Negative regulation by p70 S6 kinase of FGF-2-stimulated VEGF release through stress-activated protein kinase/c-Jun N-terminal kinase in osteoblasts.
To clarify the mechanism of VEGF release in osteoblasts, we studied whether p70 S6 kinase is involved in basic FGF-2-stimulated VEGF release in osteoblast-like MC3T3-E1 cells. In this study, we show that p70 S6 kinase activated by FGF-2 negatively regulates VEGF release through SAPK/JNK in osteoblasts.


BACKGROUND
Vascular endothelial growth factor (VEGF) plays an important role in bone metabolism. We have previously reported that fibroblast growth factor-2 (FGF-2) stimulates the release of VEGF through p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates VEGF release. However, the mechanism behind VEGF release in osteoblasts is not precisely known.


METHODS
The levels of VEGF released from MC3T3-E1 cells were measured by enzyme immunoassay. The phosphorylation of each protein kinase was analyzed by Western blotting. To knock down p70 S6 kinase in MC3T3-E1 cells, the cells were transfected with siRNA to target p70 S6 kinase.


RESULTS
FGF-2 time-dependently induced the phosphorylation of p70 S6 kinase. Rapamycin significantly enhanced the FGF-2-stimulated VEGF release and VEGF mRNA expression. The FGF-2-induced phosphorylation of p70 S6 kinase was suppressed by rapamycin. Rapamycin markedly enhanced the FGF-2-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. SP600125, a specific inhibitor of SAPK/JNK, suppressed the amplification by rapamycin of the FGF-2-stimulated VEGF release similar to the levels of FGF-2 with SP600125. Finally, downregulation of p70 S6 kinase by siRNA significantly enhanced the FGF-2-stimulated VEGF release and phosphorylation of SAPK/JNK.


CONCLUSIONS
These results strongly suggest that p70 S6 kinase limits FGF-2-stimulated VEGF release through self-regulation of SAPK/JNK, composing a negative feedback loop, in osteoblasts.
Takai
Shinji
S
Department of Pharmacy, Gifu University Graduate School of Medicine, and Department of Clinical Laboratory, National Hospital for Geriatric Medicine, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan.
Tokuda
Haruhiko
H
Hanai
Yoshiteru
Y
Harada
Atsushi
A
Yasuda
Eisuke
E
Matsushima-Nishiwaki
Rie
R
Kato
Hisaaki
H
Ogura
Shinji
S
Ohta
Toshiki
T
Kozawa
Osamu
O
D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D002478:Cells, Cultured; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D007166:Immunosuppressive Agents / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010006:Osteoblasts / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000873:Anthracenes; D007166:Immunosuppressive Agents; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D016222:Fibroblast Growth Factor 2; C432165:pyrazolanthrone; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D048031:JNK Mitogen-Activated Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D020123:Sirolimus