Activation cathepsin l

Histological examination and renal tubular injury score fig. The results showed that CPL increased renal damage and renal dysfunction during renal injury induced by sepsis. Renal cortical tissue sections were stained with specific antibodies against mouse CD68 to evaluate the degree of macrophage infiltration during renal injury after CLP surgery fig. The sham-operated mice showed the smallest interstitial macrophage staining. However, mice with CLP surgery showed significant macrophage accumulation 24 h later.

Sepsis-induced ARI is a common disease associated with high morbidity and mortality[ 16 ]. Macrophages are pleiotropia cells of the innate immune system, and their functions span host defense, cytotoxicity, clearance of apoptotic cells and promotion of tissue repair. Macrophages are also considered to be important mediators of renal injury in other experimental models of kidney disease, including transplantation, obstruction and glomerulonephritis[ 17 ].

Macrophages can be released from the bone marrow into the circulatory system and differentiate into resident macrophages to respond to certain stimuli. Inflammation is closely related to the activation of macrophages. M1 macrophages show pro-inflammatory activity, while M2 macrophages are involved in the resolution of inflammation[ 18 , 19 ]. According to their potential mechanism, M1 macrophages play a key role in antigen presentation, pro-inflammatory cytokine secretion and phagocytosis.

The rationality of CPL as a marker of infectious organ dysfunction can be supported by its strong effect on the release of immune factors, immune cells and endothelial cells production in the early stage of infection, which may be the pathogenic factor of sepsis[ 20 , 21 ].

In the past decade, many studies have confirmed that CPL can act as a chemotaxis signal of macrophages and promote the accumulation of macrophages at the infected site[ 22 ]. CPL in endothelial tissue can induce monocytes to accumulate at the infected site through calcium-dependent channels. CPL can also activate monocytes and increase the phagocytosis of macrophages[ 23 ]. In this study, the expression of CPL increased significantly 24 h after the operation and decreased in the following h, which is consistent with the following findings.

Previous studies have shown that 24 h after sepsis-induced ARI, there were a large number of M1 macrophages, so it was possible to determine the potential relationship between macrophages and CPL. Previous studies have shown that CPL induced renal tubular cell inflammation and renal endothelial cell loss in mice, which is a symptom of renal damage[ 24 ]. Studies have shown that CPL increased renal tubular injury and renal dysfunction and plays an important role in the initial inflammatory response associated with renal injury.

However little is known about the mechanisms of invasion mediated by V-ATPases. The V-ATPases are a family of ATP-dependent proton pumps whose primary role is in acidification of intracellular compartments, where they function in such processes as macromolecule degradation, receptor-mediated endocytosis and vesicular trafficking [ 5 ].

V-ATPases are also critical for bone resorption by osteoclasts, acid-base balance in the kidney, sperm maturation, insulin secretion and neurotransmitter uptake [ 5 ]. V-ATPases are upregulated in tissue samples from highly invasive pancreatic carcinomas [ 6 ] and treatment of a human cancer cell line with anti-sense oligonucleotides to the V-ATPase c subunit decreased invasion in vitro [ 7 ].

Inhibition of V-ATPase expression in hepatocellular carcinoma cells using siRNAs reduces invasiveness of these cells in vitro and metastasis in vivo [ 8 ]. V-ATPases are found intracellularly in nearly all cells, but only a few specialized cells such as osteoclasts have V-ATPases in their plasma membrane.

The mechanism by which plasma membrane V-ATPases might be contributing to a metastatic phenotype has not been worked out, but it has been proposed that cell surface V-ATPases may work by activating lysosomal proteases that are secreted by invasive cancer cells [ 10 ].

Cysteine cathepsins are a family of proteases that prototypically reside within the lysosome where they function in the degradation and recycling of proteins. Some of the members of the cysteine cathepsin family also have more specific roles within certain cell types [ 11 ].

The family of cysteine cathepsins consists of 11 proteins in humans [ 12 ] and several different cysteine cathepsins have been found in cancer cells either associated with the cell membrane or secreted from the cells [ 13 ].

Increased expression of cathepsin B has been identified as a marker for poor prognosis for cancer survival [ 14 , 15 ]. Inhibiting cathepsin B has been shown to limit metastasis to bone and lungs in breast cancer cells [ 16 , 17 ].

