Protein Levels Of Cathepsin Research Articles

SARS-CoV-2 Spike Protein Induces Time-Dependent CTSL Upregulation in HeLa Cells and Alveolarspheres.

Autophagy and lysosomal pathways are involved in the cell entry of SARS-CoV-2 virus. To infect the host cell, the spike protein of SARS-CoV-2 binds to the cell surface receptor angiotensin-converting enzyme 2 (ACE2). To allow the fusion of the viral envelope with the host cell membrane, the spike protein has to be cleaved. One possible mechanism is the endocytosis of the SARS-CoV-2-ACE2 complex and subsequent cleavage of the spike protein, mainly by the lysosomal protease cathepsin L. However, detailed molecular and dynamic insights into the role of cathepsin L in viral cell entry remain elusive. To address this, HeLa cells and iPSC-derived alveolarspheres were treated with recombinant SARS-CoV-2 spike protein, and the changes in mRNA and protein levels of cathepsins L, B, and D were monitored. Additionally, we studied the effect of cathepsin L deficiency on spike protein internalization and investigated the influence of the spike protein on cathepsin L promoters in vitro. Furthermore, we analyzed variants in the genes coding for cathepsin L, B, D, and ACE2 possibly associated with disease progression using data from Regeneron's COVID Results Browser and our own cohort of 173 patients with COVID-19, exhibiting a variant of ACE2 showing significant association with COVID-19 disease progression. Our in vitro studies revealed a significant increase in cathepsin L mRNA and protein levels following exposure to the SARS-CoV-2 spike protein in HeLa cells, accompanied by elevated mRNA levels of cathepsin B and D in alveolarspheres. Moreover, an increase in cathepsin L promoter activity was detected in vitro upon spike protein treatment. Notably, the knockout of cathepsin L resulted in reduced internalization of the spike protein. The study highlights the importance of cathepsin L and lysosomal proteases in the SARS-CoV-2 spike protein internalization and suggests the potential of lysosomal proteases as possible therapeutic targets against COVID-19 and other viral infections.

Ethyl Gallate Inhibits Bovine Viral Diarrhea Virus by Promoting IFITM3 Expression, Lysosomal Acidification and Protease Activity.

Bovine viral diarrhea virus (BVDV) is a highly contagious viral disease which causes economic losses to the cattle industry. Ethyl gallate (EG) is a phenolic acid derivative which has various potentials to modulate the host response to pathogens, such as via antioxidant activity, antibacterial activity, inhibition of the production of cell adhesion factors, and so on. This study aimed to evaluate if EG influences BVDV infection in Madin-Darby Bovine Kidney (MDBK) cells, and to understand the antiviral mechanism. Data indicated that EG effectively inhibited BVDV infection by co-treatment and post-treatment in MDBK cells with noncytotoxic doses. In addition, EG suppressed BVDV infection at an early stage of the viral life cycle by blocking entry and replication steps but not viral attachment and release. Moreover, EG strongly inhibited BVDV infection by promoting interferon-induced transmembrane protein 3 (IFITM3) expression, which localized to the cytoplasm. The protein level of cathepsin B was significantly reduced by BVDV infection, whereas with treatment with EG, it was significantly enhanced. The fluorescence intensities of acridine orange (AO) staining were significantly decreased in BVDV-infected cells but increased in EG-treated cells. Finally, Western blot and immunofluorescence analyses demonstrated that EG treatment significantly enhanced the protein levels of autophagy markers LC3 and p62. Chloroquine (CQ) significantly increased IFITM3 expression, and Rapamycin significantly decreased it. Thus, EG may regulate IFITM3 expression through autophagy. Our results showed that EG could have a solid antiviral activity on BVDV replication in MDBK cells via increased IFITM3 expression, lysosomal acidification, protease activity, and regulated autophagy. EG might have value for further development as an antiviral agent.

PINK1-mediated mitophagy contributes to glucocorticoid-induced cathepsin K production in osteocytes.

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. 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. 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. 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. 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 D Inhibits Angiogenesis in Pituitary Neuroendocrine Tumors.

