From the § Tissue Engineering Research Center
(TERC), National Institute of Advanced Industrial Science and
Technology (AIST), 1-1-1 Higashi, Tsukuba Ibaraki 305-8562, Japan, CREST Japan Science and Technology Corporation
(JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan,
** Department of Biomedical Engineering, Graduate School of Medicine,
University of Tokyo, 7-3-1, Hongo, Bunkyoku, Tokyo 113-0033, Japan,
§§ Institute of Applied Biochemistry, University
of Tsukuba, Tsukuba Ibaraki, 305-8572, Japan, and ¶¶ Liver
Cancer Institute, Shanghai Medical University, Yi Xue-Yuan Road,
Shanghai 200032, China
Received for publication, February 2, 2001, and in revised form, March 13, 2001
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ABSTRACT |
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This is the first report of a novel
serine/threonine kinase, rabbit death-associated protein (DAP)
kinase-related apoptosis-inducing protein kinase 1 (rDRAK1), involved
in osteoclast apoptosis. We searched for osteoclast-specific genes from
a cDNA library of highly enriched rabbit osteoclasts cultured on
ivory. One of the cloned genes has a high homology with human
DRAK1 (hDRAK1), which belongs to the DAP kinase subfamily of
serine/threonine kinases. By screening a rabbit osteoclast cDNA
library and 5'-RACE (rapid amplification of cDNA ends), we obtained
a full length of this cDNA, termed rDRAK1. The sequencing
data indicated that rDRAK1 has 88.0, 44.6, 38.7, and 42.3% identity
with hDRAK1, DAP kinase, DRP-1, and ZIP (zipper-interacting protein)
kinase, respectively. To clarify the role of DRAK1 in osteoclasts, we
examined the effect of three osteoclast survival factors
(interleukin-1, macrophage colony-stimulating factor, and osteoclast
differentiation-inducing factor) on rDRAK1 mRNA expression and the
effect of rDRAK1 overexpression on osteoclast apoptosis. The results
suggested that these three survival factors were proved to inhibit
rDRAK1 expression in rabbit osteoclasts. After transfection of a rDRAK1
expression vector into cultured osteoclasts, overexpressed rDRAK1 was
localized exclusively to the nuclei and induced apoptosis. Hence,
rDRAK1 may play an important role in the core apoptosis program in osteoclast.
Osteoclasts are multinucleated giant cells with the resorbing
activity of calcified tissues. Stem cells for osteoclasts are present
in the hematopoietic tissue (1). How the mononuclear stem cells fuse
and differentiate into mature osteoclasts has begun to be clarified
with the identification of osteoclast differentiation-inducing factor
(ODF)1/receptor activator of
nuclear factor- Oscteoclast culture in vitro has shown that interleukin 1 (IL-1), macrophage colony-stimulating factor (M-CSF)/colony
stimulating-factor-1, and ODF/RANKL/TRANCE/OPGL support osteoclast
survival (14). On the other hand, transforming growth factor- For several years, we have tried to clone osteoclast-specific genes
involved in osteoclast resorption (cycle) including apoptosis. For this
purpose, it was necessary to isolate primary osteoclasts of sufficient
numbers and purity for biochemical or molecular biological study.
Recently, an efficient method for the isolation of rabbit osteoclasts,
established by Kakudo et al. (18), has contributed to many
studies on osteoclasts (19-24). Using a cDNA library of highly
enriched rabbit osteoclasts, we obtained several fragments of genes
that might be related to the osteoclast resorption cycle. One of them
has high homology with hDRAK1, a member of the DAP kinase subfamily
(25, 26), and it was termed as rDRAK1. DAP kinase is a
calcium/calmodulin-regulated serine/threonine protein kinase and an
effector of Isolation of Osteoclasts--
Osteoclasts were harvested from
10-day-old Japanese white rabbits according to the method of Kakudo
et al. (18) with minor modifications. Briefly, the long
bones were minced with scissors for 15 min in Construction of an Osteoclast cDNA Library and cDNA
Cloning--
The purified rabbit osteoclasts were cultured on sliced
ivory with Tissue Distribution Determined by Slot Blot Analysis--
The
total RNA of rabbit tissue (bone marrow, brain, lung, liver, and
kidney) was isolated and further purified by the TRIzol method. A nylon
membrane (Amersham Pharmacia Biotech) of suitable size was used after
soaking in 10× standard saline citrate (SSC) for 10 min. The RNA
(10 µg) in denatured solution was heated at 65 °C for 15 min and
then immediately chilled on ice. The RNA mixture was blotted onto nylon
membrane followed by heating membrane at 80 °C for 2 h.
