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Rufy4, a protein belonging to the RUN and FYVE domain-containing protein family, participates in various cellular processes such as autophagy and intracellular trafficking. However, its role in osteoclast-mediated bone resorption remains uncertain. In this study, we investigated the expression and role of the Rufy4 gene in osteoclasts using small interfering RNA (siRNA) transfection and gene overexpression systems. Our findings revealed a significant increase in Rufy4 expression during osteoclast differentiation. Silencing Rufy4 enhanced osteoclast differentiation, intracellular cathepsin K levels, and formation of axial protrusive structures but suppressed bone resorption. Conversely, overexpressing wild-type Rufy4 in osteoclasts hindered differentiation while promoting podosome formation and bone resorption. Similarly, overexpression of a Rufy4 variant lacking the RUN domain mimics the effects of Rufy4 knockdown, significantly increasing intracellular cathepsin K levels, promoting osteoclastogenesis, and elongated axial protrusions formation, yet inhibiting bone resorption. These findings indicate that Rufy4 plays a critical role in osteoclast differentiation and bone resorption by regulating the cytoskeletal organization through its RUN domain. Our study provides new insights into the molecular mechanisms governing osteoclast activity and underscores Rufy4's potential as a novel therapeutic target for bone disorders characterized by excessive bone resorption. [ABSTRACT FROM AUTHOR]

Cathepsin‐K (CTSK) is an osteoclast‐secreted cysteine protease that efficiently cleaves extracellular matrices and promotes bone homeostasis and remodeling, making it an excellent therapeutic target. Detection of CTSK activity in complex biological samples using tailored tools such as activity‐based probes (ABPs) will aid tremendously in drug development. Here, potent and selective CTSK probes are designed and created, comparing irreversible and reversible covalent ABPs with improved recognition components and electrophiles. The newly developed CTSK ABPs precisely detect active CTSK in mouse and human cells and tissues, from diseased and healthy states such as inflamed tooth implants, osteoclasts, and lung samples, indicating changes in CTSK's activity in the pathological samples. These probes are used to study how acidic pH stimulates mature CTSK activation, specifically, its transition from pro‐form to mature form. Furthermore, this study reveals for the first time, why intact cells and cell lysate exhibit diverse CTSK activity while having equal levels of mature CTSK enzyme. Interestingly, these tools enabled the discovery of active CTSK in human osteoclast nuclei and in the nucleoli. Altogether, these novel probes are excellent research tools and can be applied in vivo to examine CTSK activity and inhibition in diverse diseases without immunogenicity hazards. [ABSTRACT FROM AUTHOR]

External cervical resorption (ECR) is an aggressive disease characterized by resorption of the tooth root structure. While the pericanalar resorption-resistant sheet (PRRS) impedes ECR progression towards the pulp, the underlying mechanisms of its protective role in human teeth remain unclear. This study aimed to elucidate the pathology of ECR in a 31-year-old female patient by employing radiographic, histological, and immunohistochemical analyses of an extracted tooth. Histological examination revealed that the PRRS comprised dentin, predentin, and reparative bone-like tissue. Notably, clastic cells were observed on the surfaces of all three tissues within the same specimens. Immunohistochemical staining for cathepsin K demonstrated diminished resorptive activity of clastic cells on predentin compared to dentin and bone-like tissue. These findings suggest a potential role for predentin in attenuating clastic cell activity, potentially serving as the final barrier safeguarding the pulp tissue.

The tooth movement is a fundamental requirement during any orthodontic treatment. This study aimed to investigate the action and mechanism of strontium ranelate (SR) in orthodontic tooth movement (OTM). Rats were given SR by gavage daily, along with lentiviral vectors interfering with circBACH1 or miR-155-5p. Three weeks later, an OTM rat model was established. RANKL and osteoprotegerin (OpG) in serum were measured. The gap between the first and second molar and root resorption were examined. Osteoclast test was used; and root condition was examined. Detection of miR-155-5p, circBACH1, CLC7 and cathepsin K was performed. The binding of circBACH1 to miR-155-5p was verified. In OTM rats, circBACH1 was elevated (p<0.05) and miR-155-5p was silenced (p<0.05). SR reduced osteoclast activity (p<0.05) and improved root resorption (p<0.05) in OTM rats, which was enhanced by silenced circBACH1 (p<0.05) or elevated miR-155-5p (p<0.05). circBACH1 inhibited miR-155-5p expression (p<0.05). Silenced miR-155-5p reversed the ameliorative effect of circBACH1 on tooth root resorption in OTM rats (all p<0.05). SR modulates circBACH1/miR-155-5p to ameliorate root resorption and tooth fixation during OTM. [ABSTRACT FROM AUTHOR]

External cervical resorption (ECR) is an aggressive disease characterized by resorption of the tooth root structure. While the pericanalar resorption-resistant sheet (PRRS) impedes ECR progression towards the pulp, the underlying mechanisms of its protective role in human teeth remain unclear. This study aimed to elucidate the pathology of ECR in a 31-year-old female patient by employing radiographic, histological, and immunohistochemical analyses of an extracted tooth. Histological examination revealed that the PRRS comprised dentin, predentin, and reparative bone-like tissue. Notably, clastic cells were observed on the surfaces of all three tissues within the same specimens. Immunohistochemical staining for cathepsin K demonstrated diminished resorptive activity of clastic cells on predentin compared to dentin and bone-like tissue. These findings suggest a potential role for predentin in attenuating clastic cell activity, potentially serving as the final barrier safeguarding the pulp tissue. [ABSTRACT FROM AUTHOR]

Abstract Cathepsin‐K (CTSK) is an osteoclast‐secreted cysteine protease that efficiently cleaves extracellular matrices and promotes bone homeostasis and remodeling, making it an excellent therapeutic target. Detection of CTSK activity in complex biological samples using tailored tools such as activity‐based probes (ABPs) will aid tremendously in drug development. Here, potent and selective CTSK probes are designed and created, comparing irreversible and reversible covalent ABPs with improved recognition components and electrophiles. The newly developed CTSK ABPs precisely detect active CTSK in mouse and human cells and tissues, from diseased and healthy states such as inflamed tooth implants, osteoclasts, and lung samples, indicating changes in CTSK's activity in the pathological samples. These probes are used to study how acidic pH stimulates mature CTSK activation, specifically, its transition from pro‐form to mature form. Furthermore, this study reveals for the first time, why intact cells and cell lysate exhibit diverse CTSK activity while having equal levels of mature CTSK enzyme. Interestingly, these tools enabled the discovery of active CTSK in human osteoclast nuclei and in the nucleoli. Altogether, these novel probes are excellent research tools and can be applied in vivo to examine CTSK activity and inhibition in diverse diseases without immunogenicity hazards.

Abstract The role of cathepsin K (CTSK) expression in the pathogenesis and progression of gastric cancer (GC) remains unclear. Hence, the primary objective of this study is to elucidate the precise expression and biological role of CTSK in GC by employing a combination of bioinformatics analysis and in vitro experiments. Our findings indicated a significant upregulation of CTSK in GC. The bioinformatics analysis revealed that GC patients with a high level of CTSK expression exhibited enrichment of hallmark gene sets associated with angiogenesis, epithelial–mesenchymal transition (EMT), inflammatory response, KRAS signaling up, TNFα signaling via KFκB, IL2-STAT5 signaling, and IL6-JAK-STAT3 signaling. Additionally, these patients demonstrated elevated levels of M2-macrophage infiltration, which was also correlated with a poorer prognosis. The results of in vitro experiments provided confirmation that the over-expression of CTSK leads to an increase in the proliferative and invasive abilities of GC cells. However, further evaluation was necessary to determine the impact of CTSK on the migration capability of these cells. Our findings suggested that CTSK has the potential to facilitate the initiation and progression of GC by augmenting the invasive capacity of GC cells, engaging in tumor-associated EMT, and fostering the establishment of an immunosuppressive tumor microenvironment (TME).