Prolactin and growth hormone can acquire anti-angiogenic properties after undergoing proteolytic cleavage by Cathepsin D and bone morphogenetic protein 1 (BMP-1) into fragments known as vasoinhibins. Little is known about the effect of vasoinhibins on angiogenesis through the involvement of key cleavage enzymes Cathepsin D and BMP-1 in pituitary neuroendocrine tumors (PitNETs, formerly pituitary adenomas). The purpose of this study was to investigate the mechanism of action of Cathepsin D and BMP-1 on angiogenesis in PitNETs compared with that of pro-angiogenic factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-2 (FGF2). A total of 43 patients were enrolled in a retrospective analysis and 22 samples were suitable for RNA extraction, including 16 nonfunctional PitNETs and six somatotroph tumors. The mRNA and protein levels of Cathepsin D, BMP-1, VEGF, and FGF2 were compared with those of von Willebrand factor, which was assessed to determine the vascularization of PitNETs. Cathepsin D and FGF2 were significantly correlated with vascularization in PitNETs. Both Cathepsin D and FGF2 are highly involved in angiogenesis in PitNETs, although the effect of Cathepsin D as an anti-angiogenic factor is dominant over that of FGF2 as a pro-angiogenic factor.

Benzyl Isothiocyanate-Induced Cytotoxicity via the Inhibition of Autophagy and Lysosomal Function in AGS Cells.

Gastric adenocarcinoma is among the top causes of cancer-related death and is one of the most commonly diagnosed carcinomas worldwide. Benzyl isothiocyanate (BITC) has been reported to inhibit the gastric cancer metastasis. In our previous study, BITC induced apoptosis in AGS cells. The purpose of the present study was to investigate the effect of BITC on autophagy mechanism in AGS cells. First, the AGS cells were treated with 5, 10, or 15 μM BITC for 24 h, followed by an analysis of the autophagy mechanism. The expression level of autophagy proteins involved in different steps of autophagy, such as LC3B, p62/SQSTM1, Atg5-Atg12, Beclin1, p-mTOR/mTOR ratio, and class III PI3K was measured in the BITC-treated cells. Lysosomal function was investigated using cathepsin activity and Bafilomycin A1, an autophagy degradation stage inhibitor. Methods including qPCR, western blotting, and immunocytochemistry were employed to detect the protein expression levels. Acridine orange staining and omnicathepsin assay were conducted to analyze the lysosomal function. siRNA transfection was performed to knock down the LC3B gene. BITC reduced the level of autophagy protein such as Beclin 1, class III PI3K, and Atg5-Atg12. BITC also induced lysosomal dysfunction which was shown as reducing cathepsin activity, protein level of cathepsin, and enlargement of acidic vesicle. Overall, the results showed that the BITC-induced AGS cell death mechanism also comprises the inhibition of the cytoprotective autophagy at both initiation and degradation steps.

α-Lipoic Acid-Plus Ameliorates Endothelial Injury by Inhibiting the Apoptosis Pathway Mediated by Intralysosomal Cathepsins in an In Vivo and In Vitro Endothelial Injury Model.

α-Lipoic acid-plus (LAP), an amine derivative of α-lipoic acid, has been reported to protect cells from oxidative stress damage by reacting with lysosomal iron and is more powerful than desferrioxamine (DFO). However, the role of LAP in experimental carotid artery intimal injury (CAII) has not yet been well investigated. Therefore, we sought to uncover the role and potential endovascular protective mechanisms of LAP in endothelial injury. In vitro, oxyhemoglobin (OxyHb) stimulation of cultured human umbilical vein endothelial cells (HUVECs) simulated intimal injury. In vivo, balloon compression injury of the carotid artery was used to establish a rat CAII model. We found that the protein levels of cathepsin B/D, ferritin, transferrin receptor (TfR), cleaved caspase-3, and Bax increased in the injured endothelium and HUVECs but were rectified by DFO and LAP treatments, as revealed by western blotting and immunofluorescence staining. Additionally, DFO and LAP decreased oxidative stress levels and endothelial cell necrosis of the damaged endothelium. Moreover, DFO and LAP significantly ameliorated the increased oxidative stress, iron level, and lactic dehydrogenase activity of HUVECs and improved the reduced HUVEC viability induced by OxyHb. More importantly, DFO and LAP significantly reduced mitochondrial damage and were beneficial for maintaining lysosomal integrity, as indicated by acridine orange (AO), Lyso-Tracker Red, JC-1, and ATPB staining in HUVECs. Finally, LAP might offer more significant endovascular protective effects than DFO. Our data suggested that LAP exerted endovascular protective effects by inhibiting the apoptosis signaling pathway mediated by intralysosomal cathepsins by reacting with excessive iron in endothelial lysosomes after intimal injury.

