Role Of Cathepsin Research Articles

The role of cathepsin D in the pathophysiology of heart failure and its potentially beneficial properties: a translational approach.

AimsCathepsin D is a ubiquitous lysosomal protease that is primarily secreted due to oxidative stress. The role of circulating cathepsin D in heart failure (HF) is unknown. The aim of this study is to determine the association between circulating cathepsin D levels and clinical outcomes in patients with HF and to investigate the biological settings that induce the release of cathepsin D in HF.Methods and resultsCathepsin D levels were studied in 2174 patients with HF from the BIOSTAT‐CHF index study. Results were validated in 1700 HF patients from the BIOSTAT‐CHF validation cohort. The primary combined outcome was all‐cause mortality and/or HF hospitalizations. Human pluripotent stem cell‐derived cardiomyocytes were subjected to hypoxic, pro‐inflammatory signalling and stretch conditions. Additionally, cathepsin D expression was inhibited by targeted short hairpin RNAs (shRNA). Higher levels of cathepsin D were independently associated with diabetes mellitus, renal failure and higher levels of interleukin‐6 and N‐terminal pro‐B‐type natriuretic peptide (P < 0.001 for all). Cathepsin D levels were independently associated with the primary combined outcome [hazard ratio (HR) per standard deviation (SD): 1.12; 95% confidence interval (CI) 1.02–1.23], which was validated in an independent cohort (HR per SD: 1.23, 95% CI 1.09–1.40). In vitro experiments demonstrated that human stem cell‐derived cardiomyocytes released cathepsin D and troponin T in response to mechanical stretch. ShRNA‐mediated silencing of cathepsin D resulted in increased necrosis, abrogated autophagy, increased stress‐induced metabolism, and increased release of troponin T from human stem cell‐derived cardiomyocytes under stress.ConclusionsCirculating cathepsin D levels are associated with HF severity and poorer outcome, and reduced levels of cathepsin D may have detrimental effects with therapeutic potential in HF.

Lost or Forgotten: The nuclear cathepsin protein isoforms in cancer.

While research into the role of cathepsins has been progressing at an exponential pace over the years, research into their respective isoform proteins has been less frenetic. In view of the functional and biological potential of such protein isoforms in model systems for cancer during their initial discovery, much later they have offered a new direction in the field of cathepsin basic and applied research. Consequently, the analysis of such isoforms has laid strong foundations in revealing other important regulatory aspects of the cathepsin proteins in general. In this review article, we address these key aspects of cathepsin isoform proteins, with particular emphasis on how they have shaped what is now known in the context of nuclear cathepsin localization and what potential these hold as nuclear-based therapeutic targets in cancer.

A Novel Role for Cathepsin S as a Potential Biomarker in Triple Negative Breast Cancer.

Cathepsin S (CTSS) has previously been implicated in a number of cancer types, where it is associated with poor clinical features and outcome. To date, patient outcome in breast cancer has not been examined with respect to this protease. Here, we carried out immunohistochemical (IHC) staining of CTSS using a breast cancer tissue microarray in patients who received adjuvant therapy. We scored CTSS expression in the epithelial and stromal compartments and evaluated the association of CTSS expression with matched clinical outcome data. We observed differences in outcome based on CTSS expression, with stromal-derived CTSS expression correlating with a poor outcome and epithelial CTSS expression associated with an improved outcome. Further subtype characterisation revealed high epithelial CTSS expression in TNBC patients with improved outcome, which remained consistent across two independent TMA cohorts. Further in silico gene expression analysis, using both in-house and publicly available datasets, confirmed these observations and suggested high CTSS expression may also be beneficial to outcome in ER-/HER2+ cancer. Furthermore, high CTSS expression was associated with the BL1 Lehmann subgroup, which is characterised by defects in DNA damage repair pathways and correlates with improved outcome. Finally, analysis of matching IHC analysis reveals an increased M1 (tumour destructive) polarisation in macrophage in patients exhibiting high epithelial CTSS expression. In conclusion, our observations suggest epithelial CTSS expression may be prognostic of improved outcome in TNBC. Improved outcome observed with HER2+ at the gene expression level furthermore suggests CTSS may be prognostic of improved outcome in ER- cancers as a whole. Lastly, from the context of these patients receiving adjuvant therapy and as a result of its association with BL1 subgroup CTSS may be elevated in patients with defects in DNA damage repair pathways, indicating it may be predictive of tumour sensitivity to DNA damaging agents.