The role of cathepsin K (CTSK) expression in the pathogenesis and progression of gastric cancer (GC) remains unclear. Hence, the primary objective of this study is to elucidate the precise expression and biological role of CTSK in GC by employing a combination of bioinformatics analysis and in vitro experiments. Our findings indicated a significant upregulation of CTSK in GC. The bioinformatics analysis revealed that GC patients with a high level of CTSK expression exhibited enrichment of hallmark gene sets associated with angiogenesis, epithelial–mesenchymal transition (EMT), inflammatory response, KRAS signaling up, TNFα signaling via KFκB, IL2-STAT5 signaling, and IL6-JAK-STAT3 signaling. Additionally, these patients demonstrated elevated levels of M2-macrophage infiltration, which was also correlated with a poorer prognosis. The results of in vitro experiments provided confirmation that the over-expression of CTSK leads to an increase in the proliferative and invasive abilities of GC cells. However, further evaluation was necessary to determine the impact of CTSK on the migration capability of these cells. Our findings suggested that CTSK has the potential to facilitate the initiation and progression of GC by augmenting the invasive capacity of GC cells, engaging in tumor-associated EMT, and fostering the establishment of an immunosuppressive tumor microenvironment (TME). [ABSTRACT FROM AUTHOR]

Background: Adamantinomatous craniopharyngiomas (ACPs) are rare benign epithelial tumours with high recurrence and poor prognosis. Biological differences between recurrent and primary ACPs that may be associated with disease recurrence and treatment have yet to be evaluated at the proteomic level. In this study, we aimed to determine the proteomic profiles of paired recurrent and primary ACP, gain biological insight into ACP recurrence, and identify potential targets for ACP treatment. Method: Patients with ACP (n = 15) or Rathke's cleft cyst (RCC; n = 7) who underwent surgery at Sanbo Brain Hospital, Capital Medical University, Beijing, China and received pathological confirmation of ACP or RCC were enrolled in this study. We conducted a proteomic analysis to investigate the characteristics of primary ACP, paired recurrent ACP, and RCC. Western blotting was used to validate our proteomic results and assess the expression of key tumour-associated proteins in recurrent and primary ACPs. Flow cytometry was performed to evaluate the exhaustion of tumour-infiltrating lymphocytes (TILs) in primary and recurrent ACP tissue samples. Immunohistochemical staining for CD3 and PD-L1 was conducted to determine differences in T-cell infiltration and the expression of immunosuppressive molecules between paired primary and recurrent ACP samples. Results: The bioinformatics analysis showed that proteins differentially expressed between recurrent and primary ACPs were significantly associated with extracellular matrix organisation and interleukin signalling. Cathepsin K, which was upregulated in recurrent ACP compared with that in primary ACP, may play a role in ACP recurrence. High infiltration of T cells and exhaustion of TILs were revealed by the flow cytometry analysis of ACP. Conclusions: This study provides a preliminary description of the proteomic differences between primary ACP, recurrent ACP, and RCC. Our findings serve as a resource for craniopharyngioma researchers and may ultimately expand existing knowledge of recurrent ACP and benefit clinical practice. [ABSTRACT FROM AUTHOR]

Objective: Compressive force has been found to be catabolic to alveolar bone during orthodontic tooth movement. This study quantified the fusion of mononuclear RAW 264.7 cells (a murine osteoclastic-like cell line) into multinucleated osteoclasts under a hydrostatic pressure-generated mechanical compression-the new model of various magnitudes and durations. Methods: RAW 264.7 cells were subjected to 0.3, 0.6 or 0.9 g/cm2 of compressive force by an acrylic cylinder custom-made by laser cutting or no compressive force for 4 days during osteoclastogenic induction. TRAP-positive multinucleated cells were quantified. For the release from force experiment, osteoclastogenesis was induced by 0.6 g/cm2 mechanical stimuli for 0, 1, 2, 3 or 4 days. Cell viability, TRAP-positive multinucleated cells, DCSTAMP and Cathepsin K (CTSK) gene expression were evaluated 4 days after release from force. Results: Compressive force at 0.6 and 0.9 g/cm2 significantly increase the number of TRAP-positive multinucleated cells (P

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

Case summaryA 9-month-old entire male domestic longhair cat presented with a history of pathological fractures, chronic musculoskeletal pain and poor growth. Multiple facial and skeletal abnormalities were identified on physical examination and advanced imaging (CT and radiographs). A variant in CTSK was identified in the affected cat following whole-exome sequencing (WES). The cat was managed symptomatically with diet, environmental modifications and analgesia.Relevance and novel informationThis is the first report of a cat with a similar clinical presentation and genetic variant to the hereditary human genetic disorder pyknodysostosis. In this case, WES was performed, which often facilitates the diagnosis of various hereditary disorders (ie, a conceptual framework for practicing feline genomic medicine). Despite the severe skeletal and appendicular abnormalities described, the cat was alive more than 2 years after its initial presentation.

Compressive force has been found to be catabolic to alveolar bone during orthodontic tooth movement. This study quantified the fusion of mononuclear RAW 264.7 cells (a murine osteoclastic-like cell line) into multinucleated osteoclasts under a hydrostatic pressure-generated mechanical compression-the new model of various magnitudes and durations. RAW 264.7 cells were subjected to 0.3, 0.6 or 0.9 g/cm2 of compressive force by an acrylic cylinder custom-made by laser cutting or no compressive force for 4 days during osteoclastogenic induction. TRAP-positive multinucleated cells were quantified. For the release from force experiment, osteoclastogenesis was induced by 0.6 g/cm2 mechanical stimuli for 0, 1, 2, 3 or 4 days. Cell viability, TRAP-positive multinucleated cells, DCSTAMP and Cathepsin K (CTSK) gene expression were evaluated 4 days after release from force. Compressive force at 0.6 and 0.9 g/cm2 significantly increase the number of TRAP-positive multinucleated cells (P < 0.05). Release from continuous mechanical compression after 4 days significantly elevated the number of TRAP-positive multinucleated cells and DCSTAMP and CTSK mRNA expression, with no adverse effects on cell viability (P < 0.05). Continuous stimulation with compressive force induced osteoclastogenesis in RAW 264.7 cells by enhancing DCSTAMP and CTSK expression, which provides new understanding of bone remodeling during orthodontic treatment. [ABSTRACT FROM AUTHOR]

