RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts.

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

Cathepsin K deficiency prevented stress-related thrombosis in a mouse FeCl3 model.

BACKGROUND: Exposure to chronic psychological stress (CPS) is a risk factor for thrombotic cardiocerebrovascular diseases (CCVDs). The expression and activity of the cysteine cathepsin K (CTSK) are upregulated in stressed cardiovascular tissues, and we investigated whether CTSK is involved in chronic stress-related thrombosis, focusing on stress serum-induced endothelial apoptosis. METHODS AND RESULTS: Eight-week-old wild-type male mice (CTSK+/+) randomly divided to non-stress and 3-week restraint stress groups received a left carotid artery iron chloride3 (FeCl3)-induced thrombosis injury for biological and morphological evaluations at specific timepoints. On day 21 post-stress/injury, the stress had enhanced the arterial thrombi weights and lengths, in addition to harmful alterations of plasma ADAMTS13, von Willebrand factor, and plasminogen activation inhibitor-1, plus injured-artery endothelial loss and CTSK protein/mRNA expression. The stressed CTSK+/+ mice had increased levels of injured arterial cleaved Notch1, Hes1, cleaved caspase8, matrix metalloproteinase-9/-2, angiotensin type 1 receptor, galactin3, p16IN4A, p22phox, gp91phox, intracellular adhesion molecule-1, TNF-α, MCP-1, and TLR-4 proteins and/or genes. Pharmacological and genetic inhibitions of CTSK ameliorated the stress-induced thrombus formation and the observed molecular and morphological changes. In cultured HUVECs, CTSK overexpression and silencing respectively increased and mitigated stressed-serum- and H2O2-induced apoptosis associated with apoptosis-related protein changes. Recombinant human CTSK degraded γ-secretase substrate in a dose-dependent manor and activated Notch1 and Hes1 expression upregulation. CONCLUSIONS: CTSK appeared to contribute to stress-related thrombosis in mice subjected to FeCl3 stress, possibly via the modulation of vascular inflammation, oxidative production and apoptosis, suggesting that CTSK could be an effective therapeutic target for CPS-related thrombotic events in patients with CCVDs.

Dipeptidyl peptidase-4 disturbs adipocyte differentiation via the negative regulation of the glucagon-like peptide-1/adiponectin-cathepsin K axis in mice under chronic stress conditions.

Exposure to chronic psychosocial stress is a risk factor for metabolic disorders. Because dipeptidyl peptidase-4 (DPP4) and cysteinyl cathepsin K (CTSK) play important roles in human pathobiology, we investigated the role(s) of DPP4 in stress-related adipocyte differentiation, with a focus on the glucagon-like peptide-1 (GLP-1)/adiponectin-CTSK axis in vivo and in vitro. Plasma and inguinal adipose tissue from non-stress wild-type (DPP4+/+), DPP4-knockout (DPP4-/-) and CTSK-knockout (CTSK-/-) mice, and stressed DPP4+/+, DPP4-/-, CTSK-/-, and DPP4+/+ mice underwent stress exposure plus GLP-1 receptor agonist exenatide loading for 2 weeks and then were analyzed for stress-related biological and/or morphological alterations. On day 14 under chronic stress, stress decreased the weights of adipose tissue and resulted in harmful changes in the plasma levels of DPP4, GLP-1, CTSK, adiponectin, and tumor necrosis factor-α proteins and the adipose tissue levels of CTSK, preadipocyte factor-1, fatty acid binding protein-4, CCAAT/enhancer binding protein-α, GLP-1 receptor, peroxisome proliferator-activated receptor-γ, perilipin2, secreted frizzled-related protein-4, Wnt5α, Wnt11 and ß-catenin proteins and/or mRNAs as well as macrophage infiltration in adipose tissue; these changes were rectified by DPP4 deletion. GLP-1 receptor activation and CTSK deletion mimic the adipose benefits of DPP4 deficiency. In vitro, CTSK silencing and overexpression respectively prevented and facilitated stress serum and oxidative stress-induced adipocyte differentiation accompanied with changes in the levels of pref-1, C/EBP-α, and PPAR-γ in 3T3-L1 cells. Thus, these findings indicated that increased DPP4 plays an essential role in stress-related adipocyte differentiation, possibly through a negative regulation of GLP-1/adiponectin-CTSK axis activation in mice under chronic stress conditions.