Dopamine promotes cathepsin B-mediated amyloid precursor protein degradation by reactive oxygen species-sensitive mechanism in neuronal cell.

Recent literature suggested an important function of native amyloid precursor protein (APP) as amine oxidase implicating in protection of brain cells from catecholamine-induced toxicity. However, any role of catecholamines on regulation of native APP has not been explored. Here we report that dopamine (DA), one of the most prominent catecholamine neurotransmitters in brain, down-modulates native APP protein in several neuronal cell types. Using SH-SY5Y cells as model, we detected no alteration of transcript expression and unaffected translation suggested that DA might induce APP degradation. We actually found that DA treatment decreased the stability of APP. Lysosomal blockers inhibited DA-induced APP degradation, but specific proteasomal blocker failed to do so. We detected the role of cathepsin B in DA-induced APP degradation by using pharmacological inhibitor and specific siRNA. We also revealed that DA could increase cathepsin B expression at both transcript and protein levels. Using antioxidant N-acetyl cysteine, we detected increased level of reactive oxygen species generation that was found responsible for induced cathepsin B expression by DA and resultant APP degradation. Our study reveals the existence of reciprocal regulation of a catecholamine and an amine oxidase implicating in brain catecholamine homeostasis.

Regulatory role of cathepsin D in degradation of advanced glycation end products by human dermal fibroblasts

Objective To investigate the regulatory role of cathepsin D (CatD) in the degradation of intracellular advanced glycation end products (AGEs) endocytosed by human dermal fibroblasts (HDFs) . Methods Cultured HDFs were treated with 1 μmol/L CA074Me (an inhibitor of CatB and CatL) , 75 μmol/L pepstatin A (an inhibitor of CatD) and 1 μmol/L MG-132 (an inhibitor of 20S proteasome) separately for 4 hours, and then cell counting kit 8 (CCK8) assay and fluorometric assay were performed to determine the cellular viability and protease activity, respectively. The cells in the CA074Me group, pepstatin A group and MG-132 group were additionally treated with AGE-bovine serum albumin (BSA) for 8 hours, and the cells in the blank control group were treated with phosphate-buffered saline (PBS) alone. After 8-hour cultivation, the cells in the above groups were subsequently reincubated with fresh culture medium containing the corresponding inhibitors for 24 hours. Then, flow cytometry was performed to assess the mean fluorescence intensity of intracellular AGE-BSA at different time points. Some other HDFs were treated with 37.5, 75 and 150 μmol/L pepstatin A and PBS separately for 4 hours, and then the cells in the 4 groups were treated with 200 mg/L AGE-BSA for 8 hours, followed by the removal of AGE-BSA from the medium and the treatment with 37.5, 75 and 150 μmol/L pepstatin A and PBS respectively. Enzyme-linked immunosorbent assay (ELISA) was conducted to measure the mean concentration of intracellular AGE-BSA at different time points, and the degradation rate of AGE was calculated. Some HDFs were divided into 3 groups: blank control group receiving no treatment, NC group transfected with an empty vector, and CatD group transfected with a CatD-overexpressing lentiviral vector. Fluorescence microscopy was conducted to estimate the transfection efficiency. Reverse transcription-PCR, Western blot analysis and fluorometric assay were performed to determine the mRNA and protein expression, and activity of CatD respectively. Then, the cells in the above 3 groups were incubated with AGE-BSA for 8 hours, followed by the removal of AGE-BSA from the medium and the treatment with fresh culture medium. The detection methods were same as the above experiment, and the degradation rate was calculated. Results The cellular proliferative activity in the 1-μmol/L CA074Me group, 75-μmol/L pepstatin A group and 1-μmol/L MG-132 group was more than 90%, and there was no significant difference between the 3 groups and the control group (100%, F=1.525, P > 0.05) . Twenty-four hours after the removal of AGE-BSA from the medium, the fluorescence intensities of intracellular AGE-BSA in the CA074Me+AGE-BSA group (275.00 ± 10.15) and MG-132+AGE-BSA group (259.00 ± 11.14) significantly decreased compared with those at the 8-hour time point (295.00 ± 6.56 and 285.67 ± 8.74 respectively; paired t test, t=4.778, 6.154 respectively, both P 0.05) . The degradation rates of intracellular AGE-BSA within 24 hours in the 37.5, 75 and 150 μmol/L pepstatin A groups were 9.64% ± 1.27%, 5.62% ± 0.47% and 3.21% ± 0.73% respectively; there were significant differences among the 3 groups (F=45.876, P 80%) of fluorescent cells. The mRNA and protein expression as well as the activity of CatD were significantly higher in the CatD group than in the blank control group and NC group (all P < 0.05) . The CatD+AGE-BSA group showed a significantly higher degradation rate of intracellular AGE-BSA within 24 hours compared with the AGE-BSA group and NC+AGE-BSA group (both P < 0.05) . Conclusion CatD can promote the degradation of intracellular AGE-BSA endocytosed by HDFs. Key words: Cathepsin D; Fibroblasts; Glycosylation end products, advanced; Degradation