Hybridization with standard hybridization buffer containing digoxigenin
(DIG)-labeled probe at a concentration of 2 µg/ml was carried out
overnight at 65 °C. After washing the membrane, the color was
developed with a DIG DNA detection kit (Roche Diagnostics). The
obtained cDNA clone was prepared as DIG-labeled probe with a DIG
DNA labeling kit. The density of the positive spots was analyzed with
NIH Image software. In this case, DIG-labeled Quantitative RT-PCR--
Total RNA was isolated from osteoclasts
using TRIzol® reagent. For reverse transcription, the
reaction mixture contained 2 µg of RNA, 2.5 µM
oligo(dT) primer, and 5 units of avian myeloblastosis virus reverse
transcriptase (AMV, TaKaRa) in a total volume of 20 µl. The reaction
was performed by incubation for 1 h at 42 °C and stopped by
heating for 5 min at 99 °C. Aliquots (0.5 µl) of the reverse
transcriptase products were amplified in the reaction mixture (20 µl)
containing LightCyclerTM-FastStart DNA Master SYBR Green I, 0.5 µM each primer, and 3 mM MgCl2
using LightCyclerTM (Roche Molecular Biochemicals). After
pre-incubation at 95 °C for 10 min, a PCR was performed with 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C
for 5 s, and elongation at 72 °C for 10 s. A single
fluorescence reading was taken in each cycle following the
elongation step. The primers used were as follows: rDRAK1,
5'-CGTGGTTGACACAGAGCAGT-3' (corresponding to 905-924) and
5'-TTCGGTTCCTGGTTTCTCAG-3' (1016-1035); rabbit glyceraldehyde-3-phosphate dehydrogenase as an internal standard, 5'-CGACATCAAGAAGGTGGTGA-3' and 5'-CCAGCATCGAAGGTAGAGGA-3'. The GenBankTM accession number for rabbit
glyceraldehyde-3-phosphate dehydrogenase is L23961. The relative rDRAK1
mRNA levels for each condition were determined by performing
quantitative RT-PCR three times for each of the six independent batches
(n = 6).
Transfection and Immunostaining--
We subcloned the
full-length rDRAK1 and LacZ into the pcDNA4/HisMaxA expression
vector (Invitrogen) to create His-tagged rDRAK1 and LacZ expression
vectors. The semi-purified osteoclasts were cultured on FBS-coated
4-well glass chamber slides (Nunc) for 24 h at 37 °C.
Transfection was done by using TfxTM-50 reagent (Promega). After the
cultivation period, the cells were incubated in the presence of
0.1-0.5 µg of His-rDRAK1 or His-LacZ in Detection of DNA Fragmentation by TUNEL--
After 6 h
of incubation in the presence of TfxTM-50 and His-rDRAK1 or His-LacZ
expression vector, TUNEL was performed with an in situ cell
death detection kit-alkaline phosphatase (Roche Diagnostics)
according to the manufacturer's instructions. Briefly, after the
cultivation, the cells were fixed with 3.7% formaldehyde, washed with
PBS, and then permeabilized in 0.1% sodium citrate containing 0.1%
Triton X-100 for 2 min on ice. After permeabilization, the cells were
incubated in a TUNEL reaction mixture comprising terminal
deoxynucleotidyl transferase for 60 min at 37 °C, washed with PBS,
and incubated in anti-fluorescein antibody conjugated with
alkaline phosphatase. After a reaction using nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP, Roche Diagnostics) and 4-toluidine salt as substrates for alkaline
phosphatase, the stained cells were analyzed under a light microscope.