Cathepsin L secretion is associated with late stage and malignant tumors [ 18 , 19 ]. Both cathepsin B and cathepsin L are known to activate extracellular proteases that degrade extracellular matrix proteins [ 12 , 20 ]. There is also in vitro evidence that cathepsin B and cathepsin L can directly degrade components of the extracellular matrix [ 19 , 21 ]. In addition, cathepsin L has been shown to degrade E-cadherin an important cell adhesion protein [ 19 ].

We wanted to investigate whether either of these cathepsins might be working with the V-ATPase to facilitate invasion by human breast cancer cells. We hypothesized that by inhibiting V-ATPases the activity of one or both of these cathepsins in the extracellular space conditioned media would decrease.

This should be due to two factors: 1 inhibiting the V-ATPase will effect vesicular trafficking thus affecting secretion and more interestingly 2 inhibiting cell surface V-ATPases will lead to decreased processing of the secreted pro form of the cathepsins to the active form thus leading to less cathepsin activity in the extracellular space.

An equivalent volume of dimethyl sulfoxide DMSO was added to control cells. Protein concentration for all samples was determined using Pierce BCA protein assay kit. Briefly, assays were done in 96 well plates where samples were added to buffer with or without the cathepsin inhibitors, CA for cathepsin B and the cathepsin L inhibitor provided with the kit Z-Phe-Phe-fluoromethylketone.

Protein concentration for all samples was determined by BCA protein assay Pierce. The blots were imaged using the Alpha Innotech Imaging system and the bands were quantified using the Alpha Innotech software.

To quantify the amount of protein seen on the Western blots, the density of bands representing different versions of the cathepsins [pre-pro 55 kDa , pro- Cat B — 39 kDa; Cat L — 38 kDa or active Cat B — 30 kDa; Cat L — 32 kDa forms] were divided by the density of the loading control bands.

To determine the activity of cathepsins both within the cell and outside of the cell, MB cells were initially grown in complete growth media for 48 h and then the media were replaced with media lacking serum.

Concanamycin A a specific inhibitor of V-ATPases was added to the cells at this time to a final concentration of nM. Control cells were treated with an equivalent volume of DMSO. Twenty four hours after treatment, the conditioned media were collected and concentrated and the cells were harvested and lysed. The conditioned media and cell lysates were then assayed for cathepsin B and cathepsin L activity using fluorescence-based cathepsin activity assays.

As shown in Fig. The activity of cathepsin B was appreciably higher than cathepsin L activity in cell lysates as well. Following treatment with concanamycin A, there was very little change in the activity of cathepsin L in conditioned media, but the activity of cathepsin B trended lower.

In addition, treatment with concanamycin A significantly decreased the activity of cathepsin B in cell lysates. The change in cathepsin activity is shown as percent reduction in Fig. However, in the conditioned media, there was significantly greather reduction in cathepsin B activity than reduction of cathepsin L following ConA treatment. Conditioned media a and cell lysates b from cells grown for 24 h in the absence 0 nM or presence nM of concanamycin A were assayed for cathepsin activity.

Cathepsins are produced initially in a pre-pro form which is cleaved to a pro-form and directed to the lysosome where they are processed into the final active form. In these metastatic breast cancer cells cathepsins B and L have been redirected to be secreted from the cell. The pro and active forms of both proteins are present in the conditioned media. The band densities were quantified and normalized to a loading control galectin. Band densities relative to the pre-pro form of cathepsins B or L.

Percent reduction of band densities following treatment with concanamycin A for cathepsins B e and L f. We quantified the change in the amount of Cat L and Cat B following ConA treatment in both conditioned media and cell lysates. The band densities were normalized to loading controls. For cathepsin B there is a significant decrease in the amount of the 55 kDa, 39 kDa, and 30 kDa forms of the protein. Since the initial discovery in bovine, a large number of cathelicidins have been characterized from an array of phylogenetically distant vertebrates Tomasinsig and Zanetti, ; Uzzell et al.

During the inflammatory response, the proform will further be processed to make functionally active by protease cleavage of the antimicrobial domain that will be released to sites of microbial infection. The proform isolated from the rabbit PMN in vitro exhibits high affinity to bind Escherichia coli and modulate the antibacterial actions of other leukocyte proteins on this Gram-negative bacterium Zarember et al.

