help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamashita, T.
Right arrow Articles by Noda, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamashita, T.
Right arrow Articles by Noda, M.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Endocrinology Vol. 143, No. 12 4711-4717
Copyright © 2002 by The Endocrine Society

ARTICLE

Double Mutations in Klotho and Osteoprotegerin Gene Loci Rescued Osteopetrotic Phenotype

Teruhito Yamashita, Seiji Okada, Kanji Higashio, Yo-ichi Nabeshima and Masaki Noda

Department of Molecular Pharmacology (T.Y., M.N.), Medical Research Institute, Tokyo Medical and Dental University, Tokyo 101-0062, Japan; Department of Development Genetics (S.O.), Chiba University Graduate School of Medicine, Chiba 260-8670, Japan; Research Institute of Life Science (K.H.), Snow Brand Milk Products Co., Ltd., Tochigi 329-0512, Japan; Department of Pathology and Tumor Biology (Y.N.), Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan; and Core Research for Evolutional Science and Technology (Y.N.), Japan Science and Technology Corp., Tokyo, Japan 102-8666

Address all correspondence and requests for reprints to: Masaki Noda, Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-0062, Japan. E-mail: noda.mph{at}mri.tmd.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Klotho gene mutant mice (klotho mice, also called kl/kl) exhibit osteopetrosis in the metaphysis of femora and tibiae and die within 3 months. We previously showed by semiquantitative RT-PCR that osteoprotegerin (opg) expression levels in klotho mice were about 2-fold higher than those in wild-type mice in the bone marrow, spleen, and lung. To examine whether the high osteoprotegerin expression levels account for the osteopetrotic phenotype in the klotho homozygous mutant mice in vivo, we made double mutant mice by crossing klotho mutant and osteoprotegerin-deficient mice. Micro computed tomography analysis in the two-dimensional sagittal planes of the metaphyses and cross-sections of femoral midshaft revealed that the abnormally high fractional trabecular bone volume in klotho homozygous mice (kl/kl; 29.71%), which was about 4-fold higher compared with that of wild-type [klotho (+/+) opg (+/+)] mice (7.81%), was rescued by the coexistence of heterozygous mutation in opg gene locus (+/-; 8.36%). Single heterozygous mutation in the opg gene locus alone (without klotho mutation) did not show phenotype (trabecular bone volume, 5.84%; not significantly different from wild type). High levels of osteoprotegerin mRNA expression in the bone marrow in klotho mutant mice were reduced by the heterozygous mutation in the opg gene locus. Furthermore, high osteoprotegerin protein levels in klotho mutant mice were also reduced by the heterozygous mutations in opg gene locus. Thus, elevated levels of osteoprotegerin in mutant mice contribute at least in part to reveal the osteopetrotic phenotype in klotho mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
KLOTHO MUTANT MICE exhibit emphysema, nerve degeneration, skin atrophy, and ectopic calcification and die within 3 months (1). These mice exhibit osteopetrosis in the metaphysis of femora and tibiae 4 wk after birth (1, 2, 3, 4). We previously showed that bone resorption is impaired in the klotho mutant mice, although bone formation was normal using a bone marrow ablation model (5). About 2-fold higher osteoprotegerin (opg) expression levels were observed in klotho mice compared with wild type on the basis of semiquantitative RT-PCR analysis using RNAs obtained from bone marrow, spleen, and lung (5). Serum osteoprotegerin levels in klotho mutant mice were also reported to be three times higher than those in wild-type mice (3). These observations suggest that klotho gene product would regulate bone resorption via modulating the expression levels of opg gene.

Osteoprotegerin is one of the TNF receptor family members (6, 7, 8, 9, 10). Osteoprotegerin expression levels are enhanced by IL-1, TNF-{alpha}, estrogen, IL-11, and leptin, whereas they are suppressed by basic fibroblast growth factor and vitamin D3 (11, 12, 13, 14, 15). Osteoprotegerin gene expression levels are also altered by aging (16, 17); osteoprotegerin knockout mice show osteopenia in both cancellous and cortical bone (18, 19), revealing that this molecule plays a critical role in bone metabolism. Osteoprotegerin gene heterozygous mutant mice exhibit moderate loss in bone density compared with that of wild-type mice, suggesting the presence of gene dosage effect.

