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(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 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. ( 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
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,
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,
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 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. ( 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.
Volume 271,
Number 21,
Issue of May 24, 1996 pp. 12511-12516
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, 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) .
)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) .
Osteoclast cDNA Library
Fresh osteoclastoma
tissue was chopped into small pieces and placed into a sterile 50-ml
centrifuge tube. The pieces were disaggregated by incubating at 37
°C for 30 min in serum-free RPMI 1640 medium (Life Technologies,
Inc.), supplemented with 3 mg/ml (w/v) type I collagenase (Sigma). A cell suspension was obtained by gently
homogenizing the remaining tissue with a plunger from a 30-ml syringe.
The osteoclastoma-derived cells were pelleted by centrifugation (400
g for 10 min) and resuspended in 6 ml of cold culture
medium (RPMI with 10% fetal calf serum, 100 units/ml penicillin, and 50
µg/ml streptomycin; Life Technologies, Inc.) to which was added 3
ml of the anti-
3 antibody, C22. Following a 30-min incubation on
ice, the cells were washed twice by centrifugation in cold RPMI medium.
After the last wash, the cells were resuspended in 10 ml of medium and
were enumerated in a hemocytometer. Dynabeads (Dynal Inc., Great Neck,
NY). coated with goat anti-mouse IgG were incubated for 30 min on ice
with the cell suspension at a density of 6 beads/osteoclast. The
bead-coated osteoclasts were immobilized on a magnet, and the uncoated
cells were removed by extensive washing with cold RPMI medium. The
osteoclast-rich suspension was then resuspended in fresh RPMI medium
and seeded into eight T250 tissue culture flasks. The cells were
cultured for 3 days prior to the extraction of mRNA using the
Invitrogen FastTrack mRNA Isolation kit. The mRNA was methylmercuric
hydroxide denatured prior to cDNA synthesis, and a directional
oligo(dT)-primed cDNA library was prepared (Stratagene, La Jolla, CA).
cDNA was size fractionated, and fragments greater than 1 kilobase were
ligated into the Uni-ZAP XR vector.In Situ Hybridization
Cryostat sections of
osteophytes and osteoclastoma tissue were processed as described
previously(31) . The sections were picked off onto
3-aminopropyltriethoxy silane (TESPA)-coated glass slides. A cDNA clone
(pBluescript SK) containing the coding region of human cathepsin K was
obtained from the osteoclast library. Clones for cathepsins B, S, and L
were obtained from stromal cell, pancreatic tumor, and human embryo
cDNA libraries, respectively. cDNA templates were linearized and then
transcribed from the T3 or the T7 promoter to generate sense and
antisense probes, respectively. Riboprobes were prepared using the
Promega (Madison, WI) In Vitro transcription kit with
[
S]thio-CTP (Amersham Corp.). Following
transcription, cDNA templates were digested with RQ1 RNase-free DNase I
(Promega), and unincorporated nucleotides were removed by
centrifugation through Quick Spin Sephadex G-50 columns (Boehringer
Mannheim). RNA transcripts with a specific activity in excess of
10
cpm/mg were used for hybridization. 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.Antibodies
For anti-peptide antibodies, rabbits
were immunized with a synthetic peptide corresponding to a sequence in
cathepsin K that has low sequence identity to other known cathepsins.