Recent literature reported that some proforms can be mapped onto the cell surface of PMN. Although extensive studies have focused on the antimicrobial domains of the cathelicidin family due to their central roles in both innate and adaptive immunity through direct antimicrobial activity and as immune modulators and mediators of inflammation, the body of evidence for their possible immune-related defense functions of CLDs has been growing in recent years.

For instance, Zaiou et al. Such inhibitory activity on cathepsin L could be associated with its structural similarity to type 2 cystatins which belong to secreted natural inhibitors of family C1 papain-like cysteine peptidases Dieckmann et al.

Given the key role of cathepsin L in antigen presentation Honey and Rudensky, , it is possible that the regulation of its activity by CLD can establish a link between innate and adaptive immunity, which will undoubtedly provide new insights into more understanding of specific and independent functions of CLD in host defense.

Mutational experiments combined with a structure complex model allow us to correlate this activity to a structurally flexible loop of PG3 CLD which could be involved in a direct interaction with cathepsin L. Biological significance of the activating effect on cathepsin L has been discussed in the context of antigen presentation.

We used the similar method described by Sanchez et al. For the detailed description of the expression and purification methods, see Supplemental material 2. Protein concentration was determined according to the biuret method Layne, The samples were mixed in an eppendorf tube with the same volume of the matrix solution. The spectra were obtained in the linear mode by summing laser shots with an ion source voltage 1 of 19 kV, ion source 2 of The instrument was calibrated externally by cytochrome c Bruker.

Spectra were measured at room temperature from to nmusing a quartz cell of 1. The CD spectra measure was performed by averaging three scans. Cathepsin L 0. Spectra were taken at a peptide concentration of 0. Firstly, we assayed the inhibitory activity of CLD-W on three proteases human neutrophil elastase, human liver cathepsin L and bovine pancreas trypsin in the presence or absence of the recombinant protein using the fluorescence-conjugated casein substrate.

As expected on the basis of their structural feature, CLD-W lacked of any detectable effect on trypsin and elastase data not shown. As presented in Fig. Concentration of cathepsin L is 0.

The assays were performed in triplicate in one experiment. Previous studies have identified the functional motifs of cystatins involved in interacting with cathepsin L, including: 1 Q x V x G motif in the first binding loop where x represents any amino acid; 2 PW in the second binding loop; 3 a glycine at the extremity of the N-terminal region Rzychon et al. However, these motifs are completely lacking in all characterized CLDs. Interestingly, both human and pig CLDs target cathepsin L either inhibiting or activating its activity.

These observations suggest that the cystatin-fold itself other than sequence conservation is the key functional determinant. We thus hypothesized that CLDs and cystatins could adopt a similar strategy to bind cathepsin L, in which the L1 and L2 loops are presumably involved. This result showed that the activating effect significantly decreased after the partial deletion of the L2 loop, which provides evidence in support of the involvement of the L2 loop in the activation of cathepsin L.

It is also worthy mentioning that the deletion of the 7-aa of the L2 loop alone was not completely sufficient to diminish its activating function, suggesting that other minor determinants of activation also may exist. Fields from two independent LAMP1 experiments are shown c. Quantitation is shown in Supplementary Figures S5a and c. Nilotinib treatment increased the amount of EEA1-positive vesicles in low-density and intermediate-density fractions fractions 1—4 , decreased the amount of LAMP1 present in high-density fractions fraction 12 and slightly reduced Rab7 expression in high-density fractions Figures 5a—d.

These data are consistent with the immunofluorescence data and indicate that nilotinib induces an accumulation of low-density early endosomes and decreases lysosome number. Thus, these data are consistent with the notion that c-Abl induces NMH1 degradation by promoting its accumulation in lysosomes Supplementary Figure S6.

Nilotinib inhibits endosome—lysosome trafficking and induces NMH1 accumulation in endosomal fractions and depletion from lysosomal fractions. Twelve fractions were collected, the indicated fractions 1 , 3 , 4 6 8 9 , 10 , 11 , 12 12 from vehicle- and nilotinib-treated cells were run on the same gel and blotted with EEA1, Rab7 or LAMP1 antibodies top.

