Accepted Article
Original Article Vitamin D receptor signaling enhances locomotive ability in mice† Sadaoki Sakai, Miho Suzuki, Yoshihito Tashiro, Keisuke Tanaka, Satoshi Takeda, Ken Aizawa, Michinori Hirata, Kenji Yogo, and Koichi Endo
Product Research Department, Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Shizuoka, Japan Corresponding Author Koichi Endo, Ph.D., Product Research Department Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan TEL: +81-550- 87-6375; FAX: +81-550-87-6782; E-mail address: [email protected] Conflict of Interest All authors are employees of Chugai Pharmaceutical Co., Ltd.
†
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jbmr.2317]
Initial Date Submitted October 17, 2013; Date Revision Submitted June 6, 2014; Date Final Disposition Set July 14, 2014
Journal of Bone and Mineral Research © 2014 American Society for Bone and Mineral Research DOI 10.1002/jbmr.2317
1
Accepted Article
Abstract
Bone fractures markedly reduce quality of life and life expectancy in elderly people. Although osteoporosis increases bone fragility, fractures frequently occur in patients with normal bone mineral density. Because most fractures occur on falling, preventing falls is another focus for reducing bone fractures. In this study, we investigated the role of vitamin D receptor (VDR) signaling in locomotive ability. In the rotarod test, physical exercise enhanced locomotive ability of WT mice by 1.6-fold, whereas exercise did not enhance locomotive ability of VDR KO mice. Compared to WT mice, VDR KO mice had smaller peripheral nerve axonal diameter and disordered AChR morphology on the extensor digitorum longus muscle. Eldecalcitol (ED-71, ELD), an analog of 1,25(OH)2D3, administered to rotarod-trained C57BL/6 mice enhanced locomotor performance compared to vehicle-treated non-trained mice. The Area of AChR cluster on the extensor digitorum longus was greater in ELD-treated mice than in vehicle-treated mice. ELD and 1,25(OH)2D3 enhanced expression of IGF-1,
myelin basic protein, and VDR in rat primary Schwann cells. VDR signaling regulates neuromuscular maintenance and enhances locomotive ability following physical exercise. Further investigation is required, but Schwann cells and the neuromuscular junction are targets of vitamin D3 signaling in locomotive ability.
2
Accepted Article
Introduction Osteoporosis is a disease characterized by low bone density and microarchitectural deterioration of bone tissue, leading to increased bone fragility and a consequent increase in fracture risk (1). Because bone fractures markedly reduce quality of life and life expectancy (2, 3), one goal of osteoporosis treatment is prevention of bone fractures. Bone is a dynamic tissue that constantly undergoes remodeling, and bone mineral density (BMD) is used as an index of osteoporosis. Most osteoporosis treatments increase BMD by inhibiting bone resorption. However, although BMD is a key factor related to bone strength, bone fractures in the elderly are common even among those with normal BMD. This fact suggests that correction of BMD is in itself not sufficient to prevent bone fractures. Besides BMD, the factor correlating most highly with fracture incidence is the risk of falling, because almost all fractures are a result of falls. Declining locomotive ability is observed in elderly people, and it is reported that a person’s tendency to fall becomes an important predictor of bone fractures later in life (4). Therefore, it is preferable that a drug for the treatment of osteoporosis not only increase bone strength but also act to prevent falls. In 2011, eldecalcitol (ELD) [ED-71; 1α,25-dihydroxy-2β-(3-hydroxypropyloxy) vitamin D3], a
1,25(OH)2D3 analog, was approved for the treatment of osteoporosis in Japan. ELD has a greater activity than alfacalcidol (ALF), a prodrug of 1,25(OH)2D3, in suppressing bone resorption as well as increasing
