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Tsuyoshi Osawa,
Rika Tsuchida,
Masashi Muramatsu,
Teppei Shimamura,
Feng Wang,
Jun-Ichi Suehiro,
Yasuharu Kanki,
Youichiro Wada,
Yasuhito Yuasa,
Hiroyuki Aburatani,
Satoru Miyano, Takashi Minami,
Tatsuhiko Kodama,
Masabumi Shibuya
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ABSTRACT: Anti-angiogenic strategies can be effective for cancer therapy, but like all therapies resistance poses a major clinical challenge. Hypoxia and nutrient starvation select for aggressive qualities that may render tumors resistant to anti-angiogenic attack. Here we show that hypoxia and nutrient starvation cooperate to drive tumor aggressiveness through epigenetic regulation of the histone demethylase JMJD1A (JHDM2A; KDM3A). In cancer cells rendered resistant to long-term hypoxia and nutrient starvation, we documented a stimulation of AKT phosphorylation, cell morphological changes, cell migration, invasion and anchorage independent growth in culture. These qualities associated in vivo with increased angiogenesis and infiltration of macrophages into tumor tissues. Through expression microarray analysis we identified a cluster of functional drivers such as VEGFA, FGF18 and JMJD1A, the latter which was upregulated in vitro under conditions of hypoxia and nutrient starvation and in vivo before activation of the angiogenic switch or the pre-refractory phase of anti-angiogenic therapy. JMJD1A inhibition suppressed tumor growth by downregulating angiogenesis and macrophage infiltration, by suppressing expression of FGF2, HGF and ANG2. Notably, JMJD1A inhibition enhanced the anti-tumor effects of the anti-VEGF compound bevacizumab and the VEGFR/KDR inhibitor sunitinib. Our results form the foundation of a strategy to attack hypoxia- and nutrient starvation-resistant cancer cells as an approach to leverage anti-angiogenic treatments and limit resistance to them.
Cancer Research 03/2013; · 7.86 Impact Factor
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ABSTRACT: COUP-TFII nuclear receptor, which is composed of a DNA-binding domain and a ligand-binding domain, exerts pleiotropic effects on development and cell differentiation by regulating the transcription of its target genes, including Cyp7a1, which plays important roles in catabolism of cholesterol in liver. While there exist multiple variants of COUP-TFII, their roles in the regulation of Cyp7a1 expression have not been elucidated. In the present study, we studied the roles of the COUP-TFII-V2 (variant 2), which lacks a DNA-binding domain, in the regulation of the transcriptional control of Cyp7a1 gene by COUP-TFII in hepatocellular carcinoma cells. We found that COUP-TFII-V2 was significantly expressed in Huh7 cells, in which Cyp7a1 was not expressed. Furthermore, knockdown of COUP-TFII-V2 enhanced endogenous Cyp7a1 expression in Huh7 cells. While COUP-TFII activates the Cyp7a1 promoter through direct binding to DNA, this activation was interfered by COUP-TFII-V2, which physically interacted with COUP-TFII and inhibited its DNA-binding ability. Chromatin immunoprecipitation assays showed that COUP-TFII-V2 inhibited the binding of endogenous COUP-TFII to the intact Cyp7a1 promoter. These results suggest that COUP-TFII-V2 negatively regulates the function of COUP-TFII by inhibiting its binding to DNA to decrease Cyp7a1 expression.
Biochemical Journal 03/2013; · 4.90 Impact Factor
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Kaori Higuchi,
Yoshikazu Nakaoka,
Wataru Shioyama,
Yoh Arita,
Takahiro Hashimoto,
Taku Yasui,
Kuniyasu Ikeoka,
Tadashi Kuroda, Takashi Minami,
Keigo Nishida,
Yasushi Fujio,
Keiko Yamauchi-Takihara,
Mikiyasu Shirai,
Naoki Mochizuki,
Issei Komuro
[show abstract]
[hide abstract]
ABSTRACT: Docking protein Grb2-associated binder 1 (Gab1) has critical roles in signal transduction of various growth factors, cytokines, and numerous other molecules. Our previous reports show that Gab1 is essential for postnatal angiogenesis through the analysis of endothelium-specific Gab1 knockout (Gab1ECKO) mice. However, the role of Gab1 in atherosclerosis remains unknown. The aim of the present study was to elucidate the role of endothelial Gab1 in vascular inflammation and atherosclerosis.