Antibody C2 was generated to a peptide corresponding to amino acids
248-260 of preprocathepsin K (AIDASLTSFQFYS). The antibody was
affinity-purified by chromatography on immobilized peptide antigen. For
anti-protein antibodies (SR-1), a rabbit was immunized with full-length
preprocathepsin K that had been overproduced in Escherichia
coli, and purified by preparative SDS-PAGE.Western Blot
Extracts of osteoclastoma were
prepared by rapid lysis in hot (60 °C) 2% SDS. Fetal rat humerus
was Dounce homogenized, followed by lysis in hot 2% SDS. Samples were
run on SDS-PAGE and blotted onto nitrocellulose. Blots were probed with
anti-cathepsin K antisera (or affinity-purified antibodies), followed
by goat anti-rabbit IgG coupled to alkaline phosphatase.Immunocytochemistry
Curettage samples of
osteoclastoma tissue were fixed overnight in 10% buffered formalin
(Baxter Diagnostics, Deerfield, IL) and then washed in 70% ethanol. The
tissue was processed for paraffin embedding using a Shandon Citadel
1000 tissue processor, and embedded in paraffin wax using a Shandon
embedding center (Pittsburgh, PA). The tissue blocks were cut, using a
Reichert Histostat rotary microtome (Warner Lambert Instruments,
Buffalo, NY), and the 7-µm sections were placed onto TESPA-coated
glass slides. The sections were dried overnight at 37 °C and were
then dewaxed in xylene and rehydrated into 0.1 M citrate
buffer (pH 6.0). The sections were then boiled for 15-30 min in
the citrate buffer by microwaving them at high power in a household
microwave oven (General Electric, Louisville, KY); the buffer was
periodically topped up to prevent the tissue from drying out. After
cooling slides to room temperature, the sections were overlaid with
either the rabbit polyclonal cathepsin-K reactive primary antibodies,
or with nonimmune serum controls, and a standard avidin/biotin alkaline
phosphatase method was performed according to the manufacturer's
protocol (DAKO, Carpenteria, CA). Positive reactivity was detected
using a post-coupling naphthol phosphate method with Fast Red-TR (Sigma) as the coupler; a red precipitate indicated
positive reactivity.
Cathepsin EST Frequency
Cathepsin K was
identified as a novel cysteine protease whose ESTs were highly abundant
in an osteoclast library from human osteoclastoma(25) .
Approximately 4% of all ESTs randomly sequenced from this library
encoded cathepsin K (223 ESTs of 5475 total). In contrast, ESTs for
cathepsin K were absent in most other libraries sequenced. For cDNA
libraries in which ESTs for cathepsin K were found, they were present
at much lower frequency than in the osteoclast cDNA library. For
example, in cDNA libraries for which greater than 1000 ESTs had been
sequenced, the frequency of ESTs for cathepsin K was: placenta,
0.0016%; white adipose, 0.06%; retina, 0.06%; colon, 0.037%;
epididymus, 0.09%; gall bladder, 0.03%; testes, 0.006%; tonsils,
0.009%; chondrosarcoma, 0.13%; ovarian cancer, 0.018%; B cell lymphoma,
0.015%; pancreatic tumor, 0.017%; prostate cancer, 0.087%, T-cell
lymphoma, 0.036%; and activated monocytes, 0.044%. Thus, data from a
number of libraries suggest that cathepsin K is abundant only in
osteoclasts.Expression of Cathepsin K mRNA
Northern blot
analysis of osteoclastoma tissue with a specific probe for cathepsin K
demonstrated a single band of approximately 2 kilobases (data not
shown). To determine which cells expressed the enzyme, in situ hybridization studies were performed in human osteoclastoma and
osteophyte. Cathepsin K was abundantly and selectively expressed in
osteoclasts and a discrete population of mononuclear cells within human
osteophyte and osteoclastoma tissue (Fig. 1, A and D). All other cell types, including stromal cells (of the
osteoclastoma), marrow cells, osteoblasts, osteocytes, and
chondrocytes, were negative. At sites of cartilage remodeling in the
osteophyte, chondroclasts also expressed cathepsin K mRNA (not shown).
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.
Expression of mRNA for Cathepsins S, L, and B
The
EST frequency suggested that cathepsins S, L, and B were expressed at a
much lower frequency in osteoclasts than cathepsin K. To confirm this, in situ hybridization studies were performed on osteoclastoma
and osteophyte sections with probes specific for these cathepsins. No
hybridization was detected in osteoclasts in osteoclastoma or
osteophyte with probes for cathepsins S, L, or B (cathepsin B shown as
representative; Fig. 1, C and F; Table 2). As expected, cathepsins B and L were highly expressed
in spleen, liver, and kidney (Table 2).