High-sugar high-fat treatment induces autophagy of retinal microvascular endothelial cells

ObjectiveTo investigate the role of high-sugar high-fat treatment in inducing autophagy of rat retinal microvascular endothelial cells. MethodsThe optimal concentrations and time points of glucose and oxidized low-density lipoprotein (ox-LDL) in inducing rat retinal microvascular endothelial cells were determined by examining the proliferate rate by CCK-8 assay. They were divided into control group (blank control), model group (treatment of 50 mM glucose and 10 μg/ml ox-LDL for 24 h), chloroquine group (treatment of 20 μM chloroquine, 50 mM glucose and 10 μg/ml ox-LDL for 24 h), resveratrol group (treatment of 50 μM resveratrol, 50 mM glucose and 10 μg/ml ox-LDL for 24 h) and MITO-Tempol group (treatment of 20 μM MITO-Tempol, 50 mM glucose and 10 μg/ml ox-LDL for 24 h). Reactive oxygen species (ROS) level in rat retinal microvascular endothelial cells induced with high sugar high-fat treatment was measured by flow cytometry. In addition, protein levels of cathepsin B and cathepsin D in rat retinal microvascular endothelial cells induced with high sugar high-fat treatment were examined by immunofluorescence, and protein levels of LC3 A/B and the autophagy substrate P62 were detected by Western blot. ResultsPrimary retinal microvascular endothelial cells were isolated from neonatal Sprague-Dawley (SD) rats. ROS level was significantly higher in model group than that of control group (P < 0.05). Compared with that of model group, ROS level was significantly reduced in chloroquine group and MITO-Tempol group, which was significantly elevated in resveratrol group (P < 0.05). Positive expressions of cathepsin B and cathepsin D were significantly reduced in model group than those of control group (P < 0.05). They were significantly elevated in chloroquine group and MITO-Tempol group, and reduced in resveratrol group than those of model group (P < 0.05). LC3 A/B and P62 were significantly upregulated in model group than those of control group (P < 0.05). Compared with those of model group, LC3 A/B and P62 were significantly downregulated in chloroquine group and MITO-Tempol group, and upregulated in resveratrol group (P < 0.05). ConclusionHigh-sugar high-fat treatment induces autophagy of rat retinal microvascular endothelial cells, which can be intervened to a certain extent by chloroquine and MITO-Tempol.

Evidence for Lysosomal Dysfunction within the Epidermis in Psoriasis and Atopic Dermatitis

Atopic dermatitis and psoriasis are frequent chronic inflammatory skin diseases. Autophagy plays a substantial role in the homeostasis of an organism. Loss or impairment of autophagy is associated with multiple diseases. To investigate the possibility that autophagy plays a role in atopic dermatitis and psoriasis, we investigated the levels of key ATG proteins in human skin specimens as well as in primary human epidermal keratinocytes exposed to inflammatory stimuli invitro. Although TNF-α facilitated the induction of autophagy in an initial phase, it reduced the levels and enzymatic activities of lysosomal cathepsins in later time periods, resulting in autophagy inhibition. Therefore, TNF-α appears to play a dual role in the regulation of autophagy. The relevance of these invitro findings was supported by the observation that the protein levels of cathepsins D and L are decreased in both psoriasis and atopic dermatitis skin specimens. Taken together, this study suggests that TNF-α blocks autophagy in keratinocytes after long-term exposure, a mechanism that may contribute to the chronicity of inflammatory diseases of the skin and, perhaps, of other organs.

Chronic Periodontitis and Alzheimer Disease: A Putative Link of Serum Proteins Identification by 2D-DIGE Proteomics.

