WDR23 prevents bone loss by promoting autophagic degradation of TRAF6 in osteoclastogenesis

Abstract

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a pivotal adaptor molecule in the receptor activator of nuclear factor-κB (RANK) and its ligand (RANKL) signaling pathways, which are essential for osteoclastogenesis. In this study, we identified WD40 repeat-containing protein 23 (WDR23), also known as DDB1-CUL4 associated factor 11 (DCAF11), as a novel binding partner of TRAF6. Our findings demonstrate that WDR23/DCAF11 acts as a negative feedback regulator of RANK/RANKL-induced osteoclastogenesis by promoting the autophagy-dependent degradation of TRAF6. Notably, RANKL induced the upregulation of WDR23 expression during osteoclastogenesis. WDR23 physically interacted with the TRAF domain of TRAF6 via the WD40 repeat domains 1 and 2 of WDR23, resulting in reduced TRAF6 protein stability by its autophagy-dependent degradation during osteoclastogenesis. By modulating TRAF6 protein levels, WDR23 attenuated RANKL signaling cascades, including nuclear factor-κB and mitogen-activated protein kinases, thereby downregulating the expression of osteoclastogenic markers, such as nuclear factor of activated T-cell c1, tartrate-resistant acid phosphatase, dendritic cell-specific transmembrane protein, V-ATPase subunit d2 and cathepsin K. Conversely, WDR23 knockdown or deficiency enhanced RANKL-induced osteoclastogenesis by preventing the autophagy-dependent degradation of TRAF6. WDR23-deficient mice exhibit an osteoporotic bone phenotype characterized by elevated osteoclast formation and reduced bone mass. Collectively, these results establish WDR23 as a key negative feedback regulator of RANKL-induced osteoclastogenesis via autophagy-mediated TRAF6 degradation and underscore its potential as a therapeutic target for bone disorders associated with aberrant osteoclast formation and function.

References

[1] Amarasekara DS, Kim S, Rho J. Regulation of osteoblast differentiation by cytokine networks. Int. J. Mol. Sci., 2021, 22: 2851

[2] Amarasekara DS, Yu J, Rho J. Bone loss triggered by the cytokine network in inflammatory autoimmune diseases. J. Immunol. Res., 2015, 2015 832127

[3] Amarasekara DS, et al.. Regulation of osteoclast differentiation by cytokine networks. Immune Netw., 2018, 18 e8

[4] Walsh MC, Lee J, Choi Y. Tumor necrosis factor receptor- associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system. Immunol. Rev., 2015, 266: 72-92

[5] Walsh MC, et al.. Osteoimmunology: interplay between the immune system and bone metabolism. Annu. Rev. Immunol., 2006, 24: 33-63

[6] Lee SH, et al.. v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation. Nat. Med., 2006, 12: 1403-1409

[7] Miyamoto T. The dendritic cell-specific transmembrane protein DC-STAMP is essential for osteoclast fusion and osteoclast bone-resorbing activity. Mod. Rheumatol., 2006, 16: 341-342

[8] Dougall WC, et al.. RANK is essential for osteoclast and lymph node development. Genes Dev., 1999, 13: 2412-2424

[9] Kong YY, et al.. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature, 1999, 397: 315-323

[10] Lomaga MA, et al.. TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev., 1999, 13: 1015-1024

[11] Lin XW, et al.. WW domain containing E3 ubiquitin protein ligase 1 (WWP1) negatively regulates TLR4-mediated TNF-alpha and IL-6 production by proteasomal degradation of TNF receptor associated factor 6 (TRAF6). PLoS One, 2013, 8: e67633

[12] McBerry C, Gonzalez RM, Shryock N, Dias A, Aliberti J. SOCS2-induced proteasome-dependent TRAF6 degradation: a common anti-inflammatory pathway for control of innate immune responses. PLoS One, 2012, 7: e38384

[13] Wang T, et al.. CHIP regulates bone mass by targeting multiple TRAF family members in bone marrow stromal cells. Bone Res., 2018, 6: 10

[14] Wu C, et al.. NLRP11 attenuates Toll-like receptor signalling by targeting TRAF6 for degradation via the ubiquitin ligase RNF19A. Nat. Commun., 2017, 8 1977

[15] Zhang H, et al.. Ubiquitin E3 ligase Itch negatively regulates osteoclast formation by promoting deubiquitination of tumor necrosis factor (TNF) receptor-associated factor 6. J. Biol. Chem., 2013, 288: 22359-22368

[16] Park HH. Structure of TRAF family: current understanding of receptor recognition. Front. Immunol., 2018, 9: 1999

[17] Yu J, et al.. Interaction of tumor necrosis factor receptor-associated factor 6 (TRAF6) and Vav3 in the receptor activator of nuclear factor kappaB (RANK) signaling complex enhances osteoclastogenesis. J. Biol. Chem., 2016, 291: 20643-20660

