Publications by authors named "Naoyuki Miura"

54 Publications

Carcinoma cells that have undergone an epithelial-mesenchymal transition differentiate into endothelial cells and contribute to tumor growth.

Oncotarget 2021 Apr 13;12(8):823-844. Epub 2021 Apr 13.

Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Hypoxia stimulates neoangiogenesis, promoting tumor outgrowth, and triggers the epithelial-mesenchymal transition (EMT), which bestows cells with mesenchymal traits and multi-lineage differentiation potential. Here, we investigated whether EMT can confer endothelial attributes upon carcinoma cells, augmenting tumor growth and vascularization. Following orthotopic implantation of MCF-7 human epithelial breast cancer cells into mice, tumors of different sizes were immunostained for markers of hypoxia and EMT. Larger tumors were well-vascularized with CD31-positive cells of human origin. Hypoxic regions, demarcated by HIF-1α staining, exhibited focal areas of E-cadherin loss and elevated levels of vimentin and the EMT-mediator FOXC2. Implantation of MCF-7 cells, co-mixed with human mammary epithelial (HMLE) cells overexpressing the EMT-inducer Snail, markedly potentiated tumor growth and vascularization, compared with MCF-7 cells injected alone or co-mixed with HMLE-vector cells. Intra-tumoral vessels contained CD31-positive cells derived from either donor cell type. FOXC2 knockdown abrogated the potentiating effects of HMLE-Snail cells on MCF-7 tumor growth and vascularization, and compromised endothelial transdifferentiation of mesenchymal cells cultured in endothelial growth medium. Hence, cells that have undergone EMT can promote tumor growth and neovascularization either indirectly, by promoting endothelial transdifferentiation of carcinoma cells, or directly, by acquiring an endothelial phenotype, with FOXC2 playing key roles in these processes.
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http://dx.doi.org/10.18632/oncotarget.27940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8057273PMC
April 2021

Atypical cadherin FAT4 orchestrates lymphatic endothelial cell polarity in response to flow.

J Clin Invest 2020 06;130(6):3315-3328

Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, South Australia, Australia.

The atypical cadherin FAT4 has established roles in the regulation of planar cell polarity and Hippo pathway signaling that are cell context dependent. The recent identification of FAT4 mutations in Hennekam syndrome, features of which include lymphedema, lymphangiectasia, and mental retardation, uncovered an important role for FAT4 in the lymphatic vasculature. Hennekam syndrome is also caused by mutations in collagen and calcium binding EGF domains 1 (CCBE1) and ADAM metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3), encoding a matrix protein and protease, respectively, that regulate activity of the key prolymphangiogenic VEGF-C/VEGFR3 signaling axis by facilitating the proteolytic cleavage and activation of VEGF-C. The fact that FAT4, CCBE1, and ADAMTS3 mutations underlie Hennekam syndrome suggested that all 3 genes might function in a common pathway. We identified FAT4 as a target gene of GATA-binding protein 2 (GATA2), a key transcriptional regulator of lymphatic vascular development and, in particular, lymphatic vessel valve development. Here, we demonstrate that FAT4 functions in a lymphatic endothelial cell-autonomous manner to control cell polarity in response to flow and is required for lymphatic vessel morphogenesis throughout development. Our data reveal a crucial role for FAT4 in lymphangiogenesis and shed light on the mechanistic basis by which FAT4 mutations underlie a human lymphedema syndrome.
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http://dx.doi.org/10.1172/JCI99027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260025PMC
June 2020

Foxc2 knock-in mice mark stage-specific Foxc2-expressing cells during mouse organogenesis.

Congenit Anom (Kyoto) 2017 Jan;57(1):24-31

Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Japan.

Foxc2, a member of the winged helix transcription factor family, is essential for eye, calvarial bone, cardiovascular and kidney development in mice. Nevertheless, how Foxc2-expressing cells and their descendent cells contribute to the development of these tissues and organs has not been elucidated. Here, we generated a Foxc2 knock-in (Foxc2 ) mouse, in which administration of estrogen receptor antagonist tamoxifen induces nuclear translocation of Cre recombinase in Foxc2-expressing cells. By crossing with ROSA-LacZ reporter mice (Foxc2 ; R26R), the fate of Foxc2 positive (Foxc2 ) cells was analyzed through LacZ staining at various embryonic stages. We found Foxc2 cell descendants in the supraoccipital and exoccipital bone in E18.5 embryos, when tamoxifen was administered at embryonic day (E) 8.5. Furthermore, Foxc2 descendant cranial neural crest cells at E8-10 were restricted to the corneal mesenchyme, while Foxc2 cell derived cardiac neural crest cells at E6-12 were found in the aorta, pulmonary trunk and valves, and endocardial cushions. Foxc2 cell descendant contributions to the glomerular podocytes in the kidney were also observed following E6.5 tamoxifen treatment. Our results are consistent with previous reports of Foxc2 expression during early embryogenesis and the Foxc2 mouse provides a tool to investigate spatiotemporal roles of Foxc2 and contributions of Foxc2 expressing cells during mouse embryogenesis.
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http://dx.doi.org/10.1111/cga.12198DOI Listing
January 2017

FOXC2 augments tumor propagation and metastasis in osteosarcoma.