Correlation analysis between the expression of cathepsin D and the accumulation of advanced glycation end products in skin tissues

Objective To determine the expression of cathepsin D and advanced glycation end products (AGEs) in skin tissues from patients of different ages or skin tissues with different degrees of sun exposure, to evaluate their correlation, and to preliminarily investigate the role of cathepsin D in the degradation and accumulation of AGEs in photoaged skin. Methods Skin tissues were collected from sun-exposed and sun-protected body sites in patients aged 15 - 20 years, 35 - 40 years, 55 - 60 years or 75 - 80 years. These skin tissues were divided into 8 groups according to age of patients and degrees of sun exposure, and there were 6 specimens in each group. Immunohistochemical and immunofluorescent methods were used to measure the expression of cathepsin D and AGEs in the skin tissues. Statistical analysis was carried out by factorial design analysis of variance, Wilcoxon rank sum test and Kruskal-Wallis rank sum test for analyzing associations of the expression of cathepsin D and AGEs with age and sun exposure, as well as by Pearson correlation analysis for assessing the correlation between cathepsin D expression and AGEs expression. Results Immunohistochemical study showed that the expression of cathepsin D markedly decreased along with the increase of age, but the accumulation of AGEs gradually increased along with the increase of age. In the same age group, the cathepsin D expression was lower in the sun-exposed skin tissues than in the sun-protected skin tissues, while the accumulation of AGEs was more in the sun-exposed skin tissues than in the sun-protected skin tissues. Factorial design analysis of variance showed that sun exposure could decrease the expression of cathepsin D (F = 58.70, P < 0.001) , but increase the accumulation of AGEs (F = 158.18, P < 0.001) . Moreover, the increase of age could lead to decreased expression of cathepsin D (F = 79.49, P < 0.001) , and increased expression of AGEs (F = 106.06, P < 0.001) . Compared with the sun-protected skin tissues, the sun-exposed skin tissues in all the age groups showed significantly lower absorbance value of cathepsin D (35 - 40 years: 0.020 ± 0.005 vs. 0.032 ± 0.005; 55 - 60 years: 0.012 ± 0.004 vs. 0.026 ± 0.002; 75 - 80 years: 0.002 ± 0.001 vs. 0.013 ± 0.004; all P < 0.001) , but higher absorbance value of AGEs (35 - 40 years: 0.030 ± 0.008 vs. 0.010 ± 0.003; 55 - 60 years: 0.066 ± 0.010 vs. 0.021 ± 0.004; 75 - 80 years: 0.085 ± 0.015 vs. 0.035 ± 0.009; all P < 0.001) except the age group of 15 - 20 years. No matter whether the skin tissues were sun-exposed or sun-protected, there were significant differences in the expression of cathepsin D and AGEs among different age groups (all P < 0.001) . The results of double immunofluorescence staining were similar to those of immunohistochemical study. Pearson correlation analysis showed that the expression of cathepsin D in the sun-exposed skin tissues was highly negatively correlated with the accumulation of AGEs (r = -0.915, P < 0.05) , while they were moderately negatively correlated in the sun-protected skin tissues (r = -0.730, P < 0.05) . Conclusions Along with the increase of age, the expression of cathepsin D in skin tissues decreased, but the expression of AGEs increased. In the sun-protected skin tissues, the expression of cathepsin D was moderately negatively correlated with the expression of AGEs, while they were highly negatively correlated in the sun-exposed skin tissues, suggesting that cathepsin D may play an important role in the degradation and accumulation of AGEs in photoaged skin. Key words: Skin aging; Cathepsin D; Glycosylation end products, advanced; Age factors; Sunlight

