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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2013 January 25; 288(4): 2201–2209.
Published online 2012 November 30. doi:  10.1074/jbc.M112.436394
PMCID: PMC3554893

Cathepsin H Mediates the Processing of Talin and Regulates Migration of Prostate Cancer Cells*An external file that holds a picture, illustration, etc.
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Abstract

The cytoskeletal protein talin, an actin- and β-integrin tail-binding protein, plays an important role in cell migration by promoting integrin activation and focal adhesion formation. Here, we show that talin is a substrate for cathepsin H (CtsH), a lysosomal cysteine protease with a strong aminopeptidase activity. Purified active CtsH sequentially cleaved a synthetic peptide representing the N terminus of the talin F0 head domain. The processing of talin by CtsH was determined also in the metastatic PC-3 prostate cancer cell line, which exhibits increased expression of CtsH. The attenuation of CtsH aminopeptidase activity by a specific inhibitor or siRNA-mediated silencing significantly reduced the migration of PC-3 cells on fibronectin and invasion through Matrigel. We found that in migrating PC-3 cells, CtsH was co-localized with talin in the focal adhesions. Furthermore, specific inhibition of CtsH increased the activation of αvβ3-integrin on PC-3 cells. We propose that CtsH-mediated processing of talin might promote cancer cell progression by affecting integrin activation and adhesion strength.

Keywords: Cell Migration, Cysteine Protease, Integrins, Molecular Cell Biology, Tumor Cell Biology

Introduction

Tumor metastases represent a major oncological clinical problem and are a leading cause of death in cancer patients. The migration of individual tumor cells through the tissue is a prerequisite for invasion and metastasis. The transmembrane receptors of the integrin family enable cell migration by mediating adhesion to the extracellular matrix (ECM).3 Integrins are activated by inside-out signaling that increases the affinity for ECM ligands. The focal adhesion (FA) protein talin-1 (referred to here as talin), which acts as an intracellular ligand, is a key participant in integrin activation and consequently in cell migration. The interaction of talin with integrin cytoplasmic regions causes conformational changes within the extracellular domains, increasing binding affinities for ECM ligands at the cell surface (13). In addition, talin functions as a molecular bridge to link the cytoplasmic domains of integrins with the actin cytoskeleton, enabling the cell to exert the force on the ECM that is required for cell traction and movement across the barriers (4, 5).

Talin is composed of an N-terminal head domain (residues 1–400), which includes the principal integrin-binding site, and a large C-terminal rod domain (residues 482–2541), which contains lateral integrin-binding sites and binding sites for actin and vinculin (6). The talin head is composed of the FERM domain, which contains the F1–F3 subdomains with a further extension toward the N terminus termed the F0 subdomain (7). Structural studies revealed that F3 contains a phosphotyrosine-binding fold that interacts with the NPXY motif present in β-integrin cytoplasmic tails (8). This interaction is thought to disrupt the salt bridge between the α- and β-integrin tails that normally keeps integrins in the low affinity state (6, 9, 10). Although the other subdomains are not directly involved in integrin binding, they cooperate with talin F3 to mediate integrin activation by targeting talin to integrins (11) and by interaction with the adjacent phospholipids of the plasma membrane (9, 12, 13).

Because of their involvement in cell migration, integrin-containing adhesion complexes are dynamic structures that undergo repeated cycles of formation and disassembly (14). Thus, accurate regulation of the talin-mediated linkage of integrins to the actin cytoskeleton in these complexes is essential for cell migration. The cleavage of talin into head and rod domains by the calcium-dependent cysteine protease calpain has a key role in FA turnover (15). The head domain stimulates integrin activation and maintains adhesions at the cell edge protrusions (16, 17).

Cathepsin H (CtsH) is a lysosomal cysteine protease with a unique aminopeptidase activity (18, 19). There is growing evidence that expression of CtsH is increased in conditions such as breast carcinoma (20), melanoma (21), gliomas (22), colorectal carcinoma (23), and prostate carcinoma (24). However, the role of CtsH in tumor progression is not well understood. In this study, we identify a CtsH as a new cysteine protease capable of proteolytic cleavage of talin. It cleaves sequentially the N terminus of the talin F0 head domain by its monoaminopeptidase activity. Through talin processing, CtsH could affect the migration of metastatic PC-3 prostate cancer cells on fibronectin and invasion through Matrigel. We show that CtsH co-localizes with talin at FAs, where it modulates the activation of αvβ3-integrins.

