From the Division of Endocrinology, Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425
Received for publication, February 7, 2000, and in revised form, November 17, 2000
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ABSTRACT |
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Superoxide production contributes to osteoclastic
bone resorption. Evidence strongly indicates that NADPH oxidase is an
enzyme system responsible for superoxide generation in osteoclasts. A membrane-bound subunit, p91, is the catalytic domain of NADPH oxidase.
However, osteoclasts from p91 knockout mice still produce superoxide at
a rate similar to that observed in wild type mice. This unexpected
phenomenon prompted us to examine the osteoclasts for an alternative to
the p91-containing oxidase. In this study, the cloning of a NADPH
oxidase subunit (Nox 4) with 578 amino acids is reported. Nox 4 has
58% similarity in amino acids with the known p91 subunit of NADPH
oxidase. Nox 4 is present and active in osteoclasts. Antisense
oligonucleotides of Nox 4 reduced osteoclastic superoxide generation as
well as resorption pit formation by osteoclasts. This new oxidase
complex was present and functional in osteoclasts from p91 knockout
mice, explaining the normal resorptive activity seen in the osteoclasts
where no p91 is present.
Osteoclast-generated superoxide directly contributes to bone
degradation. The presence of superoxide production at the
osteoclast-bone interface suggests a direct effect of superoxide in
osteoclastic bone resorption (1, 2). In addition, inhibition of
osteoclastic superoxide availability results in a reduction in bone
resorption (3, 4). Treatment with interferon NADPH oxidase, a common enzyme system that produces superoxide in white
cell phagocytes, is also present and active in osteoclasts. Several
studies suggest that NADPH oxidase is responsible for osteoclastic
superoxide production (3, 7, 8). However, in studies of p91 knockout
mice, despite the absence of the p91 catalytic subunit of NADPH
oxidase, osteoclasts generate normal amounts of superoxide. X-ray bone
density analysis demonstrates that the p91 knockout mice are not
osteopetrotic, as would be expected if there were a severe defect in
osteoclastic bone resorption (data not shown). In keeping with the
normal superoxide production found in the osteoclasts from the p91
knockout mutants, a similar finding has been reported in the lung
endothelium. Kubo et al. (9) found that lung endothelial
cells from p91 knockout animals generate normal amounts of superoxide.
In these endothelial cells, the xanthine oxidase enzyme complex was
found to be responsible for generating superoxide. In a study of human
fibroblasts, a "second" NADPH oxidase was demonstrated in cells
from a patient afflicted with chronic granulomatous disease, who had a
mutation in the p91 gene. This fibroblastic oxidase
differed immunologically and functionally from NADPH oxidase,
substantiating the presence of an alternative oxidase (10). Thus, in at
least two cell types, superoxide generation by oxidases other than
NADPH oxidase has been observed.
In this study, we have searched for an alternative oxidase responsible
for superoxide generation in the osteoclasts from p91 knockout mice. To
accomplish this, a mouse EST1
has been identified by searching a gene bank. This EST contains 46%
amino acid identity with the C-terminal portion of mouse p91. A
complete sequence was obtained by 5'-RACE. Expression of a new oxidase
subunit, Nox 4, in the osteoclasts from p91 knockout mice was
demonstrated. Finally, using antisense oligonucleotide disruption of
the production of Nox 4, we have demonstrated that expression of Nox 4 is related to osteoclastic superoxide generation and bone resorption.
Materials--
Anti-Nox 4 antibody was generated at the Medical
University of South Carolina facility using a unique peptide
(SKTLHSLSNRNNSYGTKFEY). The rabbit anti-Nox 4 antibodies were further
purified by a peptide affinity column. Preincubation of the serum with
Nox 4 peptide (1 µg/ml) resulted in undetectable signals, suggesting
the specificity of the antibody. NTB2 liquid photographic emulsion was
purchased from Eastman Kodak Co. ECL was obtained from Amersham
Pharmacia Biotech. All other reagents were purchased from Sigma.