BMD (5). In a clinical trial, ELD had superior effects over ALF in preventing vertebral and wrist fractures in osteoporotic patients with sufficient vitamin D supply (6). Although the incidence of falls was not monitored in that study, it seems that almost all wrist fractures occur after falls (7). A correlation between vitamin D supplementation and reduced risk of falls has been reported (8, 9, 10, 11). This led to the hypothesis that ELD may have some effect on reducing incidences of falls. In this study, we investigated the relationship between vitamin D3 signaling and locomotive ability
in mice on a rotarod treadmill. In addition, we investigated the role of 1,25(OH)2D3 and ELD in peripheral nerves and the neuromuscular junction which are known to decline in function with senescence. 3
Accepted Article
Materials and Methods Animals Vitamin D receptor knockout (VDR KO) mice were constructed by Dr. Kato (12) and maintained at Chugai Pharmaceutical Co., Ltd. The mice were reared under standard laboratory conditions at 20–26°C, 35–75% relative humidity, and a 12 h light:12 h dark cycle. Animals were given free access to tap water and standard rodent chow (CE-2; CLEA Japan, Tokyo Japan) for WT mice and modified CE-2 supplemented with 20% lactose, 2% calcium, and 1.25% phosphate for VDR KO mice. Male WT and VDR KO mice began rotarod training at 32 weeks old. The ELD treatment study used 9-week-old male C57BL/6 mice (Charles River Laboratories, Yokohama Japan) reared under the same conditions as for WT mice.
The experimental protocol was approved by the Institutional Animal Care and Use Committee of Chugai Pharmaceutical Co., Ltd. Rotarod exercise and locomotive ability assessment All mice were trained on a 30-mm diameter rotarod treadmill (MK-670; Muromachi Kikai, Tokyo Japan) at 15 rpm for 5 minutes, 3 times a day 4 days a week. To calculate the effect of 1,25(OH)2D3 analog treatment, ELD was administered orally to male C57BL/6 mce in short-chain fatty acid vehicle at a dose of 0.05 μg/kg/day 4 days a week for 2 weeks together with rotarod training. After the training
period, motor coordination was measured with an accelerating rotarod test to assess locomotive ability. Mice were placed individually on the rotating drum and the drum was accelerated from 3 to 30 rpm in 2 min and then kept at 30 rpm for 1 min. The time at which the mouse fell off the drum was recorded. The mice that did not fall from the drum in the 3 min were given a score of 180 seconds. The test was performed 2 times for practice and a third time to collect data for analysis. Three sequential measurements were performed at 1-min intervals. Histological analysis of mouse sciatic nerve and neuromuscular junction Sciatic nerves from WT and VDR KO mice were fixed in 2.5% glutaraldehyde. Toluidine blue staining and electron microscopy imaging were performed at the Bozo Research Center (Tokyo Japan). The
4
Accepted Article
diameters of all axons in cross sections of toluidine-blue–stained sciatic nerves were calculated by using a fluorescence microscope (BZ-9000; Keyence, Osaka Japan). Extensor digitorum longus muscles from each group were fixed with 10% formalin and stained with rhodamine-conjugated α-bungarotoxin (Invitrogen, CA, USA). Muscle fibers were carefully disentangled using forceps. AChR clusters were observed under the fluorescence microscope (BZ-9000; Keyence) and the area of AChR cluster was calculated using BZ Analyzer software (Keyence). The individual area of AChR clusters from 3 mice of each group were analyzed. Analysis of gene expression in rat nerve tissues and primary Schwann cells Real-time PCR analysis was performed on cDNA from the cerebellum, hippocampus, and sciatic nerves from old female rats. The specimens were immediately conserved in RNAlater (Qiagen, Hilden Germany) and stored at −80°C until mRNA extraction. mRNA was extracted using an RNeasy lipid tissue kit (Qiagen) and reverse transcribed using a Omniscript (Qiagen) according to manufacturer’s protocol. mRNA from rat primary Schwann cells was extracted using RNeasy (Qiagen). Real-time PCR analysis was performed with a 7500 Fast Real-Time PCR System (Applied Biosystems, CA, USA) using TaqMan probes Rn00566976_m1 for VDR, Rn00710306_m1 for IGF-1, Rn01399613_m1 for MBP, and 