Ultrasound stimulates NF-kappaB activation and iNOS expression via the Ras/Raf/MEK/ERK signaling pathway in cultured preosteoblasts.
It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and non-operatively clinical uses. Nitric oxide (NO) is a crucial early mediator in mechanically induced bone formation. Here we found that US-mediated inducible nitric oxide synthase (iNOS) expression was attenuated by Ras inhibitor (manumycin A), Raf-1 inhibitor (GW5074), MEK inhibitor (PD98059), NF-kappaB inhibitor (PDTC), and IkappaB protease inhibitor (TPCK). US-induced Ras activation was inhibited by manumycin A. Raf-1 phosphorylation at Ser(338) by US was inhibited by manumycin A and GW5074. US-induced MEK and ERK activation was inhibited by manumycin A, GW5074, and PD98059. Stimulation of preosteoblasts with US activated IkappaB kinase alpha/beta (IKK alpha/beta), IkappaBalpha phosphorylation, p65 phosphorylation at Ser(276), p65, and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. US-mediated an increase of IKK alpha/beta, IkappaBalpha, and p65 phosphorylation, kappaB-luciferase activity and p65 and p50 binding to the NF-kappaB element was inhibited by manumycin A, GW5074, and PD98059. Our results suggest that US increased iNOS expression in preosteoblasts via the Ras/Raf-1/MEK/ERK/IKKalphabeta and NF-kappaB signaling pathways.
Hou
Chun-Han
CH
Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan.
Lin
Jinn
J
Huang
Shier-Chieg
SC
Hou
Sheng-Mou
SM
Tang
Chih-Hsin
CH
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D004790:Enzyme Induction; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D017102:Fracture Healing; D017930:Genes, Reporter; D051550:I-kappa B Kinase / Q000378:metabolism; D020935:MAP Kinase Signaling System; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis* / Q000235:genetics; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051996:Transcription Factor RelA / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D014465:Ultrasonics; D048490:raf Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D047428:Protein Kinase Inhibitors; C498144:Rela protein, mouse; D051996:Transcription Factor RelA; C073701:Nfkb1 protein, mouse; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D048490:raf Kinases; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496562:Ikbkb protein, mouse; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins