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Heterotopic ossification (HO) is pathological bone formation characterized by ossification within muscle, tendons, or other soft tissues. However, the cells of origin and mechanisms involved in the pathogenesis of HO remain elusive. Here we show that deletion of suppressor of fused (Sufu) in cathepsin K-Cre-expressing (Ctsk-Cre-expressing) cells resulted in spontaneous and progressive ligament, tendon, and periarticular ossification. Lineage tracing studies and cell functional analysis demonstrated that Ctsk-Cre could label a subpopulation of tendon-derived progenitor cells (TDPCs) marked by the tendon marker Scleraxis (Scx). Ctsk+Scx+ TDPCs are enriched for tendon stem cell markers and show the highest self-renewal capacity and differentiation potential. Sufu deficiency caused enhanced chondrogenic and osteogenic differentiation of Ctsk-Cre-expressing tendon-derived cells via upregulation of Hedgehog (Hh) signaling. Furthermore, pharmacological intervention in Hh signaling using JQ1 suppressed the development of HO. Thus, our results show that Ctsk-Cre labels a subpopulation of TDPCs contributing to HO and that their cell-fate changes are driven by activation of Hh signaling.

Elevated angiopoietin-2 is associated with diverse inflammatory conditions, including sepsis, a leading global cause of mortality. During inflammation, angiopoietin-2 antagonizes the endothelium-enriched receptor Tie2 to destabilize the vasculature. In other contexts, angiopoietin-2 stimulates Tie2. The basis for context-dependent antagonism remains incompletely understood. Here, we show that inflammation-induced proteolytic cleavage of angiopoietin-2 converts this ligand from Tie2 agonist to antagonist. Conditioned media from stimulated macrophages induced endothelial angiopoietin-2 secretion. Unexpectedly, this was associated with reduction of the 75 kDa full-length protein and appearance of new 25 and 50 kDa C-terminal fragments. Peptide sequencing proposed cathepsin K as a candidate protease. Cathepsin K was necessary and sufficient to cleave angiopoietin-2. Recombinant 25 and 50 kDa angiopoietin-2 fragments (cANGPT2 25 and cANGPT2 50 ) bound and antagonized Tie2. Cathepsin K inhibition with the phase 3 small-molecule inhibitor odanacatib improved survival in distinct murine sepsis models. Full-length angiopoietin-2 enhanced survival in endotoxemic mice administered odanacatib and, conversely, increased mortality in the drug’s absence. Odanacatib’s benefit was reversed by heterologous cANGPT2 25 . Septic humans accumulated circulating angiopoietin-2 fragments, which were associated with adverse outcomes. These results identify cathepsin K as a candidate marker of sepsis and a proteolytic mechanism for the conversion of angiopoietin-2 from Tie2 agonist to antagonist, with therapeutic implications for inflammatory conditions associated with angiopoietin-2 induction.

Cathepsin K (CTSK) is secreted by osteoclasts to degrade collagen and other matrix proteins during bone resorption. Global deletion of Ctsk in mice decreases bone resorption, leading to osteopetrosis, but also increases the bone formation rate (BFR). To understand how Ctsk deletion increases the BFR, we generated osteoclast- and osteoblast-targeted Ctsk knockout mice using floxed Ctsk alleles. Targeted ablation of Ctsk in hematopoietic cells, or specifically in osteoclasts and cells of the monocyte-osteoclast lineage, resulted in increased bone volume and BFR as well as osteoclast and osteoblast numbers. In contrast, targeted deletion of Ctsk in osteoblasts had no effect on bone resorption or BFR, demonstrating that the increased BFR is osteoclast dependent. Deletion of Ctsk in osteoclasts increased their sphingosine kinase 1 (Sphk1) expression. Conditioned media from Ctsk-deficient osteoclasts, which contained elevated levels of sphingosine-1-phosphate (S1P), increased alkaline phosphatase and mineralized nodules in osteoblast cultures. An S1P1,3 receptor antagonist inhibited these responses. Osteoblasts derived from mice with Ctsk-deficient osteoclasts had an increased RANKL/OPG ratio, providing a positive feedback loop that increased the number of osteoclasts. Our data provide genetic evidence that deletion of CTSK in osteoclasts enhances bone formation in vivo by increasing the generation of osteoclast-derived S1P.

A common stage of advanced prostate cancer is castration-resistant prostate cancer (CRPC), greater understanding of which is required in order to address and solve the clinically difficult challenge. Cathepsin K (CTSK) is a cysteine protease that usually has a strong activity of degrading extracellular matrix and is related to osteoclast-mediated bone destruction. However, the mechanism of CTSK-regulation in CRPC is still unclear to us. The current study aimed to analyze the expression of differentially expressed genes (DEGs) in patient samples (from localized PC and CRPC). Interestingly, we found that CTSK to be significantly up-regulated in CRPC. Through further signal pathway enrichment analysis, we found that the IL-17 signaling pathway to be highly correlated with CTSK. The oncogenic functions of CTSK and IL-17 in CRPC were proven by a series of in vivo and in vitro experiments. Possible downstream molecules of CTSK were investigated, which could serve as control elements to regulate the expression of EMT, thereby facilitating the metastasis and excessive proliferation of PC cells. Expression of CTSK was related to high concentration of M2 tumor-associated macrophages (TAMs) M2 in CRPC. A CTSK-mediated feedback circuit between TAMs and CRPC tissues was indicated in the process of transfer, proving the possibility of CTSK could be use as an available therapeutic target for CRPC.

