[Activation of HIF-1 by bFGF in breast cancer: role of PI-3K and MEK1/ERK pathways].
OBJECTIVE
To explore the mechanism of basic fibroblast growth factor (bFGF)-mediated hypoxia inducible factor (HIF-1) activation and the down-stream signaling pathways involved.


METHODS
Human breast cancer cells of the line T47D were cultured and lysed to extract the total protein in the supernatant. The amounts of extracellular signal kinase 1/2 (ERK1/2), Akt, p38, and beta-tubulin were measured. T47D cells were inoculated into a 24-well plate, co-transfected with luciferase vector OB-HRE containing HIF-1 functional sequence (HRE) and pRL-SV40 (as inner marker), pretreated with SU5402, inhibitor of FGFR1, SB203580, inhibitors of PI-3K, PD98059, inhibitors of MEK1, or LY294002, inhibitors of p38, treated with basic fibroblast growth factor (bFGF), and then lysed. The amounts of ERK1/2, Akt, p38, and beta-tubulin were measured. Western blotting was used to detect the HIF-1alpha level in total protein. Dual luciferase assay was used to analyze the transactivity of HIF-1. The firefly/renilla luciferase ratio was measured to access the transcription activity of HIF-1. Western blotting was used to detect the expression of HIF-1alpha protein and phosphorylated Akt, ERK1/2 and p38 in whole cell extract.


RESULTS
After the addition of bFGF Western blotting showed that the and phosphorylation of Akt, ERK1/2 and p38 in the T47D cells were increased time- and dose-dependent manner, and dual luciferase assay showed that the fluorescent intensity was increased, signifying the increase of expression of HIF-1alpha protein. Ten minutes after the addition of CHX the expression of HIF-1alpha protein began to be decreased and ceased 90 minutes after. SU5402 remarkably dose-dependently blocked the bFGF-induced phosphorylation of ERK1/2, Akt and p38. 15 micromol/L LY294002 completely blocked the bFGF-induced phosphorylation of Akt. 5 micromol/L PD98059 blocked 80% of the bFGF-induced phosphorylation of ERK1/2. 10 approximately 20 micromol/L SB203580 basically blocked the bFGF-induced phosphorylation of p38. SU5402 and LY294002 100% inhibited the bFGF-induced expression of HIF-1alpha protein. However, PD98059 and SB203580 did not significantly influence the expression of HIF-1alpha protein induced by bFGF. Luciferase assay showed that SU5402 and PD98059 inhibited the bFGF-induced transcription activity of HIF-1 by 94.8% and 81.7% respectively. LY294002 not only completely inhibited the bFGF-induced transcription activity of HIF-1 but also inhibited the basic transcription of HIF-1, and SB203580 did not significantly influence the transcription activity of HIF-1.


CONCLUSIONS
bFGF activates HIF-1 via the PI-3K/Akt and MEK1/ERK pathways that co-operatively and differentially regulate this process with PI-3K/Akt pathway playing a more important role.
Shi
Yong-hong
YH
Department of Pathology, Peking University Health Science Center, Beijing 100083, China.
Wang
Yu-xiang
YX
You
Jiang-feng
JF
Heng
Wan-jie
WJ
Zhong
Hao-hao
HH
Fang
Wei-gang
WG
D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D016222:Fibroblast Growth Factor 2; D019869:Phosphatidylinositol 3-Kinases; D048052:Mitogen-Activated Protein Kinase 3; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human