Cathepsin S provokes interleukin-6 (IL-6) trans-signaling through cleavage of the IL-6 receptor in vitro.

The cytokine interleukin-6 (IL-6) fulfills its pleiotropic functions via different modes of signaling. Regenerative and anti-inflammatory activities are mediated via classic signaling, in which IL-6 binds to the membrane-bound IL-6 receptor (IL-6R). For IL-6 trans-signaling, which accounts for the pro-inflammatory properties of the cytokine, IL-6 activates its target cells via soluble forms of the IL-6R (sIL-6R). We have previously shown that the majority of sIL-6R in human serum originates from proteolytic cleavage and mapped the cleavage site of the IL-6R. The cleavage occurs between Pro-355 and Val-356, which is the same cleavage site that the metalloprotease ADAM17 uses in vitro. However, sIL-6R serum levels are unchanged in hypomorphic ADAM17ex/ex mice, making the involvement of ADAM17 questionable. In order to identify other proteases that could be relevant for sIL-6R generation in vivo, we perform a screening approach based on the known cleavage site. We identify several candidate proteases and characterize the cysteine protease cathepsin S (CTSS) in detail. We show that CTSS is able to cleave the IL-6R in vitro and that the released sIL-6R is biologically active and can induce IL-6 trans-signaling. However, CTSS does not use the Pro-355/Val-356 cleavage site, and sIL-6R serum levels are not altered in Ctss-/- mice. In conclusion, we identify a novel protease of the IL-6R that can induce IL-6 trans-signaling, but does not contribute to steady-state sIL-6R serum levels.

HMGB1 mediates homocysteine-induced endothelial cells pyroptosis via cathepsin V-dependent pathway.

Endothelial cells injury and pro-inflammation cytokines release are the initial steps of hyperhomocysteinemia (HHcy)-associated vascular inflammation. Pyroptosis is a newly identified pro-inflammation form of programmed cell death, causing cell lysis and IL-1ß release, and characterized by the caspases-induced cleavage of its effector molecule gasdermins (GSDMs). However, the effect of homocysteine (Hcy) on endothelial cells pyroptosis and the underlying mechanisms have not been fully defined. We have previously reported that Hcy induces vascular endothelial inflammation accompanied by the increase of high mobility group box-1 protein (HMGB1) and lysosomal cysteine protease cathepsin V in endothelial cells, and other studies have shown that HMGB1 or cathepsins are involved in activation of NLRP3 inflammasome and caspase-1. Here, we investigated the role of HMGB1 and cathepsin V in the process of Hcy-induced pyroptosis. We observed an increase in plasma IL-1ß levels in HHcy patients and mice models, cathepsin V inhibitor reduced the plasma IL-1ß levels and cleavage of GSDMD full-length into GSDMD N-terminal in the thoracic aorta of hyperhomocysteinemia mice. Using cultured HUVECs, we observed that Hcy promoted GSDMD N-terminal expression, silencing GSDMD or HMGB1 rescued Hcy-induced pyroptosis. HMGB1 also increased GSDMD N-terminal expression, and silencing cathepsin V reversed HMGB1-induced pyroptosis. HMGB1 could increase lysosome permeability, and silencing cathepsin V attenuated HMGB1-induced activation of caspase-1. In conclusion, this study has delineated a novel mechanism that HMGB1 mediated Hcy-induced endothelial cells pyroptosis partly via cathepsin V-dependent pathway.

Proteomic Analysis Identifies Distinct Glomerular Extracellular Matrix in Collapsing Focal Segmental Glomerulosclerosis.