Cloning--
Using the mouse p91 sequence as a query to search
the murine EST data base, a homologous sequence (accession no.
AI746441) was found, which contained 46% identity in amino acid
sequence to the C-terminal portion of mouse p91. To obtain a complete
sequence, 5'-RACE was carried out using Marathon-ready cDNA
(CLONTECH, Palo Alto, CA). PCR was performed using
primer AP 1 and the gene-specific primer
(5'-CTGCACACCCAGATAAAGTACAGTCTT-3'). A 1.5-kilobase pair PCR product
was purified and cloned into a TA vector (Invitrogen, San Diego, CA).
The clones containing the insert were sequenced to reveal the complete
sequence for the Nox 4 mRNA.
Osteoclast Culture--
Osteoclasts were generated by the
culture of mouse bone marrow cells for 1-2 weeks in Antisense Oligonucleotides--
Antisense oligonucleotides were
designed near the ATG start codon of mouse native p91
(5'-CTTCATTCACAGCCCAGTTC-3') and Nox 4 (5'-AGCTCCTCCAGGACACCGCC-3').
Antisense and the corresponding sense oligonucleotides were synthesized
as phosphorothiolated oligonucleotides and purified by high performance
liquid chromatography (Integrated DNA Technology Inc., Coralville, IA).
All oligonucleotides were designed with the help of the OLIGO program
to minimize self-complementing and dimer formation.
Osteoclasts were incubated with 25 µM oligonucleotides in
To demonstrate antisense oligonucleotide incorporation into
osteoclasts, the 32P-labeled oligonucleotide was used.
Briefly, mouse osteoclasts were cultured on chamber slides. After
removing nonadherent cells, osteoclasts were incubated with
32P-labeled antisense oligonucleotide (50,000 cpm/ml,
~0.14 nM). A slide incubated with 0.14 µM
unlabeled oligonucleotide in addition to 32P-labeled
antisense oligonucleotide was the negative control. At the end of
incubation, osteoclasts were fixed with 10% formalin and stained with
TRAP to identify multinuclear osteoclasts. Thereafter, slides were
air-dried and dipped in NTB2 liquid photographic emulsion. After
exposure within a light-tight box at 4 °C for 2-3 weeks, slides
were then developed. The autoradiograms of osteoclasts incubated with
the 32P-labeled oligonucleotide were examined under a microscope.
The transfection efficiency of 32P-labeled oligonucleotide
was determined by the percentage of the 32P-labeled
oligonucleotide incorporated into osteoclasts. After being incubated
with 32P-labeled oligonucleotide (200,000 cpm/ml),
osteoclasts were stained by TRAP and the number of osteoclasts in each
dish was determined. The radioactivity in cell lysates and supernatant
removed from these cells was measured using a scintillation counter.
The transfection efficiency is expressed as a percentage of
32P-labeled oligonucleotide incorporated per 1 × 105 osteoclasts.
Western Blot Analysis--
Osteoclasts were cultured in 10-cm
plastic culture dishes. After vigorously washing with 1×
phosphate-buffered saline, nonosteoclastic cells were removed after a
brief digestion with collagenase/dispase (1 mg/ml) for 5-10 min. The
remaining attached cells were 90% pure multinucleated osteoclasts.
These osteoclasts were incubated in a 25 µM solution of
antisense oligonucleotides for 2 days. Thereafter, the osteoclasts
(0.1-1 × 105) were lysed with 1× sample buffer.
After a brief sonication, osteoclast lysates were loaded on 8% SDS
acrylamide gel. After transfer to a polyvinylidene difluoride membrane,
the membrane was incubated with anti-Nox 4 polyclonal antibody
(1:2000). Horseradish peroxidase-conjugated secondary antibody (1:5000)
was used to identify the presence of the bound anti-Nox 4 antibody
using ECL.
