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Dystrophinopathy is caused by alterations in the dystrophin gene. The severe phenotype, Duchenne muscular dystrophy (DMD), is caused by a lack of dystrophin in skeletal muscles, resulting in necrosis and regenerating fibers, inflammatory cells, and muscle fibrosis. Progressive muscle weakness is a characteristic finding of this condition. Here, we encountered a rare case of a 10-year-old patient with asymptomatic dystrophinopathy with no dystrophin expression and investigated the reason for the absence of muscle weakness to obtain therapeutic insights for DMD. Using RNA-seq analysis, gene expression in skeletal muscles was compared among patients with asymptomatic dystrophinopathy, three patients with typical DMD, and two patients without dystrophinopathy who were leading normal daily lives. Cathepsin K (CTSK), myosin heavy chain 3 (MYH3), and nodal modulator 3-like genes exhibited a >8-fold change, whereas crystallin mu gene (CRYM) showed a <1/8-fold change in patients with typical DMD compared with their expression in the patient with asymptomatic dystrophinopathy. Additionally, CTSK and MYH3 expression exhibited a >16-fold change (P < 0.01), whereas CRYM expression showed a <1/16-fold change (P < 0.01) in patients with typical DMD compared with their expression in those without dystrophinopathy. CTSK plays an essential role in skeletal muscle loss, fibrosis, and inflammation in response to muscles injected with cardiotoxin, one of the most common reagents that induce muscle injury. Increased CTSK expression is associated with muscle injury or necrosis in patients with DMD. The lack of muscle weakness in the patient with asymptomatic dystrophinopathy might be attributed to the low CTSK expression in the muscles. To the best of our knowledge, this is the first report to demonstrate that CTSK expression was significantly higher in the skeletal muscles of patients with DMD with a typical phenotype than in those without dystrophinopathy., (© 2024 Japanese Society of Neuropathology.)

Bone morphogenetic protein two (BMP-2) has been widely used as an osteoinductive agent in the treatment of bone diseases. However, some side effects, such as osteoclast activation have emerged when it was used at high doses. In this study, by considering the osteoclast-suppressing capability of melatonin (MEL), its effect on osteoclast differentiation induced by BMP-2 was investigated. These two factors, MEL and BMP-2, were embedded into chitosan/hydroxyapatite (HAp) scaffolds that were characterized morphologically by scanning electron microscopy (SEM) and micro-computed tomography (μ-CT). Release profiles of MEL and BMP-2 from scaffolds were determined in vitro and then, the differentiation of RAW 264.7 cells to osteoclasts was investigated on the scaffolds. Results of tartrate-resistant acid phosphatase (TRAP) staining, SEM imaging and expression of cathepsin K gene showed that, in the presence of BMP-2, osteoclast differentiation increased, whereas it decreased in MEL and MEL/BMP-2 embedded scaffolds suggesting that melatonin successfully attenuated osteoclast differentiation induced by BMP-2. Thus, the MEL/BMP-2 loaded chitosan/HAp scaffolds that have dual function in enhancing bone formation and inhibiting osteoclast activity are recommended biomaterials in the field of bone regeneration.

Aim: Cathepsins are proteases that regulate a wide range of physiological processes, including protein turnover, cell signalling and antigen presentation. Recent studies have shown that cathepsins are highly upregulated in many types of tumours. Of the 15 cathepsins in humans, cathepsins V and S are abundantly expressed in the thymus, and we previously showed that the immunostaining of these cathepsins could serve as diagnostic markers for thymic epithelial tumours. However, little is known about the expression of other cathepsins in thymic epithelial tumours. To determine the diagnostic implications of cathepsins, we performed immunohistochemical analysis of cathepsin B (CTB), cathepsin D (CTD) and cathepsin K (CTK), all of which have been reported to correlate with the progression of squamous cell carcinoma., Methods: The association between cathepsin expression and clinicopathological features was evaluated in 122 cases of thymoma and thymic carcinoma., Results: CTB and CTD were frequently expressed in type A and type AB thymomas. In contrast, CTB and CTD were significantly less common in type B thymomas than in type A or AB thymomas. In type AB thymomas, the expression of CTB correlated with histological features, and was found predominantly in the type A component. Notably, CTK was expressed most commonly in thymic carcinomas, and patients who died of the disease showed increased expression of CTK., Conclusions: The expression of CTB and CTD correlated with the histological subtype of thymoma. In addition, the expression of CTK appears to be useful for the diagnosis of thymic carcinomas and as a prognostic marker., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Endodontic disease is characterized by inflammation and destruction of periapical tissues, leading to severe bone resorption and tooth loss. ATP6AP1 (Ac45) has been implicated in human immune diseases, yet the mechanism underlying how Ac45 regulates immune response and reaction in inflammatory diseases remains unknown. We generated endodontic disease mice through bacterial infection as an inflammatory disease model and used adeno-associated virus (AAV)-mediated Ac45 RNA interference knockdown to study the function of Ac45 in periapical inflammation and bone resorption. We demonstrated that the AAV small hairpin RNA targeting Ac45 (AAV-sh- Ac45 ) impaired cellular acidification, extracellular acidification, and bone resorption. Our results showed that local delivery of AAV-sh- Ac45 in periapical tissues in bacterium-induced inflammatory lesions largely reduced bone destruction, inhibited inflammation, and dramatically reduced mononuclear immune cells. T-cell, macrophage, and dendritic cell infiltration in the periapical lesion was dramatically reduced, and the periodontal ligament was protected from inflammation-induced destruction. Furthermore, AAV-sh- Ac45 significantly reduced osteoclast formation and the expression of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin-10 (IL-10), IL-12, IL-1α, IL-6, and IL-17. Interestingly, AAV-sh- Ac45 impaired mature cathepsin K secretion more significantly than that by AAV-sh- C1 and AAV-sh- CtsK Unbiased genome-wide transcriptome sequencing analysis of Ctsk -/- dendritic cells stimulated with lipopolysaccharide demonstrated that the ablation of Ctsk dramatically reduced dendritic cell-mediated inflammatory signaling. Taken together, our results indicated that AAV-sh- Ac45 simultaneously inhibits osteoclast-mediated bone resorption and attenuates dendritic cell-mediated inflammation through impairing acidification and cathepsin K secretion. Thus, Ac45 may be a novel target for therapeutic approaches to attenuate inflammation and bone erosion in endodontic disease and other inflammation-related osteolytic diseases., (Copyright © 2020 American Society for Microbiology.)

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.

The intent of the present study was to demonstrate that multiphasic silica/collagen xerogels are able to manipulate cellular processes. These xerogels were prepared by a sol-gel approach allowing the incorporation of mineral phases. The resulting nanocomposites are designed as biomaterial for bone regeneration. Human osteoclasts derived from peripheral blood mononuclear cells were cultured both indirectly and directly, either in presence of different xerogel types or on their surface, to investigate the factor with the main influence on osteoclastogenesis. To this end, the incorporation of a third phase to silica/collagen xerogels was used to affect osteoclastogenesis. In cell culture, ambient ion conditions controlled by both the degradation products of the xerogel and the bioactivity-dependent ion release and reprecipitation were shown to have the main effect on osteoclast specific enzyme tartrate-resistant acid phosphatase (TRAP) 5b. Late stage of osteoclastogenesis characterized by resorption was strongly dependent on the xerogels composition. Surface chemistry of the xerogels was displayed to play an important role in osteoclast resorption. Biphasic silica/collagen xerogels and triphasic xerogels with calcium carbonate offered widespread resorbed areas, whereas hydroxyapatite containing xerogels showed distinctly reduced resorption. The incorporation of strontium carbonate and phosphate, respectively, as third phase changed TRAP 5b activity dose-dependently and inhibited resorption within 21 days. Quantitative evaluation on osteoclast differentiation was carried out using biochemical methods (TRAP 5b, cathepsin K) and was supported by confocal laser scanning microscopy and scanning electron microscopy (SEM). Qualitative estimation of resorption was carried out by SEM., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cathepsin K, or CTSK, has been found to be involved in the peritoneal metastasis of ovarian carcinoma. However, the expression and clinicopathological significance of CTSK remains unknown in epithelial ovarian cancer (EOC). The aim of the present study was to investigate the expression of CTSK and its clinicopathological significance in EOC. CTSK expression was evaluated using immunohistochemistry in EOC tissue microarray. The expression of CTSK in EOC was displayed to be markedly higher than that of adjacent normal control. In addition, CSTK expression was shown to be remarkably associated with metastases and inferior overall prognosis of EOC. In vitro, Knock-down of CTSD was exhibited to be able to suppress migration and invasion in EOC cell lines OV-2008 but not proliferation in OV-2008. Together, our data showed that elevated CTSD in EOC can potentiate the metastasis of EOC cells, suggesting that targeting CTSD might be used as a novel therapeutic target for EOC.