EXPERIMENTAL PROCEDURES

Cell Line and Inhibition of CtsH

All cell lines used were obtained from American Type Culture Collection (Manassas, VA). The PC-3 prostate cancer cell line and the MDA-MB-231 breast tumor epithelial cell line were cultured in DMEM/nutrient mixture F-12 (1:1; Invitrogen) supplemented with antibiotics (penicillin/streptomycin; Sigma), 2 mm glutamine (Sigma), and 10% FBS (HyClone, Logan, UT). MCF-7 cells were cultured in DMEM/nutrient mixture F-12 (1:1) supplemented with 10% FBS, 10 μg/ml insulin (Sigma), 0.5 μg/ml hydrocortisone (Sigma), 20 ng/ml epidermal growth factor (Sigma), 2 mm glutamine, and antibiotics. HepG2 cells were grown in DMEM supplemented with 10% FBS, 2 mm glutamine, and antibiotics. To inhibit the intracellular activity of CtsH, PC-3 cells were incubated for 24 h with the CtsH-specific inhibitor (CTSHi) H2N-Ser(benzyloxy)-CHN2 (25), synthesized at the Faculty of Pharmacy of the University of Ljubljana (26). To knock down CtsH expression, cells were transiently transfected with CtsH-specific siRNA (HSS102489, Invitrogen) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Control cells were transfected with control siRNA-A (sc-37007, Santa Cruz Biotechnology) using the same procedure.

Antibodies

Mouse monoclonal antibody (mAb) 1D10 and sheep polyclonal antibody (pAb) against CtsH were raised by our group (27). Goat pAbs against talin (sc-7534) and against β3-integrin (sc-6627) were purchased from Santa Cruz Biotechnology. Mouse anti-actin mAb (ab3280) from Abcam (Cambridge, MA). Mouse anti-vinculin mAb (V9131) and Cy3-conjugated rabbit anti-LAMP1 mAb (L0419) were from Sigma. The HRP-conjugated secondary antibodies used were donkey anti-goat antibodies (sc-2020) purchased from Santa Cruz Biotechnology and goat anti-mouse antibodies (AP308P) obtained from Millipore. All Alexa-labeled secondary antibodies were from Molecular Probes.

Talin Cleavage Site Studies

CtsH was tested for its ability to degrade sequence VALSLKISIGNVVKTMQFEPST (synthesized by Biosynthesis Inc.), corresponding to the N-terminal region of the talin head. The peptide substrate (200 μm) was incubated with active CtsH (1.42 μm) isolated from human liver (27) for 1 h at 37 °C in phosphate buffer (pH 6.8) containing 1 mm EDTA and 2.5 mm dithiothreitol. For inhibition of CtsH, CTSHi (5 μm) was preincubated with active CtsH for 10 min at 37 °C. Each sample was separated by reverse-phase HPLC using a Gemini C18 column (5 μm, 110 Å, 150 × 46 mm; Phenomenex). Peak fractions were analyzed using a Q-Tof Premier mass spectrometer (in ESI+ mode). Sequences were assigned to proteolytic fragments on the basis of the known sequence of the undigested peptide and the determined molecular masses of the fragments.

ELISA

CtsH-specific ELISA was performed as described (27). Briefly, microtiter plates (Nunc-ImmunoTM modules) were coated overnight with 5 μg/ml sheep pAb against CtsH in 0.01 m carbonate/bicarbonate buffer (pH 9.6) at 4 °C. After blocking (2% BSA in PBS), the wells were filled with cell lysates (protein concentration was set to 0.3 mg/ml) or CtsH standards and incubated for 2 h at 37 °C. The wells were then washed and filled with HRP-conjugated mAb 1D10. Following another 2-h incubation at 37 °C, 200 μl/well 3,3′,5,5′-tetramethylbenzidine (Sigma) and 0.012% H2O2 were added for 15 min, and the reaction was stopped by the addition of 50 μl of 2 m H2SO4. Finally, the absorbance was measured at 450 nm with a Tecan Safire2 plate reader, and the concentration of CtsH was calculated from the standard curve.