Regulating BRCA1 protein stability by cathepsin S-mediated ubiquitin degradation

Cathepsin S (CTSS) is a cysteine protease that is thought to play a role in many physiological and pathological processes including tumor growth, angiogenesis, and metastasis; it has been identified as a radiation response gene. Here, we examined the role of CTSS in regulating the DNA damage response in breast cancer cells. Activating CTSS (producing the cleavage form of the protein) by radiation induced proteolytic degradation of BRCA1, which ultimately suppressed DNA double-strand break repair activity. Depletion of CTSS by RNAi or expression of a mutant type of CTSS enhanced the protein stability of BRCA1 by inhibiting its ubiquitination. CTSS interacted with the BRCT domain of BRCA1 and facilitated ubiquitin-mediated proteolytic degradation of BRCA1, which was tightly associated with decreased BRCA1-mediated DNA repair activity. Treatment with a pharmacological CTSS inhibitor inhibited proteolytic degradation of BRCA1 and restored BRCA1 function. Depletion of CTSS by shRNA delayed tumor growth in a xenograft mouse model, only in the presence of functional BRCA1. Spontaneously uced rat mammary tumors and human breast cancer tissues with high levels of CTSS expression showed low BRCA1 expression. From these data, we suggest that CTSS inhibition is a good strategy for functional restoration of BRCA1 in breast cancers with reduced BRCA1 protein stability.

Effect of the Calpain System on Volatile Flavor Compounds in the Beef Longissimus lumborum Muscle.

The present study was designed to investigate the effects of calpain system on the formation of volatile flavor compounds in Hanwoo beef. In the first experiment (exp.1), Longissimus lumborum (LL) muscle samples were injected with solutions containing 50 mM CaCl2 or 50 mM ZnCl2 and 154 mM NaCl respectively, and aged for 7 d at 4°C. In the second experiment (exp.2), the ground LL muscle was incubated with the aforementioned solutions containing cathepsin inhibitor. The injection with CaCl2 solution greatly elevated the calpain activity and concomitantly, significantly decreased the Warner-Bratzler shear force (p<0.05). The pH, meat color and cooking loss did not differ (p>0.05) between the treatment groups. A total of 51 volatile compounds were identified using the solid phase microextraction with gas chromatography (SPME-GC). Results on volatile analyses from the both experiments showed that the injection with calcium ions led to significant increase (p<0.05) concentrations of pyrazines and sulfuric compounds. These results coincide with a higher rate of protein degradation due to the CaCl2 injection as compared to the control group. Significantly (p<0.05) higher levels of lipid oxidation derived-aldehydes were found in the samples with ZnCl2. The exp.1 showed that cathepsin inhibitors had no effect on the formation of volatile flavor components after 7 d of aging. These results imply that the proteolytic activity of the calpain system is associated with generation of volatile compounds of chiller-aged beef, while the role of cathepsins is likely very limited.