CCAAT/enhancer-binding protein α (C/ebpα) is critical for osteoclastogenesis by regulating osteoclast (OC) lineage commitment and is also important for OC differentiation and function in vitro. However, the role of C/ebpα in postnatal skeletal development has not been reported owing to lethality in C/ebpα -/- mice from hypoglycemia within 8 hours after birth. Herein, we generated conditional knockout mice by deleting the C/ebpα gene in monocyte via LysM-Cre to examine its role in OC differentiation and function. C/ebpα f/f LysM-Cre mice exhibited postnatal osteopetrosis due to impaired osteoclastogenesis, OC lineage priming defects, as well as defective OC differentiation and activity. Furthermore, our ex vivo analysis demonstrated that C/ebpα conditional deletion significantly reduced OC differentiation, maturation, and activity while mildly repressing macrophage development. At the molecular level, C/ebpα deficiency significantly suppresses the expressions of OC genes associated with early stages of osteoclastogenesis as well as genes associated with OC differentiation and activity. We also identified numerous C/ebpα critical cis-regulatory elements on the Cathepsin K promoter that allow C/ebpα to significantly upregulate Cathepsin K expression during OC differentiation and activity. In pathologically induced mouse model of osteoporosis, C/ebpα deficiency can protect mice against ovariectomy-induced bone loss, uncovering a central role for C/ebpα in osteolytic diseases. Collectively, our findings have further established C/ebpα as a promising therapeutic target for bone loss by concurrently targeting OC lineage priming, differentiation, and activity. © 2017 American Society for Bone and Mineral Research., (© 2017 American Society for Bone and Mineral Research.)

Extracellular signal-regulated protein kinase 5 (ERK5) has been implicated during development and carcinogenesis. Nkx3.1-mediated Cre expression is a useful strategy to genetically manipulate the mouse prostate. While grossly normal at birth, we observed an unexpected phenotype of spinal protrusion in Nkx3.1:Cre;Erk5 fl/fl (Erk5 fl/fl ) mice by ~6-8 weeks of age. X-ray, histological and micro CT (µCT) analyses showed that 100% of male and female Erk5 fl/fl mice had a severely deformed curved thoracic spine, with an associated loss of trabecular bone volume. Although sex-specific differences were observed, histomorphometry measurements revealed that both bone resorption and bone formation parameters were increased in male Erk5 fl/fl mice compared to wild type (WT) littermates. Osteopenia occurs where the rate of bone resorption exceeds that of bone formation, so we investigated the role of the osteoclast compartment. We found that treatment of RANKL-stimulated primary bone marrow-derived macrophage (BMDM) cultures with small molecule ERK5 pathway inhibitors increased osteoclast numbers. Furthermore, osteoclast numbers and expression of osteoclast marker genes were increased in parallel with reduced Erk5 expression in cultures generated from Erk5 fl/fl mice compared to WT mice. Collectively, these results reveal a novel role for Erk5 during bone maturation and homeostasis in vivo.

Introduction: Melanoma of the skin shows a tendency to metastasize via lymph or blood secreting matrix metalloproteinases and cathepsins, which enable penetration through the dermis. Cathepsin K acts in cytoplasm of atypical melanocytes and completely cleaves internalized collagen., Materials and Methods: Expression of cathepsin K was analyzed immunohistochemically in 45 melanomas and correlated to morphological and clinical parameters., Results: During six years follow up, 13 patients developed lymph node metastases and three of them distant metastases. Positive expression of cathepsin K was found in 19 cases. In univariate regression analysis histological type, pagetoid spread, mitotic activity and cathepsin K expression were significantly connected to metastases. Cathepsin K was significantly associated to histologic type, ulceration, pagetoid spread and mitotic rate. In multiple logistic regression adjusted to these variables, cathepsin K was an independent predictor in occurrence of metastases (P=0.015). Median to the occurrence of metastases was 40 months in patients with cathepsin K positive expression and 71 months in patients with cathepsin K negative expression (P<0.001)., Conclusions: In this preliminary study positive expression of cathepsin K in melanoma of the skin is associated with other unfavorable prognostic factors. We consider cathepsin K expression in primary tumor would significantly precipitate occurrence of metastases.

Objective: The aim of this study was to explore the association between cathepsin K and the clinical characteristics of skull base chordoma (SBC)., Methods: This study included 58 paraffin-embedded samples and 85 frozen samples of 94 patients. All clinical data corresponding to these patients were available. Immunohistochemical staining and quantitative real-time polymerase chain reaction were performed. Positive rate of immunohistochemical staining slices and delta cycle threshold value of quantitative real-time polymerase chain reaction represented the cathepsin K expression level in protein and gene level separately., Results: In protein level, expression level (EL) of invasive tumors was increased compared with noninvasive tumors (P = 0.006), EL of tumors with dura erosion was increased compared with tumors without dura erosion (P = 0.001). Tumors with septa exhibited increased EL compared with tumors without septa (P = 0.001). Tumors with lobulation exhibited increased EL compared with tumors without lobulation (P = 0.000). Higher EL of cathepsin K was associated with reduced progression-free survival (PFS) (P = 0.015). In gene level, tumors with septa showed higher EL than tumors without septa (P = 0.015), and tumors with lobulation showed higher EL than tumors without lobulation (P = 0.049). Cathepsin K EL was an independent risk factor for reduced PFS, and an increased level of cathepsin K in SBC was associated with reduced PFS (P = 0.042)., Conclusions: Increased cathepsin K expression in SBC was associated with tumor invasion and reduced PFS. The cathepsin K level in SBC also was associated with tumor stage, tumor lobulation, and septa., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective . Tumor necrosis factor (TNF) increases circulating osteoclast (OC) precursors numbers by promoting their proliferation and differentiation. The aim of this study was to assess the effect of TNF inhibitors (TNFi) on the differentiation and activity of OC in rheumatoid arthritis (RA) patients. Methods. Seventeen RA patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers, in vitro OC differentiation assays, and qRT-PCR for OC specific genes was performed. Results . After TNFi therapy, patients had reduced RANKL surface expression in B-lymphocytes and the frequency of circulating classical CD14 bright CD16 - monocytes was decreased. Serum levels of sRANKL, sRANKL/OPG ratio, and CTX-I were reduced in RA patients after TNFi treatment. Moreover, after exposure to TNFi, osteoclast differentiation and activity were decreased, as well as the expression of TRAF6 and cathepsin K. Conclusion . We propose that TNFi arrests bone loss and erosion, through two pathways: direct reduction of osteoclast precursor numbers and inhibition of intracellular signaling pathways acting through TRAF6., Competing Interests: The authors declare no competing interests regarding the publication of this paper.

