not seen in non-CHD patients. These findings suggest that pro-inflammatory adipokines cooperatively accelerate CHD development and that maintaining a balance between pro- and anti-inflammatory adipokines likely protects non-CHD patients from developing CHD. Overall, our studies demonstrate that perivascular adipose tissue-secreted Angptl2 accelerates vascular inflammation and the subsequent CVD development.

Overlapping Roles of the Methylated DNA-binding Protein MBD1 and Polycomb Group Proteins in Transcriptional Repression of HOXA Genes and Heterochromatin Foci Formation

Methylated DNA binding domain (MBD) proteins and Polycomb group (PcG) proteins maintain epigenetic silencing of transcriptional activity. We report that the DNA methylation-mediated repressor MBD1 interacts with Ring1b and hPc2, the major components of Polycomb repressive complex 1. The cysteine-rich CXXC domains of MBD1 bound to Ring1b and the chromodomain of hPc2. Chromatin immunoprecipitation analysis revealed that MBD1 and hPc2 were present in silenced Homeobox A (HOXA) genes which could be reactivated by knockdown of either MBD1 or hPc2, suggesting that MBD1 and hPc2 cooperate for transcriptional repression of HOXA genes. In the nuclei of HeLa cells, MBD1 existed in close association with these PcG proteins in some heterochromatin foci, whereas an MBD1 mutant lacking the CXXC domains or an hPc2 mutant lacking the chromodomain lost this colocalization in foci. Use of the DNA demethylating agent 5-azadeoxycytidine abolished the formation of MBD1 foci but not PcG foci. Knockdown of MBD1 by small interfering RNAs did not affect the foci containing hPc2 and Ring1b, whereas the MBD1 foci were not influenced by knockdown of hPc2. These indicate that the heterochromatin foci showing MBD1 and hPc2 colocalization arise through the interaction of MBD1 and hPc2 and that the foci of MBD1 are separable from those of the PcG proteins per se. Our present findings suggest that MBD1 and PcG proteins have overlapping roles in epigenetic gene silencing and heterochromatin foci formation through their interactions.

Class A scavenger receptor (CD204) attenuates hyperoxia-induced lung injury by reducing oxidative stress.

To clarify the role of macrophage class A scavenger receptors (SR‐A, CD204) in oxidative lung injury, we examined lung tissue of SR‐A deficient (SR‐A−/−) and wild‐type (SR‐A+/+) mice in response to hyperoxic treatment. Protein levels of bronchoalveolar lavage fluid (BALF) and pulmonary oedema (wet : dry weight ratios) were higher in SR‐A−/− mice than those in SR‐A+/+ mice. Cumulative survival was significantly decreased in SR‐A−/− mice. However, there were no differences in BALF macrophage and neutrophil count between the two groups. Real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) revealed that messenger RNA (mRNA) levels of the inducible nitric oxide synthase (iNOS) were increased during hyperoxic injury, and this increase was more prominent in SR‐A−/− mice. Expression levels of iNOS in alveolar macrophages after hyperoxia in vivo and in vitro were higher in SR‐A−/− macrophages compared with SR‐A+/+ macrophages. Immunohistochemistry using anti‐nitrotyrosine antibodies revealed distinctive oxidative stress in the injured lung in both groups, but it was more remarkable in the SR‐A−/− mice. After hyperoxic treatment, pulmonary mRNA levels of tumour necrosis factor‐α(TNF‐α) were elevated more rapidly in SR‐A−/− mice than in SR‐A+/+ mice. Together these results suggest that SR‐A expression attenuates hyperoxia‐induced lung injury by reducing macrophage activation. Copyright

Modified gastric pull-up reconstructions following pharyngolaryngectomy with total esophagectomy

Reconstruction following pharyngolaryngectomy with total esophagectomy is a challenging surgery to perform. Between April 2008 and August 2012, three types of modified gastric pull-up reconstruction procedures, including a gastric tube creation combined with a free jejunal transfer (n = 7), elongated gastric tube creation with vascular anastomoses (n = 2) and pedunculated gastric tube creation with Roux-en-Y anastomosis (n = 5), were performed after pharyngolaryngectomy with total esophagectomy. To clarify feasibility of these reconstructive methods, we retrospectively analyzed the short-term outcomes. There were no graft failures. Salivary fistulae were observed in two cases after high pharyngoenteral anastomoses due to oropharyngeal extension of hypopharyngeal cancers. Overall morbidity rate was 21.4%, and no deaths occurred. Although the operation time was shortest for pedunculated gastric tube reconstructions, morbidity rates were similar among all methods. All three types of modified gastric pull-up reconstruction procedures can be performed safely. We can choose one of these methods according to the tumor status and the patient condition, understanding advantages and disadvantages of each procedure.

Different roles of Foxo1 and Foxo3 in the control of endothelial cell morphology

Foxo1, a member of the Foxo subfamily of forkhead box transcription factors, is known to be essential for progression of normal vascular development in the mouse embryos. In the cultures of endothelial cells derived from embryonic stem cells, Foxo1‐deficient endothelial cells exhibit an abnormal morphological response to vascular endothelial growth factor‐A (VEGF‐A), which is characterized by a lack of cell elongation, yet the molecular mechanisms governing endothelial cell morphology under angiogenic stimulation remain unknown. Here, we report that transforming growth actor‐β also induces endothelial cell elongation in collaboration with Foxo1 and VEGF‐A. Furthermore, tetracycline‐regulated induction of Foxo3, another member of the Foxo subfamily, into Foxo1‐null endothelial cells failed to restore abnormal morphological response to VEGF‐A at an early differentiation stage. In contrast, Foxo1 and Foxo3 exerted the same function at a late differentiation stage, i.e. enhancement of VEGF responsiveness and promotion of cell elongation. Our results provide evidence that endothelial cell morphology is regulated by several mechanisms in which Foxo1 and Foxo3 express distinct functional properties depending on differentiation stages.