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
Source: PubMed


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.

Download full-text


Available from: Mehmet Zeynel Cilek,
  • Source
    • "common feature of human solid tumors and causes tumor cells to undergo adaptive changes that enable them to survive and proliferate . Hypoxia - inducible factor 1 ( HIF - 1 ) is a master regulator of hypoxic activation of genes for angiogenesis , hormone synthesis , glycolysis and cell survival ( Hatipoglu et al . , 2009 ; Torii et al . , 2009 ) . Hatipoglu et al . ( 2009 ) identified that there is Hypoxia Response Element ( HRE ) in the promoter region of the ADAMTS1 promoter . This upregulation of ADAMTS1 gene expression obtained from hepatocarcinoma cell type could be due to these HRE elements present in the promoter of ADAMTS1 gene . Apart from hypoxia , there is nothing known the regulation of human"
    [Show abstract] [Hide abstract]
    ABSTRACT: ADAM metallopeptidase with thrombospondin type I motif, 1 (ADAMTS1) that has both antiangiogenic and aggrecanase activity was dysregulated in many pathophysiologic circumstances. However, there is limited information available on the transcriptional regulation of ADAMTS1 gene. Therefore, this study mainly aimed to identify regulatory regions important for the regulation of ADAMTS1 gene under normoxic and hypoxic conditions in human hepatoma cells (HEP3B). Cultured HEP3B cells were exposed to normal oxygen condition, and Cobalt chloride (CoCl2) induced the hypoxic condition, which is an HIF-1 inducer. The cocl2-induced hypoxic condition led to the induced ADAMTS1 mRNA and protein expression in Hepatoma cells. Differential regulation of SP1 and USF transcription factors on ADAMTS1 gene expression was determined by transcriptional activity, mRNA and protein level of ADAMTS1 gene. Ectopic expression of SP1 and USF transcription factors resulted in the decrease in ADAMTS1 transcriptional activity of all promoter constructs consistent with mRNA and protein level in normoxic condition. However, overexpression of SP1 and USF led to the increase of ADAMTS1 gene expressions at mRNA and protein level in hypoxic condition. On the other hand, C/EBPα transcription factor didn't show any statistically significant effect on ADAMTS1 gene expression at mRNA, protein and transcriptional level under normoxic and hypoxic condition. Copyright © 2015. Published by Elsevier B.V.
    Gene 08/2015; 575(1). DOI:10.1016/j.gene.2015.08.035 · 2.14 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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
  • Source
    • "Mouse -actin was amplified as a control for the PCR reaction. Quantitative real time PCR was performed using a LightCycler rapid thermal cycler system (Roche) described previously [3] [9] [10] [14] [25]. Mouse primers for PCR were designed to amplify fragments for each ADAMTS gene and -actin. "
    [Show abstract] [Hide abstract]
    ABSTRACT: ADAMTS (adisintegrin and metalloproteinase with thrombospondin motifs) proteinases are involved in a variety of biological processes such as angiogenesis, cancer and arthritis. ADAMTSs appears to be responsible for the cleavage of proteoglycans in several tissues including brain and cartilage. Chondroitin sulfate proteoglycans (CSPGs) maintains the integrity of the brain extracellular matrix and major inhibitory contributors for glial scar and neural plasticity. The activity of aggrecanases in the central nervous system (CNS) has been reported. ADAMTSs are an enzyme degrading CSPGs in the brain. However, there is a little knowledge regarding ADAMTSs in the CNS. We investigated the expression levels of ADAMTSs mRNAs by RT-PCR after spinal cord injury in mouse. Transcripts encoding 4 of the 19 known ADAMTSs were evaluated in the mouse spinal cord following injury. ADAMTS1, -5 and -9 expression levels were found to be upregulated. No change was observed in ADAMTS4 expression. By means of immunohistochemistry, ADAMTSs were detected in the astrocytes implying its cellular source in SCI. Western blot analyses indicated that aggrecanase-generated proteoglycan fragments are produced after SCI.
    Neuroscience Letters 04/2013; 544. DOI:10.1016/j.neulet.2013.02.064 · 2.03 Impact Factor
Show more