Introduction

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Bronchial and alveolar epithelial cells play an important role in the maintenance of lung structure and function. Goblet cells, Clara cells, and alveolar type II epithelial cells are major producers of components of the pulmonary secretions such as mucin and surfactant. In addition, pulmonary epithelial cells can synthesize a variety of extracellular matrix components and therefore participate in basement membrane and connective tissue metabolism (1, 2). However, little information exists about their possible role in the destructive pathway of components of the interstitial matrix.

Cysteine proteinases (such as cathepsins B, L, S, and H) are thought to be the major proteinases involved in intracellular protein degradation (3). Several studies have shown the release of enzymatically active cathepsins from bronchial epithelial cells (4) and the involvement of these proteases in tissue degeneration (7) and tumor progression (8).

A novel human cysteine protease was recently cloned by several groups (9) and named cathepsin K. It shows a high-sequence homology to the cathepsins S and L. This protease was shown to be expressed predominantly in osteoclasts (12). Recent data have demonstrated that pycnodysostosis is caused by mutations in the cathepsin K gene (15, 16). The expression of cathepsin K in the lung and in other tissues is controversial. Brömme and Okamoto (12) showed a significant messenger RNA (mRNA) expression in the lung tissue, whereas Drake and coworkers (13) described an exclusive expression in bone.

Cathepsin K seems to be the cysteine protease with the highest matrix degradation activity yet known. It was shown that cathepsin K cleaves collagen type I with higher efficiency than do cathepsin L and S. Its elastinolytic activity is higher than that of cathepsin L and pancreatic elastase (17). The capability of cathepsin K to degrade components of the extracellular matrix, especially of elastin at neutral pH, suggests an involvement of this protease in the process of tissue destruction and remodeling in the lung. Fundamental data on the occurrence and the activity of this particular enzyme are missing as yet.

Using a combination of immunologic, enzymologic, and molecular biologic methods, this study demonstrates that cathepsin K is expressed in the lung tissue and shows the cellular distribution of this enzyme.

	Materials and Methods

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Lung-tissue samples were obtained at surgery from patients with lung tumors. The samples were isolated from areas without morphologic signs of neoplastic cells. The tissue was formalin-fixed and paraffin-embedded for use in immunhistologic studies, or shock-frozen in liquid nitrogen. The alveolar epithelial cell-like tumor cell line A549 was obtained from DSMZ (Braunschweig, Germany). The cell line BEAS-2B, which was derived from virus-transformed human bronchial epithelial cells, was a kind gift from A. Gillissen (Klinikum Bergmannsheil, Bochum, Germany). Both cell lines were grown in Iscove's modified Dulbecco's medium with 10% fetal calf serum (both from PAA Laboratories, Cölbe, Germany), 1% antibiotic-antimycotic solution (Sigma, Deissenhofen, Germany) and 1 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (Life Technologies, Eggenstein, Germany).

The polyclonal anti-cathepsin K antiserum was raised in rabbits against recombinant cathepsin K as described previously (10). Recombinant cathepsin K was purified from the culture supernatant of Pichia pastoris transfected with human procathepsin K complementary DNA (cDNA) (18). For reverse transcriptase-polymerase chain reaction (RT-PCR) experiments, sense and antisense primers were designed using previously published cDNA sequences for the human cathepsins K, B, and L (12, 19). Specific sense and antisense primers were selected as follows: cathepsin K: sense primer 5'-TCCATCCATAACCTTGAGGCTT, antisense primer 5'-CCACAGCCATCATTCTCAGACACA; cathepsin B: sense primer 5'-GCCTGCAAGCTTCGATGCAC, antisense primer 5'-CTATTGGAGACGCTGTAGGA; cathepsin L: sense primer 5'-CAGGCAGGTGATGAATGGCT, antisense primer 5'-CAGGCCTCCATTATCCTGAA. These primers corresponded to cDNA fragments of 361, 464, and 324 base pair (bp) bases for the cathepsins K, B, and L, respectively. The primers were synthesized and purified by Life Technologies.

For Northern blot analyses of cathepsin K, the resulting PCR products were ³²P-labeled, using the Rediprime labeling kit (Amersham, Braunschweig, Germany).

