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Endocrinology Vol. 144, No. 12 5138-5144
Copyright © 2003 by The Endocrine Society

Deletion of Vitamin D Receptor Gene in Mice Results in Abnormal Skeletal Muscle Development with Deregulated Expression of Myoregulatory Transcription Factors

Itsuro Endo, Daisuke Inoue, Takao Mitsui, Yoshifumi Umaki, Masashi Akaike, Tatsuya Yoshizawa, Shigeaki Kato and Toshio Matsumoto

Department of Medicine and Bioregulatory Sciences (I.E., D.I., T.Mi., Y.U., M.A., T.Ma.), University of Tokushima Graduate School of Medicine, Tokushima 770-8503, Japan; and Institute of Molecular and Cellular Biosciences (T.Y., S.K.), University of Tokyo, Tokyo 113-0032, Japan

Address all correspondence and requests for reprints to: Daisuke Inoue, M.D., Department of Medicine and Bioregulatory Sciences, University of Tokushima Graduate School of Medicine, 3-18-15 Kuramoto-Cho, Tokushima 770-8503, Japan. E-mail: inoued{at}clin.med.tokushima-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although rachitic/osteomalacic myopathy caused by impaired vitamin D actions has long been described, the molecular pathogenesis remains elusive. To determine physiological roles of vitamin D actions through vitamin D receptor (VDR) in skeletal muscle development, we examined skeletal muscle in VDR gene deleted (VDR -/-) mice, an animal model of vitamin D-dependent rickets type II, for morphological changes and expression of myoregulatory transcription factors and myosin heavy chain isoforms. We found that each muscle fiber was small and variable in size in hindlimb skeletal muscle from VDR -/- mice, although overall myocyte differentiation occurred normally. These abnormalities were independent of secondary metabolic changes such as hypocalcemia and hypophosphatemia, and were accompanied by aberrantly high and persistent expression of myf5, myogenin, E2A, and early myosin heavy chain isoforms, which are normally down-regulated at earlier stages. Moreover, treatment of VDR-positive myoblastic cells with 1,25(OH)2D3 in vitro caused down-regulation of these factors. These results suggest that VDR plays a physiological role in skeletal muscle development, participating in temporally strict down-regulation of myoregulatory transcription factors. The present study can form a molecular basis of VDR actions on muscle and should help further establish the physiological roles of VDR in muscle development as well as pharmacological effects of vitamin D on muscle functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ACTIVE FORM of vitamin D, 1{alpha},25-dihydroxyvitamin D [1,25(OH)2D], is a major calcium-regulating hormone that is indispensable for maintenance of calcium and bone homeostasis and acts through binding to the vitamin D receptor (VDR) that belongs to the nuclear receptor superfamily. Various disorders with impaired vitamin D actions, including vitamin D deficiency, genetic defects in the vitamin D-activating enzyme, 25-hydroxyvitamin D 1{alpha}-hydroxylase, or in the vitamin D receptor (VDR) lead to rickets or osteomalacia characterized by hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and bone abnormalities due to mineralizing defects (1, 2, 3). Moreover, VDR is known to be expressed in a wide spectrum of tissues unrelated to calcium and bone metabolism, and accordingly, vitamin D has been shown to modulate fundamental cellular processes such as proliferation, differentiation, and survival of various cell lineages in vitro (4, 5). However, physiological relevance of such vitamin D effects in vivo has not yet been established, nor has the role of VDR.

Clinical evidence suggests that vitamin D may play a role in muscle metabolism and function. Progressive weakness and wasting of skeletal muscle have been demonstrated in patients with rickets or osteomalacia (6, 7). In addition, it has been shown that VDR is expressed at particular stages of differentiation from myoblasts to myotubes (8, 9, 10), implying that skeletal muscle may potentially be a physiological target of 1,25(OH)2D. However, the mechanism of rachitic/osteomalacic myopathy is not fully understood, and it is currently unclear whether muscle abnormalities in those patients are a direct consequence of impaired vitamin D actions in muscle or a result of secondary systemic changes such as hypocalcemia, hypophosphatemia, and elevated PTH levels in the circulation.

To address these issues in vivo, we examined morphological abnormalities of skeletal muscle in VDR gene-null mutant (VDR -/-) mice that recapitulated a human disease of vitamin D resistance, vitamin D-dependent rickets type II (11). At the same time, we investigated expression of myogenic regulatory factors such as Myf5, myogenin, MyoD, MRF4, and E2A (12, 13) that play critical roles in myoblast differentiation and skeletal muscle development. We also examined myosin heavy chain (MHC) isoforms including embryonic, neonatal, and adult fast types (14) as differentiation markers that are expressed in a stage-specific manner during muscle development. In addition, we examined 1,25(OH)2D effects on expression of these genes by a mouse myoblast cell line, C2C12, to analyze direct vitamin D actions on muscle cells in vitro. We hereby present evidence that VDR plays a pivotal role in the maintenance of homeostasis in fully differentiated skeletal muscle cells, supporting our hypothesis that muscle is a direct physiological target of VDR-dependent vitamin D actions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and cell cultures
Generation of VDR gene deleted mice has been described (11). C2C12, a mouse myoblast cell line, was purchased from Riken cell bank (Tsukuba, Japan) and maintained in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin (Life Technologies, Rockville, MD). For experiments, 80% confluent C2C12 cells were treated with vehicles alone or 10 nM 1,25(OH)2D3 for 48–96 h and harvested for further analysis.