To examine whether osteoprotegerin is involved in the osteopetrotic phenotype in the klotho homozygous mutant mice in vivo, we made double knockout mice having mutations in both klotho and osteoprotegerin gene loci.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Double knockout mice harboring mutations in both klotho and opg gene loci were obtained by intercrossing between heterozygous klotho mutant mice and heterozygous opg knockout mice. F1 heterozygous mutant mice with mutations in both of the two gene loci were obtained by mating between klotho (+/kl) females and opg (+/-) male. The klotho (+/kl) opg (+/-) mice were selected on the basis of genotyping and were maintained and mated to obtain F2 offspring used in this study. Klotho mutant mice were C3H background, and opg knockout mice were C57BL/6(B6) background. Therefore, the double mutant mice were on the mixed background. Littermates were used to minimize the impact of the background strains.

Micro-computed tomography (CT) analyses
Micro CT apparatus was used to estimate the trabecular bone volume levels as described previously (5). For measurements, left femora of 7- to 11-wk-old mice were used. For micro CT analysis, we used Musashi system (model MXCP-C80, NS-ELEX, Tokyo, Japan). Micro focus spot size of this apparatus was 6 x 8 µm. Micro CT analysis was conducted at 40 kVp and 100 µA to obtain the best contrast between bone and soft tissues. The final reconstruction of the digital data was conducted by a computer attached to the machine. The reconstructed images were in 1024 x 1024-pixel matrices, with the resolution approximately 11 µm. Trabecular bone volume in the metaphysical region of the femora was quantified by using the Luzex-F image analyzing system (Nireco, Tokyo, Japan).

Histological analysis
Femora were fixed with 4% paraformaldehyde in PBS and were decalcified with 10% EDTA for 14 d. Then, they were dehydrated and embedded in glycol methacrylate or paraffin. Sections (3 µm thick) were stained for tartrate-resistant acid phosphotase (TRAP), followed by counter-staining with hematoxylin.

Semiquantitative RT-PCR analysis
Total RNA was prepared from bone marrow using the acid guanidine phenol-chloroform-method. The RNA was subjected to reverse-transcription using Superscript (Roche Boeringer-Mannheim, Indianapolis, IN) and oligo (dT) primer. PCR primers for the cDNA amplification were designed as follows: osteoprotegerin (forward, 5'-TCC TGG CAC CTA CCT AAA ACA GCA-3'; reverse, 5'-CTA CAC TCT CGG CAT TCA CTT TGG-3'); receptor activator NF{kappa}B ligand (forward, 5'-TCA TCT CTG TGG TAG TAG TGG CTG-3'; reverse, 5'-TTA GGA GCA GTG AAC CAG TCG AAG-3'); glyceraldehyde-3-phosphate dehydrogenase (forward, 5'-ACC ACA GTC CAT GCC ATC AC-3'; reverse, 5'-TCC ACC ACC CTG TTG CTG TA-3'). The PCR products were separated on a 1.6% agarose gel and stained with ethidium bromide. The intensity of the bands was quantified using a densitometry apparatus (Velber Lumat, France).

Measurement of serum osteoprotegerin concentration by ELISA
Blood samples were centrifuged at 1500 rpm for 15 min, and serum was separated and stored at -30 C until assay. Serum samples were transferred into the ELISA-plated containing enzyme-linked monoclonal antibody raised against osteoprotegerin. After color development, absorbance at 450 nm was measured using a microplate reader (Nippon Intermed, Tokyo, Japan).

Hematopoietic analysis using flow cytometry
Fluorescent dye-conjugated monoclonal antibodies against B220 (RA3–6B2), CD43 (S7), Ig-M, Thy1.2 (53–2.1), Mac-1 (M1/70) and TER119 (Ly-76) were used to detect B lymphocytes, mature B cells, pro-B cells, T lymphocytes, myeloid cells, or erythrocytes, respectively (PharMingen, San Diego, CA). Cells in suspension were treated with a hypotonic lysing buffer to remove erythrocytes before staining. The cells in suspensions were stained with the monoclonal antibodies and propidium iodide. Stained cells were analyzed by a fluorescence-activated cell sorter (FACSCalibur, Becton Dickinson and Co., San Jose, CA).

