1. Arterioscler Thromb Vasc Biol
Positive Cross-Talk Between Hypoxia Inducible Factor-1α and Liver X Receptor α Induces Formation of Triglyceride-Loaded Foam Cells
by Tae-Young Na, Hyo-Jeong Lee, Hyeon-Jeong Oh, Seung Huh, In-Kyu Lee, and Mi-Ock Lee
Arterioscler Thromb Vasc Biol
Volume 31(12):
November 16, 2011
Copyright © American Heart Association, Inc. All rights reserved.

2. Liver X receptor α (LXRα) was induced at the transcription-level in macrophages and RAW cells under hypoxia.
Liver X receptor α (LXRα) was induced at the transcription-level in macrophages and RAW cells under hypoxia. A, Human primary macrophages obtained from healthy donors and murine RAW cells were incubated for indicated time under hypoxia. Whole cell lysates were prepared and the proteins were analyzed by Western blotting. B, RAW cells were incubated for 0, 0.5, 1, 3, or 6 hours under hypoxia. Total RNA was prepared and analyzed for expression of the indicated transcripts by qRT-PCR using specific primers. C, HeLa cells were transiently transfected with the human LXRα-promoter-Luc and were treated with 100 μmol/L desferrioxamine (DFO) and/or 1 μmol/L T17 for 18 hours (left). Schematic representation of the human LXRα promoter investigated in this study. Sticks indicate the presence of putative hypoxia response elements (right, upper). THP-1 cells were treated with 100 μmol/L DFO for 12 hours. DNA fragments that immunoprecipitated were amplified by PCR using primers that amplify the DNA fragment a, b, and c as indicated (right, lower). D, RAW cells were transfected with si-RNAs as indicated and treated with 0.1% O2 for 12 hours. Whole cell lysates were analyzed by Western blotting. *P<0.05, and **P<0.01 vs vehicle treatment (n=3). #P<0.01, and ##P<0.005 vs desferrioxamine (DFO) or T17 treatment (n=3). One representative of at least 3 independent experiments with similar results is shown. SREBP indicates sterol regulatory element binding protein; FAS, fatty acid synthase; HIF-1α, hypoxia inducible factor-1α.
Tae-Young Na et al. Arterioscler Thromb Vasc Biol. 2011;31:
Copyright © American Heart Association, Inc. All rights reserved.

3. Activation of liver X receptor α (LXRα) enhances HIF-1α at protein-level not at mRNA-level.
Activation of liver X receptor α (LXRα) enhances HIF-1α at protein-level not at mRNA-level. A, Human primary macrophages and RAW cells were treated 1 μmol/L TO (T17) for the indicated time or with the indicated concentration for 24 hours. B, RAW cells were treated with 1 μmol/L T17 for 0, 0.5, 1, 4, 9, or 24 hours. Total RNA was prepared and analyzed for expression of the indicated transcripts by qRT-PCR. C, RAW cells were transfected with Myc-LXRα or empty vector (EV) and were incubated with 100 μmol/L desferrioxamine (DFO) for 12 hours. Whole cell lysates were prepared and the proteins were analyzed by Western blotting. D, RAW cells were transfected with si-RNAs as indicated and treated with 1 μmol/L TO (T17) for 24 hours. Whole cell lysates were prepared and the proteins were analyzed by Western blotting. *P<0.05, **P<0.01 (n=3). One representative of at least 3 independent experiments with similar results is shown. SREBP indicates sterol regulatory element binding protein; FAS, fatty acid synthase; HIF-1α, hypoxia inducible factor-1α.
Tae-Young Na et al. Arterioscler Thromb Vasc Biol. 2011;31:
Copyright © American Heart Association, Inc. All rights reserved.

4. Liver X receptor α (LXRα) suppresses degradation of HIF-1α by blocking ubiquitination.
Liver X receptor α (LXRα) suppresses degradation of HIF-1α by blocking ubiquitination. A, RAW cells were transfected with empty vector (EV) or expression vector encoding Myc-LXRα. Cells transfected with EV were treated with 1 μmol/L T17 for 24 hours. At the end of treatment, 10 μmol/L cycloheximide (CHX) was added for the indicated time period. Expression of the indicated proteins was analyzed by western blot analysis (WB) (upper). The density of each protein band was determined with an image analysis system and normalized to that of the corresponding α-tubulin (lower). B, RAW cells were transfected with EV or Myc-LXRα together with HA-ubiquitin (Ub). Cells transfected with EV were treated with 0.1% O2 for 12 hours or 1 μmol/L TO (T17) for 24 hours. Cells were then treated with 10 μmol/L MG132 for 3 hours before being harvested. Whole cell lysates were immunoprecipitated (IP) and analyzed by WB. Protein level of hypoxia inducible factor-1α (HIF-1α) in whole cell lysates was determined by WB as input. C, NIH3T3 cells were transfected with expression vectors encoding GFP-PHD1, GFP-PHD2, and GFP-PHD3 with or without pCMV-Myc-LXRα as indicated. Transfected cells were incubated under hypoxia or normoxia for 12 hours. Cells were treated with or without 10 μmol/L MG132 for 3 hours before being harvested. Whole cell lysates were immunoprecipitated and analyzed by Western blotting. One representative of at least 3 independent experiments with similar results is shown.
Tae-Young Na et al. Arterioscler Thromb Vasc Biol. 2011;31:
Copyright © American Heart Association, Inc. All rights reserved.