Two-dimensional Electrophoresis

PC-3 cells were treated with 2.5 μm CTSHi or dimethyl sulfoxide (DMSO) as a control for 24 h. Afterward, cells were washed once with PBS, harvested with trypsin in PBS and 0.5% EDTA, and solubilized in lysis buffer containing 2 M thiourea (Sigma), 7 m urea (Sigma), 4% CHAPS (Sigma), 1% dithiothreitol (Sigma), 2% IPG buffer (GE Healthcare), and 1× inhibitor mixture tablet (Roche). Samples (containing 200 μg of total protein) were mixed with rehydration solution (2 m thiourea, 7 m urea, 2% CHAPS, 2% IPG buffer, and 0.002% bromphenol blue) and applied to 13-cm immobilized pH 3–10 nonlinear gradient IPG strips (GE Healthcare) according to the manufacturer's protocol. Isoelectric focusing was performed at a total of 20,000 kV-h, followed by equilibration in 6 m urea, 75 mm Tris base, 2% (w/v) SDS, 30% (v/v) glycerol, 0.002% (w/v) bromphenol blue, and 65 mm DTT. The strips were then transferred to the same solution without DTT but with iodoacetic acid (260 mm) and incubated for 15 min at room temperature with shaking. The second-dimension separation was done using a 12% SDS-polyacrylamide gel on a PROTEAN II xi 2-D cell system at 100 V for 1 h and then at 300 V until the dye front reached the bottom of the gel. Samples were transferred onto nitrocellulose membranes (GE Healthcare) following standard protocols. Membranes were blocked with 5% skimmed milk in PBS for 30 min and incubated with primary antibodies overnight at 4 °C. After washing with Tween/PBS, membranes were incubated with species-specific HRP-conjugated secondary antibodies for 2 h at room temperature. Bands were detected using SuperSignal West Dura enhanced chemiluminescent substrate (Thermo Fisher Scientific).

Immunofluorescence Microscopy

PC-3 cells were grown on glass cover slides in 24-well plates 24 h prior to the experiment. Before labeling, cells were fixed with 10% formalin for 30 min and permeabilized with 0.05% Triton X-100 in PBS (pH 7.4) for 10 min. Nonspecific staining was blocked with 3% BSA in PBS (pH 7.4). CtsH was labeled with mAb 1D10 (10 μg/ml of 3% BSA in PBS) or sheep pAb (5 μg/ml of 3% BSA in PBS). Anti-Talin, anti-vinculin, and anti-LAMP1 antibodies were used according to the manufacturers' protocols. For imaging GFP-talin, pRK5/GFP-talin-1 (28) was transiently transfected in PC-3 cells using Lipofectamine 2000. After a 2-h incubation, cells were washed three times with PBS and treated with species-specific Alexa Fluor®-labeled secondary antibodies (2:1000 (v/v)) for 2 h. After washing with PBS, a ProLong antifade kit (Molecular Probes) was mounted on the dried cover slides and allowed to dry overnight at 4 °C. Fluorescence microscopy was performed at room temperature using a Zeiss LSM 510 confocal microscope, and immersion oil was used as imaging medium. Images were analyzed using Zeiss LSM image software 3.0. The percentage of co-localization was calculated as the amount of fluorescence of the co-localizing objects in each component of the image relative to the total fluorescence in that component (29).

Real-time Cell Migration and Invasion Analysis

The invasion and migration of human PC-3 cells were monitored using an xCELLigence System real-time cell analyzer instrument (Roche Applied Science). This novel technology is based on real-time monitoring of cell invasion or migration, and it captures cell responses during the entire course of an experiment, otherwise missed if measured by conventional end point assays such as the Boyden chamber assay. The membranes of CIM-Plates (Roche Applied Science) were coated with a thick layer (20 μl/membrane) of 5 mg/ml Matrigel for an invasion assay and left to incubate for 20 min at 37 °C. For a migration assay, the membranes of CIM-Plates were incubated with 20 μg/ml fibronectin (20 μl/membrane) and left to incubate for 2 h at 37 °C. The lower compartments were filled with complete medium containing 2.5 μm CTSHi. The upper compartments were filled with serum-free medium containing the cells (3 × 104 cells/well) and 2.5 μm CTSHi. For cathepsin silencing, the cells were transfected with CtsH-specific siRNA before the experiment. The cell index (CI) reflects the electrical impedance across the interdigitated microelectrodes integrated on the lower side of the 8-μm pore membrane, caused by the cells invading through the Matrigel or fibronectin, across the membrane, and spreading on the lower side of the membrane. Dynamic CI values were monitored at 15- or 30-min intervals from the time of plating until the end of the experiment (72 h). The data were analyzed with real-time cell analyzer software (Roche Applied Science).

