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A new HIF-1 alpha variant induced by zinc ion suppresses HIF-1-mediated hypoxic responses

¹ Department of Pharmacology and Heart Research Institute, BK21 Human Life Sciences, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea
² Institute of Animal Science and Technology, College of Agriculture and Life Sciences, Seoul National University, Suwon 441-744, Korea

View larger version (39K): [in a new window]   Fig. 1. Expression of a human HIF-1 mRNA isoform induced by the zinc ion. HEK 293 cells were cultured for 4 hours in the absence (C) and presence of ZnCl2 (100, 300 and 500 µM) (Zn), CoCl2 (100 µM) (Co), or NiCl2 (300 µM) (Ni), or subjected to 4 hours of hypoxia (H). HIF-1, its isoform, and ß-actin mRNAs were amplified using RT-PCR, and analyzed by electrophoresis and ethidium bromide staining, as described in Materials and Methods (A, B, C). The molecular size of PCR product was assessed by comparing its mobility with those of DNA markers (M). These mRNA levels were analyzed by semiquantitative RT-PCR using [-32P]CTP (D). The relative amounts of the mRNAs were expressed as the ratios of the cpm value of HIF-1 or its variant fragments to ß-actin under each set of conditions (E). The results presented in each panel represent three separate experiments.

View larger version (22K): [in a new window]   Fig. 2. Schematic representation of a newly cloned HIF-1 isoform, HIF-1Z, compared with HIF-1. The 11th and 13th exons were directly joined, which generated an immediate termination codon and a new frame in the 13th exon. This alternative splicing introduces four new amino acids following the Gln553 of HIF-1. It translates into a 557 amino acid polypeptide. Compared with HIF-1, it conserves both bHLH and PAS but loses a part of the ODD domain, TAD and the NLS motif.

View larger version (53K): [in a new window]   Fig. 3. Expression of HIF-1Z protein. HIF-1Z protein was identified using anti-HIF-1 antibody 8 hours after HEK 293 cells were treated with 500 µM ZnCl2 in the absence and presence of cycloheximide (CH) (10 µg/ml). The HIF-1Z protein band (HIF-1Z) and nonspecific protein band (NS) are indicated. The molecular mass of HIF-1Z was determined to be 62 kDa on SDS-polyacrylamide gel (A). C, Control. HEK 293 cells were transfected with pcDNA3 (pcDNA) or pcDNA3-HIF-1Z (pHIF-1Z), using the calcium phosphate method. The transfected cells were subjected to normoxia (N) or hypoxia (H) for 4 hours, and then homogenized to isolate the cytosolic (Cy) and nuclear proteins (Nu). Expressed HIF-1Z and endogenous HIF-1 proteins were analyzed on 10% and 6% poylacrylamide gels (B).

View larger version (83K): [in a new window]   Fig. 4. Subcellular localization of HIF-1 and HIF-1Z. HEK 293 cells (left panel) and pHA-HIF-1Z transfectant cells (right panel) were subjected to normoxia or 4 hours of hypoxia, and then incubated with rabbit anti-HIF-1 antibody and rabbit anti-HA antibody, respectively, followed by a FITC-conjugated goat anti-rabbit antibody. The corresponding transmission images are shown in the outer columns.

View larger version (76K): [in a new window]   Fig. 5. mRNA expressions of the hypoxia-inducible genes. The mRNAs were isolated from transfected HEK 293 cells subjected to normoxia (N) or 16 hours of hypoxia (H), and analyzed by semi-quantitative RT-PCR. The results presented in each panel are representative of three separate experiments.

View larger version (21K): [in a new window]   Fig. 6. Effect of HIF-1Z on HIF-1 activity. Luciferase reporter plasmids containing the Epo enhancer (A) and the VEGF enhancer (B) were transfected into HEK 293 cells transfected with pcDNA and pHIF-1Z. After 16 hours of hypoxia, the luciferase activity of the reporter gene was measured. Each bar, in arbitrary units, represents the mean±s.d. of six experiments. HIF-1 was extracted from the nuclei of the transfected cells subjected to normoxia (N) or 4 hours of hypoxia (H), and analyzed by EMSA (C). For supershift analysis, each 1 µl of rabbit HIF-1 anti-serum, used in Fig. 3B, was added to the completed EMSA reaction mixture. HIF-1 binding (HIF-1), nonspecific binding (NS), and supershifted bands (SS) are indicated. The loading amount of nuclear extract is indicated below the corresponding result. *P<0.05 vs the normoxic control by using unpaired Student’s t-test.

View larger version (17K): [in a new window]   Fig. 7. The effect of competition by HIF-1 or ARNT with HIF-1Z upon HIF-1 activity. pHIF-1 (A) and pARNT (B) plasmids were cotransfected with the Epo enhancer reporter vector into the stable transfectant cell line of pHIF-1Z. After 16 hours of hypoxia, the luciferase activities of the reporter gene were measured. Each bar, in arbitrary units, represents the mean±s.d. of six experiments, and the loading amount of the plasmid is indicated below the corresponding result. *P<0.05 vs the normoxic control in each condition by using unpaired Student’s t-test.

View larger version (48K): [in a new window]   Fig. 8. Nuclear translocation of ARNT. ARNT protein was extracted from the nuclei and the cytosol of the stable transfectant pHIF-1Z cell line. The cells were subjected to normoxia or hypoxia (4 hours), and 5 µg of protein from nuclear extracts or 40 µg of protein from total cellular lysates were separated by SDS-PAGE followed by western blotting with goat anti-ARNT antibody.

View larger version (99K): [in a new window]   Fig. 9. Subcellular localization of ARNT. HEK 293 cells transfected with pcDNA (-HIF-1Z) or pHA-HIF-1Z (+HIF-1Z) were subjected to normoxia or 4 hours of hypoxia, and then incubated with the monoclonal anti-ARNT antibody followed by an FITC-conjugated goat anti-mouse antibody. The corresponding transmission images are shown in the outer columns.

View larger version (34K): [in a new window]   Fig. 10. Co-immunoprecipitation of HIF-1Z and ARNT. pARNT plasmid was transfected into stable cell lines transfected with pcDNA and pHA-HIF-1Z plasmids. After 4 hours of hypoxia, the cytosolic proteins were immunoprecipitated (IP) by rabbit anti-HA antibody (-HA) or goat anti-ARNT antibody (-ARNT). HA-HIF-1Z protein and ARNT protein were identified by western blotting, and are indicated by arrows.

View larger version (19K): [in a new window]   Fig. 11. Generation of HIF-1Z and the mechanism of the HIF-1Z-induced suppression of hypoxic responses.