Abstract
Itch is a ubiquitin E3 ligase that regulates protein stability. Itch(-/-) mice develop an autoimmune disease phenotype characterized by itchy skin and multiorgan inflammation. The role of Itch in the regulation of osteoclast function has not been examined. We report that Itch(-/-) bone marrow and spleen cells formed more osteoclasts than cells from WT littermates in response to receptor activator of NF-κB ligand (RANKL) and was associated with increased expression of the osteoclastogenic transcription factors c-fos and Nfatc1. Overexpression of Itch in Itch(-/-) cells rescued increased osteoclastogenesis. RANKL increased Itch expression, which can be blocked by a NF-κB inhibitor. The murine Itch promoter contains NF-κB binding sites. Overexpression of NF-κB p65 increased Itch expression, and RANKL promoted the binding of p65 onto the NF-κB binding sites in the Itch promoter. Itch(-/-) osteoclast precursors had prolonged RANKL-induced NF-κB activation and delayed TNF receptor-associated factor 6 (TRAF6) deubiquitination. In WT osteoclast precursors, Itch bound to TRAF6 and the deubiquitinating enzyme cylindromatosis. Adult Itch(-/-) mice had normal bone volume, but they had significantly increased LPS-induced osteoclastogenesis and bone resorption. Thus, Itch is a new RANKL target gene that is induced during osteoclastogenesis. Itch interacts with the deubiquitinating enzyme and is required for deubiquitination of TRAF6, thus limiting RANKL-induced osteoclast formation.
Highlights
TNF receptor-associated factor 6 (TRAF6) activity is crucial for osteoclastogenesis, which is decreased by deubiquitination
ItchϪ/Ϫ Mice Have Increased Osteoclast Formation in Vitro— To examine whether Itch regulates osteoclastogenesis, we cultured bone marrow cells or spleen cells from ItchϪ/Ϫ mice and their WT littermates with M-CSF and RANKL in osteoclastogenic assays
To determine whether the increased osteoclast formation in the absence of Itch is due to increased osteoclast precursors (OCPs) in ItchϪ/Ϫ mice, we examined the frequency of OCPs in bone marrow and spleen cells by fluorescence-activated cell sorting (FACS) analysis
Summary
TRAF6 activity is crucial for osteoclastogenesis, which is decreased by deubiquitination. After cells are stimulated by cytokines such as RANKL, IL-1, and LPS, TRAF6 promotes Lys polyubiquitination on target proteins in NF-B signaling and on itself [9] This Lys63-linked polyubiquitination on TRAF6 can be reversed by deubiquitinating enzymes (DUB) such as cylindromatosis (CYLD) and A20 [12, 13]. A20Ϫ/Ϫ mice develop spontaneous inflammation because A20 is required for the termination of IL-1R/Toll-like receptor 4 signaling in immune cells by promoting TRAF6 deubiquitination [13] These studies highlight the importance of deubiquitination as one of the molecular mechanisms to regulate TRAF6 function, which has not been well studied in osteoclast biology. Itch-regulated TRAF6 deubiquitination may represent a new mechanism for persistent NF-B activation in immune cells to account for autoimmune phenotypes of ItchϪ/Ϫ mice
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