We intercrossed Gab1ECKO mice with apolipoprotein E (ApoE) knockout (ApoEKO) mice. Six-month-old male ApoEKO/Gab1ECKO and littermate control (ApoEKO) mice were treated with angiotensin II (AngII) via an osmotic infusion mini-pump. After AngII treatment, ApoEKO/Gab1ECKO mice showed significantly enhanced atherosclerosis and aneurysm formation compared with control mice. The production of proinflammatory cytokines in the aorta was significantly enhanced in ApoEKO/Gab1ECKO mice compared with control mice. Furthermore, the expression levels of Krüppel-like factor (KLF) 2 (KLF2) and KLF4, key transcription factors for endothelial homeostasis, were significantly reduced in the aortic endothelium of ApoEKO/Gab1ECKO mice compared with those of control mice. Consistently, both vascular cell adhesion molecule-1 expression and macrophage infiltration on the aortic walls were enhanced in ApoEKO/Gab1ECKO mice compared with control mice.
Collectively, endothelial Gab1 deletion accelerates AngII-dependent vascular inflammation and atherosclerosis on ApoE-null background presumably in association with downregulation of KLF2 and KLF4.
Circulation Journal 05/2012; 76(8):2031-40. · 3.77 Impact Factor
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ABSTRACT: Histone demethylase JHDM1D (also known as KDM7A) modifies the level of methylation in histone and participates in epigenetic gene regulation; however, the role of JHDM1D in tumor progression is unknown. Here, we show that JHDM1D plays a tumor-suppressive role by regulating angiogenesis. Expression of JHDM1D was increased in mouse and human cancer cells under long-term nutrient starvation in vitro. Expression of JHDM1D mRNA was increased within avascular tumor tissue at the preangiogenic switch, along with increased expression of angiogenesis-regulating genes such as Vegf-A. Stable expression of JHDM1D cDNA or siRNA silencing of JHDM1D in cancer cells did not affect cell proliferation, anchorage-independent cell growth, or cell cycle progression in vitro. Notably, JHDM1D-expressing mouse melanoma (B16) and human cervical carcinoma (HeLa) cells exhibited significantly slower tumor growth in vivo compared with the original cells. This reduction in tumor growth was associated with decreased formation of CD31(+) blood vessels and reduced infiltration of CD11b(+) macrophage linage cells into tumor tissues. Expression of multiple angiogenic factors such as VEGF-B and angiopoietins was decreased in tumor xenografts of JHDM1D-expressing B16 and HeLa cells. Our results provide evidence that increased JHDM1D expression suppressed tumor growth by down-regulating angiogenesis under nutrient starvation.
Proceedings of the National Academy of Sciences 12/2011; 108(51):20725-9. · 9.68 Impact Factor
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Yasuhiro Yoshimatsu,
Tomoko Yamazaki,
Hajime Mihira,
Taichi Itoh,
Junichi Suehiro,
Keiko Yuki,
Kaori Harada,
Masato Morikawa,
Caname Iwata, Takashi Minami,
Yasuyuki Morishita,
Tatsuhiko Kodama,
Kohei Miyazono,
Tetsuro Watabe
[show abstract]
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ABSTRACT: Prox1 plays pivotal roles during embryonic lymphatic development and maintenance of adult lymphatic systems by modulating the expression of various lymphatic endothelial cell (LEC) markers, such as vascular endothelial growth factor receptor 3 (VEGFR3). However, the molecular mechanisms by which Prox1 transactivates its target genes remain largely unknown. Here, we identified Ets-2 as a candidate molecule that regulates the functions of Prox1. Whereas Ets-2 has been implicated in angiogenesis, its roles during lymphangiogenesis have not yet been elucidated. We found that endogenous Ets-2 interacts with Prox1 in LECs. Using an in vivo model of chronic aseptic peritonitis, we found that Ets-2 enhanced inflammatory lymphangiogenesis, whereas a dominant-negative mutant of Ets-1 suppressed it. Ets-2 also enhanced endothelial migration towards VEGF-C through induction of expression of VEGFR3 in collaboration with Prox1. Furthermore, we found that both Prox1 and Ets-2 bind to the VEGFR3 promoter in intact chromatin. These findings suggest that Ets family members function as transcriptional cofactors that enhance Prox1-induced lymphangiogenesis.
