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First published online 12 February 2003
doi: 10.1242/jcs.00318

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Intracellular localisation of human HIF-1 hydroxylases: implications for oxygen sensing

¹ Institute of Physiology, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany
² Institute of Physiology, University of Essen, Hufelandstr. 55, D-45122 Essen, Germany
³ Institute of Anatomy, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany
⁴ Max-Planck-Institute of Molecular Physiology, Dortmund, Otto-Hahn-Straße 11, D-44227 Dortmund, Germany

View larger version (30K): [in a new window]   Fig. 1. Control EGFP or HIF-1 hydroxylase. EGFP fusion proteins transiently expressed in subconfluent human U2OS osteosarcoma cells. Cells were transfected with pEGFP-N1 or with plasmids encoding PHD-EGFP or FIH-1-EGFP. Cells were fixed with cold 4% paraformaldehyde/PBS for 10 minutes. Left column, EGFP fluorescence microscopy; right column, fluorescence intensities visualised using a false colour table shading from black (lowest intensity), through blue, green, yellow, orange, red and purple to white (highest intensity).

View larger version (90K): [in a new window]   Fig. 2. Three-dimensional two-photon confocal laser scanning microscopy of PHD1, PHD2, PHD3 and FIH-1. Different EGFP fluorescence intensities of single cells were visualised in false colours as indicated by the color table (right column). Therefore, up to 64 optical slices through the transfected cells were recovered by two-photon confocal laser scanning microscopy. After reconstruction of the optical slices, the distribution of the EGFP fluorescence within a single cell was visualised in three dimensions. A cut through the cell reveals the inside distribution. An overlay of all optical slices is shown in the inserts.

View larger version (21K): [in a new window]   Fig. 3. In vitro characterisation of PHD-EGFP fusion proteins. (A) In vitro transcription/translation products detected by 8% SDS-PAGE and autoradiography. (B) An in vitro protein interaction assay. After treatment of an immobilised GalDBD- HIF-1 549-582 fusion protein with unprogrammed reticulocyte lysate or PHD or PHD-EGFP fusion protein, 35S-labelled pVHL was added. Two pVHL isoforms (24 kDa and 19 kDa) were detected because translation was initiated at two distinct ATG codons in VHL. The ability to capture pVHL indicates hydroxylation of HIF-1 Pro564. Note that reticulocyte lysate has a low but significant hydroxylating activity.

View larger version (43K): [in a new window]   Fig. 4. Effect of transiently expressed PHD fusion or FIH-1 fusion proteins on the nuclear accumulation of endogenous HIF-1 under hypoxia. PHD-EGFP fusion or FIH-1-EGFP fusion protein or control EGFP was transiently expressed in U2OS cells. The cell cultures were placed in a hypoxia workstation (1% O2) for 4 hours. Cells were then fixed in icecold methanol/acetone and immunostained for endogenous HIF-1 as detailed in Materials and Methods. The left column shows hydroxylase EGFP-fusion proteins, the middle column indirect immunofluorescence of endogenous HIF-1 and the right column (Merge) an overlay of both columns.

View larger version (15K): [in a new window]   Fig. 5. Effect of the transient overexpression of HIF-1 hydroxylases on HRE-luciferase expression. U2OS cells were cotransfected with the indicated HIF-1 hydroxylase construct and a hypoxia-inducible firefly luciferase gene. Luciferase values of three separate wells were divided by ß-galactosidase values to correct for variations of transfection efficiency and are given as means plus standard deviation.

View larger version (15K): [in a new window]   Fig. 6. HIF-1 hydroxylase expression in normoxia and responses to hypoxia, desferrioxamine (150 µM) or cobalt chloride (100 µM) treatment. U2OS total mRNA was reverse transcribed and subjected to quantitative PCR as detailed in Materials and Methods. Numbers above the bars indicate fold induction compared to normoxia. Results are given as means of four separate samples plus standard deviation.