Increasing evidence indicates Chronic Periodontitis (CP) is a comorbidity of Alzheimer’s disease (AD), which is the most common form of age-related dementia, and for the latter, effective diagnostic and treatment strategies are lacking. Although inflammation is present in both diseases, the exact mechanisms and cross-links between CP and AD are poorly understood; and a direct association between the two has not been reported. This study aimed to identify a direct serum proteins link between AD and CP. Two-dimensional differential in-gel electrophoresis was employed to analyze serum samples from 12 CP patients and 12 age-matched controls. Furthermore, to determine the molecular link between CP and AD, neuroblastoma SK-N-SH APPwt cells were treated with 1 μg/ml of lipopolysaccharide from Porphyromonas gingivalis (P.g-LPS). Ten differentially expressed proteins were identified in CP patients. Among them, nine proteins were up-regulated, and one protein was down-regulated. Of the 10 differentially expressed proteins, five proteins were reportedly involved in the pathology of AD: Cofilin-2, Cathepsin B, Clusterin, Triosephosphate isomerase, and inter-alpha-trypsin inhibitor heavy chain H4 (ITI-H4). Western blotting indicated significantly higher expression of Cofilin-2, Cathepsin B, and Clusterin and lower expression of ITI-H4 in the CP group than in the Control group. The serum concentration of Cathepsin B has a good correlation with MMSE scores. Moreover, the protein level of Cathepsin B (but not that of ADAM10 and BACE1) increased significantly along with a prominent increase in Aβ1–40 and Aβ1–42 in the cell lysates of P.g-LPS-treated SK-N-SH APPwt cells. Cathepsin B inhibition resulted in a sharp decrease in Aβ1–40 and Aβ1–42 in the cell lysates. Furthermore, TNF-α was one of the most important inflammatory cytokines for the P.g-LPS-induced Cathepsin B upregulation in SK-N-SH APPwt cells. These results show that CP and AD share an association, while Cathepsin B could be a key link between the two diseases. The discovery of the identical serum proteins provides a potential mechanism underlying the increased risk of AD in CP patients, which could be critical for elucidating the pathophysiology of AD.

Yougui pills exert osteoprotective effects on rabbit steroid-related osteonecrosis of the femoral head by activating β-catenin

ObjectiveTo investigate the effect and underlying mechanism of Yougui pills (YGPs) on steroid-related osteonecrosis of the femoral head (SONFH). MethodsMale New Zealand white rabbits were divided into three groups: control group, SONFH group and YGPs group. Rabbit SONFH was induced by methylprednisolone (MPS) combined with lipopolysaccharide (LPS). At 6 weeks post induction, the femoral heads were harvested for tissue analyses, including histopathology, mechanical test of femoral heads, micro-CT, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemistry for osteocalcin (OCN), vascular endothelial growth factor (VEGF) and β-catenin. Protein levels of cathepsin K (CTSK), phospho-glycogen synthase kinase-3 beta (p-Ser9 GSK-3β) and total glycogen synthase kinase-3 beta (GSK-3β) in femoral heads were also detected. Additionally, the serum TRAP activity was measured using enzyme-linked immunosorbent assay (ELISA). Finally, the effects of YGPs treatment on osteoclast differentiation and osteoblast formation were evaluated in vitro. ResultsThe ratio of empty lacuna was markedly lower in YGPs group than SONFH group. Micro-CT evaluation indicated that YGPs has a preventive effect on bone loss in rabbit SONFH. YGPs treatment could suppress bone resorption by reducing TRAP+ osteoclast and serum TRACP5b levels in necrotic femoral heads. Moreover, YGPs treatment could promote bone formation by up-regulating the expression of OCN, VEGF and β-catenin, while increasing load-bearing capacity of femoral heads. Interestingly, p-Ser9 GSK-3β downregulation, and CTSK upregulation in necrotic femoral head could be reversed by YGPs treatment, which also effectively inhibited RANKL-induced osteoclast differentiation and promoted osteoblast formation in vitro. ConclusionYGPs could suppress osteoclastogenesis and promote bone formation during SONFH in rabbits by activating β-catenin.

Cathepsin B inhibition ameliorates leukocyte-endothelial adhesion in the BTBR mouse model of autism.