Statistical analysis
All of the data were expressed as mean ± SD. The difference was evaluated on the basis of ANOVA and Bonferroni correction for multiple comparisons. P values less than 0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Heterozygous mutation in opg gene allele rescued osteopetrotic phenotype in the femora of klotho mice
To examine the effects of heterozygous mutation in the opg gene allele on the osteopetrotic phenotype in klotho mice, we evaluated trabecular bone in the metaphysis of femora using a micro CT apparatus (Fig. 1Go). Compared with wild-type mice (Fig. 1AGo), osteopetrotic changes were observed in klotho mutant mice (Fig. 1CGo), as reported previously. Although trabecular bone morphology in opg (+/-) mice (Fig. 1BGo) was similar to that in wild type (Fig. 1AGo), heterozygous mutation in opg gene locus rescued osteopetrotic phenotype in kl/kl mice (Fig. 1DGo).



View larger version (87K):
[in this window]
[in a new window]
  		Figure 1. Heterozygous mutation in opg gene suppressed osteopetrotic phenotype in klotho mutant mice. Two-dimensional images of the distal end of femora were obtained by micro-focus CT using bones of 7-wk-old mice. A, Klotho (+/+) opg (+/+); B, klotho (+/+) opg (+/-); C, klotho (kl/kl) opg (+/+); D, klotho (kl/kl) opg (+/-).

 
Quantification of the bone volume indicated that trabecular bone fraction in the metaphyseal region in klotho mutant mice (29.7 ± 2.8%) was about three times more than that in wild-type mice, and heterozygous mutation in opg gene locus [klotho (kl/kl) opg (+/-); 8.4 ± 2.6%] reduced such osteopetrosis (P < 0.05; Fig. 2AGo). The length of the femora (Fig. 2BGo) and body weight (Fig. 2CGo) in klotho mice were less than those in the wild-type mice. In contrast to the recovery in the trabecular bone phenotype, double mutant [klotho (kl/kl) opg (+/-)] mice exhibited more growth retardation than that of klotho mutant mice [(klotho (kl/kl) opg (+/+)].



View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. Quantification of trabecular bone volume, the length of femora, and body weight. A, Bone volume was quantified using micro CT images of the distal end of femora. Klotho (kl/kl) opg (+/-) mice exhibited similar levels of bone volume to levels in wild-type mice. B, The length of femora. C, Body weight of the mice. *, Statistically significant difference (P < 0.05).

 
The number of osteoclasts on the trabecular bone in the femora was reduced in klotho (kl/kl) opg (+/-) mice
To obtain insight into the cellular bases for the recovery (reduction) in the bone volume in klotho (kl/kl) opg (+/-) double mutant mice, we examined osteoclast features in vivo. Osteopetrosis in klotho mice was associated with reduction in osteoclast number (Fig. 3Go, D, I, and N, for low-, middle-, and high-power magnification photomicrographs, respectively) compared with wild-type (Fig. 3Go, A, F, and K for low-, middle-, and high-power magnification photomicrographs, respectively) as we previously observed. In opg-deficient mice (Fig. 3MGo), osteoclasts in the metaphyseal region were more than those in wild-type mice, as reported previously (18). In the klotho mutant background (Fig. 3Go, D, E, I, J, N, and O), heterozygous mutation in opg gene locus (Fig. 3Go, E, J, and O) resumed the appearance of osteoclasts on the trabecular bone compared with the bone in klotho mutant mice with normal opg gene locus (Fig. 3Go, D, I, and N).



View larger version (144K):
[in this window]
[in a new window]
  		Figure 3. Histology of the bone. TRAP-positive cells in the distal region of femora of klotho (kl/kl) opg (+/-) mice. Top panels (A–E) show distal end of femora of the mice. Middle panels (F–J) and bottom panels (K–O) indicate high-power views of the magnified areas adjacent to and 800 µm apart from growth plate, respectively. In the klotho (kl/kl) opg (+/-) mice (panel O), osteoclasts were observed on the trabecular bone surface similarly to those in wild-type mice (panel K). Samples were prepared from the femora of 5-wk-old mice and were stained for TRAP and with hematoxylin. A, F, and K, Klotho (+/+) opg (+/+); B, G, and L, klotho (+/+) opg (+/-); C, H, and M, klotho (+/+) opg (-/-); D, I, and N, klotho (kl/kl) opg (+/+); E, J, and O, klotho (kl/kl) opg (+/-). Arrows indicate osteoclasts.