BACKGROUND: The mechanisms leading to extracellular matrix (ECM) replacement of areas of glomerular capillaries in histologic variants of FSGS are unknown. This study used proteomics to test the hypothesis that glomerular ECM composition in collapsing FSGS (cFSGS) differs from that of other variants. METHODS: ECM proteins in glomeruli from biopsy specimens of patients with FSGS not otherwise specified (FSGS-NOS) or cFSGS and from normal controls were distinguished and quantified using mass spectrometry, verified and localized using immunohistochemistry (IHC) and confocal microscopy, and assessed for gene expression. The analysis also quantified urinary excretion of ECM proteins and peptides. RESULTS: Of 58 ECM proteins that differed in abundance between cFSGS and FSGS-NOS, 41 were more abundant in cFSGS and 17 in FSGS-NOS. IHC showed that glomerular tuft staining for cathepsin B, cathepsin C, and annexin A3 in cFSGS was significantly greater than in other FSGS variants, in minimal change disease, or in membranous nephropathy. Annexin A3 colocalized with cathepsin B and C, claudin-1, phosphorylated ERK1/2, and CD44, but not with synaptopodin, in parietal epithelial cells (PECs) infiltrating cFSGS glomeruli. Transcripts for cathepsins B and C were increased in FSGS glomeruli compared with normal controls, and urinary excretion of both cathepsins was significantly greater in cFSGS compared with FSGS-NOS. Urinary excretion of ECM-derived peptides was enhanced in cFSGS, although in silico analysis did not identify enhanced excretion of peptides derived from cathepsin B or C. CONCLUSIONS: ECM differences suggest that glomerular sclerosis in cFSGS differs from that in other FSGS variants. Infiltration of activated PECs may disrupt ECM remodeling in cFSGS. These cells and their cathepsins may be therapeutic targets.

FSGS-Causing INF2 Mutation Impairs Cleaved INF2 N-Fragment Functions in Podocytes.

BACKGROUND: Mutations in the gene encoding inverted formin-2 (INF2), a member of the formin family of actin regulatory proteins, are among the most common causes of autosomal dominant FSGS. INF2 is regulated by interaction between its N-terminal diaphanous inhibitory domain (DID) and its C-terminal diaphanous autoregulatory domain (DAD). INF2 also modulates activity of other formins, such as the mDIA subfamily, and promotes stable microtubule assembly. Why the disease-causing mutations are restricted to the N terminus and how they cause human disease has been unclear. METHODS: We examined INF2 isoforms present in podocytes and evaluated INF2 cleavage as an explanation for immunoblot findings. We evaluated the expression of INF2 N- and C-terminal fragments in human kidney disease conditions. We also investigated the localization and functions of the DID-containing N-terminal fragment in podocytes and assessed whether the FSGS-associated R218Q mutation impairs INF2 cleavage or the function of the N-fragment. RESULTS: The INF2-CAAX isoform is the predominant isoform in podocytes. INF2 is proteolytically cleaved, a process mediated by cathepsin proteases, liberating the N-terminal DID to function independently. Although the N-terminal region normally localizes to podocyte foot processes, it does not do so in the presence of FSGS-associated INF2 mutations. The C-terminal fragment localizes to the cell body irrespective of INF2 mutations. In podocytes, the N-fragment localizes to the plasma membrane, binds mDIA1, and promotes cell spreading in a cleavage-dependent way. The disease-associated R218Q mutation impairs these N-fragment functions but not INF2 cleavage. CONCLUSIONS: INF2 is cleaved into an N-terminal DID-containing fragment and a C-terminal DAD-containing fragment. Cleavage allows the N-terminal fragment to function independently and helps explain the clustering of FSGS-associated mutations.

Cathepsin S in the spinal microglia facilitates morphine-induced antinociceptive tolerance in rats.