Expression of Cathepsin K Protein
Western blot
analysis of osteoclastoma tissue with antibody against either synthetic
peptides unique to cathepsin K (antibody C2) or to intact procathepsin
K that had been expressed in E. coli (antibody SR1)
demonstrated immunoreactive bands of 38 kDa and 27 kDa (Fig. 3, lane A), consistent with the predicted size of the pro- and
mature cathepsin K, respectively. For the antipeptide antibody,
addition of an excess of the peptide immunogen prevented the detection
of these bands (Fig. 3, lane B); an excess of an
unrelated peptide had no effect (data not shown). To determine if
cathepsin K was expressed in normal tissue, fetal rat humerus was
analyzed by Western blot, and a similar pattern of expression was
observed (data not shown).
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.
) 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.
)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 initial EST clones used in this study were
discovered as part of a joint collaboration between scientists at The
Institute for Genomic Research (TIGR), SmithKline Beecham, and Human
Genome Sciences (HGS) using established EST
methods(28, 29) . These clones are part of a larger
EST project(30) . We thank Dr. R. H. Rothman (Rothman
Institute, Philadelphia, PA) for supplying bone samples, Dr. R. D.
Lackman (Jefferson Hospital, Philadelphia, PA) for supplying
osteoclastomas, and the Anatomical Gift Foundation (Folkson, GA) for
providing tissue samples. We also thank Frank McCabe for technical
assistance, Drs. John Lee and Jeremy Bradbeer for critical review of
the manuscript, and Drs. Brian Metcalf and Martin Rosenberg for
continued support. We thank scientists at the sequencing facilities of
HGS and TIGR, and the Bioinformatics staff at SmithKline Beecham. We
would also like to thank Dr. Alan Barrett, Chair of the Advisory Panel
on Peptidase Nomenclature to the Nomenclature Committee of the
International Union of Biochemistry and Molecular Biology, for promptly
responding to our request to resolve the nomenclature for this
protease.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 C. M. Stack, C. R. Caffrey, S. M. Donnelly, A. Seshaadri, J. Lowther, J. F. Tort, P. R. Collins, M. W. Robinson, W. Xu, J. H. McKerrow, et al. Structural and Functional Relationships in the Virulence-associated Cathepsin L Proteases of the Parasitic Liver Fluke, Fasciola hepatica J. Biol. Chem., April 11, 2008; 283(15): 9896 - 9908. [Abstract] [Full Text] [PDF]
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A. Ruettger, S. Schueler, J. A. Mollenhauer, and B. Wiederanders Cathepsins B, K, and L Are Regulated by a Defined Collagen Type II Peptide via Activation of Classical Protein Kinase C and p38 MAP Kinase in Articular Chondrocytes J. Biol. Chem., January 11, 2008; 283(2): 1043 - 1051. [Abstract] [Full Text] [PDF]
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J. Selent, J. Kaleta, Z. Li, G. Lalmanach, and D. Bromme Selective Inhibition of the Collagenase Activity of Cathepsin K J. Biol. Chem., June 1, 2007; 282(22): 16492 - 16501. [Abstract] [Full Text] [PDF]
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 Y. Xiao, H. Junfeng, L. Tianhong, W. Lu, C. Shulin, Z. Yu, L. Xiaohua, J. Weixia, Z. Sheng, G. Yanyun, et al. Cathepsin K in Adipocyte Differentiation and Its Potential Role in the Pathogenesis of Obesity J. Clin. Endocrinol. Metab., November 1, 2006; 91(11): 4520 - 4527. [Abstract] [Full Text] [PDF]
