Ultrasound Stimulates NF-κB 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-I inhibitor (GW5074), MEK inhibitor (PD98059), NF-kappa B inhibitor (PDTC), and I kappa B protease inhibitor (TPCK). US-induced Ras activation was inhibited by manumycin A. Raf-I 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 I kappa B kinase alpha/beta (IKK alpha/beta), I kappa B alpha phosphorylation, p65 phosphorylation at Ser(276), p65, and p50 translocation from the cytosol to the nucleus, and kappa B-luciferase activity. US-mediated an increase of IKK alpha/beta, I kappa B alpha, and p65 phosphorylation, kappa B-luciferase activity and p65 and p50 binding to the NF-kappa B element was inhibited by manumycin A, GW5074, and PD98059. Our results suggest that US increased iNOS expression in preosteoblasts via the Ras/Raf-I/MEK/ERK/IKK alpha beta and NF-kappa B signaling pathways. J. Cell. Physiol. 220: 196-203, 2009. (C) 2009 Wiley-Liss, Inc.