Anti-CTSK monoclonal antibody, clone 3G2

Recently, autophagy-related proteins were shown to regulate osteoclast mediated bone resorption, a critical process in autoimmune diseases such as rheumatoid arthritis. However, the role of autophagy-linked FYVE containing protein, WDFY3, in osteoclast biology remains elusive. WDFY3 is a master regulator in selective autophagy for clearing ubiquitinated protein aggregates and has been linked with rheumatoid arthritis. Herein, we used a series of WDFY3 transgenic mice (Wdfy3(lacZ) and Wdfy3(loxP)) to investigate the function of WDFY3 in osteoclast development and function. Our data demonstrate that WDFY3 is highly expressed at the growth plate of neonatal mice and is expressed in osteoclasts in vitro cultures. Osteoclasts derived from WDFY3 conditional knockout mice (Wdfy3(loxP/IoxP)-LysM-Cre(+)) demonstrated increased osteoclast differentiation as evidenced by higher number and enlarged size of TRAP(+) multinucleated cells. Western blot analysis also revealed up-regulation of TRAF6 and an increase in RANKL-induced NF-kappa B signaling in WDFY3-deficient bone marrow-derived macrophages compared to wild type cultures. Consistent with these observations WDFY3-deficient cells also demonstrated an increase in osteoclast-related genes Ctsk, Acp5, Mmp9 and an increase of dentine resorption in in vitro assays. Importantly, in vivo RANKL gene transfer exacerbated bone loss in WDFY3 conditional knockout mice, as evidenced by elevated serum TRAP, CTX-I and micro-CT analysis of distal femurs compared to wild type littermates. Taken together, our data highlight a novel role for WDFY3 in osteoclast development and function, which can be exploited for the treatment of musculoskeletal diseases. (C) 2016 Elsevier Ltd. All rights reserved.

Introduction: Endodontic disease, 1 of the most prevalent chronic infectious diseases worldwide, occurs when the dental pulp becomes infected and inflamed, leading to bone destruction around the tooth root, severe pain, and tooth loss. Although many studies have tried to develop therapies to alleviate the bone erosion and inflammation associated with endodontic disease, there is an urgent need for an effective treatment. Methods: In this study, we used a gene-based therapy approach by administering recombinant adeno-associated virus (AAV)-mediated Atp6v1c1 knockdown to target both periapical bone resorption and inflammation in the mouse model of endodontic disease. Results: The results showed that Atp6v1c1 knockdown is simultaneously capable of reducing bone resorption by 70% through impaired osteoclast activation, inhibiting inflammation by decreasing T-cell infiltration in the periapical lesion by 75%, and protecting the periodontal ligament from destruction caused by inflammation. Notably, AAV-mediated gene therapy of Atp6v1c1 knockdown significantly reduced proinflammatory cytokine expression, including tumor necrosis factor alpha, interleukin 1 alpha, interleukin 17, interleukin 12, and interleukin 6 levels in periapical tissues caused by bacterial infection. Quantitative real-time polymerase chain reaction revealed that Atp6v1c1 knockdown reduced osteoclast-specific functional genes (ie, Ctsk) in periapical tissues. Conclusions: Our results showed that AAV-mediated Atp6v1c1 knock-down in periapical tissues slowed endodontic disease progression, prevented bone erosion, and alleviated inflammation in the periapical tissues and periodontal ligament potentially through regulation of toll-like receptor signaling, indicating that targeting Atp6v1c1 may facilitate the design of novel therapeutic approaches to reduce inflammation and bone erosion in endodontic disease.