- Email: [email protected]
Overexpression of granulocyte colony-stimulating factor induces severe osteopenia in developing mice that is partially prevented by a diet containing vitamin K2 (menatetrenone)
Bone Vol. 30, No. 6 June 2002:880 – 885
Overexpression of Granulocyte Colony-stimulating Factor Induces Severe Osteopenia in Developing Mice That Is Partially Prevented by a Diet Containing Vitamin K2 (Menatetrenone) Y. KOKAI,1 T. WADA,2 T. ODA,1,2 H. KUWABARA,2 K. HARA,3 Y. AKIYAMA,3 S. ISHII,2 and N. SAWADA1 1 3
Departments of Pathology and 2Orthopedics, Sapporo Medical University School of Medicine, Sapporo, Japan Department of Applied Drug Research, Eisai Co., Ltd., Tokyo, Japan
and differentiation of cells of granulocytic lineage.4 Its strong effect on the increase of neutrophils in vivo provides a useful therapeutic tool for a various types of neutropenia in the clinical setting.20,22 Later studies, however, have revealed that this cytokine has effects on a variety of cell types besides neutrophilic cells, such as mobilization of hematopoietic stem cells20,24 and modulation of the immune system.6 We previously showed that overexpression of G-CSF induces severe osteopenia with dramatic increases of bone cavities and osteoclasts.18 A similar effect was also reported with long-term administration of G-CSF.10,17 This indicates that G-CSF promotes growth and differentiation of osteoclastic cells and results in stimulation of bone resorption. Moreover, a recent study has shown that de novo bone synthesis is also impaired by overexpression of G-CSF.9 Thus, G-CSF acts as a potent modulator of bone phenotypes in vivo. This effect was especially apparent when a relatively high dose of G-CSF was administered for long period in an animal model.17 Thus, special consideration is necessary when using high-dose G-CSF of long duration. Severe congenital neutropenia (SCN; or Kostmann’s syndrome) is a disorder of myelopoiesis characterized by severe neutropenia.26 Recombinant human G-CSF has substantially improved the life expectancy for children with SCN.25 Osteoporosis in SCN has been reported in a number of recent studies.1,14,16,23 Some of these patients suffered from pathologic fractures of vertebral bodies.1,23 These reports suggested a high incidence of bone mineral loss in children with SCN. However, it is not clear whether the bone loss is caused by the pathophysiology of the underlying disease or by G-CSF treatment.14,16,23 To examine possible effects of G-CSF in developing animals, we studied the bone phenotypes of G-CSF transgenic mice (G-Tg) during the developmental period. Apparent loss of bone was observed throughout the period of examination from 4 weeks through 36 weeks of age. Neither maturational retardation nor skeletal deformation was detected during the period of this study. Although a positive response to bisphosphonate therapy has been suggested in osteoporosis in SCN treated with G-CSF, the mechanism of bone remodeling for this compound has not been fully elucidated.1,14,26 There has been consideration for the long-term usage of this synthetic compound in developing children.19 To investigate another option for therapy of osteopenia in developing bone, we studied the effects of vitamin K2 (VK2, a food derivative nutrient supplement). A partial protective effect
Mice transgenic for granulocyte colony-stimulating factor (G-CSF) exhibit severe osteopenia with an increase of osteoclast number and acceleration of bone resorption in adult mice. To examine the effect of G-CSF overexpression on developing bone, bone mineral density levels were examined from 4 weeks through 36 weeks after birth. Peak bone mass was observed at around 24 weeks of age irrespective of G-CSF expression. Apparent osteopenia was observed as early as 4 weeks of age without detectable developmental retardation in bone length and skeletal structure. Morphological examination confirmed a reduction of cancellous bone and cortical bone at this early stage of life, indicating that overexpression of G-CSF results in apparent osteopenia in developing mice, similar to that in adult animals. The effect of vitamin K2 (menatetrenone) (MK4) on bone phenotypes during development was then examined. Mice were fed chow containing either 0.05 mg MK-4 per 100 g or 20.0 mg MK-4 per 100 g for 12 weeks as the control and experimental diets, respectively. This treatment did not change bone length, irrespective of the type of mouse or diet. Peripheral quantitative computed tomography (pQCT) revealed an increase of in CT value bone of MK4-treated mice. Taken together, these results indicate that overexpression of G-CSF induces an apparent reduction of bone mass and results in osteopenia in developing mice. The bone reduction was partially restored by feeding the mice MK4, suggesting a choice for treatment on the osteopenia induced by G-CSF. (Bone 30:880 – 885; 2002) © 2002 by Elsevier Science Inc. All rights reserved. Key Words: Osteoporosis; Granulocyte colony-stimulating factor (G-CSF); Vitamin K2; Developing; Transgenic mice.