Oncotarget 2016 Oct;7(42):68792-68802

Women's Cancer Program at the Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

Osteosarcoma is a highly malignant tumor that contains a small subpopulation of tumor-propagating cells (also known as tumor-initiating cells) characterized by drug resistance and high metastatic potential. The molecular mechanism by which tumor-propagating cells promote tumor growth is poorly understood. Here, we report that the transcription factor forkhead box C2 (FOXC2) is frequently expressed in human osteosarcomas and is important in maintaining osteosarcoma cells in a stem-like state. In osteosarcoma cell lines, we show that anoikis conditions stimulate FOXC2 expression. Downregulation of FOXC2 decreases anchorage-independent growth and invasion in vitro and lung metastasis in vivo, while overexpression of FOXC2 increases tumor propagation in vivo. In osteosarcoma cell lines, we demonstrate that high levels of FOXC2 are associated with and required for the expression of osteosarcoma tumor-propagating cell markers. In FOXC2 knockdown cell lines, we show that CXCR4, a downstream target of FOXC2, can restore osteosarcoma cell invasiveness and metastasis to the lung.
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http://dx.doi.org/10.18632/oncotarget.11990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5356590PMC
October 2016

Data supporting the regulation of FOXC2 in podocyte dysfunction.

Data Brief 2016 Mar 7;6:514-20. Epub 2016 Jan 7.

Department of Pathology, University of Helsinki, 00290 Helsinki, Finland.

This data article shows the expression levels of specific podocyte injury markers and podocyte slit diaphragm protein nephrin in obese and lean Zucker rat glomeruli. It also contains information on the effect of the overexpression of transcription factor FOXC2 on the ratio of F- and G-actin and the expression level of ZO-1 in differentiated human podocytes. The article also shows data on the effect of treatments of differentiated podocytes with various factors associated with obesity and diabetes on the expression level of FOXC2. The detailed interpretation of these data and other aspects of podocyte injury mediated by upregulation of FOXC2 can be found in "Overexpression of transcription factor FOXC2 in cultured human podocytes upregulates injury markers and increases motility [1].
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http://dx.doi.org/10.1016/j.dib.2015.12.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4724709PMC
March 2016

Multiple mouse models of primary lymphedema exhibit distinct defects in lymphovenous valve development.

Dev Biol 2016 Jan 2;409(1):218-233. Epub 2015 Nov 2.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA; Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. Electronic address:

Lymph is returned to the blood circulation exclusively via four lymphovenous valves (LVVs). Despite their vital importance, the architecture and development of LVVs is poorly understood. We analyzed the formation of LVVs at the molecular and ultrastructural levels during mouse embryogenesis and identified three critical steps. First, LVV-forming endothelial cells (LVV-ECs) differentiate from PROX1(+) progenitors and delaminate from the luminal side of the veins. Second, LVV-ECs aggregate, align perpendicular to the direction of lymph flow and establish lympho-venous connections. Finally, LVVs mature with the recruitment of mural cells. LVV morphogenesis is disrupted in four different mouse models of primary lymphedema and the severity of LVV defects correlate with that of lymphedema. In summary, we have provided the first and the most comprehensive analysis of LVV development. Furthermore, our work suggests that aberrant LVVs contribute to lymphedema.
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http://dx.doi.org/10.1016/j.ydbio.2015.10.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4688075PMC
January 2016

Overexpression of transcription factor FOXC2 in cultured human podocytes upregulates injury markers and increases motility.

Exp Cell Res 2016 Jan 30;340(1):32-42. Epub 2015 Oct 30.

Department of Pathology, University of Helsinki, 00290 Helsinki, Finland. Electronic address:

Obesity and diabetes-related kidney diseases associate with renal failure and cardiovascular morbidity, and represent a major health issue worldwide. However, the molecular mechanisms leading to their development remain poorly understood. We observed increased expression of transcription factor FoxC2 in the podocytes of obese Zucker rats that are insulin resistant and albuminuric. We also found that depletion of adiponectin, an adipocyte-derived hormone whose secretion is decreased in obesity, upregulated FOXC2 in differentiated human podocytes in vitro. Overexpression of FOXC2 in cultured human podocytes led to increased nuclear expression of FOXC2 associated with a change of cellular morphology. This was accompanied by upregulation of vimentin, a key mesenchymal marker, and active beta-catenin, associated with podocyte injury. We also observed re-organization of the actin cytoskeleton, disrupted localization of the tight junction protein ZO-1, and increased motility of podocytes overexpressing FOXC2. These data indicate that the expression of FOXC2 in podocytes needs to be tightly regulated, and that its overexpression induces a chain of cellular events leading to podocyte dysfunction. These changes may lead to podocyte detachment and depletion ultimately contributing to albuminuria. We also suggest a novel molecular mechanism linking obesity-induced decrease in adiponectin to podocyte dysfunction via upregulation of FOXC2.
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http://dx.doi.org/10.1016/j.yexcr.2015.10.035DOI Listing
January 2016

Genetic defects in a His-Purkinje system transcription factor, IRX3, cause lethal cardiac arrhythmias.

