| Introduction:Paget's disease?PD?features abnormal osteoclasts?OC?which sharply increase in number and size and then intensely induce bone resorption.It is a common elderly skeletal metabolism disturbance which is caused by enhanced bone resorption and followed by disorganized new bone formation.Previous studies have implicated that the pathogenic factors of PD point towards genetic susceptibility or paramyxovirus infection or combined effect.Both of them are supposed to cause the formation of"Pagetic phenotype" abnormal OC which include nearly about 100 nuclei when compared with 3-10 nuclei in normal,increase bone resorption ability and bone turnover rate and enhance the expression of transcription regulator TAF12 and the cytokine IL-6.Meanwhile,this kind of OC precursors are hypersensitive to 1,25?OH?2D3,TNF-a,and RANKL.The genetic susceptibility is closely related to sequestosomel,which encodes p62,a scaffold protein that is a component of the nuclear factor kappa-light-chain-enhancer of activated B cells?NF-?B?signaling pathway,and important in OC differentiation.However,there is little correlation between disease severity and identical genetic mutations in patients,some elderly of them don't show any symptoms of PD but with confirmed genetic mutations.Besides,genetic factors alone do not appear to be sufficient to induce PD.Jumpei Teramachi et al.reported that transfection of the p62 gene into normal OC precursors does not result in formation of pagetic-like OC in vitro.Importantly,OC from transgenic mice overexpressing the p62 mutation or p62 knock in mice do not express elevated TAF12,are not hypersensitive to 1,25?OH?2D3.and do not develop pagetic bone lesions.TAF12 acts as a co-activator of the vitamin D receptor?VDR?,and increased levels of TAF12 enhance the VDR responsivity of OC precursors from PD patients.Therefore,paramyxovirus infection takes the lead in the process of abnormal OC formation.The research regarding paramyxovirus infection in PD concentrates on the expression of measles virus nucleocapsid protein?MVNP?.Expression of the MVNP in OC can increase the number of pagetic-like OC.the production of IL-6,and the extent of bone lesions in transgenic mice.Enhanced pagetic-like OC formation are mainly through activated NF-?B signaling pathway by MVNP.Meanwhile,the high levels of IL-6 in PD of bone are caused by MVNP principally,but not by mutant p62.Furthermore,some researches have predominantly identified the relationship between canine distemper virus?CDV?and PD.Mee AP et al.reported that the RNA of CDV has been proved to be existing in pagetic bone tissue.Selby PL et al.demonstrated that CDV can infect and replicate in human OC precursor cells in a dose-dependent manner.But there are no reports to confirm which part of the structural protein in CDV play an important role during osteoclastogenesis.And it is the problem awaiting to be solved in this study.CDV is a kind of single-stranded and negative-sense RN A virus,which is a member of the genus morbillivirus family paramyxoviridae.CDV can infect a wide range of large mammals.It includes six structural proteins?nucleocapsid?N?,matrix?M?,fusion?F?,hemagglutinin?H?,phosphorus?P?,and large?L??.Viral glycoproteins?F and H?mediate virus-cell fusion and cell-cell fusion,specifically,H protein regulates the promotion of virus to cell membrane and determines the extent of cell to cell membrane,F protein acts on the fusion process of two membranes,which permits the transport of viral RNA into cytoplasm.Based on the important role of MVNP in the process of abnormal OC formation and the crucial effect of CDV glycoproteins during the fusion process of two membranes,we suspect that the viral glycoproteins play a pivotal role in cell to cell fusion during the process of abnormal OC formation.Therefore,our study is to investigate the direct effects of CDV and its glycoproteins on RANKL induced OC formation and to identify the potential underlying mechanisms.It is aim to find the mechnisms of Pagetic phenotype OC formation in Paget's disease of bone.This work further provide a