Background: Oral lichen planus (OLP) is an idiopathic T-cell-mediated mucosal inflammatory disease. Cathepsin K (Cat K) is one of the lysosomal cysteine proteases. It is involved in many pathological conditions, including osteoporosis and cancer. The expression and role of Cat K in OLP are unknown., Methods: Twenty-five oral mucosal specimens diagnosed histopathologically as OLP and fourteen healthy controls (HC) were used to study the immunohistochemical (IHC) expression of Cat K. Colocalization of Cat K with CD1a, Melan-A, CD68, CD45, mast cell tryptase (MCT), and Toll-like receptors (TLRs) 4 and 9 were studied using double IHC and/or immunofluorescence (IF) staining. Expression of Cat K was also evaluated in OLP tissue samples before and after topical tacrolimus treatment., Results: Cat K was expressed in a higher percentage of cells in the epithelial zone, and the staining intensity was stronger in the stroma in OLP compared to controls (P < 0.001). In OLP, Cat K was present mostly in melanocytes and macrophages and sporadically in basal keratinocytes, endothelial cells, and extracellularly. Cat K was found also in some fibroblasts in HC and OLP samples. Coexpression of Cat K and TLRs 4 and 9 was seen in some dendritic cells (presumably melanocytes) and macrophages. In OLP, tacrolimus treatment reduced the expression of Cat K in the epithelium but increased it in the stroma., Conclusions: These results suggest that Cat K is involved in the pathogenesis of OLP. Cat K possibly takes part in the modulation of matrix molecules and cellular receptors., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Little is known about the effects of tensile forces on osteoclastogenesis by human monocytes in the absence of mechanosensitive cells, including osteoblasts and fibroblasts. In this study we consider the effects of tensile force on osteoclastogenesis in human monocytes. The cells were treated with receptor activator of nuclear factor κB ligand (RANKL) to promote osteoclastogenesis. Then,expression and secretion of cathepsin K were examined. RANKL and the formation of osteoclasts during the osteoclast differentiation process under continual tensile stress were evaluated by Western blot. It was also found that -100 kPa or lower induces RANKL-enhanced tartrate-resistant acid phosphatase activity in a dose-dependent manner. Furthermore, an increased tensile force raises the expression and secretion of cathepsin K elevated by RANKL, and is concurrent with the increase of TNF-receptor-associated factor 6 induction and nuclear factor κB activation. Overall, the current report demonstrates that tensile force reinforces RANKL-induced osteoclastogenesis by retarding osteoclast differentiation. The tensile force is able to modify every cell through dose-dependent in vitro RANKL-mediated osteoclastogenesis, affecting the fusion of preosteoclasts and function of osteoclasts. However, tensile force increased TNF-receptor-associated factor 6 expression. These results are in vitro findings and were obtained under a condition of tensile force. The current results help us to better understand the cellular roles of human macrophage populations in osteoclastogenesis as well as in alveolar bone remodeling when there is tensile stress.

This study was designed to investigate the effect of 10-hydroxycamptothecin (10-HCPT) on osteoclast formation. RAW264.7 cells were cultured in vitro with 100 ng/ml receptor activator for nuclear factor-κ B ligand (RANKL) and 30 ng/ml recombinant macrophage colony stimulating factor (M-CSF), and 10-HCPT with different solubilities were added. After five-day cultivation, tartrate-resistant acid phosphatase (TRAP) staining was used to observe the number of osteoclasts. mRNA expression of osteoclast-specific genes, such as TRAP, cathepsin K (CTSK) and matrix metalloproteinase protease 9 (MMP-9), was detected by real-time Polymerase Chain Reaction (PCR). The effect of 10-HCPT on the proliferation activity of RAW264.7 cells was detected using Cell Counting Kit-8 (CCK-8). CCK-8 detection showed that 10-HCPT with a certain concentration (1 ng/ml to 5 ng/ml) had no effect on cell proliferation (P>0.05); 10-HCPT could inhibit the generation of osteoclasts. With the increase of the concentration of 10-HCPT, the number of osteoclasts generated from cells cultured with 10-HCPT [1 ng/ml (86±11.14), 2 ng/ml (66.67±7.51), 5ng/ml (27.67±6.51)] was much lower than that of the control group (145±8.19), and the difference was statistically significant (all P=0, P less than 0.05); mRNA expression of osteoclast-specific gene TRAP [1 ng/ml (24.38±0.68), 2 ng/ml (20.09±1.86), 5 ng/ml (6.23±0.53)], CTSK [1 ng/ml (10.08±0.81), 2 ng/ml (7.30±0.30), 5 ng/ml (3.20±0.56)] and MMP-9 [1 ng/ml (43.54±6.96), 2 ng/ml (28.28±5.83), 5 ng/ml (11.07±2.53)] was much lower than that of the groups added with RANKL and M-CSF only (all P=0, P less than 0.05), and with the increase of concentration of 10-HCPT, the expression of osteoclast-specific genes showed a decreasing tendency. All the findings suggest that 10-HCPT can inhibit the formation of osteoclasts by reducing the expression of osteoclast-specific genes such as TRAP, CTSK and MMP-9.

Aim: To assess the beneficial effects of JiangTang XiaoKe (JTXK) granule on the bone metabolism in high fat diet (HFD) fed KK-Ay diabetic mice., Materials and Methods: The KK-Ay mice were used as a diabetic model, while C57BL/6 mice were utilized as the non-diabetic control. The left tibia was used for determining bone mineral density (BMD) and bone ash coefficient. The HE and alizarin red S staining of femur were employed to evaluate bone pathology and calcium deposition. The expressions of alkaline phosphatase (ALP), insulin growth factor 1 (IGF-1) and cathepsin K were assessed by western blotting and immunohistochemical staining., Key Findings: JTXK granule significantly improved the bone ash coefficient, the distribution of trabecular bone and the calcification nodules deposition in KK-Ay mice with diabetes. IGF-1 and ALP expressions were significantly decreased, and cathepsin K expression was dramatically increased in the HFD fed KK-Ay diabetic model mice, which can be reversed by JTXK granule treatment. JTXK granule at medium or high dosage was more efficient in improving diabetic bone quality when compared with that in mice with a low dosage. However, the BMD values in each group of KK-Ay diabetic mice were not significantly different., Significance: We demonstrate that cathepsin K expression is increased in KK-Ay diabetic mouse model. JTXK granule treatment inhibits osteoclastic bone resorption and promotes the new bone formation by decreasing cathepsin K activity and increasing IGF-1 and ALP levels. These changes may contribute to the increase of bone strength and thus reducing the risk of bone fractures., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of this study was to investigate the effect of local administration of odanacatib (ODN) on orthodontic root resorption and the status of alveolar bone metabolism in rat molars. All specimens were scanned using microcomputed tomography and then the raw images were reconstructed. The total volume of the root resorption craters of the 60 g-NS (normal saline) group was higher than in the 60 g-ODN group and the control group. In the 60 g-NS group, the bone volume fraction values of alveolar bone were significantly decreased compared with the other 2 groups. There were no significant differences in the bone volume fraction values of the tibiae among the 3 groups. The results of tartrate-resistant acid phosphatase-positive (TRAP+) numbers showed that there was no difference between the 60 g-NS group and the 60 g-ODN group. The expression of cathepsin K was decreased significantly in the 60 g-ODN group. These results indicate that ODN reduces orthodontics-induced external root resorption and increases alveolar bone metabolism. This may be because ODN inhibits the activity of odontoclasts, but maintains the quantity of odontoclasts and enhances bone formation. ODN promotes local alveolar bone metabolism, but does not affect systemic bone metabolism.

