Autophagy-linked FYVE containing protein WDFY3 interacts with TRAF6 and modulates RANKL-induced osteoclastogenesis

Abstract

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 (Wdfy3lacZ and Wdfy3loxP) 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 (Wdfy3loxP/loxP-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-κB 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.

Loss of WDFY3 ameliorates severity of serum transfer-induced arthritis independently of autophagy

“…The deletion efficiency of WDFY3 in macrophages and osteoclasts derived from Wdfy3-cKO mice has been previously described [20]. We observed reduced joint swelling in Wdfy3-cKO mice compared to wild type littermates with serum transfer-induced arthritis suggesting that loss of WDFY3 in myeloid cells leads to a protective phenotype in arthritis.…”

“…WDFY4 has been associated with rheumatoid arthritis [26] and systemic lupus erythematosus [8] pathology in human genome-wide association studies (GWAS) studies, and WDFY3 has been detected in RA synovial fibroblasts [18] and osteoclasts [19]. Recently we showed that WDFY3 interacts with TRAF6 in RANKL-induced osteoclastogenesis in the absence of inflammation [20]. …”

“…The functional interaction between SQSTM1 and WDFY3 has been clearly documented in human osteoclasts [19], and we recently showed a novel role of WDFY3 in RANKL-induced osteoclastogenesis in the absence of inflammation [20]. To investigate the role of WDFY3 in arthritis we employed the K/BxN serum transfer-induced arthritis model, where anti-glucose-6-phosphate isomerase autoantibodies induce joint specific inflammation that closely resembles the rheumatoid arthritis pathologies in humans [21].…”

T Cell–Independent Mechanisms Associated with Neutrophil Extracellular Trap Formation and Selective Autophagy in IL-17A–Mediated Epidermal Hyperplasia

“…IL-23R GFP reporter mice crossed on RAG1 background and the Wdfy3 floxed mice were previously described [27],[28],[29]. All mice were used between 8–12 wks of age.…”

Autophagy and autoimmunity

“…Deletion of SQSTM1 ubiquitin-binding domain (UBD) leads to increased osteoclast differentiation and function suggesting that SQSTM1 may regulate osteoclastogenesis via multiple pathways [77]. Interaction of autophagy-related proteins SQSTM1 and WDFY3 has been observed in human osteoclasts [78], and we recently showed that WDFY3 deficient mice (WDFY3 fl/fl LysM Cre + ) show enhanced osteoclastogenesis and RANKL-mediated bone resorption in vitro and in vivo assays via TRAF6 dependent activation of NF-κB [79]. Another autophagy-related protein, optineurin (OPTN) also negatively regulates osteoclastogenesis by modulating NF-κB and IFN-β signaling [80].…”