Objective: To investigate the clinicopathological, immunohistochemical and molecular genetic characteristics of TFE3-rearranged perivascular epithelioid cell tumor (PEComa). Methods: Eight cases of PEComa with TFE3 rearrangement diagnosed in the First Affiliated Hospital of Air Force Medical University from January 2014 to July 2022 were collected. Three were consultation cases and 5 were collected from our hospital; 7 cases were resection specimens and 1 case was a needle biopsy specimen. Routine histolopathological analysis, immunohistochemical staining, fluorescence in situ hybridization (FISH) and the next-generation sequencing were performed. Clinical data were collected and the prognosis was assessed. Results: The 8 patients consisted of 5 females and 3 males with a median age of 45 years (ranged from 25 to 65 years). The tumor location included 1 uterus, 1 liver, 1 urachus, 2 kidneys, 1 abdominal cavity, 1 colon, and 1 retroperitoneum (3 subsequent recurrences in the abdominal cavity, pelvis and ovary, and abdominal cavity, respectively). Morphologically, the tumor cells were uniform and epithelioid with translucent or eosinophilic cytoplasm. They were arranged in nests or sheets, most of which were separated by thin-walled blood vessels. There were no papillary structures, and no overt smooth muscle or fat components. Atypical features were seen in 3 cases, with bizarre nuclei and tumor giant cells. Large areas of necrosis were visible, and mitosis was common (up to 28/50 HPF). Melanin deposition was present in 3 cases. Immunohistochemical staining showed diffuse and strong positivity for TFE3 in 8/8 cases and for HMB45 in 6/8 cases; focal positivity for Cathepsin K and Melan-A in 6/8 cases and for SMA in 2/8 of cases. All cases were negative for CKpan, PAX8 and Desmin. TFE3 gene break-apart was detected by FISH in all 8 cases, 4 of which underwent next-generation sequencing, and it revealed that 2 cases presented with SFPQ::TFE3 fusion, 1 case with ASPSCR1::TFE3 fusion, and 1 case with no chimeric fusion. Seven cases were followed up for 4-94 months. All cases were alive; 4 cases were disease-free, 2 cases showed recurrence, and 1 case had metastasis at initial diagnosis. Conclusions: TFE3-rearranged PEComa has unique histomorphological, immunohistochemical and molecular characteristics. The biological behavior is aggressive, which could lead to recurrence and metastasis, and warrants close clinical follow-up.
目的： 探讨TFE3重排的血管周上皮样细胞肿瘤（perivascular epithelioid cell tumor，PEComa）的临床病理特征、免疫表型、分子遗传学特征及预后。 方法： 收集空军军医大学第一附属医院2014年1月至2022年7月诊断的8例TFE3重排的PEComa，3例为会诊病例，5例为本院标本，其中7例为手术切除标本，1例为穿刺活检标本。对其进行常规组织学分析、免疫组织化学染色、荧光原位杂交（FISH）及二代测序检测，并进行预后随访。 结果： 8例患者中，5例为女性，3例为男性，年龄25~65岁（中位年龄45岁）。发病部位：子宫1例，肝脏1例，脐尿管1例，肾脏2例，腹腔1例，结肠1例，腹膜后1例（后3次复发，部位分别为腹腔、盆腔和卵巢、腹腔）。组织形态学：肿瘤细胞呈上皮样，大小形态较一致，胞质透亮或嗜酸，巢片状排列，大部分区域由纤细的薄壁血管分隔形成巢泡状，未见乳头状结构，无明显的平滑肌及脂肪成分；3例细胞异型性显著，可见奇异形核及瘤巨细胞并伴有大片坏死，核分裂象多见（最多约28个/50 HPF）。3例可见黑色素颗粒沉着。免疫组织化学染色结果：所有病例均弥漫一致强阳性表达TFE3，8例均表达HMB45（2例灶状阳性），6例（6/8）表达Cathepsin K和Melan A，2例（2/8）表达平滑肌肌动蛋白（均为灶状阳性），所有病例均不表达广谱细胞角蛋白、PAX8和结蛋白。8例FISH均检测到TFE3基因断裂，其中4例进行了二代测序，显示2例具有SFPQ：：TFE3融合，1例具有ASPSCR1：：TFE3融合，另1例未检出。7例随访到有效信息，随访时间4~94个月，4例无病生存，2例复发，1例初诊时已出现转移，所有患者均未死亡。 结论： TFE3重排的PEComa具有独特的组织形态学特征、免疫表型及分子特征，具有侵袭性的生物学行为，个别病例表现出高度恶性的生物学行为，可出现复发和转移，应提示临床进行密切随访。.