The hormonally active form of vitamin D3, 1α,25-(OH)2D3, has an important role in bone metabolism. This study examined the effects of 1α,25-(OH)2D3 on the ability of two cytokines, receptor activator of nuclear factor-κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), to induce RAW 264.7 cells to form osteoclasts. A TRAP histochemical staining assay and bone resorption analysis were used to identify the rate of formation and activity of osteoclasts. The numbers of osteoclasts formed, and their bone resorption activity, was enhanced by the addition of 1α,25-(OH)2D3. The expression levels of osteoclast-specific proteins that are essential for bone resorption, integrin β3, V-ATPase, CAII, CTSK, TRAP and MMP-9, were detected by western blotting. During 48 h, the expression levels of all these proteins significantly increased. Quantitative real-time polymerase chain reaction was used to determine the expression levels of the transcription factors, c-Fos and NFATcl. The expression levels of c-Fos and NFATc1 also increased 24h after treatment with 1α,25-(OH)2D3. These results suggest that 1α,25-(OH)2D3 can regulate bone metabolism by directly enhancing the formation and maturation of osteoclasts., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Scoparone, one of the bioactive components of Artemisia capillaris Thunb, has various biological properties including immunosuppressive, hepatoprotective, anti-allergic, anti-inflammatory, and antioxidant effects. This study aims at evaluating the anti-osteoporotic effect of scoparone and its underlying mechanism in vitro. Scoparone demonstrated potent cellular antioxidant capacity. It was also found that scoparone inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and suppressed cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression via c-jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)/p38-mediated c-Fos-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway. During osteoclast differentiation, the production of general reactive oxygen species (ROS) and superoxide anions was dose-dependently attenuated by scoparone. In addition, scoparone diminished NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) expression and activation via the tumor necrosis factor receptor-associated factor 6 (TRAF6)-cSrc-phosphatidylinositol 3-kinase (PI3k) signaling pathway and prevented the disruption of mitochondrial electron transport chain system. Furthermore, scoparone augmented the expression of superoxide dismutase 1 (SOD1) and catalase (CAT). The overall results indicate that the inhibitory effect of scoparone on RANKL-induced osteoclast differentiation is attributed to the suppressive effect on ROS and superoxide anion production by inhibiting Nox1 expression and activation and protecting the mitochondrial electron transport chain system and the scavenging effect of ROS resulting from elevated SOD1 and CAT expression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Root resorption is a common complication after replantation following traumatic dental avulsion. Endodontic therapy combined with local and intracanal medications aims to avoid osteoclastic activity. In such cases, the application of alendronate (ALN), a bisphosphonate widely used for the treatment of bone disorders, could be of clinical relevance. This study evaluated alendronate biocompatibility on periodontal ligament cells as well as its effects on an in vitro osteoclastogenesis model., Methods: Alendronate cytotoxicity (10(-3) to 10(-9) mol/L) in human periodontal ligament fibroblasts, human osteogenic sarcoma cells, and murine osteoclastic precursors (RAW 264.7) was analyzed using cell number determination, cell viability, and proliferation assays. ALN (10(-6) to 10(-12) mol/L) effects on RANKL-induced osteoclastogenesis of RAW cells were assessed by tartrate-resistant acid phosphatase (TRAP) staining and activity and real-time polymerase chain reaction., Results: ALN at higher concentrations was cytotoxic for all cell types, inhibiting significantly the proliferation of human osteogenic sarcoma cells and human periodontal ligament fibroblasts (≥10(-5) mol/L). TRAP activity and expression of the osteoclast markers TRAP and cathepsin K by RAW-derived osteoclasts decreased significantly with ALN at low concentrations, reaching the maximum effect at 10(-10) mol/L., Conclusions: We showed that ALN at very low concentrations is an effective inhibitor of RANKL-generated osteoclasts, without causing cytotoxic effects on their precursors or periapical cells. ALN at such concentrations might be useful to prevent replacement resorption in avulsed teeth., (Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

We report the first case of inflammatory variant of hepatic angiomyolipoma (AML) with expression of transcription factor E3 (TFE3) protein but negativity for HMB45 and melan A in a 62-year-old female. Imaging studies revealed a tumor in the left lobe of liver, sized 5.8 cm in maximum diameter. Microscopically, the lesion was composed of large polygonal or epithelioid cells with copious eosinophilic granular cytoplasm. There was a very prominent stromal lymphoplasmacytic infiltrate. Immunohistochemically, the tumor cells showed very strong and diffuse positivity for smooth muscle actin, and cathepsin K, while S-100 protein, keratin, desmin, HMB45 and Melan-A are negative. However, there was multifocal and very convincing nuclear positivity for TFE3, thus confirms the diagnosis.

During long bone development and post-natal growth, the cartilaginous model of the skeleton is progressively replaced by bone, a process known as endochondral ossification. In the primary spongiosa, osteoclasts degrade the mineralized cartilage produced by hypertrophic chondrocytes to generate cartilage trabeculae that osteoblasts embed in bone matrix. This leads to the formation of the trabecular bone network of the secondary spongiosa that will undergo continuous remodeling. Osteoclasts are specialized in mineralized tissue degradation, with the combined ability to solubilize hydroxyapatite and to degrade extracellular matrix proteins. We reported previously that osteoclasts lacking Dock5 could not degrade bone due to abnormal podosome organization and absence of sealing zone formation. Consequently, adult Dock5(-/-) mice have increased trabecular bone mass. We used Dock5(-/-) mice to further investigate the different functions of osteoclast during endochondral bone formation. We show that long bones are overall morphologically normal in developing and growing Dock5(-/-) mice. We demonstrate that Dock5(-/-) mice also have normal hypertrophic cartilage and cartilage trabecular network. Conversely, trabecular bone volume increased progressively in the secondary spongiosa of Dock5(-/-) growing mice as compared to Dock5(+/+) animals, even though their osteoclast numbers were the same. In vitro, we show that Dock5(-/-) osteoclasts do present acidic compartments at the ventral plasma membrane and produce normal amounts of active MMP9, TRAP and CtsK for matrix protein degradation but they are unable to solubilize minerals. These observations reveal that contrarily to bone resorption, the ability of osteoclasts to dissolve minerals is dispensable for the degradation of mineralized hypertrophic cartilage during endochondral bone formation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Due to some severe side effects or lack of efficacy of currently used synthetic drugs, such as bisphosphonates (BPs), the search for new therapeutic agents that can more effectively prevent and treat osteoporosis (OP) has been an increasingly important topic of research. In this study, the low-molecular weight hyaluronan (LMW-HA, 50 kDa) produced by enzymatic degradation of high-molecular weight hyaluronan (HMW-HA, 1922 kDa) from Streptococcus zooepidemicus was evaluated in vitro for its anti-osteoclastogenic potentials using RAW 264.7 murine macrophage cells. LMW-HA (25-200 μg/ml) dose-dependently inhibited the receptor activator of NF-κB ligand (RANKL)-induced tartrate-resistance acid phosphatase (TRAP) activity and the formation of multinucleated osteoclasts. Western blot analysis showed that LMW-HA reduced the RANKL-induced expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), gelsolin and c-Src-proline-rich tyrosine kinase 2 suggesting that it could inhibit actin ring formation of osteoclast cells. In addition, LMW-HA inhibited the bone resorption activity of osteoclastic cells by dose-dependently attenuating the RANKL-induced expression of carbonic anhydrase II and integrin β3. RT-PCR analysis showed that LMW-HA dose-dependently decreased the expression of osteoclast-specific genes, such as matrix metalloproteinase 9 (MMP-9) and cathepsin K, suggesting that it has potential to inhibit the differentiation of osteoclastic cells. Taken collectively, these results suggested that LMW-HA (50 kDa) has significant anti-osteoporotic activity in vitro and may be used as a potent functional ingredient in health beneficial foods or as a therapeutic agent to prevent or treat OP., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Major advances in highly active antiretroviral therapies (HAART) have extended the lives of people living with HIV, but there still remains an increased risk of death by cardiovascular diseases (CVD). HIV proteins have been shown to contribute to cardiovascular dysfunction with effects on the different cell types that comprise the arterial wall. In particular, HIV-1 transactivating factor (Tat) has been shown to bind to endothelial cells inducing a range of responses that contribute to vascular dysfunction. It is well established that hemodynamics also play an important role in endothelial cell mediated atherosclerotic development. When exposed to low or oscillatory shear stress, such as that found at branches and bifurcations, endothelial cells contribute to proteolytic vascular remodeling by upregulating cathepsins, potent elastases and collagenases that contribute to altered biomechanics and plaque formation. Mechanisms to understand the influence of Tat on shear stress mediated vascular remodeling have not been fully elucidated. Using an in vivo HIV-Tg mouse model and an in vitro cone and plate shear stress bioreactor to actuate physiologically relevant pro-atherogenic or atheroprotective shear stress on human aortic endothelial cells, we have shown synergism between HIV proteins and pro-atherogenic shear stress to increase endothelial cell expression of the powerful protease cathepsin K, and may implicate this protease in accelerated CVD in people living with HIV.