Superoxide Production--
Superoxide generation by individual
osteoclasts was determined by NBT assays (1). Briefly, osteoclasts were
incubated with NBT solution containing 2.0 mg/ml NBT, 35% FBS in RPMI
1640 medium for 1 h at 37 °C. After incubation, osteoclasts
were fixed in 10% formalin. Stained osteoclasts were identified as
multinuclear giant cells. Twenty randomly selected NBT-stained
osteoclasts from each treatment were analyzed. The NBT staining
intensity of individual osteoclasts was measured and quantified by
microdensitometry using an inverted microscope (Olympus) equipped with
a CCD camera. The camera images were displayed on a high resolution
monitor (Sony), stored in a microcomputer (ALR, Inc., Irvine, CA), and analyzed using densitometry software (Cue 2d, Opelco). The specificity of the NBT reduction assay for measuring osteoclastic superoxide generation has been demonstrated previously (1, 4).
A Pit Assay--
Bone resorption was determined by a pit assay
as described (12). Osteoclasts were isolated from the bone marrow of
4-6-day-old neonatal mice and placed on bovine bone slices for 0.5-1
h. After removing nonadherent cells by vigorous washing, osteoclasts
attached to the bone slices were treated with antisense and sense
oligonucleotides for 2 days. Triplicates of bone slices were kept for
each experimental condition. At the end of incubation, the cells on the
bone slices were first removed by gentle scraping and further treated
with 1 M NH4OH (1 ml/well) for 30 min. Bone
slices were then cleaned by untrasonication and stained with
hematoxylin (0.1%) for 35-45 s. The resorption pits were identified
using bright field reflected light microscopy. The plan area of
resorption pits was measured and quantified using the Cue 2 (C2D) image
analysis system (Opelco, Washington, DC) linked to the microscope.
Statistical analysis of the results was performed using Student's
t test.
Calvarial 45Ca Release Assay--
Bone resorption
determined by a 45Ca release assay was performed (4).
Briefly, 2-day-old mouse pups received subcutaneous injections of 0.5 µCi of 45CaCl2. Two days after injection,
calvaria were removed and preincubated in 1 ml of BGJb medium with 10%
FBS at 37 °C for 24 h to remove rapidly exchangeable
45Ca. After two rinses with the medium, the calvaria were
halved at the sagittal suture. One half of each calvarium was placed in
BGJb medium with 5% FBS containing DPI (1 µM) or PTH (3 µg/ml). The other half of the calvarium was incubated with the medium only. The calvaria were incubated in 5% CO2 at 37 °C
for 2 days. The 45Ca released into the medium was counted.
The calvarial bones were treated with 10% trichloric acid at 4 °C
for 24 h, and the trichloric acid digests were counted.
The resorption activity is expressed as a percentage of
45Ca released into the medium, calculated by the ratio of
the counts/min in the medium divided by the total counts/min
(counts/min in the medium plus counts/min from bone digests). The
results of the resorption assay are reported as the ratio of the
experimental to the control percentage of 45Ca release.
The Presence of an Alternative Enzyme Responsible for
SuperOxide Generation in p91(
The hypothesis that an alternative oxidase is present in osteoclasts
from the p91( A Complete Sequence of Nox 4--
Using the mouse p91 sequence as
a query to search the murine EST data base, a homologous sequence
(accession no. AI746441) was found that had 46% identity with the
C-terminal portion of mouse p91. A complete mRNA sequence (Nox 4, GenBankTM accession no. AF218723) was obtained by 5'-RACE. The
predicted amino acid sequence is shown in Fig.
2. Nox 4 has a predicted sequence containing 578 amino acids compared with 570 residues in murine p91.
The two sequences have 58% similarity. Conserved regions containing
FAD (13, 14) and a NADPH binding site (14, 15) are observed in Nox 4 (Fig. 2). Four histidine residues, which represent the conserved amino
acid for a heme binding site (16), are also present in Nox 4.
Expression of Nox 4--
The expression of Nox 4 in osteoclasts
from both p91( Effect of Antisense Oligonucleotide on Osteoclasts--
When
cultured osteoclasts were incubated with antisense oligonucleotide for
2 days, no significant difference in osteoclast viability was observed.