Introduction Granulocyte colony-stimulating factor (G-CSF) promotes growth
Address for correspondence and reprints: Dr. Yasuo Kokai, Department of Pathology, Sapporo Medical University School of Medicine, S1W17, Chuo-ku, Sapporo, 060-8556, Japan. E-mail: [email protected] © 2002 by Elsevier Science Inc. All rights reserved.
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of a VK2-containing diet on this type of bone loss was observed. Collectively, G-CSF downmodulates bone mass in the developmental stage of postnatal life as well as in adult life. The relative usefulness of vitamin K2 was also examined. Materials and Methods Mice Production and maintenance of G-CSF transgenic mice were as previously described.8,24 At 3 weeks of age, mice were examined for their genotypes as described and kept under specific pathogen-free conditions in our animal laboratory. All experiments were conducted under the guidance and approval of the Animal Experimental Laboratory at Sapporo Medical University School of Medicine. Reagents All reagents were purchased from Wako Biochemicals (Tokyo, Japan) unless otherwise indicated. Morphological Examination Bones were cleaned to remove soft tissues and radiologically examined with soft X-rays (Sofron Type SRO-M50, Soken, Tokyo, Japan). The tissues were then fixed with 10% formaldehyde for 24 h, decalcified in 5% formic acid, and embedded in paraffin. The 5 m sections were stained with hematoxylineosin. DEXA, pQCT, and CT Bone mineral density (BMD) of the tibiae was measured by dual-energy X-ray absorptiometry (DEXA) using a QDR-1000w Plus Densitometer (Hologic, Inc., Waltham, MA) that was calibrated with a hydroxyapatite phantom of the human lumbar spine. The femora or tibiae were fixed with 100% ethanol and soft tissues were removed. Whole femora and tibiae were scanned and measurement was performed with a customized rodent whole body software package (Hologic). The scans were done with a 1.270-mm-diameter collimator, 0.762 mm line spacing, 0.380 mm point resolution, and an acquisition time of 9 min. Values were expressed as BMD (mg/cm2). The conditions of peripheral quantative computed tomography (pQCT) (XCTmScope, Stratec, Germany) used in this study were determined by preliminary study to adapt them specifically to the murine tibiae. In brief, cancellous bone was determined as the fraction of the CT value under 430 mg/cm3. Cortical bone was detected as a fraction of the CT value of 690 mg/cm3 or more. Microcomputed tomography (CT) was kindly performed by Asahikasei Co., Ltd. (Tokyo, Japan), using an MCT-12505MF device (Hitachi Medical Corp., Kashiwa, Japan). Slices were made at a thickness of 20 m in the axial planes of the bone. Diet and Feeding Starting at 8 weeks of age, mice were given free access to food containing 0.5% calcium with vitamin K (MK-4) at 0.05 mg MK-4 per 100 g or 20.0 mg MK-4 per 100 g as the control and experimental diets, respectively.11 After 12 weeks of treatment, mice were killed and prepared for further examination.
Figure 1. BMD of G-Tg during development. BMD was examined by DEXA. As early as 4 weeks of age, G-Tg exhibited reduced BMD, and the decrease of BMD was observed throughout the period of examination (4 –36 weeks of age). Peak bone mass was observed at 24 weeks in both transgenic and control mice (filled squares: control; filled circles: G-Tg).
Statistical Analysis At least four mice were examined in each experiment and identical protocols were repeated three times. The mean response of each experimental group was compared with its simultaneous control by the unpaired Student’s t-test. Analysis of variance was used to compare the mean responses of the experimental and control groups. Results Bone Mineral Density in G-CSF Transgenic Mice During Development Femora from each mouse were studied for BMD by DEXA. As early as 4 weeks of age, the G-CSF transgenic mice (G-Tg) exhibited significantly low BMD (Figure 1). The reduction of BMD was detected throughout the examination period (4 –36 weeks after birth). Peak bone mass was observed at around 24 weeks of age in both G-Tg and littermate control (Lm) mice. Although reduced BMD was observed even at 4 weeks in G-Tg, bone length appeared to be unchanged at this developmental stage (Figure 2). Morphological Analysis of G-Tg Bone at 4 Weeks of Age The radiological appearance of the femur at 4 weeks of age is shown in Figure 3. Decreased density of bone and normal size were observed. Deformities were not detectable in the transgenic bone. As shown in Figure 4, histological findings revealed a dramatic decrease of trabecular bone and cortical bone in the G-Tg compared with the Lm mice. An apparent increase of neutrophils was observed in the marrow cavity in the G-Tg mice. Effects of Diet Containing MK4 on Osteopenia in Developing G-Tg Mice As shown in Figure 1, the peak bone mass of G-Tg and Lm was observed at about 24 weeks after birth, indicating that bone
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Figure 2. Femur length of G-Tg at 4 weeks. Length of the femur was measured at 4 weeks. G-Tg did not show detectable growth retardation as measured by femur length (N.S., not significant). In adult mice (from 8 to 32 weeks), the bone length of G-Tg has been reported to be unaltered compared with the normal controls.18
developed until this age. At 8 weeks of age, mice began to be fed a diet containing MK-4, which was continued for 12 weeks. MK-4 treatment did not change bone development as examined by bone length in the whole tibia or the length of the midportion of the tibia (Figure 5), nor was there a detectable change of BMD (Figure 6). To examine the bone phenotype in detail, we employed pQCT. The design of the scan is depicted in Figure 7. A site-specific increase of the CT value in mice treated with MK-4 (20.0 mg per 100 g) was detected (Figure 8). Although the CT value in slice 4 did not show any detectable change, in slice 3, a portion relatively rich with cancellous bone,
Figure 3. Radiological appearance of G-Tg femur. Decreased density of developmental bone (4 weeks after birth) but normal size was observed in the G-Tg femur compared with a control littermate.