Recent studies have demonstrated that cathepsin K seems to be a powerful marker in identifying the microphthalmia associated transcription factor (MITF) family tumors such as renal perivascular epithelioid cell neoplasms (PEComas), alveolar soft part sarcoma, and translocation-associated renal cell carcinomas. However, the expression of cathepsin K in melanocytic lesions has not been well characterized. Our aim was to investigate the expression of cathepsin K in a wide histological spectrum of melanocytic lesions and to evaluate its potential diagnostic and molecular target therapy usefulness in comparison with other commonly used markers. 143 consecutive melanocytic lesions were selected for study including 56 primary malignant melanomas, 62 metastatic melanomas, and 25 benign nevi (16 intradermal melanocytic nevi and 9 compound melanocytic nevi). 107 of the 118 (91%) primary and metastatic melanomas displayed a high percentage of cells with moderately to strongly positive reactions for cathepsin K (mean 82%; range 0-95%). MITF, HMB45, Melan-A, and S100 were expressed in 85, 76, 78 and 96% of cases, respectively, with various percentages of positive cells (mean, 63, 49, 55 and 86%; range 0-90, 0-80, 0-90 and 0-95%). Among the benign nevi, cathepsin K, MITF, HMB45, Melan-A, and S100 were expressed in 88, 80, 36, 68 and 100% of cases, respectively. Cathepsin K appears to be consistently and strongly expressed in melanocytic lesions and valuable in distinguishing malignant melanomas from the majority of human cancers.

The microphthalmia-associated transcription factor (MITF) is required for terminal osteoclast differentiation and is a signaling effector engaged by macrophage colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). MITF exerts its regulatory functions through its association with cofactors. Discovering the identity of its various partners will provide insights into the mechanisms governing gene expression during osteoclastogenesis. Here, we demonstrate that the proto-oncogene fused in sarcoma (FUS), the chromatin remodeling ATPase BRG1, and MITF form a trimeric complex that is regulated by phosphorylation of MITF at Ser-307 by p38 MAPK during osteoclast differentiation. FUS was recruited to MITF target gene promoters Acp5 and Ctsk during osteoclast differentiation, and FUS knockdown abolished efficient transcription of Acp5 and Ctsk. Furthermore, sumoylation of MITF at Lys-316, known to negatively regulate MITF transcriptional activity, inhibited MITF interactions with FUS and BRG1 in a p38 MAPK phosphorylation-dependent manner. These results demonstrate that FUS is a coregulator of MITF activity and provide new insights into how the RANKL/p38 MAPK signaling nexus controls gene expression in osteoclasts.

Evidence from human and animal studies has documented elevated levels of lysosomal cysteine protease cathepsin K in failing hearts. Here, we hypothesized that ablation of cathepsin K mitigates pressure overload-induced cardiac hypertrophy. Cathepsin K knockout mice and their wild-type littermates were subjected to abdominal aortic constriction, resulting in cardiac remodeling (heart weight, cardiomyocyte size, left ventricular wall thickness, and end diastolic and end systolic dimensions) and decreased fractional shortening, the effects of which were significantly attenuated or ablated by cathepsin K knockout. Pressure overload dampened cardiomyocyte contractile function along with decreased resting Ca2+ levels and delayed Ca2+ clearance, which were partly resolved by cathepsin K knockout. Cardiac mammalian target of rapamycin and extracellular signal-regulated kinases (ERK) signaling cascades were upregulated by pressure overload, the effects of which were attenuated by cathepsin K knockout. In cultured H9c2 myoblast cells, silencing of cathepsin K blunted, whereas cathepsin K transfection mimicked phenylephrine-induced hypertrophic response, along with elevated phosphorylation of mammalian target of rapamycin and ERK. In addition, cathepsin K protein levels were markedly elevated in human hearts of end-stage dilated cardiomyopathy. Collectively, our data suggest that cathepsin K ablation mitigates pressure overload-induced hypertrophy, possibly via inhibition of the mammalian target of rapamycin and ERK pathways.

The pathological changes of subchondral bone during osteoarthritis (OA) development in the temporomandibular joint (TMJ) are poorly understood. In the present study, we investigated the longitudinal alterations of subchondral bone using a rat TMJ-OA model developed in our laboratory. Changes in bone mass were examined by micro-CT, and changes in osteoblast and osteoclast activities were analyzed by real-time PCR, immunohistochemistry, and TRAP staining. Subchondral bone loss was detected from 8 weeks after dental occlusion alteration and reached the maximum at 12 weeks, followed by a repair phase until 32 weeks. Although bone mass increased at late stages, poor mechanical structure and lower bone mineral density (BMD) were found in these rats. The numbers of TRAP-positive cells were increased at 12 weeks, while the numbers of osteocalcin-expressing cells were increased at both 12 and 32 weeks. Levels of mRNA expression of TRAP and cathepsin K were increased at 12 weeks, while levels of ALP and osteocalcin were increased at both 12 and 32 weeks. These findings demonstrated that there is an active bone remodeling in subchondral bone in TMJs in response to alteration in occlusion, although new bone was formed with lower BMD and poor mechanical properties.

Rheumatoid synovial fibroblasts (RSF) are activated by toll-like receptor (TLR) signaling pathways during the pathogenesis of rheumatoid arthritis (RA). Cathepsin K is highly expressed by RSF, and is known to play a key role in the degradation of type I and type II collagen. Cathepsin K is considered to be implicated in the degradation of bone and cartilage in RA. Recent observations have shown that hyaluronan (HA) is an important inhibitor of inflammation. In the present study, we show that lipopolysaccharide (LPS) stimulation significantly increases cathepsin K expression by real-time PCR and western blotting analysis via a TLR-4 signaling pathway. Furthermore, we demonstrate that HA suppresses LPS-induced cathepsin K expression, which is dependent on CD44 but not intercellular adhesion molecule-1 (ICAM-1) interaction. We also show that HA suppresses LPS-induced matrix metalloproteinase-1 (MMP-1) expression, which is dependent on both CD44 and ICAM-1 interaction. We conclude that the anti-inflammatory effect of HA occurs through crosstalk between more than one HA receptor. Our study provides evidence for HA mediated suppression of LPS-induced cathepsin K and MMP-1 expression, supporting a protective effect of HA in RA., (Copyright © 2012 Elsevier Inc. All rights reserved.)

C1q deficiency is the strongest known risk factor for SLE (systemic lupus erythematosus) but its endogenous cellular origin remains limitedly understood. In the present study we investigate the production of C1q by both cultured and endogenous bone osteoclasts. Blood monocytes were cultured with RANKL (receptor activator of nuclear factor κB ligand) and M-CSF (macrophage colony-stimulating factor) to generate osteoclasts and these cells expressed C1Q mRNA and also secreted C1q protein. Intracellular C1q was detectable in developing osteoclasts at day 3 by Western blotting and was also detectable by flow cytometry. By immunofluorescence microscopy, C1q was preferentially detected in immature osteoclasts. By multiple detection methods, C1q expression was markedly increased after IFNγ (interferon γ) treatment. By immunohistochemistry, C1q was also detected in endogenous bone osteoclasts. When osteoclasts were cultured on immobilized C1q, these cells exhibited 2-7-fold increases in the expression of signature osteoclast genes [TRAP (tartrate-resistant acid phosphatase), cathepsin K, calcitonin receptor, carbonic anhydrase II and NFATc1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1)], suggesting an osteoclastogenic capability. This is the first report of C1q production by osteoclasts. Its ability to enhance osteoclast development implies reduced osteoclastogenesis in patients with SLE as they often experience decreased C1q levels. This is consistent with the non-erosive nature of lupus arthritis.