5. Liver X receptor α (LXRα) interacts with hypoxia inducible factor-1α (HIF-1α) and enhances transactivation function of HIF-1α.
Liver X receptor α (LXRα) interacts with hypoxia inducible factor-1α (HIF-1α) and enhances transactivation function of HIF-1α. A, RAW cells incubated under hypoxia for 12 hours. Whole cell lysates were immunoprecipitated (IP) with the indicated antibodies, and precipitates were probed by Western blot (WB) analysis. B, NIH3T3 cells were transfected with Gal4-tk-luc/pGal4-LXRα together with increasing amount of VP16-HIF-1α. C, RAW cells were transfected with expression vectors encoding GST-HIF-1α (left) or FLAG-LXRα (right) with the indicated LXRα or HIF-1α truncates, respectively. Whole cell lysates were IP and analyzed by WB as indicated. EV indicates empty vector; ODD, oxygen-dependent degradation domain; ID, inhibitory domain; CTAD, C-terminal activation domain, NT, N-terminal domain; DBD, DNA binding domain; LBD, ligand binding domain. Subcellular localization of the truncated proteins were shown in the Supplemental Figure X. D, RAW cells were transfected with FLAG-LXRαNT, FLAG-LXRαDBD, or FLAG-LXRαLBD. Whole cell lysates were prepared and the proteins were analyzed by Western blotting. E, NIH3T3 cells were transfected with Gal4-tk-luc and pGal4-HIF-1α together with the indicated LXRα truncates (left). Or cells were transfected with pGal4-LXRα and HIF-1α expression vector and were treated with 1 μmol/L TO (T17), 100 μmol/L CoCl2, or 100 μmol/L desferrioxamine (DFO) (right). *P<0.05, **P<0.01 (n=3). F, RAW cells were treated with 1 μmol/L T17 or 0.1% O2 for 12 hours. DNA fragments that contain flanking region of the HIF-1α binding sites on the mouse promoter of Glut-1 and erythropoietin (EPO) 3′-UTR genes were IP with the indicated antibodies were amplified by PCR. One representative of at least 3 independent experiments with similar results is shown.
Tae-Young Na et al. Arterioscler Thromb Vasc Biol. 2011;31:
Copyright © American Heart Association, Inc. All rights reserved.

6. Hypoxia and TO901317 induce lipogenesis in macrophages.
Hypoxia and TO induce lipogenesis in macrophages. A, RAW cells were incubated under hypoxia and/or treated with 1 μmol/L TO (T17). B, RAW cells were transfected with si-RNAs and were treated with 0.1% O2 for 24 hours and/or 1 μmol/L T17 for 96 hours. At the end of treatment, lipid droplets were stained using Nile-red, and fluorescence was determined by flow cytometry. C, RAW cells were incubated with 100 μmol/L desferrioxamine (DFO) for 12 hours or 1 μmol/L TO for 48 hours. Cell pellets and culture media were analyzed for triglycerides and total cholesterol. D, RAW cells were incubated with 100 μmol/L DFO for 12 hours or 1 μmol/L TO (T17) for 24 hours (left). RAW cells were transfected with the indicated si-RNAs. After 3 hours of transfection, the cells were treated with 22(S)-hydroxycholesterol (HC) or vehicle for 24 hours (right). Total RNA was prepared and analyzed for expression of the indicated transcripts by qRT-PCR. *P<0.05, **P<0.01, and ***P<0.001 vs vehicle treatment (n=3). #P<0.01, and ##P<0.005 vs DFO or T17 treatment (n=3). One representative of at least 3 independent experiments with similar results is shown.
Tae-Young Na et al. Arterioscler Thromb Vasc Biol. 2011;31:
Copyright © American Heart Association, Inc. All rights reserved.

7. Increases in expression level of liver X receptor α (LXRα) and hypoxia inducible factor-1α (HIF-1α) after treatment of inflammatory cytokines.
Increases in expression level of liver X receptor α (LXRα) and hypoxia inducible factor-1α (HIF-1α) after treatment of inflammatory cytokines. A, RAW cells were treated with 10 ng/mL IL-1β or TNFα for the indicated period. B, RAW cells were transfected with si-control or si-LXRα and treated with 10 ng/mL IL-1β or TNFα for 24 hours. The whole cell lysates were analyzed for expression of the indicated proteins by western blot analysis. One representative of at least 3 independent experiments with similar results is shown. C, Schematic model for cross-talk of HIF-1α and LXRα in the foam cell formation of human atherosclerotic lesions. SREBP indicates sterol regulatory element binding protein; FAS, fatty acid synthase.
Tae-Young Na et al. Arterioscler Thromb Vasc Biol. 2011;31:
Copyright © American Heart Association, Inc. All rights reserved.