Boric Acid Inhibits RANKL-Stimulated Osteoclastogenesis In Vitro and Attenuates LPS-Induced Bone Loss In Vivo.

Boron and boric acid (BA) can promote osteogenic differentiation and reduce bone resorption, which controls bone growth and maintenance of bone tissue. It has been reported that BA activates PERK-eIF2α signaling to induce cytoplasmic stress granules and cell senescence in human prostate DU-145 cells. However, whether BA can affect osteoclasts formation and LPS-induced inflammatory bone loss, and the role of the PERK-eIF2α pathway in the process, remains unknown. In vitro, RAW264.7 cells were pre-treated with boric acid (BA, 1, 10, 100 µmol/L) for 4 h, and then incubated with receptor activator of nuclear factor-kappaB ligand (RANKL, 50 ng/mL) in the presence or absence of BA for 5 days. CCK-8 and tartrate-resistant acid phosphatase (TRAP) were used to examine cell viability, osteoclastogenesis, and bone resorption; quantitative real-time PCR was performed to examine mRNA levels of c-Fos, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), TRAP, and cathepsin K; western blotting was used to examine protein expressions of glucose-regulated protein 78 (GRP78), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), phosphorylated PERK (p-PERK), eukaryotic initiation factor 2α (eIF2α), and phosphorylated eIF2α (p-eIF2α). In vivo, lipopolysaccharide (LPS)-induced bone loss model in mice was established, and micro-computed tomography (micro-CT) scanning, bone biochemical analysis, and osteoclastogenic cytokines were detected to evaluate the effect of BA on LPS-induced bone loss. In our vitro results showed that BA treatment for 5 days inhibited osteoclasts formation as well as osteoclastic bone resorption in a dose-dependent manner. The expression of osteoclasts marker genes c-Fos, NFATc1, TRAP, and cathepsin K were attenuated by BA. Immunoblotting analysis demonstrated that BA attenuated RANKL-induced PERK-eIF2α pathway activation. The in vivo data indicated that BA significantly prevented lipopolysaccharide (LPS)-induced bone loss. Our findings strongly suggest that BA may be a promising agent for the treatment of bone destructive diseases caused by excessive osteoclastogenesis.

[Icariin inhibits thioacetamide-induced osteoclast differentiation through RANKL-p38/ERK-NFAT pathway].

This study aims to investigate the therapeutic effect of icariin(ICA) on thioacetamide(TAA)-induced femoral osteolysis in rats. RAW264.7 cells were treated with TAA and ICA. Cell counting kit-8(CCK-8) assay was used to detect cell proliferation, and tartrate-resistant acid phosphatase(TRAP) staining to examine the formation of osteoclasts. The expression of TRAP, cathepsin K, c-FOS, and NFATc1 in RAW264.7 cells was determined by Western blot and immunofluorescence method. Thirty-two SD rats were randomized into the control group, TAA group(intraperitoneal injection of TAA at 300 mg·kg~(-1)), ICA group(gavage of ICA at 600 mg·kg~(-1)) and TAA + ICA group(intraperitoneal injection of TAA at 300 mg·kg~(-1) and gavage of ICA at 600 mg·kg~(-1)). Administration was performed every other day for 6 weeks. Body weight and length of femur were recorded at execution. Pathological injury and osteoclast differentiation of femur were observed based on hematoxylin-eosin(HE) staining and TRAP staining, and the changes of bone metabolism-related indexes alkaline phosphatase(ALP), calcium(Ca), phosphorus(P), magnesium(Mg), and cross-linked N-telopeptide of type Ⅰ collagen(NTX-Ⅰ) in serum were detected. Three-point bending test and micro-CT were applied to evaluate the quality of femur, and Western blot to detect the levels of osteoclast-related proteins TRAP, cathepsin K, RANK, RANKL, p38, p-p38, ERK, p-ERK, JNK, p-JNK, c-Fos, and NFATc1. The results showed ICA could inhibit TAA-induced production of TRAP-positive cells, the expression of osteoclast-related proteins, and nuclear translocation of NFATc1. ICA alleviated the weight loss, reduction of femur length, and growth inhibition induced by TAA in SD rats. ICA ameliorated the decline of femur elastic modulus caused by TAA and significantly restored trabecular bone mineral density(BMD), trabecular pattern factor(Tb.Pf), trabecular number(Tb.N), trabecular thickness(Tb.Th), and structure model index(SMI), thus improving bone structure. Western blot results showed ICA suppressed femoral osteoclast differentiation induced by TAA through RANKL-p38/ERK-NFATc1 signaling pathway. ICA inhibits osteoclast differentiation and prevents TAA-induced osteolysis by down-regulating RANKL-p38/ERK-NFAT signaling pathway.