Cathepsins: innate immune proteases that regulate viral entry into host cells

Cathepsins are group of endolysosomal proteases that regulate the mechanisms of innate and adaptive immunity, including cell adhesion and migration, antigen processing and presentation and resistance to several viral infections. Some cathepsins are required for Toll-like receptor (TLR)3, TLR7 and TLR9 cleavage and the formation of functional receptors that participate in sensing viral nucleic acids. Moreover, cathepsins directly stimulate or inhibit cytokine secretion involved in the regulation of antiviral innate immune response. Recent findings underline the important role of cathepsins in the entry of filoviruses, reoviruses, retroviruses and other types of viruses into the host cell. Many enveloped viruses require the presence of cathepsins for efficient fusion with membranes of infected cells, and the inhibition of their activity results in a significant reduction of virus replication. In addition, many viruses utilize conserved cellular mechanisms, such as endocytosis or low pH within the endosome, for efficient penetration into the cell interior, disassembly of viral capsid, and other stages of productive viral replication cycle. Therefore, a better understanding of the functional role of cathepsin proteases in the pathogenesis of viral infections should lead to the development of novel therapeutics for a variety of particularly dangerous human pathogens.

Role of cathepsin S In periodontal wound healing\u2013an in vitro study on human PDL cells

BackgroundCathepsin S is a cysteine protease, which is expressed in human periodontal ligament (PDL) cells under inflammatory and infectious conditions. This in vitro study was established to investigate the effect of cathepsin S on PDL cell wound closure.MethodsAn in vitro wound healing assay was used to monitor wound closure in wounded PDL cell monolayers for 72 h in the presence and absence of cathepsin S. In addition, the effects of cathepsin S on specific markers for apoptosis and proliferation were studied at transcriptional level. Changes in the proliferation rate due to cathepsin S stimulation were analyzed by an XTT assay, and the actions of cathepsin S on cell migration were investigated via live cell tracking. Additionally, PDL cell monolayers were treated with a toll-like receptor 2 agonist in the presence and absence of a cathepsin inhibitor to examine if periodontal bacteria can alter wound closure via cathepsins.ResultsCathepsin S enhanced significantly the in vitro wound healing rate by inducing proliferation and by increasing the speed of cell migration, but had no effect on apoptosis. Moreover, the toll-like receptor 2 agonist enhanced significantly the wound closure and this stimulatory effect was dependent on cathepsins.ConclusionsOur findings provide original evidence that cathepsin S stimulates PDL cell proliferation and migration and, thereby, wound closure, suggesting that this cysteine protease might play a critical role in periodontal remodeling and healing. In addition, cathepsins might be exploited by periodontal bacteria to regulate critical PDL cell functions.

Cathepsin D deficiency delays central nervous system myelination by inhibiting proteolipid protein trafficking from late endosome/lysosome to plasma membrane

This study aimed to investigate the role of cathepsin D (CathD) in central nervous system (CNS) myelination and its possible mechanism. By using CathD knockout mice in conjunction with immunohistochemistry, immunocytochemistry and western blot assays, the myelination of the CNS and the development of oligodendrocyte lineage cells in vivo and in vitro were observed. Endocytosis assays, real-time-lapse experiments and total internal reflection fluorescence microscopy were used to demonstrate the location and movement of proteolipid protein in oligodendrocyte lineage cells. In addition, the relevant molecular mechanism was explored by immunoprecipitation. The increase in Fluoromyelin Green staining and proteolipid protein expression was not significant in the corpus callosum of CathD−/− mice at the age of P11, P14 and P24. Proteolipid protein expression was weak at each time point and was mostly accumulated around the nucleus. The number of oligodendrocyte lineage cells (olig2+) and mature oligodendrocytes (CC1+) significantly decreased between P14 and P24. In the oligodendrocyte precursor cell culture of CathD−/− mice, the morphology of myelin basic protein-positive mature oligodendrocytes was simple while oligodendrocyte precursor cells showed delayed differentiation into mature oligodendrocytes. Moreover, more proteolipid protein gathered in late endosomes/lysosomes (LEs/Ls) and fewer reached the plasma membrane. Immunohistochemistry and immunoelectron microscopy analysis showed that CathD, proteolipid protein and VAMP7 could bind with each other, whereas VAMP7 and proteolipid protein colocalized with CathD in late endosome/lysosome. The findings of this paper suggest that CathD may have an important role in the myelination of CNS, presumably by altering the trafficking of proteolipid protein.

