Biomedical subjects

Misao Suzuki

Publications and source records attributed to Misao Suzuki.

At least 19 recordsLinked to original sources

PubMed2007

Beta-defensin overexpression induces progressive muscle degeneration in mice.

Defensins comprise a family of cationic antimicrobial peptides characterized by conserved cysteine residues. They are produced in various organs including skeletal muscle and are identified as key elements in the host defense system as potent effectors. At the same time, defensins have potential roles in the regulation of inflammation and, furthermore, can exert cytotoxic effects on several mammalian cells. Here, we developed transgenic mice overexpressing mouse beta-defensin-6 to explore the pathophysiological roles of the defensin family as a novel mediator of inflammatory tissue injury. Unexpectedly, the transgenic mice showed short lifespan, poor growth, and progressive myofiber degeneration with functional muscle impairment, predominant centronucleated myofibers, and elevated serum creatine kinase activity, as seen in human muscular dystrophy. Furthermore, some of the transgenic myofibers showed IkappaBalpha accumulation, which would be related to the myofiber apoptosis of limb-girdle muscular dystrophy type 2A. The present findings may unravel a concealed linkage between the innate immune system and the pathophysiology of degenerative diseases.

Aging↗

PubMed2006

A neuronal identity code for the odorant receptor-specific and activity-dependent axon sorting.

In the mouse, olfactory sensory neurons (OSNs) expressing the same odorant receptor (OR) converge their axons to a specific set of glomeruli in the olfactory bulb. To study how OR-instructed axonal fasciculation is controlled, we searched for genes whose expression profiles are correlated with the expressed ORs. Using the transgenic mouse in which the majority of OSNs express a particular OR, we identified such genes coding for the homophilic adhesive molecules Kirrel2/Kirrel3 and repulsive molecules ephrin-A5/EphA5. In the CNGA2 knockout mouse, where the odor-evoked cation influx is disrupted, Kirrel2 and EphA5 were downregulated, while Kirrel3 and ephrin-A5 were upregulated, indicating that these genes are transcribed in an activity-dependent manner. Mosaic analysis demonstrated that gain of function of these genes generates duplicated glomeruli. We propose that a specific set of adhesive/repulsive molecules, whose expression levels are determined by OR molecules, regulate the axonal fasciculation of OSNs during the process of glomerular map formation.

Animals↗

PubMed2006

Odorant receptor-derived cAMP signals direct axonal targeting.

In mammals, odorant receptors (ORs) direct the axons of olfactory sensory neurons (OSNs) toward targets in the olfactory bulb. We show that cyclic adenosine monophosphate (cAMP) signals that regulate the expression of axon guidance molecules are essential for the OR-instructed axonal projection. Genetic manipulations of ORs, stimulatory G protein, cAMP-dependent protein kinase, and cAMP response element-binding protein shifted the axonal projection sites along the anteriorposterior axis in the olfactory bulb. Thus, it is the OR-derived cAMP signals, rather than direct action of OR molecules, that determine the target destinations of OSNs.

Animals↗

PubMed2006

Smooth muscle-specific dystrophin expression improves aberrant vasoregulation in mdx mice.

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disease caused by mutations of the gene encoding the cytoskeletal protein dystrophin. Therapeutic options for DMD are limited because the pathogenetic mechanism by which dystrophin deficiency produces the clinical phenotype remains obscure. Recent reports of abnormal alpha-adrenergic vasoregulation in the exercising muscles of DMD patients and in the mdx mouse, an animal model of DMD, prompted us to hypothesize that the dystrophin-deficient smooth muscle contributes to the vascular and dystrophic phenotypes of DMD. To test this, we generated transgenic mdx mice that express dystrophin only in smooth muscle (SMTg/mdx). We found that alpha-adrenergic vasoconstriction was markedly attenuated in the contracting hindlimbs of C57BL/10 wild-type mice, an effect that was mediated by nitric oxide (NO) and was severely impaired in the mdx mice. SMTg/mdx mice showed an intermediate phenotype, with partial restoration of the NO-dependent modulation of alpha-adrenergic vasoconstriction in active muscle. In addition, the elevated serum creatine kinase levels observed in mdx mice were significantly reduced in SMTg/mdx mice. This is the first report of a functional role of dystrophin in vascular smooth muscle.