Oral microbial extracellular DNA initiates periodontitis through gingival degradation by fibroblast-derived cathepsin K in mice.

Periodontitis is a highly prevalent disease leading to uncontrolled osteoclastic jawbone resorption and ultimately edentulism; however, the disease onset mechanism has not been fully elucidated. Here we propose a mechanism for initial pathology based on results obtained using a recently developed Osteoadsorptive Fluogenic Sentinel (OFS) probe that emits a fluorescent signal triggered by cathepsin K (Ctsk) activity. In a ligature-induced mouse model of periodontitis, a strong OFS signal is observed before the establishment of chronic inflammation and bone resorption. Single cell RNA sequencing shows gingival fibroblasts to be the primary cellular source of early Ctsk. The in vivo OFS signal is activated when Toll-Like Receptor 9 (TLR9) ligand or oral biofilm extracellular DNA (eDNA) is topically applied to the mouse palatal gingiva. This previously unrecognized interaction between oral microbial eDNA and Ctsk of gingival fibroblasts provides a pathological mechanism for disease initiation and a strategic basis for early diagnosis and treatment of periodontitis.

Effects of local vs systemic administration of CXCR4 inhibitor AMD3100 on orthodontic tooth movement in rats.

INTRODUCTION: Chemokines play pivotal roles in orthodontic tooth movement (OTM) through osteoclast-mediated bone resorption, but the underlying mechanism remains unclear. We aimed to elucidate the effects of serial local vs systemic administration of the chemokine receptor CXCR4 antagonist AMD3100 on OTM. METHODS: The maxillary first molar (M1) of rats was moved mesially using a 10 g of force nickel-titanium coil spring. The injections were performed every other day with phosphate-buffered saline as a control, whereas local and systemic animals were injected with AMD3100 at the buccal palatal mucosa adjacent to M1 and subcutaneously, respectively. OTM distance and alveolar bone were examined by microcomputed tomography and histologic analysis. Osteoclast numbers were quantified using TRAP staining. Cathepsin K and stromal cell-derived factor-1 (SDF-1) were evaluated using immunohistochemistry. Reverse transcriptase polymerase chain reaction for cathepsin K, Runx2, SDF-1, CXCR4, RANKL, and OPG were also examined. RESULTS: OTM and osteoclast numbers were significantly decreased in the local and systemic groups compared with the control group, whereas there was no significant difference among the experimental groups. Local administration inhibited molar but not incisor movement. Trabecular thickness and trabecular spacing of the alveolar bone significantly increased, and trabecular number significantly decreased in the systemic group compared with the control group, whereas local injection also affected bone quality in the same tendency as a systemic injection. AMD3100 significantly downregulated the mRNA expression levels of cathepsin K, Runx2, SDF-1, RANKL, and RANKL/OPG ratio in both experimental groups. CONCLUSIONS: Local administration of AMD3100 can control initial OTM and diminish bone resorption processes during OTM via inhibition of the SDF-1/CXCR4 axis, similar to the systemic administration.