Cloning of rDRAK1--
We have cloned several gene fragments of
osteoclast, which might be related to each resorption process
(27). One of these fragments has a sequence of 291 base pairs located
at the 3'-terminus as shown in Fig. 1. By
comparison with the nucleotide data base of BLAST, it was found to have
high homology with hDRAK1, in which the catalytic domain is related to
that of DAP kinase, a serine/threonine kinase involved in apoptosis. In
this paper, we focus on this gene and its role in osteoclast apoptosis.
To obtain a full-length copy of the fragment related to hDRAK1,
screening of a rabbit osteoclast cDNA library and 5'-RACE were
performed. This cDNA library was constructed from osteoclasts
attached to ivory and included 1.7 × 106 independent
clones. For 5'-RACE, the RNA extracted from bone marrow cells was used
as a template. The predicted amino acid sequence of the obtained
full-length cDNA, termed rDRAK1, is 397 residues long (Fig. 1) and
is 88.0, 58.4, 44.6, 38.7, and 42.3% identical to hDRAK1, hDRAK2, DAP
kinase, DRP-1, and ZIP kinase (25, 26, 28-30), respectively. The
putative kinase domain of rDRAK1 is located at the N terminus and
contains all 11 subdomains conserved among serine/threonine kinases
(Fig. 2).
Tissue Distribution of rDRAK1--
Comparing rDRAK1 expression in
six kinds of tissue (bone marrow, brain, heart, lung, liver, and
kidney), the expression is highest in bone marrow as shown in Fig.
3. This result is consistent with the
presence of mature osteoclasts in bone marrow tissue. DRAK1 expression
levels in brain and lung are similar, and the expression in heart is
the weakest.
Effect of Osteoclast Survival Factors on mRNA Expressions of
rDRAK1--
To elucidate the role of DRAK1 in osteoclast apoptosis, it
is necessary to examine the effect of osteoclast survival factors on
DRAK1 mRNA expression. However, to carry out such a series of
experiments, the osteoclast mRNA from 10 rabbits was
needed. The usual semi-quantitative RT-PCR method was not adequate.
Therefore, we adopted a quantitative RT-PCR method for this
analysis, as described under "Experimental Procedures," using a
LightCyclerTM system and reagents (Roche Molecular Biochemicals).
Typical examples of the use of this system are cited in the references
(31, 32). We chose IL-1, M-CSF, and ODF as typical osteoclast survival factors.
Fig. 4A shows the effect of
the osteoclast survival factors on DRAK1 mRNA expression. The three
factors inhibited DRAK1 expression by 51, 34, and 57% (IL-1, M-CSF,
and ODF, respectively). We observed the inhibition of DRAK1 expression
by survival factors to be partial. With survival factors, DRAK1 is
still expressed in osteoclasts. At the same time, optical microscopic
images are shown in Fig. 4B, suggesting that those three
factors induced spreading of osteoclasts.
Cellular Localization of Ectopically Expressed rDRAK1 in
Osteoclasts--
It has been reported that hDRAKs are exclusively
localized to the nuclei (25). To investigate the cellular localization of rDRAK1, the expression plasmids His-rDRAK1 and His-LacZ were transiently transfected into osteoclasts. After a cultivation period,
the His-tagged proteins were detected by indirect immunostaining with anti-His monoclonal antibody and fluorescein
isothiocyanate-conjugated secondary antibody. All nuclei could be
simultaneously visualized by staining with propidium iodide. As seen in
Fig. 5, a-c, the fluorescent
signals showed that LacZ localized in the cytoplasm but not in the
nucleus. On the other hand, in rDRAK1-transfected osteoclasts, the
fluorescent signals and the nuclei stained by propidium iodide
overlapped as shown in Fig. 5, d-f. These results indicated
that rDRAK1 is exclusively localized to the nuclei.