new research direction that F and H proteins in CDV should be considered as a trigger during the pathogenesis of Paget's disease.Meanwhile,our results are very important to research the transboundary infectious disease between CDV and human.Materials and methods:1.Immunofluorescence analysisRAW264.7 cells or PBMC were cultured in DMEM or aMEM.After the first day,they were pre-treated with or without CDV for 2 hours.The cells were then stimulated with?20 ng/ml?mouse RANKL or?100 ng/ml?human RANKL for 5 days or 7 days.After that cells were fixed with 80%ice-cold acetone.Subsequently,cells were incubated with antibodies against CDV.Cells were washed,and then added FITC-conjugated antibodies.The cells were observed using a fluorescence microscope after washing.Lentivirus infected RAW264.7?LV RAW264.7?cells were treated in the same way of RAW264.7 cells but without CDV infection.2.Osteoclast differentiation,TRAP staining and pit formation assayRAW264.7 cells or PBMC were cultured in the same way above.After 7 days or 10 days,TRAP staining was used to evaluate OC differentiation.LV RAW264.7 cells were treated in the same way like RAW264.7 cells but without CDV infection.Pit formation assay was performed using the osteo assay surface multiple well plates.RAW264.7 cells or PBMC were performed like the one mentioned before.After that,plates were stained with Von Kossa to increase the contrast between pits and surface coating and observed under a light microscope,LV RAW264.7 cells were treated in the same way like RAW264.7 cells but without CDV infection.3.Western blot analysisRAW264.7 cells,PBMC or LV RAW264.7 cells were performed like the one mentioned before.Then the total proteins were collected for NFATcl and c-Fos immunoblotting.Then the total proteins were collected after RANKL stimulation for 0,5,15 or 30 min.After that,the total proteins were collected for NF-?B,ERK,p38 and JNK immunoblotting.4.Real-time PCR analysis and enzyme-linked immunosorbent assayR.A.W264.7 cells,PBMC or LV RAW264.7 cells were performed like the one mentioned before.During the course of cell culture,inhibitors of SB203580 and Bay 11-7082 were added in a medium.Total RNA was isolated.Quantitative real-time PCR analysis was performed to test mRNA expression of CK,MMP-9,TRAP,and IL-6.The concentration of IL-6 from cell supernatant was determined using a mouse and human IL-6 ELISA k t.Results:1.Immunofluorescence assay provided the conclusive proof that CDV can infect and replicate in RAW264.7 mouse monocyte cell line,primary human PBMC and their further fused OC.2.Both CDV and F+H significantly promoted OC formation and bone resorption ability induced by RANKL.3.CDV and F+H significantly upregulated the phosphorylation of NF-?B and MAPKs induced by RANKL,respectively.4.CDV and F+H upregulate the expression of CK,MMP-9,TRAP and IL-6.Meanwhile,CK,MMP-9,and TRAP are mainly expressed through NF-?B signaling pathway but IL-6 is mainly expressed by p38 signaling pathway.5.Without RANKL stimulation,both CDV and F+H slightly induced OC-like cells formation in RAW264.7 cell line even in the presence of NF-?B inhibitor.6.F+H upregulate OC differentiation and formation through NF-?B signaling pathway,and induce OC precursor cells merging dependent on the function of glycoproteins themselves.7.F and H proteins play a pivotal role in CDV supporting Pagetic phenotype OC formation.Conclusions:1.CDV significantly promoted mouse OC and human OC differentiation and bone resorption through activating NF-?B signaling pathway.Meanwhile,Pagetic phenotype OC inflammatory factor IL-6 was increased too.2.F+H showed the same effect as CDV.F+H significantly promoted OC differentiation and bone resorption through activating NF-?B signaling pathway.Meanwhile,Pagetic phenotype OC inflammatory factor IL-6 was increased too.3.F+H induce OC precursor cells merging dependent on the function of glycoproteins themselves.Therefore,CDV and CDV glycoproteins can upregulate OC differentiation and activity through independent NF-?B signaling pathway. |
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