Determination of CtsH Aminopeptidase Activity

PC-3 cells were grown in 12-well plates coated with fibronectin 24 h prior to the experiment. Cell lysates of PC-3 cells were prepared in lysis buffer containing 0.05 m HEPES (pH 5.5), 1 mm EDTA, 0.150 m NaCl, and 1% Triton X-100, and total protein concentration was determined by the Bradford method. The CtsH activity in cell lysates was determined using the fluorogenic CtsH-specific aminopeptidase substrate l-arginine-7-amido-4-methylcoumarin (l-Arg-AMC) (30). 5 μl of 20 μm l-Arg-AMC was added to the wells of a black microplate. The reaction was initiated with 95 μl of lysate sample (final protein concentration of 0.3 mg/ml) in assay buffer (100 mm phosphate buffer (pH 6.8) containing 5 mm cysteine and 1.5 mm EDTA). Prior to the assay, the lysates were incubated in assay buffer for 5 min at 37 °C. Formation of fluorescent degradation products was monitored continuously at an excitation of 380 ± 20 nm and an emission of 460 ± 10 nm on a Tecan Safire2 spectrofluorometer. All measurements were performed in triplicate.

FACS Analysis of 5-Carboxyfluorescein-labeled Cyclo-RGDyK Peptide Binding to the PC-3 Cell Line

PC-3 cells were resuspended with trypsin and washed once with cold PBS. One million cells in a final volume of 200 μl were resuspended in 5 μm precooled 5-carboxyfluorescein-labeled cyclo-RGDyK (cyclo-Arg-Gly-Asp-d-Tyr-Lys) peptide solution (AnaSpec) in PBS and incubated for 30 min at 4 °C. Peptide solutions were removed, and pellets were carefully rinsed twice with PBS. Cells were then rapidly analyzed by flow cytometry. The results are reported as density plots showing 5-carboxyfluorescein fluorescence and PAC1 staining and as mean fluorescence intensity of 5-carboxyfluorescein-labeled cyclo-RGDyK binding. All measurements were performed in triplicate.

Western Blotting

Cell lysates (50 μg of total cell proteins) were heated at 100 °C in reducing sample buffer (Laemmli sample buffer, Bio-Rad) for 10 min, separated on 12% SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk in 0.05% Tween/PBS for 30 min and incubated with primary antibodies overnight at 4 °C. The primary antibodies used were goat anti-β3-integrin pAb, anti-actin mAb, anti-CtsH mAb 1D10, and goat anti-talin pAb. After washing with 0.05% Tween/PBS, the membrane was incubated with species-specific HRP-conjugated secondary antibodies for 2 h at room temperature. Bands were detected using the SuperSignal West Dura enhanced chemiluminescent substrate.

Statistical Analysis

SPSS PC software (release 13.0) was used for statistical analysis. Statistical significance was evaluated by Student's t test. p values of <0.05 were considered to be statistically significant.

RESULTS

CtsH Sequentially Cleaves the N-terminal Region of Talin

To determine whether talin is a substrate for CtsH, a synthetic peptide (VALSLKISIGNVVKTMQFEPST) corresponding to the N-terminal region of the talin head was incubated in vitro with purified active CtsH (26), and the reaction products were separated by HPLC. In the presence of CtsH, the peak representing the original peptide was decreased, and numerous new peaks were detected (Fig. 1A). Inhibition with the specific cell-permeable covalent inhibitor of CtsH (CTSHi) (30) completely abolished CtsH proteolytic activity, and the peak was identical to that of the original peptide. Using mass spectrometry analysis, we identified 26 different cleavage products (Fig. 1B). The results clearly show the N-terminal sequential cleavage of the synthetic peptide, indicating that CtsH processes the N-terminal region of talin primarily by its monoaminopeptidase activity. A previous report showed that CtsH is not able to hydrolyze substrates by its aminopeptidase activity if proline is present at the S1′ position (19). This suggests that CtsH could sequentially remove only the first 18 N-terminal amino acids of talin, stopping at Glu-19–Pro-20. We have confirmed the sequential cleavage of the first 16 amino acids, whereas the cleavage of Gln-17 and Phe-18 was not detected presumably due to the shortness of the peptides (17–22 and 18–22 amino acids).