A novel role for cathepsin K in periosteal osteoclast precursors during fracture repair

Osteoporosis management is currently centered around bisphosphonates, which inhibit osteoclast (OC) bone resorption but do not affect bone formation. This reduces fracture risk, but fails to restore healthy bone remodeling. Studies in animal models showed that cathepsin K (CatK) inhibition by genetic deletion or chemical inhibitors maintained bone formation while abrogating resorption during bone remodeling and stimulated periosteal bone modeling. Recently, periosteal mononuclear tartrate-resistant acid phosphatase-positive (TRAP+ ) osteoclast precursors (OCPs) were shown to augment angiogenesis-coupled osteogenesis. CatK gene deletion increased osteoblast differentiation via enhanced OCP and OC secretion of platelet-derived growth factor (PDGF)-BB and sphingosine 1 phosphate. The effects of periosteum-derived OCPs on bone remodeling are unknown, particularly with regard to fracture repair. We hypothesized that periosteal OCPs derived from CatK-null (Ctsk-/- ) mice may enhance periosteal bone formation during fracture repair. We found fewer periosteal OCPs in Ctsk-/- mice under homeostatic conditions; however, after fracture, this population increased in number relative to that seen in wild-type (WT) mice. Enhanced TRAP staining and greater expression of PDGF-BB were observed in fractured Ctsk-/- femurs relative to WT femurs. This early pattern of augmented PDGF-BB expression in Ctsk-/- mice may contribute to improved fracture healing by enhancing callus mineralization in Ctsk-/- mice.

PPARα-Target Gene Expression Requires TIS21/BTG2 Gene in Liver of the C57BL/6 Mice under Fasting Condition.

The TIS21/BTG2/PC3 gene belongs to the antiproliferative gene (APRO) family and exhibits tumor suppressive activity. However, here we report that TIS21 controls lipid metabolism, rather than cell proliferation, under fasting condition. Using microarray analysis, whole gene expression changes were investigated in liver of TIS21 knockout (TIS21-KO) mice after 20 h fasting and compared with wild type (WT). Peroxisome proliferator-activated receptor alpha (PPARα) target gene expression was almost absent in contrast to increased lipid synthesis in the TIS21-KO mice compared to WT mice. Immunohistochemistry with hematoxylin and eosin staining revealed that lipid deposition was focal in the TIS21-KO liver as opposed to the diffuse and homogeneous pattern in the WT liver after 24 h starvation. In addition, cathepsin E expression was over 10 times higher in the TIS21-KO liver than that in the WT, as opposed to the significant reduction of thioltransferase in both adult and fetal livers. At present, we cannot account for the role of cathepsin E. However, downregulation of glutaredoxin 2 thioltransferase expression might affect hypoxic damage in the TIS21-KO liver. We suggest that the TIS21/BTG2 gene might be essential to maintain energy metabolism and reducing power in the liver under fasting condition.

Erratum: Evaluation of carcinoma-associated fibroblasts in oral squamous cell carcinoma: a potential role for cathepsin K

Erratum: Evaluation of carcinoma-associated fibroblasts in oral squamous cell carcinoma: a potential role for cathepsin K

Progress in the study on roles of cathepsin in hypertension

Cardiovascular remodeling or dysfunction-induced abnormal cardiac output, blood volume and peripheral vascular resistance is an important pathophysiological mechanism for the occurrence and development of hypertension. Cathepsins are widely expressed in human various tissue and cells and they are involved in the pathogenesis of hypertension through activation of renin - angiotensin system, degradation of cytoplasmic matrix, proliferation of smooth muscle cell and hypertrophy of myocyte. The clinical studies have found that cathepsins can be used as a biomarker for hypertension. In recent years, the studies on the functions and mechanisms of cathepsins have provided a new sight and strategy for treatment of hypertension.

