Ultrasound Stimulates Cyclooxygenase-2 Expression and Increases Bone Formation through Integrin, Focal Adhesion Kinase, Phosphatidylinositol 3-Kinase, and Akt Pathway in Osteoblasts

Abstract

It has been shown that ultrasound (US) stimulation accelerates fracture healing in animal models and in clinical studies. Here we found that US stimulation transiently increased the surface expression of ␣2, ␣5, ␤1, and ␤3 integrins in cultured osteoblasts, as shown by flow cytometric analysis and immunofluorescence staining. US stimulation increased prostaglandin E 2 formation and the protein and mRNA levels of cyclooxygenase-2 (COX-2). At the mechanistic level, anti-integrin ␣5␤1 and ␣v␤3 antibodies or rhodostomin, a snake venom disintegrin, attenuated the US-induced COX-2 expression. Phosphatidylinositol 3-kinase (PI3K) inhibitors 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and wortmannin also inhibited the potentiating action of US. US stimulation increased the phosphorylation of focal adhesion kinase (FAK), extracellular signal-regulated kinases (ERK), p85 subunit of PI3K, and serine 473 of Akt. COX-2 promoter activity was enhanced by US stimulation in cells transfected with pCOX2-Luc. Cotransfection with dominant-negative mutant of FAK(Y397F), p85(⌬p85), Akt(K179A), or ERK2(K52R) inhibited the potentiating action of US on COX-2 promoter activity. Expression of mineralized nodule was lower in dominant-negative mutants of FAK, p85, and Akt-transfected clones than in vector-transfected control cells. Taken together, our results provide evidence that US stimulation increases COX-2 expression and promotes bone formation in osteoblasts via the integrin/ FAK/PI3K/Akt and ERK signaling pathway.

Ultrasound treatment for accelerating fracture healing of the distal radius. A control study

“…Several studies indicate that LIPUS can be applied to accelerate the fracture healing by influencing all four stages of fracture healing [18][19][20][21][22] Moreover, there was no difference in reposition between the two groups (p >0.05) in the present study, and the therapeutic effects of LIPUS were not satisfactory. The possible reasons were not difficult to explore.…”

Low-intensity Ultrasound Increases FAK, ERK-1/2, and IRS-1 Expression of Intact Rat Bones in a Noncumulative Manner

“…How long-term LIPUS induced sustained increased P-FAK and P-ERK-1/2 is unknown. One mechanical stimulation increases P-FAK and P-ERK-1/2 for 4 hours [4,14,26,36], whereas our long-term LIPUS not only increased P-FAK and P-ERK-1/2, but also prolonged their activation for 15 hours at 7 and 14 days (only ERK-1/2). P-FAK and P-ERK-1/2 peaks coincided with their expression peaks, supporting the observation that sustained P-FAK and P-ERK-1/2 elevation are proportional to the elevation of their expression [46].…”

“…LIPUS augmented FAK and ERK-1/2 expression at 7 and 14 days, respectively, indicating long-term LIPUS not only influences cytoplasmic reactions [9,36,45], but also affects nuclear reactions, which induce FAK and ERK-1/2 synthesis. Seven days after the augmentation of FAK (at 14 days) and ERK-1/2 (at 21 days) expression, their expression decreased, showing a noncumulative effect of LIPUS on intact bone and suggesting there is a regulatory mechanism in bone cells, similar to negative feedback, that is activated after the augmentation of FAK and ERK-1/2 expression.…”

“…Because LIPUS stimulates fracture repair but does not increase bone mineral density on growing bone or prevent osteoporosis after spinal cord injury and estrogen deprivation [21,35,41,42,44], we questioned whether LIPUS on intact bone would regulate FAK and ERK-1/2 expression and activation, which are proteins that act on mechanotransduction signaling and induce osteogenesis [36]. In addition, because mechanical loading acts Fig.…”

“…P-FAK couples with various proteins to activate mechanotransduction signaling pathways. P-FAK/ERK-1/2 is one important mechanotransduction signaling pathway that promotes bone formation [4,25,36].…”