The substrates benzyloxycarbonyl-phenyl-arginyl-7-amido-4-methylcoumarin (Z-FR-AMC), benzyloxycarbonyl-glycyl-prolyl-arginyl - 7 - amido - 4 - methylcoumarin (Z-GPR-AMC), benzyloxycarbonyl-glycyl-prolyl-arginyl-4-methoxy--naphtylamine (Z-GPR-4MNA), and benzyloxycarbonyl-arginyl-arginyl - 4 - methoxy -  - naphtylamine (Z-RR-4MNA) were obtained from Bachem (Heidelberg, Germany). The cysteine protease inhibitors L-trans-epoxysuccinyl-Leu-4-guanidinobutylamide (E64) and L-trans-epoxysuccinyl-Ile-Pro-OH propylamide (CA074) were obtained from Sigma and Bachem, respectively.

Northern Blot Analysis

PolyA mRNA was prepared with Dynabeads (Dynal, Oslo, Norway) according to the manufacturer's instructions. mRNA (2 µg) from the epithelial cell lines A549 and BEAS-2B was electrophoresed on a formalin gel (1.2%) and the RNA was blotted onto a nylon membrane. Cathepsin K mRNA was detected by a radioactively labeled cDNA probe (310 bp) for 4 h at 65°C in Rapid Hyb Buffer (Amersham). The blots were washed twice and exposed to Kodak BioMax MS-1 film at 70°C for 14 h. Following hybridization with cathepsin K, blots were stripped and rehybridized with -actin as a semiquantitative control. These blots were exposed for 4 h.

PCR Analysis

Total RNA was isolated using the RNeasy kit (Qiagene, Hilden, Germany). The RT reaction and the PCR amplification were done exactly as described by Alcorn and associates (20) using the thermocycler PTC-200 (MJ Research, Inc., Watertown, MA) and touchdown cycling protocol. The amplified products were verified by their predicted sizes. In addition, random samples were digested with appropriate restriction enzymes (SalI, Sau3aI, and BglII). All products were visualized after electrophoresis on an ethidium bromide-stained 2% agarose gel (MetaPhor agarose; FMC, Rockland ME).

Flow Cytometric Analysis

Cells were permeabilized using the Fix & Perm Kit (An der Grub, Kaumberg, Austria) and incubated with the cathepsin K antiserum (diluted 1:10) for 60 min. The samples were washed and incubated with fluorescein isothiocyanate (FITC)-conjugated rabbit antiserum (DIANOVA, Hamburg, Germany) for 60 min. For specificity control experiments, cathepsin K antiserum was preincubated with saturating amounts of purified cathepsin K for 60 min and used for intracellular staining as indicated. The samples were analyzed by means of flow cytometry (FACSCalibur; Becton Dickinson, Heidelberg, Germany).

Western Blot Analysis

Frozen samples of lung tissue were crushed in liquid nitrogen before addition of lysis buffer (50 mM Tris, 0.75% Triton X-100, 10 mg/ml phenylmethylsulfonyl fluoride, 1 mg/ml Aprotinin, and 1 mg/ml Leupeptin. The tissue samples were incubated for 20 min on ice, reducing sample buffer was added, and the probes were boiled for 5 min before being loaded onto a 15% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) gel. After separation, the proteins were transferred to an enhanced chemiluminescence (ECL)-nitrocellulose membrane (Amersham). Nonspecific binding was blocked by overnight incubation in sodium phosphate buffer (PBS) containing 5% nonfat dried milk. The transfers were then incubated for 90 min with cathepsin K antiserum (1:5,000 in PBS/Tween). The blots were washed and incubated for 90 min with peroxidase-labeled antirabbit F(ab)₂ (1:5,000), and visualized using the chemiluminescent SuperSignal substrate (Pierce, Rockford, IL). For detection of cystatin B expression, the blots were stained with a rabbit anti-cystatin B-antibody (1:1,000; Quartett, Berlin, Germany). The blots were developed using the ProtoBlot Western Blot AP System (Promega, Madison, WI).