Overexpression of rDRAK1 Induced Apoptosis in
Osteoclasts--
Most of the DAP kinase family has the ability to
induce apoptosis in various cells (25, 26, 28, 29, 33). It was reported
that DAP kinase participates in tumor necrosis factor- This is the first report of a novel serine/threonine kinase,
rDRAK1, involved in osteoclast apoptosis. In the past 5 years, much
work has been done relating to osteoclast apoptosis of the basic and
clinical level, with many studies using bisphosphonates. Bisphosphonates are known to inhibit bone resorption and to be therapeutically effective in diseases involving increased bone turnover, such as Paget's disease. Apoptosis has proved to be a major
mechanism whereby bisphosphonates reduce osteoclast numbers and
activity (12, 35). Further study of bisphosphonate-induced signaling
and its relation to the mevalonate pathway (36-39), as well as caspase
cleavage of MST1 kinase (mammalian STE20-like kinase 1) (38), is
underway. Moreover, the action of bisphosphonates in bone metastasis
has attracted much attention in cancer researchers (40, 41).
Suppression of osteoclast activity is a primary approach to inhibiting
bone metastasis, and bisphosphonates, specific inhibitors of
osteoclasts, have been used widely for treatment of bone metastasis in
cancer patients. Despite the clinical and basic importance of
bisphosphonates in osteoclast apoptosis, the molecular mechanism
inducing apoptosis has not been fully elucidated, and no report has
been published regarding the core apoptosis program in osteoclasts. In
addition to bisphosphonates, estrogen (13, 15), high extracellular
calcium ions (42, 43), and mechanical force (44) have been reported to
induce osteoclast apoptosis.
IL-1 (45, 46), M-CSF/colony stimulating-factor-1 (47), and
ODF/RANKL/TRANCE/OPGL (48), by contrast, have been reported to support
osteoclast survival by preventing apoptosis. Jimi et al.
(45) found that IL-1 transiently activates transcription factor NF- DRAK1 and DRAK2 were first cloned by Sanjo et al. (25) in
1998 as novel serine/threonine kinases. Both DRAKs are composed of an
N-terminal catalytic domain and a C-terminal domain responsible for the
regulation of kinase activity. The kinase activity of DRAK1 is
significantly stronger than that of DRAK2. How, specifically, is DRAK1
expressed in osteoclasts? Sanjo et al. (25) reported that
overexpression of DRAK1 induced morphological changes in NIH3T3 cells
that were associated with apoptosis but not in COS-7 cells, suggesting
that the sensitivity to DRAK1-induced apoptosis differed depending on
cell type. According to our results, DRAK1 is strongly expressed in
bone marrow tissues. Moreover, DRAK1 is expressed weakly or not at all
in osteoblasts (data not shown); however, it is expressed strongly in
osteoclasts. These results suggest that DRAK1 is closely involved in
the regulation of osteoclastogenesis and osteoclast apoptosis. How does
DRAK1 act in osteoclast apoptosis? DRP-1 (28), DRAK1/2, and ZIP kinase
(29) form a DAP kinase subfamily of serine/threonine kinases that
mediate apoptosis. As shown in Fig. 2, all the members have a similar
kinase domain at their N terminus. DAP kinase (26, 34, 51) and DRP-1
have a calcium/calmodulin regulatory region and are localized to the cytoplasm, whereas DRAK1/2 and ZIP kinase have no calmodulin region and
are localized to the nuclei. Overexpression in the wild type and kinase
inactive mutant suggests that their kinase activity triggers apoptosis.