 
Heterozygous mutations in opg gene locus suppressed elevated osteoprotegerin mRNA and protein expression levels in the bone marrow cells in klotho (kl/kl) mice
To elucidate molecular mechanisms of the reduction in the levels of osteopetrosis seen in klotho (kl/kl) mutant mice by the additional mutation in opg gene loci, we examined whether the elevated expression of osteoprotegerin mRNA and protein in klotho mice was reduced by the heterozygous mutation in the opg allele. Semiquantitative RT-PCR analysis showed that osteoprotegerin transcript levels in klotho (kl/kl) opg (+/+) mice (Fig. 4AGo; P < 0.05) were about 7-fold more than those in wild-type as we reported previously (5). Heterozygous mutation in opg gene locus significantly suppressed such elevation (Fig. 4AGo, filled bar; P < 0.05). To address whether alterations in osteoprotegerin mRNA levels were translated into those in protein expression levels, osteoprotegerin protein concentration in serum was measured. As shown in Fig. 4BGo, osteoprotegerin protein levels were elevated in klotho mutant mice, and heterozygous mutation in opg locus suppressed the osteoprotegerin protein levels in the serum of klotho mice.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 4. Expression levels of osteoprotegerin mRNA and protein in the bone marrow cells. A, Expression levels of osteoprotegerin mRNA in the bone marrow cells from klotho (kl/kl) opg (+/-) were decreased to one third that of klotho (kl/kl) opg (+/+) mice. Total RNA was extracted from bone marrow obtained from the femora of 4-wk-old mice and was subjected to examination by semiquantitative RT-PCR analysis. B, Serum osteoprotegerin concentration in klotho (kl/kl) opg (+/-) was reduced to levels similar to those in wild-type mice. *, Statistically significant difference (P < 0.05).

 
Heterozygous mutation in opg locus of klotho (kl/kl) mice does not resume normal B lymphopoietic development
Klotho mutant mice also exhibited B lymphocytopenia in the bone marrow (20, 21). To examine the effect of opg-heterozygous mutation on the alterations in hematopoietic cells, we analyzed the lymphocyte populations using the cells in the bone marrow, spleen, and peripheral blood. As reported previously, klotho mutant mice showed reduction in the B cell populations (Fig. 5Go, the second column from the right). However, heterozygous mutation on opg locus did not recover such reduction in B cell population (Fig. 5Go, rightmost column of panels). Rather, there was a tendency to slightly reduce the size of B cell population by introducing the heterozygous mutation in opg locus. As control, the sizes of myeloid/erythroid cell populations were similar, regardless of the genotypes.



View larger version (77K):
[in this window]
[in a new window]
  		Figure 5. Flow cytometric analysis of blood cell populations. Flow cytometry was conducted using bone marrow cells from the mice. These data are a representative profile of the lymphocyte populations in the bone marrow cells from 5-wk-old mice. Antibodies including B220, CD43, Ig-M, Thy-1, Mac-1, and TER119 were used to label B lymphocytes, mature B cells, pro-B cells, T lymphocytes, myeloid cells, and erythrocytes, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrated in this study that osteopetrotic phenotype in klotho mutant mice was at least partially rescued by the heterozygous mutation in the opg gene locus. This observation was associated with the reduction in the levels of osteoprotegerin mRNA in the bone marrow. Furthermore, osteoprotegerin protein levels were enhanced by the mutation in klotho gene locus, whereas elevation of such osteoprotegerin protein levels was suppressed by the heterozygous mutation in the opg gene locus. These observations indicated that previously observed enhancement of opg gene expression in the klotho mutant mice could at least in part be involved in the occurrence of the osteopetrosis phenotype in these mice.

Despite the partial rescue of the osteopetrosis phenotype by heterozygous mutation in the opg locus in the klotho mutant background mice, such rescue effect was not observed in terms of the reduction in the size of the B cell population in klotho mutant mice. Similarly, other phenotypes observed in the klotho mutant mice, such as growth retardation and shortening in the longevity of life, were not affected by the presence of heterozygous mutation in the opg locus. Thus, our results show dissociation between the striking effects on bone and the lack of parallel effects on the lymphocytes. These data suggest that, depending on the specific organs, klotho gene products may play different roles in association with the tissue-specific factors. In the case of bone, klotho gene product may be upstream of the opg gene. Alternately, these two genes may normally function in parallel, and loss of one may result in a compensatory change in expression of the other.

