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Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University (T.Y., H.Y., K.T., N.K., M.N.), Tokyo 101-0062; the Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University (Y.-i.N.), Kyoto 606-8501; and the Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Saitama 332-0012, Japan
Address all correspondence and requests for reprints to: Dr. Masaki Noda, Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3–10, Kanda-Surugadai 2-Chome, Chiyoda-ku, Tokyo, 101-0062, Japan. E-mail: noda.mph{at}mri.tmd.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The klotho gene is expressed at high levels in kidney (1), and it is postulated that a soluble form of Klotho protein could exist and act as a humoral factor (3, 4). Although the expression of the klotho gene has not been detected in the bones of wild-type mice even by RT-PCR analysis, the presence of abnormalities in the bones of klotho mutant mice suggests that Klotho protein could act as a humoral signaling molecule. Although klotho mutant mice were first reported to exhibit osteopenia in the diaphyseal cortical bones (1), the presence of osteopetrotic phenotype, including high levels of trabecular bones in the epiphyseal and metaphyseal regions, in klotho mutant mice (5, 6) indicates that the effect of the lack of Klotho protein may not be a simple reduction of the levels of overall bone mass. These diverse features raised the question of whether the klotho gene defect affects bone resorption in vivo. Although it is desirable to examine the direct effects of Klotho protein on osteoclasts, difficulty in obtaining the Klotho protein hampered this direction of research. Therefore, we were forced to use in vivo assays to evaluate the effect of the klotho gene defect on bone resorption. Bone marrow ablation in the long bones of rodents causes vigorous new bone formation within the first week and then subsequent rapid bone resorption in the second week to regenerate bone marrow with normal levels of trabecular bones. It is a highly reproducible in vivo assay to evaluate bone formation and resorption (7, 8, 9).
We found that bone resorption that occurred in the second week postsurgery was significantly impaired in klotho mutant mice, indicating the presence of a defect in bone resorption in vivo. We also observed site-specific morphological changes in osteoclasts and high levels of osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) messenger RNA (mRNA) expression in the ablated femora in klotho mutant mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Marrow ablation procedure
Either klotho mutant homozygotes or wild-type mice
(4–6 weeks old) were used for experiments. At least four animals were
used per group. Bone marrow was ablated in the right femora of each
animal as follows. Under general anesthesia, a hole was made in the
intercondylar regions of the femora by inserting a 26-gauge needle.
Then the content of the bone marrow was removed by using no. 10–30
dental files in a gradient manner and finally by inserting a 0.6-mm
diameter Kirschner wire up to the proximal ends of the femora to ensure
completion of marrow ablation. The left femora were untreated and were
used as an internal control. Animals were killed either at the time of
surgery or 1 and 2 weeks postsurgery, and soft x-ray radiographs of the
bones were taken before processing the bones for histological
examination. Half of the bones were subjected to histological
examination, and the other half were used for RNA preparation.
Tissue preparation and RNA extraction
Femora were fixed with 4% paraformaldehyde in PBS (pH 7.4) and
were subsequently subjected to microcomputed tomography (microCT)
examination before histological analysis. For RT-PCR analysis, femora
were collected before operation and at 1 and 2 weeks postsurgery, and
marrow tissues were flushed out from them to extract total RNA
according to the acid guanidine isothiocyanate-phenol/chloroform
method.
MicroCT analysis
The bones were subjected to microCT analysis using Musashi
(model NXCP-C80, NS-ELEX, Tokyo, Japan). This apparatus is equipped
with a microfocus x-ray tube with a spot size of 6 x 8 µm.
Analysis was conducted at 40 kVp and 100 µA to obtain the best
contrast between bones and soft tissues. The digital data were
reconstructed to obtain CT images in 1024 x 1024 pixel matrices.
The resolution was approximately 11 µm. Trabecular bone volume in a
square area of 30 x 300 pixels (0.28 x 2.8 mm) in the
metaphyseal region of the femora was quantitated using the Luzex-F
Image analyzing system (NIRECO, Tokyo, Japan).
Histological analysis
After microCT examination, femora were decalcified in 10% EDTA
(pH 7.4), dehydrated, and then embedded in glycol methacrylate.