As shown in Fig. 5, the majority of cells consist of TRAP-positive osteoclasts (panel B)
and uptake of 32P-labeled oligonucleotides is observed in
osteoclasts (panel A). The efficiency of
osteoclastic uptake of 32P-labeled oligonucleotide is
~10%. Expression of Nox 4 protein in osteoclasts was undetectable
after incubation with Nox 4 antisense oligonucleotide, compared with
the control (Fig. 6).
Effect of Nox 4 Antisense Oligonucleotides on Superoxide Generation
and Bone Resorption--
Osteoclastic superoxide generation was
determined using an NBT assay. The addition of Nox 4 antisense
oligonucleotide resulted in an 80% reduction of superoxide production
for both C57Bl/6 and p91(
The degree of inhibition of bone resorption by antisense
oligonucleotides is shown in Tables II
and III. The number of bone resorption pits was reduced to ~50% when
antisense oligonucleotides of Nox 4 and p91 were included in the
incubation medium (Fig. 8). The average
resorption area for C57Bl/6 osteoclasts was reduced by 47% and 46%,
respectively, by Nox 4 antisense (p < 0.01) and p91
antisense (p < 0.01). It is important to note that a
combination of both the p91 and Nox 4 antisense oligonucleotides
reduced resorption by 61% compared with the control and by an
additional 27% compared with the Nox 4 alone. Furthermore, incubation
with the antisense oligonucleotides reduced the bone resorptive
activity of p91( Effect of DPI on Superoxide Generation and Bone
Resorption--
The p91 subunit is a FAD-containing flavoprotein that
transfers electrons to oxygen, producing superoxide. A flavoprotein inhibitor, DPI, has been shown to inhibit NADPH oxidase activity as a
consequence of binding to the flavoprotein covalently, thus interrupting the electron transferring process (17, 18). The sequence
of Nox 4 reveals two conserved regions for FAD binding. To determine
whether the Nox 4 complex requires the activity of the FAD-containing
region for the production of superoxide, DPI inhibition was performed.
Table IV shows that DPI inhibited not only osteoclastic superoxide generation, but also bone resorption in
both the wild type and p91( These studies demonstrate that Nox 4 is a p91-like protein that
produces osteoclastic superoxide in p91( Generation of superoxide is a common function of phagocytes and
osteoclasts. Superoxide produced by phagocytes plays an important role
in bacterial killing and in normal host protection (19). Osteoclasts
generate superoxide that is necessary for bone resorption. Activated
osteoclasts generate a ruffled-border space adjacent to bone which
serves as the active site for bone resorption. Osteoclasts secrete
hydrogen ions, proteinases, and superoxide into the ruffled border
space to excavate a resorption pit or lacuna on the bone surface.
Although an 80% decrease in superoxide generation was observed when
antisense oligonucleotides were used to inhibit the production of the
oxidase, bone resorption declined by ~50%. Antisense
oligonucleotides block superoxide generation, but acid production and
protease secretion from osteoclasts are presumably not altered. Thus,
osteoclasts continue to degrade bone, but the amount of bone resorption
is decreased (2, 4). In previous studies (4), Ries et al.
reported that pits obtained in the presence of a superoxide scavenger
still contained dangling collagen fibrils, while pits formed by control
osteoclasts, in the absence of the scavenger, had a smooth surface. In
the study, osteoclasts in the presence of the scavenger formed only one
pit. Osteoclasts without the scavenger moved to form multiple pits.
Thus, bone resorption activity is not halted in the absence of
superoxide, but only reduced. This explains why there is a correlation
between reduced superoxide production and bone resorption in the
absence of a one to one relationship.