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.

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.

Human cathepsin K (CatK) stands out as a promising target for the treatment of osteoporosis, considering its role in degrading the bone matrix. Given the small and shallow S2 subsite of CatK and considering its preference for proline or hydroxyproline, we now propose the rigidification of the leucine fragment found at the P2 position in a dipeptidyl-based inhibitor, generating rigid proline-based analogs. Accordingly, with these new proline-based peptidomimetics inhibitors, we selectively inhibited CatK against other human cathepsins (B, L and S). Among these new ligands, the most active one exhibited a high affinity (pKi = 7.3 - 50.1 nM) for CatK and no inhibition over the other cathepsins. This specific inhibitor harbors two novel substituents never employed in other CatK inhibitors: the trifluoromethylpyrazole and the 4-methylproline at P3 and P2 positions. These results broaden and advance the path toward new potent and selective inhibitors for CatK.

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.

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).

OBJECTIVE: Airway epithelial cells (AECs) regulate the activation of epithelial-mesenchymal trophic units (EMTUs) during airway remodelling through secretion of signalling mediators. However, the major trigger and the intrinsic pathogenesis of airway remodelling is still obscure.
METHODS: The differing expressed genes in airway epithelia related to airway remodelling were screened and verified by RNA-sequencing and signalling pathway analysis. Then, the effects of increased cathepsin K (CTSK) in airway epithelia on airway remodelling and EMTU activation were identified both in vitro and in vivo, and the molecular mechanism was elucidated in the EMTU model. The potential of CTSK as an an effective biomarker of airway remodelling was analysed in an asthma cohort of differing severity. Finally, an inhibitor of CTSK was administered for potential therapeutic intervention for airway remodelling in asthma.
RESULTS: The expression of CTSK in airway epithelia increased significantly along with the development of airway remodelling in a house dust mite (HDM)-stressed asthma model. Increased secretion of CTSK from airway epithelia induced the activation of EMTUs by activation of the PAR2-mediated pathway. Blockade of CTSK inhibited EMTU activation and alleviated airway remodelling as an effective intervention target of airway remodelling.
CONCLUSIONS: Increased expression of CTSK in airway epithelia is involved in the development of airway remodelling in asthma through EMTU activation, mediated partly through the PAR2-mediated signalling pathway. CTSK is a potential biomarker for airway remodelling, and may also be a useful intervention target for airway remodelling in asthma patients.

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

N6-methyladenosine (m6A) modification, a eukaryotic messenger RNA modification catalyzed by methyltransferase-like 3 (METTL3), plays a pivotal role in stem cell fate determination. Calvarial bone development and maintenance are orchestrated by the cranial sutures. Cathepsin K (CTSK)-positive calvarial stem cells (CSCs) contribute to mice calvarial ossification. However, the role of m6A modification in regulating Ctsk+ lineage cells during calvarial development remains elusive. Here, we showed that METTL3 was colocalized with cranial nonosteoclastic Ctsk+ lineage cells, which were also associated with GLI1 expression. During neonatal development, depletion of Mettl3 in the Ctsk+ lineage cells delayed suture formation and decreased mineralization. During adulthood maintenance, loss of Mettl3 in the Ctsk+ lineage cells impaired calvarial bone formation, which was featured by the increased bone porosity, enhanced bone marrow cavity, and decreased number of osteocytes with the less-developed cellular outline. The analysis of methylated RNA immunoprecipitation sequencing and RNA sequencing data indicated that loss of METTL3 reduced Hedgehog (Hh) signaling pathway. Restoration of Hh signaling pathway by crossing Sufufl/+ alleles or by local administration of SAG21 partially rescued the abnormity. Our data indicate that METTL3 modulates Ctsk+ lineage cells supporting calvarial bone formation by regulating the Hh signaling pathway, providing new insights for clinical treatment of skull vault osseous diseases.

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