Five-micron thick sections were prepared and stained for
tartrate-resistant acid phosphatase (TRAP) followed by staining with
toluidine blue. Histomorphometry was conducted to quantify the number
of osteoclasts and osteoclast surface as defined by Parfitt et
al. (10). The size and area of more than 60 osteoclasts were
measured using the sections containing TRAP-positive MNCs. Ferret’s
minimum distance between two parallel lines was used as an indicator of
the height of osteoclasts. To evaluate osteoclast number and size, two
square areas (230 x 230 µm/area) in each of the three separate
regions were examined as described below. The first region was adjacent
to the growth plate (GP), being 100 µm away from the boundary between
hypertropic chondrocytes and ossified matrix. The second and third
regions were 800 and 2000 µm away from this boundary
respectively.
Semiquantitative RT-PCR analysis
RT-PCR analysis was performed using primers specific to OPG/OCIF
and RANKL/TRANCE/ODF/OPGL genes and one tube RT-PCR system (Roche Molecular Biochemicals, Mannheim, Germany). The first DNA
synthesis was conducted using AMV reverse transcriptase at 55 C for 30
min followed by denaturation at 94 C for 30 sec, annealing at 55 C for
30 sec, and polymerization at 68 C for 45 sec. Subsequently, the
amplification cycle was repeated 30–38 times. The cycle number was
determined so that the PCR product levels were amplified within a
linear range. Ethidium bromide-stained DNA bands were quantitated using
an image analyzer Bio-1D system (VILBER LOURMAT, France). As a
control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels
were also estimated by RT-PCR. The levels of OPG/OCIF or
RANKL/TRANCE/ODF/OPGL relative to GAPDH expression levels were
calculated and compared between klotho mutant and wild-type
mice.
Oligomer sets used for OPG/OCIF, RANKL/TRANCE/ODF/OPGL and GAPDH were 5'-GCA CAT TTG GCC TCC TGC TAA TTC-3' (forward, OPG) and 5'-ACT CTC GGC ATT CAC TTT GGT CCC-3' (reverse, OPG), 5'-TCA TCT CTG TGG TAG TAG TGG CTG-3' (forward, RANKL) and 5'-TTA GGA GCA GTG AAC CAG TCG AAG-3' (reverse, RANKL), and 5'-ACC ACA GTC CAT GCC ATC AC-3' (forward, GAPDH) and 5'-TCC ACC ACC CTG TTG CTG TA-3' (reverse, GAPDH), respectively.
Statistical evaluation
The results were presented as the mean ± SEM.
Comparison between the values for ablated and control bones in
individual animals were evaluated using paired Wilcoxon’s signed rank
test. The mean values at each time point were analyzed with
Mann-Whitney’s U test. P < 0.05 was considered
significantly different.
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Results |
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The changes in radiopacity could be due to the alteration in cortical
bones, trabecular bones, or both. To visualize the changes in
trabecular bone structures and those in cortical bone thickness in the
femora after bone marrow ablation, we conducted microCT in the
epiphyseal to metaphyseal regions of femora within the midsagittal
plane. MicroCT examination demonstrated that ablated bone marrow cavity
of the right femora (Fig. 1
, B and F) was
filled with newly formed trabecular bones that were more abundant than
the trabecular bones in untreated (left) femora (Fig. 1, A and E) at 1
week postsurgery in both klotho mutant (Fig. 1, B and A) and
wild-type mice (Fig. 1, F and E), respectively. Cortical bone thickness
was not altered by bone marrow ablation in both klotho
mutant (Fig. 1, B compared with A) and wild-type mice (Fig. 1, F
compared with E), indicating that the major reason for the increase in
radiopacity is due to trabecular bone formation in the ablated marrow
space. By 2 weeks after operation, trabecular bones in the ablated
femora of wild-type mice were resorbed, and the pattern and amount of
these bones were similar between control and ablated femora (Fig. 1, G
vs. H) as reported previously. In contrast, ablated (right)
femur of klotho mutant mice even at 2 weeks postsurgery
showed abundant trabecular bones compared with nonablated (left) femora
(Fig. 1, D vs. C). Cortical thickness again was not altered
in either (klotho mutant and wild-type) genotype at 2 weeks
after bone marrow ablation (Fig. 1, D and H) and was similar to the
cortical morphology at 1 week (Fig. 1, B vs. F).