Microglia-derived lysosomal cysteine protease cathepsin S (CatS) is increasingly recognized as important mediators to exaggerate nociceptive signaling. However, the patterns and functional roles of CatS in morphine tolerance have never been investigated. Here, we showed that mature form of CatS was exclusively upregulated in the spinal microglia following chronic morphine treatment. Pharmacological blockade of CatS before each morphine treatment prolonged the efficacy of morphine analgesia. Correspondingly, inhibition of CatS suppressed activation of spinal microglia and phosphorylated p38 MAPK. Finally, intrathecal injection of selective microglia inhibitor minocycline reduced upregulation of mature CatS induced by chronic morphine treatment. Our data provide novel insight into the cellular mechanisms underlying morphine antinociceptive tolerance and highlight CatS as a therapeutic target for preventing morphine tolerance.

Hydrogen sulfide inhibits ATP-induced neuroinflammation and Aß1-42 synthesis by suppressing the activation of STAT3 and cathepsin S.

Neuroinflammation and excessive ß-amyloid1-42 (Aß1-42) generation contribute to the pathogenesis of Alzheimer's disease (AD). Emerging evidence has demonstrated that hydrogen sulfide (H2S), an endogenous gasotransmitter, produces therapeutic effects in AD; however, the underlying mechanisms remain largely elusive. In the present study, we investigated the effects of H2S on exogenous ATP-induced inflammation and Aß1-42 production in both BV-2 and primary cultured microglial cells and analyzed the potential mechanism(s) mediating these effects. Our results showed that NaHS, an H2S donor, inhibited exogenous ATP-stimulated inflammatory responses as manifested by the reduction of pro-inflammatory cytokines, ROS and activation of nuclear factor-κB (NF-κB) pathway. Furthermore, NaHS also suppressed the enhanced production of Aß1-42 induced by exogenous ATP, which is probably due to its inhibitory effect on exogenous ATP-boosted expression of amyloid precursor protein (APP) and activation of ß- and γ-secretase enzymes. Thereafter, we found that exogenous ATP-induced inflammation and Aß1-42 production requires the activation of signal transducer and activator of transcription 3 (STAT3) and cathepsin S (Cat S) as inhibition of the activity of either proteins attenuated the effect of exogenous ATP. Intriguingly, NaHS suppressed exogenous ATP-induced phosphorylation of STAT3 and the activation of Cat S. In addition, we observed that NaHS led to the persulfidation of Cat S at cysteine-25. Importantly, mutation of cysteine-25 into serine attenuated the activity of Cat S stimulated by exogenous ATP and subsequent inflammation and Aß1-42 production, indicating its involvement in H2S-mediated effect. Taken together, our data provide a novel understanding of H2S-mediated effect on neuroinflammation and Aß1-42 production by suppressing the activation of STAT3 and Cat S.

Vibrio alginolyticus VepA Induces Lysosomal Membrane Permeability and Cathepsin-Independent Cell Death.

The bacterium Vibrio alginolyticus, an opportunistic pathogen in humans, has a type III secretion system (T3SS) that is responsible for its cytotoxicity toward eukaryotic cells. The effector of T3SS that is responsible for the cytotoxicity had not been identified. Here we demonstrate that VepA, a homolog of the T3SS effector in V. parahaemolyticus, is required for cytotoxicity in V. alginolyticus. VepA induces lysosomal membrane permeabilization, and it allows the leakage of only small molecules into the cytosol. Our findings revealed that VepA induces cathepsin-independent cell death in mammalian cells. The ferrous ion, one of the small molecules in the lysosome contents, appears to be involved in the cell death caused by V. alginolyticus VepA.

Role of cathepsin S In periodontal wound healing-an in vitro study on human PDL cells.

BACKGROUND: Cathepsin 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. METHODS: An 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. RESULTS: Cathepsin 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. CONCLUSIONS: Our 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.

[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.

Role of Cathepsin S in Periodontal Inflammation and Infection.

