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AHR (Acute hypoxia responsive element) As a New Tool Detecting Acute Hypoxia
Author(s) -
CILEK Mehmet Zeynel,
HIROHATA Satoshi,
HATIPOGLU Omer Faruk,
NINOMIYA Yoshifumi
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.483.1
Subject(s) - hypoxia (environmental) , umbilical vein , transfection , green fluorescent protein , paraformaldehyde , flow cytometry , microbiology and biotechnology , chemistry , andrology , biology , medicine , in vitro , gene , pathology , biochemistry , oxygen , organic chemistry
Objective Previously we reported an interesting gene that transiently induced in the endothelium in myocardial infarction which may be a hypoxia‐inducible gene expressed by endothelial cells. Its expression was limited to a few hours’ hypoxia. We tested if this switch element (we named this sequence as AHR (Acute hypoxia responsive element) for this induction can be used for detecting acute hypoxia. Methods We prepared several different constructs with different length of AHR promoter then performed luciferase assay in hypoxic cells. We identified the responsible AHR activated by acute hypoxia. We made green fluorescence protein (GFP) expressing construct (AHR‐GFP) under the control of AHR. We transfected AHR‐GFP construct into human umbilical vein endothelial cells (HUVECs), and examined GFP production under 30 minutes, 1, 3, 6, 12 or 24 hours of hypoxia respectively. After incubation, cells were fixed with 4% paraformaldehyde (PFA) and observed under the fluorescence microscope. Results When AHR‐GFP –transfected HUVEC was cultured in normoxic condition, GFP was very slightly observed. In contrast to this, the number of GFP‐positive HUVEC increased at 3h (~10‐fold compared to normoxia) under hypoxic stimulation. The induction of AHR under hypoxia returned to the normoxic level at 24h of hypoxia. Conclusions AHR works under acute hypoxia and may be used as a specific tool for detecting acute hypoxia.