Eur Heart J 2016 May 1;37(18):1469-75. Epub 2015 Oct 1.

Department of Bio-Informational Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

Aim: Ventricular fibrillation (VF), the main cause of sudden cardiac death (SCD), occurs most frequently in the acute phase of myocardial infarction: a certain fraction of VF, however, develops in an apparently healthy heart, referred as idiopathic VF. The contribution of perturbation in the fast conduction system in the ventricle, the His-Purkinje system, for idiopathic VF has been implicated, but the underlying mechanism remains unknown. Irx3/IRX3 encodes a transcription factor specifically expressed in the His-Purkinje system in the heart. Genetic deletion of Irx3 provides a mouse model of ventricular fast conduction disturbance without anatomical or contraction abnormalities. The aim of this study was to examine the link between perturbed His-Purkinje system and idiopathic VF in Irx3-null mice, and to search for IRX3 genetic defects in idiopathic VF patients in human.

Methods And Results: Telemetry electrocardiogram recording showed that Irx3-deleted mice developed frequent ventricular tachyarrhythmias mostly at night. Ventricular tachyarrhythmias were enhanced by exercise and sympathetic nerve activation. In human, the sequence analysis of IRX3 exons in 130 probands of idiopathic VF without SCN5A mutations revealed two novel IRX3 mutations, 1262G>C (R421P) and 1453C>A (P485T). Ventricular fibrillation associated with physical activities in both probands with IRX3 mutations. In HL-1 cells and neonatal mouse ventricular myocytes, IRX3 transfection up-regulated SCN5A and connexin-40 mRNA, which was attenuated by IRX3 mutations.

Conclusion: IRX3 genetic defects and resultant functional perturbation in the His-Purkinje system are novel genetic risk factors of idiopathic VF, and would improve risk stratification and preventive therapy for SCD in otherwise healthy hearts.
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http://dx.doi.org/10.1093/eurheartj/ehv449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914882PMC
May 2016

FOXC2 and fluid shear stress stabilize postnatal lymphatic vasculature.

J Clin Invest 2015 Oct 21;125(10):3861-77. Epub 2015 Sep 21.

Biomechanical forces, such as fluid shear stress, govern multiple aspects of endothelial cell biology. In blood vessels, disturbed flow is associated with vascular diseases, such as atherosclerosis, and promotes endothelial cell proliferation and apoptosis. Here, we identified an important role for disturbed flow in lymphatic vessels, in which it cooperates with the transcription factor FOXC2 to ensure lifelong stability of the lymphatic vasculature. In cultured lymphatic endothelial cells, FOXC2 inactivation conferred abnormal shear stress sensing, promoting junction disassembly and entry into the cell cycle. Loss of FOXC2-dependent quiescence was mediated by the Hippo pathway transcriptional coactivator TAZ and, ultimately, led to cell death. In murine models, inducible deletion of Foxc2 within the lymphatic vasculature led to cell-cell junction defects, regression of valves, and focal vascular lumen collapse, which triggered generalized lymphatic vascular dysfunction and lethality. Together, our work describes a fundamental mechanism by which FOXC2 and oscillatory shear stress maintain lymphatic endothelial cell quiescence through intercellular junction and cytoskeleton stabilization and provides an essential link between biomechanical forces and endothelial cell identity that is necessary for postnatal vessel homeostasis. As FOXC2 is mutated in lymphedema-distichiasis syndrome, our data also underscore the role of impaired mechanotransduction in the pathology of this hereditary human disease.
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http://dx.doi.org/10.1172/JCI80454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4607114PMC
October 2015

Foxc2 in pharyngeal arch mesenchyme is important for aortic arch artery remodelling and ventricular septum formation.

Biomed Res 2015 ;36(4):235-45

Department of Biochemistry, Hamamatsu University School of Medicine.