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 M. K. Kim, H. D. Kim, J. H. Park, J. I. Lim, J. S. Yang, W. Y. Kwak, S. Y. Sung, H. J. Kim, S. H. Kim, C. H. Lee, et al. An Orally Active Cathepsin K Inhibitor, Furan-2-Carboxylic Acid, 1-{1-[4-Fluoro-2-(2-oxo-pyrrolidin-1-yl)-phenyl]-3-oxo-piperidin-4-ylcarbamoyl}-cyclohexyl)-amide (OST-4077), Inhibits Osteoclast Activity in Vitro and Bone Loss in Ovariectomized Rats J. Pharmacol. Exp. Ther., August 1, 2006; 318(2): 555 - 562. [Abstract] [Full Text] [PDF]
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J. Ljusberg, Y. Wang, P. Lang, M. Norgard, R. Dodds, K. Hultenby, B. Ek-Rylander, and G. Andersson Proteolytic Excision of a Repressive Loop Domain in Tartrate-resistant Acid Phosphatase by Cathepsin K in Osteoclasts J. Biol. Chem., August 5, 2005; 280(31): 28370 - 28381. [Abstract] [Full Text] [PDF]
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 Z. Dong, R. D. Bonfil, S. Chinni, X. Deng, J. C. Trindade Filho, M. Bernardo, U. Vaishampayan, M. Che, B. F. Sloane, S. Sheng, et al. Matrix Metalloproteinase Activity and Osteoclasts in Experimental Prostate Cancer Bone Metastasis Tissue Am. J. Pathol., April 1, 2005; 166(4): 1173 - 1186. [Abstract] [Full Text] [PDF]
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J. H.N. Lindeman, R. Hanemaaijer, A. Mulder, P.D. S. Dijkstra, K. Szuhai, D. Bromme, J. H. Verheijen, and P. C.W. Hogendoorn Cathepsin K Is the Principal Protease in Giant Cell Tumor of Bone Am. J. Pathol., August 1, 2004; 165(2): 593 - 600. [Abstract] [Full Text] [PDF]
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P. R. Collins, C. M. Stack, S. M. O'Neill, S. Doyle, T. Ryan, G. P. Brennan, A. Mousley, M. Stewart, A. G. Maule, J. P. Dalton, et al. Cathepsin L1, the Major Protease Involved in Liver Fluke (Fasciola hepatica) Virulence: PROPEPTIDE CLEAVAGE SITES AND AUTOACTIVATION OF THE ZYMOGEN SECRETED FROM GASTRODERMAL CELLS J. Biol. Chem., April 23, 2004; 279(17): 17038 - 17046. [Abstract] [Full Text] [PDF]
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Z. Li, Y. Yasuda, W. Li, M. Bogyo, N. Katz, R. E. Gordon, G. B. Fields, and D. Bromme Regulation of Collagenase Activities of Human Cathepsins by Glycosaminoglycans J. Biol. Chem., February 13, 2004; 279(7): 5470 - 5479. [Abstract] [Full Text] [PDF]
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H. Hotokezaka, E. Sakai, K. Kanaoka, K. Saito, K.-i. Matsuo, H. Kitaura, N. Yoshida, and K. Nakayama U0126 and PD98059, Specific Inhibitors of MEK, Accelerate Differentiation of RAW264.7 Cells into Osteoclast-like Cells J. Biol. Chem., November 27, 2002; 277(49): 47366 - 47372. [Abstract] [Full Text] [PDF]
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Z. Li, W.-S. Hou, C. R. Escalante-Torres, B. D. Gelb, and D. Bromme Collagenase Activity of Cathepsin K Depends on Complex Formation with Chondroitin Sulfate J. Biol. Chem., August 2, 2002; 277(32): 28669 - 28676. [Abstract] [Full Text] [PDF]
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 D.P. Dickinson CYSTEINE PEPTIDASES OF MAMMALS: THEIR BIOLOGICAL ROLES AND POTENTIAL EFFECTS IN THE ORAL CAVITY AND OTHER TISSUES IN HEALTH AND DISEASE Critical Reviews in Oral Biology & Medicine, May 1, 2002; 13(3): 238 - 275. [Abstract] [Full Text] [PDF]
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 V. Parikka, P. Lehenkari, M.-L. Sassi, J. Halleen, J. Risteli, P. Harkonen, and H. K. Vaananen Estrogen Reduces the Depth of Resorption Pits by Disturbing the Organic Bone Matrix Degradation Activity of Mature Osteoclasts Endocrinology, December 1, 2001; 142(12): 5371 - 5378. [Abstract] [Full Text] [PDF]
