(Received for publication, October 27, 1995; and in revised form, March 13, 1996)
From the
Random high throughput sequencing of a human osteoclast cDNA library was employed to identify novel osteoclast-expressed genes. Of the 5475 ESTs obtained, approximately 4% encoded cathepsin K, a novel cysteine protease homologous to cathepsins S and L; ESTs for other cathepsins were rare. In addition, ESTs for cathepsin K were absent or at low frequency in cDNA libraries from numerous other tissues and cells. In situ hybridization in osteoclastoma and osteophyte confirmed that cathepsin K mRNA was highly expressed selectively in osteoclasts; cathepsins S, L, and B were not detectable. Cathepsin K was not detected by in situ hybridization in a panel of other tissues. Western blot of human osteoclastoma or fetal rat humerus demonstrated bands of 38 and 27 kDa, consistent with sizes predicted for pro- and mature cathepsin K. Immunolocalization in osteoclastoma and osteophyte showed intense punctate staining of cathepsin K exclusively in osteoclasts, with a polar distribution that was more intense at the bone surface. The abundant expression of cathepsin K selectively in osteoclasts strongly suggests that it plays a specialized role in bone resorption. Furthermore, the data suggest that random sequencing of ESTs from cDNA libraries is a valuable approach for identifying novel cell-selective genes.
Bone is composed of a protein matrix in which spindle- or plate-shaped crystals of hydroxyapatite are incorporated(1) . The matrix is approximately 90% Type I collagen, but also contains a number of non-collagenous proteins such as osteocalcin, osteopontin, and bone sialoprotein. It has been recognized for many years that bone resorption requires both dissolution of the inorganic mineral component (acidic microenvironment) and degradation of the protein matrix (protease activity). This has led to extensive efforts to identify the protease(s) responsible for osteoclast-mediated bone resorption. However, since osteoclasts are very rare cells and no appropriate osteoclast cell model has been identified, standard biochemical approaches for identification of the protease(s) have proven to be very difficult.
A number of studies have suggested that a cysteine
protease(s) is involved in bone resorption. For example, several known
cathepsins have collagenolytic activity under acidic
conditions(2) , a property that is predicted to be required for
the enzyme(s) secreted from the osteoclast into the acidic resorption
lacunae. In addition, classical inhibitors of cysteine proteases, such
as leupeptin, Z-Phe-Ala-CHN, E-64, and cystatin,
have demonstrated activity at preventing osteoclast-mediated bone
resorption in in vitro models(3, 4, 5, 6, 7, 8) . Z-Phe-Ala-CHN
and leupeptin have also shown
activity in vivo in a murine hypercalcemia model of bone
resorption(4) . Based upon observed substrate and inhibitor
preferences, as well as immunological reactivity, several groups have
suggested that cathepsins B or L, or a closely related enzyme, are
likely to be responsible for osteoclast-mediated resorption (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) .
Recently a novel member of the papain family of cysteine proteases
has been cloned that is most homologous to cathepsins S and
L(22, 23, 24, 25, 26, 27) .
Clones for this enzyme were first identified in cDNA libraries of
rabbit (22) and human (25, 27) osteoclasts,
suggesting that it was selectively expressed in osteoclasts. This novel
cathepsin has been referred to as OC2 (22) or cathepsins
O(23) , K(27) , X(26) , or O2 (24) ; we
refer to it as cathepsin K. ()The approach that we used to
identify cathepsin K was to partially sequence large numbers of
randomly chosen clones from an osteoclast cDNA library(25) . By
comparing homology to known sequences, the expressed sequence tags
(ESTs) (
)obtained from this technique provide a valuable
approach for identification of novel expressed
genes(28, 29, 30) .
In the present study, cellular expression of cathepsin K was examined by in situ hybridization in multiple tissues and compared with expression of cathepsins S, B, and L. In addition, specific anti-cathepsin K antibodies were generated and used to demonstrate expression and cellular localization of cathepsin K protein. The data clearly show that cathepsin K is abundantly and selectively expressed in osteoclasts, and that it displays a cellular localization consistent with an involvement of the enzyme in bone resorption. Furthermore, the data indicate that cathepsins S, B, and L, which had been proposed to be involved in bone resorption, are either expressed at very low levels or are absent in osteoclasts.