Of great interest, patients with osteopetrosis have reduced, not absent
bone resorption as evidenced by N-telopeptide excretion (6). The
defects in superoxide generation described in patients with
osteopetrosis are related to the mechanisms that regulate oxidase
activity, such as the regulatory components of the oxidase (20) or the
ability of cells to depolarize, triggering the activation of oxidases
(21). Based upon current data, no explanation exists for the absence of
reduced bone resorption in the p91 knockout animal or humans with
chronic granulomatous disease. Perhaps it is possible to up-regulate
Nox 4 (or its human counterpart) when the NADPH oxidase is defective,
as suggested in the increased amount of Nox 4 mRNA present in p91
knockout animals (Fig. 3).
In addition to being expressed and active in osteoclasts, the
Nox4-containing oxidase does not appear to be stimulated to have the
"oxidative burst" phenomenon observed in phagocytes, but rather a
steady level of superoxide production (data not shown). Kinetic
experiments have suggested that the NADPH oxidase is stimulated in
bursts, both in white cells (22) and in osteoclasts (23). Thus, in the
wild type animals, the greater bone resorption may result from a burst
of superoxide production after the initial exposure to PTH, a time when
the bulk of calcium release occurs in the calvarial calcium release
assay (24). Thus, by 48 h of incubation, a greater calcium release
would be seen in the wild type cultures than in the
p91( Not only does superoxide directly contribute to bone resorption by
facilitating the degradation of bone matrix proteins (4), it is also
involved in the activation and formation of osteoclasts (25). A number
of reports have suggested that the types of physiological functions for
the superoxide generated by different cell types are linked with
cellular signaling and activation (26-29). Superoxide has been shown
to play a central role in the activation of the transcription factor
NF- A group at Emory University (31) reported an alternative oxidase
(MOX) in human (AF127763) and rat (AF152963) tissues. The murine Nox 4 protein, reported here, has 56% similarity to MOX found in humans and
54% to that of the rat. The existence of oxidases, other than NADPH
oxidase, in different species and tissues suggests that there is a
biological role for these oxidases, perhaps in generating the
appropriate amount or the targeted amounts of superoxide. The link
between osteoclastic superoxide production and bone resorption has been
underscored in a variety of studies (1-4, 8, 25). The absence of
defective bone resorption by osteoclasts derived from p91 knockout
animals would suggest that NADPH oxidase is not necessary for normal
bone resorption. NADPH oxidase has been considered to be the oxidase
responsible for all superoxide generation by osteoclasts (3, 7, 8). The data presented here demonstrate that at least one additional oxidase, Nox 4, contributes to superoxide production and bone resorption by both
p91 knockout and wild type murine osteoclasts.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, a stimulator of NADPH oxidase activity, corrects defective osteoclastic function in osteopetrotic, microphthalmic mice in vivo and in calvaria
cultured from these animals (5). In patients with osteopetrosis,
increased bone resorption was documented by a decrease in medullary
bone (6). Therefore, superoxide generation at the osteoclast-bone interface is necessary for optimal levels of bone resorption.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimal
essential medium, 10% FBS, and 1% penicillin containing 1 × 10
8 M 1,25-dihydroxyvitamin D3
and 25 ng/ml macrophage colony-stimulating factor (11). The medium was
changed twice a week. Nonosteoclastic cells were removed by vigorous
washings. Additional contaminating cells were removed by a brief
digestion (5-10 min) with lipase and collagenase (1 mg/ml). We
generated 10,000-100,000 osteoclasts from 10 mice. This cell
population was 90% pure osteoclasts. These osteoclasts stained
positively with TRAP and were able to make resorptive pits on bovine
bone slices (Fig. 8).
-minimal essential medium with 5% FBS. After 2 days of incubation, osteoclast viability was determined by exclusion of trypan blue from
the cytoplasm.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
)
Mutants--
As shown by Table I,
osteoclasts from p91(
/
) mutants produced an amount of
superoxide similar to that produced by osteoclasts from wild type mice.
In addition, no significant difference in bone resorption was found by
comparing cultures from p91(
/
) and C57/Bl6 mice.