Study the effect of CTSD interaction proteins in invasion and metastasis of nasopharyngeal carcinoma

Background: Cathepsin D is a lysosomal aspartyl protease. Our previous studies have suggested that cathepsin D plays an important role in invasion and metastasis of nasopharyngeal carcinoma (NPC). Methods: To identify proteins that interact with cathepsin D and gain an insight into the role of cathepsin D in invasion and metastasis of NPC, co-immunoprecipitation (co-IP) combined with mass spectrometry (MS)-based proteomics approach was used. Cathepsin D associated proteins were identified by MS. Protein-protein interaction network were analyzed by bioinformatics including gene ontology (GO), function clustering. And co-IP and western blotting confirmation were for protein-protein interaction. The expression of cathepsin D, epidermal growth factor receptor (EGFR) and heat-shock protein 90A (HSP90A) in NPC were detected by immunohistochemistry (IHC). And the invasion and metastasis capability were detected by Transwell invasion assay. Results: One hundred and forty-one cathepsin D associated proteins were identified, including EGFR and HSP90A, proteins clearly associated with tumor invasion and metastasis. The interaction of these two proteins with cathepsin D was further validated by co-IP followed by western blotting. The 141 proteins were classified into 12 function-related groups. Protein-protein interaction network analysis indicated that cathepsin D might have a significance on invasion and metastasis of NPC by interacting with EGFR and HSP90A. Indeed, cathepsin D/EGFR/HSP90A could form complexes in NPC cells. In addition, overexpression of cathepsin D in NPC cells not only up-regulated EGFR and HSP90A, but also increased the invasive ability of NPC cells. Conclusions: Cathepsin D could enhance the invasion and metastasis capability of NPC cells may through binding to EGFR and HSP90A and triggering the activation of the signaling pathways.

A role of cathepsin L gene in innate immune response of crayfish (Procambarus clarkii)

Cathepsin L is one of the crucial enzyme superfamilies and involved in the immune responses. In the present study, cathepsin L gene from the red crayfish Procambarus clarkii, named PcCTSL, was cloned and characterized. The cDNA fragment of PcCTSL was 1026 bp in length, which encoded a putative protein of 341 amino acid residues with a molecular weight of 37.884 kDa. The theoretical isoelectric point was 5.218. The prepro-cathepsin L was comprised of a typical signal peptide (Met1-Ala18), a prodomain proregion peptide (Trp29-Phe89) and a mature peptide (Leu124-Leu340). Homology analysis indicated that PcCTSL exhibited 53.2%–87.1% identity to other selected species. The recombinant protein of PcCTSL was successfully expressed in Escherichia coli and rabbit anti-PcCTSL polyclonal antibodies were prepared. Real-time quantitative reverse transcription-PCR (qPCR) analysis revealed that the PcCTSL was expressed in all examined tissues, while the greatest mRNA level was observed in hepatopancreas. The expression of PcCTSL mRNA was clearly up regulated in hepatopancreas after challenge by lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid (Poly I:C). RNA interference of PcCTSL affected the gene expression of members of the Toll pathway. Our results suggest that the PcCTSL may play an important role to defend P. clarkii against the pathogens infection.

Upregulation of cathepsin C expression contributes to endothelial chymase activation in preeclampsia.

Chymase is an ACE (angiotensin-converting enzyme)-independent angiotensin II-forming enzyme whose expression is increased in the maternal vascular endothelium in preeclampsia. However, mechanisms underlying chymase activation in preeclampsia remain unclear. Cathepsin C is a key enzyme in the activation of several serine proteases including chymase. In this study, we determined whether increased cathepsin C expression/activity might be responsible for the upregulation of chymase expression in preeclampsia. Maternal vascular cathepsin C, chymase and ACE expression were examined through immunohistochemical staining of subcutaneous fat tissue sections of normal and preeclamptic pregnant women. The role of cathepsin C in endothelial chymase and ACE expression was determined in cells treated with cathepsin C. Consequences of chymase activation were then determined by measurement of angiotensin II production in cells treated with the ACE inhibitor captopril and the chymase inhibitor chymostatin, separately and in combination. Expression of both cathepsin C and chymase, but not ACE expression, was markedly increased in the maternal vascular endothelium in subjects with preeclampsia compared with normal pregnant controls. Exogenous cathepsin C induced a dose-dependent increase in expression of mature cathepsin C and chymase, but not ACE, in endothelial cells. Moreover, angiotensin II production was significantly inhibited in cells treated with captopril or chymostatin alone and was further inhibited in cells treated with both inhibitors. These results suggest that cathepsin C upregulation induces chymase activation and subsequently promotes angiotensin II generation in endothelial cells. These data also provide evidence of upregulation of the cathepsin C-chymase-angiotensin signaling axis in maternal vasculature in preeclampsia.