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Figure 4. Histological appearance of developing G-Tg bone. Hematoxylin-eosin-stained specimens revealed dramatic decreases of trabecular bone and cortical bone in the G-Tg compared with control littermate at 4 weeks after birth. An apparent increase of neutrophils was observed in the marrow cavity of G-Tg. (A,C) Control. (B,D) G-Tg [original magnification (A,B) ⫻40, (C,D) ⫻200].
Figure 5. Effect of MK4 on bone length of G-Tg and control mice. An MK-4 containing diet was given for 12 weeks and the bone length was determined. The tibial length and length of the midportion (50% of bone length; see Figure 7) showed no significant difference between the MK-4 containing diet and control diet.
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Figure 6. Effect of MK4 diet on BMD of G-Tg bone. BMD of tibiae obtained from G-Tg fed MK-4-containing [Vit.K2(⫹)] and control [Vit.K2(⫺)] diets showed no detectable difference between the two treatments (N.S., not significant).
revealed an increased QCT value (235.4 ⫾ 3.5 vs. 310.8 ⫾ 23.6, p ⫽ 0.0195; peel mode 2 at a threshold of 430 mg/cm3). Reconstruction of bone based on the CT value showed increases of cancellous and cortical bone-like structure as shown in Figure 9. Although all slices of 1, 2, 3, and 4 showed increased bone, only in slice 3 was it statistically significant. The change of bone phenotype was further examined by CT; Figure 10. This examination also showed apparent increases of cortical bone and trabecular bone after MK-4 treatment (Figure 10). Discussion In the present study, we showed that overexpression of G-CSF resulted in severe osteopenia in developing mice without detectable skeletal deformity, indicating a potent effect of G-CSF on bone loss prior to the period of peak bone mass. This effect may be relevant in children with severe congenital neutropenia (SCN). Although a synergistic effect with G-CSF and the under-
Figure 7. Design of pQCT measurement of tibiae. To examine the bone phenotype in detail, we employed pQCT. Tibiae were examined at each slice level (slices 1– 4). The pQCT value is expressed as milligrams per cubic centimeter (CORTBD, cortical total bone density).
Figure 8. A site-specific increase of pQCT value in mice treated with MK-4. MK-4 treatment caused a significant increase of the pQCT value in slice 3. This increase was not detected in slice 4, indicating a site-specific effect of MK-4 treatment on the pQCT value.
lying disease may occur in the osteopenia of SCN children, G-CSF alone seemed to be a potent modulator of the bone phenotype in vivo. Overexpression of G-CSF induces an increase of osteoclast number and results in acceleration of bone resorption.18 The increase of osteoclast number was found to be strongly linked with upregulation of granulocyte-macrophage colony forming units (CFU-GM; Oda et al., manuscript in preparation).24 The growth and differentiation of osteoclasts have been extensively studied, although the cellular mechanism has not been fully elucidated.13 The osteoclast is believed to develop as a precursor derived from CFU-GM. A number of cytokines, including interleukin-3 (IL-3), IL-6, GM-CSF, and G-CSF, have been reported to promote numbers of CFU-GM. However, none of these cytokines, except G-CSF, can induce bone loss or increase bone resorption in vivo.7,21 It is conceivable that G-CSF may play a role in regulating differentiation of osteoclasts from hematopoietic precursors.12 We recently reported that overexpression of G-CSF downregulates de novo bone synthesis by impairing the initial induction of mesenchymal cells.9 This suggests that G-CSF regulates bone metabolism by modulating both osteoclast and mesenchymal system. This activity is particularly important when G-CSF is employed at the developmental stage of life. Because G-CSF is indispensable for maintaining the number and activity of neutrophils in SCN patients, it is crucial to employ G-CSF for this type of neutropenia. To prevent possible bone loss in this particular condition, some reports have examined the effect of bisphosphonate on bone loss during treatment of SCN with
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Figure 10. Microcomputed tomographic results for MK4-treated bone. The change of bone phenotype was further examined by CT. Apparent increases of both cortical and trabecular bone in MK-4-treated mice [VK(⫹) observed in slice 3 (Figure 7) vs. control (VK(⫺)].