Animals↗

PubMed2005

A novel murine gene, Sickle tail, linked to the Danforth's short tail locus, is required for normal development of the intervertebral disc.

We established the mutant mouse line, B6;CB-SktGtAyu8021IMEG (SktGt), through gene-trap mutagenesis in embryonic stem cells. The novel gene identified, called Sickle tail (Skt), is composed of 19 exons and encodes a protein of 1352 amino acids. Expression of a reporter gene was detected in the notochord during embryogenesis and in the nucleus pulposus of mice. Compression of some of the nuclei pulposi in the intervertebral discs (IVDs) appeared at embryonic day (E) 17.5, resulting in a kinky-tail phenotype showing defects in the nucleus pulposus and annulus fibrosus of IVDs in SktGt/Gt mice. These phenotypes were different from those in Danforth's short tail (Sd) mice in which the nucleus pulposus was totally absent and replaced by peripheral fibers similar to those seen in the annulus fibrosus in all IVDs. The Skt gene maps to the proximal part of mouse chromosome 2, near the Sd locus. The genetic distance between them was 0.95 cM. The number of vertebrae in both [Sd +/+ SktGt] and [Sd SktGt/+ +] compound heterozygotes was less than that of Sd heterozygotes. Furthermore, the enhancer trap locus Etl4lacZ, which was previously reported to be an allele of Sd, was located in the third intron of the Skt gene.

Amino Acid Sequence↗

PubMed2005

Expression of the mouse Fgf15 gene is directly initiated by Sonic hedgehog signaling in the diencephalon and midbrain.

Sonic hedgehog (Shh) is a secreted molecule that is thought to regulate tissue growth and patterning in vertebrate embryos. Although it has been reported that Gli transcription factors mediate Shh signaling to the nucleus, little is known about developmental target genes of Gli. In the previous genetic study, we showed that Shh is required for Fgf15 expression in the diencephalon and midbrain. Here, we examined whether Fgf15 is a direct target of Shh signaling through Gli. Shh was expressed in the midline cells and Fgf15 in the medial region of the diencephalon/midbrain by the seven-somite stage. The Fgf15 expression domain coincided with that of Gli1 and overlapped with that of Gli2 at this stage. Fgf15 expression in the diencephalon/midbrain was greatly reduced in the seven-somite Shh mutant embryos. Transgenic analysis showed that the 3.6-kb 5'-flanking region of the Fgf15 gene is sufficient for induction of Fgf15 in the medial/ventral diencephalon/midbrain. Luciferase assay showed that the 3.6-kb Fgf15 enhancer/promoter was activated by Gli2. A Gli-binding site was located 1 kb upstream of the transcription start site and was required for expression in the medial/ventral diencephalon/midbrain in transgenic embryos and for activation in luciferase assay. These findings indicate that Fgf15 is directly regulated by Shh signaling through Gli proteins.

Amino Acid Motifs↗

PubMed2004

Cre-loxP-controlled periodic Aurora-A overexpression induces mitotic abnormalities and hyperplasia in mammary glands of mouse models.

