Activation of hypoxia-inducible factor-1alpha is necessary for lysophosphatidic acid-induced vascular endothelial growth factor expression.
OBJECTIVE
Lysophosphatidic acid (LPA) plays an important role in mediating cell proliferation, survival, and tumor invasion and angiogenesis. This bioactive phospholipid at the concentration in ascitic fluid stimulates the growth of malignant ovarian tumors by increasing the expression of vascular endothelial growth factor (VEGF). In the present study, we investigated whether LPA activates hypoxia inducible factor-1 (HIF-1), a key transcriptional complex in tumor progression and metastasis, thereby increasing the expression of VEGF.


METHODS
Immunoblotting, reverse transcription-PCR, ELISA, immunofluorescence, and chromatin immunoprecipitation assay were used to examine the expression of VEGF and HIF-1alpha in various cancer cells. Specific HIF-1alpha small interfering RNA was transfected to various cancer cells to determine the role of HIF-1alpha in LPA-induced VEGF expression.


RESULTS
LPA induced expressions of VEGF and HIF-1alpha in OVCAR-3, CAOV-3, PC-3, and SK-Hep1 cells but not in SKOV-3 and Hep-3B cells. In OVCAR-3 and PC-3 cells, the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin/p70S6K and p42/p44 mitogen-activated protein kinase pathways were required for LPA-induced HIF-1alpha and VEGF expressions, whereas only the phosphoinositide 3-kinase/mammalian target of rapamycin/p70S6K pathway was important in SK-Hep1 cells. Immunofluorescence microscopy assay showed translocation of HIF-1alpha to nucleus by LPA, and chromatin immunoprecipitation assay revealed the binding of HIF-1alpha to the promoter of VEGF by LPA. Importantly, we found that small interfering RNA-induced reduction of HIF-1alpha expression significantly attenuated VEGF expression by LPA.


CONCLUSIONS
Our results show for the first time that LPA induces VEGF via HIF-1alpha activation and reveal a critical role of HIF-1alpha in LPA-induced cancer cell proliferation and angiogenesis.
Lee
Jangsoon
J
College of Medicine, Konyang University, Daejeon, Korea.
Park
Soon Young
SY
Lee
Eun Kyung
EK
Park
Chang Gyo
CG
Chung
Hyun Cheol
HC
Rha
Sun Young
SY
Kim
Yong Kee
YK
Bae
Gyu-Un
GU
Kim
Bum Kyeong
BK
Han
Jeung-Whan
JW
Lee
Hoi Young
HY
D015153:Blotting, Western; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D047468:Immunoprecipitation; D008246:Lysophospholipids / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D009369:Neoplasms / Q000378:metabolism*; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis*
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008246:Lysophospholipids; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C032881:lysophosphatidic acid