Sistema adesivo universal: análise da interface adesiva e degradação colagenolítica em dentina submetida à diferentes protocolos de condicionamento ácido / Universal adhesive system: analysis of adhesive interface and collagenolytic degradation of demineralized dentin submitted to different acid etching ptotocols

O objetivo desse estudo foi analisar a interface adesiva do sistema adesivo Single Bond Universal (SBU) em dentina submetida à diferentes protocolos de condicionamento ácido em 24 h e 12 meses. E a degradação colagenolítica (DC) mediada por metaloproteinases (MMPs) e Catepsina-K (CAT-K) em tempo imediato. Esse estudo foi conduzido em 3 etapas: 1) Caracterização química da dentina em FTIR; 2) DC por meio de fragmentos do Telelopeptídeo Carboxiterminal do Colágeno Tipo I (ICTP) e do Terminal C do Telopeptídeo ligado ao Colágeno Tipo I (CTX) e a resistência à tração (RT) da do colágeno; 3) Análise da interface adesiva através da resistência de união (RU), análise de fratura, microscopia eletrônica de varredura (MEV) e nanoinfiltração (NI). Para FTIR foram utilizados 6 discos de dentina, divididos em 2 grupos: 1) Ácido fosfórico 32 %15 s (AF), 2) Ácido poliacrílico 25 % 10 s (AP). Para análise da DC, 12 discos de dentina foram completamente desmineralizados e divididos em 3 grupos: 1) AF, 2) AP e 3) Água deionizada (Controle) 15 s. Após, foram incubados e armazenados por 1 semana. Seguindo-se a análise da concentração de proteína total (PT). 50 µl da solução de incubação foram utilizadas para analisar ICTP e CTX. As concentrações foram calculadas em relação à PT. Para RT, foram testados 36 palitos obtidos dos discos de colágeno. Para RU foram utilizados 48 dentes, divididos em 2 grupos, de acordo com o período de armazenamento, divididos em três subgrupos: 1) AF, 2) AP e 3) Autocondicionante SBU 20 s (SE). Os dentes foram restaurados e armazenados em água destilada 37 °C. Após, foram submetidos ao teste de microtração e análise de fraturas. Para as análises MEV e NI foram utilizados 2 espécimes de cada subgrupo. Para análise estatística utilizou-se ANOVA 1-Fator, ANOVA- 2 Fatores e teste de Tukey (α=0.05). Para FTIR, AF reduziu a quantidade de fosfato e carbonato quando comparado ao AP. Para DC, a liberação de ICTPPT para AF foi significantemente maior do que para AP (p < 0,05). Não houve diferença na liberação de CTXPT para AF e AP (p > 0,05). Para RT não houve diferença entre AP e Controle, porém, apresentaram valores maiores do que AF (p<0,05). Para RU em MPa, não houve diferença estatisticamente significativa para todos os tratamentos nos diferentes períodos de análise (p < 0,05). A análise de fraturas evidenciou a predominância de fraturas adesivas e mistas. MEV mostrou melhor qualidade da interface após 12 meses para AF e AP. Após 12 meses apenas SE não apresentou NI. Assim sendo, o autocondicionamento ainda parece ser a melhor opção para sistemas adesivos universais que possuam monômeros funcionais em sua composição.(AU)