As all 24 fractions could not be run on one gel and fractions 2,5 and 7 were missing from the first gel, consecutive vehicle- and nilotinib-treated endosomal fractions 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 were rerun on a second gel.

Western blots from one of three experiments are shown. Two independent experiments are shown for NMH1. In contrast, expression of exogenous NMH1 into invasive cancer cells inhibited matrigel invasion, anchorage-independent growth and metastasis. In control vector-infected s cells, imatinib treatment significantly reduced matrigel and three-dimensional invasion; however, this effect was rescued in cells expressing the NMH1 shRNA Figure 6e and f , indicating that NMH1 upregulation is required for imatinib to inhibit invasion.

Representative fields were photographed. Lysates from a representative experiment were probed with antibodies. Non-invasive and invasive cells were scored and invasive index calculated cells with invasive extensions versus the total number of cells multiplied by Arrows denote invading cells.

Fluorescent surface lung metastases were quantitated on Day IVIS flux values for all mice right. Cores of bad quality were not scored. Scores were grouped into high, medium and low scores. Representative cores left and immunohistochemical score distributions right are shown. Here, we show that cathepsins, proteases with known roles in invasion and metastasis, directly cleave and degrade NMH1, which not only explains how NMH1 expression is lost, but also provides new insight into how intracellular cathepsins contribute to invasion and metastasis degradation of metastasis suppressors.

Interestingly, c-Abl also activates cathepsins by promoting endosome maturation, which is important as abnormal vesicular trafficking has a key role in cancer progression.

Interestingly, silencing Arg had no affect on cathepsin activation in s cells, but it did decrease procathepsin expression, and prevent NMH1 degradation. Thus, cathepsin proforms, which are transcriptionally upregulated by both c-Abl and Arg, also may be involved in NMH1 degradation. Alternatively, Arg may affect a different step in vesicular trafficking, or may promote activation of another cathepsin, not yet identified. Lysosomal proteases degrade plasma membrane-bound proteins for example, RTKs, E-cadherin via clathrin-mediated endocytosis followed by trafficking to early endosomes.

Drosophila NM23 awd promotes synaptic vesicle internalization and endocytosis of adherens junction components. Agents that induce upregulation of NMH1 are currently being pursued in the clinic.

Leakiness was detected in untreated cells; Shield1 treatment further increased expression. The Arg kinase assay antibody was previously described. Immunoblotting using fractions or cell lysates in RIPA buffer 57 was performed using the protocols of the antibody manufacturers.

Kinase assays were performed as previously described. Pathologists scored tissue microarrays blindly on a microscope, except the melanoma array, which they scored at the computer. Mice were treated with vehicle 0. Some data were normalized to vehicle or scrambled control and analyzed with one-sample t -tests.

Two-tailed values are shown; 0. Multiple mechanisms underlie metastasis suppressor function of NMH1 in melanoma. Naunyn Schmiedebergs Arch Pharmacol ; : — Clinical-translational approaches to the NmH1 metastasis suppressor. Clin Cancer Res ; 14 : — Smith SC, Theodorescu D. Learning therapeutic lessons from metastasis suppressor proteins. Nat Rev Cancer ; 9 : — Saha A, Robertson ES. Functional modulation of the metastatic suppressor NmH1 by oncogenic viruses. FEBS Lett ; : — The role of metastasis suppressor genes in metastatic dormancy.

APMIS ; : — Implication of metastasis suppressor NMH1 in maintaining adherens junctions and limiting the invasive potential of human cancer cells. Cancer Res ; 70 : — The NmH1 metastasis suppressor as a translational target.

Eur J Cancer ; 46 : — Cysteine cathepsins: from structure, function and regulation to new frontiers. Biochim Biophys Acta ; : 68— Specialized roles for cysteine cathepsins in health and disease.

J Clin Invest ; : — ABL1 fusion genes in hematological malignancies: a review. Eur J Haematol ; 86 : — Srinivasan D, Plattner R. Activation of Abl tyrosine kinases promotes invasion of aggressive breast cancer cells. Cancer Res ; 66 : — Aggressive breast cancer cells are dependent on activated Abl kinases for proliferation, anchorage-independent growth and survival. Oncogene ; 27 : —