Chemicals and antibodies
All the reagents were obtained from Sigma (St. Louis, MO) unless otherwise indicated. Antibodies against Myf5, MyoD, MRF4, E2A, Id1, and Id2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), those against myogenin and MHC of embryonic type from American Research Products, Inc. (Belmont, MA), and those against MHC of neonatal and adult fast type from Medac Diagnostika (Hamburg, Germany).

Histological analysis
Samples were isolated from hindlimb skeletal muscle of 3- and 8-wk-old VDR knockout (-/-) mice and wild-type control littermates, rapidly frozen in liquid nitrogen-cooled isopentane (2-methylbutane), sectioned, and stored in liquid nitrogen. Muscle tissue sections were subjected to hematoxylin/eosin staining as follows. Serial sections of frozen muscle with 6-µm thickness were first incubated with Mayer’s hematoxylin solution for 5 min, washed in distilled deionized water, and then incubated with 0.5% eosin solution for 3 min. Sections were washed three times in distilled deionized water and once in ethanol, dehydrated, and mounted. Diameters of muscle fibers were measured in photomicrographs of hematoxylin/eosin-stained muscle tissue sections.

Immunostaining
Serial sections of frozen muscle with 6-µm thickness were fixed for 20 min with PBS containing 4% paraformaldehyde and first incubated with a primary antibody at room temperature for 1 h. Sections were washed three times in PBS, incubated with biotin-conjugated secondary antibodies for 1 h, and then with ABC solution (Vector Laboratories, Burlingame, CA) diluted 100-fold in PBS. For detection, the samples were incubated for 5–20 min with 0.5 mg/ml diaminobenzidine solution or p-nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution (Life Technologies, Rockville, MD), washed twice in distilled deionized water, mounted, and observed under a microscope.

RT-PCR analysis
Total RNA from skeletal muscle tissues and C2C12 cells were isolated by using RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) or TRIzol reagent (Invitrogen, Carlsbad, CA). One microgram total RNA was reverse-transcribed by incubating in a 20-µl reaction containing random primers (Promega; 0.5 mg/ml, 2 µl), reverse transcriptase buffer (Promega; 2 µl), deoxynucleoside triphosphates (2.5 mM each), 20 U RNase inhibitor (Promega), and 20 U reverse transcriptase (Promega) for 10 min at room temperature, 60 min at 42 C, and 5 min at 95 C. PCR was performed using various sets of primers shown in Table 1Go. One microliter of 20 µl reverse transcription reactions was denatured for 2 min at 95 C, followed by 28–35 cycles (except for 23 cycles with glyceraldehyde-3-phosphate dehydrogenase) of amplification: 2 min at 95 C, 30 sec at 57–61 C, and 30 sec at 72 C. PCR products were electrophoretically separated on 2% agarose gels and visualized with ethidium bromide staining.


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  		TABLE 1. List of PCR primers used in this study