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

To observe the effects of liraglutide (analog of glucagon-like peptide 1 (GLP-1)) on atrial natriuretic peptide (ANP) secretion and atrial dynamics, an ex vivo isolated rat atrial perfusion model was used to determine atrial ANP secretion and pulse pressure. DPP-4−/− mice were also established in vivo. ANP levels were determined by radioimmunoassay; GLP-1 content was determined by Elisa. The expression levels of GLP-1 receptor (GLP-1R), PI3K/AKT/mTOR, piezo 1, and cathepsin K were analyzed by Western blot. In the clinical study, patients with acute coronary syndrome (ACS) had low levels of plasma GLP-1 but relatively high levels of plasma ANP. In ex vivo (3.2 nmol/L) and in vivo (30 μg/kg) models, liraglutide significantly decreased ANP levels and atrial pulse pressure. Exendin9–39 alone (GLP-1R antagonist) reversibly significantly increased ANP secretion, and the reduction effect of liraglutide on the secret ion of ANP was significantly alleviated by Exendin9–39. Exendin9–39 demonstrated slightly decreased atrial pulse pressure; however, combined liraglutide and Exendin9–39 significantly decreased atrial pulse pressure. Ly294002 (PI3K/AKT inhibitor) inhibited the increase of ANP secretion by liraglutide for a short time, while Ly294002 didn't counteract the decrease in pulse pressure by liraglutide in atrial dynamics studies. Liraglutide increased the expression of GLP-1R and PI3K/AKT/mTOR in isolated rat atria and the hearts of mice in vivo, whereas Exendin9–39 reversibly reduced the expression of GLP-1R and PI3K/AKT/mTOR. Piezo 1 was significantly decreased in wild type and DPP-4−/− mouse heart or isolated rat atria after being treated with liraglutide. Cathepsin K expression was only decreased in in vivo model hearts. Liraglutide can inhibit ANP secretion while decreasing atrial pulse pressure mediated by GLP-1R. Liraglutide probably plays a role in the reduction of ANP secretion via the PI3K/AKT/mTOR signaling pathway. Piezo 1 and cathepsin K may be involved in the liraglutide mechanism of reduction.

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.

This retrospective case-control study examined the relationship between the serum and synovial levels of cathepsin G (CatG) and cathepsin K (CatK) in patients with psoriatic arthritis (PsA) and their association with disease activity. Methods: This case-control study involved 156 PsA patients, 50 patients with gonarthrosis (GoA), and 30 healthy controls. The target parameters were measured using enzyme-linked immunosorbent assay (ELISA) kits. The serum levels of CatG and CatK were found to be significantly higher in PsA patients compared to both control groups (p < 0.001). Moreover, they could distinguish PsA patients from healthy controls with 100% accuracy. Synovial fluid CatG and CatK were positively associated with the following indicators of disease activity: the VAS (rs = 0.362, rs = 0.391); the DAPSA (rs = 0.191, rs = 0.182); and the mCPDAI (rs = 0.378, rs = 0.313). Our results suggest that serum and synovial fluid CatG and CatK levels could serve as biomarkers for PsA. In PsA patients with synovial fluid crystals, elevated synovial CatG levels demonstrated a sensitivity of 89.54% and a specificity of 86.00% in distinguishing them from PsA patients without crystals. Similarly, elevated synovial CatK levels had a sensitivity of 93.67% and a specificity of 94.34% for distinguishing PsA patients with synovial fluid crystals from those without. Furthermore, the synovial fluid levels of both CatG and CatK showed positive associations with key indicators of disease activity, including the visual analog scale (VAS) (rs = 0.362, rs = 0.391), the disease activity in psoriatic arthritis (DAPSA) (rs = 0.191, rs = 0.182), and the modified composite psoriatic disease activity index (mCPDAI) (rs = 0.378, rs = 0.313). In conclusion, our findings suggest that the serum and synovial fluid levels of CatG and CatK hold promise as potential biomarkers for assessing disease activity in psoriatic arthritis. [ABSTRACT FROM AUTHOR]

Clear cell tumors of the lung (CCTL), or “sugar tumors” of lung, are very uncommon lesions and are mostly benign perivascular epithelioid cell (PEC) tumors with no specific morphologic features. Fewer than 100 cases have been reported; the aggressive nature demonstrated in sporadic reports has rarely been described in the literature. Although the course is generally described as benign, eight reported cases showed malignant behavior. We report a case of a PEC with a malignant presentation in a young man, correlating the main characteristics of the tumor with other cases reported in the literature to better elucidate this rare presentation. We also performed a literature review of reports on benign and malignant CCTL cases, with a focus on clinical, imaging, and immunohistochemical differentiation. CCTLs are rare tumors that require histopathological and immunohistochemical confirmation; to date, criteria that can predict malignant evolution are lacking. [ABSTRACT FROM AUTHOR]

We describe the design and synthesis of OFS-1, an Osteoadsorptive Fluorogenic Sentinel imaging probe that is adsorbed by hydroxyapatite (HAp) and bone mineral surfaces, where it generates an external fluorescent signal in response to osteoclast-secreted cathepsin K (Ctsk). The probe consists of a bone-anchoring bisphosphonate moiety connected to a Förster resonance energy transfer (FRET) internally quenched fluorescent (IQF) dye pair, linked by a Ctsk peptide substrate, GHPGGPQG. Key structural features contributing to the effectiveness of OFS-1 were defined by structure-activity relationship (SAR) and modeling studies comparing OFS-1 with two cognates, OFS-2 and OFS-3. In solution or when preadsorbed on HAp, OFS-1 exhibited strong fluorescence when exposed to Ctsk (2.5-20 nM). Time-lapse photomicrographs obtained after seeding human osteoclasts onto HAp-coated well plates containing preadsorbed OFS-1 revealed bright fluorescence at the periphery of resorbing cells. OFS-1 administered systemically detected early osteolysis colocalized with orthotopic engraftment of RPMI-8226-Luc human multiple myeloma cells at a metastatic skeletal site in a humanized mouse model. OFS-1 is thus a promising new imaging tool for detecting abnormal bone resorption.

Abstract Background Salivary gland carcinomas (SGCs) represent various groups of tumors that demonstrate marked diversity in their prognosis owing to different histology and clinical characteristics. One of the poor prognostic indicators is distant metastasis which is considered the major reason for death in SGC patients. Discovering new biomarkers is urgently required to aid in the detection of cancer onset and progression. Cathepsin K (CTSK), the lysosomal cysteine protease has a principal role in cancer invasion and progression through interaction with the tumor microenvironment, degradation of extracellular membrane proteins and destruction of the elastic lamina of blood vessels. In the English literature, little information was present about the role of CTSK in SGCs. The current study aimed to assess the immunohistochemical expression of CTSK in SGCs and correlate its expression to different clinicopathologic parameters. Methods The retrospective study applied to 45 cases of SGCs categorized as high-grade (33 cases) and low-grade SGCs (12 cases) following the criteria of WHO classification (2017) of head and neck tumors. All patients` clinicopathological and follow-up records were retrieved. The following statistical tests were used to study the variance of CTSK expression in SGCs concerning different clinicopathological parameters; Pearson`s Chi-square test, unpaired two-tailed student t-test, One-way ANOVA, and Post Hoc tests. Disease-free survival (DFS) and Overall survival (OS) were calculated and displayed with the Kaplan–Meier strategy and analyzed with the log-rank test. Univariate and multivariate survival analyses were performed with Cox regression. A P-value lesser than 0.05 was considered statistically significant. Results Strong CTSK expression was significantly related to high-grade SGCs (P = 0.000), large infiltrating carcinomas (P = 0.000), presence of nodal (P = 0.041) and distant metastasis (P = 0.009), advanced TNM clinical stage (P = 0.000), the incidence of recurrence (P = 0.009), and reduced DFS (P = 0.006). Distant metastasis was the independent predictor for DFS using Cox regression model. Conclusions CTSK has a great role in cancer progression by triggering many signaling pathways. Its level in cancerous tissue is considered an effective index for predicting the severity and prognosis of cancer. Therefore, we indicate its utility as a prognostic tool and therapeutic target for cancer treatment. Trial registration Retrospectively registered.