Immunohistochemistry

Paraffin-embedded tissue blocks were cut, using a microtome, and the 5-µm sections were placed on coated glass slides. The sections were dried overnight, dewaxed in xylene, and rehydrated into 0.1 M Tris buffer. After three washing steps in Tris buffer, the sections were overlaid either with the rabbit cathepsin K antiserum (1:2,000) or with antiserum preincubated with saturating amounts of purified cathepsin K (negative control). A standard avidin-biotin complex method (alkaline phosphatase) was performed according to the manufacturer's protocol (Vectastain Kit; Boehringer Ingelheim, Heidelberg, Germany). New fuchsin was used a substrate. Sections were counterstained with hematoxylin and mounted in gelatin.

Enzymatic Activity of Cell Lysates

Pellets of the cell lines A549 and BEAS-2B were lysed in assay buffer (50 mM sodium acetate buffer, pH 5.5, containing 2.5 mM dithiothreitol [DTT] and 2.5 mM ethylenediaminetetraacetic acid [EDTA]) with 1% Triton X-100. The samples were centrifuged at 14,000 × g for 10 min, and the resulting supernatants were used for determination of enzymatic activity as described by Kirschke and Wiederanders (21), with minor modifications. Briefly, supernatants of 5 × 10⁵ cells were incubated with one of the methylcoumarylamide substrates, Z-FR-AMC (5 µM) or Z-GPR-AMC (80 µM), for 1,000 s, and the reaction was terminated by the addition of stop buffer (3). The resulting fluorescence was measured using a Fluorolite microplate reader (Dynex, Chantilly, VA).

Cytochemical Cathepsin K Activity

Cathepsin K activity was detected at the cellular level according to the method of Dolbeare and Smith (22) by means of the substrate Z-GPR-4MNA (1 mg/ml) and the cathepsin B-specific inhibitor CA074 (10 µM). In control experiments, the cathepsin B activity was visualized with the substrate Z-RR-4MNA (1 mM). The liberated 4MNA was precipitated and detected by 5-nitro-salicylaldehyde (Sigma). The reaction buffer was 0.1 M PBS, pH 6.0, supplemented with 1.3 mM EDTA, 1 mM DTT, and 3 mM L-cysteine. The cells were harvested from cultures and seeded on glass slips. After an incubation period of 24 h, the cells were fixed in 1% formaldehyde for 10 min at room temperature, washed in the reaction buffer, and incubated with the substrate solution at 37°C for 20 min. The reaction was stopped by several washes with PBS. The slips were mounted on glass slides. The fluorescence was monitored with an Axiovert microscope (Zeiss, Jena, Germany) equipped with a BP 450-490/FT 510/LP 515-565.

	Results

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The cathepsin K expression was characterized at mRNA, protein, and enzymatic levels using different molecular biologic, immunochemical, and biochemical methods.

Expression of Cathepsin K mRNA

The DNA transcription is the first prerequisite of protein expression. The mRNA synthesis is strongly regulated for the majority of proteins. To characterize the expression of the cathepsin K mRNA, lung epithelial cell lines and lung tissues were analyzed using PCR and Northern blotting.

Northern blot analyses of the mRNA isolated from the epithelial cell lines BEAS-2B and A549 with a specific probe for cathepsin K demonstrated a single band of 1.7 kb (Figure 1). For estimation of the relative amounts of the cathepsin K mRNA expression in comparison with the cathepsins B and L, RT-PCR analyses of the cell lines were performed. As documented in Figure 2A, the level of the cathepsin K expression was significantly lower than those of the cathepsins B and L in BEAS-2B cells and lower than that of cathepsin L in A549 cells. Figure 2B shows the mRNA expression of the cathepsins B, L, and K in lung tissues of two patients. The PCR shown in the figures was performed from the same RNA preparation for the three cathepsins and -actin. One representative experiment is shown, out of the three runs of each PCR for each cathepsin and -actin.

View larger version (50K): [in this window] [in a new window]   Figure 1.   Northern blot analysis for human cathepsin K and - actin in A549 (A) and BEAS-2B (B) cells. Equal amounts (2 µg/ lane) of mRNA were electrophoresed on a formalin gel, blotted, and hybridized with 32P-cDNA probes.

View larger version (42K): [in this window] [in a new window]   Figure 2.   Semiquantitative RT-PCR analysis of cathepsin B (B), cathepsin L (L), cathepsin K (K), and -actin (A) mRNA expressed by A549 and BEAS-2B cells (A) and lung tissues of two patients (B). The target product sizes were verified using mol wt markers (M).