Real-time analysis of osteoclast resorption and fusion dynamics in response to bone resorption inhibitors.

Cathepsin K (CatK), an essential collagenase in osteoclasts (OCs), is a potential therapeutic target for the treatment of osteoporosis. Using live-cell imaging, we monitored the bone resorptive behaviour of OCs during dose-dependent inhibition of CatK by an ectosteric (Tanshinone IIA sulfonate) and an active site inhibitor (odanacatib). CatK inhibition caused drastic reductions in the overall resorption speed of OCs. At IC50 CatK-inhibitor concentration, OCs reduced about 40% of their trench-forming capacity and at fourfold IC50 concentrations, a > 95% reduction was observed. The majority of CatK-inhibited OCs (~ 75%) were involved in resorption-migration-resorption episodes forming adjacent pits, while ~ 25% were stagnating OCs which remained associated with the same excavation. We also observed fusions of OCs during the resorption process both in control and inhibitor-treated conditions, which increased their resorption speeds by 30-50%. Inhibitor IC50-concentrations increased OC-fusion by twofold. Nevertheless, more fusion could not counterweigh the overall loss of resorption activity by inhibitors. Using an activity-based probe, we demonstrated the presence of active CatK at the resorbing front in pits and trenches. In conclusion, our data document how OCs respond to CatK-inhibition with respect to movement, bone resorption activity, and their attempt to compensate for inhibition by activating fusion.

Establishment and validation of an efficient method for the 3D culture of osteoclasts in vitro.

INTRODUCTION: Osteoclasts (OCs) play a crucial role in maintaining bone health. Changes in OC activity are linked to different bone diseases, making them an intriguing focus for research. However, most studies on OCs have relied on 2D cultures, limiting our understanding of their behavior. Yet, there's a lack of knowledge regarding platforms that effectively support osteoclast formation in 3D cultures. METHODS: In our investigation, we explored the capacity of collagen and GelMA hydrogels to facilitate osteoclast development in 3D culture settings. We assessed the osteoclast development by using different hydrogels and cell seeding strategies and optimizing cell seeding density and cytokine concentration. The osteoclast development in 3D cultures was further validated by biochemical assays and immunochemical staining. RESULTS: Our findings revealed that 0.3 % (w/v) collagen was conducive to osteoclast formation in both 2D and 3D cultures, demonstrated by increased multinucleation and higher TRAP activity compared to 0.6 % collagen and 5 % to 10 % (w/v) GelMA hydrogels. Additionally, we devised a "sandwich" technique using collagen substrates and augmented the initial macrophage seeding density and doubling cytokine concentrations, significantly enhancing the efficiency of OC culture in 3D conditions. Notably, we validated osteoclasts derived from macrophages in our 3D cultures express key osteoclast markers like cathepsin K and TRAP. CONCLUSIONS: To conclude, our study contributes to establishing an effective method for cultivating osteoclasts in 3D environments in vitro. This innovative approach not only promises a more physiologically relevant platform to study osteoclast behavior during bone remodeling but also holds potential for applications in bone tissue engineering. CLINICAL SIGNIFICANCE: This study introduces an efficient method for cultivating osteoclasts in 3D environments in vitro. It offers a more physiologically relevant platform to investigate osteoclast behavior and holds promise to advance research in bone biology and regenerative dentistry.

[Deficiency of cathepsin K improves ischemic angiogenesis in high-fat diet fed mice].

The present study aims to investigate the effect of cathepsin K (CatK) on ischemic angiogenesis in high-fat diet fed mice. The mice were subjected to unilateral hindlimb ischemic surgery, and the ischemic blood flow was measured with a laser Doppler blood flow imager. Immunohistochemical staining was used to observe the quantity of new capillaries in the ischemic lower extremity, and Western blot was used to detect the expression of insulin receptor substrate-1 (IRS-1), p-Akt, Akt and vascular endothelial growth factor (VEGF). Firstly, the effect of high-fat diet on ischemic angiogenesis was observed in wild-type mice, which were randomly divided into control group and high-fat diet group and were fed with normal diet or 60% high-fat diet respectively for 16 weeks. The results showed the body weight and the plasma CatK concentration of the high-fat diet group was significantly increased compared with the control group (P < 0.05), and the blood flow recovery of the high-fat diet group was significantly lower than control group (P < 0.05). Then, wild-type and CatK knock out (CatK-/-) mice were both fed with high-fat diet to further observe the effect and mechanism of CatK on ischemic angiogenesis under high-fat diet. The results showed that the blood flow recovery in the CatK-/- group was significantly greater than the wild-type group, and the number of CD31 positive cells was significantly increased (P < 0.05). At the same time, the protein expression levels of IRS-1, p-Akt and VEGF in the ischemic skeletal muscle were significantly increased in the CatK-/- group compared with the wild-type group (P < 0.05). These results suggest that the deficiency of CatK improves ischemic angiogenesis in high-fat diet fed mice through IRS-1-Akt-VEGF signaling pathway.