Cathepsin S is a cysteine protease and regulator of autophagy with possible involvement in periodontitis. The objective of this study was to investigate whether cathepsin S is involved in the pathogenesis of periodontal diseases. Human periodontal fibroblasts were cultured under inflammatory and infectious conditions elicited by interleukin-1ß and Fusobacterium nucleatum, respectively. An array-based approach was used to analyze differential expression of autophagy-associated genes. Cathepsin S was upregulated most strongly and thus further studied in vitro at gene and protein levels. In vivo, gingival tissue biopsies from rats with ligature-induced periodontitis and from periodontitis patients were also analyzed at transcriptional and protein levels. Multiple gene expression changes due to interleukin-1ß and F. nucleatum were observed in vitro. Both stimulants caused a significant cathepsin S upregulation. A significantly elevated cathepsin S expression in gingival biopsies from rats with experimental periodontitis was found in vivo, as compared to that from control. Gingival biopsies from periodontitis patients showed a significantly higher cathepsin S expression than those from healthy gingiva. Our findings provide original evidence that cathepsin S is increased in periodontal cells and tissues under inflammatory and infectious conditions, suggesting a critical role of this autophagy-associated molecule in the pathogenesis of periodontitis.

Papel das proteases na erosão dentinária / The role of proteases on dental erosion

Na dentina, a matriz orgânica desmineralizada tem um papel protetor contra desafios erosivos subsequentes. Porém, essa camada pode ser degradada por proteases, como as metaloproteinases da matriz (MMPs) e cisteína catepsinas (CCs). Recentemente, o uso de inibidores de proteases da matriz surgiu como uma importante ferramenta preventiva contra a erosão dentinária. Entretanto, o(s) mecanismo(s) exato(s) pelo(s) qual(is) os inibidores de proteases podem prevenir a erosão dentinária, bem como os tipos de proteases mais envolvidas neste processo ainda não são completamente conhecidos. O projeto foi desenvolvido em 2 subprojetos, com os seguintes objetivos: A)Subprojeto 1:Avaliar o papel das proteases na progressão da erosão dentária; B)subprojeto 2: Testar o potencial inibitório do NaF em CCs dentinárias. Para cumprir esses objetivos, foram utilizadas dentina de terceiros molares humanos para a preparação dos espécimes. A)Subprojeto1:Blocos de dentina (4 X 4 x 2 mm) (n=119) foram obtidos de raízes. Os espécimes foram divididos em 7 grupos de acordo com o seu tratamento (E-64, inibidor especifico II de catepsinas B, clorexidina, galardina NaF, placebo) ou sem tratamento, géis foram aplicados uma única vez sobre a superfície e feito o desafio erosivo (90s, 4x por dia por 5 dias) e feita analise perfilométrica. Os espécimes foram incubadas em solução contendo colagenase de Clostridium histolyticum tipo VII por 96hrs e então feita uma segunda analise perfilometrica para se determinar a espessura da MOD. Dois espécimes foram separados para análise de microscopia eletrônica de varredura. B)Subprojeto 2: Palitos de dentina (6 mm X 2 mm X 1 mm) (n=60) foram cortados da porção médio coronária dos dentes e completamente desmineralizados por imersão em EDTA 0,5 M (pH7,4) por 30 dias e lavados em água deionizada sob constante agitação a 4ºC por 72 h. Os espécimes foram divididos em 6 grupos (E-64, NaF e controle negativo, pH 5,5 ou 7,2) e incubados em saliva artificial contendo seus respectivos inibidores por 24 h 7 dias e 21 dias; ao termino de cada período, os espécimes eram pesados para avaliar a perda de massa e analisada a presença de CTX. A)Subprojeto 1: a perda de tecido desmineralizado (m, média± SD) foi: CHX 8,4±1,7b, Gala 8,6±1,9b, IECB 9,6±1,4a, E64 9,9±1,3a, NaF 9,9±1,7a, P 10,9±2,2a, ST 11,0±1,5a. A perda de tecido mineralizado foi: CHX 15,4±2,2b, Gala 16,0±1,8b, IECB 17,6±2,4a, E64 17,6±2,0a, NaF 17,3±2,8a, P 19,1±2,1a, ST 18,9±2,4a. Os inibidores de MMP reduziu significativamente a perda de matriz orgânica e tecido mineralizado em comparação com os outros grupos (p<0,05). Não foi achada diferença significante entre a espessura da matriz orgânica desmineralizada remanescente (p=0,845). B)Subprojeto 2: Na perda de massa houve diferença significante em relação ao inibidor (F=20,047, p<0,0001) e tempo de incubação (F=222,462, p<0,0001) com significante interação entre esses critérios, nos período de menor tempo de incubação, a perda foi similar para todos os grupos testados, no período de maior tempo de incubação, o grupo contendo NaF demostrou os melhores resultados. Na analise de CTX, houve diferença significante em relação aos inibidores (F46,543, p<0,0001), pH (F=14,836, p<0,0004) e tempo de incubação (F=161,438, p<0,0001) com significante interação entre esses critérios, como ocorrido na perda de massa, não houve diferença estatística nos períodos de menor incubação. No período de maior tempo de incubação, mais uma vez o grupo NaF mostrou os melhores resultados. No valor acumulado de CTX, os grupos E64 e controle negativo tiveram os maiores valores de CTX acumulado, o grupo NaF, independente do pH mostrou redução significante em relação aos demais grupos. Após analise dos resultados dos dois subprojetos, podemos indicar que as MMPs são as proteases de maior importância na progressão da erosão dentinária, assim, sua inibição é de maior importância para a redução desta patologia. Mesmo as CCs não exercendo papel direto para a progressão da erosão, elas são efetivas na cascata da ativação de outras proteases, como as próprias MMPs. Com isso, sua inibição também pode ser importante para a redução indireta da progressão da erosão. Neste presente estudo, pudemos comprovar que o NaF tem potencial inibitório sobre as CCs dentinárias, assim, sugerindo um novo inbidor de CCs. Com os resultados deste estudo, podemos afirmar que as MMPs são as principais proteases na progressão da erosão dentinária e que o NaF tem potencial inibitório nas CCs dentinárias.(AU)