4352338E-0608007 for GAPD (Applied Biosystems). Measurement of serum calcium concentration Mouse serum was separated from whole blood, and serum calcium concentration was measured by using a 7170S Automatic Analyzer (Hitachi High-Technologies, Tokyo, Japan). Primary Schwann cell culture Rat primary Schwann cell culture was performed according to the methods of a previous report (13). Briefly, sciatic nerves were resected from 3-day-old neonatal rats and digested with 3 mg/mL collagenase (Wako Pure Chemical Industries, Osaka, Japan) for 15 min at 37°C, followed by digestion with 3 mg/mL collagenase and 25 mg/mL trypsin (Sigma Aldrich, MO, USA) for 15 min at 37°C. Dissociated sciatic nerves were cultured in 10% FBS DMEM with 10 μM of cytosine β-D
arabinofuranoside (AraC) for 3 days to remove mesenchymal cells. For immunostaining, cells were
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Accepted Article
cultured on cover glass for 2 days and fixed with 4% paraformaldehyde. Fixed rat Schwann cells were stained with polyclonal rabbit anti S-100 antibody (DakoCytomation, Glostrup, Denmark) and visualized with DyLight 488 anti-rabbit IgG (H+L) (Vector Laboratories, CA, USA). Nuclei were stained with Prolong Gold antifade reagent with DAPI (Invitrogen). Immunofluorescent observation was performed with a fluorescence microscope (BZ-9000; Keyence) fitted with a ×40 objective lens. Statistical analysis All values are shown as means + standard deviation (SD). Statistical analysis was performed by analysis of variance (ANOVA) on the SAS statistical analysis software package (SAS Institute Japan, Tokyo, Japan). The significance of differences was determined using unpaired t-test, Wilcoxon test, Dunnett’s multiple comparison test, and Tukey’s test. For all statistical analyses, p
Original Article Vitamin D receptor signaling enhances locomotive ability in mice† Sadaoki Sakai, Miho Suzuki, Yoshihito Tashiro, Keisuke Tanaka, Satoshi Takeda, Ken Aizawa, Michinori Hirata, Kenji Yogo, and Koichi Endo
Product Research Department, Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Shizuoka, Japan Corresponding Author Koichi Endo, Ph.D., Product Research Department Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan TEL: +81-550- 87-6375; FAX: +81-550-87-6782; E-mail address: [email protected] Conflict of Interest All authors are employees of Chugai Pharmaceutical Co., Ltd.
†
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jbmr.2317]
Initial Date Submitted October 17, 2013; Date Revision Submitted June 6, 2014; Date Final Disposition Set July 14, 2014
Journal of Bone and Mineral Research © 2014 American Society for Bone and Mineral Research DOI 10.1002/jbmr.2317
1
Accepted Article
Abstract
Bone fractures markedly reduce quality of life and life expectancy in elderly people. Although osteoporosis increases bone fragility, fractures frequently occur in patients with normal bone mineral density. Because most fractures occur on falling, preventing falls is another focus for reducing bone fractures. In this study, we investigated the role of vitamin D receptor (VDR) signaling in locomotive ability. In the rotarod test, physical exercise enhanced locomotive ability of WT mice by 1.6-fold, whereas exercise did not enhance locomotive ability of VDR KO mice. Compared to WT mice, VDR KO mice had smaller peripheral nerve axonal diameter and disordered AChR morphology on the extensor digitorum longus muscle. Eldecalcitol (ED-71, ELD), an analog of 1,25(OH)2D3, administered to rotarod-trained C57BL/6 mice enhanced locomotor performance compared to vehicle-treated non-trained mice. The Area of AChR cluster on the extensor digitorum longus was greater in ELD-treated mice than in vehicle-treated mice. ELD and 1,25(OH)2D3 enhanced expression of IGF-1,
myelin basic protein, and VDR in rat primary Schwann cells. VDR signaling regulates neuromuscular maintenance and enhances locomotive ability following physical exercise. Further investigation is required, but Schwann cells and the neuromuscular junction are targets of vitamin D3 signaling in locomotive ability.
