Effect of RhoU Silencing on Osteoclast Differentiation

ZHENG Jiaming;HE Shuangjiang;ZHAO Hongyan;SONG Ruilong;LIU Zongping;College of Veterinary Medicine, Yangzhou University;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses;

RhoU;;RAW 264.7 cells;;osteoclast;;Rho GTPases;;cytoskeleton

In order to study the effect of RhoU(Wrch1) in osteoclast(OC) differentiation, we used RAW 264.7 cells(mononuclear macrophage cell line) as the basis for transfection of negative lentivirus and siRhoU lentivirus and established stable cell lines. In the presence of macrophage colony-stimulating factor(M-CSF) and nuclear factor-κB receptor activating factor ligand(RANKL), the negative control group and the RhoU-silencing group were induced for 3 or 4 days, respectively. The expression of RhoU and osteoclast-specific protein and its mRNA was detected by Western blot and quantitative polymerase chain reaction(qRT-PCR), respectively. The formation of osteoclasts was observed by tartrate acid phosphatase(TRAP) staining. The morphology of osteoclasts and their precursor cells was observed by confocal laser scanning microscope. The results showed that compared with the negative control group, the RhoU gene mRNA and protein expression in the RhoU-silencing group was significantly decreased(P<0.01).The number and area formation of osteoclasts were significantly decreased(P<0.01). The formation of filopodia in osteoclast precursor cells was blocked and it lacked complete sealing zone. The expression of osteoclast-specific gene mRNA and protein was significantly decreased(P<0.01 or P<0.05), while the TRAP gene mRNA was not changed(P>0.05). In conclusion, silencing RhoU can inhibit the differentiation of osteoclasts, inhibiting the formation of filamentous pseudopods in precursor cells and affecting their fusion may be the main mechanism.