The Preliminary Exploration Of The New Signaling Protein(s) Mediating Osteoclastogenesis And The New Therapeutic Target For Myeloma Bone Disease

Objective:The enhanced RANKL expression and over activation of RANKL/RANKsignaling pathway play acritical role in the development of myeloma bone disease(MBD),by promoting osteoclast (OC) differentiation and function. Therefore,targetingRANKL/RANK signaling to inhibit OC formation and activity offers a effectivetherapeutic approach for MBD. Our previous studies have indentified a novel motif inRANK protein: IVVY. It is a essential site and mediates a essential signaling pathwayfor OC formation. In this study, we are going to identify the down stream signalingprotein(s) which interacts with the IVVY motif and mediates OC differentiation, alsoget new insight in the mechanism of OC formation. Then, this novel down streamsignaling protein or novel signaling pathway may offer a specific therapeutic targetfor MBD.Methods:1. Construct and identify the cDNA library of mouse macrophage which is suitable for expression in yeast.2. Screen the cDNA library of mouse macrophage by yeast two-hybridizationexperiments to identify the downstream signaling candidate protein(s) interacting withthe IVVY motif of RANK protein.3. To confirm the expressing level of candidate protein(s) and it’s the localizationin cells by Western Blotting and the immunofluorescence imaging with confocalmicroscopy.4. To confirm the interaction between RANK and candidate protein(s), weperformed two-hybridization experiments in yeast and coimmunoprecipitation (coIP)in mammalian cells. Then we constructed a mutant Bait protein in which the IVVYmotif was mutated into IVAF. We repeated the two-hybridization and coIPexperiments with candidate protein(s) and this mutant Bait to further confirm theinteraction between the IVVY motif and candidate protein(s).5. Silence the candidate protein(s) by RNAi to identify its (their) function tomediate osteoclast formation.6．We used coIP,siRNA and siRNA rescue technique to identify the bindingdomain and function domain of RYBP. And also we used ChIP to exploretheepigenetic mechanisms of osteoclastogenesis mediated by RYBP.Results:1. A yeast expression library was constructed successfully with a titer of3.24×106cfu／m1, total clones of3.89×107cfu, the range of insertion size is0.2to5.6Kb,a mean insertion size is about2.27kb and the percentage of recombinant clones is95．83％(23／24)．All of these data shows this library meets the requirements ofstandard library.2. Using yeast two-hybird screening, four candidate proteins probablyinteracting with IVVY motif in RANK protein were obtained, including Ring1andYY1binding protein (RYBP), ATP-binding cassette, E2F transcription factor andheat-shock protein8. The clone coding RYBP was screened out frequently andquickly. So RYBP is the most probable candidate.3. When BMMs were treated with M-CSF and RANKL, they differentiated into osteoclasts. During the whole course, all the cells expressed high level of RYBP whenanalized by Western Blotting. When analized by confocal laser scaning microscopy,RYBP was also expressed in high level and moved from nucleus to membranes thenback to nucleus during the course of OC differentiation. These data suggest thatRYBP is assosiated with osteoclast formation.4. The yeast strain AH l09was co-transformed with plasmids of BD-Bait andAD-RYBP to directly confirm the interaction between RYBP and RANK.We notonly got positive colonies to verified the interaction, but when the AD-RYBPplasmids were diluted to1000times we also got the positive cononies.5. In293T cells, RYBP could immunopreciptitate with Bait-IVVY but failed todo so with mutant Bait-IVAF, indicating that the interaction between RANK regionand RYBP is specifically mediated by the IVVY motif.6. The selected siRNA significantly suppressed RYBP expression in osteoclastprecursors (BMMs) and the siRNA-mediated down-regulation of RYBP expressioninhibited osteoclastogenesis.These data suggest that RYBP is functionally involved inthe RANK motif-mediated osteoclastogenesis.7. We identified2binding domains of RYBP which interact with the IVVYmotif of RANK to mediate osteoclastogenesis. The explore of the function domainsand it’s epigenetic mechanisms of RYBP which mediate osteoclastogenesis are going on.Conclution:In this study, based on the new motif IVVY which we have identified before, weconstructed a cDNA library of macrophage (OC precursor),then applied some advancedtechniques, such as yeast two-hybrid screening, immunoprecipitation,immunofluorescence imaging, RNA interference, and so on, to identify the downstreamsignaling protein(s) which interacts with the IVVY motif and mediates OCdifferentiation. We identified a new protein, RYBP, which not only interacts with theIVVY motif but is also functionally involved in osteoclastogenesis. And we identifiedthe binding domains of RYBP which interact with the IVVY motif of RANK tomediate osteoclastogenesis.We get new insight in the signaling pathway inRANKL/RANK system and the mechanism of OC formation. Then, the domains of RYBP and their epigenetic mechanisms to mediateosteoclastogenesis may offer aspecific therapeutic target for MBD.