In the dentine, the demineralized organic matrix has a protector part against the following erosive challenges. Nevertheless, this layer can be degraded by proteases, like the matrix metalloproteinases (MMPS) and cystein cathepsins (CCs). Recently, the use of proteases of the matrix´s inhibitors, emerged as an important preventive tool against the dentinária erosion. However, the exact mechanisms from which the inhibitors of the proteases may prevent the dentin erosion, as much as the kinds of proteases more involved in this process are not completely known yet. Therefore, the general objective of this project was to investigate the part of the two main proteases of the matrix (MMPs and CCs) in the dental erosion. The project was developed in 2 subprojects, with the following objectives: A)Subproject 1: Evaluate the part of the proteases in the progression of the dental erosion; B)subproject 2: To test the NaF inhibitory potencial in the dentin CCs. To accomplish these objectives, human third molar dentin were used for the preparation of the specimens, obtained in the surgery and urgency clinics of FOB-USP (subproject 1) or granted by the University of Oulu (subproject 2). A) Subproject 1: Dentine blocks 4 X 4 X 2 mm) (n=119) were obtained from the roots of the obtained teeth. The specimens were divided in 7 groups according with their treatment. Gels containing inhibitors (E-64, specific cathepsin B inhibitor II, chlorhexidine, galardin NaF, placebo), or without treatment, were produced, applied only one time over the surface and made the erosive challenge (90s, 4x a day for 5 days) and made profilometric analysis. The specimens were incubated in a solution containing collagenase of Clostridium histolyticum type VII for 96 hours and then a second profilometric analysis was made to determine the thickness of the MOD. Two specimens were separated for the electronic microscopy scan analysis. B) Subproject 2: Dentine sticks (6 mm X 2 mm X 1 mm) (n=60) were cut from the medium coronary portion of the teeth and completely demineralized by immersion in EDTA 0,5 M (pH7,4) ifor 30 days and washed in deionized water under constant agitation in 4º C for 72 hours. The specimens were divided in 6 groups (divided by inhibitors: E-64, NaF and negative control, pH 5,5 or 7,2) and incubated in artificial saliva containing their respective inhibitors for 24 hours, 7 days and 21 days; by the end of each period, the specimens were weighted to evaluate the loss of mass and analised the presence of CTX. A)Subproject 1: the loss of demineralized tissue (m, média± SD) was : CHX 8,4±1,7b, Gala 8,6±1,9b, IECB 9,6±1,4a, E64 9,9±1,3a, NaF 9,9±1,7a, P 10,9±2,2a, ST 11,0±1,5a. The loss of demineralized tissue was: CHX 15,4±2,2b, Gala 16,0±1,8b, IECB 17,6±2,4a, E64 17,6±2,0a, NaF 17,3±2,8a, P 19,1±2,1a, ST 18,9±2,4a. The MMP inhibitors reduced significantly the loss of organic matrix and demineralized tissue in comparison with other groups (p<0,05). There was no significant difference found between the thickness of the remaining demineralized organic matrix.(p=0,845). B)Subproject: In the loss of mass, there was a significant difference in relation to the inhibitor (F=20,047, p<0,0001) and incubation time (F=222,462, p<0,0001) with significant interaction between these criteria, in the periods of lesser time of incubation, the loss was similar for all the tested groups, in the period of higher time of incubation, the group containing NaF demonstrated the best results. In the analysis of CTX, there was significant difference in relation the inhibitors (F46,543, p<0,0001), pH (F=14,836, p<0,0004) and time of incubation (F=161,438, p<0,0001)with significant interaction between these criteria, as occurred in the mass loss, there was no statistic difference in the period of lesser incubation. In the period of higher time of incubation, once again, the NaF group demonstrated the best results. The CTX accumulated value, the E64 groups and negative control had the greater accumulated values of CTX, the NaF group, regardlessof the pH, demonstrated significant reduction in relation to the other groups. After the analysisof the results of both subprojects, we can indicate that the MMPs are the proteases of greater importance in the progression of the dentin erosion, thus, its inhibition is of graeter importance for the reduction of this pathology. Even the CCs don´t playing the part directly for the progression of erosion, they are effective in the cascade of the activation of other proteases, like the MMPs themselves. In this manner, its inhibition can also be important for the indirect reduction of the progression of the erosion. In this present study, we can prove that the NaF has inhibiting potential over the dentin CCs, thus, suggesting a new inhibitor of CCs. With the results of this study, we can affirm that the MMPs are the main proteases in the progression of the dentin erosion and that the NaF has inhibiting potential in the dentin CCs.(AU)

