title: Rho-kinase limits FGF-2-stimulated VEGF release in osteoblasts.
abstract: We previously reported that basic fibroblast growth factor (FGF-2) stimulates the release of vascular endothelial growth factor (VEGF) via 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 the VEGF release in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is involved in FGF-2-stimulated VEGF release in MC3T3-E1 cells. FGF-2 induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase. Y27632, a specific inhibitor of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly enhanced the FGF-2-stimulated VEGF release. Fasudil, another Rho-kinase inhibitor, also amplified the VEGF release. FGF-2 significantly stimulated VEGF accumulation and fasudil enhanced FGF-2-stimulated VEGF accumulation also in whole cell lysates. Neither Y27632 nor fasudil affected the phosphorylation levels of p44/p42 MAP kinase or p38 MAP kinase. Y27632 and fasudil markedly strengthened the FGF-2-induced phosphorylation of SAPK/JNK. Y27632 as well as fasudil enhanced FGF-2-stimulated VEGF release and Y27632 enhanced the FGF-2-induced phosphorylation levels of SAPK/JNK also in human osteoblasts. These results strongly suggest that Rho-kinase negatively regulates FGF-2-stimulated VEGF release in osteoblasts.
authors.affiliation: Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Natsume
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Tokuda
authors.forename: Seiji
authors.initials: S
authors.lastname: Adachi
authors.forename: Shinji
authors.initials: S
authors.lastname: Takai
authors.forename: Rie
authors.initials: R
authors.lastname: Matsushima-Nishiwaki
authors.forename: Kenji
authors.initials: K
authors.lastname: Kato
authors.forename: Chiho
authors.initials: C
authors.lastname: Minamitani
authors.forename: Shunpei
authors.initials: S
authors.lastname: Niida
authors.forename: Jun
authors.initials: J
authors.lastname: Mizutani
authors.forename: Osamu
authors.initials: O
authors.lastname: Kozawa
authors.forename: Takanobu
authors.initials: T
authors.lastname: Otsuka
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D006801:Humans; D007124:Immunoenzyme Techniques; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D051379:Mice; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D000577:Amides; D004791:Enzyme Inhibitors; D011725:Pyridines; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; C108830:Y 27632; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D043263:Myosin-Light-Chain Phosphatase; C510645:Ppp1r12a protein, mouse; C049347:fasudil