The forkhead box C2 (Foxc2) protein is a member of the forkhead/winged helix transcription factor family and plays an essential role in cardiovascular development. Previous studies showed that Foxc2 null mouse embryos die during midgestation or just after birth with severe cardiovascular defects, including interruption, coarctation of the aortic arch and ventricular septal defects. These are also seen in human congenital heart disease. However, the tissue specific role of Foxc2 in aortic arch remodelling is not yet fully understood. Here we show that Foxc2 is expressed in a restricted pattern in several cell populations, including the mesenchyme and endothelium of pharyngeal arch arteries, which are important for cardiovascular development. In this study, we use a conditional knockout approach to examine the tissue specific role of Foxc2 in aortic arch remodelling. We demonstrate that mouse embryos lacking Foxc2 in Nkx2.5-expressing mesenchyme and endothelium of pharyngeal arch arteries display aortic arch interruption type B and ventricular septal defects. In contrast, conditional deletion of Foxc2 in Tie2-expressing endothelial cells does not result in aortic arch or ventricular septal defects, but does result in embryonic lethality due to peripheral oedema. Our data therefore provide for a detailed understanding of the role of mesenchymal Foxc2 in aortic arch remodelling and in the development of ventricular septum.
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http://dx.doi.org/10.2220/biomedres.36.235DOI Listing
May 2016

GATA2 is required for lymphatic vessel valve development and maintenance.

J Clin Invest 2015 Aug 27;125(8):2979-94. Epub 2015 Jul 27.

Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema.
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http://dx.doi.org/10.1172/JCI78888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4563742PMC
August 2015

Pol I-transcribed hepatitis C virus genome RNA replicates, produces an infectious virus and leads to severe hepatic steatosis in transgenic mice.

Biomed Res 2015 ;36(3):159-67

Department of Biochemistry, Hamamatsu University School of Medicine.

Patients chronically infected with hepatitis C virus (HCV) are at risk of developing end-stage liver disease and hepatocellular carcinoma. Development of drugs to inhibit hepatocyte damage and a vaccine against HCV is hampered by the lack of a small animal model. We generated mice in which the viral genome RNA was always present in the hepatocytes using a special transgene. Here we show that the HCV genome RNA transcribed by Pol I polymerase can replicate and produce infectious viruses in mice. We obtained a transgenic mouse with 200 copies per haploid which we named the A line mouse. It produced ~ 3 × 10(6) HCV RNA copies/mL serum, which is at the comparable level as patients with chronic HCV infection. This mouse was immunotolerant to HCV and showed hepatic steatosis without any necroinflammation at the age of 6 months or hepatocellular carcinoma at the age of 15 months. Thus, the A line mouse can be used as an animal model for chronic HCV infection. This will enable better study of the abnormalities in metabolism and signal transduction in infected hepatocytes, and development of drugs that cure abnormalities.
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http://dx.doi.org/10.2220/biomedres.36.159DOI Listing
March 2016

Cdk5 controls lymphatic vessel development and function by phosphorylation of Foxc2.

Nat Commun 2015 Jun 1;6:7274. Epub 2015 Jun 1.

Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University, 81377 Munich, Germany.

The lymphatic system maintains tissue fluid balance, and dysfunction of lymphatic vessels and valves causes human lymphedema syndromes. Yet, our knowledge of the molecular mechanisms underlying lymphatic vessel development is still limited. Here, we show that cyclin-dependent kinase 5 (Cdk5) is an essential regulator of lymphatic vessel development. Endothelial-specific Cdk5 knockdown causes congenital lymphatic dysfunction and lymphedema due to defective lymphatic vessel patterning and valve formation. We identify the transcription factor Foxc2 as a key substrate of Cdk5 in the lymphatic vasculature, mechanistically linking Cdk5 to lymphatic development and valve morphogenesis. Collectively, our findings show that Cdk5-Foxc2 interaction represents a critical regulator of lymphatic vessel development and the transcriptional network underlying lymphatic vascular remodeling.
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http://dx.doi.org/10.1038/ncomms8274DOI Listing
June 2015

Effects of Hydroxy Groups in the A-Ring on the Anti-proteasome Activity of Flavone.

Biol Pharm Bull 2015 25;38(6):935-40. Epub 2015 Mar 25.

Department of Biochemistry, Hamamatsu University School of Medicine.

The ubiquitin-proteasome pathway plays an important role in regulating apoptosis and the cell cycle. Recently, proteasome inhibitors have been shown to have antitumor effects and have been used in anticancer therapy for several cancers such as multiple myeloma. Although some flavones, such as apigenin, chrysin and luteolin, have a specific role in the inhibition of proteasome activity and induced apoptosis in some reports, these findings did not address all flavone types. To further investigate the proteasome-inhibitory mechanism of flavonoids, we examined the inhibitory activity of 5,6,7-trihydroxyflavone, baicalein and 5,6,7,4'-tetrahydroxyflavone, scutellarein on extracted proteasomes from mice and cancer cells. Unlike the other flavones, baicalein and scutellarein did not inhibit proteasome activity or accumulate levels of ubiquitinated proteins. These results indicate that flavones with hydroxy groups at positions 5, 6 and 7 of the A-ring lack the anti-proteasome function.
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http://dx.doi.org/10.1248/bpb.b15-00018DOI Listing
February 2016

Control of retinoid levels by CYP26B1 is important for lymphatic vascular development in the mouse embryo.