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W.-S. Hou, Z. Li, R. E. Gordon, K. Chan, M. J. Klein, R. Levy, M. Keysser, G. Keyszer, and D. Bromme Cathepsin K Is a Critical Protease in Synovial Fibroblast-Mediated Collagen Degradation Am. J. Pathol., December 1, 2001; 159(6): 2167 - 2177. [Abstract] [Full Text] [PDF]
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 Y. Suzuki, Y. Tsutsumi, M. Nakagawa, H. Suzuki, K. Matsushita, M. Beppu, H. Aoki, Y. Ichikawa, and Y. Mizushima Osteoclast-like cells in an in vitro model of bone destruction by rheumatoid synovium Rheumatology, June 1, 2001; 40(6): 673 - 682. [Abstract] [Full Text] [PDF]
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C. Rocken, B. Stix, D. Bromme, S. Ansorge, A. Roessner, and F. Buhling A Putative Role for Cathepsin K in Degradation of AA and AL Amyloidosis Am. J. Pathol., March 1, 2001; 158(3): 1029 - 1038. [Abstract] [Full Text] [PDF]
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 C Faucheux, S Nesbitt, M Horton, and J Price Cells in regenerating deer antler cartilage provide a microenvironment that supports osteoclast differentiation J. Exp. Biol., January 2, 2001; 204(3): 443 - 455. [Abstract] [PDF]
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T. Nakase, E. Takeuchi, K. Sugamoto, M. Kaneko, T. Tomita, A. Myoui, Y. Uchiyama, T. Ochi, and H. Yoshikawa Involvement of multinucleated giant cells synthesizing cathepsin K in calcified tendinitis of the rotator cuff tendons Rheumatology, October 1, 2000; 39(10): 1074 - 1077. [Abstract] [Full Text] [PDF]
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 A. Punturieri, S. Filippov, E. Allen, I. Caras, R. Murray, V. Reddy, and S. J. Weiss Regulation of Elastinolytic Cysteine Proteinase Activity in Normal and Cathepsin K-deficient Human Macrophages J. Exp. Med., September 11, 2000; 192(6): 789 - 800. [Abstract] [Full Text] [PDF]
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 M. T. Moran, J. P. Schofield, A. R. Hayman, G.-P. Shi, E. Young, and T. M. Cox Pathologic gene expression in Gaucher disease: up-regulation of cysteine proteinases including osteoclastic cathepsin K Blood, September 1, 2000; 96(5): 1969 - 1978. [Abstract] [Full Text] [PDF]
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 C Tepel, D Bromme, V Herzog, and K Brix Cathepsin K in thyroid epithelial cells: sequence, localization and possible function in extracellular proteolysis of thyroglobulin J. Cell Sci., January 12, 2000; 113(24): 4487 - 4498. [Abstract] [PDF]
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H. Vaananen, H Zhao, M Mulari, and J. Halleen The cell biology of osteoclast function J. Cell Sci., January 2, 2000; 113(3): 377 - 381. [Abstract] [PDF]
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Y. Fujita, K. Nakata, N. Yasui, Y. Matsui, E. Kataoka, K. Hiroshima, R.-i. Shiba, and T. Ochi Novel Mutations of the Cathepsin K Gene in Patients with Pycnodysostosis and Their Characterization J. Clin. Endocrinol. Metab., January 1, 2000; 85(1): 425 - 431. [Abstract] [Full Text]
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B. S. Lee, S. L. Gluck, and L. S. Holliday Interaction between Vacuolar H+-ATPase and Microfilaments during Osteoclast Activation J. Biol. Chem., October 8, 1999; 274(41): 29164 - 29171. [Abstract] [Full Text] [PDF]
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 V. EVERTS, W. KORPER, D. C. JANSEN, J. STEINFORT, I. LAMMERSE, S. HEERA, A. J. P. DOCHERTY, and W. BEERTSEN Functional heterogeneity of osteoclasts: matrix metalloproteinases participate in osteoclastic resorption of calvarial bone but not in resorption of long bone FASEB J, July 1, 1999; 13(10): 1219 - 1230. [Abstract] [Full Text]