Among, DAP, DRP-1, DRAK1/2, and ZIP kinase, the noncatalytic C-terminal
regions are structurally different and do not share any homology. DAP
kinase contains two known domains characterized by eight ankyrin
repeats and the death domain, whereas ZIP kinase has a leucine zipper
domain at its C-terminal end. These domains are known to mediate
protein-protein interaction. It is suggested that DAP kinase is
activated by the formation of a homodimer or binding to regulatory
molecule(s) and ZIP kinase is activated when it homodimerizes through
the leucine zipper domain. The C-terminal end of DRP-1 is also
essential for its dimerization, and the homodimerization is a
requirement for the functionality of this kinase in apoptosis. By
analogy with other DAP kinases, the noncatalytic C-terminal region of
DRAK1 may function as an interaction domain important for induction of
apoptosis through homodimerization or binding to some regulatory
molecule(s). Our DRAK1 overexpression experiment suggests that DRAK1
was exclusively localized to the nuclei, consistent with the results of
Sanjo et al. (25), and at the same time, apoptosis was
clearly observed by TUNEL. This means that DRAK1 triggers osteoclast
apoptosis in the nuclei by its kinase activity, perhaps through
homodimerization or binding to specific regulatory molecules. According
to our results, rDRAK1 is expressed in active osteoclasts, even
with survival factors, which also suggests the existence of an
inhibitory (regulatory) molecule for rDRAK1-induced apoptosis. However,
the upstream cascade of the DAP kinase family including DRAK1
and their substrates are unknown. In general, serine/threonine kinases function as molecular gatekeepers guarding entry into the death pathway, and their substrates can either enhance or inhibit
susceptibility to apoptosis (52). Moreover, no evidence has been
presented that kinases are required for the execution of apoptosis. The caspase family appears to be the executioners of apoptosis, although none has been shown to be regulated by phosphorylation. It is likely
that kinases play important roles in signaling cascades linking
exogenous stimuli to the core apoptosis program. rDRAK1 might be a
candidate for a key effective molecule of the apoptotic signaling
cascade, and more experiments on DRAK1 might open the way for
elucidating the core apoptosis program in osteoclasts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (NF-
B) ligand (RANKL)/tumor necrosis
factor-related activation-induced cytokine (TRANCE)/osteoprotegerin ligand (OPGL) (2-5), and osteoclastogenesis inhibitory factor (OCIF)/osteoprotegerin (OPG)/tumor necrosis factor receptor-like molecule 1 (TR1) (6-8). After maturation, osteoclasts undergo a
multistep resorption process (resorption cycle) that involves matrix
recognition, osteoclast attachment, polarization and formation of a
sealing zone on the bone, and resorption itself, with final detachment
and possible cell death (9, 10). Apoptosis of osteoclasts is a hot
topic experimentally and clinically in bone biology for the following
reasons. It has been reported that osteoclasts die by apoptosis at the
end of the bone resorption process (11) and that control of apoptosis
might potentially represent a key step in the regulation of bone
resorption (remodeling). It has been suggested that the drugs with the
most potential in the treatment of osteoporosis, bisphosphonates (12)
and estrogens (13), act as inducers of osteoclast apoptosis.
and
OCIF/OPG/TR1 have been reported to inhibit osteoclast survival (13,
15-17). However, the kinetics of osteoclast apoptosis is not yet clarified.