Aurora-A, a serine/threonine mitotic kinase, was reported to be overexpressed in various human cancers, and its overexpression induces aneuploidy, centrosome amplification and tumorigenic transformation in cultured human and rodent cells. However, the underlying mechanisms and pathological settings by which Aurora-A promotes tumorigenesis are largely unknown. Here, we created a transgenic mouse model to investigate the involvement of Aurora-A overexpression in the development of mammary glands and tumorigenesis using a Cre-loxP system. The conditional expression of Aurora-A resulted in significantly increased binucleated cell formation and apoptosis in the mammary epithelium. The surviving mammary epithelial cells composed hyperplastic areas after a short latency. Induction of Aurora-A overexpression in mouse embryonic fibroblasts prepared from the transgenic mice also led to aberrant mitosis and binucleated cell formation followed by apoptosis. The levels of p53 protein were remarkably increased in these Aurora-A-overexpressing cells, and the apoptosis was significantly suppressed by deletion of p53. Given that no malignant tumor formation was found in the Aurora-A-overexpressing mouse model after a long latency, additional factors, such as p53 inactivation, are required for the tumorigenesis of Aurora-A-overexpressing mammary epithelium. Our findings indicated that this mouse model is a useful system to study the physiological roles of Aurora-A and the genetic pathways of Aurora-A-induced carcinogenesis.

Animals↗

PubMed2004

Human parvovirus B19 transgenic mice become susceptible to polyarthritis.

Human parvovirus B19 (B19) often causes acute polyarthritis in adults. In this paper, we analyzed nucleotide sequences of the B19 genome of patients with rheumatoid arthritis (RA), and then introduced the nonstructural protein 1 (NS1) gene of B19 into C57BL/6 mice that had a genetic origin not susceptible to arthritis. The transgenic mice developed no lesions spontaneously, but were susceptible to type II collagen (CII)-induced arthritis. B19 NS1 was expressed in synovial cells on the articular lesions that were histologically characteristic of granulomatous synovitis and pannus formation in cartilage and bone. Serum levels of anti-CII Abs and TNF-alpha increased in NS1 transgenic mice to the same levels as those of DBA/1 mice, which were susceptible to polyarthritis. Stimulation with CII increased secretion of Th1-type- and Th2-type cytokines in NS1 transgenic mice, indicating that a nonpermissive H-2(b) haplotype in the wild type of C57BL/6 mice can be made susceptible to polyarthritis through the expression of NS1. This study is the first to show that a viral agent from the joints in humans can cause CII-induced arthritis resembling RA.

Animals↗

PubMed2004

Conditional activation of RhoA suppresses the epithelial to mesenchymal transition at the primitive streak during mouse gastrulation.

Gastrulation is a pivotal event of mouse early embryogenesis. In telencephalin (TLCN)-Cre mice carrying the Cre recombinase gene inserted into the translational initiation site of the TLCN gene, Cre-mediated recombination took place at the postimplantation stage. To examine the role of RhoA signaling in early embryogenesis, we produced Rho36 mice carrying constitutively active RhoA(G14V) gene inducible by Cre recombinase and crossed with TLCN-Cre mice. In doubly transgenic embryos at the gastrulation stage, there appeared an abnormal bulge of cells protruded from the primitive streak region into the amniotic cavity. The bulged cell mass expressed the epiblast marker gene Oct3 and E-cadherin, but not the primitive streak marker gene T except for the basal portion. These results suggest that the conditional activation of RhoA signaling suppressed the epithelial to mesenchymal transition at the primitive streak during mouse gastrulation.

Allantois↗

PubMed2004

Cysteine 10 is a key residue in amyloidogenesis of human transthyretin Val30Met.

Type I familial amyloidotic polyneuropathy (FAP), a systemic amyloidosis, is characterized by aggregation of variant transthyretin (TTR Val30Met) into stable, insoluble fibrils. This aggregation is caused by genetic and environmental factors. Genetic factors have been studied extensively. However, little is known about environmental or physiological factors involved in the disease process, and their identification may be important for development of effective treatment. X-ray crystallography of normal and amyloidogenic human TTR Val30Met in type I FAP showed that the -SH side chain of cysteine at position 10 (Cys10) forms a hydrogen bond with Gly57 in normal TTR but not in TTR Val30Met. This result suggests a crucial role for the free Cys10 residue and possible involvement of physiological factors affecting Cys residue reactivity in TTR amyloidogenesis. To analyze amyloidogenesis in vivo, our group generated murine FAP models by transgenic technology, with human TTR Val30Met. The three lines of transgenic mice expressed amyloidogenic mutant TTR (Cys10/Met30), wild-type TTR (Cys10/Val30), and artificial Cys-free mutant TTR (Ser10/Met30). Histochemical investigation showed deposition of amyloid derived from human TTR only in amyloidogenic mutant TTR (Cys10/Met30) mice. Thus, the -SH residue in Cys10 plays a crucial role in TTR Val30Met amyloidogenesis in vivo. These data suggest the possibility of innovative treatment via physiological factors modulating Cys10 residue reactivity.