Heparan sulfate selectively inhibits the collagenase activity of cathepsin K.

Cathepsin K (CtsK) is a cysteine protease with potent collagenase activity. CtsK is highly expressed by bone-resorbing osteoclasts and plays an essential role in resorption of bone matrix. Although CtsK is known to bind heparan sulfate (HS), the structural details of the interaction, and how HS regulates the biological functions of CtsK, remains largely unknown. In this report, we discovered that HS is a multifaceted regulator of the structure and function of CtsK. Structurally, HS forms a highly stable complex with CtsK and induces its dimerization. Co-crystal structures of CtsK with bound HS oligosaccharides reveal the location of the HS binding site and suggest how HS may support dimerization. Functionally, HS plays a dual role in regulating the enzymatic activity of CtsK. While it preserves the peptidase activity of CtsK by stabilizing its active conformation, it inhibits the collagenase activity of CtsK in a sulfation level-dependent manner. These opposing effects can be explained by our finding that the HS binding site is remote from the active site, which allows HS to specifically inhibit the collagenase activity without affecting the peptidase activity. At last, we show that structurally defined HS oligosaccharides effectively block osteoclast resorption of bone in vitro without inhibiting osteoclast differentiation, which suggests that HS-based oligosaccharide might be explored as a new class of selective CtsK inhibitor for many diseases involving exaggerated bone resorption.

Inhibition of cysteine protease disturbs the topological relationship between bone resorption and formation in vitro.

INTRODUCTION: Osteoporosis is a global health issue. Bisphosphonates that are commonly used to treat osteoporosis suppress both bone resorption and subsequent bone formation. Inhibition of cathepsin K, a cysteine proteinase secreted by osteoclasts, was reported to suppress bone resorption while preserving or increasing bone formation. Analyses of the different effects of antiresorptive reagents such as bisphosphonates and cysteine proteinase inhibitors will contribute to the understanding of the mechanisms underlying bone remodeling. MATERIALS AND METHODS: Our team has developed an in vitro system in which bone remodeling can be temporally observed at the cellular level by 2-photon microscopy. We used this system in the present study to examine the effects of the cysteine proteinase inhibitor E-64 and those of zoledronic acid on bone remodeling. RESULTS: In the control group, the amount of the reduction and the increase in the matrix were correlated in each region of interest, indicating the topological and quantitative coordination of bone resorption and formation. Parameters for osteoblasts, osteoclasts, and matrix resorption/formation were also correlated. E-64 disrupted the correlation between resorption and formation by potentially inhibiting the emergence of spherical osteoblasts, which are speculated to be reversal cells in the resorption sites. CONCLUSION: These new findings help clarify coupling mechanisms and will contribute to the development of new drugs for osteoporosis.

Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) Immunohistochemistry Can Be a Useful Ancillary Tool to Identify Perivascular Epithelioid Cell Tumor.

Perivascular epithelioid cell tumors (PEComas) are rare mesenchymal tumors that express smooth muscle and melanocytic makers. Diagnosis of PEComas can be challenging due to focal or lost expression of traditional immunohistochemical markers, limited availability of molecular testing, and morphological overlap with much more common smooth muscle tumors. This study evaluates the use of glycoprotein nonmetastatic melanoma protein B (GPNMB) immunohistochemical staining as a surrogate marker for TSC1/2/MTOR alteration or TFE3 rearrangement to differentiate PEComas from other mesenchymal tumors. Cathepsin K was also assessed for comparison. A total of 399 tumors, including PEComas, alveolar soft part sarcomas, and other histologic PEComa mimics, were analyzed using GPNMB and cathepsin K immunohistochemistry. GPNMB expression was seen in all PEComas and alveolar soft part sarcomas with the majority showing diffuse and moderate-to-strong labeling, whereas other sarcomas were negative or showed focal labeling. When a cutoff of diffuse and at least moderate staining was used, GPNMB demonstrated 95% sensitivity and 97% specificity in distinguishing PEComas from leiomyosarcoma, well-differentiated/dedifferentiated liposarcomas, and undifferentiated pleomorphic sarcomas. Cathepsin K with a cutoff of any labeling had lower sensitivity (78%) and similar specificity (94%) to GPNMB. This study highlights GPNMB as a highly sensitive marker for PEComas and suggests its potential use as an ancillary tool within a panel of markers for accurate classification of these tumors.

