title: Ras induction of superoxide activates ERK-dependent angiogenic transcription factor HIF-1alpha and VEGF-A expression in shock wave-stimulated osteoblasts.
abstract: Vascular endothelial growth factor (VEGF) released by osteoblasts plays an important role in angiogenesis and endochondral ossification during bone formation. In animal studies, we have reported that shock waves (SW) can promote osteogenic differentiation of mesenchymal stem cells through superoxide-mediated signal transduction (Wang, F. S., Wang, C. J., Sheen-Chen, S. M., Kuo, Y. R., Chen, R. F., and Yang, K. D. (2002) J. Biol. Chem. 277, 10931-10937) and vascularization of the bone-tendon junction. Here, we found that SW elevation of VEGF-A expression in human osteoblasts to be mediated by Ras-induced superoxide and ERK-dependent HIF-1alpha activation. SW treatment (0.16 mJ/mm(2), 1 Hz, 500 impulses) rapidly activated Ras protein (15 min) and Rac1 protein (30 min) and increased superoxide production in 30 min and VEGF mRNA expression in 6 h. Early scavenging of superoxide, but not nitric oxide, peroxide hydrogen, or prostaglandin E(2), reduced SW-augmented VEGF-A levels. Inhibition of superoxide production by diphenyliodonium, an NADPH oxidase inhibitor, was found to suppress VEGF-A expression. Transfection of osteoblasts with a dominant negative (S17N) Ras mutant abrogated the SW enhancement of Rac1 activation, superoxide synthesis, and VEGF expression. Further studies demonstrated that SW significantly promoted ERK activation in 1 h and HIF-1alpha phosphorylation and HIF-1alpha binding to VEGF promoter in 3 h. In support of the observation that superoxide mediated the SW-induced ERK activation and HIF-1alpha transactivation, we further demonstrated that scavenging of superoxide by superoxide dismutase and inhibition of ERK activity by PD98059 decreased HIF-1alpha activation and VEGF-A levels. Moreover, culture medium harvested from SW-treated osteoblasts increased vessel number of chick chorioallantoic membrane. Superoxide dismutase pretreatment and anti-VEGF-A antibody neutralization reduced the promoting effect of conditioned medium on angiogenesis. Thus, modulation of redox reaction by SW may have some positive effect on angiogenesis during bone regeneration.
authors.affiliation: Department of Medical Research, Chang Gung Memorial Hospital, Kaohsiung 833, Taiwan.
authors.forename: Feng-Sheng
authors.initials: FS
authors.lastname: Wang
authors.forename: Ching-Jen
authors.initials: CJ
authors.lastname: Wang
authors.forename: Yeung-Jen
authors.initials: YJ
authors.lastname: Chen
authors.forename: Per-Rong
authors.initials: PR
authors.lastname: Chang
authors.forename: Yu-Ting
authors.initials: YT
authors.lastname: Huang
authors.forename: Yi-Chih
authors.initials: YC
authors.lastname: Sun
authors.forename: Hueng-Chen
authors.initials: HC
authors.lastname: Huang
authors.forename: Ya-Ju
authors.initials: YJ
authors.lastname: Yang
authors.forename: Kuender D
authors.initials: KD
authors.lastname: Yang
mesh_terms: D002454:Cell Differentiation; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004558:Electric Stimulation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009389:Neovascularization, Pathologic; D018919:Neovascularization, Physiologic; D009569:Nitric Oxide / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism* / Q000473:pathology*; D010084:Oxidation-Reduction; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D005419:Flavonoids; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D014157:Transcription Factors; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D013481:Superoxides; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020830:rac1 GTP-Binding Protein; D018631:ras Proteins; D015232:Dinoprostone; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one