Journal of Cell Science 08/2011; 124(Pt 16):2753-62. · 6.11 Impact Factor
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Yasuharu Kanki,
Takahide Kohro,
Shuying Jiang,
Shuichi Tsutsumi,
Imari Mimura,
Jun-Ichi Suehiro,
Youichiro Wada,
Yoshihiro Ohta,
Sigeo Ihara,
Hiroko Iwanari,
Makoto Naito,
Takao Hamakubo,
Hiroyuki Aburatani,
Tatsuhiko Kodama, Takashi Minami
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ABSTRACT: GATA2 is well recognized as a key transcription factor and regulator of cell-type specificity and differentiation. Here, we carried out comparative chromatin immunoprecipitation with comprehensive sequencing (ChIP-seq) to determine genome-wide occupancy of GATA2 in endothelial cells and erythroids, and compared the occupancy to the respective gene expression profile in each cell type. Although GATA2 was commonly expressed in both cell types, different GATA2 bindings and distinct cell-specific gene expressions were observed. By using the ChIP-seq with epigenetic histone modifications and chromatin conformation capture assays; we elucidated the mechanistic regulation of endothelial-specific GATA2-mediated endomucin gene expression, that was regulated by the endothelial-specific chromatin loop with a GATA2-associated distal enhancer and core promoter. Knockdown of endomucin markedly attenuated endothelial cell growth, migration and tube formation. Moreover, abrogation of GATA2 in endothelium demonstrated not only a reduction of endothelial-specific markers, but also induction of mesenchymal transition promoting gene expression. Our findings provide new insights into the correlation of endothelial-expressed GATA2 binding, epigenetic modification, and the determination of endothelial cell specificity.
The EMBO Journal 06/2011; 30(13):2582-95. · 9.20 Impact Factor
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ABSTRACT: Endothelial cell activation and dysfunction underlie many vascular disorders, including atherosclerosis and inflammation. Here, we show that interleukin-4 (IL-4) markedly induced vascular cell adhesion molecule 1 (VCAM-1), both in cultured endothelial cells and in the intact endothelium in mice. Combined treatment with IL-4 and tumor necrosis factor alpha (TNF-α) resulted in further, sustained induction of VCAM-1 expression. IL-4-mediated induction of VCAM-1 and secondary monocyte adhesion was predominantly regulated by the transcription factor STAT6. Genome-wide survey of IL-4-mediated STAT6 binding from sequential chromatin-immunoprecipitation with deep sequencing (chromatin immunoprecipitation sequencing [ChIP-seq]) in endothelial cells revealed regions of transient and sustained transcription factor binding. Through the combination of DNA microarrays and ChIP-seq at the same time points, the majority of IL-4-responsive genes were shown to be STAT6 dependent and associated with direct STAT6 binding to their promoter. IL-4-mediated stable binding of STAT6 led to sustained target gene expression. Moreover, our strategy led to the identification of a novel functionally important STAT6 binding site within 16 kb upstream of the VCAM-1 gene. Taken together, these findings support a critical role for STAT6 in mediating IL-4 signal transduction in endothelial cells. Identification of a novel IL-4-mediated VCAM-1 enhancer may provide a foundation for targeted therapy in vascular disease.
Molecular and cellular biology 04/2011; 31(11):2196-209. · 6.06 Impact Factor
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Wataru Shioyama,
Yoshikazu Nakaoka,
Kaori Higuchi, Takashi Minami,
Yoshiaki Taniyama,
Keigo Nishida,
Hiroyasu Kidoya,
Takashi Sonobe,
Hisamichi Naito,
Yoh Arita, [......],
Tadashi Kuroda,
Yasushi Fujio,
Mikiyasu Shirai,
Nobuyuki Takakura,
Ryuichi Morishita,
Keiko Yamauchi-Takihara,
Tatsuhiko Kodama,
Toshio Hirano,
Naoki Mochizuki,
Issei Komuro
[show abstract]
[hide abstract]
ABSTRACT: Grb2-associated binder (Gab) docking proteins, consisting of Gab1, Gab2, and Gab3, have crucial roles in growth factor-dependent signaling. Various proangiogenic growth factors regulate angiogenesis and endothelial function. However, the roles of Gab proteins in angiogenesis remain elusive.