We obtained klotho (kl/kl) opg (+/-) mutant mice by intercrossing between klotho (+/kl) opg (+/-) female and male mice and showed that klotho (kl/kl) opg (+/-) genotype suppressed the osteopetrotic phenotype in the metaphyseal region of the femora in klotho mice. Although we tried to obtain klotho mutant mice with homozygous opg-deficient background (-/-), no pups were available due to possible embryonic death or early death after birth. The reason for such observation is not known. Possibly, klotho gene product may participate in a certain signaling network operating to maintain biological systems. However, because klotho gene product has not yet been available as a protein, direct action of the klotho gene product cannot be examined at this point. In fact, how klotho protein acts in bone has yet to be elucidated. It is still possible that the bone phenotype may not be a primary effect of loss of klotho, but rather just the consequence of disrupted physiology.

Klotho mutant mice exhibit increased serum concentrations of calcium and phosphorus (22); however, high calcium levels could regulate serum concentrations of calcitonin and PTH of klotho mice normally. In contrast, even in the presence of the high calcium concentrations, the levels of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and the expression of 25-hydroxyvitamin D1 {alpha}-hydroxylase gene in klotho mice serum were significantly higher than those in wild type. Administered 1,25-(OH)2D3 failed to down-regulate 25-hydroxyvitamin D1 {alpha}-hydroxylase gene expression and to up-regulate 24-hydroxylase and vitamin D receptor genes (22). Because 1,25-(OH)2D3 enhances osteoprotegerin expression, it is possible that abnormal 1,25-(OH)2D3 signaling pathway is involved in the elevation of osteoprotegerin levels in klotho mice.

In conclusion, high levels of osteoprotegerin expression observed in klotho mutant mice are at least in part responsible for the osteopetrotic phenotype in the cancellous bone in klotho mutant mice.


   Acknowledgments
 


   Footnotes
 
This research was supported by the grants-in-aid received from the Japanese Ministry of Education (14207056, 14034214, 14028022, 12557123, 13045011, 13216034), grants from Japan Space forum, NASDA, Japan Society for Promotion of Science (JSPS, Research for the Future Program, Genome Science), and a JSPS research grant for foreign postdoctoral fellow.

Abbreviations: CT, Computed tomography; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; opg, osteoprotegerin; TRAP, tartrate-resistant acid phosphotase.

Received June 7, 2002.