Follicle-stimulating hormone accelerates osteoclast migration by enhancing methyltransferase-like 3-mediated m6A methylation of cathepsin K.

Follicle-stimulating hormone (FSH) accelerates osteoporosis in postmenopausal women, while the underlying mechanism remains uncharacterized. N6-methyladenosine (m6A) is one of the most important regulations in the development of osteoporosis. In this study, we aimed to investigate the role of FSH in m6A modification and osteoclast function. Here, we showed that FSH upregulated m6A levels in osteoclasts via stimulating methyltransferase-like 3 (METTL3) protein expression. FSH enhanced osteoclast migration, while the knockdown of METTL3 eliminated this enhancement. Both MeRIP-seq and RNA sequencing identified that cathepsin K (CTSK) is the potential downstream target of METTL3. Knockdown of CTSK reduced FSH-upregulated osteoclast migration. Furthermore, silencing METTL3 decreased CTSK mRNA stability. Finally, FSH induced phosphorylation of cyclic-AMP response element-binding protein (CREB), while silencing of CREB attenuated the effects of FSH on the promoter transcriptional activity of Mettl3 and CTSK/METTL3 protein. Taken together, these findings indicate that FSH promotes osteoclast migration via the CREB/METTL3/CTSK signaling pathway, which may provide a potential target for suppressing osteoclast mobility and postmenopausal osteoporosis therapy.

Real-Time Live Imaging of Osteoclast Activation via Cathepsin K Activity in Bone Diseases.

Intravital fluorescence imaging of functional osteoclasts within their intact disease context provides valuable insights into the intricate biology at the microscopic level, facilitating the development of therapeutic approaches for osteoclast-associated bone diseases. However, there is a lack of studies investigating osteoclast activity within deep-seated bone lesions using appropriate fluorescent probes, despite the advantages offered by the multi-photon excitation system in enhancing deep tissue imaging resolution. In this study, we report on the intravital tracking of osteoclast activity in three distinct murine bone disease models. We utilized a cathepsin K (CatK)-responsive two-photon fluorogenic probe (CatKP1), which exhibited a notable fluorescence turn-on response in the presence of active CatK. By utilizing CatKP1, we successfully monitored a significant increase in osteoclast activity in hindlimb long bones and its attenuation through pharmacological intervention without sacrificing mice. Thus, our findings highlight the efficacy of CatKP1 as a valuable tool for unraveling pathological osteoclast behavior and exploring novel therapeutic strategies.

The activity, distribution, and colocalization of cathepsin K and matrix metalloproteases in intact and eroded dentin.

OBJECTIVES: This study aimed to assess the activity, distribution, and colocalization of cathepsin K (catK) and matrix metalloproteases (MMPs) in both intact and eroded dentin in vitro. MATERIALS AND METHODS: Eroded dentin was obtained by consecutive treatment with 5% citric acid (pH = 2.3) for 7 days, while intact dentin remained untreated. Pulverized dentin powder (1.0 g) was extracted from both intact and eroded dentin using 5 mL of 50 mM Tris-HCl buffer (0.2 g/1 mL, pH = 7.4) for 60 h to measure the activity of catK and MMPs spectrofluorometrically. In addition, three 200-µm-thick dentin slices were prepared from intact and eroded dentin for double-labeling immunofluorescence to evaluate the distribution and colocalization of catK and MMPs (MMP-2 and MMP-9). The distribution and colocalization of enzymes were analyzed using inverted confocal laser scanning microscopy (CLSM), with colocalization rates quantified using Leica Application Suite Advanced Fluorescent (LAS AF) software. One-way analysis of variance (ANOVA) was used to analyze the fluorescence data related to enzyme activity (α = 0.05). RESULTS: The activity of catK and MMPs was significantly increased in eroded dentin compared with intact dentin. After erosive attacks, catK, MMP-2, and MMP-9 were prominently localized in the eroded regions. The colocalization rates of catK with MMP-2 and MMP-9 were 13- and 26-fold higher in eroded dentin, respectively, than in intact dentin. CONCLUSIONS: Erosive attacks amplified the activity of catK and MMPs in dentin while also altering their distribution patterns. Colocalization between catK and MMPs increased following erosive attacks. CLINICAL RELEVANCE: CatK, MMP-2, and MMP-9 likely play synergistic roles in the pathophysiology of dentin erosion.

