Koichiro Abe

ER stress-mediated apoptosis in a new mouse model of osteogenesis imperfecta

Osteogenesis imperfecta is an inherited disorder characterized by increased bone fragility, fractures, and osteoporosis, and most cases are caused by mutations affecting the type I collagen genes. Here, we describe a new mouse model for Osteogenesis imperfecta termed Aga2 (abnormal gait 2) that was isolated from the Munich N-ethyl-N-nitrosourea mutagenesis program and exhibited phenotypic variability, including reduced bone mass, multiple fractures, and early lethality. The causal gene was mapped to Chromosome 11 by linkage analysis, and a C-terminal frameshift mutation was identified in the Col1a1 (procollagen type I, alpha 1) gene as the cause of the disorder. Aga2 heterozygous animals had markedly increased bone turnover and a disrupted native collagen network. Further studies showed that abnormal proα1(I) chains accumulated intracellularly in Aga2/+ dermal fibroblasts and were poorly secreted extracellularly. This was associated with the induction of an endoplasmic reticulum stress-specific unfolded protein response involving upregulation of BiP, Hsp47, and Gadd153 with caspases-12 and −3 activation and apoptosis of osteoblasts both in vitro and in vivo. These studies resulted in the identification of a new model for Osteogenesis imperfecta, and identified a role for intracellular modulation of the endoplasmic reticulum stress-associated unfolded protein response machinery toward osteoblast apoptosis during the pathogenesis of disease.

Enhancement of Intervertebral Disc Cell Senescence by WNT/β-Catenin Signaling–Induced Matrix Metalloproteinase Expression

OBJECTIVE To determine whether intervertebral disc (IVD) cells express β-catenin and to assess the role of the WNT/β-catenin signaling pathway in cellular senescence and aggrecan synthesis. METHODS The expression of β-catenin messenger RNA (mRNA) and protein in rat IVD cells was assessed by using several real-time reverse transcription-polymerase chain reaction, Western blot, immunohistochemical, and immunofluorescence analyses. The effect of WNT/β-catenin on nucleus pulposus (NP) cells was examined by transfection experiments, an MTT assay, senescence-associated β-galactosidase staining, a cell cycle analysis, and a transforming growth factor (TGFβ)/bone morphogenetic protein (BMP) pathway-focused microarray analysis. RESULTS We found that β-catenin mRNA and protein were expressed in discs in vivo and that rat NP cells exhibited increased β-catenin mRNA and protein upon stimulation with lithium chloride, a known activator of WNT signaling. LiCl treatment inhibited the proliferation of NP cells in a time- and dose-dependent manner. In addition, there was an increased level of cellular senescence in LiCl-treated cells. Long-term treatment with LiCl induced cell cycle arrest and promoted subsequent apoptosis in NP cells. Activation of WNT/β-catenin signaling also regulated the expression of aggrecan. We also demonstrated that WNT/β-catenin signaling induced the expression of matrix metalloproteinases (MMPs) and TGFβ in NP cells. CONCLUSION The activation of WNT/β-catenin signaling promotes cellular senescence and may modulate MMP and TGFβ signaling in NP cells. We hypothesize that the activation of WNT/β-catenin signaling may lead to an increased breakdown of the matrix, thereby promoting IVD degeneration.

A transposon-based chromosomal engineering method to survey a large cis-regulatory landscape in mice

A large cis-regulatory landscape is a common feature of vertebrate genomes, particularly at key developmental gene loci with finely tuned expression patterns. Existing genetic tools for surveying large genomic regions of interest spanning over hundreds of kilobases are limited. Here we propose a chromosomal engineering strategy exploiting the local hopping trait of the Sleeping Beauty transposon in the mouse genome. We generated embryonic stem cells with a targeted integration of the transposon vector, carrying an enhancer-detecting lacZ reporter and loxP cassette, into the developmentally critical Pax1 gene locus, followed by efficient local transpositions, nested deletion formation and derivation of embryos by tetraploid complementation. Comparative reporter expression analysis among different insertion/deletion embryos substantially facilitated long-range cis-regulatory element mapping in the genomic neighborhood and demonstrated the potential of the transposon-based approach as a versatile tool for exploration of defined genomic intervals of functional or clinical relevance, such as disease-associated microdeletions.

The relationship between the Wnt/β-catenin and TGF-β/BMP signals in the intervertebral disc cell.