References
Figure 9. Reconstruction of bone based on pQCT value. Visualization of pQCT value of each slice reveals an apparent increase in the pQCT value in slice 3. Although slices 1, 2, and 4 also showed increases in this value, they were not significant. This reconstruction suggests that increased pQCT may be detectable in both trabecular and cortical bone.
G-CSF.1,14 Bisphosphonate may be the strongest bioactive synthetic component to inhibit bone resorption.19 However, it is not clear whether this strong activity influences bone remodeling, especially in children, who are right in the middle of development of the skeleton.5 As another possible choice for the treatment of bone loss by G-CSF, we investigated the effect of vitamin K2 (menatetrenone) on the bone phenotype. Of interest, this food derivative showed a positive effect on bone mass. It is unclear as to why vitamin K2 did not increase bone formation when administered to adults.2 Because vitamin K2 is a relatively mild food supplement,3 this may be applicable for the prevention of bone loss by G-CSF in developing bone.15 In summary, we have presented data strongly suggesting that in vivo activity of G-CSF is linked to bone loss in developing bone without detectable skeletal deformity. Thus, special attention should be paid to this phenomenon when this cytokine is employed in childhood. In addition, study of the effects on developing bone is essential in the use of inhibitors of bone resorption due to bone remodeling and development.
1. Bishop N. J., Williams D. M., Compston J. C., Stirling D. M., and Prentice A. Osteoporosis in severe congenital neutropenia treated with granulocyte colonystimulating factor. Br J Haematol 89:927–928; 1995. 2. Booth S. L., Tucker K. L., Chen H., Hannan M. T., Gagnon D. R., Cupples L. A., Wilson P. W., Ordovas J., Schaefer E. J., Dawson-Hughes B., and Kiel D. P. Dietary vitamin K intakes are associated with hip fracture but not with bone mineral density in elderly men and women. Am J Clin Nutr 71:1201– 1208; 2000. 3. Feskanich D., Weber P., Willett W. C., Rockett H., Booth S. L., and Colditz G. A. Vitamin K intake and hip fractures in women: A prospective study. Am J Clin Nutr 69:74 –79; 1999. 4. Glaspy J. A., and Golde D. W. Granulocyte colony-stimulating factor (G-CSF): Preclinical and clinical studies. Semin Oncol 19:386 –394; 1992. 5. Kauffman R. P., Overton T. H., Shiflett M., and Jennings J. C. Osteoporosis in children and adolescent girls: Case report of idiopathic juvenile osteoporosis and review of the literature. Obstet Gynecol Surv 56:492–504; 2001. 6. Kawamura H., Kawamura T., Kokai Y., Mori M., Matsuura A., Oya H., Honda S., Suzuki S., Weerashinghe A., Watanabe H., and Abo T. Expansion of extrathymic T cells as well as granulocytes in the liver and other organs of granulocyte-colony stimulating factor transgenic mice: Why they lost the ability of hybrid resistance. J Immunol 162:5957–5964; 1999. 7. Kitamura H., Kawata H., Takahashi F., Higuchi Y., Furuichi T., and Ohkawa H. Bone marrow neutrophilia and suppressed bone turnover in human interleukin-6 trangenic mice. A cellular relationship among hematopoietic cells, osteoblasts, and osteoclasts mediated by stromal cells in bone marrow. Am J Pathol 147:1682–1692; 1995. 8. Kokai Y., Kaneko H., Izuka K., Matsuura A., and Fujimoto J. Inhibition of resistance to hemopoietic allo-grafts in granulocyte colony-stimulating factor transgenic mice. Eur J Immunol 26:115–119; 1996. 9. Kuwabara H., Wada T., Oda T., Yoshikawa H., Sawada N., Kokai Y., and Ishii S. Overexpression of the granulocyte colony-stimulating factor gene impairs bone morphogenic protein responsiveness in mice. Lab Invest 81:1133–1141; 2001. 10. Lee M. Y., Fukunaga R., Lee T. J., Lottsfeldt J. L., and Nagata S. Bone modulation in sustained hematopoietic stimulation in mice. Blood 77:2135– 2141; 1991. 11. Mawatari T., Miura H., Higaki H., Moro-Oka T., Kurata K., Murakami T., and Iwamoto Y. Effect of vitamin K2 on three-dimensional trabecular microarchitecture in ovariectomized rats. J Bone Miner Res 15:1810 –1817; 2000. 12. Purton L. E., Lee M. Y., and Torok-Storb B. Normal human peripheral blood mononuclear cells mobilized with granulocyte colony-stimulating factor have increased osteoclastogenic potential compared to nonmobilized blood. Blood 87:1802–1808; 1996. 13. Roodman G. D. Cell biology of the osteoclast. Exp Hematol 27:1229 –1241; 1999.
