Abstract
It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and clinical studies. Nitric oxide (NO) is a crucial early mediator in mechanically induced bone formation. Here we found that US stimulation increased NO formation and the protein level of inducible nitric-oxide synthase (iNOS). US-mediated iNOS expression was attenuated by anti-integrin alpha5beta1 or beta1 antibodies but not anti-integrin alphavbeta3 or beta3 antibodies or focal adhesion kinase mutant. Integrin-linked kinase (ILK) inhibitor (KP-392), Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-[(R)-2-O-methyl-3-O-octadecylcarbonate]) or mammalian target of rapamycin (mTOR) inhibitor (rapamycin) also inhibited the potentiating action of US. US stimulation increased the kinase activity of ILK and phosphorylation of Akt and mTOR. Furthermore, US stimulation also increased the stability and activity of HIF-1 protein. The binding of HIF-1alpha to the HRE elements on the iNOS promoter was enhanced by US stimulation. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that both ILK/Akt and mTOR signaling pathway were potentially required for US-induced HIF-1alpha activation and subsequent iNOS up-regulation. Taken together, our results provide evidence that US stimulation up-regulates iNOS expression in osteoblasts by an HIF-1alpha-dependent mechanism involving the activation of ILK/Akt and mTOR pathways via integrin receptor.
Highlights
Distinguishes itself by being non-invasive and easy to apply
Effect of Ultrasound Stimulation on inducible nitric-oxide synthase (iNOS) Expression in Osteoblasts—It has been demonstrated that pulse application of low intensity US increased nitric oxide (NO) release via up-regulation of iNOS [5], which is important for mechanically induced bone formation [27]
We investigated the effect of US stimulation on the iNOS expression in osteoblasts
Summary
Distinguishes itself by being non-invasive and easy to apply. Low intensity levels are used to accelerate fracture healing and are considered neither thermal nor destructive. We found that US stimulation increased iNOS expression and promoted NO production in MC3T3-E1 cells in a HIF-dependent manner involving the activation ILK/Akt/mTOR through the 1 integrin receptor. C, cells were pretreated with mAb against 1 integrin (20 g/ml), KP-392 (30 M), Akt inhibitor (3 M) for 30 min or transfected with ILK siRNA for 24 h followed by exposure to US for 20 min, and mTOR phosphorylation was determined 30 min after US stimulation.
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