Osteoclasts, bone-specified multinucleated cells produced by monocyte/macrophage, are involved in numerous bone destructive diseases such as arthritis, osteoporosis, and inflammation-induced bone loss. The osteoclast differentiation mechanism suggests a possible strategy to treat bone diseases. In this regard, we recently examined the in vivo impact of kalkitoxin (KT), a marine product obtained from the marine cyanobacterium Moorena producens (previously Lyngbya majuscula), on the macrophage colony-stimulating factor (M-CSF) and on the receptor activator of nuclear factor κB ligand (RANKL)-stimulated in vitro osteoclastogenesis and inflammation-mediated bone loss. We have now examined the molecular mechanism of KT in greater detail. KT decreased RANKL-induced bone marrow-derived macrophages (BMMs) tartrate-resistant acid phosphatase (TRAP)-multinucleated cells at a late stage. Likewise, KT suppressed RANKL-induced pit area and actin ring formation in BMM cells. Additionally, KT inhibited several RANKL-induced genes such as cathepsin K, matrix metalloproteinase (MMP-9), TRAP, and dendritic cell-specific transmembrane protein (DC-STAMP). In line with these results, RANKL stimulated both genes and protein expression of c-Fos and nuclear factor of activated T cells (NFATc1), and this was also suppressed by KT. Moreover, KT markedly decreased RANKL-induced p-ERK1/2 and p-JNK pathways at different time points. As a result, KT prevented inflammatory bone loss in mice, such as bone mineral density (BMD) and osteoclast differentiation markers. These experiments demonstrated that KT markedly inhibited osteoclast formation and inflammatory bone loss through NFATc1 and mitogen-activated protein kinase (MAPK) signaling pathways. Therefore, KT may have potential as a treatment for destructive bone diseases.

Atherosclerosis is a chronic inflammatory disease, occurring preferentially in bifurcation, branching, and bending of blood vessels exposed to disturbed flow. Disturbed flow in atheroprone areas activates elevated proteases, degrading elastin lamellae and collagenous matrix, resulting in endothelial dysfunction and vascular remodeling. As a mediator for extracellular matrix protein degradation, cathepsin K (CTSK) was directly regulated by hemodynamics and contributed to atherosclerosis. The mechanism of CTSK responding to disturbed flow and contributing to disturbed flow-induced atherosclerosis is unclear. In this study, the partial carotid ligation model of mice and in vitro disturbed shear stress model were constructed to explore the contribution and potential mechanism of CTSK in atherosclerosis. Our results indicated that CTSK elevated in the disturbed flow area in vivo and in vitro along with endothelial inflammation and atherogenesis. Additionally, the expression of integrin αvβ3 was upregulated in these atheroprone areas. We found that inhibition of the integrin αvβ3-cytoskeleton pathway could significantly block the activation of NF-κB and the expression of CTSK. Collectively, our findings unraveled that disturbed flow induces increased CTSK expression, and contributes to endothelial inflammation and vascular remodeling, leading to atherogenesis eventually. This study is helpful to provide new enlightenment for the therapy of atherosclerosis.

Bone disorders having different causes, such as metabolic, genetic, and environmental, are well known and each of them has a characteristic clinical and radiographic appearance. Pycnodysostosis is one such rare bone disease that manifests as generalized osteosclerosis of the skeleton because of decreased bone turnover. Some of the unique radiographic and clinical features distinguish it from other bone disorders, such as osteogenesis imperfect, osteopetrosis, and cleidocranial dysplasia. Recognition of these signs is important to make a diagnosis and prevent further complications. We report a case of a 24-year-old male, who presented with a chief complaint of dirty and malaligned teeth and was unhappy with his facial appearance. General examination features which drew attention were short stature, beaking of the nose, and frontal and parietal bossing. Radiographically, open fontanelles, wormian bones, and dental abnormalities were evident.

Background: Glucocorticoid (GC) is one of frequently used anti-inflammatory agents, but its administration is unfortunately accompanied with bone loss. Although sporadic studies indicated that osteocytes are subject to a series of pathological changes under GC stress, including overexpression of cathepsin K, the definite role of osteocytes in GC-induced bone loss remains largely unclear. Methods: Gene expression of Ctsk and protein levels of cathepsin K were assessed in MLO-Y4 cell lines exposed to dexamethasone (Dex) of different time (0, 12, 24 hours) and dose (0, 10−8 and 10−6 M) courses by RT-qPCR and western blotting, respectively. Confocal imaging and immunostaining were then performed to evaluate the effects of osteocyte-derived cathepsin K on type I collagen in a primary osteocyte ex vivo culture system. MitoTracker Red was used to stain mitochondria for mitochondria morphology assessment and JC-1 assay was employed to evaluate the mitochondria membrane potential in MLO-Y4 cells following Dex treatment. Activation of PINK1-mediated mitophagy was evaluated by immunostaining of the PINK1 protein and CytoID assay. Mdivi-1 was used to inhibit mitophagy and siRNAs were used for the inhibition of Pink1 and Atg5. Results: GC triggered osteocytes to produce excessive cathepsin K which in turn led to the degradation of type I collagen in the extracellular matrix in a primary osteocyte ex vivo culture system. Meanwhile, GC administration increased mitochondrial fission and membrane depolarization in osteocytes. Further, the activation of PINK1-mediated mitophagy was demonstrated to be responsible for the diminishment of dysfunctional mitochondria in osteocytes. Examination of relationship between mitophagy and cathepsin K production revealed that inhibition of mitophagy via knocking down Pink1 gene abolished the GC-triggered cathepsin K production. Interestingly, GC’s activation effect towards cathepsin K via mitophagy was found to be independent on the canonical autophagy as this effect was not impeded when inhibiting the canonical autophagy via Atg5 suppression. Conclusion: GC-induced PINK1-mediated mitophagy substantially modulates the production of cathepsin K in osteocytes, which could be an underlying mechanism by which osteocytes contribute to the extracellular matrix degradation during bone loss. The Translational potential of this article: Findings of the current study indicate a possible role of osteocyte mitophagy in GC-induced bone loss, which provides a potential therapeutic approach to alleviate GC-induced osteoporosis by targeting PINK1-mediated osteocytic mitophagy.

Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-β-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.

AimsTissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the zinc-dependent matrix metalloproteinases (MMP) and A disintegrin and metalloproteinases (ADAM) involved in extracellular matrix modulation. The present study aims to develop the TIMPs as biologics for osteoclast-related disorders.MethodsWe examine the inhibitory effect of a high affinity, glycosyl-phosphatidylinositol-anchored TIMP variant named ‘T1PrαTACE’ on receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced osteoclast differentiation.ResultsOsteoclast progenitor cells transduced with T1PrαTACE failed to form tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts or exhibit bone-resorbing activity following treatment with RANKL. At the messenger RNA level, T1PrαTACE strongly attenuated expression of key osteoclast marker genes that included TRAP, cathepsin K, osteoclast stimulatory transmembrane protein (OC-STAMP), dendritic cell-specific transmembrane protein (DC-STAMP), osteoclast-associated receptor (OSCAR), and ATPase H+-transporting V0 subunit d2 (ATP6V0D2) by blocking autoamplification of nuclear factor of activated T cells 1 (NFATc1), the osteoclastogenic transcription factor. T1PrαTACE selectively extended p44/42 mitogen-activated protein kinase activation, an action that may have interrupted terminal differentiation of osteoclasts. Inhibition studies with broad-spectrum hydroxamate inhibitors confirmed that the anti-resorptive activity of T1PrαTACE was not reliant on its metalloproteinase-inhibitory activity.ConclusionT1PrαTACE disrupts the RANKL-NFATc1 signalling pathway, which leads to osteoclast dysfunction. As a novel candidate in the prevention of osteoclastogenesis, the TIMP could potentially be developed for the treatment of osteoclast-related disorders such as osteoporosis.Cite this article: Bone Joint Res 2022;11(11):763–776.