Bone Vol. 30, No. 6 June 2002:880 – 885 14. Sekhar R. V., Culbert S., Hoots W. K., Klein M. J., Zietz H., and Vassilopoulou-Sellin R. Severe osteopenia in a young boy with Kostmann’s congenital neutropenia treated with granulocyte colony-stimulating factor: Suggested therapeutic approach. Pediatrics 108:E54; 2001. 15. Shirasaki M., Shiraki Y., Aoki C., and Miura M. Vitamin K2 (menatetrenone) effectively prevents fractures and sustains lumbar bone mineral density in osteoporosis. J Bone Miner Res 15:515–521; 2000. 16. Simon M., Lengfelder E., Reiter S., and Hehlmann R. Osteoporosis in severe congenital neutropenia: Inherent to the disease or a sequela of G-CSF treatment. Am J Hematol 52:127; 1996. 17. Suzuki M., Adachi K., Sugimoto T., Nakayama H., and Doi K. The development of bone changes induced in rats by recombinant human granulocyte colony-stimulating factor is suppressed by bisphosphonate. Histol Histopathol 14:679 –686; 1999. 18. Takahashi T., Wada T., Mori M., Kokai Y., and Ishii S. Overexpression of the granulocyte colony-stimulating factor gene leads to osteoporosis in mice. Lab Invest 74:827–834; 1996. 19. Theriault R. L., and Hortobagyi G. N. The evolving role of bisphosphonates. Semin Oncol 28:284 –290; 2001. 20. To L. B., Haylock D. N., Simmons P. J., and Juttner C. A. The biology and clinical uses of blood stem cells. Blood 89:2233–2258; 1997. 21. Vargas S. J., Naprta A., Lee S. K., Kalinowski J., Kawaguchi C. C., Raisz L. G., and Lorenzo J. A. Lack of evidence for an increase in interleukin-6
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expression in adult murine bone, bone marrow, and marrow stromal cell cultures after ovariectomy. J Bone Miner Res 11:1926 –1934; 1996. Wada T., Isu K., Takeda N., Usui M., Ishii S., and Yamawaki S. A preliminary report of neoadjuvant chemotherapy NSH-7 study in osteosarcoma: Preoperative salvage chemotherapy based on clinical tumor response and the use of granulocyte colony-stimulating factor. Oncology 53:221–227; 1996. Yakisan E., Schirg E., Zeidler C., Bishop N. J., Reiter A., Hirt A., Riehm H., and Welte K. High incidence of significant bone loss in patients with severe congenital neutropenia (Kostmann’s syndrome). J Pediatr 131:592–597; 1997. Yamada T., Kaneko H., Iizuka K., Matsubayashi Y., Kokai Y., and Fujimoto J. Elevation of lymphocyte and hematopoietic stem cell numbers in mice transgenic for human granulocyte CSF. Lab Invest 74:384 –394; 1996. Zeidler C., Reiter A., Yakisan E., Koci B., Riehm H., and Welte K. Long-term treatment with recombinant human granulocyte colony stimulating factor in patients with severe congenital neutropenia [in German]. Klin Padiatr 205: 264 –271; 1993. Zeidler C., Schwinzer B., and Welte K. Severe congenital neutropenia: Trends in diagnosis and therapy [in German]. Klin Padiatr 212:145–152; 2000.