To elucidate the role of Gab proteins in postnatal angiogenesis.
Endothelium-specific Gab1 knockout (Gab1ECKO) mice were viable and showed no obvious defects in vascular development. Therefore, we analyzed a hindlimb ischemia (HLI) model of control, Gab1ECKO, or conventional Gab2 knockout (Gab2KO) mice. Intriguingly, impaired blood flow recovery and necrosis in the operated limb was observed in all of Gab1ECKO, but not in control or Gab2KO mice. Among several proangiogenic growth factors, hepatocyte growth factor (HGF) induced the most prominent tyrosine phosphorylation of Gab1 and subsequent complex formation of Gab1 with SHP2 (Src homology-2-containing protein tyrosine phosphatase 2) and phosphatidylinositol 3-kinase subunit p85 in human endothelial cells (ECs). Gab1-SHP2 complex was required for HGF-induced migration and proliferation of ECs via extracellular signal-regulated kinase (ERK)1/2 pathway and for HGF-induced stabilization of ECs via ERK5. In contrast, Gab1-p85 complex regulated activation of AKT and contributed partially to migration of ECs after HGF stimulation. Microarray analysis demonstrated that HGF upregulated angiogenesis-related genes such as KLF2 (Krüppel-like factor 2) and Egr1 (early growth response 1) via Gab1-SHP2 complex in human ECs. In Gab1ECKO mice, gene transfer of vascular endothelial growth factor, but not HGF, improved blood flow recovery and ameliorated limb necrosis after HLI.
Gab1 is essential for postnatal angiogenesis after ischemia via HGF/c-Met signaling.
Circulation Research 02/2011; 108(6):664-75. · 9.49 Impact Factor
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Ju Liu,
Lei Yuan,
Grietje Molema,
Erzsébet Regan,
Lauren Janes,
David Beeler,
Katherine C Spokes,
Yoshiaki Okada, Takashi Minami,
Peter Oettgen,
William C Aird
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ABSTRACT: A region of the human von Willebrand factor (VWF) gene between -2812 and the end of the first intron (termed vWF2) was previously shown to direct expression in the endothelium of capillaries and a subset of larger blood vessels in the heart and skeletal muscle. Here, our goal was to delineate the DNA sequences responsible for this effect. A series of constructs containing deletions or mutations of vWF2 coupled to LacZ were targeted to the Hprt locus of mice, and the resulting animals were analyzed for reporter gene expression. The findings demonstrate that DNA sequences between -843 and -620 are necessary for expression in capillary but not large vessel endothelium in heart and skeletal muscle. Further, expression of VWF in capillaries and larger vessels of both tissues required the presence of a native or heterologous intron. In vitro assays implicated a role for ERG-binding ETS motif at -56 in mediating basal expression of VWF. In Hprt-targeted mice, mutation of the ETS consensus motif resulted in loss of LacZ expression in the endothelium of the heart and skeletal muscle. Together, these data indicate that distinct DNA modules regulate vascular bed-specific expression of VWF.
Blood 10/2010; 117(1):342-51. · 9.90 Impact Factor
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ABSTRACT: PSF1 (partner of sld five 1) is an evolutionarily conserved DNA replication factor implicated in DNA replication in lower species that is strongly expressed in a wide range of normal stem cell populations and progenitor cell populations. Because stem and progenitor cells possess high proliferative capacity, we hypothesized that PSF1 may play an important role in tumor growth. To begin to investigate PSF1 function in cancer cells, we cloned the mouse PSF1 promoter and generated lung and colon carcinoma cells that stably express a PSF1 promoter-reporter gene. Reporter expression in cells correlated with endogenous PSF1 mRNA expression. In a tumor cell xenograft model, high levels of reporter expression correlated with high proliferative activity, serial transplantation potential, and metastatic capability. Notably, cancer cells expressing reporter levels localized to perivascular regions in tumors and displayed expression signatures related to embryonic stem cells. RNAi-mediated silencing of endogenous PSF1 inhibited cancer cell growth by disrupting DNA synthesis and chromosomal segregation. These findings implicate PSF1 in tumorigenesis and offer initial evidence of its potential as a theranostic target.