Chronic cathepsin inhibition by E-64 in Dahl salt-sensitive rats.

Cysteine cathepsins are lysosomal enzymes expressed in the kidneys and other tissues, and are involved in the maturation and breakdown of cellular proteins. They have been shown to be integrally involved in the progression of many cardiovascular and renal diseases. The goal of this study was to determine the involvement of cysteine cathepsins in the development of salt-sensitive hypertension and associated kidney damage. In our experiments, Dahl salt-sensitive (SS) rats were fed an 8% high salt NaCl diet and intravenously infused with the irreversible cysteine cathepsin inhibitor E-64 (1 mg/day) or the vehicle (control). Both the control and E-64 infused groups developed significant hypertension and kidney damage, and no difference of the mean arterial pressure and the hypertension-associated albuminuria was observed between the groups. We next tested basal calcium levels in the podocytes of both control and infused groups using confocal calcium imaging. Basal calcium did not differ between the groups, indicative of the lack of a protective or aggravating influence by the cathepsin inhibition. The efficacy of E-64 was tested in Western blotting. Our findings corresponded to the previously reported, E-64 induced increase in cathepsin B and L abundance. We conclude that the inhibition of cysteine cathepsins by E-64 does not have any effects on the blood pressure development and kidney damage, at least under the studied conditions of this model of SS hypertension.