Toll-like receptor 9-positive plasmacytoid dendritic cells promote Th17 immune responses in oral lichen planus stimulated by epithelium-derived cathepsin K.

Oral lichen planus (OLP) is a chronic inflammatory disease associated with T cell infiltration. The crosstalk between oral epithelium and mucosal T cells was considered to be crucial in the pathogenesis of OLP. Here, we selectively extracted the normal epithelium (NE) and lesional epithelium (LE) of buccal mucosa specimens from three patients with OLP by laser capture microdissection due to identify the pathogenic factors. Cathepsin K (CTSK) was identified as one of common upregulated genes in the LE by DNA microarray. Immunohistochemically, CTSK was distinctly detected in and around the LE, while it was rarely seen in the NE. Recent studies showed that CTSK enhanced Toll-like receptor 9 (TLR9) signaling in antigen-presenting cells, leading to Th17 cell differentiation. TLR9 expression mainly co-localized with CD123+ plasmacytoid dendritic cells (pDCs). The number of RORγt-positive cells correlated with that of CTSK-positive cells in OLP tissues. CD123+ pDCs induced the production of Th17-related cytokines (IL-6, IL-23, and TGF-ß) upon stimulation with TLR9 agonist CpG DNA. Moreover, single cell RNA-sequencing analysis revealed that TLR9-positive pDCs enhanced in genes associated with Th17 cell differentiation in comparison with TLR9-negative pDCs. CTSK could induce Th17-related production of CD123+ pDCs via TLR9 signaling to promote the pathogenesis of OLP.

Comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins.

BACKGROUND: Ever since their discovery, induced pluripotent stem cells (iPSCs) have been extensively differentiated into a large variety of cell types. However, a limited amount of work has been dedicated to differentiating iPSCs into osteoclasts. While several differentiation protocols have been published, it remains unclear which protocols or differentiation methods are preferable regarding the differentiation of osteoclasts. METHODS: In this study, we compared the osteoclastogenesis capacity of a peripheral blood mononuclear cell (PBMC)-derived iPSC line to a fibroblast-derived iPSC line in conjunction with either embryoid body-based or monolayer-based differentiation strategies. Both cell lines and differentiation protocols were investigated regarding their ability to generate osteoclasts and their inherent robustness and ease of use. The ability of both cell lines to remain undifferentiated while propagating using a feeder-free system was assessed using alkaline phosphatase staining. This was followed by evaluating mesodermal differentiation and the characterization of hematopoietic progenitor cells using flow cytometry. Finally, osteoclast yield and functionality based on resorptive activity, Cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression were assessed. The results were validated using qRT-PCR throughout the differentiation stages. RESULTS: Embryoid body-based differentiation yielded CD45+, CD14+, CD11b+ subpopulations which in turn differentiated into osteoclasts which demonstrated TRAP positivity, Cathepsin K expression and mineral resorptive capabilities. This was regardless of which iPSC line was used. Monolayer-based differentiation yielded lower quantities of hematopoietic cells that were mostly CD34+ and did not subsequently differentiate into osteoclasts. CONCLUSIONS: The outcome of this study demonstrates the successful differentiation of osteoclasts from iPSCs in conjunction with the embryoid-based differentiation method, while the monolayer-based method did not yield osteoclasts. No differences were observed regarding osteoclast differentiation between the PBMC and fibroblast-derived iPSC lines.

Enhanced BMP signaling in Cathepsin K-positive tendon progenitors induces heterotopic ossification.

