Cell-penetrating H3 tail peptides suppress osteoclast differentiation via inhibition of MMP-9-mediated histone H3 clipping.

Osteoclast-mediated bone resorption is closely linked to bone formation, and any disruption in this process can lead to diseases such as osteoporosis. Matrix metalloproteinase-9 (MMP-9) is a nuclear histone protease that specifically cleaves the N-terminal tail (NT) of histone H3 at osteoclastogenic gene loci, thereby facilitating transcription and osteoclast differentiation. In this study, we developed tandem H3 tail peptides (residues 1-40) fused to a cell-penetrating peptide (CPP) to serve as competitive inhibitors of MMP-9. Both wild-type (WT) and P16G mutant peptides effectively localized to the nucleus, inhibited RANKLinduced osteoclast differentiation, and downregulated MMP-9 target genes (Xpr1, Nfatc1) as well as osteoclast-specific genes (Ctsk, Mmp9, Trap, Oscar) without impacting precursor proliferation. Mechanistically, both peptides inhibited MMP-9- mediated H3 NT clipping and decreased H3K18 acetylation by competitively binding to p300, thus disrupting a crucial epigenetic step necessary for histone clipping. While WT and P16G peptides exhibited similar binding affinities to MMP-9, the P16G mutant was resistant to proteolytic cleavage, allowing it to remain associated with MMP-9 for a longer period, which resulted in more effective inhibition of H3 NT clipping and osteoclastogenesis. Collectively, these findings indicate that histone H3 tail-derived peptides inhibit osteoclast differentiation by simultaneously targeting p300-dependent histone acetylation and MMP-9-mediated histone proteolysis. Our study provides valuable mechanistic insights into the epigenetic regulation of osteoclastogenesis and emphasizes engineered histone-derived peptides as a promising class of selective therapeutic inhibitors for osteoclast-driven bone diseases. [BMB Reports 2026; 59(2): 169-175].