Background: Salivary gland carcinomas (SGCs) represent various groups of tumors that demonstrate marked diversity in their prognosis owing to different histology and clinical characteristics. One of the poor prognostic indicators is distant metastasis which is considered the major reason for death in SGC patients. Discovering new biomarkers is urgently required to aid in the detection of cancer onset and progression. Cathepsin K (CTSK), the lysosomal cysteine protease has a principal role in cancer invasion and progression through interaction with the tumor microenvironment, degradation of extracellular membrane proteins and destruction of the elastic lamina of blood vessels. In the English literature, little information was present about the role of CTSK in SGCs. The current study aimed to assess the immunohistochemical expression of CTSK in SGCs and correlate its expression to different clinicopathologic parameters. Methods: The retrospective study applied to 45 cases of SGCs categorized as high-grade (33 cases) and low-grade SGCs (12 cases) following the criteria of WHO classification (2017) of head and neck tumors. All patients' clinicopathological and follow-up records were retrieved. The following statistical tests were used to study the variance of CTSK expression in SGCs concerning different clinicopathological parameters; Pearson's Chi-square test, unpaired two-tailed student t-test, One-way ANOVA, and Post Hoc tests. Disease-free survival (DFS) and Overall survival (OS) were calculated and displayed with the Kaplan–Meier strategy and analyzed with the log-rank test. Univariate and multivariate survival analyses were performed with Cox regression. A P-value lesser than 0.05 was considered statistically significant. Results: Strong CTSK expression was significantly related to high-grade SGCs (P = 0.000), large infiltrating carcinomas (P = 0.000), presence of nodal (P = 0.041) and distant metastasis (P = 0.009), advanced TNM clinical stage (P = 0.000), the incidence of recurrence (P = 0.009), and reduced DFS (P = 0.006). Distant metastasis was the independent predictor for DFS using Cox regression model. Conclusions: CTSK has a great role in cancer progression by triggering many signaling pathways. Its level in cancerous tissue is considered an effective index for predicting the severity and prognosis of cancer. Therefore, we indicate its utility as a prognostic tool and therapeutic target for cancer treatment. Trial registration: Retrospectively registered. [ABSTRACT FROM AUTHOR]

Background: Cathepsin K, which is involved in bone resorption, is a good target for treating osteoporosis, but no clinically approved medicine has been developed. Recently, allosteric inhibitors with high specificity and few side effects have been attracting attention for use in new medicines. Methods: Cathepsin K inhibitors were isolated from the methanol extract of Chamaecrista nomame (Leguminosae) using cathepsin K inhibition activity-assisted multi-step chromatography. Standard kinetic analysis was employed to examine the mechanism of cathepsin K inhibition when an isolated inhibitor and its derivative were used. The allosteric binding of these cathepsin K inhibitors was supported by a docking study using AutoDock vina. Combinations of allosteric cathepsin K inhibitors expected to bind to different allosteric sites were examined by means of cathepsin K inhibition assay. Results: Two types of cathepsin K inhibitors were identified in the methanol extract of Chamaecrista nomame. One type consisted of cassiaoccidentalin B and torachrysone 8-β-gentiobioside, and inhibited both cathepsin K and B with similar inhibitory potential, while the other type of inhibitor consisted of pheophytin a, and inhibited cathepsin K but not cathepsin B, suggesting that pheophytin a binds to an allosteric site of cathepsin K. Kinetic analysis of inhibitory activity suggested that pheophytin a and its derivative, pheophorbide b, bind allosterically to cathepsin K. This possibility was supported by a docking study on cathepsin K. The cathepsin K inhibitory activity of pheophytin a and pheophorbide b was enhanced by combining them with the allosteric inhibitors NSC 13345 and NSC94914, which bind to other allosteric sites on cathepsin K. Conclusions: Different allosteric inhibitors that bind to different sites in combination, as shown in this study, may be useful for designing new allosteric inhibitory drugs with high specificity and few side effects. [ABSTRACT FROM AUTHOR]

Simple Summary: Osteoporosis, an age-related disease, develops in part because the rate of new bone formation does not catch up with the increased bone destruction caused by osteoclasts. A full understanding of how osteoclasts function is essential to identify novel drug targets for osteoporosis treatment. Previously, we demonstrated that mice lacking leucine rich repeat kinase 1 (LRRK1) had high bone mass due to defective resorption caused by dysfunctional osteoclasts. Little is known about how LRRK1 regulates osteoclast activity. Here, we found that LRRK1-deficient osteoclasts had an altered distribution of the acidic vacuoles/lysosomes, unlike the wild-type osteoclast. The lysosomes remained in the cytoplasm away from the extracellular lacunae of the bone. The apparent F-actin ring was large, but the sealing zone was weak. Albeit CTSK and v-ATPase were comparably expressed in LRRK1 deficient osteoclasts, CTSK and v-ATPase, two lysosome-associated proteins, were not stationed on the ruffled border. Our data indicate the LRRK1 positively controls osteoclast activity via regulating lysosomal distribution, acid secretion, and protease exocytosis. We previously demonstrated that mice with targeted deletion of the leucine repeat rich kinase 1 (Lrrk1) gene were osteopetrotic due to the failure of osteoclasts to resorb bone. To determine how LRRK1 regulates osteoclast activity, we examined the intracellular and extracellular acidification with an acidotropic probe, acridine orange, in live osteoclasts on bone slices. We examined lysosome distribution in osteoclasts by localization of LAMP-2, cathepsin K, and v-ATPase by immunofluorescent staining with specific antibodies. We found that both vertical and horizontal cross-sectional images of the wild-type (WT) osteoclasts showed orange-staining of the intracellular acidic vacuoles/lysosomes dispersed to the ruffled border. By contrast, the LRRK1 deficient osteoclasts exhibited fluorescent orange staining in the cytoplasm away from the extracellular lacunae because of an altered distribution of the acidic vacuoles/lysosomes. In addition, WT osteoclasts displayed a peripheral distribution of LAMP-2 positive lysosomes with a typical actin ring. The clustered F-actin constitutes a peripheral sealing zone and a ruffled border which was stretched out into a resorption pit. The LAMP-2 positive lysosomes were also distributed to the sealing zone, and the cell was associated with a resorption pit. By contrast, LRRK1-deficient osteoclasts showed diffused F-actin throughout the cytoplasm. The sealing zone was weak and not associated with a resorption pit. LAMP-2 positive lysosomes were also diffuse in the cytoplasm and were not distributed to the ruffled border. Although the LRRK1-deficient osteoclast expressed normal levels of cathepsin K and v-ATPase, the lysosomal-associated cathepsin K and v-ATPase were not accumulated at the ruffled border in Lrrk1 KO osteoclasts. Our data indicate that LRRK1 controls osteoclast activity by regulating lysosomal distribution, acid secretion, and protease exocytosis. [ABSTRACT FROM AUTHOR]

Clear cell tumors of the lung (CCTL), or “sugar tumors” of lung, are very uncommon lesions and are mostly benign perivascular epithelioid cell (PEC) tumors with no specific morphologic features. Fewer than 100 cases have been reported; the aggressive nature demonstrated in sporadic reports has rarely been described in the literature. Although the course is generally described as benign, eight reported cases showed malignant behavior. We report a case of a PEC with a malignant presentation in a young man, correlating the main characteristics of the tumor with other cases reported in the literature to better elucidate this rare presentation. We also performed a literature review of reports on benign and malignant CCTL cases, with a focus on clinical, imaging, and immunohistochemical differentiation. CCTLs are rare tumors that require histopathological and immunohistochemical confirmation; to date, criteria that can predict malignant evolution are lacking.