FIGURE 1.
CtsH mediates the processing of talin. A, direct cleavage of talin by CtsH in vitro. The synthetic peptide corresponding to the first 22 N-terminal amino acids of talin (talin(1–22)) was incubated with or without active CtsH in CtsH activation ...

Next, CtsH-specific ELISA was used to compare the expression of CtsH in four different human cancer cell lines: a prostate cancer cell line (PC-3), two breast cancer cell lines (MDA-MB-231 and MCF-7), and a liver carcinoma cell line (HepG2) (Fig. 1C). Because PC-3 cells expressed the highest levels of CtsH (428.3 ± 56.7 ng/mg), this cell line was used in the subsequent experiments. To assess whether talin is cleaved by CtsH in vivo, PC-3 cells were treated with CTSHi. The cell lysates of control and CTSHi-treated cells were separated by two-dimensional electrophoresis, and talin was detected by immunoblotting (Fig. 1D). Three spots representing unmodified talin and cleaved talin isoforms were identified in the control sample, whereas only unmodified talin was present when the cells were treated with CTSHi. Presumably, the two spots representing the cleaved isoforms were detected by isoelectric focusing due to the proteolytic removal of two positively charged lysine residues (Lys-6 and Lys-14) (Fig. 1B), explaining the more acidic isoelectric points. The resolution of conventional one-dimensional electrophoresis followed by Western blotting was not high enough to detect the processed isoforms of talin in the PC-3 cell lysate; however, the single bands confirm that the antibody against talin is monospecific (Fig. 1E and supplemental Fig. S1A).

Co-localization of CtsH and Talin at the Leading Edge of PC-3 Cells

The spreading of PC-3 cells on fibronectin, the representative ECM component, induced significant changes in CtsH aminopeptidase activity and its intracellular localization. CtsH activity was increased together with the coating concentration of fibronectin (Fig. 2A). The coating concentration that was used in the subsequent experiments (20 μg/ml) induced a 24.3 ± 3.2% increase in CtsH aminopeptidase activity. In addition, we observed that part of the perinuclear CtsH was translocated toward the cell membrane during PC-3 cell spreading on fibronectin (Fig. 2B). The translocated CtsH co-localized with the FA marker vinculin (31).

FIGURE 2.
Co-localization of CtsH and talin at the cell leading edge. A, PC-3 cells were grown for 24 h on different concentrations of fibronectin (FN), and the activity of CtsH was analyzed using the CtsH-specific aminopeptidase substrate l-Arg-AMC. The results ...

To further confirm the potential intracellular interaction of CtsH and talin, we examined if they are co-localized in PC-3 cells. Talin is a structural component and thus a common resident of FAs that form at the leading edge of migratory cells (32). PC-3 cells expressing GFP-talin were left to migrate on fibronectin and fixed and stained for CtsH. Detailed examination revealed the localization of CtsH at the membrane-proximal ends of talin-delimited FAs (Fig. 2C). The co-localization of CtsH with talin at the cell leading edge was further confirmed by co-staining CtsH with endogenous talin (Fig. 2D), suggesting that CtsH processing of talin occurs directly at FAs. According to the quantitative co-localization analysis, 35.7 ± 10.7% of total CtsH was co-localized with talin, and 31.2 ± 4.5% of total talin was co-localized with CtsH in PC-3 cells (Fig. 2E). The monospecificity of the utilized anti-talin and anti-CtsH antibodies was confirmed by Western blotting performed on the PC-3 cell lysate (supplemental Fig. S1, A and B). Additionally, we showed that only a minor fraction (13.2 ± 5.1%) of total cellular CtsH co-localized with the lysosomal marker LAMP1 (supplemental Fig. S2).