The aim of this study was to analyze the adhesive interface of the Single Bond Universal (SBU) to dentin submitted to different acid etching protocols in 24 h and 12 months, and the collagenolytic degradation (CD) by matrix metalloproteinases (MMPs) and Cathepsin-K (CAT-K) in the immediate time. This study was divided into three stages: 1) Dentin chemical characterization by FTIR; 2) CD by release of the collagen telopeptide fragment cross-linked carboxyterminal telopeptide of type I collagen (ICTP), and C-terminal crosslinked telopeptide of type I collagen (CTX) and ultimate tensile strength (UTS); 3) Analysis of the adhesive interface by microtensile bond strength (µTBS), failure mode, scanning electron microscopy (SEM) of the AI, and nanoleakage by SEM (NL). For FTIR, six dentin disks were divided into two groups: 1) Phosphoric acid 15 s (PA), 2) Polyacrylic acid 10 s (PAA). For CD twelve dentin disks were completely demineralized, then were divided into 3 groups 1) PA, 2) PAA, and 3) deionized water (Control) for 15 s. All disks were incubated in a buffered solution (BS) for 1 week. Total protein (TP) concentrations were measured using Nanodrop™ at 280 nm. 50 µl of BS was used to analyze solubilized telopeptide fragments using ICTP and CTX . ICTP and CTX average ratios were calculated in relation to TP concentration (ICTPtp and CTXtp). For UTS, 36 dentin beams obtained from collagen disks were tested. For µTBS, forty-eight teeth were divided into two groups according to the period of storage, then subdivided into three subgroups: 1) PA, 2) PAA, and 3) Self-etch 20 s (SE). After, composite build up, the specimens were stored in distilled water at 37 °C. Two specimens of each group were used for SEM analysis of AI and NL. Data were analyzed by one-way ANOVA, two-way ANOVA and Tukey tests (p<0.05). According to the results of the FTIR etching with PA reduced the amount of phosphate and carbonate when compared to PAA. ICTPtp release of PA was significantly higher A than PAA (p > 0,05). CTXtp showed no difference between the PA and PAA (p < 0,05). For UTS there was no difference between PAA and control, but they were significantly higher (p<0.05) than PA. For µTBS in MPa, there is no statistical difference among all the etching protocols tested, as well in both storage periods of analysis (p < 0,05). The most prevalent failure mode were adhesives associated with mixed. SEM analysis highlighted a better quality of AI after 12 months for PA and PAA. However, after 12 months only SE did not show NL. Then, the self-etching protocol seems to be a better choice regarding universal adhesive systems which have functional monomers in their blend(AU)

Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer.

BACKGROUND: Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and -4 are highly expressed, but PAR-3 shows low expression and unclear functions. METHODS: Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFß monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells. RESULTS: We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and -4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFß in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer. CONCLUSIONS: Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells.

The silencing of cathepsin K used in gene therapy for periodontal disease reveals the role of cathepsin K in chronic infection and inflammation.

BACKGROUND AND OBJECTIVE: Periodontitis is a severe chronic inflammatory disease and one of the most prevalent non-communicable chronic diseases that affects the majority of the world's adult population. While great efforts have been devoted toward understanding the pathogenesis of periodontitis, there remains a pressing need for developing potent therapeutic strategies for targeting this dreadful disease. In this study, we utilized adeno-associated virus (AAV) expressing cathepsin K (Ctsk) small hairpin (sh)RNA (AAV-sh-Ctsk) to silence Ctsk in vivo and subsequently evaluated its impact in periodontitis as a potential therapeutic strategy for this disease. MATERIAL AND METHODS: We used a known mouse model of periodontitis, in which wild-type BALB/cJ mice were infected with Porphyromonas gingivalis W50 in the maxillary and mandibular periodontium to induce the disease. AAV-sh-Ctsk was then administrated locally into the periodontal tissues in vivo, followed by analyses to assess progression of the disease. RESULTS: AAV-mediated Ctsk silencing drastically protected mice (> 80%) from P. gingivalis-induced bone resorption by osteoclasts. In addition, AAV-sh-Ctsk administration drastically reduced inflammation by impacting the expression of many inflammatory cytokines as well as T-cell and dendritic cell numbers in periodontal lesions. CONCLUSION: AAV-mediated Ctsk silencing can simultaneously target both the inflammation and bone resorption associated with periodontitis through its inhibitory effect on immune cells and osteoclast function. Thereby, AAV-sh-Ctsk administration can efficiently protect against periodontal tissue damage and alveolar bone loss, establishing this AAV-mediated local silencing of Ctsk as an important therapeutic strategy for effectively treating periodontal disease.

Using agents that suppress bone remodeling to treat or prevent joint disease: quo vadis?