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 F. Bühling, A. Gerber, C. Häckel, S. Krüger, T. Köhnlein, D. Brömme, D. Reinhold, S. Ansorge, and T. Welte Expression of Cathepsin K in Lung Epithelial Cells Am. J. Respir. Cell Mol. Biol., April 1, 1999; 20(4): 612 - 619. [Abstract] [Full Text]
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 L. D. Greller and F. L. Tobin Detecting Selective Expression of Genes and Proteins Genome Res., March 1, 1999; 9(3): 282 - 296. [Abstract] [Full Text]
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 P. Saftig, E. Hunziker, O. Wehmeyer, S. Jones, A. Boyde, W. Rommerskirch, J. D. Moritz, P. Schu, and K. von Figura Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice PNAS, November 10, 1998; 95(23): 13453 - 13458. [Abstract] [Full Text] [PDF]
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S. K. Thompson, S. M. Halbert, M. J. Bossard, T. A. Tomaszek, M. A. Levy, B. Zhao, W. W. Smith, S. S. Abdel-Meguid, C. A. Janson, K. J. D'Alessio, et al. Design of potent and selective human cathepsin K inhibitors that span the active site PNAS, December 23, 1997; 94(26): 14249 - 14254. [Abstract] [Full Text] [PDF]
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G. Spizz and P. J. Blackshear Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme J. Biol. Chem., September 19, 1997; 272(38): 23833 - 23842. [Abstract] [Full Text] [PDF]
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L. S. Holliday, H. G. Welgus, C. J. Fliszar, G. M. Veith, J. J. Jeffrey, and S. L. Gluck Initiation of Osteoclast Bone Resorption by Interstitial Collagenase J. Biol. Chem., August 29, 1997; 272(35): 22053 - 22058. [Abstract] [Full Text] [PDF]
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C. S. Kumar, I. E. James, A. Wong, V. Mwangi, J. A. Feild, P. Nuthulaganti, J. R. Connor, C. Eichman, F. Ali, S. M. Hwang, et al. Cloning and Characterization of a Novel Integrin beta 3 Subunit J. Biol. Chem., June 27, 1997; 272(26): 16390 - 16397. [Abstract] [Full Text] [PDF]
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M. S. McQueney, B. Y. Amegadzie, K. D'Alessio, C. R. Hanning, M. M. McLaughlin, D. McNulty, S. A. Carr, C. Ijames, J. Kurdyla, and C. S. Jones Autocatalytic Activation of Human Cathepsin K J. Biol. Chem., May 23, 1997; 272(21): 13955 - 13960. [Abstract] [Full Text] [PDF]
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T. Inui, O. Ishibashi, T. Inaoka, Y. Origane, M. Kumegawa, T. Kokubo, and T. Yamamura Cathepsin K Antisense Oligodeoxynucleotide Inhibits Osteoclastic Bone Resorption J. Biol. Chem., March 28, 1997; 272(13): 8109 - 8112. [Abstract] [Full Text] [PDF]
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M R Johnson, M H Polymeropoulos, H L Vos, R I Ortiz de Luna, and C A Francomano A nonsense mutation in the cathepsin K gene observed in a family with pycnodysostosis. Genome Res., November 1, 1996; 6(11): 1050 - 1055. [Abstract] [PDF]
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M. J. Bossard, T. A. Tomaszek, S. K. Thompson, B. Y. Amegadzie, C. R. Hanning, C. Jones, J. T. Kurdyla, D. E. McNulty, F. H. Drake, M. Gowen, et al. Proteolytic Activity of Human Osteoclast Cathepsin K J. Biol. Chem., May 24, 1996; 271(21): 12517 - 12524. [Abstract] [Full Text] [PDF]
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I. E. James, R. W. Marquis, S. M. Blake, S. M. Hwang, C. J. Gress, Y. Ru, D. Zembryki, D. S. Yamashita, M. S. McQueney, T. A. Tomaszek, et al. Potent and Selective Cathepsin L Inhibitors Do Not Inhibit Human Osteoclast Resorption in Vitro J. Biol. Chem., April 6, 2001; 276(15): 11507 - 11511. [Abstract] [Full Text] [PDF]
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