Background: CD200-CD200R plays a critical role in regulating the human tumor microenvironment, but its role in cervical cancer remains unclear. Methods: A total of 62 paraffin blocks of tumor tissues were collected from cervical cancer patients. Expression of CD200 and cathepsin K (CTSK) in cancer tissues and para-cancerous tissues was analyzed by immunohistochemistry. Stably transfected CD200 cells were established in HeLa and SiHa cells. Human THP-1 monocytes were induced to differentiate into M2 macrophages. HeLa and SiHa cells were cultured in conditioned medium from M2 macrophages to observe the effects of CD200-CD200R on invasion, CTSK, p65NF-κB, and cisplatin or paclitaxel sensitivity in cervical cancer cells. HeLa cells were injected to induce xenograft tumors in mice, and a CTSK inhibitor, MK-0822, was used to confirm the regulation of CTSK and paclitaxel sensitivity by CD200-CD200R in vivo. Results: A significant decrease in CD200 and CTSK expression was found in tumor cancer tissues compared with para-cancerous tissues. Only CD200 overexpression did not affect cervical cell invasion, but CD200-CD200R could enhance the cell invasion and resistance to cisplatin or paclitaxel. Meanwhile, expression of CTSK and p-p65NF-κB in cancer cells stably transfected with CD200 was obviously increased after culture in conditioned medium from M2 macrophages compared with transfection with the plasmid control. In vivo, CTSK inhibition significantly suppressed the effects of CD200-CD200R overexpression on the response to paclitaxel by suppressing the CTSK-mediated NF-κB pathway. Conclusions: CD200-CD200R regulates CTSK-mediated NF-κB pathway to affect cisplatin or paclitaxel sensitivity in cervical cancer, which provides a possible immunotherapeutic target and combination strategy for advanced cervical cancer.

Purpose of reviewIn perilacunar/canalicular remodeling (PLR), osteocytes dynamically resorb, and then replace, the organic and mineral components of the pericellular extracellular matrix. Given the enormous surface area of the osteocyte lacuna-canalicular network (LCN), PLR is important for maintaining homeostasis of the skeleton. The goal of this review is to examine the motivations and critical considerations for the analysis of PLR, in both in vitro and in vivo systems.Recent findingsMorphological approaches alone are insufficient to elucidate the complex mechanisms regulating PLR in the healthy skeleton and in disease. Understanding the role and regulation of PLR will require the incorporation of standardized PLR outcomes as a routine part of skeletal phenotyping, as well as the development of improved molecular and cellular outcomes. Current PLR outcomes assess PLR enzyme expression, the LCN, and bone matrix composition and organization, among others. Here, we discuss current PLR outcomes and how they have been applied to study PLR induction and suppression in vitro and in vivo. Given the role of PLR in skeletal health and disease, integrated analysis of PLR has potential to elucidate new mechanisms by which osteocytes participate in skeletal health and disease.

Cancer, a common malignant disease, is one of the predominant causes of diseases that lead to death. Additionally, cancer is often detected in advanced stages and cannot be radically cured. Consequently, there is an urgent need for reliable and easily detectable markers to identify and monitor cancer onset and progression as early as possible. Our aim was to systematically review the relevant roles of cathepsin K (CTSK) in various possible cancers in existing studies. CTSK, a well-known key enzyme in the bone resorption process and most studied for its roles in the effective degradation of the bone extracellular matrix, is expressed in various organs. Nowadays, CTSK has been involved in various cancers such as prostate cancer, breast cancer, bone cancer, renal carcinoma, lung cancer and other cancers. In addition, CTSK can promote tumor cells proliferation, invasion and migration, and its mechanism may be related to RANK/RANKL, TGF-β, mTOR and the Wnt/β-catenin signaling pathway. Clinically, some progress has been made with the use of cathepsin K inhibitors in the treatment of certain cancers. This paper reviewed our current understanding of the possible roles of CTSK in various cancers and discussed its potential as a biomarker and/or novel molecular target for various cancers.

Objective To investigate the role of cathepsin K (CatK) in cardiac dysfunction in alcoholic cardiomyopathy (ACM) mice and its damage mechanism. Methods Forty-eight C57BL/6J male mice (8 weeks old) were randomly divided into control group (n=12, fed with isocaloric maltodextrin control liquid diet) and model group (n=36, fed with 4% ethanol Lieber-DeCarli liquid diet). After 8 weeks, the mice in the model group identified with decreased cardiac systolic function by echocardiography were regarded as ACM mice, and they were randomly divided into ACM group, sh-NC group, and sh-CatK group, with 12 mice in each group. The mice in the sh-NC group and the sh-CatK group were injected with 1×109 vg (100 μL) of recombinant adenoviruses containing scramble-shRNA or CatK-shRNA via tail vein, respectively. At the end of the 12th week, cardiac function was measured by M-mode echocardiography. The expression of CatK and Nox4 was detected by immunohistochemical assay. In in vitro study, H9c2 cardiomyocytes were used to evaluate the effect of CatK silencing on alcohol-induced cardiomyocyte injury. Western blotting was used to analyze the protein expression of AMPK/Nox4 signaling pathway. Flow cytometry was employed to analyze the apoptosis of H9c2 cells. The levels of reactive oxygen species in mouse myocardium and H9c2 cells cultured were measured using fluorescent dye dihydroethidium. Results The surface area of myocardial cells, area of myocardial fibrosis, protein levels of CatK and Nox4 and production of superoxide were significantly increased in the ACM group compared with the control group (P < 0.05), and those indicators in the sh-CatK group were significantly decreased than the ACM group (P < 0.05). Sh-CatK transfection significantly reversed the above changes in ACM mice (P < 0.05). In vitro results showed that the expression of CatK protein in H9c2 cells was increased with the prolonged exposure to ethanol (P < 0.05). CatK inhibition significantly reduced the apoptostic rate and oxidative stress levels (P < 0.05), and up-regulated AMPK phosphorylation in H9c2 cells (P < 0.05), resulting in decreased expression of Nox4 protein, collagen COL-1 and TGF-β (P < 0.05). Conclusion CatK is involved in the pathological process of cardiac dysfunction in the ACM mice, and its mechanism might be related to the promotion of oxidative stress mediated by the AMPK/Nox4 signaling pathway.

Abstract Objectives To evaluate the effects of matrix metalloproteinase (MMP) and cathepsin K (catK) inhibitors on resistance to dentin erosion. Methodology A total of 96 dentin specimens (3×3×2 mm) were prepared and randomly assigned into four groups (n=24): deionized water (DW); 1 µM odanacatib (ODN, catK inhibitor); 1 mM 1,10-phenanthroline (PHEN, MMP inhibitor); and 1 µM odanacatib + 1 mM 1,10-phenanthroline (COM). Each group was further divided into two subgroups for the application of treatment solutions before (PRE) and after erosive challenges (POST). All specimens were subjected to four daily erosive challenges for 5 d. For each erosive challenge, the specimens in subgroup PRE were immersed in the respective solutions before cola drinks, while the specimens in subgroup POST were immersed in the respective solutions after cola drinks (the immersion duration was 5 min in both cases). All specimens were stored in artificial saliva at 37°C between erosive challenges. The erosive dentin loss (EDL) was measured by profilometry. The residual demineralized organic matrix (DOM) of specimens was removed using type VII collagenase and evaluated by profilometry. Both the EDL and thickness of the residual DOM were statistically analyzed by two-way analysis of variance (ANOVA) and Bonferroni’s test (α=0.05). The surface topography and transverse sections of the specimens were observed using SEM. MMPs and catK were immunolabeled in the eroded dentin and in situ zymography was performed to evaluate the enzyme activity. Results Significantly lower EDL was found in the groups ODN, PHEN, and COM than in the control group (all p0.05). The application sequence showed no significant effect on the EDL of the tested groups (p=0.310). A significantly thicker DOM was observed in the group ODN than in the control group regardless of the application sequence (both p