Heterotopic ossification (HO) is abnormal bone growth in soft tissues that results from injury, trauma, and rare genetic disorders. Bone morphogenetic proteins (BMPs) are critical osteogenic regulators which are involved in HO. However, it remains unclear how BMP signaling interacts with other extracellular stimuli to form HO. To address this question, using the Cre-loxP recombination system in mice, we conditionally expressed the constitutively activated BMP type I receptor ALK2 with a Q207D mutation (Ca-ALK2) in Cathepsin K-Cre labeled tendon progenitors (hereafter "Ca-Alk2:Ctsk-Cre"). Ca-Alk2:Ctsk-Cre mice were viable but they formed spontaneous HO in the Achilles tendon. Histological and molecular marker analysis revealed that HO is formed via endochondral ossification. Ectopic chondrogenesis coincided with enhanced GLI1 production, suggesting that elevated Hedgehog (Hh) signaling is involved in the pathogenesis of HO. Interestingly, focal adhesion kinase, a critical mediator for the mechanotransduction pathway, was also activated in Ca-Alk2:Ctsk-Cre mice. Our findings suggest that enhanced BMP signaling may elevate Hh and mechanotransduction pathways, thereby causing HO in the regions of the Achilles tendon.

Sfrp4 is required to maintain Ctsk-lineage periosteal stem cell niche function.

We have previously reported that the cortical bone thinning seen in mice lacking the Wnt signaling antagonist Sfrp4 is due in part to impaired periosteal apposition. The periosteum contains cells which function as a reservoir of stem cells and contribute to cortical bone expansion, homeostasis, and repair. However, the local or paracrine factors that govern stem cells within the periosteal niche remain elusive. Cathepsin K (Ctsk), together with additional stem cell surface markers, marks a subset of periosteal stem cells (PSCs) which possess self-renewal ability and inducible multipotency. Sfrp4 is expressed in periosteal Ctsk-lineage cells, and Sfrp4 global deletion decreases the pool of PSCs, impairs their clonal multipotency for differentiation into osteoblasts and chondrocytes and formation of bone organoids. Bulk RNA sequencing analysis of Ctsk-lineage PSCs demonstrated that Sfrp4 deletion down-regulates signaling pathways associated with skeletal development, positive regulation of bone mineralization, and wound healing. Supporting these findings, Sfrp4 deletion hampers the periosteal response to bone injury and impairs Ctsk-lineage periosteal cell recruitment. Ctsk-lineage PSCs express the PTH receptor and PTH treatment increases the % of PSCs, a response not seen in the absence of Sfrp4. Importantly, in the absence of Sfrp4, PTH-dependent increase in cortical thickness and periosteal bone formation is markedly impaired. Thus, this study provides insights into the regulation of a specific population of periosteal cells by a secreted local factor, and shows a central role for Sfrp4 in the regulation of Ctsk-lineage periosteal stem cell differentiation and function.

Centrosome clustering control in osteoclasts through CCR5-mediated signaling.

Osteoclasts uniquely resorb calcified bone matrices. To exert their function, mature osteoclasts maintain the cellular polarity and directional vesicle trafficking to and from the resorbing bone surface. However, the regulatory mechanisms and pathophysiological relevance of these processes remain largely unexplored. Bone histomorphometric analyses in Ccr5-deficient mice showed abnormalities in the morphology and functional phenotype of their osteoclasts, compared to wild type mice. We observed disorganized clustering of nuclei, as well as centrosomes that organize the microtubule network, which was concomitant with impaired cathepsin K secretion in cultured Ccr5-deficient osteoclasts. Intriguingly, forced expression of constitutively active Rho or Rac restored these cytoskeletal phenotypes with recovery of cathepsin K secretion. Furthermore, a gene-disease enrichment analysis identified that PLEKHM1, a responsible gene for osteopetrosis, which regulates lysosomal trafficking in osteoclasts, was regulated by CCR5. These experimental results highlighted that CCR5-mediated signaling served as an intracellular organizer for centrosome clustering in osteoclasts, which was involved in the pathophysiology of bone metabolism.

Staphylococcus aureus enhances osteoclast differentiation and bone resorption by stimulating the NLRP3 inflammasome pathway.