Degeneration of the lumbar intervertebral disc (IVD) is a cause of low back pain. In osteoarthritis patients, an increase in β‐catenin accumulation has been reported. However, the molecular mechanisms involved in IVD remain unclear. In the present study, we examined the relationship of Wnt/β‐catenin and transforming growth factor‐β (TGF‐β)/bone morphogenetic protein (BMP) signals in the IVDs. We found that treatment of nucleus pulposus (NP) cells with the Wnt/β‐catenin activator lithium chloride (LiCl) results in the increased expression of β‐catenin mRNA and protein, and cell proliferation is decreased due to the activation of the Wnt/β‐catenin signals through the suppression of c‐myc and cyclin‐D1. In addition, T‐cell‐specific transcription factor (TCF) promoter activity was found to increase the following stimulation with LiCl alone, and was further increased when BMP2 was added, in comparison to the control group. We further observed the effects of treatment with PD98059, a specific inhibitor of the mitogen‐activated protein kinase pathway, on TCF promoter activity in NP cells. These effects were largely attenuated by PD98059. Moreover, when transfected IVDs were co‐transfected with R‐Smad expression plasmids, there was a significant decrease in TCF reporter activity. We thereafter evaluated the effects of increased Wnt/β‐catenin activity on the transcriptional activity of the Smad binding element (SBE). As a result, LiCl suppressed the activity of SBE reporter activity. The present study demonstrates for the first time that there are opposing effects between the Wnt/β‐catenin and TGF‐β/BMP signals in IVDs, which is consistent with the Wnt/β‐catenin signals contributing to the pathogenesis of IVD degeneration. J. Cell. Physiol. 226: 1139–1148, 2011.

Characterization of phospholipase C gamma enzymes with gain-of-function mutations.

Phospholipase Cγ isozymes (PLCγ1 and PLCγ2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLCγ2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLCγ1 and PLCγ2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLCγ2 without increasing Rac binding. Importantly, the activation of the ALI14-PLCγ2 and corresponding PLCγ1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.

The role of nuclear hormone receptors in cutaneous wound repair

The cutaneous wound repair process involves balancing a dynamic series of events ranging from inflammation, oxidative stress, cell migration, proliferation, survival and differentiation. A complex series of secreted trophic factors, cytokines, surface and intracellular proteins are expressed in a temporospatial manner to restore skin integrity after wounding. Impaired initiation, maintenance or termination of the tissue repair processes can lead to perturbed healing, necrosis, fibrosis or even cancer. Nuclear hormone receptors (NHRs) in the cutaneous environment regulate tissue repair processes such as fibroplasia and angiogenesis. Defects in functional NHRs and their ligands are associated with the clinical phenotypes of chronic non‐healing wounds and skin endocrine disorders. The functional relationship between NHRs and skin niche cells such as epidermal keratinocytes and dermal fibroblasts is pivotal for successful wound closure and permanent repair. The aim of this review is to delineate the cutaneous effects and cross‐talk of various nuclear receptors upon injury towards functional tissue restoration. Copyright

A Genetic Screen for Modifiers of the Delta1-Dependent Notch Signaling Function in the Mouse

The Notch signaling pathway is an evolutionarily conserved transduction pathway involved in embryonic patterning and regulation of cell fates during development. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, which are also involved in distinct human diseases. Delta1 is one of the known ligands of the Notch receptors. Mice homozygous for a loss-of-function allele of the Delta1 gene Dll1lacZ/lacZ die during embryonic development. Here, we present the results of two phenotype-driven modifier screens. Heterozygous Dll1lacZ knockout animals were crossed with ENU-mutagenized mice and screened for dysmorphological, clinical chemical, and immunological variants that are dependent on the Delta1 loss-of-function allele. First, we show that mutagenized heterozygous Dll1lacZ offspring have reduced body weight and altered specific clinical chemical parameters, including changes in metabolites and electrolytes relevant for kidney function. In our mutagenesis screen we have successfully generated 35 new mutant lines. Of major interest are 7 mutant lines that exhibit a Dll1lacZ/+-dependent phenotype. These mutant mouse lines provide excellent in vivo tools for studying the role of Notch signaling in kidney and liver function, cholesterol and iron metabolism, cell-fate decisions, and during maturation of T cells in the immune system.

A novel N‐ethyl‐N‐nitrosourea–induced mutation in phospholipase Cγ2 causes inflammatory arthritis, metabolic defects, and male infertility in vitro in a murine model