The Cre/lox system is a fundamental tool for functional genomic studies, and a number of Cre lines have been generated to target genes of interest spatially and temporally in defined cells or tissues; this approach has greatly expanded our knowledge of gene functions. However, the limitations of this system have recently been recognized, and we must address the challenge of so‐called nonspecific/off‐target effects when a Cre line is utilized to investigate a gene of interest. For example, cathepsin K (Ctsk) has been used as a specific osteoclast marker, and Cre driven by its promoter is widely utilized for osteoclast investigations. However, Ctsk‐Cre expression has recently been identified in other cell types, such as osteocytes, periosteal stem cells, and tenocytes. To better understand Ctsk‐Cre expression and ensure appropriate use of this Cre line, we performed a comprehensive analysis of Ctsk‐Cre expression at the single‐cell level in major organs and tissues using two green fluorescent protein (GFP) reporters (ROSA nT‐nG and ROSA tdT) and a tissue clearing technique in young and aging mice. The expression profile was further verified by immunofluorescence staining and droplet digital RT‐PCR. The results demonstrate that Ctsk‐Cre is expressed not only in osteoclasts but also at various levels in osteoblast lineage cells and other major organs/tissues, particularly in the brain, kidney, pancreas, and blood vessels. Furthermore, Ctsk‐Cre expression increases markedly in the bone marrow, skeletal muscle, and intervertebral discs in aging mice. These data will be valuable for accurately interpreting data obtained from in vivo studies using Ctsk‐Cre mice to avoid potentially misleading conclusions. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Abstract Background Cachexia is a complicated metabolic disorder that is characterize by progressive atrophy of skeletal muscle. Cathepsin K (CTSK) is a widely expressed cysteine protease that has garnered attention because of its enzymatic and non‐enzymatic functions in signalling in various pathological conditions. Here, we examined whether CTSK participates in cancer‐induced skeletal muscle loss and dysfunction, focusing on protein metabolic imbalance. Methods Male 9‐week‐old wild‐type (CTSK+/+, n = 10) and CTSK‐knockout (CTSK−/−, n = 10) mice were injected subcutaneously with Lewis lung carcinoma cells (LLC; 5 × 105) or saline, respectively. The mice were then subjected to muscle mass and muscle function measurements. HE staining, immunostaining, quantitative polymerase chain reaction, enzyme‐linked immunosorbent assay, and western blotting were used to explore the CTSK expression and IRS1/Akt pathway in the gastrocnemius muscle at various time points. In vitro measurements included CTSK expression, IRS1/Akt pathway‐related target molecule expressions, and the diameter of C2C12 myotubes with or without LLC‐conditioned medium (LCM). An IRS1 ubiquitin assay, and truncation, co‐immunoprecipitation, and co‐localization experiments were also performed. Results CTSK+/+ cachectic animals exhibited loss of skeletal muscle mass (muscle weight loss of 15%, n = 10, P 15%, n = 10, P 30%, n = 5, P

This retrospective case-control study examined the relationship between the serum and synovial levels of cathepsin G (CatG) and cathepsin K (CatK) in patients with psoriatic arthritis (PsA) and their association with disease activity. Methods: This case-control study involved 156 PsA patients, 50 patients with gonarthrosis (GoA), and 30 healthy controls. The target parameters were measured using enzyme-linked immunosorbent assay (ELISA) kits. The serum levels of CatG and CatK were found to be significantly higher in PsA patients compared to both control groups (p < 0.001). Moreover, they could distinguish PsA patients from healthy controls with 100% accuracy. Synovial fluid CatG and CatK were positively associated with the following indicators of disease activity: the VAS (rs = 0.362, rs = 0.391); the DAPSA (rs = 0.191, rs = 0.182); and the mCPDAI (rs = 0.378, rs = 0.313). Our results suggest that serum and synovial fluid CatG and CatK levels could serve as biomarkers for PsA. In PsA patients with synovial fluid crystals, elevated synovial CatG levels demonstrated a sensitivity of 89.54% and a specificity of 86.00% in distinguishing them from PsA patients without crystals. Similarly, elevated synovial CatK levels had a sensitivity of 93.67% and a specificity of 94.34% for distinguishing PsA patients with synovial fluid crystals from those without. Furthermore, the synovial fluid levels of both CatG and CatK showed positive associations with key indicators of disease activity, including the visual analog scale (VAS) (rs = 0.362, rs = 0.391), the disease activity in psoriatic arthritis (DAPSA) (rs = 0.191, rs = 0.182), and the modified composite psoriatic disease activity index (mCPDAI) (rs = 0.378, rs = 0.313). In conclusion, our findings suggest that the serum and synovial fluid levels of CatG and CatK hold promise as potential biomarkers for assessing disease activity in psoriatic arthritis.

ABSTRACT The Cre/lox system is a fundamental tool for functional genomic studies, and a number of Cre lines have been generated to target genes of interest spatially and temporally in defined cells or tissues; this approach has greatly expanded our knowledge of gene functions. However, the limitations of this system have recently been recognized, and we must address the challenge of so‐called nonspecific/off‐target effects when a Cre line is utilized to investigate a gene of interest. For example, cathepsin K (Ctsk) has been used as a specific osteoclast marker, and Cre driven by its promoter is widely utilized for osteoclast investigations. However, Ctsk‐Cre expression has recently been identified in other cell types, such as osteocytes, periosteal stem cells, and tenocytes. To better understand Ctsk‐Cre expression and ensure appropriate use of this Cre line, we performed a comprehensive analysis of Ctsk‐Cre expression at the single‐cell level in major organs and tissues using two green fluorescent protein (GFP) reporters (ROSA nT‐nG and ROSA tdT) and a tissue clearing technique in young and aging mice. The expression profile was further verified by immunofluorescence staining and droplet digital RT‐PCR. The results demonstrate that Ctsk‐Cre is expressed not only in osteoclasts but also at various levels in osteoblast lineage cells and other major organs/tissues, particularly in the brain, kidney, pancreas, and blood vessels. Furthermore, Ctsk‐Cre expression increases markedly in the bone marrow, skeletal muscle, and intervertebral discs in aging mice. These data will be valuable for accurately interpreting data obtained from in vivo studies using Ctsk‐Cre mice to avoid potentially misleading conclusions. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile GÜ1303 and a 3-cyano-3-aza-β-amino acid GÜ2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of GÜ2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs. [ABSTRACT FROM AUTHOR]

Cathepsin K (CatK) is a part of the family of cysteine proteases involved in many important processes, including the degradation activity of collagen 1 and elastin in bone resorption. Changes in levels of CatK are associated with various pathological conditions, primarily related to bone and cartilage degradation, such as pycnodysostosis (associated with CatK deficiency), osteoporosis, and osteoarthritis (associated with CatK overexpression). Recently, the increased secretion of CatK is being highly correlated to vascular inflammation, hypersensitivity pneumonitis, Wegener granulomatosis, berylliosis, tuberculosis, as well as with tumor progression. Due to the wide spectrum of diseases in which CatK is involved, the design and validation of active site-specific inhibitors has been a subject of keen interest in pharmaceutical companies in recent decades. In this review, we summarized the molecular background of CatK and its involvement in various diseases, as well as its clinical significance for diagnosis and therapy. [ABSTRACT FROM AUTHOR]