Dev Biol 2014 Feb 19;386(1):25-33. Epub 2013 Dec 19.

Institute for Molecular Bioscience, The University of Queensland, Brisbane QLD 4072, Australia. Electronic address:

During embryogenesis, lymphatic endothelial progenitor cells first arise from a subset of blood vascular endothelial cells in the dorsolateral aspects of the cardinal veins. The molecular cues responsible for defining the regionalisation of such a discrete pool of progenitors remain uncharacterised. Here we identify a novel function for CYP26B1, an enzyme known to play a role in tissue morphogenesis by fine-tuning retinoic acid (RA) concentration, in regulating lymphangiogenesis. Cyp26b1-null mice, in which RA levels are elevated, exhibited an increased number of lymphatic endothelial progenitor cells in the cardinal veins, together with hyperplastic, blood filled lymph sacs and hyperplastic dermal lymphatic vessels. Conversely, mice over-expressing Cyp26b1 had hypoplastic lymph sacs and lymphatic vessels. Our data suggest that RA clearance by CYP26B1 in the vicinity of lymphatic endothelial progenitor cells is important for determining the position and size of the progenitor pool specified. Our studies identify a genetic pathway that underpins the architecture of the developing lymphatics and define CYP26B1 as a novel modulator of lymphatic vascular patterning.
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http://dx.doi.org/10.1016/j.ydbio.2013.12.008DOI Listing
February 2014

Phosphorylation regulates FOXC2-mediated transcription in lymphatic endothelial cells.

Mol Cell Biol 2013 Oct 22;33(19):3749-61. Epub 2013 Jul 22.

Department of Oncology, CHUV, and Department of Biochemistry, University of Lausanne, Epalinges, Switzerland.

One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in the human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. We report here that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild-type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development.
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http://dx.doi.org/10.1128/MCB.01387-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811871PMC
October 2013

FOXC2 expression links epithelial-mesenchymal transition and stem cell properties in breast cancer.

Cancer Res 2013 Mar 1;73(6):1981-92. Epub 2013 Feb 1.

Department of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

Resistance to chemotherapy and metastases are the major causes of breast cancer-related mortality. Moreover, cancer stem cells (CSC) play critical roles in cancer progression and treatment resistance. Previously, it was found that CSC-like cells can be generated by aberrant activation of epithelial-mesenchymal transition (EMT), thereby making anti-EMT strategies a novel therapeutic option for treatment of aggressive breast cancers. Here, we report that the transcription factor FOXC2 induced in response to multiple EMT signaling pathways as well as elevated in stem cell-enriched factions is a critical determinant of mesenchymal and stem cell properties, in cells induced to undergo EMT- and CSC-enriched breast cancer cell lines. More specifically, attenuation of FOXC2 expression using lentiviral short hairpin RNA led to inhibition of the mesenchymal phenotype and associated invasive and stem cell properties, which included reduced mammosphere-forming ability and tumor initiation. Whereas, overexpression of FOXC2 was sufficient to induce CSC properties and spontaneous metastasis in transformed human mammary epithelial cells. Furthermore, a FOXC2-induced gene expression signature was enriched in the claudin-low/basal B breast tumor subtype that contains EMT and CSC features. Having identified PDGFR-β to be regulated by FOXC2, we show that the U.S. Food and Drug Administration-approved PDGFR inhibitor, sunitinib, targets FOXC2-expressing tumor cells leading to reduced CSC and metastatic properties. Thus, FOXC2 or its associated gene expression program may provide an effective target for anti-EMT-based therapies for the treatment of claudin-low/basal B breast tumors or other EMT-/CSC-enriched tumors.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-2962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3602160PMC
March 2013

Baicalin and scutellarin are proteasome inhibitors that specifically target chymotrypsin-like catalytic activity.

Phytother Res 2013 Sep 12;27(9):1362-7. Epub 2012 Nov 12.

Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Japan.

Baicalin and scutellarin are the major active principal flavonoids extracted from the Chinese herbal medicines Scutellaria baicalensis and Erigeron breviscapus (Vant.) Hand-Mazz. It has recently been reported that baicalin and scutellarin have antitumor activity. However, the mechanisms of action are unknown. We previously reported that some flavonoids have a specific role in the inhibition of the activity of proteasome subunits and induced apoptosis in tumor cells. To further investigate these pharmacological effects, we examined the inhibitory activity of baicalin and scutellarin on the extracted proteasomes from mice and cancer cells. Using fluorogenic substrates for proteasome catalytic subunits, we found that baicalin and scutellarin specifically inhibited chymotrypsin-like activity but did not inhibit trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities. These data suggested that baicalin and scutellarin specifically inhibit chymotrypsin-like catalytic activity in the proteasome.
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http://dx.doi.org/10.1002/ptr.4878DOI Listing
September 2013

Generation and characterization of Tbx1-AmCyan1 transgenic reporter mouse line that selectively labels developing thymus primordium.