In situ hybridization was performed by a modification of the method of
Zeller and Rogers(32) , as follows. Cryosections were fixed in
4% paraformaldehyde for 5 min, washed, dehydrated, and frozen at
-20 °C. Prior to hybridization sections were rehydrated in
phosphate-buffered saline containing 2 mg/ml glycine and rinsed in
phosphate-buffered saline. Demineralization was in 0.2 N HCl
for 20 min, followed by acetylation in 0.25% acetic anhydride, 0.1 M triethanolamine. Finally, sections were washed twice in 2
SSC (20
SSC: 3 M NaCl, 0.3 M sodium
citrate, pH 7.0), dehydrated in 30, 60, 80, 95, and 100% ethanol, and
air-dried. Sections were used immediately for hybridization in buffer
consisting of 2 parts hybridization mix B (1.2 M NaCl, 20
mM Tris-HCl, pH 7.5, 4 mM EDTA, 2
Denhardt's solution, 1 mg/ml yeast tRNA, 200 µg/ml poly(A))
(Pharmacia Biotech Inc.), 2 parts deionized formamide, and 1 part 50%
dextran sulfate. Dithiothreitol was added to a final concentration of
50 mM, and probe concentration was 2
10
cpm/µl. Hybridization was carried out at 42 °C for 4 h in
a well sealed moist chamber. Post-hybridization washes were as follows:
at least twice in prewarmed 2
SSC, 50% formamide, 0.1%
-mercaptoethanol for 15 min at 50 °C; once in 20 µg/ml
boiled ribonuclease A in 0.5 M NaCl, 10 mM Tris-HCl,
pH 8.0, for 30 min at 37 °C; at least twice in prewarmed 2
SSC, 50% formamide, 0.1%
-mercaptoethanol for 15 min at 50 °C;
twice in prewarmed 0.1
SSC, 1%
-mercaptoethanol for 15 min
at 50 °C. Sections were dehydrated in the following: 0.6 M NaCl, 30% ethanol; 0.6 M NaCl, 60% ethanol; 80%, 95%, and
100% ethanol for 2 min each. Air-dried slides were coated in Amersham
LM-1 emulsion and exposed at 4 °C for 2 weeks. The slides were
developed in a Kodak developer and counterstained with methylene blue.
The extent of the hybridization signal was assessed by the
autoradiographic grain density over the cell.
Alternatively, cryostat sections were used to demonstrate the reactivity of the anti-cathepsin K antibodies. Sections (8 µm) of human tissues were cut using a Bright's cryostat (Bright Instrument Co., Huntingdon, United Kingdom) equipped with a tungsten-tipped steel knife (ARP, Cheshire, United Kingdom). The sections (undecalcified adult osteophytic bone and rheumatoid synovium from osteoarthritic and rheumatoid femoral heads, respectively; post-mortem specimens of human kidney, spleen, liver, lung, heart, skin, and colon) were placed onto TESPA-coated slides and air-dried for at least 15 min. Tissues were fixed in 10% formalin for 10 min and then washed in citrate buffer (pH 6.0) immediately before boiling them in the citrate buffer for 15 min in the microwave. The remainder of the technique was performed as outlined above.
In contrast to the abundance of ESTs for cathepsin K, ESTs for other cathepsins were rare in the osteoclast library. Only two ESTs (0.036%) for cathepsin B were identified from the osteoclast library, and one EST (0.018%) for cathepsin S was found. No ESTs for cathepsin L were found, and no other ESTs for cysteine proteases were represented in the library. Thus, ESTs for cathepsin K represented greater than 98% of the total cysteine protease ESTs in the human osteoclast cDNA library.
Figure 1:
In situ hybridization. Sections were hybridized to the probes indicated,
followed by methylene blue counterstain (original magnification,
20). A, cathepsin K antisense probe in a section of
human osteoclastoma tissue. Osteoclasts (large arrowheads) and
a small population of mononuclear cells (small arrowheads)
demonstrated strong cathepsin K mRNA expression. B, serial
section of A probed with the cathepsin K sense strand. C, cathepsin B mRNA expression in section of osteoclastoma.
Osteoclasts (large arrowheads) did not demonstrate expression;
however, associated mononuclear cells (small arrowheads) demonstrated
strong cathepsin B mRNA expression. D, cathepsin K antisense
probe in a section of human osteophyte. Osteoclasts resorbing or
adjacent to bone (B) demonstrated selective and strong
cathepsin K mRNA expression (arrowheads). E, serial
section of D probed with the cathepsin K sense strand. F, cathepsin B antisense probe in a section of human
osteophyte. Osteoclasts (large arrowheads) resorbing bone
demonstrated no cathepsin B mRNA
expression.
To determine the expression of cathepsin K in other cell types, a panel of human tissues was tested by in situ hybridization. Cathepsin K mRNA was not detected in any of the tissues tested (Table 1).
Figure 3: Cathepsin K protein expression in osteoclastoma. Human osteoclastoma lysate was separated by SDS-PAGE (12%) and blotted onto nitrocellulose. The blot was probed with an antibody raised against a synthetic peptide from a unique region of the predicted amino acid sequence of human cathepsin K (antibody C-2). In lane A, immunoreactive bands of 38 and 27 kDa are observed. Lane B demonstrates that these immunoreactive bands can be competed with 3 µg/ml peptide antigen.