Inhibition of CtsH Reduces PC-3 Cell Migration and Invasion

First, the ability of the inhibitor CTSHi and of siRNA-mediated silencing of CtsH to efficiently reduce the aminopeptidase activity of CtsH was confirmed using l-Arg-AMC. The treatment of PC-3 cells with 2.5 μm CTSHi or CtsH-specific siRNA reduced CtsH aminopeptidase activity by 62.7 ± 2.3 and 38.3 ± 0.7%, respectively (Fig. 3A). The effect of CtsH inhibition on the migration and invasion of PC-3 cells was analyzed by xCELLigence, a highly quantitative real-time monitoring system. As shown by the slopes (1/h), representing the cell migrating ability, the migration of PC-3 cells across fibronectin-coated Transwell membrane was reduced by 68.4 ± 4.1% in the presence of CTSHi and by 48.9 ± 18.1% if CtsH activity was regulated by siRNA (Fig. 3B and supplemental Fig. S3). Furthermore, we showed that CtsH is important also for the invasion of PC-3 cells through a thick layer of Matrigel, as this process was significantly attenuated by CTSHi (Fig. 3C and supplemental Fig. S3). Finally, we compared the role of CtsH in the migrating ability of different human cancer cell lines in a single xCELLigence experiment (Fig. 3D). The transmigration of MDA-MB-231 cells with a very high metastatic potential (33) was fast, as the cells crossed the fibronectin-coated Transwell membrane in the first 10 h (Fig. 3D, Migration plot D). The transmigration of PC-3 cells started only after 16 h and slowed down in the 45 h of the experiment. The presence of CTSHi significantly reduced the migration of both cell lines; however, the reduction was less prominent in MDA-MB-231 cells (only 13% reduction) than in PC-3 cells (63% reduction). As expected, the noninvasive MCF-7 (33) and HepG2 (34) cells were not able to transmigrate across the membrane.

FIGURE 3.
Role of CtsH in PC-3 cell migration and invasion. A, the CtsH inhibition by CTSHi (2.5 μm) and by siRNA-mediated silencing was determined with the CtsH-specific substrate l-Arg-AMC. The results are presented as means ± S.D. of two independent ...

CtsH Modulates Integrin Activation

Because previous work indicated that migration of PC-3 cells depends on αvβ3-integrins (35), we studied the ability of CtsH to affect integrin activation in migrating PC-3 cells. PC-3 cells spreading on fibronectin were pretreated with CTSHi, and αVβ3 activation was evaluated by flow cytometry using the fluorescently labeled activation-specific cyclo-RGDyK peptide (36). Remarkably, the inhibition of CtsH increased αVβ3 activation in dose-dependent manner. The population expressing active integrins was 25.7% for control cells and increased up to 45.3% for cells pretreated with 2.5 μm CTSHi (Fig. 4, A and B). Because the total protein levels of β3-integrins were not altered (Fig. 4C), CtsH inhibition influenced the conversion of inactive integrins to their active conformation.

FIGURE 4.
Effect of CtsH inhibition on αVβ3-integrin activation. PC-3 cells growing on fibronectin were preincubated (24 h) with CTSHi (1 and 2.5 μm) or DMSO as a control. Active integrins were stained for flow cytometry with the labeled ...

DISCUSSION

The proteolytic activity of cysteine cathepsins is known to affect the fate of many intracellular as well as extracellular targets, and deregulation of these functions is associated with a number of disease states, including cancer (37). Most of the research to date has focused on cathepsins B and L, whereas other members of the family such as the aminopeptidase cathepsin H have been less studied, and their role in cancer progression remains to be clarified. CtsH was proposed to participate in cancer metastasis by degradation of ECM components (38); however, its monoaminopeptidase activity implies that this enzyme might also have more specific functions.

In this study, we have provided evidence that CtsH is able to cleave the cytoskeletal protein talin, which is known to mediate cell migration by activating integrins (39). CtsH efficiently processed the peptide corresponding to the N terminus of the talin F0 head domain primarily by its monoaminopeptidase activity. The processing of talin was confirmed in vivo using the PC-3 human prostate cancer cell line. This cell line was selected because of its invasive and metastatic behavior (40) as well as high CtsH protein expression and aminopeptidase activity (30). The levels of CtsH in PC-3 cells were the highest among all of the human cancer cell lines tested in this study.

Previous studies demonstrated that close contact of prostate cancer cells with ECM ligands might affect the localization and activity of cysteine cathepsins (41). For instance, cathepsin B has a predominantly perinuclear distribution in PC-3 cells grown on uncoated surfaces but a more peripheral staining in PC-3 cells plated on a thin layer of Matrigel (42). Similarly, we showed that the spreading of PC-3 cells on a fibronectin-coated surface induced increased aminopeptidase activity and the translocation of CtsH toward the cell membrane of the leading edge.