Role of cathepsin K in the development of chronic subdural hematoma

Despite extensive investigations, the process of development of chronic subdural hematoma (CSDH) is not known. The present study aims to investigate CSDH by measuring biomarkers in it, gas analysis, and immunohistochemical examination. A total of 42 patients with symptomatic CSDH who underwent burr-hole drainage were enrolled. Intraoperatively, hematoma fluid and peripheral venous blood (PVCSDH) were simultaneously collected. As controls, peripheral venous blood (PVControl) and intracranial cerebrospinal fluid (CSF) were collected from other subjects during other surgeries. CatK, lipocalin-type prostaglandin D synthase (PGDS), and cystatin C (CysC) present in these specimens were measured using enzyme-linked immunosorbent assay. Data obtained were statistically analyzed after age correction. In 15 patients, gas analysis was performed for CSDH and PVCSDH. Furthermore, immunohistochemical examination for the outer membrane was performed for four patients. CatK, PGDS, and CysC levels were markedly elevated in the CSF and CSDH. CatK levels in PVCSDH were significantly higher than in PVControl (P<0.0001). In contrast, CysC levels in PVCSDH were significantly lower than in PVControl (P=0.004). The gas analysis revealed that the internal environment of CSDH is characterized by marked hypoxia, hypoglycemia, and lactic acidosis. Furthermore, the outer membrane consistently showed a diffuse staining for CatK. Based on these, CatK was thought to play a role in the development of CSDH, with the levels in peripheral venous blood elevated in patients with CSDH.

Abstract 2952: The novel cathepsin L/K inhibitors KGP94 and KGP207 prevent M0 to M2 macrophage differentiation and macrophage-mediated pro-tumor functions

Abstract Tumor-associated macrophages play many important roles in tumor progression, including facilitating tumor cell invasion and angiogenesis. Due to their numerous pro-tumor functions, tumor-associated macrophages represent an important cell population in the design of targeted anti-cancer therapies. Interleukin-4-mediated activation of macrophages resulted in an M2-like, or pro-tumor macrophage phenotype, often associated with increased expression of proteases including the lysosomal cathepsin L. Cathepsin L is important in both macrophage and tumor cell invasion, whereby secreted cathepsin L degrades the extracellular matrix, allowing for cell infiltration. The present study examined the role of cathepsin L in M0 to M2 differentiation and macrophage-mediated tumor cell invasion using the novel cathepsin L/K inhibitors KGP94 and KGP207 [Dr. Kevin Pinney, Baylor University]. KGP94 and KGP207 prevented M2 macrophage motility and invasion in in vitro migration and invasion assays. Moreover, KGP94 and KGP207 reduced macrophage-stimulated invasion of 4T1 murine breast cancer cells. Together, these data suggested that cathepsin L was necessary for macrophage invasion and motility and for macrophage-mediated invasion of breast cancer cells. KGP94 and KGP207 treatment partially prevented IL-4-stimulated M0 to M2 differentiation of macrophages as determined by a decrease in the IL-4-induced expression of the M2 marker Arginase-1 upon drug treatment. Additionally, exogenous recombinant cathepsin L partially stimulated the expression of Arginase-1 in M0 macrophages. Together, these data suggest that cathepsin L plays a role in macrophage M0 to M2 differentiation. In conclusion, the novel cathepsin L/K inhibitors KGP94 and KGP207 prevented M0 to M2 differentiation, macrophage invasion, and macrophage-stimulated invasion of breast cancer cells. These data highlight the importance of cathepsin L in macrophage functions and suggest that cathepsin L inhibition is a viable approach for the treatment of tumors associated with high macrophage infiltration. Citation Format: Samantha S. Dykes, Dietmar W. Siemann. The novel cathepsin L/K inhibitors KGP94 and KGP207 prevent M0 to M2 macrophage differentiation and macrophage-mediated pro-tumor functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2952. doi:10.1158/1538-7445.AM2017-2952