2
Accepted Article
Introduction Osteoporosis is a disease characterized by low bone density and microarchitectural deterioration of bone tissue, leading to increased bone fragility and a consequent increase in fracture risk (1). Because bone fractures markedly reduce quality of life and life expectancy (2, 3), one goal of osteoporosis treatment is prevention of bone fractures. Bone is a dynamic tissue that constantly undergoes remodeling, and bone mineral density (BMD) is used as an index of osteoporosis. Most osteoporosis treatments increase BMD by inhibiting bone resorption. However, although BMD is a key factor related to bone strength, bone fractures in the elderly are common even among those with normal BMD. This fact suggests that correction of BMD is in itself not sufficient to prevent bone fractures. Besides BMD, the factor correlating most highly with fracture incidence is the risk of falling, because almost all fractures are a result of falls. Declining locomotive ability is observed in elderly people, and it is reported that a person’s tendency to fall becomes an important predictor of bone fractures later in life (4). Therefore, it is preferable that a drug for the treatment of osteoporosis not only increase bone strength but also act to prevent falls. In 2011, eldecalcitol (ELD) [ED-71; 1α,25-dihydroxy-2β-(3-hydroxypropyloxy) vitamin D3], a
1,25(OH)2D3 analog, was approved for the treatment of osteoporosis in Japan. ELD has a greater activity than alfacalcidol (ALF), a prodrug of 1,25(OH)2D3, in suppressing bone resorption as well as increasing
BMD (5). In a clinical trial, ELD had superior effects over ALF in preventing vertebral and wrist fractures in osteoporotic patients with sufficient vitamin D supply (6). Although the incidence of falls was not monitored in that study, it seems that almost all wrist fractures occur after falls (7). A correlation between vitamin D supplementation and reduced risk of falls has been reported (8, 9, 10, 11). This led to the hypothesis that ELD may have some effect on reducing incidences of falls. In this study, we investigated the relationship between vitamin D3 signaling and locomotive ability
in mice on a rotarod treadmill. In addition, we investigated the role of 1,25(OH)2D3 and ELD in peripheral nerves and the neuromuscular junction which are known to decline in function with senescence. 3
Accepted Article
Materials and Methods Animals Vitamin D receptor knockout (VDR KO) mice were constructed by Dr. Kato (12) and maintained at Chugai Pharmaceutical Co., Ltd. The mice were reared under standard laboratory conditions at 20–26°C, 35–75% relative humidity, and a 12 h light:12 h dark cycle. Animals were given free access to tap water and standard rodent chow (CE-2; CLEA Japan, Tokyo Japan) for WT mice and modified CE-2 supplemented with 20% lactose, 2% calcium, and 1.25% phosphate for VDR KO mice. Male WT and VDR KO mice began rotarod training at 32 weeks old. The ELD treatment study used 9-week-old male C57BL/6 mice (Charles River Laboratories, Yokohama Japan) reared under the same conditions as for WT mice.