Ethanol has been known to induce osteopenia. However, the cellular and molecular mechanisms responsible for its effect have not been well characterized. This study investigated the effects of ethanol on bone metabolism and osteoclastogenesis using rats fed an ethanol-containing liquid diet (35% of calories from ethanol) for 3 weeks. Ethanol increased the activities of bone tartrate-resistant acid phosphatase (TRAP) and cathepsin K, without affecting the levels of serum osteocalcin or bone alkaline phosphatase activity. Histological analysis showed an increased number of osteoclasts in the proximal tibia, but no significant change in the number of osteoblasts. The mRNA levels of receptor for activation of NF-κB (RANK), c-fos, c-jun, TRAP and cathepsin K were significantly increased, although those of macrophage colony-stimulating factor and c-fms were unaltered. The mRNA and protein levels of PU.1 and microphthalmia-associated trascription factor (MITF) also increased. Further, the osteoclastic differentiation of bone marrow-derived macrophage/monocyte precursor cells (BMMs) in vitro was stimulated by ethanol. The increased osteoclastogenesis of BMMs was associated with increased levels of RANK, PU.1 and MITF expression, activated extracellular signal-regulated kinase (ERK), and reactive oxygen species (ROS). Higher lipid peroxide levels and lower glutathione levels were also observed in the serum of the ethanol-fed rats. These results suggested that ethanol promoted osteoclastogenesis by increasing RANK expression through increases in the production of ROS, activation of ERK and expression of PU.1 and MITF.

IL-20 is a proinflammatory cytokine involved in rheumatoid arthritis, atherosclerosis, and stroke. However, little is known about its role in breast cancer. We explored the function of IL-20 in tumor growth and metastasis, as well as in clinical outcome. Tumor expression of IL-20 was assessed by immunohistochemical staining among 198 patients with invasive ductal carcinoma of the breast, using available clinical and survival data. IL-20 expression was associated with advanced tumor stage, greater tumor metastasis, and worse survival. Reverse transcription quantitative polymerase chain reaction showed that clinical breast tumor tissue expressed higher levels of IL-20 and its receptors than did nontumorous breast tissue. IL-20 was also highly expressed in breast cancer bone-metastasis tissue. In vitro, IL-20 upregulated matrix metalloproteinase-9, matrix metalloproteinase-12, cathepsin K, and cathepsin G, and enhanced proliferation and migration of breast cancer cells, which were inhibited by anti-IL-20 mAb 7E. In vivo, we generated murine models to evaluate the therapeutic potential of 7E, using luminescence intensity, radiological scans, and micro-computed tomography. 7E reduced tumor growth, suppressed bone colonization, diminished tumor-mediated osteolysis, and lessened bone density decrement in mice injected with breast cancer cells. In conclusion, our results suggest that IL-20 plays pivotal roles in the tumor progression of breast cancer. IL-20 expression in breast cancer tissue is associated with a poor clinical outcome. Anti-IL-20 mAb 7E suppressed bone colonization and decreased osteolytic bone lesions. Therefore, IL-20 may be a novel target in treating breast tumor-induced osteolysis.

Background: Cathepsin K (CatK) is a lysosomal protease with collagenolytic activity, and its secretion by osteoclasts is responsible for degrading organic bone matrix. People with pathologic bone resorption have higher circulating CatK concentrations., Hypothesis: Canine osteosarcoma (OS) cells will possess CatK, and its secretion will be cytokine inducible. Circulating CatK concentrations will be increased in dogs with OS, and will be a surrogate marker of bone resorption., Animals: Fifty-one dogs with appendicular OS and 18 age- and weight-matched healthy control dogs., Methods: In a prospective study, expressions of CatK mRNA and protein were investigated in OS cells. The inducible secretion and proteolytic activity of CatK from OS cells was assessed in vitro. Serum CatK concentrations were quantified in normal dogs and dogs with OS and its utility as a bone resorption marker was evaluated in dogs with OS treated with palliative radiation and antiresorptive agents., Results: Canine OS cells contain preformed CatK within cytoplasmic vesicles. In OS cells, TGFβ1 induced the secretion of CatK, which degraded bone-derived type I collagen in vitro. CatK concentrations were higher in dogs with OS than healthy dogs (11.3 ± 5.2 pmol/L versus 8.1 ± 5.0 pmol/L, P = .03). In a subset of dogs with OS, pretreatment CatK concentrations gradually decreased after palliative radiation and antiresorptive treatment, from 9.3 ± 3.2 pmol/L to 5.0 ± 3.1 pmol/L, P = .03., Conclusions and Clinical Importance: Canine OS is associated with pathologic bone resorption, and CatK inhibitors might aid in the management of canine OS-related malignant osteolysis., (Copyright © 2011 by the American College of Veterinary Internal Medicine.)

RUNX1 is a critical transcription factor during embryogenesis and neoplastic disease. To identify novel transcriptional targets of RUNX1 in the context of chromatin, we performed genome wide location analysis (ChIP-on-chip). Here we report that SERPINB13, a gene downregulated in head and neck cancers, is a novel RUNX1transcriptional target. RUNX1 binds the SERPINB13 promoter in chromatin to repress its transcription. Mutation of either RUNX1 binding site in the SERPINB13 promoter increased the activity of the promoter. Finally, overexpression of RUNX1 and concomitant decrease in SERPINB13 expression led to increased activity of cathepsin K, an enzyme inhibited by SERPINB13. These data demonstrate that RUNX1 is an important regulator of SERPINB13 and cathepsin K activity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Consumption of almonds has been associated with increased bone mineral density, but the direct effects of almonds on bone cells are not known. We determined whether serum obtained following the consumption of a meal containing 60 g of almonds affects human osteoclast formation, function, and gene expression in vitro. Human osteoclast precursors were cultured in medium containing 10% serum obtained from 14 healthy subjects at baseline and 4 hours following the consumption of 3 test meals containing almonds, potatoes, and rice and balanced for macronutrient composition. Osteoclast formation was determined by the number of tartrate-resistant acid phosphatase (TRAP)(+) multinucleated cells, and osteoclast function was assessed by measuring TRAP activity in the culture medium and calcium released from OsteoAssay (Lonza Walkersville, Walkersville, MD, USA) plates. The expression of cathepsin K, receptor activator of nuclear factor kB, and matrix metalloproteinase-9 genes was measured by real-time reverse transcriptase-polymerase chain reaction. Compared with serum obtained at baseline, serum obtained 4 hours following the consumption of the almond meal reduced osteoclast formation by approximately 20%, TRAP activity by approximately 15%, calcium release by approximately 65%, and the expression of cathepsin K, receptor activator of nuclear factor kB, and matrix metalloproteinase-9 by 13% to 23%. No effects were observed with serum obtained from the other test meals. Serum obtained 4 hours following the consumption of an almond meal inhibits osteoclast formation, function, and gene expression in cultured human osteoclast precursors, and provides evidence for a positive effect of almonds on bone health., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Unlabelled: This study investigated the role of macrophage migration inhibitory factor (MIF) in fracture repair using MIF gene-deficient mice (MIF KO). Fracture healing was delayed in MIF KO, and this was mainly due to the delay in the mineralization of osteoid within the fracture callus., Introduction: We previously reported that the expression of macrophage migration inhibitory factor (MIF) was up-regulated during the fracture healing process in rats. However, its role in the pathophysiology of this process remained unclear. The aim of the present study was to clarify the role of MIF in the fracture healing process using MIF gene-deficient mice (MIF KO)., Methods: Bone repair in wild-type mice (WT) and MIF KO (n = 70, respectively) was investigated using a tibia fracture model. Radiographic, biomechanical, histological, bone histomorphometric, and molecular analyses were performed., Results: Post-fracture biomechanical testing showed that maximum load and stiffness were significantly lower in MIF KO than in WT on day 42. However, similar levels were observed between the two groups on day 84. Bone histomorphometric analysis revealed significantly higher osteoid volume, a lower mineral apposition rate, and smaller numbers of osteoclasts in the MIF KO callus compared to the WT callus. The messenger ribonucleic acid expressions of matrix metalloproteinase (MMP)-2, membranous type 1-MMP, cathepsin K, and tissue nonspecific alkaline phosphatase were found to be significantly suppressed in the MIF KO callus., Conclusion: The results of the present study suggest that delayed fracture healing in MIF KO was mainly attributable to a delay in osteoid mineralization.

