Targeting NAE1 suppresses osteoclastogenesis via dual regulation of ferritinophagy and ACSL3-mediated ferroptosis

Neddylation regulates diverse cellular processes, yet its role in osteoclast-mediated bone resorption is poorly understood. Here, we identify NAE1 (NEDD8 activating enzyme E1 subunit 1)-mediated neddylation as a critical regulator of postmenopausal osteoporosis and osteoclast differentiation through two distinct regulatory mechanisms. Pharmacological inhibition of Nae1 or myeloid-specific genetic ablation of Nae1 attenuated osteoclastogenesis in vitro and ameliorated ovariectomy (OVX)-induced osteoporosis in vivo without impairing osteoblast function. Mechanistically, Nae1 depletion disrupted intracellular iron metabolism, thereby suppressing ferritinophagy initiation in osteoclast precursors. Concurrently, integrated transcriptomics and affinity purification-mass spectrometry revealed ACSL3 as a direct neddylation substrate. Nae1-mediated neddylation modulates monounsaturated fatty acid (MUFA) biosynthesis, regulating the sensitivity of bone marrow-derived macrophages (BMDMs) to ferroptosis. This dual regulatory mechanism coordinately governs ferritinophagy initiation in iron metabolism and the sensitivity to ferroptosis mediated by ACSL3 neddylation, thereby critically influencing osteoclastogenesis. Clinically, serum MUFA levels positively correlated with bone mineral density (r = 0.329, p < 0.05). These findings support MLN4924, a clinical-stage NAE inhibitor, as a potential therapeutic strategy for osteoporosis and define an Nae1-ACSL3-MUFA-ferroptosis axis regulating osteoclast metabolism.Abbreviations: 4-HNE: 4-hydroxynonenal; ACP5/TRAP: acid phosphatase, tartrate resistant; ACSL3: acyl-CoA synthetase long chain family member 3; ACSL4: acyl-CoA synthetase long chain family member 4; BGLAP/OCN: bone gamma-carboxyglutamate protein; BMD: bone mineral density; BMDMs: bone marrow-derived macrophages; BV/TV: bone volume per total volume; CHX: cycloheximide; cKO: conditional knockout; co-IP: co-immunoprecipitation; CTSK: cathepsin K; DFO: deferoxamine; MDS: myelodysplastic syndrome; MUFA: monounsaturated fatty acid; NAE1: NEDD8 activating enzyme E1 subunit 1; NCOA4: nuclear receptor coactivator 4; NEDD8: NEDD8 ubiquitin like modifier; NFE2L2: NFE2 like bZIP transcription factor 2; NFATC1: nuclear factor of activated T cells 1; OC: osteoclast; OVX: ovariectomy; PUFA: polyunsaturated fatty acid; ROS: reactive oxygen species; RUNX2: RUNX family transcription factor 2; SLC40A1: solute carrier family 40 member 1; SLC7A11: solute carrier family 7 member 11; Tb.N: trabecular number; Tb.Sp: trabecular separation; Tb.Th: trabecular thickness; TFRC: transferrin receptor; TNFSF11/RANKL: TNF superfamily member 11; UBE2M: ubiquitin conjugating enzyme E2 M.