Amyloidosis, Familial↗

PubMed2004

Autoimmunity and glomerulonephritis in mice with targeted deletion of the serum amyloid P component gene: SAP deficiency or strain combination?

Human serum amyloid P component (SAP) binds avidly to DNA, chromatin and apoptotic cells in vitro and in vivo. 129/Sv x C57BL/6 mice with targeted deletion of the SAP gene spontaneously develop antinuclear autoantibodies and immune complex glomerulonephritis. SAP-deficient animals, created by backcrossing the 129/Sv SAP gene deletion into pure line C57BL/6 mice and studied here for the first time, also spontaneously developed broad spectrum antinuclear autoimmunity and proliferative immune complex glomerulonephritis but without proteinuria, renal failure, or increased morbidity or mortality. Mice hemizygous for the SAP gene deletion had an intermediate autoimmune phenotype. Injected apoptotic cells and isolated chromatin were more immunogenic in SAP(-/-) mice than in wild-type mice. In contrast, SAP-deficient pure line 129/Sv mice did not produce significant autoantibodies either spontaneously or when immunized with extrinsic chromatin or apoptotic cells, indicating that loss of tolerance is markedly strain dependent. However, SAP deficiency in C57BL/6 mice only marginally affected plasma clearance of exogenous chromatin and had no effect on distribution of exogenous nucleosomes between the liver and kidneys, which were the only tissue sites of catabolism. Furthermore, transgenic expression of human SAP in the C57BL/6 SAP knockout mice did not abrogate the autoimmune phenotype. This may reflect the different binding affinities of mouse and human SAP for nuclear autoantigens and/or the heterologous nature of transgenic human SAP in the mouse. Alternatively, the autoimmunity may be independent of SAP deficiency and caused by expression of 129/Sv chromosome 1 genes in the C57BL/6 background.

Animals↗

PubMed2004

Dilated cardiomyopathy caused by aberrant endoplasmic reticulum quality control in mutant KDEL receptor transgenic mice.

Aberrant protein folding beyond the capacity of endoplasmic reticulum (ER) quality control leads to stress response in the ER. The Lys-Asp-Glu-Leu (KDEL) receptor, a retrieval receptor for ER chaperones in the early secretory pathway, contributes to ER quality control. To elucidate the function of the KDEL receptor in vivo, we established transgenic mice expressing a mutant KDEL receptor. We found that the mutant KDEL receptor sensitized cells to ER stress and that the mutant mice developed dilated cardiomyopathy. Ultrastructural analyses revealed expanded sarcoplasmic reticulums and protein aggregates that obstructed the adjacent transverse tubules of the mutant cardiomyocytes. Cardiomyocytes from the mutant mice were sensitive to ER stress when treated with tunicamycin and showed a functional defect in the L-type Ca(2+) current. We observed ubiquitinated protein aggregates, enhanced expression of CHOP (a death-related transcriptional factor expressed upon ER stress), and apoptosis in the mutant hearts. These findings suggest that impairment of the KDEL receptor disturbs ER quality control, resulting in accumulation of misfolded proteins in the ER in an in vivo system, and that the dilated cardiomyopathy found in the mutant KDEL receptor transgenic mice is associated with ER stress.