Osteoclastic superoxide generation and bone resorption in C57Bl/6
and p91(/
) mice
/
) mutant was supported by a Southern blot
(Fig. 1). In this experiment, a
32-P-labeled cDNA fragment of the mouse p91 gene,
encoding a FAD binding region, was hybridized to genomic DNA digested
by a series of restriction endonucleases. Multiple bands were observed
when the membrane was washed under conditions of low stringency.
Moreover, some bands, identified under conditions of low stringency,
disappeared when a high stringency wash was performed (lanes
1, 3, 4, and 5). The
disappearance of the bands under high stringency conditions suggested
the presence of a homologous, but nonidentical sequence(s) to the mouse
p91.
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Fig. 1.
Demonstration of the presence of p91
homologues by a Southern blot. Mouse genomic DNA was extracted and
10 µg of DNA was digested with restriction enzymes (1,
AccI; 2, EcoNI; 3,
NcoI; 4, BamHI; 5,
EcoRI). After separation by 0.7% agarose gel
electrophoresis and transfer to a nylon membrane, the blot was
hybridized with 32P-labeled mouse p91 probe (350 base pairs
of FAD binding region). A low stringency wash was done at 2× SSC at
50 °C. After exposure to the film, the blot was washed again at high
stringency (0.1× SSC at 60 °C). Note that some bands disappeared
when high stringency washing was performed (lanes
1 and 3-5).
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Fig. 2.
A comparison of the predicted amino acid
sequence of Nox 4 (GenBankTM accession no AF218723) with mouse p91
(U43384). The asterisks indicate conserved histidine
residues that are responsible for heme group binding. The conserved FAD
binding regions are underlined as 1a and
1b. The conserved substrate NADPH binding region is
underlined as 2a. The plus
signs indicate a similar property between two amino
acids.
/
) and C57bl/6
mice was determined by reverse transcription-PCR. As shown in Fig.
3, Nox 4 was expressed in osteoclasts
from both animals. Densitometric analysis showed that osteoclasts from
p91 knockout mutants contained 2.5 times as much of Nox4 as osteoclasts
from the wild type mice. The expression of Nox 4 in leukocytes was also
observed in both p91(
/
) and
C57bl/6 mice (data not shown). In addition to its expression in
osteoclasts and leukocytes, Nox 4 mRNA was expressed in the mouse
kidney, liver, and lung (Fig. 4). In
contrast, p91 mRNA was highly expressed in the mouse spleen (data
not shown), suggesting that Nox 4 may have functions that are different
from those of p91.
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Fig. 3.
Expression of Nox 4 in osteoclasts.
Mouse osteoclasts were obtained from bone marrow cultures in the
presence of Vit. D3 and M- macrophage colony-stimulating
factor. After removing nonadherent cells by vigorous washing and
collagenase/dispase digestion, osteoclasts, identified as multinuclear
giant cells, were isolated by micromanipulation. Total RNA was
extracted and precipitated with the aid of glycogen. Reverse
transcription-PCR was performed using specific primers according to the
sequence of Nox 4. The PCR product of 300 base pairs is observed in
both C57Bl/6 and p91( /
) mouse osteoclasts
(A). Specificity of the PCR product was confirmed by
Southern blot (data not shown). B indicates the
densitometric analysis of PCR products. Data shown here
represent one of two separate experiments.
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Fig. 4.
Tissue distribution of Nox 4. Poly(A)
RNA was isolated from indicated mouse tissues. After separation by
electrophoresis and transfer to a nylon membrane, the blot was
hybridized with 32P-labeled Nox 4 probe.
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Fig. 5.
Localization of 32P-labeled
antisense oligonucleotide in osteoclasts. Osteoclasts were
cultured on chamber slides. After removing nonadherent cells,
32P-labeled antisense oligonucleotides were added at 50,000 cpm/well (0.14 nM). Osteoclast uptake of radioactivity was
determined by autoradiography. Dense silver grains were accumulated in
every osteoclast (A), suggesting that
32P-labeled antisense oligonucleotide was incorporated into
osteoclasts. A slide containing an excess of unlabeled antisense
oligonucleotide (0.14 µM) in addition to radioactive
oligonucleotide was shown in panel B. Osteoclasts
were identified as TRAP-positive giant cells and 90% of remaining
cells are osteoclasts (magnification, ×10). The lack of grains
over osteoclasts with excess unlabeled oligonucleotide demonstrated the
specificity of the experiment (B).
