Ultrasound induces hypoxia-inducible factor-1 activation and inducible nitric-oxide synthase expression through the integrin/integrin-linked kinase/Akt/mammalian target of rapamycin pathway in osteoblasts.
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.
Tang
Chih-Hsin
CH
Department of Pharmacology, College of Medicine, China Medical University, Taichung 404, Taiwan.
Lu
Dah-Yuu
DY
Tan
Tzu-Wei
TW
Fu
Wen-Mei
WM
Yang
Rong-Sen
RS
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002460:Cell Line; D004195:Disease Models, Animal; D051417:Focal Adhesion Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D017102:Fracture Healing / Q000502:physiology; D050723:Fractures, Bone / Q000201:enzymology / Q000473:pathology / Q000628:therapy; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D039081:Integrin alpha5beta1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D039302:Integrin alphaVbeta3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis*; D010006:Osteoblasts / Q000201:enzymology* / Q000473:pathology; D011485:Protein Binding / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020218:Response Elements; D015398:Signal Transduction / Q000187:drug effects; D013010:Sonication; D058570:TOR Serine-Threonine Kinases; D015854:Up-Regulation / Q000187:drug effects; D000099200:Scaffold Protein ILK
D000906:Antibodies; D051417:Focal Adhesion Kinase 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D039081:Integrin alpha5beta1; D039302:Integrin alphaVbeta3; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D000099200:Scaffold Protein ILK; C497443:Hif1a protein, mouse; C496317:Nos2 protein, mouse; C546843:mTOR protein, mouse; C496051:Ptk2 protein, mouse