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). One week after bone
marrow ablation, BV/TV levels in the right femora were increased to
about 23% and 22% in klotho mutant mice and wild-type
mice, respectively (Fig. 2B, closed bars). By 2 weeks after
operation, BV/TV in the ablated side in wild-type (Fig. 2C, +/+,
closed bar) was reduced to about 8%, a level similar to
that in the control (left) side (Fig. 2C, +/+, open
bar). In contrast, in klotho mutant mice, the
trabecular bone volume in the right femora still remained about 23%
even at 2 weeks postsurgery (Fig. 2C, kl/kl, closed
bar). This BV/TV value of the ablated femur of klotho
mutant mice was significantly higher than that in the left femora (Fig. 2C, kl/kl, open bar; P <
0.05).
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For examination of osteoclast number and shape, we chose day 8
postsurgery for the morphological evaluation, because it was hard to
investigate the three regions immediately after or 2 weeks after
surgery in wild-type mice due to the absence by removal or
disappearance of proper amount of trabecular bones, respectively. The
numbers of TRAP-positive MNCs on the surface of trabecular bones
(N.Oc/BS) in klotho mutant mice in the regions 100 or 800
µm away from the GP were slightly lower (20% and 35%, respectively)
than those in wild-type mice, but the difference was not statistically
significant. In contrast, TRAP-positive MNCs number (N.Oc/BS) in
klotho mutant mice was reduced by more than 80% in the
regions 2000 µm away from the GP in the marrow of the ablated femur
on day 8 compared with that in wild-type mice (Table 1; N.Oc/BS, 1.4 ± 1.4 cells/mm in
klotho mutant vs. 8.8 ± 1.5 cells/mm in the
wild-type; P < 0.05). Osteoclast surface was also
reduced in klotho mutant mice in a site-specific manner in
the region 2000 µm away from the GP (Table 1B; Oc.S/BS, 2.9 ±
2.9 in klotho mutant vs. 24.6 ± 5.1 in
wild-type mice; P < 0.05), as it was not observed at
the 100 µm region. These data indicated that osteoclast number
in klotho mutant mice was reduced in a site-specific manner,
at least at 8 days postsurgery.
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, B and F, respectively). Quantitation also
indicated a similar area size (the area of individual TRAP-positive
MNCs was measured) and height (maximum height of the osteoclasts in the
axis vertical to the bone surface) of osteoclasts in the region 100
µm away from GP in wild-type and klotho mutant mice (Fig. 4, 100 µm away from the GP). In contrast,
the mean size and height of osteoclasts in the region 2000 µm away
from the growth plate in klotho mutant mice were reduced by
42% (P < 0.05) and 60% (P < 0.05),
respectively (Fig. 4; 2000 µm away from the GP), compared with those
of the osteoclasts in the corresponding regions in wild-type mice.
Intermediate levels of reduction in osteoclast size and height were
seen in the region 800 µm away from GP in klotho mutant
compared with those in wild-type mice (Fig. 4; 800 µm away from the
GP).
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). In the preoperation period, OPG/OCIF mRNA levels in
klotho mutant mice were more than 50% of those in the
wild-type (kl/kl, 1.56 ± 0.15; wild-type, 1.00 ±
0.09; P < 0.05). At 1 week postsurgery, both
klotho mutant mice and wild-type resumed trabecular bones
and marrow tissues in the ablated (right) femora. These bone marrow
tissues in klotho mutant and wild-type expressed slightly
more OPG/OCIF mRNA at 1 week (kl/kl, 2.07 ± 0.64;
wild-type, 1.44 ± 0.06; Fig. 5B) than
presurgery levels. Two weeks after ablation, OPG/OCIF gene expression
in the marrow tissues of the ablated (right) femur of klotho
mice stayed at high levels (2.15 ± 0.47), whereas in wild-type
mice OPG/OCIF expression levels were reduced significantly (0.51
± 0.22; Fig. 5B). We also examined RANKL mRNA expression levels and
found that they were similar throughout these experiments (Fig. 5, C
and D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To our knowledge this is the first study to examine the dynamic changes in the levels of OPG/OCIF expression in bone marrow ablation experiments even in wild-type mice. In wild-type mice, vigorous bone formation and then resorption take place in the first and second weeks, respectively, after bone marrow ablation, and these distinct periods coincide with the over 40% elevation (statistically significant, P < 0.05) of the OPG/OCIF expression levels and then 50% reduction (statistically significant, P < 0.05) relative to the presurgery period, respectively. The increase in OPG expression in the wild-type mice at 1 week after ablation in the presence of no change in RANKL expression is interesting. We do not have any explanation for this mechanism at this point. It might reflect an increase in the OPG-expressing cell population or enhancement of OPG expression by injury-related cytokines in the healing marrow, which has relatively high cellularity and may be rich in injury-related cytokines at this time point.