Cathepsin K Protein Expression

The cathepsin K protein expression in lysates of cell lines and lung tissues was studied by flow cytometry, immunohistochemistry, and Western blotting.

In the lung epithelial cell lines A549 and BEAS-2B, a method for cytoplasmic immunostaining of cathepsin K and their flow cytometric detection was established. The presence of the cathepsin K protein could be demonstrated in both cell types (Figure 3). In Western blots of the cell lines A549 and BEAS-2B, significant amounts of cystatin B could be visualized (Figure 4).

View larger version (13K): [in this window] [in a new window]   Figure 3.   Flow cytometric detection of cathepsin K in BEAS-2B and A549 cells. Permeabilized cells were incubated with cathepsin K-antiserum (solid lines), followed by a FITC-labeled antirabbit antiserum. In negative control experiments, the antiserum was preincubated with saturating amounts of purified cathepsin K (fine lines).

View larger version (25K): [in this window] [in a new window]   Figure 4.   Western blot analysis of cystatin B expression in the cell lines A549 (1) and BEAS-2B (2). The immunoreactive band at about 12 kD corresponds to the expected mol wt of cystatin B.

In lysates of normal human lung tissue, the cathepsin K antiserum hybridized with major immunoreactive bands at molecular weights of 46, 27, and 12 kD (Figure 5). These bands correspond to the expected molecular weights of procathepsin K (46 kD) and mature cathepsin K (27 kD) (10, 14). It could be assumed that the 12-kD band corresponds to the propeptide.

View larger version (34K): [in this window] [in a new window]   Figure 5.   Western blot analysis of cathepsin K in human lung tissues. The lung-tissue lysates were separated by SDS-PAGE (15%) and electroblotted on a nitrocellulose membrane. The immunoreactive bands at 46 and 27 kD are consistent with pro- and mature forms of cathepsin K, respectively. Tissue lysates of two patients are shown.

Using a specific cathepsin K antiserum, immunoreactivity was tested in paraffin-embedded lung tissues of six patients. Distinct immunoreactions were abundant in ciliated and nonciliated bronchial epithelial cells (Figures 6A and 6C). The specificity of the immunoreaction was shown in control experiments in which the antiserum was preincubated with purified cathepsin K; no staining was detectable in these slides (Figure 6B).

View larger version (84K): [in this window] [in a new window]   Figure 6.   Immunohistochemical demonstration of cathepsin K expression in ciliated (A) as well as nonciliated (C ) bronchial epithelial cells. In control experiments, the anti-cathepsin K antibody was preincubated with saturating amounts of purified cathepsin K (B). Bar = 20 µm.

Specific Enzymatic Activity

For determination of the resulting enzymatic cathepsin K activities, lysates of the cell lines BEAS-2B and A459 were incubated with Z-GPR-AMC, which was shown to be specific for cathepsin K (23). The Michaelis constant (K_m) value for this substrate, determined using standard procedures, was 44 µM. The substrate Z-GPR-AMC was found not to be cleaved by the cathepsins L and S (data not shown). Cathepsin B cleaved this substrate at rates at least 50-fold lower than the cleaving rates for cathepsin K. Parallel samples of the cell lysates were incubated with Z-FR-AMC. To characterize further the enzymatic activity, the cell lysates were incubated with both Z-GPR-AMC and Z-FR-AMC in the presence of the inhibitors E64, a cysteine protease inhibitor, and CA074, which was specific for cathepsin B (data not shown). As documented in Figure 7, the cleavage of Z-GPR-AMC and Z-FR-AMC was strongly inhibited by E64 and CA074. These results show that the enzymatic activity measured in the cell lysates results exclusively from a cathepsin B-like cysteine protease.

View larger version (40K): [in this window] [in a new window]   Figure 7.   Cathepsin activity of BEAS-2B and A549 cells. The proteolytic activity was evaluated using the substrates (A) Z-FR-AMC (cathepsins L, K, and B) and (B) Z-GPR-AMC (cathepsins K and B) as described in MATERIALS AND METHODS. The specificity of the enzymatic reaction was evaluated with the inhibitors E64 (16 µM, cysteine proteases) and CA074 (16 µM, cathepsin B-like proteases).