The experimental protocol was approved by the Institutional Animal Care and Use Committee of Chugai Pharmaceutical Co., Ltd. Rotarod exercise and locomotive ability assessment All mice were trained on a 30-mm diameter rotarod treadmill (MK-670; Muromachi Kikai, Tokyo Japan) at 15 rpm for 5 minutes, 3 times a day 4 days a week. To calculate the effect of 1,25(OH)2D3 analog treatment, ELD was administered orally to male C57BL/6 mce in short-chain fatty acid vehicle at a dose of 0.05 μg/kg/day 4 days a week for 2 weeks together with rotarod training. After the training
period, motor coordination was measured with an accelerating rotarod test to assess locomotive ability. Mice were placed individually on the rotating drum and the drum was accelerated from 3 to 30 rpm in 2 min and then kept at 30 rpm for 1 min. The time at which the mouse fell off the drum was recorded. The mice that did not fall from the drum in the 3 min were given a score of 180 seconds. The test was performed 2 times for practice and a third time to collect data for analysis. Three sequential measurements were performed at 1-min intervals. Histological analysis of mouse sciatic nerve and neuromuscular junction Sciatic nerves from WT and VDR KO mice were fixed in 2.5% glutaraldehyde. Toluidine blue staining and electron microscopy imaging were performed at the Bozo Research Center (Tokyo Japan). The
4
Accepted Article
diameters of all axons in cross sections of toluidine-blue–stained sciatic nerves were calculated by using a fluorescence microscope (BZ-9000; Keyence, Osaka Japan). Extensor digitorum longus muscles from each group were fixed with 10% formalin and stained with rhodamine-conjugated α-bungarotoxin (Invitrogen, CA, USA). Muscle fibers were carefully disentangled using forceps. AChR clusters were observed under the fluorescence microscope (BZ-9000; Keyence) and the area of AChR cluster was calculated using BZ Analyzer software (Keyence). The individual area of AChR clusters from 3 mice of each group were analyzed. Analysis of gene expression in rat nerve tissues and primary Schwann cells Real-time PCR analysis was performed on cDNA from the cerebellum, hippocampus, and sciatic nerves from old female rats. The specimens were immediately conserved in RNAlater (Qiagen, Hilden Germany) and stored at −80°C until mRNA extraction. mRNA was extracted using an RNeasy lipid tissue kit (Qiagen) and reverse transcribed using a Omniscript (Qiagen) according to manufacturer’s protocol. mRNA from rat primary Schwann cells was extracted using RNeasy (Qiagen). Real-time PCR analysis was performed with a 7500 Fast Real-Time PCR System (Applied Biosystems, CA, USA) using TaqMan probes Rn00566976_m1 for VDR, Rn00710306_m1 for IGF-1, Rn01399613_m1 for MBP, and 4352338E-0608007 for GAPD (Applied Biosystems). Measurement of serum calcium concentration Mouse serum was separated from whole blood, and serum calcium concentration was measured by using a 7170S Automatic Analyzer (Hitachi High-Technologies, Tokyo, Japan). Primary Schwann cell culture Rat primary Schwann cell culture was performed according to the methods of a previous report (13). Briefly, sciatic nerves were resected from 3-day-old neonatal rats and digested with 3 mg/mL collagenase (Wako Pure Chemical Industries, Osaka, Japan) for 15 min at 37°C, followed by digestion with 3 mg/mL collagenase and 25 mg/mL trypsin (Sigma Aldrich, MO, USA) for 15 min at 37°C. Dissociated sciatic nerves were cultured in 10% FBS DMEM with 10 μM of cytosine β-D
arabinofuranoside (AraC) for 3 days to remove mesenchymal cells. For immunostaining, cells were
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Accepted Article
cultured on cover glass for 2 days and fixed with 4% paraformaldehyde. Fixed rat Schwann cells were stained with polyclonal rabbit anti S-100 antibody (DakoCytomation, Glostrup, Denmark) and visualized with DyLight 488 anti-rabbit IgG (H+L) (Vector Laboratories, CA, USA). Nuclei were stained with Prolong Gold antifade reagent with DAPI (Invitrogen). Immunofluorescent observation was performed with a fluorescence microscope (BZ-9000; Keyence) fitted with a ×40 objective lens. Statistical analysis All values are shown as means + standard deviation (SD). Statistical analysis was performed by analysis of variance (ANOVA) on the SAS statistical analysis software package (SAS Institute Japan, Tokyo, Japan). The significance of differences was determined using unpaired t-test, Wilcoxon test, Dunnett’s multiple comparison test, and Tukey’s test. For all statistical analyses, p