Treatment of osteoarthritis (OA) with antiremodeling agents has had a mixed record of results. It is likely that remodeling suppression is only effective when used in the early phases of OA, before significant progression. Animal and human studies largely bear this out. Treatment of young mice with a RANKL inhibitor suppresses bone resorption and prevents OA progression. Likewise, bisphosphonate treatments in rodents and rabbits with induced injury or inflammatory arthritis, reduced cartilage degeneration when administered preemptively, but later administration did not. The increased prevalence of OA in women after the menopause, and presence of estrogen receptors in joint tissues, suggests that treatment with estrogens or Selective Estrogen Receptor Modulators may be effective. However, in clinical trials of knee and hip, results show decreased or increased risk for OA, or no effect. Raloxifene had positive effects in animal models, but no effect in human studies. More recent potential treatments such as strontium ranelate or cathepsin-K inhibitors may be effective, but may work directly on the cartilage rather than through their well-known effects on bone. The conclusion from these studies is that anti-remodeling agents must be administered pre-emptively or in the very early stages of disease to be effective. This means that better imaging techniques or identification of early structural changes in bone that occur before progressive cartilage destruction must be developed. (AU)

Expression of cathepsin K in periodontitis and in gingival fibroblasts.

OBJECTIVE: To study non-osteoclastic sources of cathepsin K in periodontitis. MATERIALS AND METHODS: Tissue samples were obtained from 10 otherwise healthy periodontitis pati-ents during routine periodontal flap operations and 10 systemically and periodontally healthy individuals who underwent extraction operations for retained third molars. Methods used were immunohistochemistry, image analysis, immunofluorescence double-staining, gingival fibroblast culture, tumour necrosis factor-α (TNF-α) stimulation and Western blotting. RESULTS: Macrophage-like cells, fibroblast-like cells, vascular endothelial cells and gingival epithelial cells were more intensively stained for cathepsin K and also more frequent in periodontitis than in controls (665 ± 104 vs 258 ± 40 cells mm(-2) , P < 0.01). Some cathepsin K(+) cells in periodontal tissues were CD68(+) , but some were CD68(-) and probably fibroblasts. Indeed, in gingival fibroblast culture, resting fibroblasts released cathepsin K, more 43 kD procathepsin K than 29 kD active cathepsin K. TNF-α increased the release of the activated cathepsin K 4- to 5-fold. CONCLUSIONS: Results suggest that GCF-cathepsin K is not only osteoclast-derived, but in periodontitis, also other cells contribute to it. GCF-cathepsin K, perhaps together with intracellular, lysosomal collagenolytically active cathepsin K in fibroblasts, macrophages and gingival epithelial cells, can contribute to the loss of attachment and destruction of the periodontal ligament.

Can quaternary ammonium methacrylates inhibit matrix MMPs and cathepsins?

OBJECTIVE: Dentin matrices release ICTP and CTX fragments during collagen degradation. ICTP fragments are known to be produced by MMPs. CTX fragments are thought to come from cathepsin K activity. The purpose of this study was to determine if quaternary methacrylates (QAMs) can inhibit matrix MMPs and cathepsins. METHODS: Dentin beams were demineralizated, and dried to constant weight. Beams were incubated with rh-cathepsin B, K, L or S for 24h at pH 7.4 to identify which cathepsins release CTX at neutral pH. Beams were dipped in ATA, an antimicrobial QAM to determine if it can inhibit dentin matrix proteases. Other beams were dipped in another QAM (MDPB) to determine if it produced similar inhibition of dentin proteases. RESULTS: Only beams incubated with cathepsin K lost more dry mass than the controls and released CTX. Dentin beams dipped in ATA and incubated for 1 week at pH 7.4, showed a concentration-dependent reduction in weight-loss. There was no change in ICTP release from control values, meaning that ATA did not inhibit MMPs. Media concentrations of CTX fell significantly at 15wt% ATA indicating that ATA inhibits capthesins. Beams dipped in increasing concentrations of MDPB lost progressively less mass, showing that MDPB is a protease-inhibitor. ICTP released from controls or beams exposed to low concentrations were the same, while 5 or 10% MDPB significantly lowered ICTP production. CTX levels were strongly inhibited by 2.5-10% MDPB, indicating that MDPB is a potent inhibitor of both MMPs and cathepsin K. SIGNIFICANCE: CTX seems to be released from dentin matrix only by cathepsin K. MMPs and cathepsin K and B may all contribute to matrix degradation.