[18] Lamothe B, et al.. The RING domain and first zinc finger of TRAF6 coordinate signaling by interleukin-1, lipopolysaccharide, and RANKL. J. Biol. Chem., 2008, 283: 24871-24880

[19] Chen ZJ. Ubiquitin signalling in the NF-kappaB pathway. Nat. Cell Biol., 2005, 7: 758-765

[20] Montaseri A, et al.. The role of autophagy in osteoclast differentiation and bone resorption function. Biomolecules, 2020, 10: 1398

[21] Busch J, et al.. TRAF6 phosphorylation prevents its autophagic degradation and re-shapes LPS-triggered signaling networks. Cancers, 2021, 13: 3618

[22] Deng T, et al.. TRAF6 autophagic degradation by avibirnavirus VP3 inhibits antiviral innate immunity via blocking NFKB/NF-kappaB activation. Autophagy, 2022, 18: 2781-2798

[23] Paul PK, Kumar A. TRAF6 coordinates the activation of autophagy and ubiquitin-proteasome systems in atrophying skeletal muscle. Autophagy, 2011, 7: 555-556

[24] Wang J, et al.. The role of autophagy in bone metabolism and clinical significance. Autophagy, 2023, 19: 2409-2427

[25] Wu DJ, et al.. Autophagy-linked FYVE containing protein WDFY3 interacts with TRAF6 and modulates RANKL-induced osteoclastogenesis. J. Autoimmun., 2016, 73: 73-84

[26] Chen RH, Chen YH, Huang TY. Ubiquitin-mediated regulation of autophagy. J. Biomed. Sci., 2019, 26: 80

[27] Wang YT, et al.. K48/K63-linked polyubiquitination of ATG9A by TRAF6 E3 ligase regulates oxidative stress-induced autophagy. Cell Rep., 2022, 38 110354

[28] Zachari M, Ganley IG. The mammalian ULK1 complex and autophagy initiation. Essays Biochem., 2017, 61: 585-596

[29] Angers S, et al.. Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery. Nature, 2006, 443: 590-593

[30] Choe KP, Przybysz AJ, Strange K. The WD40 repeat protein WDR-23 functions with the CUL4/DDB1 ubiquitin ligase to regulate nuclear abundance and activity of SKN-1 in Caenorhabditis elegans. Mol. Cell Biol., 2009, 29: 2704-2715

[31] Smith TF. Diversity of WD-repeat proteins. Subcell. Biochem, 2008, 48: 20-30

[32] Stirnimann CU, Petsalaki E, Russell RB, Muller CW. WD40 proteins propel cellular networks. Trends Biochem. Sci., 2010, 35: 565-574

[33] Higa LA, Mihaylov IS, Banks DP, Zheng J, Zhang H. Radiation-mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint. Nat. Cell Biol., 2003, 5: 1008-1015

[34] Lo JY, Spatola BN, Curran SP. WDR23 regulates NRF2 independently of KEAP1. PLoS Genet., 2017, 13: e1006762

[35] Chen Z, et al.. CRL4B(DCAF11) E3 ligase targets p21 for degradation to control cell cycle progression in human osteosarcoma cells. Sci. Rep., 2017, 7 1175

[36] Djakbarova U, Marzluff WF, Koseoglu MM. DDB1 and CUL4 associated factor 11 (DCAF11) mediates degradation of Stem-loop binding protein at the end of S phase. Cell Cycle, 2016, 15: 1986-1996

[37] Le R, et al.. Dcaf11 activates Zscan4-mediated alternative telomere lengthening in early embryos and embryonic stem cells. Cell Stem Cell, 2021, 28: 732-747 e739

[38] Wang K, et al.. Phosphorylation at Ser68 facilitates DCAF11-mediated ubiquitination and degradation of CENP-A during the cell cycle. Cell Rep., 2021, 37 109987

[39] Kannan M, et al.. WD40-repeat 47, a microtubule-associated protein, is essential for brain development and autophagy. Proc. Natl. Acad. Sci. USA, 2017, 114: E9308-E9317

[40] Kim YI, Nam IK, Um JY, Choe SK. Regulatory role of Wdr24 in autophagy activity during zebrafish embryogenesis. Mol. Cell Toxicol., 2019, 15: 85-92

[41] Xiong Q, et al.. WDR45 mutation impairs the autophagic degradation of transferrin receptor and promotes ferroptosis. Front. Mol. Biosci., 2021, 8 645831

[42] Li Y, Li S, Wu H. Ubiquitination-proteasome system (UPS) and autophagy two main protein degradation machineries in response to cell stress. Cells, 2022, 11: 851