The intracellular activity of cathepsin K was further verified by enzyme cytochemistry. The cathepsin K-reactive sites in the A549 and the BEAS-2B (data not shown) cells were visualized with the substrate Z-GPR-4MNA and the inhibitor CA074. Interestingly, a significant perinuclear enzymatic activity was shown (Figure 8A). This activity persisted after incubation in the presence of the inhibitor CA074 (Figure 8B). However, the cleavage of the cathepsin B-specific substrate Z-RR-4MNA (Figure 8C) was strongly suppressed by the same inhibitor (Figure 8D). These results suggest that the substrate Z-GPR-4MNA is cleaved by cathepsin K intracellularly.

View larger version (117K): [in this window] [in a new window]   Figure 8.   Enzyme cytochemistry of cathepsin K in A549 cells. Specific cathepsin K as well as cathepsin B activities were detected by the fluorogenic substrates Z-GPR-4MNA (A and B) and Z-RR-4MNA (C and D) in the absence (A, C ) and presence (B, D) of the inhibitor CA074 (10 µM). Bar = 20 µm.

	Discussion

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Cathepsin K is a recently identified member of the papain family of cysteine proteinases that appears to play an important role in matrix degradation (12, 24). This enzyme shows strong proteolytic activity at acidic pH and, in comparison with the cathepsins B and L, significant activity at neutral pH (10, 23). It has been demonstrated to degrade elastin and type I collagen as well as denatured type I collagen. Northern blot analyses have documented that cathepsin K is abundantly expressed in osteoclasts (9, 13, 14). Cathepsin K mRNA expression was also shown in lung tissues (9, 12), in monocyte-derived macrophages (13, 19) and in subpopulations of mononuclear marrow cells and breast carcinoma cells (13, 14, 25).

Collagen and elastin fibers form a network around the alveoli and the bronchial ducts, exerting tension in the alveolar and bronchial walls to counter the outward tension. Whereas elastin synthesized in childhood ordinarily lasts a lifetime (26), the collagen synthesis and degradation is regulated more rapidly (27). Many lung diseases are characterized by a modified extracellular matrix metabolism. Destruction of connective tissue is a crucial process in the mechanism of adult respiratory distress syndrome and other acute inflammatory diseases of the lung. The damage to the extracellular matrix is immediately followed by an upregulation of collagen de novo synthesis and the repair of elastin fibers (28). Lack of tight regulation of these processes may lead to fibrosis of the lung with potential irreversible dysfunction. A number of studies showed the role of elastase, released from infiltrating neutrophil granulocytes, in the inflammation-dependent tissue destruction. It was shown that matrix-metalloproteinases released from alveolar macrophages and epithelial cells may also play an important role in the pathogenesis of several lung diseases (31). Less information exists about the role of cysteine proteinases released from bronchial and alveolar epithelial cells.

Considering the first data concerning the expression in the lung and the unusual high collagenolytic and elastinolytic activity of the newly described cathepsin K, our aim was to study the expression and the proteolytic activity of this cysteine protease, particularly in lung epithelial cells.

Here we have presented evidence that human bronchial and alveolar epithelial cells express both the mRNA and the protein of cathepsin K. Initially we studied the mRNA expression of transformed lung epithelial cells. The cell line A549 is an epithelial lung tumor cell line with features of alveolar type II epithelial cells (35). The cell line BEAS-2B was established by viral transformation of human bronchial epithelial cells (36). In Northern blot analyses we detected the expression of a single 1.7-kb mRNA transcript in both cell lines. RT-PCR studies of the relative mRNA levels in lung tissues and cell lines suggested that the cathepsin K mRNA is expressed at somewhat lower levels than the mRNAs of cathepsins B and L. Although the PCRs were performed from the same RNA preparation, only a semiquantitative evaluation of the number of mRNA transcripts was possible because of methodologic limitations. The results correlate well to the results published by Brömme and Okamoto showing a borderline cathepsin K mRNA expression in A549 cells (12). These data clearly show that, in contrast to the osteoclastic cells, the cathepsin K mRNA is not dominant in lung epithelial cells. To investigate the relevance of our findings in nontransformed lung tissues, we studied the cathepsin K mRNA expression in human lung tissues with the help of PCR analyses. These experiments revealed, like the findings in the cell lines, a lower level of mRNA for cathepsin K than for the cathepsins B and L.