Accepted for publication August 26, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, Nabeshima YI 1997 Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 278:45–51[CrossRef]
  2. Yamashita T, Nifuji A, Furuya K, Nabeshima Y, Noda M 1998 Elongation of the epiphyseal trabecular bone in transgenic mice carrying a klotho gene locus mutation that leads to a syndrome resembling aging. J Endocrinol 159:1–8[Abstract]
  3. Kawaguchi H, Manabe N, Miyaura C, Chikuda H, Nakamura K, Kuro-o M 1999 Independent impairment of osteoblast and osteoclast differentiation in klotho mouse exhibiting low-turnover osteopenia. J Clin Invest 104:229–237[Medline]
  4. Shiraki-Iida T, Aizawa H, Matsumura Y, Sekine S, Iida A, Anazawa H, Nagai R, Kuro-o M, Nabeshima Y 1998 Structure of the mouse klotho gene and its two transcripts encoding membrane and secreted protein. FEBS Lett 424:6–10[CrossRef][Medline]
  5. Yamashita T, Yoshitake H, Tsuji K, Kawaguchi N, Nabeshima Y, Noda M 2000 Retardation in bone resorption after bone marrow ablation in klotho mutant mice. Endocrinology 141:438–445[Abstract/Free Full Text]
  6. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill D, Pattison W, Campbell P, Sander S, Van G, Tarpley J, Derby P, Lee R, Boyle WJ 1997 Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:309–319[CrossRef][Medline]
  7. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T 1998 Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95:3597–3602[Abstract/Free Full Text]
  8. Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, Morony S, Oliveira-dos-Santos AJ, Van G, Itie A, Khoo W, Wakeham A, Dunstan CR, Lacey DL, Mak TW, Boyle WJ, Penninger JM 1999 OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397:315–323[CrossRef][Medline]
  9. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ 1998 Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176[CrossRef][Medline]
  10. Li J, Sarosi I, Yan XQ, Morony S, Capparelli C, Tan HL, McCabe S, Elliott R, Scully S, Van G, Kaufman S, Juan SC, Sun Y, Tarpley J, Martin L, Christensen K, McCabe J, Kostenuik P, Hsu H, Fletcher F, Dunstan CR, Lacey DL, Boyle WJ 2000 RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc Natl Acad Sci USA 97:1566–1571[Abstract/Free Full Text]
  11. Horwood NJ, Elliott J, Martin TJ, Gillespie MT 1998 Osteotropic agents regulate the expression of osteoclast differentiation factor and osteoprotegerin in osteoblastic stromal cells. Endocrinology 139:4743–4746[Abstract/Free Full Text]
  12. Hofbauer LC, Lacey DL, Dunstan CR, Spelsberg TC, Riggs BL, Khosla S 1999 Interleukin-1ß and tumor necrosis factor-{alpha}, but not interleukin-6, stimulate osteoprotegerin ligand gene expression in human osteoblastic cells. Bone 25:255–259[Medline]
  13. Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Spelsberg TC, Riggs BL 1999 Estrogen stimulates gene expression and protein production of osteoprotegerin in human osteoblastic cells. Endocrinology 140:4367–4370[Abstract/Free Full Text]
  14. Nakagawa N, Yasuda H, Yano K, Mochizuki S, Kobayashi N, Fujimoto H, Yamaguchi K, Shima N, Morinaga T, Higashio K 1999 Basic fibroblast growth factor inhibits osteoclast formation induced by 1{alpha}, 25-dihydroxyvitamin D(3) through suppressing the production of osteoclast differentiation factor. Biochem Biophys Res Commun 265:45–50[CrossRef][Medline]
  15. Sakata M, Shiba H, Komatsuzawa H, Fujita T, Ohta K, Sugai M, Suginaka H, Kurihara H 1999 Expression of osteoprotegerin (osteoclastogenesis inhibitory factor) in cultures of human dental mesenchymal cells and epithelial cells. J Bone Miner Res 14:1486–1492[CrossRef][Medline]
  16. Yano K, Tsuda E, Washida N, Kobayashi F, Goto M, Harada A, Ikeda K, Higashio K, Yamada Y 1999 Immunological characterization of circulating osteoprotegerin/osteoclastogenesis inhibitory factor: increased serum concentrations in postmenopausal women with osteoporosis. J Bone Miner Res 14:518–527[CrossRef][Medline]
  17. Makhluf HA, Mueller SM, Mizuno S, Glowacki J 2000 Age-related decline in osteoprotegerin expression by human bone marrow cells cultured in three-dimensional collagen sponges. Biochem Biophys Res Commun 268:669–672[CrossRef][Medline]
  18. Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu W, Lacey DL, Boyle WJ, Simonet WS 1998 osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 12:1260–1268[Abstract/Free Full Text]
  19. Mizuno A, Amizuka N, Irie K, Murakami A, Fujise N, Kanno T, Sato Y, Nakagawa N, Yasuda H, Mochizuki S, Gomibuchi T, Yano K, Shima N, Washida N, Tsuda E, Morinaga T, Higashio K, Ozawa H 1998 Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin. Biochem Biophys Res Commun 247:610–615[CrossRef][Medline]
  20. Manabe N, Kawaguchi H, Chikuda H, Miyaura C, Inada M, Nagai R, Nabeshima Y, Nakamura K, Sinclair AM, Scheuermann RH, Kuro-o M 2001 Connection between B lymphocyte and osteoclast differentiation pathways. J Immunol 167:2625–2631[Abstract/Free Full Text]
  21. Okada S, Yoshida T, Hong Z, Ishii G, Hatano M, Kuro OM, Nabeshima Y, Tokuhisa T 2000 Impairment of B lymphopoiesis in precocious aging (klotho) mice. Int Immunol 12:861–871[Abstract/Free Full Text]
  22. Yoshida T, Fujimori T, Nabeshima Y 2002 Mediation of unusually high concentrations of 1,25-dihydroxyvitamin D in homozygous klotho mutant mice by increased expression of renal 1{alpha}-hydroxylase gene. Endocrinology 143:683–689[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J EndocrinolHome page
E Mrak, I Villa, R Lanzi, M Losa, F Guidobono, and A Rubinacci
Growth hormone stimulates osteoprotegerin expression and secretion in human osteoblast-like cells
J. Endocrinol., March 1, 2007; 192(3): 639 - 645.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
P. Collin-Osdoby
Regulation of Vascular Calcification by Osteoclast Regulatory Factors RANKL and Osteoprotegerin
Circ. Res., November 26, 2004; 95(11): 1046 - 1057.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamashita, T.
Right arrow Articles by Noda, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamashita, T.
Right arrow Articles by Noda, M.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals