Abstract 1

The role of SQSTM1/p62 in RANKL-activated signaling pathways and osteoclast formation

Kirk HM Yip, Ming H. Zheng, and Jiake Xu

Molecular Orthopaedic Laboratory, School of Surgery and Pathology, University of Western Australia, Nedlands, WA 6009, Australia

Paget's disease of bone (PDB) is a chronic lytic bone disorder that exhibits an increased number of nuclei and enhanced activities per cell compared with normal osteoclasts, causing bone pain, deformities, and fractures in patients. Recently, PDB has been reported to be associated with several C-terminal mutations of sequestosome 1 (SQSTM1), also known as p62. However, the role of p62 in osteoclast formation and in RANKL signaling pathway has not been well characterized. In this study, we showed that overexpression of p62 C-terminal deletion with the ubiquitin associated domain (UBA) in preosteoclastic RAW264.7 cells resulted in higher expression of TRAF6 protein, an adaptor molecule of the RANKL/RANK-activated signaling pathway, and upon RANKL stimulation, exhibited enhanced multinucleation and osteoclast forming in-vitro. Interestingly, over expression of the p62 mutant protein resulted in retarded RANKL-induced NF->KB activation, but enhanced NF-AT protein abundance and activity, as well as prolonged phosphorylation of ERK. Using confocal microscopy, we observed that EYFP-p62 was localized to aggregate structures in the cytoplasm and partially co-localized with late-lysosome. In contrast, EYFP-p62 UBA domain deletion mutant was diffusely expressed thorough out the cytoplasm and did not co-localized with late-lysosome. Taken together, our findings indicate that deletion of UBA domain of p62 results in enhanced osteoclastogenesis via the modulation of RANKL-activated signaling pathways in osteoclastic precursor cells.

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