Re-identification of special motif regulating osteoclast differentiation in RANK / 中国病理生理杂志

ABSTRACT

AIM: To re-identify the special motif regulating osteoclast(OC)differentiation in receptor activator of nuclear factor kappa B(RANK)to provide evidences for studying the mechanism of OC differentiation.METHODS: Eight amino acids were mutated(from DIIVVYVS into ELLAAFAA)in the fragment between the 533th and the 540th amino acids in RANK cytoplasmic domain.Eight mutant TNFR1/RANK chimeras,each consists of TNFR1(tumor necrosis factor receptor 1)extracellular domain linked to transmembrane domain and cytoplasmic domain of RANK with one amino acid mutated in cytoplasmic domain was constructed by point mutation method.After the eight mutant chimeras were finished,they were packed with plat E cell line to produce the retrovirus expressing mutant TNFR1/RANK.The bone marrow macrophages(BMMs),isolated from TNFR1/R2 double knockout mice,were infected with retrovirus derived from different mutants and infected BMMs which did not differentiated into OCs were inspected after stimulated by TNF-? and M-CSF.The fragment consisted of different amino acids in TNFR1/RANK chimeras,which couldn't induce OC formation after mutated,may be the special motif regulating OC differentiation.RESULTS: We found that all BMMs transfected by TNFR1/RANK-533,TNFR1/RANK-539 or TNFR1/RANK-540 differentiated into OCs,indicating that none of amino acids D533,V539 or S540 had an effect on OC differentiation.A fewer of BMMs transfected by TNFR1/RANK-534 differentiated into OCs,indicating that I534 had a partial effect on OC formation.Most importantly,BMMs transfected TNFR1/RANK-535,TNFR1/RANK-536,TNFR1/RANK-537 or TNFR1/RANK-538 did not differentiated into OCs,indicating each of amino acids I535,V536,V537 and Y538 played a pivotal role in OC differentiation.CONCLUSION: The amino acid fragment consists of I534,I535,V536,V537 and Y538 may be the special motif regulating OC differentiation in RANK.