Autism spectrum disorder (ASD) is a wide range of neurodevelopmental disorders involving deficits in social interaction and communication. Unfortunately, autism remains a scientific and clinical challenge owing to the lack of understanding the cellular and molecular mechanisms underlying it. This study aimed to investigate the pathophysiological mechanism underlying leukocyte-endothelial adhesion in autism-related neurovascular inflammation. Male BTBR T+tf/J mice were used as an autism model. The dynamic pattern of leukocyte-endothelial adhesion in mouse cerebral vessels was detected by two-photon laser scanning microscopy (TPLSM). Using FACS, RT-PCR, and Western blotting, we explored the expression of cell adhesion molecules, the mRNA expression of endothelial chemokine, the protein levels of cathepsin B, and inflammatory mediators. We found a significant increase in leukocyte-endothelial adhesion in BTBR mice, accompanied by elevated expression of the adhesion molecule neutrophils CD11b and endothelial ICAM-1. Our data further indicate that elevated neutrophil cathepsin B levels contribute to elevated endothelial chemokine CXCL7 levels in BTBR mice. The pharmacological inhibition of cathepsin B reverses the enhanced leukocyte-endothelial adhesion in the cerebral vessels of autistic mice. Our results revealed the prominent role of cathepsin B in modulating leukocyte-endothelial adhesion during autism-related neurovascular inflammation and identified a promising novel approach for autism treatment.

Cathepsin K knockout protects against cardiac dysfunction in diabetic mice

Diabetes is a major risk factor for cardiovascular disease and the lysosomal cysteine protease cathepsin K plays a critical role in cardiac pathophysiology. To expand upon our previous findings, we tested the hypothesis that, knockout of cathepsin K protects against diabetes-associated cardiac anomalies. Wild-type and cathepsin K knockout mice were rendered diabetic by streptozotocin (STZ) injections. Body weight, organ mass, fasting blood glucose, energy expenditure, cardiac geometry and function, cardiac histomorphology, glutathione levels and protein levels of cathepsin K and those associated with Ca2+ handling, calcineurin/NFAT signaling, insulin signaling, cardiac apoptosis and fibrosis were determined. STZ-induced diabetic mice exhibited distinct cardiac dysfunction, dampened intracellular calcium handling, alterations in cardiac morphology, and elevated cardiomyocyte apoptosis, which were mitigated in the cathepsin K knockout mice. Additionally, cathepsin K knockout mice attenuated cardiac oxidative stress and calcineurin/NFAT signaling in diabetic mice. In cultured H9c2 myoblasts, pharmacological inhibition of cathepsin K, or treatment with calcineurin inhibitor rescued cells from high-glucose triggered oxidative stress and apoptosis. Therefore, cathepsin K may represent a potential target in treating diabetes-associated cardiac dysfunction.

Pulsed electromagnetic field inhibits RANKL-dependent osteoclastic differentiation in RAW264.7 cells through the Ca2+-calcineurin-NFATc1 signaling pathway

Pulsed electromagnetic field (PEMF) has been reported to improve bone healing in osteoporosis patients. However, the precise mechanism has remained largely unknown. This study aimed to investigate the effects of PEMF on nuclear factor κB ligand (RANKL)-dependent osteoclastic differentiation and the Ca2+-calcineurin-NFATc1 signaling pathway in RAW264.7 cells in vitro. Treating RAW264.7 cells with RANKL for 4 days induced osteoclastic differentiation in vitro, and the formation of multinucleated osteoclasts, bone resorption-pit formation, tartrate-resistant acid phosphatase (TRAP) activity and the protein levels of cathepsin K, TRAP, Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and matrix metalloproteinase 9 (MMP-9) were significantly decreased. The mRNA levels of specific genes related to osteoclastogenesis (TRAP, NFATc1, CTSK and MMP-9) were also reduced. Moreover, the oscillations of intracellular Ca2+ in RANKL-dependent RAW264.7 cells were suppressed by PEMF, as well as by inhibitors of membrane and store-operated Ca2+ channels. Meanwhile, calcineurin activity was increased, although its protein level was not changed. PEMF increased phospho-NFATc1 in the cytosol while suppressing the nuclear translocation of NFATc1, thus inhibiting osteoclastic differentiation by suppressing the Ca2+-calcineurin-NFATc1 signaling pathway. Although many questions remain unresolved, to our knowledge, this is the first report demonstrating that PEMF is beneficial against RANKL-dependent osteoclastic differentiation in RAW264.7 cells in vitro via inhibiting the Ca2+-calcineurin-NFATc1 signaling pathway.