Date Received: November 29, 2001 Date Revised: December 25, 2001 Date Accepted: February 1, 2002
Overexpression of Granulocyte Colony-stimulating Factor Induces Severe Osteopenia in Developing Mice That Is Partially Prevented by a Diet Containing Vitamin K2 (Menatetrenone) Y. KOKAI,1 T. WADA,2 T. ODA,1,2 H. KUWABARA,2 K. HARA,3 Y. AKIYAMA,3 S. ISHII,2 and N. SAWADA1 1 3
Departments of Pathology and 2Orthopedics, Sapporo Medical University School of Medicine, Sapporo, Japan Department of Applied Drug Research, Eisai Co., Ltd., Tokyo, Japan
and differentiation of cells of granulocytic lineage.4 Its strong effect on the increase of neutrophils in vivo provides a useful therapeutic tool for a various types of neutropenia in the clinical setting.20,22 Later studies, however, have revealed that this cytokine has effects on a variety of cell types besides neutrophilic cells, such as mobilization of hematopoietic stem cells20,24 and modulation of the immune system.6 We previously showed that overexpression of G-CSF induces severe osteopenia with dramatic increases of bone cavities and osteoclasts.18 A similar effect was also reported with long-term administration of G-CSF.10,17 This indicates that G-CSF promotes growth and differentiation of osteoclastic cells and results in stimulation of bone resorption. Moreover, a recent study has shown that de novo bone synthesis is also impaired by overexpression of G-CSF.9 Thus, G-CSF acts as a potent modulator of bone phenotypes in vivo. This effect was especially apparent when a relatively high dose of G-CSF was administered for long period in an animal model.17 Thus, special consideration is necessary when using high-dose G-CSF of long duration. Severe congenital neutropenia (SCN; or Kostmann’s syndrome) is a disorder of myelopoiesis characterized by severe neutropenia.26 Recombinant human G-CSF has substantially improved the life expectancy for children with SCN.25 Osteoporosis in SCN has been reported in a number of recent studies.1,14,16,23 Some of these patients suffered from pathologic fractures of vertebral bodies.1,23 These reports suggested a high incidence of bone mineral loss in children with SCN. However, it is not clear whether the bone loss is caused by the pathophysiology of the underlying disease or by G-CSF treatment.14,16,23 To examine possible effects of G-CSF in developing animals, we studied the bone phenotypes of G-CSF transgenic mice (G-Tg) during the developmental period. Apparent loss of bone was observed throughout the period of examination from 4 weeks through 36 weeks of age. Neither maturational retardation nor skeletal deformation was detected during the period of this study. Although a positive response to bisphosphonate therapy has been suggested in osteoporosis in SCN treated with G-CSF, the mechanism of bone remodeling for this compound has not been fully elucidated.1,14,26 There has been consideration for the long-term usage of this synthetic compound in developing children.19 To investigate another option for therapy of osteopenia in developing bone, we studied the effects of vitamin K2 (VK2, a food derivative nutrient supplement). A partial protective effect
Mice transgenic for granulocyte colony-stimulating factor (G-CSF) exhibit severe osteopenia with an increase of osteoclast number and acceleration of bone resorption in adult mice. To examine the effect of G-CSF overexpression on developing bone, bone mineral density levels were examined from 4 weeks through 36 weeks after birth. Peak bone mass was observed at around 24 weeks of age irrespective of G-CSF expression. Apparent osteopenia was observed as early as 4 weeks of age without detectable developmental retardation in bone length and skeletal structure. Morphological examination confirmed a reduction of cancellous bone and cortical bone at this early stage of life, indicating that overexpression of G-CSF results in apparent osteopenia in developing mice, similar to that in adult animals. The effect of vitamin K2 (menatetrenone) (MK4) on bone phenotypes during development was then examined. Mice were fed chow containing either 0.05 mg MK-4 per 100 g or 20.0 mg MK-4 per 100 g for 12 weeks as the control and experimental diets, respectively. This treatment did not change bone length, irrespective of the type of mouse or diet. Peripheral quantitative computed tomography (pQCT) revealed an increase of in CT value bone of MK4-treated mice. Taken together, these results indicate that overexpression of G-CSF induces an apparent reduction of bone mass and results in osteopenia in developing mice. The bone reduction was partially restored by feeding the mice MK4, suggesting a choice for treatment on the osteopenia induced by G-CSF. (Bone 30:880 – 885; 2002) © 2002 by Elsevier Science Inc. All rights reserved. Key Words: Osteoporosis; Granulocyte colony-stimulating factor (G-CSF); Vitamin K2; Developing; Transgenic mice.
Introduction Granulocyte colony-stimulating factor (G-CSF) promotes growth
Address for correspondence and reprints: Dr. Yasuo Kokai, Department of Pathology, Sapporo Medical University School of Medicine, S1W17, Chuo-ku, Sapporo, 060-8556, Japan. E-mail: [email protected] © 2002 by Elsevier Science Inc. All rights reserved.
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of a VK2-containing diet on this type of bone loss was observed. Collectively, G-CSF downmodulates bone mass in the developmental stage of postnatal life as well as in adult life. The relative usefulness of vitamin K2 was also examined. Materials and Methods Mice Production and maintenance of G-CSF transgenic mice were as previously described.8,24 At 3 weeks of age, mice were examined for their genotypes as described and kept under specific pathogen-free conditions in our animal laboratory. All experiments were conducted under the guidance and approval of the Animal Experimental Laboratory at Sapporo Medical University School of Medicine. Reagents All reagents were purchased from Wako Biochemicals (Tokyo, Japan) unless otherwise indicated. Morphological Examination Bones were cleaned to remove soft tissues and radiologically examined with soft X-rays (Sofron Type SRO-M50, Soken, Tokyo, Japan). The tissues were then fixed with 10% formaldehyde for 24 h, decalcified in 5% formic acid, and embedded in paraffin. The 5 m sections were stained with hematoxylineosin. DEXA, pQCT, and CT Bone mineral density (BMD) of the tibiae was measured by dual-energy X-ray absorptiometry (DEXA) using a QDR-1000w Plus Densitometer (Hologic, Inc., Waltham, MA) that was calibrated with a hydroxyapatite phantom of the human lumbar spine. The femora or tibiae were fixed with 100% ethanol and soft tissues were removed. Whole femora and tibiae were scanned and measurement was performed with a customized rodent whole body software package (Hologic). The scans were done with a 1.270-mm-diameter collimator, 0.762 mm line spacing, 0.380 mm point resolution, and an acquisition time of 9 min. Values were expressed as BMD (mg/cm2). The conditions of peripheral quantative computed tomography (pQCT) (XCTmScope, Stratec, Germany) used in this study were determined by preliminary study to adapt them specifically to the murine tibiae. In brief, cancellous bone was determined as the fraction of the CT value under 430 mg/cm3. Cortical bone was detected as a fraction of the CT value of 690 mg/cm3 or more. Microcomputed tomography (CT) was kindly performed by Asahikasei Co., Ltd. (Tokyo, Japan), using an MCT-12505MF device (Hitachi Medical Corp., Kashiwa, Japan). Slices were made at a thickness of 20 m in the axial planes of the bone. Diet and Feeding Starting at 8 weeks of age, mice were given free access to food containing 0.5% calcium with vitamin K (MK-4) at 0.05 mg MK-4 per 100 g or 20.0 mg MK-4 per 100 g as the control and experimental diets, respectively.11 After 12 weeks of treatment, mice were killed and prepared for further examination.