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Fig. 6.
Effect of antisense oligonucleotide on Nox 4 protein expression. P91( /
) osteoclasts were
incubated with or without 25 µM Nox 4 antisense
oligonucleotide for 2 days. Osteoclasts (0.1-1 × 105) were lysed with 1× sample buffer and the presence of
Nox 4 protein was examined by Western blot. Lysates from white blood
cells (100 µg) was included as a positive control (lane
1). A light band is observed in the osteoclast control
sample (lane 2), while no detectable Nox 4 protein is present in samples treated with Nox 4 antisense
oligonucleotide (lane 3).
/
)
osteoclasts (Fig. 7). A similar
inhibition of superoxide production was observed in earlier studies,
using the superoxide scavenger desferal manganese complex (4).
Moreover, calcitonin, an inhibitor of osteoclastic function, blocks
over 90% of superoxide production.
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Fig. 7.
Effect of antisense oligonucleotide on
osteoclastic superoxide generation. Osteoclasts were incubated
with 25 µM amounts of indicated oligonucleotides for 2 days. Superoxide generation by individual osteoclasts was determined
with a NBT assay. Twenty NBT-stained osteoclasts from each treatment
were randomly selected and the NBT staining intensity of individual
osteoclasts was quantified by microdensitometry. *, p < 0.001. SS, sense strand; AS, antisense strand;
DMnC, superoxide scavenger, desferal manganese complex;
CT, calcitonin.
/
) mutants
(Table III). These data suggest that both
oxidases are active in osteoclast from the wild type mice and
contribute to bone resorption. In addition, Nox 4 remains active in the
osteoclasts from p91 knockout animals.
Effect of antisense oligonucleotide on C57Bl/6 osteoclastic bone
resorption
,
NS vs. control. AS, antisense strand; SS, sense strand;
DMnC, superoxide scavenger, desferal manganese complex; CT, calcitonin.
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Fig. 8.
Bone resorption pits generated by
osteoclasts. Osteoclasts were isolated from
p91( /
) neonatal mouse pups and
placed on the bone slices for 0.5-1 h. After removing nonadherent
cells, 25 µM Nox 4 antisense oligonucleotides were added
and incubated for 2 days. Resorption pits on bone slices were
visualized by bright field reflected light microscopy after hematoxylin
staining. Numerous resorption lacunae formed by osteoclasts are readily
identifiable in a control slice (A). A few resorption
lacunae are observed when bone slices were treated with antisense
oligonucleotide (B).
Effect of antisense oligonucleotide on p91 knockout osteoclastic bone
resorption
,
NS vs. control. SS, sense strand; AS, antisense strand; CT,
calcitonin.
/
) mice. These data strongly
suggest that a FAD-containing protein was responsible for the majority
of the osteoclastic superoxide production in both wild type and
p91(
/
) mice. The addition of PTH to the culture medium
stimulated superoxide production and bone resorption. The PTH effect
was markedly diminished by DPI in both types of osteoclasts.
Effect of DPI on osteoclastic superoxide production and bone resorption
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) mice. The
presence of this novel oxidase explains the ability of
p91(
/
) osteoclasts to resorb bone normally. In wild
type mice, both p91 and Nox 4 are expressed in osteoclasts. It is
unclear why murine osteoclasts contain two enzymes that are involved in
generating superoxide. Both enzymes may be active in the osteoclasts,
due to the need for the production of large quantities of specifically targeted superoxide by the osteoclasts. Alternatively, the relatively low concentrations of Nox 4 in normal osteoclasts (Fig. 4) may suggest
that Nox 4 is only expressed in large quantities when the NADPH oxidase
is absent or nonfunctional (a 2.5-fold greater amount in
p91(
/
) than in the wild type). It is also possible that
the involvement of two enzyme systems is needed to effectively regulate
osteoclastic superoxide production, allowing for sustained and
controlled bone resorption.