Animals↗

PubMed2004

Establishment of an efficient BAC transgenesis protocol and its application to functional characterization of the mouse Brachyury locus.

Transgenesis using large DNA such as YAC or BAC has extended the range of applications in functional genomics. Here we describe an efficient BAC transgenesis protocol using a simple BAC DNA preparation method adopted from YAC DNA purification methods. This method allowed us to isolate BAC DNA from small scale culture of BAC-containing cells in sufficient quantity and purity for microinjection. More than 40 founders have been produced with linearized BAC DNA prepared by this method, and 85% of them contained intact BAC transgenes. In contrast, when circular BAC DNA was injected, an approximately three-fold reduction of transgene integration rate was observed and fewer intact transgene integrations were obtained. A line of transgenic mice carrying a 170-kb BAC clone generated in this way successfully rescued tail and embryonic lethality phenotypes of the mouse Brachyury (T) mutants, further demonstrating the utility of this method in functional analysis of the mouse genome.

Animals↗

PubMed2003

Characterization of the pufferfish Otx2 cis-regulators reveals evolutionarily conserved genetic mechanisms for vertebrate head specification.

The Otx2 gene, containing a highly conserved paired-type homeobox, plays a pivotal role in the development of the rostral head throughout vertebrates. Precise regulation of the temporal and spatial expression of Otx2 is likely to be crucial for proper head specification. However, regulatory mechanisms of Otx2 expression remain largely unknown. In this study, the Otx2 genome of the puffer fish Fugu rubripes, which has been proposed as a model vertebrate owing to its highly compact genome, was cloned. Consistently, Fugu Otx2 possesses introns threefold smaller in size than those of the mouse Otx2 gene. Otx2 mRNA was transcribed after MBT, and expressed in the rostral head region throughout the segmentation and pharyngula periods of wild-type Fugu embryos. To elucidate regulatory mechanisms of Otx2 expression, the expression of Otx2-lacZ reporter genes nearly covering the Fugu Otx2 locus, from -30.5 to +38.5 kb, was analyzed, by generating transgenic mice. Subsequently, seven independent cis-regulators were identified over an expanse of 60 kb; these regulators are involved in the mediation of spatiotemporally distinct subdomains of Otx2 expression. Additionally, these expression domains appear to coincide with local signaling centers and developing sense organs. Interestingly, most domains do not overlap with one another, which implies that cis-regulators for redundant expression may be abolished exclusively in the pufferfish so as to reduce its genome size. Moreover, these cis-regions were also able to direct expression in zebrafish embryos equivalent to that observed in transgenic mice. Further comparative sequence analysis of mouse and pufferfish intergenic regions revealed eight highly conserved elements within these cis-regulators. Therefore, we propose that, in vertebrate evolution, the Otx2 promoter acquires multiple, spatiotemporally specific cis-regulators in order to precisely control highly coordinated processes in head development.

Amino Acid Sequence↗

PubMed2003

Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse.

In the mouse olfactory system, each olfactory sensory neuron (OSN) expresses only one odorant receptor (OR) gene in a monoallelic and mutually exclusive manner. Such expression forms the genetic basis for OR-instructed axonal projection of OSNs to the olfactory bulb of the brain during development. Here, we identify an upstream cis-acting DNA region that activates the OR gene cluster in mouse and allows the expression of only one OR gene within the cluster. Deletion of the coding region of the expressed OR gene or a naturally occurring frame-shift mutation allows a second OR gene to be expressed. We propose that stochastic activation of only one OR gene within the cluster and negative feedback regulation by that OR gene product are necessary to ensure the one receptor-one neuron rule.

Alleles↗

PubMed2003

Generation of a transgenic animal model of hyperthyroid Graves' disease.