Cancer Research 02/2010; 70(3):1215-24. · 7.86 Impact Factor
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ABSTRACT: Endothelial cell activation and dysfunction underlie many vascular disorders, including atherosclerosis, tumor growth, and sepsis. Endothelial cell activation, in turn, is mediated primarily at the level of gene transcription. Here, we show that in response to several activation agonists, including vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha, and thrombin, endothelial cells demonstrate rapid and profound induction of the early growth response (Egr) genes egr-1 and egr-3. In VEGF-treated endothelial cells, induction of Egr-3 was far greater and more prolonged compared with Egr-1. VEGF-mediated stimulation of Egr-3 involved the inducible binding of NFATc, serum response factor, and CREB to their respective consensus motifs in the upstream promoter region of Egr-3. Knockdown of Egr-3 markedly impaired VEGF-mediated proliferation, migration, and tube formation of endothelial cells and blocked VEGF-induced monocyte adhesion. Egr-3 knockdown abrogated VEGF-mediated vascular outgrowth from ex vivo aortic rings and attenuated Matrigel plug vascularization and melanoma tumor growth in vivo. Together, these findings suggest that Egr-3 is a critical determinant of VEGF signaling in activated endothelial cells. Thus, Egr-3 represents a potential therapeutic target in VEGF-mediated vasculopathic diseases.
Blood 11/2009; 115(12):2520-32. · 9.90 Impact Factor
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Haihua Song,
Jun-ichi Suehiro,
Yasuharu Kanki,
Yoshiko Kawai,
Kenji Inoue,
Hiroyuki Daida,
Kiichiro Yano,
Toshio Ohhashi,
Peter Oettgen,
William C. Aird,
Tatsuhiko Kodama, Takashi Minami
[show abstract]
[hide abstract]
ABSTRACT: Endothelial phenotypes are highly regulated in space and time by both transcriptional and post-transcriptional mechanisms.
There is increasing evidence that the GATA family of transcription factors function as signal transducers, coupling changes
in the extracellular environment to changes in downstream target gene expression. Here we show that human primary endothelial
cells derived from large blood vessels express GATA2, -3, and -6. Of these factors, GATA3 was expressed at the highest levels.
In DNA microarrays of human umbilical vein endothelial cells (HUVEC), small interfering RNA-mediated knockdown of GATA3 resulted
in reduced expression of genes associated with angiogenesis, including Tie2. At a functional level, GATA3 knockdown inhibited
angiopoietin (Ang)-1-mediated but not vascular endothelial cell growth factor (VEGF)-mediated AKT signaling, cell migration,
survival, and tube formation. In electrophoretic gel mobility shift assays and chromatin immunoprecipitation, GATA3 was shown
to bind to regulatory regions within the 5′-untranslated region of the Tie2 gene. In co-immunoprecipitation and co-transfection assays, GATA3 and the Ets transcription factor, ELF1, physically interacted
and synergized to transactivate the Tie2 promoter. GATA3 knockdown blocked the ability of Ang-1 to attenuate vascular endothelial cell growth factor stimulation of
vascular cell adhesion molecule-1 expression and monocytic cell adhesion. Moreover, exposure of human umbilical vein endothelial
cells to tumor necrosis factor-α resulted in marked down-regulation of GATA3 expression and reduction in Tie2 expression.
Together, these findings suggest that GATA3 is indispensable for Ang-1-Tie2-mediated signaling in large vessel endothelial
cells.