[43] Nazio F, et al.. mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6. Nat. Cell Biol., 2013, 15: 406-416

[44] Shi CS, Kehrl JH. TRAF6 and A20 regulate lysine 63-linked ubiquitination of Beclin-1 to control TLR4-induced autophagy. Sci. Signal, 2010, 3: ra42

[45] Kim MJ, et al.. USP15 negatively regulates lung cancer progression through the TRAF6-BECN1 signaling axis for autophagy induction. Cell Death Dis., 2022, 13 348

[46] Wu H, et al.. TRAF6 inhibits colorectal cancer metastasis through regulating selective autophagic CTNNB1/beta-catenin degradation and is targeted for GSK3B/GSK3beta-mediated phosphorylation and degradation. Autophagy, 2019, 15: 1506-1522

[47] Ma Q, et al.. TRAF6 triggers Mycobacterium-infected host autophagy through Rab7 ubiquitination. Cell Death Discov., 2023, 9: 427

[48] Paul PK, et al.. Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice. J. Cell Biol., 2010, 191: 1395-1411

[49] Antonioli M, et al.. AMBRA1 interplay with cullin E3 ubiquitin ligases regulates autophagy dynamics. Dev. Cell, 2014, 31: 734-746

[50] Cheng J, Bin X, Tang Z. Cullin-RING Ligase 4 in cancer: structure, functions, and mechanisms. Biochim Biophys. Acta Rev. Cancer, 2024, 1879: 189169

[51] Park JY, Kim S, Sohn HY, Koh YH, Jo C. TFEB activates Nrf2 by repressing its E3 ubiquitin ligase DCAF11 and promoting phosphorylation of p62. Sci. Rep., 2019, 9 14354

[52] Kanzaki H, Shinohara F, Kajiya M, Kodama T. The Keap1/Nrf2 protein axis plays a role in osteoclast differentiation by regulating intracellular reactive oxygen species signaling. J. Biol. Chem., 2013, 288: 23009-23020

[53] Yang Y, et al.. Nrf2 Mitigates RANKL and M-CSF induced osteoclast differentiation via ROS-dependent mechanisms. Antioxidants, 2023, 12: 2094

[54] Walsh MC, Kim GK, Maurizio PL, Molnar EE, Choi Y. TRAF6 autoubiquitination-independent activation of the NFkappaB and MAPK pathways in response to IL-1 and RANKL. PLoS One, 2008, 3: e4064

[55] Min Y, Kim MJ, Lee S, Chun E, Lee KY. Inhibition of TRAF6 ubiquitin-ligase activity by PRDX1 leads to inhibition of NFκB activation and autophagy activation. Autophagy, 2018, 14: 1347-1358

[56] Park Y, et al.. Destablilization of TRAF6 by DRAK1 suppresses tumor growth and metastasis in cervical cancer cells. Cancer Res., 2020, 80: 2537-2549

[57] Novack DV. Role of NF-kappaB in the skeleton. Cell Res., 2011, 21: 169-182

[58] Wu M, Wu S, Chen W, Li YP. The roles and regulatory mechanisms of TGF-beta and BMP signaling in bone and cartilage development, homeostasis and disease. Cell Res., 2024, 34: 101-123

[59] Lee JY, et al.. HDAC6 controls autophagosome maturation essential for ubiquitin-selective quality-control autophagy. EMBO J., 2010, 29: 969-980

[60] Hwang SH, Han BI, Lee M. Knockout of ATG5 leads to malignant cell transformation and resistance to Src family kinase inhibitor PP2. J. Cell Physiol., 2018, 233: 506-515

[61] Shin B, et al.. Secretion of a truncated osteopetrosis-associated transmembrane protein 1 (OSTM1) mutant inhibits osteoclastogenesis through down-regulation of the B lymphocyte-induced maturation protein 1 (BLIMP1)-nuclear factor of activated T cells c1 (NFATc1) axis. J. Biol. Chem., 2014, 289: 35868-35881

[62] Park ES, et al.. TDAG51 promotes transcription factor FoxO1 activity during LPS-induced inflammatory responses. EMBO J., 2023, 42 e111867

[63] Jeon H, et al.. 1,3-dibenzyl-5-fluorouracil prevents ovariectomy-induced bone loss by suppressing osteoclast differentiation. Immune Netw., 2022, 22 e43

[64] Yu J, et al.. Pax5 negatively regulates osteoclastogenesis through downregulation of Blimp1. Int. J. Mol. Sci., 2021, 22: 2097

[65] Yu J, et al.. Generation of an osteoblast-based artificial niche that supports in vitro B lymphopoiesis. Exp. Mol. Med., 2017, 49 e400

Funding

National Research Foundation of Korea (NRF)(NRF-2019R1A2C1084311)

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