Objective: Mesenchymal stem cells (MSCs) have been proposed to be a useful tool for treatment of rheumatoid arthritis (RA), not only because of their multipotency but also because of their immunosuppressive effect on lymphocytes, dendritic cells, and other proinflammatory cells. Since bone destruction caused by activated osteoclasts occurs in RA, we undertook the present study to investigate the effect of MSCs on osteoclast function and differentiation in order to evaluate their potential use in RA therapy., Methods: Human MSCs and peripheral blood mononuclear cells were cultured under cell-cell contact-free conditions with osteoclast induction medium. Differentiation into osteoclast-like cells was determined by tartrate-resistant acid phosphatase staining and expression of osteoclast differentiation markers., Results: The number of osteoclast-like cells was decreased and expression of cathepsin K and nuclear factor of activated T cells c1 (NF-ATc1) was down-regulated by the addition of either MSCs or a conditioned medium obtained from MSCs. Osteoprotegerin (OPG) was constitutively produced by MSCs and inhibited osteoclastogenesis. However, osteoclast differentiation was not fully recovered upon treatment with either anti-OPG antibody or OPG small interfering RNA, suggesting that OPG had only a partial role in the inhibitory effect of MSCs. Moreover, bone-resorbing activity of osteoclast-like cells was partially recovered by addition of anti-OPG antibody into the conditioned medium., Conclusion: The present results indicate that human MSCs constitutively produce OPG, resulting in inhibition of osteoclastogenesis and expression of NF-ATc1 and cathepsin K in the absence of cell-cell contact. Therefore, we conclude that human MSCs exert a suppressive effect on osteoclastogenesis, which may be beneficial in inhibition of joint damage in RA., (Copyright © 2011 by the American College of Rheumatology.)

Background and Purpose: The aim of this study was to test if corticotomy-induced osteoclastogenesis and bone remodeling underlie orthodontic tooth movement and how selective alveolar decortication enhances the rate of tooth movement., Materials and Methods: A total of 114 Sprague-Dawley rats were included in 3 treatment groups: selective alveolar decortication alone (SADc); tooth movement alone (TM); and "combined" therapy (SADc + TM). Surgery was performed around the buccal and palatal aspects of the left maxillary first molar tooth and included 5 decortication dots on each side. Tooth movement was performed on the first molar using a 25-g Sentalloy spring. Measurements were done at baseline (day 0: no treatment rendered) and on days 3, 7, 14, 21, 28 and 42. Microcomputed tomography, Faxitron analyses, and quantitative real-time polymerase chain reaction (q-PCR) of expressed mRNAs were used to assess changes., Results: The combined group showed increased tooth movement (P = 0.04) at 7 days compared with the tooth movement group with significantly decreased bone volume (62%; P = 0.016) and bone mineral content (63%; P = 0.015). RNA markers of osteoclastic cells and key osteoclastic regulators (M-CSF [macrophage colony-stimulating factor], RANKL [receptor activator of nuclear factor kappa-B ligand], OPG [osteoprotegerin], calcitonin receptor [CTR], TRACP-5b [tartrate-resistant acid phosphatase 5b], cathepsin K [Ctsk]) all showed expression indicating increased osteoclastogenesis in the combined group. RNA markers of osteoblastic cells (OPN [osteopontin], BSP [bone sialoprotein], OCN [osteocalcin]) also showed increased anabolic activity in response to the combination of alveolar decortication and tooth movement., Conclusions: The data suggest that the alveolar decortication enhances the rate of tooth movement during the initial tooth displacement phase; this results in a coupled mechanism of bone resorption and bone formation during the earlier stages of treatment, and this mechanism underlies the rapid orthodontic tooth movement., (Copyright © 2011 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.)

This study examined how interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6r) influence osteoclastic differentiation through the function of chondrocytes. Chondrocytes were cultured with or without IL-6 and/or sIL-6r in the presence or absence of NS398, a specific inhibitor of cyclooxygenase (COX)-2, for up to 28 days. Chondrocytes were also cultured with or without IL-6 and sIL-6r for 28 days, and the conditioned medium from cells cultured without IL-6 and sIL-6r was used to induce differentiation of RAW264.7 cells into osteoclast precursors. Osteoclastic differentiation was assessed by tartrate-resistant acid phosphatase (TRAP) staining. Expression of osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), COX-2, and prostaglandin E(2) (PGE(2)) increased in cells exposed to IL-6 and sIL-6r, whereas expression of macrophage colony-stimulating factor (M-CSF) and bone resorption-related enzymes decreased. NS398 blocked the stimulatory/suppressive effects of IL-6 and sIL-6r on the expression of OPG, RANKL, and M-CSF. Fewer TRAP-positive multinucleated cells were detected after treatment with conditioned medium from IL-6- and sIL-6r-treated chondrocytes than after treatment with conditioned medium from untreated chondrocytes. These results suggest that IL-6 and sIL-6r interfere with osteoclast function through the involvement of chondrocytes. Specifically, they appear to suppress the differentiation of osteoclast precursors into osteoclasts by inducing chondrocytic PGE(2) production, which, in turn, increases OPG secretion and decreases M-CSF secretion by chondrocytes.

Background- The molecular mechanisms that determine the localized formation of thin-capped atheromata in the coronary arteries remain unknown. This study tested the hypothesis that low endothelial shear stress augments the expression of matrix-degrading proteases and thereby promotes the formation of thin-capped atheromata. Methods and Results- Intravascular ultrasound-based, geometrically correct 3-dimensional reconstruction of the coronary arteries of 12 swine was performed in vivo 23 weeks after initiation of diabetes mellitus and a hyperlipidemic diet. Local endothelial shear stress was calculated in plaque-free subsegments of interest (n=142) with computational fluid dynamics. At week 30, the coronary arteries (n=31) were harvested and the same subsegments were identified. The messenger RNA and protein expression and elastolytic activity of selected elastases and their endogenous inhibitors were assessed. Subsegments with low preceding endothelial shear stress at week 23 showed reduced endothelial coverage, enhanced lipid accumulation, and intense infiltration of activated inflammatory cells at week 30. These lesions showed increased expression of messenger RNAs encoding matrix metalloproteinase-2, -9, and -12, and cathepsins K and S relative to their endogenous inhibitors and increased elastolytic activity. Expression of these enzymes correlated positively with the severity of internal elastic lamina fragmentation. Thin-capped atheromata developed in regions with lower preceding endothelial shear stress and had reduced endothelial coverage, intense lipid and inflammatory cell accumulation, enhanced messenger RNA expression and elastolytic activity of MMPs and cathepsins, and severe internal elastic lamina fragmentation. Conclusions- Low endothelial shear stress induces endothelial discontinuity and accumulation of activated inflammatory cells, thereby augmenting the expression and activity of elastases in the intima and shifting the balance with their inhibitors toward matrix breakdown. Our results provide new insight into the mechanisms of regional formation of plaques with thin fibrous caps.