Journal of Biological Chemistry 10/2009; 284(42):29109-29124. · 4.77 Impact Factor
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Enjing Jin,
Ju Liu,
Jun-Ichi Suehiro,
Lei Yuan,
Yoshiaki Okada,
Vesna Nikolova-Krstevski,
Kiichiro Yano,
Lauren Janes,
David Beeler,
Katherine C Spokes,
Dan Li,
Erzsébet Regan,
Shou-Ching Shih,
Peter Oettgen, Takashi Minami,
William C Aird
[show abstract]
[hide abstract]
ABSTRACT: Vascular endothelial growth factor receptor 1 (VEGFR1) is a marker for endothelial-specific gene expression. We previously reported that the human VEGFR1 promoter (between -748 and +284) contains information for expression in the intact endothelium of transgenic mice. The objective of this study was to dissect the cis-regulatory elements underlying VEGFR1 promoter activity in vitro and in vivo. In primary endothelial cells, binding sites for E74-like factor 1 (ELF-1; between -49 and -52), cyclic adenosine monophosphate response element binding (CREB; between -74 and -81), and early growth response factor 1/3 (EGR-1/3; between -16 to -25) were shown to play a positive role in gene transcription, whereas a putative E26 transformation-specificsequence (ETS) motif between -36 and -39 had a net negative effect on promoter activity. When targeted to the Hprt locus of mice, mutations of the ELF-1 binding site and the CRE element reduced promoter activity in the embryonic vasculature and resulted in a virtual loss of expression in adult endothelium. Postnatally, the EGR binding site mutant displayed significantly reduced promoter activity in a subset of vascular beds. In contrast, mutation of the -39 ETS site resulted in increased LacZ staining in multiple vascular beds. Together, these results provide new insights into the transcriptional regulatory mechanisms of VEGFR1.
Blood 10/2009; 114(27):5557-66. · 9.90 Impact Factor
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Youichiro Wada,
Yoshihiro Ohta,
Meng Xu,
Shuichi Tsutsumi, Takashi Minami,
Kenji Inoue,
Daisuke Komura,
Jun'ichi Kitakami,
Nobuhiko Oshida,
Argyris Papantonis, [......],
Imari Mimura,
Kazuki Yamamoto,
Chikage Mataki,
Takao Hamakubo,
Katsuhiko Shirahige,
Hiroyuki Aburatani,
Hiroshi Kimura,
Tatsuhiko Kodama,
Peter R Cook,
Sigeo Ihara
[show abstract]
[hide abstract]
ABSTRACT: Genome-wide studies reveal that transcription by RNA polymerase II (Pol II) is dynamically regulated. To obtain a comprehensive view of a single transcription cycle, we switched on transcription of five long human genes (>100 kbp) with tumor necrosis factor-alpha (TNFalpha) and monitored (using microarrays, RNA fluorescence in situ hybridization, and chromatin immunoprecipitation) the appearance of nascent RNA, changes in binding of Pol II and two insulators (the cohesin subunit RAD21 and the CCCTC-binding factor CTCF), and modifications of histone H3. Activation triggers a wave of transcription that sweeps along the genes at approximately 3.1 kbp/min; splicing occurs cotranscriptionally, a major checkpoint acts several kilobases downstream of the transcription start site to regulate polymerase transit, and Pol II tends to stall at cohesin/CTCF binding sites.
Proceedings of the National Academy of Sciences 10/2009; 106(43):18357-61. · 9.68 Impact Factor
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ABSTRACT: Down syndrome critical region gene 1 (DSCR-1) short variant (DSCR-1s) is an inhibitor of calcineurin/NFAT signaling encoded by exons 4-7 of DSCR1. We previously reported that VEGF induces DSCR-1s expression in endothelial cells, which in turn negatively feeds back to attenuate endothelial cell activation. Here, in order to characterize the role of the promoter that drives DSCR-1s expression in mediating inducible expression in vivo and to determine the functional relevance of DSCR-1s in inflammation, we targeted a DNA construct containing 1.7 kb of the human DSCR1s promoter coupled to the lacZ reporter to the hypoxanthine guanine phosphoribosyl transferase (Hprt) locus of mice. We determined that lacZ was uniformly expressed in the endothelium of transgenic embryos but was markedly downregulated postnatally. Systemic administration of VEGF or LPS in adult mice resulted in cyclosporine A-sensitive reactivation of the DSCR1s promoter and endogenous gene expression in a subset of organs, including the heart and brain. The DSCR1s promoter was similarly induced in the endothelium of tumor xenografts. In a mouse model of endotoxemia, DSCR-1s-deficient mice demonstrated increased sepsis mortality, whereas adenovirus-mediated DSCR-1s overexpression protected against LPS-induced lethality. Collectively, these data suggest that the DSCR1s promoter directs vascular bed-specific expression in activated endothelium and that DSCR-1s serves to dampen the host response to infection.