-interferon-mediated apoptosis in HeLa cells (26). To
demonstrate the role of rDRAK1 in rabbit osteoclast apoptosis, we
identified the full sequence of rDRAK1 and verified the effect of
typical osteoclast survival factors (IL-1, M-CSF, and ODF) on DRAK1
mRNA expression by a quantitative reverse transcription polymerase
chain reaction (RT-PCR) method. We also examined the overexpressed
rDRAK1-inducing apoptosis in rabbit osteoclasts by terminal
deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end
labeling (TUNEL).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimum essential
medium (
-MEM, Life Technologies, Inc.) supplemented with 5% fetal
bovine serum (FBS, Life Technologies, Inc.). The unfractioned cells
were collected and seeded on collagen gel (Nitta Gelatin Inc.)-coated
dishes. After 3 h of incubation, the cells were treated with
phosphate-buffered saline (PBS) containing 0.001% Pronase (Sigma) and
0.02% EDTA for 15 min at room temperature to remove the nonadherent
hematopoietic cells; this was followed by treatment with 0.01%
collagenase (Wako Pure Chemical Industries, Ltd.) for 5 min at room
temperature to further remove most of the stromal cells. Finally,
osteoclasts were obtained in suspension by treatment with 0.1%
collagenase at 37 °C for 10 min. The yield and purity of osteoclasts
from one rabbit was 1-3 × 104 and more than 90%,
respectively. Purified osteoclasts were seeded on round ivory slices
(6.5-mm radius) in four kinds of medium:
-MEM with 5% FBS
supplemented with 1.0 ng/ml IL-1 (mouse recombinant, Life Technologies,
Inc.), 50 ng/ml M-CSF (mouse recombinant, Calbiochem), or 500 ng/ml ODF
(mouse recombinant, Calbiochem) and no supplement. When the cells were
prepared for transfection, the use of 0.001% Pronase, 0.02% EDTA and
0.01% collagenase was omitted. After 3 h of incubation, the
nonadherent cells were washed out with PBS, and the osteoclasts were
then collected with 0.1% collagenase. This short procedure yielded
2-5 × 104 osteoclasts/rabbit with 30-50% purity.
-MEM containing 5% FBS for 3 h at 37 °C. Total
RNA was then extracted and further purified with TRIzol®
reagents (Life Technologies, Inc.) according to the manufacturer's instructions. Following reverse transcription of the RNA, we
constructed a cDNA library of highly enriched rabbit osteoclasts
cultured on ivory by using THE SMARTTM cDNA Library Construction
Kit (CLONTECH). The cDNA fragment cloned into
The pCR-TRAP vector (GenHunter Co.) was sequenced using an automated
DNA sequencer (ABI model 310, PerkinElmer Applied Biosystems). Then,
the full-length cDNA of the gene was obtained by screening the
rabbit osteoclast cDNA library and the 5'-rapid amplification of
cDNA ends (5'-RACE) according to the manufacturer's instructions
(Life Technologies, Inc.). The cDNA obtained was labeled with a DIG
DNA labeling kit (Roche Diagnostics) and used as probe for tissue
distribution. The determined DNA sequences were subjected to a homology
search against the BLAST nucleotide sequence data base.
-actin was used as an
internal standard. Each experiment was done twice (two batches using
two rabbits) and independently (n = 2).
-MEM containing 5% FBS
and 100 nM TfxTM-50 and then cultured for 6 h at
37 °C. After 6 h of incubation, the cells were fixed with 3.7% formaldehyde (TAAB Laboratories Ltd.) and were blocked with 5% skim milk containing 0.05% Triton X-100. They were then incubated with
mouse anti-6-His monoclonal antibody (Babco Covance Co.) at 4 °C
overnight. After being washed with 0.1% Tween 20 in PBS three times,
the cells were stained with goat anti-mouse fluorescein isothiocyanate-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for 2 h at room temperature. After three more
washes, the nuclei were stained with 10 µg/ml propidium iodide (PI,
Sigma), and the samples were embedded in 0.1% paraphenylenediamine
(Sigma) in glycerol. The cells were then observed under a laser
confocal scanning microscope (MRC-600 UV, Bio-Rad).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide sequence and deduced amino acid
sequence of rDRAK1 cDNA. The nucleotide and amino acid
sequences of rDRAK1 cDNA are shown. The stop codon is indicated by
an asterisk. The initial DNA fragment used for
cDNA cloning is underlined. The numbering of
the nucleotides and amino acids is indicated on the
right.
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Fig. 2.