Graves' disease (GD) is an organ-specific autoimmune disease characterized by hyperthyroidism. Agonistic anti-thyrotropin receptor antibodies (thyroid-stimulating antibodies, TSAb), which mimic the thyrotropin (TSH) action, are thought to cause GD. The precise immunological mechanism of TSAb production, however, remains elusive. Previous immunization approaches using TSH receptor led to transient hyperthyroidism, but did not seem sufficient for comprehensive understanding of the development of autoimmune responses. To create GD-related autoimmunity in mice, we here generated TSAb-transgenic mice in which a patient-derived TSAb is expressed in B cells. Expression of the human TSAb in mice resulted in various manifestations of hyperthyroidism including increased free thyroxine levels with concomitantly decreased TSH levels, increased thyroid uptake of technetium pertechnetate, hyperthermia and thyroid hyperplasia. We found a correlation between the serum levels of human TSAb immunoglobulin and free thyroxine. In addition, conventional B cells expressing the TSAb were partially deleted in the periphery while B1 cells expressing the TSAb persisted and accumulated in the peritoneal cavity, a finding consistent with previous demonstrations that the maintenance of B1 cells plays an important role in the development of autoimmune diseases. Thus, our transgenic mouse may provide a novel and useful animal model for elucidating the pathogenesis and pathophysiology of GD.

Animals↗

PubMed2003

Myeloproliferative stem cell disorders by deregulated Rap1 activation in SPA-1-deficient mice.

SPA-1 (signal-induced proliferation-associated gene-1) is a principal Rap1 GTPase-activating protein in hematopoietic progenitors. SPA-1-deficient mice developed a spectrum of myeloid disorders that resembled human chronic myelogenous leukemia (CML) in chronic phase, CML in blast crisis, and myelodysplastic syndrome as well as anemia. Preleukemic SPA-1-deficient mice revealed selective expansion of marrow pluripotential hematopoietic progenitors, which showed abnormal Rap1GTP accumulation. Overexpression of an active form of Rap1 promoted the proliferation of normal hematopoietic progenitors, while SPA-1 overexpression markedly suppressed it. Furthermore, restoring SPA-1 gene in a SPA-1-deficient leukemic blast cell line resulted in the dissolution of Rap1GTP accumulation and concomitant loss of the leukemogenicity in vivo. These results unveiled a role of Rap1 in myeloproliferative stem cell disorders and a tumor suppressor function of SPA-1.

Animals↗

PubMed2003

Temperature-dependent, neurotrophic factor-elicited, neuronal differentiation in adrenal chromaffin cell line immortalized with temperature-sensitive SV40 T-antigen.

We established adrenal medullary cell lines from transgenic mice expressing an oncogene, the temperature-sensitive simian virus 40 large T-antigen, under the control of the tyrosine hydroxylase promoter. A clonal cell line, named tsAM5D, conditionally grew at a permissive temperature of 33 degrees C and exhibited the dopaminergic chromaffin cell phenotype as exemplified by the expression pattern of mRNA for catecholamine-synthesizing enzymes and secretory vesicle-associated proteins. tsAM5D cells proliferated at the permissive temperature in response to basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). At a non-permissive temperature of 39 degrees C, bFGF and CNTF acted synergistically to differentiate tsAM5D cells into neuron-like cells. In addition, tsAM5D cells caused to differentiate by bFGF plus CNTF at 39 degrees C became dependent solely on nerve growth factor for their survival and showed markedly enhanced neurite outgrowth. In the presence of bFGF and CNTF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal marker genes including neuron-specific enolase, growth-associated protein-43, microtubule-associated protein 2, neurofilament, and p75 neurotrophin receptor, indicating that the cells underwent neuronal differentiation. Thus, we demonstrated that tsAM5D cells could proliferate at permissive 33 degrees C, and also had the capacity to terminally differentiate into neuron-like cells in response to bFGF and CNTF when the oncogene was inactivated by shifting the temperature to non-permissive 39 degrees C. These results suggest that tsAM5D cells should be a good tool to allow a detailed study of mechanisms regulating neuronal differentiation.

Adrenal Gland Neoplasms↗