Macrophage Migration Inhibitory Factor Activates Hypoxia-Inducible Factor in a p53-Dependent Manner

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静脈麻酔薬バルビツレイトは低酸素誘導性因子1の活性化を阻害する

京都大学0048新制・課程博士博士(医学)甲第15225号医博第3425号新制||医||979(附属図書館)27703京都大学大学院医学研究科外科系専攻(主査)教授 小池 薫, 教授 乾 賢一, 教授 淀井 淳司学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

Involvement of p53 in MIF-induced HIF-1 activation.

<p>(A and B) MDA-MB-231 cells and Saos-2 cells were exposed to 20% or 1% O₂ conditions with or without rhMIF treatment for 4 h and then harvested for immunoblot assay for p53 (A) and HIF-1α and HIF-1β protein (B). (C and D) MCF-7 cells and Saos-2 cells were transfected with p2.1 reporter and pRL-SV40 (C). MCF-7 cells transfected with pSUPER (EV) or pSUPER-p53(p53) were transfected with p2.1 reporter and pRL-SV40 (D). Cells were exposed to 20% and 1% O₂ conditions with or without rhMIF treatment and then harvested for luciferase assay. Fold induction of relative luciferase activity was calculated. A normalized mean value±S.D. of three independent transfections is shown as fold induction. *; <i>p</i><0.05 compared to respective control (ANOVA). (E) MCF-7 and MDA-MB-231 cells transfected with pFLAG-MIF-wt plasmid were exposed to 1% O₂ and then cells were harvested. 500 µg of lysates were incubated with anti-FLAG affinity agarose beads and captured protein was eluted and analyzed by Western blot with anti-p53 antibody. 50 µg of lysate were analyzed by Western blot with anti-p53 antibody.</p

Involvement of MIF in HIF-1 regulation in MCF-7 cells.

<p>HIF-1α protein stabilization and transactivation activity induced by MIF is dependent on p53- and MAPK-dependent signaling pathway.</p

Effect of MIF on HIF-1-dependent gene expressions.

<p>(A) MCF-7 cells were transfected with the indicated plasmids and exposed to 20% or 1% O₂ for 12 h. Then total RNA was isolated. Expression of VEGF, GLUT1, HIF-1α, and 18S rRNA was analyzed by RT-PCR using specific primer pairs. (B, C, D, and E) MCF-7 cells were transfected with pRL-SV40 encoding <i>Renilla</i> luciferase, FLAG-tagged MIF expression vector (B, C, and E), and one of the following plasmids encoding firefly luciferase: HRE reporter p2.1 (<i>B, C, and E</i>) or <i>VEGF</i> promoter reporter pVEGF-KpnI-Luc (<i>D</i>). (F) Constructs encoding the GAL4 DNA-binding domain (amino acids 1–147) fused to the indicated amino acids of HIF-1α were analyzed for their ability to transactivate reporter gene GAL4E1bLuc containing five GAL4-binding sites. MCF-7 cells were co-transfected with pRL-SV40 (50 ng), GAL4E1bLuc (100 ng), GAL4-HIF-1α fusion protein expression plasmids (100 ng), and FLAG-tagged MIF expression vector or empty vector (EV) (200 ng). Cells were treated with 100 µM of DFX or the indicated dose of rhMIF (F) and exposed to 20% or 1% O₂ conditions for 18 h and then harvested. Results shown represent mean±S.D. of three independent transfections. *; <i>p</i><0.05 compared to respective controls without MIF treatment (ANOVA).</p

Effect of MIF on HIF-1 protein expression in MCF-7 cells.

<p>(A and B) MCF-7 cells were transfected with 2 µg of p3xFLAG-CMV-10 plasmid (EV) or pFLAG-MIF plasmids (A and B lane 4; 2 µg, B lane 3; 0.5 µg). (C) MCF-7 cells were transfected by the plasmids for expression of FLAG tag (EV) and FLAG-tagged MIF and selected by puromycin resistance. Two clones of MCF-7 cells stably expressing FLAG tag (EV) and FLAG-tagged MIF (MIF#1 and MIF#2 cells) were established. (D) MCF-7 cells were treated with the indicated doses of bacterially produced recombinant human (rh)MIF. (E) MCF-7 cells were treated with rhMIF. Cells were exposed to 20% (A, B, C, and D), 5% (C), or 1% (A, B, C, and D) O₂ conditions or treated with 100 µM DFX (E) for 4 h. Cell were harvested for immunoblot assay for MIF (A and C), HIF-1α and HIF-1β protein (A, B, C, D, and E). Representative immunoblots are shown. Intensity of respective bands were analyzed densitometrically and fold induction to respective controls (lane1) are plotted accordingly as mean±S.D. (n = 3) *<i>p</i><0.05 compared to 20% O₂ conditions without MIF (lane 1) (ANOVA).</p

Effect of MIF-gene silencing on HIF-1 activity.

<p>(A, B, and D) MCF-7 cells were transfected with siRNA against MIF, TRX, CD74, or green fluorescence protein (GFP) as a negative control. Cells were exposed to 20% or 1% O₂ conditions and were harvested for immunoblot assay for MIF or TRX (left panels) or HIF-1α and HIF-1β protein expression (right panels). Representative immunoblots are shown. Intensity of respective bands were analyzed densitometrically and fold induction to lane 1 are plotted accordingly as mean±S.D. (n = 3) (A, B, and C). *<i>p</i><0.05 compared to 20% O₂ conditions without treatment (ANOVA). (C) MCF-7 cells were pre-treated with 10 µM α-tocopherol or 10 mM NAC and then exposed to 20% or 1% O₂ conditions for 4 h. Cells were harvested for immunoblot assay for HIF-1α and HIF-1β protein. (D) siRNAs for GFP or CD74 were introduced into MCF-7 cells. The cells were exposed to 1% O₂ with or without rhMIF treatment. Cells were harvested for immunoblot assay for MIF HIF-1α protein. (E) MCF-7 cells were transfected with HRE reporter p2.1 and pRL-SV40 encoding <i>Renilla</i> luciferase. Cells were treated with the indicated dose of rhMIF and exposed to 20% or 1% O₂ conditions for 18 h and then harvested. Results shown represent mean±S.D. of three independent transfections. *; <i>p</i><0.05 compared to respective controls (ANOVA).</p