Transgenic Res 2013 Jun 2;22(3):659-66. Epub 2012 Nov 2.

Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192, Shizuoka, Japan.

Thymus development is a complicated process that includes highly dynamic morphological changes and reciprocal tissue interactions between endoderm-derived epithelial cells of the anterior foregut and neural crest-derived mesenchymal cells. We generated and characterized a Tbx1-AmCyan1 reporter transgenic mouse to visualize thymus precursor cells during early embryonic development. In transgenic embryos, AmCyan1 fluorescence was specifically detected in the endoderm of the developing 3rd and 4th pharyngeal pouches and later in thymus epithelium until E14.5. Cells expressing AmCyan1 that were isolated based on AmCyan1 fluorescence expressed endodermal, thymic, and parathyroid markers, but they did not express neural crest or endothelial markers; these findings indicated that this transgenic mouse strain could be used to collect thymic or parathyroid precursor cells or both. We also showed that in nude mice, which exhibit defects in thymus development, the thymus precursors were clearly labeled with AmCyan1. In summary, these AmCyan1-fluorescent transgenic mice are useful for investigating early thymus development.
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http://dx.doi.org/10.1007/s11248-012-9664-5DOI Listing
June 2013

The regulation of endogenous retinoic acid level through CYP26B1 is required for elevation of palatal shelves.

Dev Dyn 2012 Nov 25;241(11):1744-56. Epub 2012 Sep 25.

Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Background: In previous studies, we investigated the effects of excess retinoic acid (RA) during palatogenesis by RA administration to pregnant mice. In the present study, we deleted Cyp26b1, one of the RA-degrading enzymes, to further study the effects of excess RA in the normal developing palate and to understand how endogenous levels of RA are regulated.

Results: Excess RA, due to the absence of Cyp26b1, targets cells in the bend region of the palatal shelves and inhibits their horizontal elevation, leading to cleft palate. An organ culture of Cyp26b1-/- palatal shelves after tongue removal did not rescue the impaired elevation of the palatal shelves. The expression of Fgf10, Bmp2, and Tbx1, important molecules in palatal development, was down-regulated. Cell proliferation was decreased in the bend region of palatal shelves. Tongue muscles were hypoplastic and/or missing in Cyp26b1-/- mice.

Conclusions: We demonstrated that CYP26B1 is essential during palatogenesis. Excess RA due to the lack of Cyp26b1 suppresses the expression of key regulators of palate development in the bend region, resulting in a failure in the horizontal elevation of the palatal shelves. The regulation of RA signaling through CYP26B1 is also necessary for the development of tongue musculature and for tongue depression.
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http://dx.doi.org/10.1002/dvdy.23862DOI Listing
November 2012

Screening of natural medicines that efficiently activate neurite outgrowth in PC12 cells in C2C12-cultured medium.

Biomed Res 2012 Feb;33(1):25-33

Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Hamamatsu 431-3192, Japan.

We have studied the effects of natural medicines on neurite outgrowth in PC12D cells in a cultured medium of C2C12 cells. Derived from mouse myoblasts, the C2C12 cells secrete neurotrophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). The secretion of these neurotrophins from C2C12 cells stimulate neurite outgrowth in PC12D cells. We have screened a total of 120 samples and found five natural medicines: Trichosanthes Root, Asiasarum Root, Lycium Bark, Sinomenium Stem, and Dictamni radicis Cortex, that enhance the activity of C2C12-cultured medium to stimulate neurite outgrowth in PC12D cells. These natural medicines promoted not only neurite outgrowth but also stabilized the neurite formation in PC12D cells for several days. RT-PCR analysis showed that NGF was significantly increased with Trichosanthes and Lycium Bark. However, BDNF was slightly decreased with Lycium Bark, Sinomenium Stem, and Dictamni radicis Cortex. NT-3 was increased slightly by all of these natural medicines except Sinomenium Stem. All these five natural medicines significantly increased the number and length of neurites in PC12D cells in co-culture with C2C12 cells.
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http://dx.doi.org/10.2220/biomedres.33.25DOI Listing
February 2012

Mechanotransduction, PROX1, and FOXC2 cooperate to control connexin37 and calcineurin during lymphatic-valve formation.

Dev Cell 2012 Feb 2;22(2):430-45. Epub 2012 Feb 2.

Division of Experimental Oncology, Multidisciplinary Oncology Center, University Hospital of Lausanne, Switzerland.