Interconnection between cathepsin protein levels and thyroid hormone transporters in human thyroid epithelial cells

Interconnection between cathepsin protein levels and thyroid hormone transporters in human thyroid epithelial cells

Knockdown of Cathepsin L promotes radiosensitivity of glioma stem cells both in vivo and in vitro

The presence of glioma stem cells (GSCs) in tumor is relevant for glioma treatment resistance. This study assessed whether knockdown of Cathepsin L can influence GSC growth, tumor radiosensitivity, and clinical outcome. Protein levels of Cathepsin L and stem cell markers (CD133 and Nestin) were analyzed in samples from 90 gliomas of different WHO grades and 6 normal brain tissues by immunohistochemistry. Two glioma stem cell lines with overexpressed Cathepsin L were stably transfected with Cathepsin L short hairpin RNA expression vectors. The effects of Cathepsin L inhibition on radiosensitivity, self-renewal, stemness, DNA damage, and apoptosis were evaluated. In addition, an intracranial animal model and subcutaneous tumor xenografts in nude mice were used to assess tumor response to Cathepsin L inhibition in vivo. Our results proved that expressions of Cathepsin L and CD133, but not of Nestin, correlated with malignant grades of glioma tissues. GSCs with high Cathepsin L and CD133 co-expression were extraordinarily radioresistant. Cathepsin L inhibition with radiotherapy significantly reduced GSC growth, promoted apoptosis, and improved radiosensitivity. Knockdown of Cathepsin L resulted in a dramatic reduction of CD133 expression, as well as the decreased phosphorylation of DNA repair checkpoint proteins (ATM and DNA-PKcs). Furthermore, combination of Cathepsin L inhibition and radiotherapy potently blocked tumor growth and decreased blood vessel formation in vivo. Taken together, these findings suggest Cathepsin L as a promising therapeutic target for clinical therapy in GBM patients.

Cathepsin K knockout alleviates aging-induced cardiac dysfunction.

Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca2+ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca2+ release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis.

The Neuroprotection of Lysosomotropic Agents in Experimental Subarachnoid Hemorrhage Probably Involving the Apoptosis Pathway Triggering by Cathepsins via Chelating Intralysosomal Iron.

α-Lipoic acid-plus (LAP), an amine derivative of α-lipoic acid (LA), could protect cells against oxidant challenges via chelating intralysosomal iron. However, the application of LAP in experimental subarachnoid hemorrhage (SAH) is still not well known. This study was designed to evaluate the potential neuroprotection of LAP on the early brain injury (EBI) and the underlying mechanisms in a rat model of SAH. The SAH models were induced in Sprague-Dawley rats. LA and LAP were oral administration and lasted for 72 h once a day. The brain tissue samples were obtained for assay at 72 h after SAH. In experiment 1, we found that lysosome amounts in neurons decreased significantly in SAH group, and LAP (100 mg/kg) could stabilize lysosomal membrane markedly based on lysosomal-associated membrane protein-1 (LAMP-1) expression in neurons by immunofluorescence. Hence, the LAP dosages of 100 and 150 mg/kg were applied in experiment 2. Firstly, Western blot analysis showed that the protein levels of cathepsin B/D, caspase-3, Bax, ferritin, and heme-oxygenase-1 (HO-1) markedly increased after SAH, which were further confirmed by double immunofluorescence staining and reversed by LA and LAP treatments. In addition, LA and LAP also reduced oxidative stress and iron deposition in brain tissue. Furthermore, LA and LAP significantly ameliorated brain edema, blood-brain barrier injury, cortical apoptosis, and neurological behavior impairment induced by SAH. Finally, it is noteworthy that LAP exerted more significant effects than LA on these parameters as described above. LAP probably exerted neuroprotective effects via targeting lysosomes and chelating intralysosomal iron in EBI post-SAH in rats.

Ascorbic Acid Modulation of Iron Homeostasis and Lysosomal Function in Trabecular Meshwork Cells