Cathepsin S Cleavage of Protease-Activated Receptor-2 on Endothelial Cells Promotes Microvascular Diabetes Complications.

Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia-induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68(+) intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage-derived circulating PAR2 agonist and mediator of endothelial dysfunction-related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases.

Differential role of endogenous cathepsin and microorganism in texture softening of ice-stored grass carp (Ctenopharyngodon idella) fillets.

BACKGROUND: Texture deterioration often negatively affects sensory attributes and commercial values of ice-stored fish fillets. The mechanism of softening of fish fillets during chilling storage is not fully resolved. Grass carp is a predominant freshwater fish species in China. The objective of the present study was to investigate the differential role of endogenous cathepsin and microorganisms in texture softening of ice-stored grass carp fillets. RESULTS: The fillets were immersed in either NaN3 solution to reduce microbial activity or in iodoacetic acid solution to exclude cathepsin activity before ice storage. Treatment with NaN3 reduced microbial load of fillets below 2 log CFU g(-1) muscle during the entire storage period, and had no significant influence on the cathepsin activity and proteolysis. But the shear force of fillets treated with NaN3 decreased by 66% after 21 days of storage. Meanwhile, treatment with iodoacetic acid inactivated cathepsin B and B + L but did not significantly affect the microbial growth of fillets. Compared to NaN3 treatment, iodoacetic acid effectively alleviated softening and inhibited the increase in TCA-soluble peptides during storage. CONCLUSION: This study demonstrated that proteolysis induced by endogenous cathepsins, rather than microorganisms, plays an important role in texture softening of ice-stored grass carp fillets. © 2015 Society of Chemical Industry.

Roles of cathepsins in pancreatic cancer.

Pancreatic cancer is an aggressive disease with rapid invasion and metastasis. Extracellular matrix degrading enzymes play an important role in cancer cell invasion and migration. Cathepsins are a group of proteolytic enzymes, which are responsible for the matrix turnover. Among the cathepsins, more number of studies have focused upon cysteine cathepsins. The function and activities of these enzymes are interwoven and their interplay causes the activation of one another by following a proteolytic cascade. This review focuses on differential expression of cathepsins in different types of pancreatic cancer and controls, importance of cathepsins in various phenomena responsible for tumorigenesis and its spread in experimental and human studies. Thus, cathepsins and its expression in pancreatic cancer may be used as potential biomarkers and may prove to be important therapeutic targets if tested clinically.

Possible role of proteases in preconditioning of brain cells to pathological conditions.

Preconditioning (PC) is one of the most effective strategies to reduce the severity of cell damage, in particular of nervous tissue cells. Although PC mechanisms are studied insufficiently, it is clear that proteases are involved in them, but their role has yet been not studied in detail. In this work, some mechanisms of a potential recruiting of proteases in PC are considered. Our attention is mainly focused on the protease families of caspases and cathepsins and on protease receptors. We present evidence that just these proteins are involved in the PC of brain cells. A hypothesis is proposed that secreted cathepsin B is involved in the realization of PC through activation of PAR2 receptor.

A novel cysteine cathepsin inhibitor yields macrophage cell death and mammary tumor regression.