The Journal of clinical investigation 09/2009; 119(8):2257-70. · 15.39 Impact Factor
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Haihua Song,
Jun-ichi Suehiro,
Yasuharu Kanki,
Yoshiko Kawai,
Kenji Inoue,
Hiroyuki Daida,
Kiichiro Yano,
Toshio Ohhashi,
Peter Oettgen,
William C Aird,
Tatsuhiko Kodama, Takashi Minami
[show abstract]
[hide abstract]
ABSTRACT: Endothelial phenotypes are highly regulated in space and time by both transcriptional and post-transcriptional mechanisms. There is increasing evidence that the GATA family of transcription factors function as signal transducers, coupling changes in the extracellular environment to changes in downstream target gene expression. Here we show that human primary endothelial cells derived from large blood vessels express GATA2, -3, and -6. Of these factors, GATA3 was expressed at the highest levels. In DNA microarrays of human umbilical vein endothelial cells (HUVEC), small interfering RNA-mediated knockdown of GATA3 resulted in reduced expression of genes associated with angiogenesis, including Tie2. At a functional level, GATA3 knockdown inhibited angiopoietin (Ang)-1-mediated but not vascular endothelial cell growth factor (VEGF)-mediated AKT signaling, cell migration, survival, and tube formation. In electrophoretic gel mobility shift assays and chromatin immunoprecipitation, GATA3 was shown to bind to regulatory regions within the 5'-untranslated region of the Tie2 gene. In co-immunoprecipitation and co-transfection assays, GATA3 and the Ets transcription factor, ELF1, physically interacted and synergized to transactivate the Tie2 promoter. GATA3 knockdown blocked the ability of Ang-1 to attenuate vascular endothelial cell growth factor stimulation of vascular cell adhesion molecule-1 expression and monocytic cell adhesion. Moreover, exposure of human umbilical vein endothelial cells to tumor necrosis factor-alpha resulted in marked down-regulation of GATA3 expression and reduction in Tie2 expression. Together, these findings suggest that GATA3 is indispensable for Ang-1-Tie2-mediated signaling in large vessel endothelial cells.
Journal of Biological Chemistry 09/2009; 284(42):29109-24. · 4.77 Impact Factor
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Kwan-Hyuck Baek,
Alexander Zaslavsky,
Ryan C Lynch,
Carmella Britt,
Yoshiaki Okada,
Richard J Siarey,
M William Lensch,
In-Hyun Park,
Sam S Yoon, Takashi Minami,
Julie R Korenberg,
Judah Folkman,
George Q Daley,
William C Aird,
Zygmunt Galdzicki,
Sandra Ryeom
[show abstract]
[hide abstract]
ABSTRACT: The incidence of many cancer types is significantly reduced in individuals with Down's syndrome, and it is thought that this broad cancer protection is conferred by the increased expression of one or more of the 231 supernumerary genes on the extra copy of chromosome 21. One such gene is Down's syndrome candidate region-1 (DSCR1, also known as RCAN1), which encodes a protein that suppresses vascular endothelial growth factor (VEGF)-mediated angiogenic signalling by the calcineurin pathway. Here we show that DSCR1 is increased in Down's syndrome tissues and in a mouse model of Down's syndrome. Furthermore, we show that the modest increase in expression afforded by a single extra transgenic copy of Dscr1 is sufficient to confer significant suppression of tumour growth in mice, and that such resistance is a consequence of a deficit in tumour angiogenesis arising from suppression of the calcineurin pathway. We also provide evidence that attenuation of calcineurin activity by DSCR1, together with another chromosome 21 gene Dyrk1a, may be sufficient to markedly diminish angiogenesis. These data provide a mechanism for the reduced cancer incidence in Down's syndrome and identify the calcineurin signalling pathway, and its regulators DSCR1 and DYRK1A, as potential therapeutic targets in cancers arising in all individuals.