Deletion of Ptpn11 in a newly defined mesenchymal progenitor population in the perichondral groove of Ranvier leads to metachondromatosis by increasing Indian hedgehog expression and activating hedgehog signalling, a process that can be reversed with the use of hedgehog pathway inhibitors. A progenitor cell for bone tumours Inactivation of PTPN11 , which encodes the phosphatase SHP2, is the cause of the inherited cartilage tumour disorder metachondromatosis. Using a mouse model, Wentian Yang and colleagues show that deletion of the Ptpn11 gene in osteoclasts leads to metachondromatosis that originates in a newly identified mesenchymal progenitor population in the perichondrial groove of Ranvier. Ptpn11 loss acts by activating hedgehog signalling, and metachondromatosis can be ameliorated in this model by hedgehog pathway inhibitors. Therefore in contrast to other tumour types where it promotes tumour formation, Ptpn11 suppresses tumorigenesis in cartilage tissue. The tyrosine phosphatase SHP2, encoded by PTPN11 , is required for the survival, proliferation and differentiation of various cell types 1 , 2 . Germline activating mutations in PTPN11 cause Noonan syndrome, whereas somatic PTPN11 mutations cause childhood myeloproliferative disease and contribute to some solid tumours. Recently, heterozygous inactivating mutations in PTPN11 were found in metachondromatosis, a rare inherited disorder featuring multiple exostoses, enchondromas, joint destruction and bony deformities 3 , 4 . The detailed pathogenesis of this disorder has remained unclear. Here we use a conditional knockout (floxed) Ptpn11 allele ( Ptpn11 fl ) and Cre recombinase transgenic mice to delete Ptpn11 specifically in monocytes, macrophages and osteoclasts (lysozyme M-Cre; LysMCre) or in cathepsin K (Ctsk)-expressing cells, previously thought to be osteoclasts. LysMCre;Ptpn11 fl/fl mice had mild osteopetrosis. Notably, however, CtskCre;Ptpn11 fl/fl mice developed features very similar to metachondromatosis. Lineage tracing revealed a novel population of CtskCre-expressing cells in the perichondrial groove of Ranvier that display markers and functional properties consistent with mesenchymal progenitors. Chondroid neoplasms arise from these cells and show decreased extracellular signal-regulated kinase (ERK) pathway activation, increased Indian hedgehog (Ihh) and parathyroid hormone-related protein (Pthrp, also known as Pthlh) expression and excessive proliferation. Shp2-deficient chondroprogenitors had decreased fibroblast growth factor-evoked ERK activation and enhanced Ihh and Pthrp expression, whereas fibroblast growth factor receptor (FGFR) or mitogen-activated protein kinase kinase (MEK) inhibitor treatment of chondroid cells increased Ihh and Pthrp expression. Importantly, smoothened inhibitor treatment ameliorated metachondromatosis features in CtskCre;Ptpn11 fl/fl mice. Thus, in contrast to its pro-oncogenic role in haematopoietic and epithelial cells, Ptpn11 is a tumour suppressor in cartilage, acting through a FGFR/MEK/ERK-dependent pathway in a novel progenitor cell population to prevent excessive Ihh production.

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 mice have a striking phenotype with a 5-fold increase in bone volume compared with WT. RNA-seq analysis of , and osteoclasts revealed their distinct functions in gene expression regulation, including reduced expression, increased expression, and in signaling pathways activity regulation. Western blots and qPCR data validated these changes. ATAC-seq profiling of , and osteoclasts indicated the changes resulted from reduced chromatin openness in the promoter region of and increased chromatin openness in Nfatc1 promoter in osteoclasts compared to that in osteoclasts of WT, and . 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 and mice, suggesting that Ctsk may epigenetically regulate distinctive groups of genes' expression by regulating proteolysis of H3K27me3. 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.