It has previously been reported that low-energy laser irradiation stimulated the velocity of tooth movement via the receptor activator of nuclear factor kappa B (RANK)/RANK ligand and the macrophage colony-stimulating factor/its receptor (c-Fms) systems. Matrix metalloproteinase (MMP)-9, cathepsin K, and alpha(v) beta(3) [alpha(v)beta3] integrin are essential for osteoclastogenesis; therefore, the present study was designed to examine the effects of low-energy laser irradiation on the expression of MMP-9, cathepsin K, and alpha(v)beta3 integrin during experimental tooth movement. Fifty male, 6-week-old Wistar strain rats were used in the experiment. A total force of 10g was applied to the rat molars to induce tooth movement. A Ga-Al-As diode laser was used to irradiate the area around the moving tooth and, after 7 days, the amount of tooth movement was measured. To determine the amount of tooth movement, plaster models of the maxillae were made using a silicone impression material before (day 0) and after tooth movement (days 1, 2, 3, 4, and 7). The models were scanned using a contact-type three-dimensional (3-D) measurement apparatus. Immunohistochemical staining for MMP-9, cathepsin K, and integrin subunits of alpha(v)beta3 was performed. Intergroup comparisons of the average values were conducted with a Mann-Whitney U-test for tooth movement and the number of tartrate-resistant acid phosphatase (TRAP), MMP-9, cathepsin K, and integrin subunits of alpha(v)beta3-positive cells. In the laser-irradiated group, the amount of tooth movement was significantly greater than that in the non-irradiated group at the end of the experiment (P < 0.05). Cells positively stained with TRAP, MMP-9, cathepsin K, and integrin subunits of alpha(v)beta3 were found to be significantly increased in the irradiated group on days 2-7 compared with those in the non-irradiated group (P < 0.05). These findings suggest that low-energy laser irradiation facilitates the velocity of tooth movement and MMP-9, cathepsin K, and integrin subunits of alpha(v)beta3 expression in rats.

Interleukin-17 (IL-17) is produced exclusively by activated T cells and neutrophils, and stimulates osteoclastic bone resorption via osteoblasts by inducing the expression of "receptor activator of NF-kappaB (RANK) ligand" (RANKL). However, the direct effects of IL-17 on the differentiation of osteoclast precursors into osteoclasts and on the function of osteoclasts have not been clarified. Therefore, we examined the effects of IL-17A on the differentiation of osteoclast precursors using RAW264.7 cells and also on the expression of carbonic anhydrase II (CA II), cathepsin K, matrix metalloproteinases-9 (MMP-9), RANK, c-fms, and IL-17 receptors in these cells. The cells were cultured with or without 0.1, 1.0, 10 or 50 ng/mL IL-17 in the presence of soluble RANKL for up to 10 days. The CA II, cathepsin K, and MMP-9 mRNA and protein expression levels were examined using real-time PCR and Western blotting, respectively. The mRNA expression levels of RANK, c-fms, and IL-17 receptors were monitored by real-time PCR. Osteoclast differentiation was estimated using tartrate-resistant acid phosphatase (TRAP) staining of the cells. TRAP-positive cells were observed after day 5 of culture, and the number of cells decreased in the presence of 10 and 50 ng/mL IL-17A at days 5 and 7. In the presence of IL-17A, the expressions of cathepsin K, MMP-9 and c-fms decreased markedly on days 5 and/or 7 of culture, whereas the expression of CA II and IL-17 receptor (type A) increased remarkably at days 3 and 7, respectively. The expression of RANK and IL-17 receptor (type C) was not affected by the addition of IL-17A. These results suggest that the differentiation of osteoclast precursors into osteoclasts is suppressed at high concentrations of IL-17A. Furthermore, IL-17A suppresses the hydrolysis of matrix proteins during bone resorption by decreasing the production of cathepsin K and MMP-9 in osteoclasts., (Copyright (c) 2009 Elsevier Masson SAS. All rights reserved.)

Previous studies have suggested that the biodegradability of octacalcium phosphate-collagen (OCP/Col) composite by osteoclasts is accelerated in association with mechanical stress suffered by the host tissue around the implant. The present study was designed to investigate whether alleviation of mechanical stress restores the bone regenerative properties of OCP/Col, as previously shown in nonload-bearing sites. OCP/Col discs supported with a polytetrafluoroethylene (PTFE) ring, which has a higher modulus than OCP/Col, were implanted in a rat subperiosteal pocket for up to 12 weeks. The structural features of the implant and biological responses were analyzed by X-ray diffraction, Fourier transform infrared spectroscopy, histomorphometry, histochemistry, and tissue mRNA expression around the implants. The effect of compression was analyzed using mouse stromal ST-2 cells by deforming the cell-seeded OCP/Col discs in vitro with or without a PTFE ring. The results clearly indicated the restoration of bone formation by the alleviation of mechanical stress and the upregulation of osteoblast-related genes, such as osterix on the other hand, the implantation of OCP/Col on calvaria or in an in vitro test without PTFE support resulted in the upregulation of osteoclast-related genes, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K, in the tissues or receptor activator of the nuclear factor-kappaB ligand (RANKL) in ST-2 cells. The results confirmed that calvaria augmentation is enhanced by implanting OCP/Col if suitable conditions regarding mechanical stress are provided.

Bone metastasis is a hallmark of advanced prostate and breast cancers, yet the critical factors behind attraction of tumors to the skeleton have not been validated. Here, we investigated the involvement of cathepsin K in the progression of prostate tumors in the bone, which occurs both by direct degradation of bone matrix collagen I and by cleavage of other factors in the bone microenvironment. Our results demonstrated that bone marrow-derived cathepsin K is capable of processing and thereby modulating SPARC, a protein implicated in bone metastasis and inflammation. The coincident up-regulation of SPARC and cathepsin K occurred both in vivo in experimental prostate bone tumors, and in vitro in co-cultures of bone marrow stromal cells with PC3 prostate carcinoma cells. PC3-bone marrow stromal cell interaction increased secretion and processing of SPARC, as did co-cultures of bone marrow stromal cells with two other cancer cell lines. In addition, bone marrow stromal cells that were either deficient in cathepsin K or treated with cathepsin K inhibitors had significantly reduced secretion and cleavage of SPARC. Increases in secretion of pro-inflammatory cytokines (ie, interleukin-6, -8) coincident with overexpression of cathepsin K suggest possible mechanisms by which this enzyme contributes to tumor progression in the bone. This is the first study implicating bone marrow cathepsin K in regulation of biological activity of SPARC in bone metastasis.

Crohn's disease is a chronic inflammatory bowel disease, whose aetiology and pathogenesis are still unknown. The occurrence of epithelioid granulomas is one characteristic feature of the disease since these lesions are found in the bowel wall in 50-87% of colectomy specimens. Although granulomas are not pathognomonic, their identification is considered a relevant element for diagnosis. Cathepsin-k, a papain-like cysteine protease, is involved in bone remodelling, and has been widely used as a immunohistochemical marker for the in situ detection of osteoclasts. Interestingly, the expression of this potent protease is also significantly increased in stimulated tissue macrophages, epithelioid cells and granulomas, but is not expressed in resident tissue macrophages. In the present study, we evaluated Cathepsin-k expression as a diagnostic tool in the identification of small granulomas in Crohn's disease. Formalin-fixed and paraffin-embedded samples of 10 cases of Crohn's disease were collected from surgical ileo-colic resections followed by comparison of Cathepsin-k and CD68 immunoreactivity. Granulomas were identified in 4 of 10 cases examined in haematoxylin & eosin preparations. Cathepsin-k enabled the identification of small granulomas (with a diameter between 100 and 200 microm) in 6 of 10 cases, mainly localized within the submucosa and muscular layers. When compared to CD68, Cathepsin-k immunoreactivity was generally absent or only weakly expressed in resting tissue macrophages, thus allowing better identification of activated epithelioid cells. Based on these results, Cathepsin-k appears to be a reliable tool for the precise and rapid identification of small epithelioid granulomas in Crohn's disease.