Immunolocalization of cathepsin K using antibody SR1 in osteoclastoma tissue demonstrated abundant staining in osteoclasts and showed a punctate, granular distribution that was very often localized to a single pole of the osteoclasts (Fig. 2A, large arrowheads). A small population of mononuclear cells (potentially representing an osteoclast precursor population) also demonstrated reactivity (Fig. 2A, small arrowheads). Surrounding stromal cells were negative for cathepsin K. Immunolocalization with antibody C2 demonstrated similar results (data not shown). No staining could be detected in any cells on the nonimmune serum control slides (Fig. 2B).
Figure 2:
Immunolocalization of cathepsin K.
Sections were probed with the antisera indicated, followed by labeled
streptavidin-biotin and staining with Mayer's hematoxylin. A, osteoclasts (large arrowheads) and a minor
population of mononuclear cells (small arrowheads) in
osteoclastoma demonstrated strong staining with anti-cathepsin K
antibody (SR1). In many of the multinucleated osteoclasts, the staining
was polarized to one edge of the cytoplasm (asterisks).
Original magnification, 20. B, no reactivity was
detected in a section of osteoclastoma probed with pre-immune serum.
Original magnification,
20. C, strong anti-cathepsin K
antibody (SR1) staining was detected on osteoclasts apposed (large
arrowheads) to and away from (arrowheads) the bone
surface in a section human osteophytic bone. The staining was most
intense at the apical surface of the majority of osteoclasts apposed to
the bone surface. No reactivity was observed in osteoblasts, osteocytes
and the majority of cells within the bone marrow space. Original
magnification,
10. D, higher magnification of C to highlight the polarized staining of SR1 in osteoclasts apposed
to the bone surface (arrows). Original magnification,
40.
In osteophyte, a similar pattern of cathepsin K reactivity was detected in osteoclasts apposed to the surface of bone (Fig. 2, C and D). The osteoclasts showed a distinct polarity of staining that was more intense toward the apical surface of resorbing osteoclasts. Cathepsin K expression also appeared to be restricted to osteoclasts, since other bone marrow cells, chondrocytes, osteoblasts, osteocytes, and connective tissue cells did not demonstrate reactivity (Fig. 2, C and D; Table 1).
In contrast to the immunoreactivity observed in osteoclasts, cathepsin K protein expression was not detected in the panel of other human tissues analyzed (Table 1).
Previous studies have consistently demonstrated that
inhibitors of cysteine proteases are very effective at preventing
osteoclast-mediated bone resorption, and have clearly implicated a
cathepsin(s) as a key mediator of this
process(3, 4, 5, 6, 7) .
Delaisse et al.(3) tested a series of protease
inhibitors in a mouse bone organ culture system and found that
inhibitors of cysteine proteases (e.g., leupeptin and Z-Phe-Ala-CHN) reduced bone resorption, while
serine protease inhibitors were ineffective. A follow-up study by the
same group showed that E-64 and leupeptin were also effective at
preventing bone resorption in vivo, as measured by acute
changes in serum calcium in rats on calcium-deficient
diets(4) . Based upon the activity of the enzyme, this group
classified the enzyme responsible as cathepsin B. Cystatin, an
endogenous cysteine protease inhibitor, was shown to prevent
parathyroid hormone-stimulated bone resorption in mouse
calvariae(7) . Detailed studies demonstrated that the number
and volume of resorption pits were decreased in the presence of
cysteine protease inhibitors, while the surface area of the pits was
unaffected(5) . Hill et al.(6) confirmed
these findings on resorption pit parameters and suggested that
cathepsins B, L, or S were involved. Thus, data from several studies
indicated that inhibitors of cysteine proteases were very effective at
preventing bone resorption, and strongly suggested that a cysteine
protease(s) plays an essential role in the process.
In the present study, an enriched population of human osteoclasts was used to prepare a cDNA library that was subjected to high throughput random sequencing of clones. Among the genes identified was a novel cysteine protease that is highly related to cathepsins S and L (25) . A striking finding was the high frequency of ESTs for this enzyme in the osteoclast library and its relative lack of expression in other libraries, suggesting that this enzyme may be expressed selectively in osteoclasts. Surprisingly, ESTs for other cysteine proteases were nearly absent from the osteoclast library. To determine whether frequency of ESTs in the osteoclast library reflects expression levels in vivo, in situ hybridization studies on human tissues were performed. The results confirm that cathepsin K mRNA is highly abundant in osteoclasts and is not detectable in cells from other human tissues. These studies also confirmed that cathepsins S, B, and L are either absent or expressed at very low levels in osteoclasts.
