Study On The Pulsed Electromagnetic Fields Promoted Bone Formation By Primary Cilia And BMP/BMPRII/Smad1/5/8 Signaling Pathway

Osteoporosis(Osteoporosis,OP) is a systemic bone disease with characteristic decreased bone density, loss of bone mass and increased risk of fracture. Currently, there is no osteoporosis drugs with high efficacy and low side effects. Pulsed electromagnetic fields(PEMFs) had been considered as a promising therapy for a wide range of bone diseases. We have reported that PEMFs promoted osteogenesis with appropriate frequencies, intensities, duty ratio and the duration of exposure can significantly promote differentiation and mineralization of osteoblast, and increase bone mass and prevent bone resorption in animal models. However, the action mechanism of PEMFs in preventing bone loss is still elusive. In current study we attempt to find the mechanism of its action to lay the foundation for PEMFs clinical application. Control of bone metabolism requires a tightly orchestrated interplay among osteoblasts, osteoclasts and osteocytes. We isolated osteoblasts and osteocytes from neonatal SD rat skull tissue and identified with HE, immunohistochemistry, immunofluorescence and western blot. We found that osteocytes were star shaped or dendrite while osteoblasts were spindle. Osteoblasts had a higher ALP activity and expression of COL-Ι and OC, and osteoblasts were more sensitive than osteocytes to osteogenesis stimulators. So we use osteoblasts as the base of follow studuies. Previously we reported that 50 Hz 0.6mT PEMFs stimulated osteoblastic differentiation and mineralization in a primary cilium-dependent manner, but did not know the reason. In the present study, we found that the PEMFs promoted osteogenic differentiation by activating bone morphogenetic protein BMP-Smad1/5/8 signaling on the condition that primary cilia were normal.Firstly, The ROBs were treated by PEMFs for different time and expression BMP2, p-Smad1/5/8, Smad1/5/8 protein were detected. It found that BMP-2, p-Smad1/5/8 significantly increased compared with the control, and p-Smad1/5/8 was positively localized in the nucleus. To determine whether PEMFs promoted the osteogenic differentiation of osteoblasts through BMP-Smad1/5/8 signaling, ROBs were pretreated with Noggin(NOG), an antagonist of BMPs, or LDN-193189(LDN), an inhibitor of BMPRI kinases, and found that the PEMFs promoted increase of ALP activity and other osteogenesis makers were abolished after BMP signaling blocking. These results confirmed that PEMFs stimulated osteoblastic differentiation and maturation by activating BMP-Smad1/5/8 signal pathway.Secondly, to examine whether primary cilia is needed in the PEMF-induced activation of BMP-Smad1/5/8 pathway, we blocked ciliogenesis using small interfering RNA(siRNA) sequence targeting IFT88. We found that PEMF-induced activation of BMP-Smad1/5/8 pathway was inhibited. Next, in order to find the connection between BMP-Smad1/5/8 pathway and primary cilium, we detected the expression and ciliary localization of BMPRs with or without PEMFs treatment. It was found that the expression levels of BMPRII and BMPRIB were significantly increased by PEMFs, and immunofluorescence confocal colocalization analyses indicated that BMPRII localized at the bases of primary cilia. Besides, we found the increase of ciliary localization of BMPRII were blocked after the primary cilia abrogation, while there no similar effect were detedcted in BMPRIB. To find the importance of BMPRII in PEMFs induced osteogenesis, we found blocked BMPRII expression using the RNA interference and various osteogenetic makers were detected. We found the PEMFs induced the increase of ALP activity, expression of COL-Ι, Runx-2, Osx, and formation of mineralized nodules were all inhibited by the silencing of BMPRII. These results indicated that BMPRII played an indispensable role in the PEMF-induced osteogenic effect on osteoblasts.In conclusion, these results indicated PEMFs stimulate osteogenic differentiation and maturation of osteoblast by primary cilium-mediated upregulation of BMPRII and subsequently activation of BMP-Smad1/5/8 signaling, and that BMPRII is the key component linking primary cilia and BMP-Smad1/5/8 pathway.