Knockout and Double Knockout of Cathepsin K and Mmp9 reveals a novel function of Cathepsin K as a regulator of osteoclast gene expression and bone homeostasis

Cathepsins play a role in regulation of cell function through their presence in the cell nucleus. However, the role of Cathepsin K (Ctsk) as an epigenetic regulator in osteoclasts remains unknown. Our data demonstrated that Ctsk(-/-)Mmp9(-/-) mice have a striking phenotype with a 5-fold increase in bone volume compared with WT. RNA-seq analysis of Ctsk(-/-), Mmp9(-/-) and Ctsk(-/-)/Mmp9(-/-) osteoclasts revealed their distinct functions in gene expression regulation, including reduced Cebpa expression, increased Nfatc1 expression, and in signaling pathways activity regulation. Western blots and qPCR data validated these changes. ATAC-seq profiling of Ctsk(-/-), Mmp9(-/-), and Ctsk(-/-)/Mmp9(-/-) osteoclasts indicated the changes resulted from reduced chromatin openness in the promoter region of Cebpa and increased chromatin openness in Nfatc1 promoter in Ctsk(-/-)/Mmp9(-/-) osteoclasts compared to that in osteoclasts of WT, Ctsk(/-) and Mmp9(-/-). We found co-localization of Ctsk with c-Fos and cleavage of H3K27me3 in wild-type osteoclasts. Remarkably, cleavage of H3K27me3 was blocked in osteoclasts of Ctsk(-/-) and Ctsk(-/-)/Mmp9(-/-) mice, suggesting that Ctsk may epigenetically regulate distinctive groups of genes' expression by regulating proteolysis of H3K27me3. Ctsk(-/-)/Mmp9(-/-) double knockout dramatically protects against ovariectomy induced bone loss. We found that Ctsk may function as an essential epigenetic regulator in modulating levels of H3K27me3 in osteoclast activation and maintaining bone homeostasis. Our study revealed complementary and unique functions of Ctsk as epigenetic regulators for maintaining osteoclast activation and bone homeostasis by orchestrating multiple signaling pathways and targeting both Ctsk and Mmp9 is a novel therapeutic approach for osteolytic diseases such as osteoporosis.