In addition, specific antibodies to cathepsin K were used to demonstrate for the first time expression of the protein. Western blotting showed expression of the enzyme in extracts of osteoclastoma as well as normal bone tissues, as demonstrated by immunoreactivity in fetal rat humerus. The mobility of cathepsin K on SDS-PAGE suggests that the enzyme is expressed as a 38-kDa proenzyme and that it is processed to a 27-kDa mature form (Fig. 3). Studies with purified cathepsin K have demonstrated that the 38-kDa proenzyme is inactive, and protease activity correlates with the appearance of the 27-kDa mature enzyme(34) . Immunohistochemistry confirmed the abundant expression of cathepsin K selectively in osteoclasts. Furthermore, the subcellular localization of cathepsin K at the osteoclast surface adjacent to the bone further supports a role of the enzyme in the bone resorption process. Thus, the abundant, selective expression of cathepsin K, coupled with the apparent lack of other cysteine proteases, strongly suggests that this enzyme plays a key role in osteoclast-mediated bone resorption.
Although previous studies have shown remarkable agreement that a cathepsin(s) is involved in bone resorption, identification of the protease(s) has been a very difficult problem, since osteoclasts are very rare cells and no appropriate osteoclast cell model has been identified. These previous studies have attempted to identify the cathepsin involved in bone resorption by immunolocalization(11, 12, 13, 14, 15, 17) or histochemically(16, 18, 19) . Contrary to our observations, these studies have suggested that cathepsins B and L are expressed by osteoclasts. However, the approaches taken in these studies necessarily relied on reagents for previously known cathepsins. Because cathepsin K is highly homologous to cathepsins L and B and is similar in size, cross-reactivity with cathepsin K by the antibodies used in earlier studies is possible. In addition, since cathepsin K may have similar enzymatic properties and substrate preferences as other cathepsins, interpretation of histochemical data is also difficult.
Another approach that has been taken to identify the relevant protease(s) involved in bone resorption has been purification of protease activity. Delaisse (9) purified protease activity from mouse calvariae and found three main peaks of activity, which they suggested were cathepsins B, L, and an unknown protease with an apparent mass of 70 kDa by gel chromatography. Page et al.(10) used osteoclastoma tissue as an enriched source of osteoclasts for purification. They found six peaks of activity, each of which showed characteristics consistent with cathepsin B. As with the immunolocalization and histochemical studies, however, it is difficult to determine whether these protease activities may have been due to cathepsin K, or even an enzyme derived from cells other than osteoclasts.
Tezuka et al.(22) cloned the rabbit homolog of cathepsin K, OC-2, from a rabbit osteoclast cDNA library. They demonstrated expression of OC-2 mRNA in the osteoclast by in situ hybridization of bone tissue. This group has also recently reported the sequence of the human enzyme(27) . Li et al.(33) have also recently reported cloning of cathepsin K from an osteoclast cDNA library, and Bromme et al.(24) cloned the gene from a human spleen library. Each group indicated that there was abundant expression in osteoclasts, although Bromme et al.(24) also reported expression of cathepsin K mRNA in ovary. Shi et al.(23) also cloned human cathepsin K, but from a human monocyte-derived macrophage library. They demonstrated proteolytic cleavage of fibrinogen when the enzyme was transiently transfected into COS cells. It is of interest that they were unable to detect cathepsin K from freshly isolated monocytes, suggesting that it was the extended culture conditions that led to induction of cathepsin K mRNA. Our inability to detect cathepsin K in rheumatoid synovium, which has high levels of macrophages, is consistent with the lack of expression of cathepsin K in macrophages under normal conditions.
In addition to osteoclasts, our data
indicate that cathepsin K was expressed in two other populations of
cells. At sites of cartilage remodeling in osteophyte, chondroclasts
expressed high levels of cathepsin K. This is not surprising, as these
cells are related to or identical to osteoclasts. The data also
indicate that cathepsin K is expressed in a population of mononuclear
cells within the osteoclastoma tissue. Further characterization of this
cell population has demonstrated that these cells possess a number of
markers of the osteoclast phenotype, and are capable of forming
resorption pits in vitro. ()Thus, in addition to
being highly expressed in mature osteoclasts, the enzyme may represent
an excellent marker for the osteoclast precursor population as well.
The ability to sequence large number of clones from an osteoclast library has provided a valuable approach for discovery of novel osteoclast proteins and led to the identification of a novel cathepsin. In addition, the availability of data from multiple human cDNA libraries has allowed us to compare the frequency of ESTs for cathepsin K from a number of cells and tissues. EST frequency suggested abundant osteoclast-selective expression of cathepsin K, and this has been confirmed by both in situ hybridization and immunohistochemistry. The results suggest that cathepsin K may play a specialized, and perhaps essential, role in osteoclast-mediated bone resorption. Selective inhibitors of cathepsin K may be useful in treatment of diseases of excessive bone loss, such as osteoporosis.