Although cysteine cathepsins have been identified as key regulators of cancer growth, their specific role in tumor development remains unclear. Recent studies have shown that high activity levels of tumor cathepsins are primarily a result of increased cathepsin activity in cancer-promoting tumor-associated macrophages (TAMs). To further investigate the role of cysteine cathepsin activity in normal and polarized macrophages, we established in vitro and in vivo models of macrophage differentiation and polarization and used a novel cysteine cathepsin inhibitor, GB111-NH2, to block the activity of cathepsins B, L and S. Here we show that in vitro, cysteine cathepsin inhibition yields both apoptosis and proliferation of macrophages, owing to increased oxidative stress. Proteomic analysis of cathepsin- inhibited macrophages demonstrates inhibition of autophagy, suggesting a likely cause of elevated reactive oxygen species (ROS) levels. In vivo models of mammary cancer further show that cathepsin inhibition yields TAM death owing to increased ROS levels. Strikingly, apoptosis in TAMs yields a seemingly cell non-autonomous death of neighboring cancer cells, and regression of the primary growth. These results show that cysteine cathepsin inhibitors can specifically trigger macrophage cell death and may function as an effective anticancer therapy in tumors with high levels of TAMs.

Matrix metalloproteinases and other matrix proteinases in relation to cariology: the era of 'dentin degradomics'.

Dentin organic matrix, with type I collagen as the main component, is exposed after demineralization in dentinal caries, erosion or acidic conditioning during adhesive composite restorative treatment. This exposed matrix is prone to slow hydrolytic degradation by host collagenolytic enzymes, matrix metalloproteinases (MMPs) and cysteine cathepsins. Here we review the recent findings demonstrating that inhibition of salivary or dentin endogenous collagenolytic enzymes may provide preventive means against progression of caries or erosion, just as they have been shown to retain the integrity and improve the longevity of resin composite filling bonding to dentin. This paper also presents the case that the organic matrix in caries-affected dentin may not be preserved as intact as previously considered. In partially demineralized dentin, MMPs and cysteine cathepsins with the ability to cleave off the terminal non-helical ends of collagen molecules (telopeptides) may lead to the gradual loss of intramolecular gap areas. This would seriously compromise the matrix ability for intrafibrillar remineralization, which is considered essential in restoring the dentin's mechanical properties. More detailed data of the enzymes responsible and their detailed function in dentin-destructive conditions may not only help to find new and better preventive means, but better preservation of demineralized dentin collagenous matrix may also facilitate true biological remineralization for the better restoration of tooth structural and mechanical integrity and mechanical properties.

Role of dentin MMPs in caries progression and bond stability.

Dentin can be described as a biological composite with collagen matrix embedded with nanosized hydroxyapatite mineral crystallites. Matrix metalloproteinases (MMPs) and cysteine cathepsins are families of endopeptidases. Enzymes of both families are present in dentin and collectively capable of degrading virtually all extracellular matrix components. This review describes these enzymes and their presence in dentin, mainly focusing on their role in dentin caries pathogenesis and loss of collagen in the adhesive hybrid layer under composite restorations. MMPs and cysteine cathepsins present in saliva, mineralized dentin, and/or dentinal fluid may affect the dentin caries process at the early phases of demineralization. Changes in collagen and noncollagenous protein structure may participate in observed decreases in mechanical properties of caries-affected dentin and reduce the ability of caries-affected dentin to remineralize. These endogenous enzymes also remain entrapped within the hybrid layer during the resin infiltration process, and the acidic bonding agents themselves (irrespective of whether they are etch-and-rinse or self-etch) can activate these endogenous protease proforms. Since resin impregnation is frequently incomplete, denuded collagen matrices associated with free water (which serves as a collagen cleavage reagent for these endogenous hydrolase enzymes) can be enzymatically disrupted, finally contributing to the degradation of the hybrid layer. There are multiple in vitro and in vivo reports showing that the longevity of the adhesive interface is increased when nonspecific enzyme-inhibiting strategies are used. Different chemicals (i.e., chlorhexidine, galardin, and benzalkonium chloride) or collagen cross-linker agents have been successfully employed as therapeutic primers in the bonding procedure. In addition, the incorporation of enzyme inhibitors (i.e., quaternary ammonium methacrylates) into the resin blends has been recently promoted. This review will describe MMP functions in caries and hybrid layer degradation and explore the potential therapeutic role of MMP inhibitors for the development of improved intervention strategies for MMP-related oral diseases.