Objectives The mechanisms underlying the effects of Toll‐like receptor 9 (TLR9) and autophagy on rheumatoid arthritis (RA)‐aggravated periodontitis are unclear. We aimed to explore a novel target, cathepsin K (Ctsk)‐mediated TLR9‐related autophagy, during the progress of periodontitis with RA. Materials and Methods DBA/J1 mouse model of periodontitis with RA was created by local colonization of Porphyromonas gingivalis (Pg) and injection of collagen. The expression of Ctsk was inhibited by adeno‐associated virus (AAV). Micro‐CT, immunohistochemistry (IHC), Western blot and quantitative real‐time polymerase chain reaction (qRT‐PCR) were used to detect the expression of TLR9‐related autophagy in periodontitis with RA. Small interfering RNA (siRNA) and CpG oligodeoxynucleotides (CpG ODN) were applied in macrophages. Western blot, immunofluorescence (IF) and qRT‐PCR were used to verify the in vivo results. Results RA can promote periodontitis bone destruction in the lesion area, while inhibiting Ctsk could effectively alleviate this effect. The infiltration of macrophages, TLR9, autophagy proteins (TFEB and LC3) and inflammatory cytokines increased in the periodontitis‐with‐RA group and was reduced by the inhibition of Ctsk in the periodontal region. Macrophage stimulation confirmed the in vivo results. With the activation of TLR9 by CpG ODN, inhibition of Ctsk could suppress both TLR9 downstream signalling proteins and autophagy‐related proteins. Conclusions This study advanced a novel role for Ctsk in TLR9 and autophagy to explain the interaction between periodontitis and RA.

Background Matrix-metalloproteinases 9 (MMP-9) belongs to the class of matrix metalloproteinases whose main function is to degrade and remodel the extracellular matrix (ECM). MMP-9 has been shown to be an integral part of many diseases where modulation of the ECM is a key step such as cancer, osteoporosis and fibrosis. MMP-9 is secreted as a latent pro-enzyme that requires activation in the extracellular space. Therefore, identifying physiological and molecular contexts, which can activate MMP-9 is important. Results Acidification of osteoclast-conditioned media to pH 5 resulted in a fragment with a size corresponding to active MMP-9. Also, treatment of recombinant proMMP-9 with recombinant cathepsin K (CTSK) at pH 5 yielded a fragment that corresponded to the molecular weight of active MMP-9, and showed MMP-9 activity. This activation was abrogated in the presence of CTSK inhibitor indicating that CTSK was responsible for the activation of pro-MMP-9. Knocking down CTSK in MDA-MB-231 cells also diminished MMP-9 activity compared to wild type control. Conclusions Here we provide the first evidence that CTSK can cleave and activate MMP-9 in acidic environments such as seen in tumors and during bone resorption. This finding provides a key link between CTSK expression in tumors and bone and ECM remodeling, through MMP-9 activation. This novel mechanism to activate MMP-9 through extracellular physiological changes elucidated in this study reveals a protease-signaling network involving CTSK and MMP-9 and provides the impetus to explore ECM proteases as physiological markers and pharmacological targets.

Betaone is a variety of barley developed by the Korea Rural Development Administration. This study investigated the anti-osteoporosis effects of Betaone barley water extract (B1W) on ovariectomy (OVX)-induced bone loss in mice. To elucidate its mechanism, the effect of B1W on osteoclasts was assessed by measuring the protein expression of nuclear factor-activated T cells c1 (NFATc1), the expression of genes involved in osteoclast differentiation, and bone pit assays. B1W (300 mg/kg/day) significantly increased bone mineral density and bone volume fraction, but decreased trabecular separation compared to the OVX group. B1W also showed a trend towards decreasing serum C-telopeptide of collagen type 1 levels in OVX mice. Additionally, B1W reduced the expression of NFATc1 and downregulated the mRNA expression levels of various marker genes such as c-Fos, tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), dendritic cell-specific transmembrane protein (DC-STAMP), and osteoclast-associated Ig-like receptor (OSCAR). B1W reduced the osteoclast activity in the receptor activator of nuclear factor-κB ligand (RANKL)-treated osteoclasts by inhibiting the mitogen-activated protein kinase (MAPK) pathway. Based on the results, B1W can be considered a useful candidate for a therapeutic agent for treating conditions of bone loss and could also be used as an ingredient in health supplements.

This study assessed the novel concept that osteoclast-derived Grem1 has regulatory functions in the skeletal response to calcium stress using an osteoclastic Grem1 conditional knockout (cKO) mouse model. The calcium stress was initiated by feeding cKO mutants and wildtype (WT) littermates a calcium-deficient diet for 2 weeks. Deletion of Grem1 in mature osteoclasts did not affect developmental bone growth nor basal bone turnover. In response to calcium depletion, male cKO mutants showed greater increases in osteoclastic resorption and trabecular bone loss than male WT littermates, indicating an enhanced skeletal sensitivity to calcium depletion in male mutants. The enhanced sensitivity to calcium depletion was sex-dependent, as female cKO mutants showed lower increases in osteoclastic resorption and bone loss than female WT littermates as well as male cKO mutants. The sex disparity in osteoclastic resorption response to calcium stress was intrinsic to osteoclasts since osteoclasts of male but not female cKO mutants showed greater in vitro bone resorption activity than osteoclasts of WT littermates of respective sex. Male cKO mutants displayed smaller bone formation response to calcium depletion than male WT littermates, while female mutants showed bigger bone formation response than female WT littermates, indicating that cKO mutants also displayed sex disparity in bone formation response. The sex disparity in bone formation response was not caused by intrinsic differences in osteoblasts but might be due to sex-dependent differential osteoclastic release of osteogenic factors. In summary, osteoclast-derived gremlin-1 has complicated and sex-dependent regulatory roles in skeletal response to calcium stress.