After bone marrow ablation in klotho mutant mice, OPG/OCIF expression was increased by about 25% (from 1.56 ± 0.15 to 2.07 ± 0.64) in the first week. However, a striking difference compared with the wild-type mice was the absence of the reduction in the levels of OPG/OCIF expression (2.15 ± 0.47) in the second week. RANKL (TRANCE/ODF/OPGL) expression levels, on the other hand, were not altered in klotho mutant and wild-type mice during the 2-week course of the bone marrow ablation experiments. These data indicated clearly the abnormal regulation of OPG/OCIF expression in klotho mutant mice. In addition, our data indicated a correlation between OPG/OCIF levels and those in bone resorption in the bone marrow ablation model. Klotho protein is predicted to be an enzyme-like molecule; however, its direct function is not known. Our data indicated for the first time that at least in bone, klotho mutation results in an increase in the levels of an important cytokine regulator, OPG/OCIF.
Site-specific reduction in the number and size of osteoclasts in
klotho mutant mice
Quantitation of osteoclast number in the three distinct regions of
the epiphyseal trabecular bones indicated that klotho mutant
mice showed interesting site-specific reduction in osteoclast number in
the most distal region relative to the GP. This could explain at least
in part why klotho mutant mice show elongation of the
trabecular bones. This site-specific reduction of osteoclast number was
not observed in wild-type mice, as the numbers of osteoclasts in the
three regions are virtually similar (100 µm, 8.5 ± 1.3; 800
µm, 6.8 ± 0.1; 2000 µm, 8.8 ± 1.5). These osteoclast
numbers were similar to the number in the nonablated side in wild-type
mice (6.2 ± 1.4). We also examined osteoclast number in the
nonablated bones in klotho mice, and our data reveal that
the dependence of the reduction in osteoclast number on the relative
distance from the growth plate can be observed in nonablated bones in
klotho mice but not in wild-type animals (data not shown),
confirming that retardation of the bone resorption associated with
klotho mutant abnormality with regard to the number of
osteoclasts is a specific feature of the klotho mutant
mice.
Osteoclastogenesis in the bone marrow after bone marrow ablation is clearly a local phenomenon, and hence, it is certainly possible that a study of osteoclastogenesis in response to some local factors might reveal a defect. An in vitro osteoclastogenesis study has shown that not only systemic factors such as vitamin D and PTH, but also local factors such as interleukin-1, interleukin-6 (including other gp130-dependent cytokines), PGE2, and tumor necrosis factor are capable of promoting osteoclastogenesis. It appears, therefore, that many, if not all, of these stimuli eventually act to stimulate expression of RANKL. RANKL functions as a local factor that directly binds to its receptor, RANK, being expressed on the surface of the progenitor cells for osteoclasts. We, therefore, examined the levels of RANKL in the ablated marrow. However, as shown in the RT-PCR analyses, RANKL expression levels were not altered significantly. These observations suggest that certain local factors independent from RANKL may be involved in the reduction of bone resorption in klotho mutant mice.
High levels of OPG/OCIF expression in klotho mutant mice compared with wild-type mice could partly explain the reduction in the number and size of osteoclasts in klotho mutant mice. However, as OPG/OCIF appears to be expressed in both skeletal and nonskeletal tissues, and it could act as a humoral factor systemically (11, 12), site-specific reduction in osteoclastic number and size in the klotho mutant mice cannot be explained by the increase in OPG/OCIF expression measured using the entire marrow tissues to give averaged values (including all three locations). In fact, our preliminary experiments indicated that OPG expression levels in lung and spleen of klotho mutant mice were higher than those in wild-type animals (data not shown).
Questions regarding whether OPG/OCIF could act somehow site specifically in klotho mutant mice or whether OPG/OCIF expression is also site specific in the bone marrow tissues depending on the distance from the growth plate in these mutant, but not in wild-type, mice require future investigation. There is also a possibility that if more bone is being formed in the klotho mutant mice than in wild-type mice at 2 weeks, less of the surface may be available for resorption and so account for the reduced osteoclast number. To address this point, we measured osteoblast surface in all three sites. The results indicated that osteoblast surface values at 100, 800, and 2000 µm away from the GP in wild-type mice (n = 5) were 44.8 ± 3.5%, 69.9 ± 13.3%, and 57.4 ±10.2%, respectively, and those in klotho mutant mice (n = 4) were 25.9 ± 7.7%, 50.9 ± 16.5%, and 81.3 ± 12.4% respectively. The ratios of klotho mice over wild-type mice at each of the three regions, 100, 800, and 2000 µm away from the GP, were 0.58, 0.73, and 1.42, respectively. However, statistical evaluation did not show that the difference was statistically significant.