Figure 1. BMD of G-Tg during development. BMD was examined by DEXA. As early as 4 weeks of age, G-Tg exhibited reduced BMD, and the decrease of BMD was observed throughout the period of examination (4 –36 weeks of age). Peak bone mass was observed at 24 weeks in both transgenic and control mice (filled squares: control; filled circles: G-Tg).
Statistical Analysis At least four mice were examined in each experiment and identical protocols were repeated three times. The mean response of each experimental group was compared with its simultaneous control by the unpaired Student’s t-test. Analysis of variance was used to compare the mean responses of the experimental and control groups. Results Bone Mineral Density in G-CSF Transgenic Mice During Development Femora from each mouse were studied for BMD by DEXA. As early as 4 weeks of age, the G-CSF transgenic mice (G-Tg) exhibited significantly low BMD (Figure 1). The reduction of BMD was detected throughout the examination period (4 –36 weeks after birth). Peak bone mass was observed at around 24 weeks of age in both G-Tg and littermate control (Lm) mice. Although reduced BMD was observed even at 4 weeks in G-Tg, bone length appeared to be unchanged at this developmental stage (Figure 2). Morphological Analysis of G-Tg Bone at 4 Weeks of Age The radiological appearance of the femur at 4 weeks of age is shown in Figure 3. Decreased density of bone and normal size were observed. Deformities were not detectable in the transgenic bone. As shown in Figure 4, histological findings revealed a dramatic decrease of trabecular bone and cortical bone in the G-Tg compared with the Lm mice. An apparent increase of neutrophils was observed in the marrow cavity in the G-Tg mice. Effects of Diet Containing MK4 on Osteopenia in Developing G-Tg Mice As shown in Figure 1, the peak bone mass of G-Tg and Lm was observed at about 24 weeks after birth, indicating that bone
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Figure 2. Femur length of G-Tg at 4 weeks. Length of the femur was measured at 4 weeks. G-Tg did not show detectable growth retardation as measured by femur length (N.S., not significant). In adult mice (from 8 to 32 weeks), the bone length of G-Tg has been reported to be unaltered compared with the normal controls.18
developed until this age. At 8 weeks of age, mice began to be fed a diet containing MK-4, which was continued for 12 weeks. MK-4 treatment did not change bone development as examined by bone length in the whole tibia or the length of the midportion of the tibia (Figure 5), nor was there a detectable change of BMD (Figure 6). To examine the bone phenotype in detail, we employed pQCT. The design of the scan is depicted in Figure 7. A site-specific increase of the CT value in mice treated with MK-4 (20.0 mg per 100 g) was detected (Figure 8). Although the CT value in slice 4 did not show any detectable change, in slice 3, a portion relatively rich with cancellous bone,
Figure 3. Radiological appearance of G-Tg femur. Decreased density of developmental bone (4 weeks after birth) but normal size was observed in the G-Tg femur compared with a control littermate.
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Figure 4. Histological appearance of developing G-Tg bone. Hematoxylin-eosin-stained specimens revealed dramatic decreases of trabecular bone and cortical bone in the G-Tg compared with control littermate at 4 weeks after birth. An apparent increase of neutrophils was observed in the marrow cavity of G-Tg. (A,C) Control. (B,D) G-Tg [original magnification (A,B) ⫻40, (C,D) ⫻200].
Figure 5. Effect of MK4 on bone length of G-Tg and control mice. An MK-4 containing diet was given for 12 weeks and the bone length was determined. The tibial length and length of the midportion (50% of bone length; see Figure 7) showed no significant difference between the MK-4 containing diet and control diet.