Lymphatic valves are essential for efficient lymphatic transport, but the mechanisms of early lymphatic-valve morphogenesis and the role of biomechanical forces are not well understood. We found that the transcription factors PROX1 and FOXC2, highly expressed from the onset of valve formation, mediate segregation of lymphatic-valve-forming cells and cell mechanosensory responses to shear stress in vitro. Mechanistically, PROX1, FOXC2, and flow coordinately control expression of the gap junction protein connexin37 and activation of calcineurin/NFAT signaling. Connexin37 and calcineurin are required for the assembly and delimitation of lymphatic valve territory during development and for its postnatal maintenance. We propose a model in which regionally increased levels/activation states of transcription factors cooperate with mechanotransduction to induce a discrete cell-signaling pattern and morphogenetic event, such as formation of lymphatic valves. Our results also provide molecular insights into the role of endothelial cell identity in the regulation of vascular mechanotransduction.
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http://dx.doi.org/10.1016/j.devcel.2011.12.020DOI Listing
February 2012

Liver tumor formation by a mutant retinoblastoma protein in the transgenic mice is caused by an upregulation of c-Myc target genes.

Biochem Biophys Res Commun 2012 Jan 11;417(1):601-6. Epub 2011 Dec 11.

Department of Biochemistry, Hamamatsu University School of Medicine, Higashi-ku, Hamamatsu, Japan.

The retinoblastoma (Rb) tumor suppressor encodes a nuclear phosphoprotein that regulates cellular proliferation, apoptosis and differentiation. In order to adapt itself to these biological functions, Rb is subjected to modification cycle, phosphorylation and dephosphorylation. To directly determine the effect of phosphorylation-resistant Rb on liver development and function, we generated transgenic mice expressing phosphorylation-resistant human mutant Rb (mt-Rb) under the control of the rat hepatocyte nuclear factor-1 gene promoter/enhancer. Expression of mt-Rb in the liver resulted in macroscopic neoplastic nodules (adenomas) with ∼50% incidence within 15 months old. Interestingly, quantitative reverse transcriptase-PCR analysis showed that c-Myc was up-regulated in the liver of mt-Rb transgenic mice irrespective of having tumor tissues or no tumor. In tumor tissues, several c-Myc target genes, Foxm1, c-Jun, c-Fos, Bmi1 and Skp2, were also up-regulated dramatically. We determined whether mt-Rb activated the Myc promoter in the HTP9 cells and demonstrated that mt-Rb acted as an inhibitor of wild-type Rb-induced repression on the Myc promoter. Our results suggest that continued upregulation of c-Myc target genes promotes the liver tumor formation after about 1 year of age.
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http://dx.doi.org/10.1016/j.bbrc.2011.12.014DOI Listing
January 2012

VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling.

Nat Cell Biol 2011 Sep 11;13(10):1202-13. Epub 2011 Sep 11.

Molecular/Cancer Biology Laboratory, Institute for Molecular Medicine Finland, Research Programs Unit and Department of Pathology, Haartman Institute, Biomedicum Helsinki, PO Box 63 (Haartmaninkatu 8), 00014 University of Helsinki, Finland.

Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2(+/-);Vegfr3(+/-) compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261765PMC
http://dx.doi.org/10.1038/ncb2331DOI Listing
September 2011

Gluteal compartment syndrome due to prolonged immobilization after alcohol intoxication: a case report.

Tokai J Exp Clin Med 2011 Jul 20;36(2):25-8. Epub 2011 Jul 20.

Department of Emergency and Critical Care of Medicine, Odawara Municipal Hospital, 49 Kuno, Odawara-City, Kanagawa 250-8558, Japan.

Gluteal compartment syndrome is a relatively rare condition that mostly result from atraumatic causes such as prolonged immobilization due to drug abuse or alcoholic intoxication and incorrect positioning during surgical procedures rather than traumatic causes. Early diagnosis is difficult and sometimes delayed or overlooked because of poor physical signs resulting from altered mental status and inappropriate diagnosis by clinicians. It has been reported that more than half of the cases of gluteal compartment syndrome are associated with crush syndrome and sciatic nerve palsy. Early diagnosis and immediate fasciotomy are necessary to improve the functional prognosis. Here, we report the case of a patient with gluteal compartment syndrome caused by prolonged immobilization after acute alcoholic intoxication. After disease onset, the patient developed complications of crush syndrome and sciatic nerve palsy, but immediate fasciotomy improved his condition.
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July 2011

Expression of human factors CD81, claudin-1, scavenger receptor, and occludin in mouse hepatocytes does not confer susceptibility to HCV entry.

Biomed Res 2011 Apr;32(2):143-50

Department of Biochemistry, Hamamatsu University School of Medicine, Handa-yama, Higashi-ku, Hamamatsu, Shizuoka, Japan.

