Previous studies have demonstrated that sulforaphane (SFN) is a promising agent against osteoclastic bone destruction. However, the mechanism underlying its anti-osteoclastogenic activity is still unclear. Herein, for the first time, we explored the potential role of autophagy in SFN-mediated anti-osteoclastogenesis in vitro and in vivo. We established an osteoclastogenesis model using receptor activator of nuclear factor kappa-β ligand (RANKL)-induced RAW264.7 cells and bone marrow macrophages (BMMs). Tartrate-resistant acid phosphatase (TRAP) staining showed the formation of osteoclasts. We observed autophagosomes by transmission electron microscopy (TEM). In vitro, we found that SFN inhibited osteoclastogenesis (number of osteoclasts: 22.67 ± 0.88 in the SFN (0) group vs. 20.33 ± 1.45 in the SFN (1 μM) group vs. 13.00 ± 1.00 in the SFN (2.5 μM) group vs. 6.66 ± 1.20 in the SFN (2.5 μM) group), decreased the number of autophagosomes, and suppressed the accumulation of several autophagic proteins in osteoclast precursors. The activation of autophagy by rapamycin (RAP) almost reversed the SFN-elicited anti-osteoclastogenesis (number of osteoclasts: 22.67 ± 0.88 in the control group vs. 13.00 ± 1.00 in the SFN group vs. 17.33 ± 0.33 in the SFN+RAP group). Furthermore, Western blot (WB) analysis revealed that SFN inhibited the phosphorylation of c-Jun N-terminal kinase (JNK). The JNK activator anisomycin significantly promoted autophagy, whereas the inhibitor SP600125 markedly suppressed autophagic activation in pre-osteoclasts. Microcomputed tomography (CT), immunohistochemistry (IHC), and immunofluorescence (IF) were used to analyze the results in vivo. Consistent with the in vitro results, we found that the administration of SFN could decrease the number of osteoclasts and the expression of autophagic light chain 3 (LC3) and protect against lipopolysaccharide (LPS)-induced calvarial erosion. Our findings highlight autophagy as a crucial mechanism of SFN-mediated anti-osteoclastogenesis and show that the JNK signaling pathway participates in this process.

Osteoclasts, through their excessive production, are the primary cause of postmenopausal osteoporosis. However, the influence of metabolism on osteoclastogenesis remains poorly understood. This study reveals that protein lactylation plays a critical role in osteoclast differentiation. The ovariectomized (OVX) mouse model was used to investigate osteoporosis by examining glycolysis and lactate levels during osteoclast differentiation. Bone resorption was assessed through histomorphometric analysis. The effects of elevating lactate levels were tested both endogenously via exercise and exogenously through sodium lactate (NaLac) administration. Protein lactylation, focusing on histone modifications, was analyzed, and key osteoclastogenesis genes, including cathepsin K (Ctsk), matrix metalloproteinase 9 (Mmp9), and matrix metalloproteinase 12 (Mmp12), were quantified. The enzymes responsible for lactylation and delactylation were identified through cleavage under targets and tagmentation (CUT&Tag) and RNA-Seq analyses. Our findings showed that glycolysis and lactate levels were reduced during osteoclast differentiation in the OVX model, despite increased bone resorption. Elevating lactate through exercise or sodium lactate supplementation increased protein lactylation and mitigated OVX-induced bone loss. Mechanistically, lactate enhanced histone H3 lysine-18 lactylation (H3K18la), which suppressed osteoclast differentiation by downregulating key osteoclastogenesis genes like Ctsk, Mmp9, and Mmp12. Alanyl-tRNA synthetase 1 (AARS1) was identified as the lactylation “writer” that mediates H3K18la, with sirtuin 6 (SIRT6) acting as an “eraser” in a regulatory circuit. Lactate suppresses osteoclast differentiation and alleviates osteoporosis through histone H3K18 lactylation, which downregulates osteoclastogenic genes including Ctsk, Mmp9, and Mmp12. The dynamic regulation of H3K18la involves AARS1 as the lactylation “writer”and SIRT6 as an “eraser”. This study reveals an epigenetic mechanism by which lactate regulates osteoclast function and suggests that exercise-induced lactate elevation or lactate supplementation may represent a viable therapeutic strategy for postmenopausal osteoporosis.

Submucous cleft palate (SMCP) is a congenital craniofacial malformation. While TBX22 is a known pathogenic gene, the potential role of TBX15 in SMCP remains unclear. This study aimed to investigate the genetic etiology of SMCP in a multigenerational family and explore the possible involvement of TBX15. Whole-exome sequencing (WES) was performed on a 4-generation Chinese family with SMCP. Functional characterization of the identified variant was conducted through protein localization and transcriptional activity assays. Furthermore, single-nucleus RNA sequencing (snRNA-seq) of developing mouse palate tissues and immunofluorescence staining were employed to analyse the expression and role of Tbx15 during palatogenesis. WES identified a heterozygous nonsense variant (c.C1231T, p.Gln411Ter) in the terminal exon of TBX15. This variant is predicted to produce a truncated protein. Functional analyses demonstrated that the mutant TBX15 protein exhibits aberrant cytoplasmic mis-localization and significantly reduced transcriptional activity. SnRNA-seq revealed high expression of Tbx15 in a specific mesenchymal cell population during mouse palate development, with the highest expression observed at E15.5. Immunofluorescence confirmed colocalization of TBX15 with osteogenic markers in the developing palate, implicating it in intramembranous bone formation. This study provides the first evidence that a loss-of-function variant in TBX15 is associated with SMCP. The findings suggest that TBX15 plays a critical role in palatal osteogenesis, potentially through mechanisms overlapping with TBX22, and implicate TBX15 as a potential novel candidate gene for SMCP, suggesting a possible expansion of the genetic spectrum of this condition. The results suggest a potential expansion of the genetic spectrum of SMCP, offering new insights for molecular diagnosis and genetic counselling. Understanding TBX15's role in palate bone development may inform future research into therapeutic strategies.