Site-specific reduction of osteoclast number and size is a unique phenomena in klotho mutant mice. This type of site specificity has not been described in other osteopetrotic mice or in drug-induced osteopetrosis. Therefore, our observation of site-specific phenomena regarding osteoclasts may give a clue to understanding of the molecular function of the Klotho protein.
Trabecular bone formation after bone marrow ablation is normal in
klotho mutant mice
In this paper we report that a bone marrow ablation study in
klotho mutant mice revealed relatively normal bone formation
in the first week before the observation of impaired bone resorption in
the second week. After ablation of bone marrow, bone marrow tissues
regenerate in a highly reproducible manner starting with hematoma
formation just after operation. One week after marrow ablation,
trabecular bone volume reaches at its maximal level, and then vigorous
bone resorption takes place within the second week. Two weeks later,
bone marrow in the ablated bones returns to normal morphology that is
indistinguishable from that in untreated-side femur in wild-type mice
(7, 8).
As mentioned, at least in the marrow ablation experiments, osteoblastic function in klotho mutant mice is normal. However, in the initial examinations (1, 5, 6), the cortical bones in klotho mutant mice were osteopenic. It appears, therefore, that the loss of klotho gene expression would not affect rapid bone formation, such as that taking place in the marrow ablation study, but it could affect slow or long term bone formation and/or remodeling to maintain steady state levels of cortical bones. This possibility still does not contradict our observation that OPG/OCIF expression levels are relatively high in klotho mutant mice, as OPG/OCIF per se does not influence osteoblastic function directly.
It has been proposed that osteoblasts and osteoclasts activate the functions of each other. We propose that klotho deficiency enhances OPG/OCIF expression levels for a long term, and these sustained high levels of OPG/OCIF might suppress osteoclasts, resulting in the long term reduction of as an yet unknown osteoclast-derived signal(s) that might, in turn, activate osteoblastic function. If this hypothesis is true, certain osteoblastic functions, such as those in the normal remodeling phase under the control of the signals elicited by osteoclasts, would be affected, whereas other osteoblastic functions unrelated to the influence from osteoclastic activity, such as the formation of new trabecular bones in the first week of the recovery phase after bone marrow ablation, would not be affected by the loss of klotho gene expression.
High osteoprotegerin expression levels in klotho mutant
mice
OPG/OCIF has been shown to be an inhibitor of osteoclastogenesis
and osteoclastic function (11, 12, 13, 14). However, its expression has been
observed in many nonskeletal tissues, such as kidney, heart, placenta,
liver, and intestine, in addition to bone, cartilage, and bone marrow
(11). As the klotho gene is not expressed in bone but is
highly expressed in kidney and to a lesser extent in other soft
tissues, it is intriguing to determine whether the link between
klotho expression and OPG/OCIF expression can be observed in
one of these extraskeletal tissues, such as kidney, in that Klotho
protein might suppress OPG/OCIF expression in wild-type animals. We are
currently trying to obtain Klotho protein to investigate its direct
action on the regulation of OPG/OCIF expression. Alternatively,
OPG/OCIF expression is regulated by vitamin D3
(suppression) or ionized calcium and estrogen (activation). We
cannot exclude the possibility that the klotho gene product
may also act through modulation of such regulators as indirect
pathways. As klotho mutant mice show defects in gonadal
development and possibly are in low estrogen status, OPG/OCIF
up-regulation would not occur directly through estrogen
regulation. Future studies should elucidate the mechanism of the
enhancement of OPG/OCIF expression by the loss of the product of the
klotho gene.
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Acknowledgments |
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Footnotes |
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Received July 9, 1999.
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This article has been cited by other articles:
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  T. Yamashita, S. Okada, K. Higashio, Y.-i. Nabeshima, and M. Noda Double Mutations in Klotho and Osteoprotegerin Gene Loci Rescued Osteopetrotic Phenotype Endocrinology, December 1, 2002; 143(12): 4711 - 4717. [Abstract] [Full Text] [PDF]
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