FA formation is initiated upon the binding of integrins to ECM ligands (e.g. fibronectin, vitronectin, collagen) along the cell periphery usually at the leading edge of a cell. Talin is one of the best characterized FA proteins and is involved in the initiation of FA formation by recruiting adaptor proteins to sites of force application (43, 44). We examined the subcellular localization of talin and CtsH in PC-3 cells spreading on fibronectin. As expected, talin was enriched at FAs that were forming along the cell membrane of the leading edge. Our results show that CtsH was co-localized with talin at FAs, implying that these structures might be the site where the N-terminal processing of talin occurs.

The attenuation of CtsH activity by specific inhibition or silencing was shown to significantly reduce the migration of PC-3 cells through fibronectin-coated Transwell membranes and invasion through Matrigel. Notably, CTSHi was considerably less effective in reducing the migration potential of highly metastatic MDA-MB-231 breast cancer cells, which express less CtsH than PC-3 cells.

Talin plays an important role in the establishment and maintenance of integrin-cytoskeleton connections, and loss of talin expression leads to impaired cell adhesion, spreading, and migration (32). A recent study showed that the regulation of talin levels by overexpression or siRNA silencing greatly affects prostate cancer cell migration and invasion (45). Because we identified talin as an intracellular CtsH target in PC-3 cells, we propose that CtsH could regulate prostate cancer cell migration by cleaving talin. However, we cannot exclude the possibility that CtsH affects other migration-associated targets.

Cysteine cathepsins such as cathepsins B, L, and S promote cancer cell invasion by extracellular and intracellular degradation of ECM components (46, 47). The weak endopeptidase activity makes it unlikely that CtsH plays an important role in the direct degradation of the ECM (48). In contrast to other cysteine cathepsins, which are predominantly lysosomal, a study on macrophages showed that the majority of intracellular CtsH activity in macrophages is concentrated in the early endosomes (49). The maintenance of CtsH in the endosomes was shown to be mannose 6-phosphate-independent; however, the exact mechanism remains to be clarified. Additionally, the co-localization analysis of CtsH and the lysosomal marker LAMP1 confirmed our recent study showing that, also in PC-3 prostate cancer cells, lysosomes are not the prevalent localization site of CtsH (50). This suggests that CtsH does not play a major role in the terminal lysosomal degradation of ECM components.

It has been reported that αvβ3-integrin is associated with malignancy and invasion of primary prostate cancer cells (51). Our results demonstrate that the specific inhibition of CtsH promotes the activation of αvβ3-integrin on migrating PC-3 cells. The binding of talin to the β-subunit cytoplasmic tail is a common final step in the integrin activation process (1); thus, it is not unexpected that talin modifications affect the activation of β3-integrins and the dynamics of FAs. For example, the ubiquitylation of the talin head by the E3 ubiquitin ligase Smurf1 leads to talin degradation and subsequently to FA disassembly. This process is regulated by another post-translational modification, Cdk5-dependent phosphorylation of the talin head at Ser-425 (52). Similarly, our results suggest that integrin activation could be influenced by the processing of talin F0 by CtsH aminopeptidase activity. Although the F0 domain is not part of the FERM domain of the talin head, which binds directly to β-integrin tails and to adjacent cell membrane phospholipids, it was shown that it is also important for the activation of β3-integrins (11). The role of the F0 domain in integrin activation remains to be clarified; however, it was proposed that it might affect integrin activation by interacting with other FA components or membrane-associated proteins (13).

In conclusion, we have identified the cytoskeletal protein talin as a specific substrate of CtsH that could be associated with regulation of β3-integrin receptors, FA strength, and cell migration. Because our studies show that CtsH is able to promote the migration and invasion of PC-3 human prostate cancer cells, we suggest that CtsH-specific inhibitors might possess anti-metastatic potential.

Acknowledgment

We thank Dr. Nace Zidar (Faculty of Pharmacy, University of Ljubljana) for synthesizing CTSHi.

*This work was supported by Slovenian Research Agency Grants P4 0127 and J4 4123 (to J. K.).

An external file that holds a picture, illustration, etc.
Object name is sbox.jpgThis article contains supplemental Figs. S1–S3.

3The abbreviations used are:

ECM
extracellular matrix
FA
focal adhesion
CtsH
cathepsin H
CTSHi
CtsH cell-permeable covalent inhibitor
mAb
monoclonal antibody
pAb
polyclonal antibody
DMSO
dimethyl sulfoxide
CI
cell index
l-Arg-AMC
l-arginine-7-amido-4-methylcoumarin.

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