Nature 06/2009; 459(7250):1126-30. · 36.28 Impact Factor
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Kwan-Hyuck Baek,
Alexander Zaslavsky,
Ryan C. Lynch,
Carmella Britt,
Yoshiaki Okada,
Richard J. Siarey,
M. William Lensch,
In-Hyun Park,
Sam S. Yoon, Takashi Minami,
Julie R. Korenberg,
Judah Folkman,
George Q. Daley,
William C. Aird,
Zygmunt Galdzicki,
Sandra Ryeom
Nature 05/2009; 459(7250):1126-1130. · 36.28 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Angiopoietin-1 (Ang1) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group have recently shown that Ang1 and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts and further demonstrated that the former selectively induces expression of Krüppel-like factor 2 (KLF2), a transcription factor involved in vascular quiescence. Here, we investigated the mechanism of how Ang1/Tie2 signal induces KLF2 expression to clarify the role of KLF2 in Ang1/Tie2 signal-mediated vascular quiescence. Ang1 stimulated KLF2 promoter-driven reporter gene expression in endothelial cells, whereas it failed when a myocyte enhancer factor 2 (MEF2)-binding site of KLF2 promoter was mutated. Depletion of MEF2 by siRNAs abolished Ang1-induced KLF2 expression, indicating the requirement of MEF2 in KLF2 induction by Ang1. Constitutive active phosphoinositide 3-kinase (PI3K) and AKT increased the MEF2-dependent reporter gene expression by enhancing its transcriptional activity and stimulated the KLF2 promoter activity cooperatively with MEF2. Consistently, inhibition of either PI3K or AKT and depletion of AKT abrogated Ang1-induced KLF2 expression. In addition, we confirmed the dispensability of extracellular signal-regulated kinase 5 (ERK5) for Ang1-induced KLF2 expression. Furthermore, depletion of KLF2 resulted in the loss of the inhibitory effect of Ang1 on vascular endothelial growth factor (VEGF)-mediated expression of vascular cell adhesion molecule-1 in endothelial cells and VEGF-mediated monocyte adhesion to endothelial cells. Collectively, these findings indicate that Ang1/Tie2 signal stimulates transcriptional activity of MEF2 through a PI3K/AKT pathway to induce KLF2 expression, which may counteract VEGF-mediated inflammatory responses.
Journal of Biological Chemistry 01/2009; 284(9):5592-601. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Recently, we reported that the forkhead transcription factor, FKHR/FOXO1, is required for vascular endothelial growth factor (VEGF)-mediated upregulation of a number of genes in endothelial cells. Here, we tested the hypothesis that hepatocyte growth factor (HGF), a potent activator of PI3K-Akt in endothelial cells, is capable of depleting the nucleus of FKHR/FOXO1 and thus inhibiting VEGF induction of this class of genes.
Incubation of human coronary artery endothelial cells with HGF induced prolonged PI3K/Akt-dependent phosphorylation and nuclear exclusion of FKHR/FOXO1. HGF-mediated inhibition of FKHR/FOXO1 activity resulted in secondary attenuation of VEGF-induced expression of FKHR/FOXO1-dependent genes including vascular cell adhesion molecule-1, manganese superoxide dismutase, endothelial specific molecule-1, CBP/p300 interacting transactivator with ED-rich tail-2, bone morphogenetic protein-2, matrix metalloproteinase (MMP)-10, and MGC5618. At a functional level, preincubation of HGF resulted in inhibition of VEGF-induced vascular cell adhesion molecule (VCAM)-1-mediated monocyte adhesion to endothelial cells. HGF-mediated inhibition of VEGF-inducible VCAM-1 expression and monocyte adhesion was reversed by overexpression of constitutively active phosphorylation-resistant triple mutant (TM)-FKHR.
These findings suggest that physiological agonists of PI3K-Akt signaling pathway may modulate VEGF-FKHR/FOXO1-dependent gene expression in endothelial cells. The data underscore the importance of the "set point" of the endothelial cell when considering mechanisms of signal transduction.
Arteriosclerosis Thrombosis and Vascular Biology 10/2008; 28(11):2042-8. · 6.37 Impact Factor