The protein expression of cathepsin K was demonstrated by means of flow cytometry. Using the specific cathepsin K antiserum, we documented the intracellular localization of this protein. This methodical approach may be helpful in future studies concerning the regulation of cathepsin expression.

The Western blot analyses of lung-tissue lysates showed a specific expression of three major immunoreactive bands that correspond to the pro- and the mature forms of the protease. Different molecular weights of the proenzyme between 37 and 46 kD are shown by several groups. In giant cell tumors beside a dominant 39-kD procathepsin, Littlewood-Evans and colleagues showed a distinct staining for cathepsin K at 46 kD (14). The variation in the molecular weights of the proenzyme published by different groups could result from different molecular forms of the protease, as shown for cathepsin B (37), or from methodologic limitations in definition of the molecular weights using SDS-PAGE. It could be suggested that the 12-kD band was the cleaved propeptide. The staining of a protein at 20 kD was inconsistent in tissue lysates from different patients. Further experiments must be performed to characterize the 12- and 20-kD proteins.

Immunohistochemical analyses of paraffin-embedded lung tissues revealed a specific staining of bronchial epithelial cells as well as single alveolar epithelial cells. The immunoreactivity of the antibody could be blocked by preincubation with purified cathepsin K. The results were verified using two different antisera raised against recombinant cathepsin K from P. pastoris (18) and a synthetic peptide corresponding to the cathepsin K sequence as described by Drake and associates (13). Especially within the bronchial epithelial cells, part of the immunoreactivity was localized in the apical part of the cells near the cell membrane. Burnett and coworkers have shown a secretion of procathepsin B by bronchial epithelial cells (4). Pardo and coworkers (33) studied the secretion of gelatinases by alveolar epithelial cells. Their zymographic figures clearly show a gelatinolytic activity at molecular weights below 50 kD, indicating a distinct enzymatic activity in addition to the activity of gelatinase A at molecular weights higher than 50 kD (33). These data, together with the described gelatinolytic activity of cathepsin K, suggest a possible secretion of the enzyme into the extracellular space.

In other studies concerning the cathepsin K expression in human embryonal lung tissues, the expression pattern in bronchial epithelial cells was confirmed by in situ hybridization (C. Häckel, manuscript submitted).

We extended our study to investigate whether these epithelial cells exhibit a significant enzymatic cathepsin K activity. Aibe and coworkers showed that the substrate Z-GPR-AMC is cleaved exclusively by cathepsin K (23). Our own studies, revealing a K_m of 44 µM for cathepsin K, no cleavage by the cathepsins S and L, and more than 50-fold lower cleavage rates for cathepsin B, supported these findings. The substrate Z-GPR-AMC was shown to be specific for cathepsin K, and Z-FR-AMC was cleaved by cathepsins L, B, and K (3). The inhibitor CA074 selectively suppressed the enzymatic activity of cathepsin B but not the activity of cathepsins K and L. The inhibitor E64 suppressed the activity of cathepsins B, L, and K. Using a combination of these substrates and inhibitors it was possible to measure the enzymatic activity of cathepsins K, B, and L in parallel. Our data show that in lysates of the epithelial cell lines, only the enzymatic activity of cathepsin B was detectable. The activities of cathepsins K and L could not be detected. Previous studies (38) showed significantly lower inhibition constant (K_i) values for the inhibition of cathepsin L by the cystatins A and B in comparison with cathepsin B. Our measurements show K_i values at subnanomolar levels for cathepsin K (D. Brömme, unpublished). As documented in Figure 4, bronchial epithelial cells do express cystatin B. Therefore, the data suggest that in cell lysates the activity of cathepsin K as well as cathepsin L could be inhibited by these cystatins. On the other hand, cytochemical detection revealed a significant specific activity of cathepsin K localized in cellular compartments without specific inhibitors, which was not inhibited by CA074 (Figure 8). Further studies will clarify the functional significance of cathepsin K in the epithelial collagenolytic and elastinolytic activity mediated by cysteine proteases.

In summary, our results demonstrate that bronchial and alveolar epithelial cells do express cathepsin K. These data are consistent with the observation that these cells actively participated in the extracellular matrix turnover. The functional significance in physiologic and pathologic processes in the lung as well as the regulation of the expression of cathepsin K remain to be investigated.