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Figure 6. Effect of MK4 diet on BMD of G-Tg bone. BMD of tibiae obtained from G-Tg fed MK-4-containing [Vit.K2(⫹)] and control [Vit.K2(⫺)] diets showed no detectable difference between the two treatments (N.S., not significant).
revealed an increased QCT value (235.4 ⫾ 3.5 vs. 310.8 ⫾ 23.6, p ⫽ 0.0195; peel mode 2 at a threshold of 430 mg/cm3). Reconstruction of bone based on the CT value showed increases of cancellous and cortical bone-like structure as shown in Figure 9. Although all slices of 1, 2, 3, and 4 showed increased bone, only in slice 3 was it statistically significant. The change of bone phenotype was further examined by CT; Figure 10. This examination also showed apparent increases of cortical bone and trabecular bone after MK-4 treatment (Figure 10). Discussion In the present study, we showed that overexpression of G-CSF resulted in severe osteopenia in developing mice without detectable skeletal deformity, indicating a potent effect of G-CSF on bone loss prior to the period of peak bone mass. This effect may be relevant in children with severe congenital neutropenia (SCN). Although a synergistic effect with G-CSF and the under-
Figure 7. Design of pQCT measurement of tibiae. To examine the bone phenotype in detail, we employed pQCT. Tibiae were examined at each slice level (slices 1– 4). The pQCT value is expressed as milligrams per cubic centimeter (CORTBD, cortical total bone density).
Figure 8. A site-specific increase of pQCT value in mice treated with MK-4. MK-4 treatment caused a significant increase of the pQCT value in slice 3. This increase was not detected in slice 4, indicating a site-specific effect of MK-4 treatment on the pQCT value.
lying disease may occur in the osteopenia of SCN children, G-CSF alone seemed to be a potent modulator of the bone phenotype in vivo. Overexpression of G-CSF induces an increase of osteoclast number and results in acceleration of bone resorption.18 The increase of osteoclast number was found to be strongly linked with upregulation of granulocyte-macrophage colony forming units (CFU-GM; Oda et al., manuscript in preparation).24 The growth and differentiation of osteoclasts have been extensively studied, although the cellular mechanism has not been fully elucidated.13 The osteoclast is believed to develop as a precursor derived from CFU-GM. A number of cytokines, including interleukin-3 (IL-3), IL-6, GM-CSF, and G-CSF, have been reported to promote numbers of CFU-GM. However, none of these cytokines, except G-CSF, can induce bone loss or increase bone resorption in vivo.7,21 It is conceivable that G-CSF may play a role in regulating differentiation of osteoclasts from hematopoietic precursors.12 We recently reported that overexpression of G-CSF downregulates de novo bone synthesis by impairing the initial induction of mesenchymal cells.9 This suggests that G-CSF regulates bone metabolism by modulating both osteoclast and mesenchymal system. This activity is particularly important when G-CSF is employed at the developmental stage of life. Because G-CSF is indispensable for maintaining the number and activity of neutrophils in SCN patients, it is crucial to employ G-CSF for this type of neutropenia. To prevent possible bone loss in this particular condition, some reports have examined the effect of bisphosphonate on bone loss during treatment of SCN with
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Figure 10. Microcomputed tomographic results for MK4-treated bone. The change of bone phenotype was further examined by CT. Apparent increases of both cortical and trabecular bone in MK-4-treated mice [VK(⫹) observed in slice 3 (Figure 7) vs. control (VK(⫺)].
References
Figure 9. Reconstruction of bone based on pQCT value. Visualization of pQCT value of each slice reveals an apparent increase in the pQCT value in slice 3. Although slices 1, 2, and 4 also showed increases in this value, they were not significant. This reconstruction suggests that increased pQCT may be detectable in both trabecular and cortical bone.
G-CSF.1,14 Bisphosphonate may be the strongest bioactive synthetic component to inhibit bone resorption.19 However, it is not clear whether this strong activity influences bone remodeling, especially in children, who are right in the middle of development of the skeleton.5 As another possible choice for the treatment of bone loss by G-CSF, we investigated the effect of vitamin K2 (menatetrenone) on the bone phenotype. Of interest, this food derivative showed a positive effect on bone mass. It is unclear as to why vitamin K2 did not increase bone formation when administered to adults.2 Because vitamin K2 is a relatively mild food supplement,3 this may be applicable for the prevention of bone loss by G-CSF in developing bone.15 In summary, we have presented data strongly suggesting that in vivo activity of G-CSF is linked to bone loss in developing bone without detectable skeletal deformity. Thus, special attention should be paid to this phenomenon when this cytokine is employed in childhood. In addition, study of the effects on developing bone is essential in the use of inhibitors of bone resorption due to bone remodeling and development.
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Date Received: November 29, 2001 Date Revised: December 25, 2001 Date Accepted: February 1, 2002