/
) osteoclasts, despite a similar, post-stimulation
level of superoxide production. The difference in the kinetics of the
two enzymes would explain the difference in the amount of calcium
released, but does not detract from the fact that PTH stimulates
superoxide production and bone resorption significantly in both sets of
cultures. The fact that the superoxide and the bone resorptive activity were decreased in osteoclasts from each animal by DPI, suggested that
the stimulation of bone resorption required superoxide production. Since the p91(
/
) animals do not contain NADPH oxidase,
the only conclusion to be drawn is that there was stimulation of bone
resorption by the activity of the alternative oxidase. These
observations are in keeping with the inhibition seen during the
antisense overexpression, demonstrating that both the p91 and the Nox 4 contribute to osteoclastic superoxide production and bone resorption.
B in osteoclasts (30). NF-
B enhances the transcription of
genes signaling osteoclastic activation. Thus, besides their
destructive properties, superoxide and its related free radicals may be
involved in mediation of cellular differentiation and activation.
Modification of cellular differentiation and activation might explain
why many nonphagocytic cells such as lymphocytes, fibroblasts, and
endothelial cells produce superoxide.
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The nomenclature for a variety of non-myeloid NADPH oxidases has developed rapidly over the past 2 years. In Trends. Biochem. Sci. (25, 459-461, 2000), Lambeth et al. established a standardized nomenclature for the NADPH oxidases. Lambeth et al. sites our gene sequence (GenBankTM accession number NM_015760, originally registered as AF218723 with the GenBankTM by Yang and Key in December, 1999) as the murine NOX4. Later, the identical gene sequence was registered by Geiszt et al. (GenBankTM AF261944) in April, 2000. The mouse NOX4 sequence was first published by Geiszt et al. in Proc. Natl. Acad. Sci. U. S. A. 97, 8010-8014, 2000 (manuscript first submitted, March, 2000) and is the subject of the current report (Yang et al., manuscript first submitted February, 2000). Additionally, Geiszt et al. described the homologous human NOX4 sequence (GenBankTM AF261943, registered on April, 2000) in their publication in the Proc. Natl. Acad. Sci. U. S. A. 87, 8010-8014, 2000. The same sequence for human NOX4 was registered by Cheng and Lambeth et al. with the GenBankTM (AF254621) on April, 2000 and reported by Lambeth et al. in Trends Biochem. Sci. 25, 459-461, 2000. Shiose et al. published work describing the same human NOX4 sequence (GenBankTM AB041035, registered in March, 2000) in J. Biol. Chem. 276, 1417-1423, 2001 (manuscript first submitted August, 2000).
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FOOTNOTES |
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* This work was supported by GCRC in Medical University of South Carolina and National Institutes of Health Grant RO1-AR41463.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) AF218723.
To whom correspondence should be addressed: 316 CSB, Pediatric
Endocrinology, Medical University of South Carolina, 171 Ashley Ave.,
Charleston, SC 29425. Tel.: 843-792-1346; Fax: 843-792-0548; E-mail: yangs@musc.edu.
Published, JBC Papers in Press, November 29, 2000, DOI 10.1074/jbc.M001004200
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ABBREVIATIONS |
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The abbreviations used are: EST, expressed sequence tag; DPI, diphenylene iodonium; FAD, flavin adenine dinucleotide; 5'-RACE, 5'-rapid amplification of cDNA ends; NBT, nitro blue tetrazolium; Nox 4, NADPH oxidase 4 (mouse); p91, a 91-kDa subunit of NADPH oxidase; PTH, parathyroid hormone; PCR, polymerase chain reaction; TRAP, tartrate-resistant acid phosphatase.
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