ADAMTS1 Is a Unique Hypoxic Early Response Gene Expressed by Endothelial Cells

Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2009; 284(24):16325-33. DOI: 10.1074/jbc.M109.001313
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ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is a member of the matrix metalloproteinase family. We have previously reported that ADAMTS1 was strongly expressed in myocardial infarction. In this study, we investigated whether hypoxia induced ADAMTS1 and investigated its regulatory mechanism. In hypoxia, the expression level of ADAMTS1 mRNA and protein rapidly increased in endothelial cells, but not in other cell types. Interestingly, the induction of ADAMTS1 by hypoxia was transient, whereas vascular endothelial growth factor induction by hypoxia in human umbilical vein endothelial cells (HUVEC) increased in a time-dependent manner. CoCl2, a transition metal that mimics hypoxia, induced ADAMTS1 in HUVEC. The phosphatidylinositol 3-kinase inhibitor LY294002 dose-dependently inhibited the increase of ADAMTS1 mRNA expression in hypoxia. We characterized the promoter region of ADAMTS1, and the secreted luciferase assay system demonstrated that hypoxia induced luciferase secretion in the culture medium 4.6-fold in HUVEC. In the promoter region of ADAMTS1, we found at least three putative hypoxia-inducible factor (HIF) binding sites, and the chromatin immunoprecipitation assay revealed HIF-1 binding to HIF binding sites in the promoter region of ADAMTS1 under hypoxia. Recombinant ADAMTS1 protein promoted the migration of HUVEC under hypoxic conditions. In summary, we found that ADAMTS1 is transiently induced by hypoxia in endothelial cells, and its transcription is mediated by HIF-1 binding. Our data indicate that ADAMTS1 is a novel acute hypoxia-inducible gene.

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Available from: Mehmet Zeynel Cilek, Oct 09, 2015
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    • "Different exposure times to hypoxic and/or hyperglycemic conditions were selected to simulate both early and late cellular and molecular responses following ischaemia (Thygesen et al. 2007). Of notice, a qRT-PCR study on HUVEC cultured for 48 h under different hypoxic conditions (1% O2, 5% CO2, with balanced N2, or 150 µM CoCl2) has demonstrated that human ATP synthase, H-transporting, mitochondrial F0 complex, subunit B1 (ATP5F1), RPLP0, and ribosomal protein, large, P2 (RPLP2) were suitable reference genes (Hatipoglu et al. 2009). In contrast, we applied shorter incubation times (1, 3, and 12 h) and identified two reference genes, namely RPLP0 and TFRC as the most stably expressed under both euglycaemia and hyperglycemia, in the presence or absence of hypoxia. "
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    ABSTRACT: Human umbilical vein endothelial cell (HUVEC)-based gene expression studies carried out under hypoxia and/ or hyperglycemia bear huge potential in modelling endothelial cell response in cardiovascular disease and diabetes. However, such studies require reference genes that are stable across the whole range of experimental conditions. These reference genes have not been comprehensively defined to date. We applied human genome-wide microarrays and quantitative real-time PCR (qRT-PCR) on RNA obtained from primary HUVEC cultures that were incubated for 24 h either in euglycemic or hyperglycemic conditions and then subjected to short-term CoCl2-induced hypoxia of either 1, 3 or 12 h. Using whole-transcript arrays, we selected ten commonly used reference genes with no significant expression variation across 8 different conditions. These genes were ranked using NormFinder software according to their stability values. Consequently, five genes were selected for validation by quantitative real-time PCR (qRT-PCR). These were: ribosomal protein large P0 (RPLP0), transferrin receptor (TFRC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), and β-actin (ACTB). All five genes displayed stable expression under hyperglycemia. However, only RPLP0 and TFRC genes were stable under hypoxia up to 12 h. Under hyperglycemia combined with hypoxia up to 12 hours the expression of RPLP0, TFRC, GUSB and ACTB genes remained unchanged. Our findings strongly confirm that RPLP0 and TFRC are the most suitable reference genes for HUVEC gene expression experiments subjected to hypoxia and/or hyperglycemia for the given experimental conditions. We provide further evidence that even commonly known references genes require experimental validation for all conditions involved.
    G3-Genes Genomes Genetics 09/2014; 4(11). DOI:10.1534/g3.114.013102 · 3.20 Impact Factor
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    • "Loss of programmed cell death 4 (Pdcd4), a tumor suppressor, increases the expression of lysyl oxidase and hypoxia-induced breast cancer cell invasion through a HIF-1-independent mechanism [34]. A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) is a hypoxic early response gene in endothelial cells that promotes migration under hypoxia [35]. Hypoxia activates the expression of angiotensin converting enzyme (ACE) and inhibits ACE2 expression in human pulmonary smooth muscle cells [36]. "
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    ABSTRACT: Hypoxia is an important microenvironmental factor that induces cancer metastasis. Hypoxia/hypoxia-inducible factor-1alpha (HIF-1alpha) regulates many important steps of the metastatic processes, especially epithelial-mesenchymal transition (EMT) that is one of the crucial mechanisms to cause early stage of tumor metastasis. To have a better understanding of the mechanism of hypoxia-regulated metastasis, various hypoxia/HIF-1alpha-regulated target genes are categorized into different classes including transcription factors, histone modifiers, enzymes, receptors, kinases, small GTPases, transporters, adhesion molecules, surface molecules, membrane proteins, and microRNAs. Different roles of these target genes are described with regards to their relationship to hypoxia-induced metastasis. We hope that this review will provide a framework for further exploration of hypoxia/HIF-1alpha-regulated target genes and a comprehensive view of the metastatic picture induced by hypoxia.
    Journal of Biomedical Science 12/2012; 19(1):102. DOI:10.1186/1423-0127-19-102 · 2.76 Impact Factor
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    • "Electroporation of HUVEC was performed using a Microporator (BMS-MP-100; Microporator), in accordance with the manufacturer's instructions (Hatipoglu et al., 2009a). Briefly, cells were removed from the plate with trypsin, centrifuged and dissolved in serum-free medium. "
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    ABSTRACT: ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is an early immediate gene. We have previously reported that ADAMTS1 was strongly induced by hypoxia. In this study, we investigated whether ADAMTS1 promoter-driven reporter signal is detectable by acute hypoxia. We constructed the GFP (green fluorescent protein) expression vector [AHR (acute hypoxia-response sequence)-GFP] under the control of ADAMTS1 promoter and compared it with the constitutive GFP-expressing vector under the control of CMV (cytomegalovirus promoter-GFP). We transduced AHR-GFP and examined whether GFP signals can be detected under the acute hypoxia. When the human umbilical vein [HUVEC (human umbilical vein endothelial cells)] was transduced under normoxia, there were few GFP signals, while CMV-GFP showed considerable GFP signals. When HUVEC was stimulated with hypoxia, GFP signals from AHR-GFP gene were induced under hypoxic conditions. Notably, the GFP signals peaked at 3 h under hypoxia. In ischaemic hind limb model, transduced AHR-GFP showed hypoxic induction of GFP signals. In summary, we have demonstrated that the AHR system induced the reporter gene expression by acute hypoxia, and its induction is transient. This is the first report showing the unique acute hypoxia-activated gene expression system.
    Cell Biology International 01/2011; 35(1):1-8. DOI:10.1042/CBI20100290 · 1.93 Impact Factor
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