BACKGROUND: Osteomyelitis is one of the most challenging infectious diseases and is mainly caused by Staphylococcus aureus (S. aureus). In this study, we analyzed the effect of S. aureus on osteoclast differentiation and its possible molecular mechanism. METHODS: We cultured RAW 264.7 cells with live S. aureus for 5 days. We assessed cell viability and the formation of resorption pits. We tested the NLRP3 inflammasome signaling pathways and measured the mRNA expression levels of osteoclastspecific genes, including TRAP, MMP9, cathepsin K, calcitonin receptor and ATP6V0d2. Furthermore, we analyzed the protein expression levels of the protein in the NF-κB and p38 MAPK signaling pathways to clarify the signaling pathways by which S. aureus promotes osteoclast differentiation. RESULTS: Staphylococcus aureus induced NLRP3 inflammasome activation. S. aureus promoted bone resorption and enhanced the expression of osteoclastspecific genes, such as TRAP, MMP9, cathepsin K, calcitonin receptor and ATP6V0d2. MCC950 was used to inhibit NLRP3 inflammasome activity. Osteoclast differentiation and the expression of osteoclastspecific genes induced by S. aureus were inhibited by MCC950 pretreatment. The degradation of IκBα and phosphorylation of P65 were increased under the induction of S. aureus, but proteins in the p38 MAPK signaling pathway did not change significantly. CONCLUSION: Staphylococcus aureus induces osteoclast differentiation and promotes bone resorption in vitro, and the NLRP3 inflammasome signaling pathway plays a significant role in this process. S. aureus-induced NLRP3 inflammasome activation was mainly dependent on the NF-κB signaling pathway during osteoclastogenesis.

Innovative workflow for the identification of cathepsin K cleavage sites in type I collagen.

Since the late 1990s, cathepsin K cleavage sites in type I collagen have been extensively studied due to its ability to release bone resorption biomarkers such as CTX and NTX. However, gel-based methods and N-sequencing used in these studies lack sensitivity, especially for small to medium peptides. In this work, we propose a degradomics mass spectrometry-based workflow that combines protein digestion, Nano-LC-UDMSE, and several software tools to identify cathepsin K cleavage sites. This workflow not only identified previously known cleavage sites, but also discovered new ones. Multiple cleavage hotspots were found and described in type I α1 and type I α2 collagen, many of which coincided with pyridinoline crosslinks, known to stabilize the triple helix. Our results allowed us to establish a chronology of digestion and conclude that cathepsin K preferentially cleaves the extremities of type I collagen before the helical part. We also found that cathepsin K preferentially cleaves amino acid residues with long and hydrophobic lateral chains at the beginning of digestion, whereas no preferred amino acid residues were identified later in the digestion. In conclusion, our workflow successfully identified new cleavage sites and can be easily applied to other proteins or proteases.

Discovery of CTSK+ Periosteal Stem Cells Mediating Bone Repair in Orbital Reconstruction.

Purpose: The purpose of this study was to explore the role of cathepsin K positive (CTSK+) periosteal stem cells (PSCs) in orbital bone repair and to clarify the source of endogenous stem cells for orbital bone self-repair. Methods: Periosteum samples obtained by clinical orbital bone repair surgery were analyzed, after which immunofluorescence and immunohistochemical staining were used to detect the content of bone marrow-derived cells and CTSK+ PSCs in periosteum as well as the mobilization of PSCs. CTSK+ PSCs were characterized by flow cytometry. Transcriptome sequencing was used to compare the transcriptomic characteristics of CTSK+ PSCs and bone marrow mesenchymal stem cells (BMSCs). Results: The orbital periosteum contained CTSK+CD200+ cell lineage, including CD200+CD105- PSCs and CD200+CD105+ progenitor cells. CTSK and osteocalcin (OCN) colocalized in the inner layer of the orbital periosteum, suggesting the osteogenic differentiation potential of CTSK+ PSCs. CTSK expression was much higher in periosteum after mobilization. Immunofluorescence showed low amounts of scattered CD31+ and CD45+ cells in the orbital periosteum. The stem cell characteristics of CTSK+ PSCs were verified by multidirectional differentiation. Flow cytometry found CD200+CD105- CTSK+ PSCs and CD200variantCD105+ progenitor cells. Transcriptome sequencing of CTSK+ PSCs and BMSCs found 3613 differential genes with significant differences. Gene Ontology (GO) analysis showed the differences between the two types of stem cells, revealing that PSCs were more suitable for intramembranous osteogenesis. Conclusions: CTSK+ PSCs may be endogenous stem cells for orbital bone repair. They are mobilized after orbital fracture and have unique features suitable for intramembranous osteogenesis, completely different from BMSCs.