Alignment of amino acid sequences of rDRAK1,
hDRAK1/2, DAP kinase, DRP-1, and ZIP kinase. The sequences of
rDRAK1, hDRAK1/2, DAP kinase, DRP-1, and ZIP kinase with
cDNA-deduced amino acid sequences are described (25, 26, 28, 29,
30). The 11 subdomains are conserved among these kinases. Identical
amino acid residues are indicated by solid boxes. Gaps are
indicated by hyphens. The GenBankTM
accession numbers for rDRAK1, hDRAK1, hDRAK2, DAP kinase, DRP-1, and
ZIP kinase in DDBJ/GenBankTM/EMBL are AB042195, AB011420,
NM004226, NM004938, NM014326, and AB022341, respectively.
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Fig. 3.
Tissue distribution of rDRAK1 in rabbit.
The relative rDRAK1 mRNA levels in rabbit tissues (bone marrow,
brain, heart, lung, liver, and kidney) are shown. The RNA of each
tissue was hybridized on nylon membrane with a DIG-labeled
DRAK1 probe and subjected to color development with a DIG DNA detection
kit. A -actin probe was used as an internal standard. Each
experiment was done twice (two batches using two rabbits) independently
(n = 2). The data are given as the mean ± S.E.
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Fig. 4.
Effect of osteoclast survival factors on
mRNA expression of DRAK1 (A) and osteoclast
morphology (B). Highly enriched osteoclasts were
cultured on ivory slices for 3 h in the four kinds of medium:
-MEM with 5% FBS supplemented with 1.0 ng/ml IL-1, 50 ng/ml M-CSF,
or 500 ng/ml ODF, and no supplement. Total RNA was isolated by the
TRIzol method. The relative rDRAK1 mRNA levels for each condition
were determined by performing quantitative RT-PCR three times for each
of six independent batches (n = 6) as described under
"Experimental Procedures." A, rDRAK1 mRNA level;
B, optical microscopic images of osteoclasts.
Bar, 10 µm. The data are shown as the mean value ± S.E.
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Fig. 5.
Cellular localization of the ectopic
expression of rDRAK1 on osteoclasts. Osteoclasts were transfected
with 0.5 µg of pcDNA4/HisMaxA-rDRAK1 (His-rDRAK1) or LacZ (His-LacZ)
plasmid, which contain His tag. Six hours post-transfection, the cells
were immunostained using mouse anti-6-His monoclonal antibody followed
by goat anti-mouse fluorescein isothiocyanate-conjugated secondary
antibody. Micrographs show epitope staining (left) of LacZ
(a-c) or rDRAK1 (d-f), propidium iodide
staining (middle), and a composite of the two panels
(right). Bar, 50 µm.
and
Fas-induced apoptosis (34). To investigate whether rDRAK1 induces
apoptosis in osteoclasts, we performed a TUNEL assay to detect DNA
fragmentation after transfection. As shown in Fig. 6, the nuclei of rDRAK1-transfected
osteoclasts were stained strongly, indicating that the nucleus
condensed and was then fragmentated. Chromatin condensation followed by
fragmentation is a specific morphological characteristic of apoptosis;
therefore, rDRAK1 induced apoptosis in osteoclasts when it was
overexpressed. On the other hand, overexpression of His-LacZ did not
affect the DNA fragmentation or the nuclear morphology of
osteoclasts.
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Fig. 6.
rDRAK1-induced osteoclast apoptosis.
Osteoclasts were transfected and stained by TUNEL assay as described
under "Experimental Procedures." Micrographs show the results of
the TUNEL assay to detect DNA fragmentation. Arrows show
condensed chromatin. Overexpression of His-LacZ did not affect the
morphology of osteoclasts (left), but that of His-rDRAK1
induced an apoptotic nuclear morphology (right).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
found in the osteoclast nuclei and involved in the survival of
osteoclasts. M-CSF plays important roles throughout the life of the
osteoclast including in proliferation, differentiation, migration,
chemotaxis, and survival (49). Both IL-1 and M-CSF induce the
multinucleation of pre-osteoclasts through their respective receptors;
however, actin ring formation (a functional marker of osteoclasts) and
pit-forming activity of multinucleated cells have been observed in
pre-osteoclast culture treated with IL-1 but not with M-CSF (46). These
results suggest that IL-1 induces the bone resorbing activity of
osteoclasts but M-CSF does not. Okahashi et al. (50)
reported the involvement of caspase in the regulation of osteoclast
survival. They found that two peptide inhibitors of caspase extended
the survival time of osteoclast-like cells and the effect was enhanced
by co-addition with IL-1 or M-CSF. They suggest that caspase-3 is
particularly important in osteoclast apoptosis. ODF targets osteoclast
precursors and osteoclasts to enhance differentiation and activation.