No suitable mouse model is available for studying chronic liver disease caused by hepatitis C virus (HCV). CD81, claudin-1, scavenger receptor class B type I, and occludin were recently reported to be the important factors in HCV entry into hepatocytes. We made transgenic mice (Alb-CCSO) expressing the four human proteins and examined whether HCV from a patient serum or HCV pseudoparticles (HCVpp) were capable of infecting them. HCV was not detected in the mouse serum after injecting the mice with HCV from a patient serum. We also found no indications of HCVpp entry into primary hepatocytes from Alb-CCSO mice. In addition, HCV-infectible Hep3B cells were fused with HCV-resistant primary mouse hepatocytes and the fused cells showed 35-fold lower infectivity compared to wild-type Hep3B cells, indicating that primary mouse hepatocytes have the inhibitory factor(s) in HCVpp entry. Our results suggest that the expression of the human factors does not confer susceptibility to HCV entry into the liver.
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http://dx.doi.org/10.2220/biomedres.32.143DOI Listing
April 2011

Irxl1 mutant mice show reduced tendon differentiation and no patterning defects in musculoskeletal system development.

Genesis 2011 Jan 22;49(1):2-9. Epub 2010 Dec 22.

Irxl1 (Iroquois-related homeobox like-1) is a newly identified three amino-acid loop extension (TALE) homeobox gene, which is expressed in various mesoderm-derived tissues, particularly in the progenitors of the musculoskeletal system. To analyze the roles of Irxl1 during embryonic development, we generated mice carrying a null allele of Irxl1. Mice homozygous for the targeted allele were viable, fertile, and showed reduced tendon differentiation. Skeletal morphology and skeletal muscle weight in Irxl1-knockout mice appeared normal. Expression patterns of several marker genes for cartilage, tendon, and muscle progenitors in homozygous mutant embryos were unchanged. These results suggest that Irxl1 is required for the tendon differentiation but dispensable for the patterning of the musculoskeletal system in development.
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http://dx.doi.org/10.1002/dvg.20688DOI Listing
January 2011

The mouse forkhead gene Foxp2 modulates expression of the lung genes.

Life Sci 2010 Jul 27;87(1-2):17-25. Epub 2010 May 27.

Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu, Japan.

Aims: Foxp2 is expressed in the lung during mouse development. A monoclonal anti-mouse Foxp2 antibody was created to determine the expression pattern in the developing lung. Next, transcriptional control of two lung genes, CC10 and surfactant protein C (SPC) genes, by Foxp2 was investigated in H441 and A549 cells. Thirdly, expression patterns of Foxp2 and Foxf2 were compared in the developing lung. Finally, Foxp2 expression was determined in the Foxf2-null mice.

Main Methods: Immunohistochemical staining and in situ hybridization were applied to the sections of lungs in the developing embryos.

Key Findings: Monoclonal anti-Foxp2 antibody demonstrated that Foxp2 was expressed in the bronchial epithelium at E10.5 and its expression became restricted to the distal portion of the elongating bronchiolar epithelium and finally to type II alveolar epithelial cells around birth and in the adult. Foxp2 activated the SPC gene promoter in the presence of Nkx2.1 in A549 cells while it repressed the CC10 gene promoter in H441 cells. Next, the expression domains of the Foxp2 and Foxf2 were found to be exclusive in the lung. Finally, the expression of Foxp2 did not change in the lung of Foxf2-null mice.

Significance: The Foxp2 protein is expressed in the growing distal edge of airway epithelium. When the bronchiolus elongates, Foxp2 suppresses CC10 expression. When the lung alveolus is formed, Foxp2 modulates the Nkx2.1-mediated SPC expression in type II alveolar cells. Foxp2 and Foxf2 independently play distinct roles in the alveoli and the mesenchyme, respectively.
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http://dx.doi.org/10.1016/j.lfs.2010.05.009DOI Listing
July 2010

A unique expression pattern of Tbx10 in the hindbrain as revealed by Tbx10(LacZ) allele.

Genesis 2010 May;48(5):295-302

To study the expression/function of Tbx10, a T-box gene, Tbx10(LacZ/+) mice were established by replacing the T-box coding region with a LacZ gene. X-gal staining showed that LacZ(+) cells were localized to two-cell populations in rhombomere 4 and rhombomere 6. No significant differences in the locations of LacZ(+) cells were found between Tbx10(LacZ/+) and Tbx10(LacZ/LacZ) mice, and the Tbx10(LacZ/LacZ) mice were viable and fertile. We found that the LacZ(+) cells are present in both embryonic and adult mice. Histological studies suggest that the rhombomere 4-derived LacZ(+) cells are a subpopulation of the ventral interneurons in the pons.
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http://dx.doi.org/10.1002/dvg.20615DOI Listing
May 2010