OBJECTIVE It is difficult to identify a single causative factor for inflammatory arthritis because of the multifactorial nature of the disease. This study was undertaken to dissect the molecular complexity of systemic inflammatory disease, utilizing a combined approach of mutagenesis and systematic phenotype screening in a murine model. METHODS In a large-scale N-ethyl-N-nitrosourea mutagenesis project, the Ali14 mutant mouse strain was established because of dominant inheritance of spontaneous swelling and inflammation of the hind paws. Genetic mapping and subsequent candidate gene sequencing were conducted to find the causative gene, and systematic phenotyping of Ali14/+ mice was performed in the German Mouse Clinic. RESULTS A novel missense mutation in the phospholipase Cγ2 gene (Plcg2) was identified in Ali14/+ mice. Because of the hyperreactive external entry of calcium observed in cultured B cells and other in vitro experiments, the Ali14 mutation is thought to be a novel gain-of-function allele of Plcg2. Findings from systematic screening of Ali14/+ mice demonstrated various phenotypic changes: an abnormally high T cell:B cell ratio, up-regulation of Ig, alterations in body composition, and a reduction in cholesterol and triglyceride levels in peripheral blood. In addition, spermatozoa from Ali14/+ mice failed to fertilize eggs in vitro, despite the normal fertility of the Ali14/+ male mice in vivo. CONCLUSION These results suggest that the Plcg2-mediated pathways play a crucial role in various metabolic and sperm functions, in addition to initiating and maintaining the immune system. These findings may indicate the importance of the Ali14/+ mouse strain as a model for systemic inflammatory diseases and inflammation-related metabolic changes in humans.

Arterial spin labeling in patients with chronic cerebral artery steno-occlusive disease: correlation with 15O-PET

Background Heterogeneity of arterial transit time due to cerebral artery steno-occlusive lesions hampers accurate regional cerebral blood flow measurement by arterial spin labeling (ASL). Purpose To assess the feasibility of regional cerebral blood flow measurement by ASL with multiple-delay time sampling in patients with steno-occlusive diseases by comparing with positron emission tomography (PET), and to determine whether regional arterial transit time measured by this ASL technique is correlated with regional mean transit time, a PET index of perfusion pressure. Material and Methods Sixteen patients with steno-occlusive diseases received both ASL and 15O-PET. The mean regional cerebral blood flow measured by ASL and PET, regional arterial transit time by ASL, and regional mean transit time by PET were obtained by a region-of-interest analysis. Correlation between regional cerebral blood flow by ASL and that by PET, and correlation between regional arterial transit time by ASL and regional mean transit time by PET were tested using Pearsons correlation coefficient for both absolute and relative values. A multivariate regression analysis was performed to test whether regional arterial transit time by ASL was a significant contributor in modeling regional mean transit time by PET after controlling the effect of regional cerebral blood flow by ASL. Results A significant positive correlation was found between regional cerebral blood flow by ASL and that by PET for both absolute (r = 0.520, P < 0.0001) and relative (r = 0.691, P < 0.0001) values. A significant positive correlation was found between regional arterial transit time by ASL and regional mean transit time by PET both for absolute (r = 0.369, P = 0.0002) and relative (r = 0.443, P < 0.0001) values. The regression analysis revealed that regional arterial transit time by ASL was a significant contributor in modeling regional mean transit time by PET after controlling regional cerebral blood flow by ASL (P = 0.0011). Conclusion The feasibility of regional cerebral blood flow measurement using ASL with multiple-delay time sampling was confirmed in patients with cerebral artery steno-occlusive diseases. Moreover, it was suggested that mapping of regional arterial transit time has the potential to detect hemodynamic impairment.

New ENU-induced semidominant mutation, Ali18, causes inflammatory arthritis, dermatitis, and osteoporosis in the mouse

Inflammation is a complex cellular and humoral response against trauma and infection, and its presence leads to destruction of tissue in humans. The mechanisms that initiate inflammatory diseases remain largely unknown because of complex interactions between multiple genetic and environmental factors during pathogenesis. Animal models for human diseases offer dissection of complex pathogenesis by inbred genetic backgrounds and controlled circumstances. In this article we report a chemically induced new mutation, Ali18 (Abnormal limb), as a mouse model for inflammatory arthritis and dermatitis. Ali18/+ mice exhibit rubor and swelling of footpads in hindlimbs in adults. In Ali18/Ali18 mice, the digits in forelimbs and hindlimbs and tails were necrotic and/or deformed by severe swelling. Histologic analysis revealed infiltration of mixed populations of inflammatory cells into bone marrow, peripheral joints, and skin in the affected areas of Ali18/Ali18 mice. In addition, generalized osteoporosis-like phenotypes were confirmed by dual energy X-ray absorptiometry (DXA), microcomputed tomography (μCT), and peripheral quantitative computed tomography (pQCT) in homozygous animals. Whereas the Ali18 mutation was mapped to a single locus, the phenotype presentation was altered by complex modifier effects from other inbred genetic backgrounds. Detailed analysis of the Ali18 phenotype and identification of the mutation and its modifier genes may provide molecular insights into the complex nature of inflammatory diseases and the relationship between inflammation and bone metabolism.