Extracellular cathepsin K exerts antimicrobial activity and is protective against chronic intestinal inflammation in mice.

OBJECTIVE: Cathepsin K is a lysosomal cysteine protease that has pleiotropic roles in bone resorption, arthritis, atherosclerosis, blood pressure regulation, obesity and cancer. Recently, it was demonstrated that cathepsin K-deficient (Ctsk(-/-) ) mice are less susceptible to experimental autoimmune arthritis and encephalomyelitis, which implies a functional role for cathepsin K in chronic inflammatory responses. Here, the authors address the relevance of cathepsin K in the intestinal immune response during chronic intestinal inflammation. DESIGN: Chronic colitis was induced by administration of 2% dextran sodium sulphate (DSS) in distilled water. Mice were assessed for disease severity, histopathology and endoscopic appearance. Furthermore, DSS-exposed Ctsk(-/-) mice were treated by rectal administration of recombinant cathepsin K. Intestinal microflora was assessed by real-time PCR and 16srDNA molecular fingerprinting of ileal and colonic mucosal and faecal samples. RESULTS: Using Ctsk(-/-) mice, the authors demonstrate a protective role of cathepsin K against chronic DSS colitis. Dissecting the underlying mechanisms the authors found cathepsin K to be present in intestinal goblet cells and the mucin layer. Furthermore, a direct cathepsin K-mediated bactericidal activity against intestinal bacteria was demonstrated, which potentially explains the alteration of intestinal microbiota observed in Ctsk(-/-) mice. Rectal administration of recombinant cathepsin K in DSS-treated Ctsk(-/-) mice ameliorates the severity of intestinal inflammation. CONCLUSION: These data identify extracellular cathepsin K as an intestinal antibacterial factor with anti-inflammatory potential and suggest that topical administration of cathepsin K might provide a therapeutic option for patients with inflammatory bowel disease.

Processing of CXCL12 by different osteoblast-secreted cathepsins.

Hematopoietic stem and progenitor cells (HSPCs) are known to reside in specialized niches at the endosteum in the trabecular bone. Osteoblasts are the major cell type of the endosteal niche. It is well established that secreted proteases are involved in cytokine-induced mobilization processes that release stem cell from their niches. However, migratory processes such as the regular trafficking of HSPCs between their niches and the periphery are not fully understood. In the current study we analyzed whether osteoblast-secreted cysteine cathepsins are able to reduce the direct interaction of HSPCs with bone-forming osteoblasts. Isolated human osteoblasts were shown to secrete proteolytically active cysteine cathepsins, such as cathepsins B, K, L, and X. All of these cathepsins were able to digest, although with different efficacy, the chemokine CXCL12, which is known to be important for retaining HSPCs in their niches. Of the 4 identified cathepsins, only cathepsin X was able to reduce binding of HSPCs to osteoblasts. Interestingly, nonactivated pro-cathepsin X and mature cathepsin X did not interfere with HSPC-osteoblast interactions. Only pro-cathepsin X treated with dithiothreitol, which unfolds but does not lead to full maturation of cathepsin X, significantly reduced HSPC adhesion to osteoblasts. These observations argue for a role of the accessible cathepsin X prodomain in diminishing cell binding. Our findings strongly suggest that the cysteine cathepsins B, K, and L constitutively secreted by osteoblasts are part of the fine-tuned regulation of CXCL12 in the bone marrow, whereas pro-cathepsin X with its prodomain can affect HSPC trafficking in the niche.