To investigate the antioxidant properties and biological functions of ascorbic acid (AA) on trabecular meshwork (TM) cells. Primary cultures of porcine TM cells were supplemented for 10 days with increasing concentrations of AA. Antioxidant properties against cytotoxic effect of H2O2 were evaluated by monitoring cell viability. Redox-active iron was quantified using calcein-AM. Intracellular reactive oxygen species (iROS) production was quantified using H2DCFDA. Ferritin and cathepsin protein levels were analyzed by Western blot. Autophagy was evaluated by monitoring lipidation of LC3-I to LC3-II. Lysosomal proteolysis and cathepsins activities were quantified using specific fluorogenic substrates. AA exerts a dual effect against oxidative stress in TM cells, acting as an anti-oxidant or a pro-oxidant, depending on the concentration used. The pro-oxidant effect of AA was mediated by free intracellular iron and correlated with increased protein levels of ferritin and elevated iROS. In contrast, antioxidant properties correlated with lower ferritin and basal iROS content. Ascorbic acid supplementation also caused induction of autophagy, as well as increased lysosomal proteolysis, with the latter resulting from higher proteolytic activation of lysosomal cathepsins in treated cultures. Our results suggest that the reported decrease of AA levels in plasma and aqueous humor can compromise lysosomal degradation in the outflow pathway cells with aging and contribute to the pathogenesis of glaucoma. Restoration of physiological levels of vitamin C inside the cells might improve their ability to degrade proteins within the lysosomal compartment and recover tissue function.

Cathepsin X promotes 6-hydroxydopamine-induced apoptosis of PC12 and SH-SY5Y cells

The cysteine carboxypeptidase cathepsin X is an important player in degenerative processes under normal ageing and pathological conditions. In the present study, we investigated the potential role of cathepsin X in 6-hydroxydopamine (6-OHDA)-induced toxicity in the pheochromocytoma cell line PC12 and neuroblastoma cell line SH-SY5Y. Cells exposed to 6-OHDA demonstrated alterations in the protein level of cathepsin X and activity of cathepsin X. Downregulation of cathepsin X expression by siRNA attenuated the neuronal death caused by 6-OHDA. Treatment with specific cathepsin X inhibitor AMS36 protected cells against 6-OHDA mediated cytotoxicity, resulting in reduced cell death and apoptosis. Furthermore, AMS36 reversed 6-OHDA-induced loss of tyrosine hydroxylase and attenuated 6-OHDA-induced activation of caspase-3, triggering apoptosis, intracellular generation of reactive oxygen species and mitochondrial dysfunction, including the release of cytochrome c and an imbalanced Bax/Bcl-2 ratio. Moreover, AMS36 interfered with NF-κB activation by blocking degradation of IκBα, preventing NF-κB translocation to the nucleus. Our data provide the first evidence that inhibition of cathepsin X protects both, PC12 and SH-SY5Y cells against 6-OHDA toxicity and indicate that cathepsin X may be responsible for dopamine neuron death, involved in the pathogenic cascade event for the neurodegenerative disorders, such as Parkinson's disease.

Inhibition of lysosomal function in macrophages incubated with elevated glucose concentrations: A potential contributory factor in diabetes-associated atherosclerosis

ObjectivePeople with diabetes have an elevated risk of atherosclerosis. The accumulation of lipid within macrophage cells in the artery wall is believed to arise via the uptake and subsequent processing of modified low-density lipoproteins (LDL) via the endo-lysosomal system. In this study the effects of prolonged exposure to elevated glucose upon macrophage lysosomal function was examined to determine whether this contributes to modulated protein catabolism. MethodsHuman monocytes were isolated from white-cell concentrates and differentiated, in vitro, into monocyte-derived macrophages over 11 days in medium containing 5–30 mmol/L glucose. Murine macrophage-like J774A.1 cells were incubated similarly. Lysosomal cathepsin (B, D, L and S) and acid lipase activities were assessed using fluorogenic substrates; cathepsin protein levels were examined by Western blotting. Lysosomal numbers were examined using the lysomotropic fluorescent dye LysoTracker DND-99, measurement of aryl sulfatase activity, and quantification of lysosome-associated membrane glycoprotein-1 (LAMP-1) by Western blotting. ResultsExposure to elevated glucose, but not mannitol, resulted in a concentration-dependent decrease in the activity, and to a lesser extent protein levels, of four lysosomal cathepsins. Acid lipase activity was also significantly reduced. Arysulfatase activity, LAMP-1 levels and lysosomal numbers were also decreased at the highest glucose concentrations, though to a lesser extent. ConclusionLong term exposure of human and murine macrophage cells to elevated glucose levels result in a depression of lysosomal proteolytic and lipase activities. This may result in decreased clearance and cellular accumulation of (lipo)proteins and contribute to the accumulation of modified proteins and lipids in diabetes-associated atherosclerosis.