We describe a case in which a 41‐year‐old pregnant female with pycnodysostosis presented for elective cesarean section at week 37 + 5 of pregnancy. This is the first reported case of pycnodysostosis during pregnancy in Oman, to the author's best knowledge. [ABSTRACT FROM AUTHOR]

Igor Aleksander Bednarski,1 Magdalena Ci&aogon;&zdot;y&nacute;ska,2 Jacek Kabzi&nacute;ski,3 Ireneusz Majsterek,3 Dorota Sobolewska-Sztychny,1 Joanna Narbutt,1 Aleksandra Lesiak1 1Department of Dermatology, Pediatric Dermatology and Dermatological Oncology, Medical University of Lodz, Lodz, 91-347, Poland; 2Nicolaus Copernicus Multidisciplinary Centre for Oncology and Traumatology, Lodz, 93-513, Poland; 3Department of Chemistry and Clinical Biochemistry, Medical University of Lodz, Lodz, 90-136, PolandCorrespondence: Igor Aleksander BednarskiDepartment of Dermatology, Pediatric Dermatology and Dermatological Oncology, Medical University of Lodz, Kniaziewicza 1/5, Lodz, 91-347, PolandEmail igorbednarskiv@gmail.comIntroduction: Photoaging is a premature skin aging developing secondarily to the excessive exposure to ultraviolet radiation. Due to its complexity, an exact mechanism of photoaging has not been found yet; however, recent research has shown two new emerging players in this process – cathepsin K and progerin.Aim: To evaluate how different wavelengths of ultraviolet radiation (UVA, narrowband UVB and broadband UVB) influence cathepsin K and progerin protein and mRNA expression in dermal cultured fibroblasts.Materials and Methods: Primary human dermal fibroblasts (Detroit 551, ATCC CCL-110) were cultured and irradiated with UVA, narrowband UVB (UVBnb) and broadband UVB (UVBwb). Fibroblasts were irradiated with 2 protocols: single high-dose exposure to UVR with protein/mRNA extraction immediately after exposure, 24 h after exposure and 48 h after exposure, and repeated (0 h, 24 h and 48 h) low-dose exposure to UVR with protein/mRNA extraction 48 h after first exposure.Results: Single high doses of UVA, UVBwb and UVBnb resulted in decreased expression of cathepsin K and progerin protein/mRNA in all subsequent time points. Repeated exposure to low doses of UVA results in significant increase of progerin mRNA and significant decrease of progerin protein after 48 h, but repeated exposure to UVBwb and UVBnb resulted in decreased progerin mRNA and protein expression. Repeated exposure to UVA, UVBwb and UVBnb resulted in decreased cathepsin K protein and mRNA expression.Conclusion: The results suggest that there could be another progerin/cathepsin K regulatory pathway, which has not been described yet. Being contradictory with previous research, the influence of ultraviolet radiation on progerin and cathepsin K needs to be further elucidated.Keywords: progerin, cathepsin K, photoaging

Abstract We describe a case in which a 41‐year‐old pregnant female with pycnodysostosis presented for elective cesarean section at week 37 + 5 of pregnancy. This is the first reported case of pycnodysostosis during pregnancy in Oman, to the author's best knowledge.

The treatment of periodontitis can be very challenging due to its complex etiologies. A new pharmacologic strategy entitled “host-modulation therapy,” has been introduced to improve periodontal treatment outcomes. Supposedly, a multifunctional drug with the potential for bacterial infection prevention, host-response modulation and bone healing promotion would be a promising option for periodontitis therapy, but related studies remain substantially lacking. In this study, we successfully conjugated tetracycline with odanacatib (a selective inhibitor of cathepsin K) to construct a multifunctional drug (TC-ODN). We discovered that TC-ODN could promote macrophages polarizing toward anti-inflammatory phenotype and promote osteogenesis of PDLSCs under inflammatory microenvironment. In vivo, TC-ODN could be absorbed and distributed specifically to the bone after systemic administration, and accumulation of TC-ODN increased bone mineral density in ovariectomized rats. Importantly, periodontal administration of TC-ODN could successfully promote bone healing in periodontitis rats with alveolar bone loss. The findings in our study uncovered the excellent biocompatibility and multifunction of TC-ODN, including bone-targeted accumulation, immunoregulation, anti-inflammatory activity and promotion of bone healing, which might contribute to the clinical treatment of periodontitis.

Background: Cathepsin K (CTSK) is a protease that degrades type I collagen and extracellular matrix, thereby contributing to bone resorption and tumor invasion. Some pituitary adenomas (PAs) could invade the sphenoid sinus (SS) and cavernous sinus (CS). Purpose: This retrospective cohort study aimed to study the expression of tumoral biomarkers (CTSK, MMP9, MMP2, TIMP2, and PTTG1) and evaluate their clinical significance in non-functioning pituitary adenomas (NFPAs) with different invasion patterns. Methods: We assessed the expression levels of candidate invasion-specific protein biomarkers CTSK, MMP9, MMP2, TIMP2, and PTTG1 by immunohistochemical staining in paraffin-embedded NFPA tumor tissues. Variations in staining intensity were analyzed in cases with SS and CS invasion and non-invasive NFPAs. Results: We found that the levels of CTSK were higher in PA cases with SS invasion than that in PA cases with CS invasion (95.57 ± 31.57 vs. 65.29 ± 29.64, P < 0.001), and the expression of MMP9 and MMP2 was higher in CS-invasive cases than that in SS-invasive cases (145.02 ± 49.25 vs. 111.80 ± 51.37, P = 0.002, and 138.67 ± 52.06 vs. 108.30 ± 41.70, P = 0.002). Multiple Cox regression demonstrated that higher CTSK expression (P=0.011), subtotal resection (P<0.001), invasion (P=0.037), and larger tumor diameter (P=0.001) were independent risk factors for recurrence. A positive correlation was observed between CTSK expression and tumor size (r=0.671, p<0.001). There was no significant difference in TIMP2 and PTTG1 levels between CS-and SSFrontiers invasive cases (97.42± 39.80 vs. 102.10± 43.22, P = 0.58 and 13.89 ± 4.59 vs. 12.56 ± 3.96, P = 0.14). Conclusion: Our data indicated that CTSK has the potential as a marker for SS invasion of PAs, whereas MMP9 and MMP2 may be markers for CS invasion. And CTSK may play an important role in tumor relapse. [ABSTRACT FROM AUTHOR]

Cancer, a common malignant disease, is one of the predominant causes of diseases that lead to death. Additionally, cancer is often detected in advanced stages and cannot be radically cured. Consequently, there is an urgent need for reliable and easily detectable markers to identify and monitor cancer onset and progression as early as possible. Our aim was to systematically review the relevant roles of cathepsin K (CTSK) in various possible cancers in existing studies. CTSK, a well-known key enzyme in the bone resorption process and most studied for its roles in the effective degradation of the bone extracellular matrix, is expressed in various organs. Nowadays, CTSK has been involved in various cancers such as prostate cancer, breast cancer, bone cancer, renal carcinoma, lung cancer and other cancers. In addition, CTSK can promote tumor cells proliferation, invasion and migration, and its mechanism may be related to RANK/RANKL, TGF-β, mTOR and the Wnt/β-catenin signaling pathway. Clinically, some progress has been made with the use of cathepsin K inhibitors in the treatment of certain cancers. This paper reviewed our current understanding of the possible roles of CTSK in various cancers and discussed its potential as a biomarker and/or novel molecular target for various cancers. [ABSTRACT FROM AUTHOR]