The effect of ODF on osteoclast survival was reported by Lacey et
al. (48). According to their report, apoptotic change with
elevated caspase-3 activity was inhibited by a combination of caspase-3
inhibitor and ODF. The signaling/survival pathways of both ODF and
M-CSF are required for optimal osteoclast survival. They also
reported that M-CSF maintained NF-
B activation and increased the
expression of bcl-2 but had no effect on c-Jun
N-terminal kinase (JNK) kinase activation. In contrast, ODF enhanced
both NF-
B and JNK kinase activation and increased the expression of
c-src but not bcl-2. Therefore ODF is essential
but not sufficient for osteoclast survival, and endogenous M-CSF levels
are insufficient to maintain osteoclast viability in the absence of
ODF. These findings indicate that IL-1, M-CSF, and ODF support
osteoclast survival but differ in their manner or mechanism. The
results outlined in Fig. 4 show that the expression of DRAK1 was
inhibited partially by each of the three survival factors, IL-1, M-CSF,
and ODF. This finding would imply that DRAK1 contributes to the
survival mechanism through NF-
B activation or apoptosis induced by
caspase-3 activation. The inhibitory effects on the mRNA expression
of DRAK1 might therefore be a common way for maintaining osteoclast survival.
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ACKNOWLEDGEMENTS |
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We thank Dr. W. Chen (Shanghai Medical University) for supporting the sequencing work, Dr. T. Inui of Osaka Bioscience Institute for helpful suggestions on osteoclast treatment, and Dr. T. Ushida and T. Tateishi of the University of Tokyo for encouragement throughout the course of this work. We thank Dr. T. Imamura of the National Institute of Bioscience and Human Technology (NIBH) for generously giving permission to use the LightCyclerTM system.
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FOOTNOTES |
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* This work was supported in part by Grant JSPS-RFTF96I00202 for the "Research for the Future Program" from the Japan Society for the Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB042195 (rDRAK1).
The first three authors contributed equally to this work.
¶ Domestic Research fellow, Japan Science and Technology Corporation (JST).
To whom correspondence should be addressed. Tel.:
+81-298-61-2559; Fax: +81-298-61-2565; E-mail:
t.uemura@aist.go.jp.
Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.M101023200
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ABBREVIATIONS |
---|
The abbreviations used are:
DAP kinase, death-associated protein kinase;
DRAK, DAP kinase-related
apoptosis-inducing protein kinase;
RACE, the rapid amplification of
cDNA ends protocol;
DRP-1, DAP kinase-related protein 1;
ZIP
kinase, zipper-interacting protein kinase;
IL-1, interleukin 1;
M-CSF, macrophage colony stimulating factor;
ODF, osteoclast differentiation
inducing factor;
NF-B, nuclear factor-
B;
RANKL, receptor
activator of NF-
B ligand;
TRANCE, tumor necrosis factor-related
activation-induced cytokine;
OPG, osteoprotegerin;
OPGL, OPG ligand;
OCIF, osteoclastogenesis inhibitory factor;
TR1, tumor necrosis factor
receptor-like molecule 1;
CSF-1, colony stimulating factor 1;
TGF-
, transforming growth factor-
;
RT-PCR, reverse transcription
polymerase chain reaction;
TUNEL, terminal deoxynucleotidyl transferase
(TdT)-mediated dUTP-biotin nick end labeling;
-MEM,
-minimum
essential medium;
FBS, fetal bovine serum;
PBS, phosphate-buffered
saline;
DIG, digoxigenin..
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