 
Northern blot analysis
Total RNA (20 µg) was separated on a 1% agarose gel containing 6% formaldehyde and transferred to HYBOND+ nylon membrane (Amersham, Little Chalfont, Buckinghamshire, UK) by capillary action in 20x sodium chloride-sodium citrate (SSC) buffer (3 M NaCl, 0.3 M sodium citrate, pH 7.0). RNA was cross-linked to the membrane using the UV Cross-Linker (model CX-2000, UVP, Upland, CA). An equal amount of RNA loading and transfer was confirmed by ethidium bromide staining and UV visualization of ribosomal RNAs (data not shown). Hybridization was performed with a nonisotopic digoxigenin labeling system using DIG PCR Probe Synthesis Kit and DIG Easy Hyb (Roche Diagnostics, Indianapolis, IN). Briefly, the membranes were first prehybridized in the DIG Easy Hyb buffer for 30 min at 42 C, then hybridized in DIG Easy Hyb buffer with an appropriate probe generated by DIG PCR Probe Synthesis Kit for 16 h at 42 C, washed twice in 2x SSC/0.1% sodium dodecyl sulfate at room temperature for 5 min and twice in 0.2% SSC/0.1% sodium dodecyl sulfate at 50 C for 15 min, and exposed to films. PCR primers used to generate cDNA probes for Northern blot analysis were as follows: 5'-gcatgcaaggtgtgtaagaggaag-3' and 5'-ggctgttttctggacatcaggaca-3' for myogenin (593 base); 5'-aagagaggtatcctgaccctgaag-3' and 5'-cttgatcttcatggtgctaggagc-3' for ß-actin (801 base); and 5'-aaccaagctttcgagacgctcaag-3' and 5'-aaaagaacaggcagaggagaaccc-3' for Myf5 (664 base). The expected size of the obtained cDNA probes is shown in parentheses. In some experiments, poly A+ RNA was obtained with PolyATract mRNA Isolation systems (Promega) following the manufacturer’s instruction and analyzed by Northern blot analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Abnormal skeletal muscle development in VDR -/- mice
To test a hypothesis that VDR has a physiological role in skeletal muscle development, we first examined skeletal muscle tissues from VDR -/- mice for morphological abnormalities. As previously described (11), the VDR -/- mice grow normally until weaning and thereafter develop various metabolic abnormalities including hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and bone deformity as typical features of rickets. At the age of 3 wk, there were no significant differences between VDR-/- and VDR+/+ mice in body weight or serum concentrations of calcium, phosphate, alkaline phosphatase, 25(OH)D, 24,25(OH)D or 1,25(OH)2D. To exclude any deleterious effects of such secondary systemic metabolic changes on muscle, we analyzed 3-wk-old mice just before weaning that showed no apparent biochemical or morphological abnormalities. As shown in Fig. 1Go, each muscle fiber obtained from quadriceps femoris muscle of VDR -/- mice (Fig. 1AGo) appeared smaller than that of wild-type (VDR+/+) mice (Fig. 1BGo) at 3 wk. Quantitative analysis showed that skeletal muscle cell diameters in VDR -/- mice at 3 wk were significantly decreased by approximately 20% on the average and appeared to be more widely distributed compared with those in wild-type mice (Fig. 2Go). The morphological changes were more prominent in 8-wk-old VDR -/- mice (Fig. 1CGo) compared with VDR+/+ controls of the same age (Fig. 1DGo), suggesting either a progressive nature of the abnormalities caused by the absence of VDR or additive effects of systemic metabolic changes already present at this age. Neither degenerative nor necrotic changes were observed in VDR -/- skeletal muscle. Similar results were obtained with biceps femoris, medial gastrocnemius, anterior tibial, and soleus muscles, indicating that the muscle abnormalities in VDR -/- mice occurred diffusely without any preference to type I or type II fibers. These results demonstrate that VDR is involved in physiological regulation of skeletal muscle development. Our observations further suggested that although overall differentiation steps into myocytes occurred normally, the absence of VDR caused abnormalities probably in late stages of myocyte maturation and/or in metabolism of mature myocytes.



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  		FIG. 1. Morphological abnormalities of skeletal muscle tissue from VDR -/- mice. Three- or 8-wk-old VDR -/- and +/+ (wild-type littermate) mice were euthanized, and quadriceps femoris muscle tissues were obtained. Fresh-frozen sections were stained with hematoxylin/eosin as described in Materials and Methods and observed under microscope. Scale bar, 20 µm. A, Three-week-old VDR -/- mice; B, 3-wk-old VDR +/+ mice; C, 8-wk-old VDR -/- mice; D, 8-wk-old VDR +/+ mice. Similar changes in muscle fiber size were also observed in biceps femoris, medial gastrocnemius, anterior tibial, and soleus muscles (data not shown).

 


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  		FIG. 2. VDR -/- muscle fibers are small and variable in size. Diameter of muscle fibers in 3-wk-old VDR -/- (A) and VDR +/+ (B) mice was measured in microphotograph of the hematoxylin/eosin-stained tissue sections. Two hundred cells were randomly counted, and data were expressed in histogram and as mean size ± SD. *, Significantly different from VDR +/+ wild-type littermates.

 
Deregulated expression of myogenic regulatory factors in VDR -/- mice
To obtain insight into the mechanism of muscle abnormalities observed in VDR -/- mice, we examined expression of myogenic differentiation factors including MyoD family of transcription factors with muscle contractile proteins, i.e. embryonic, neonatal, and adult fast (type II) isoforms of MHC. Immunohistochemical analysis of quadriceps femoris muscle from 3-wk-old mice revealed persistently increased expression of myf5 (Fig. 3AGo), E2A (Fig. 3BGo), and myogenin (Fig. 3CGo), all of which were minimally expressed in muscle from VDR +/+ mice at this age (Fig. 3Go, F–H). No apparent differences were observed in expression of MyoD (Fig. 3Go, D and I) and MRF4 (Fig. 3Go, E and J). Consistent with the deregulated expression of myogenic transcription factors that control muscle phenotype, we also observed aberrantly increased expression of embryonic (Fig. 4AGo) and neonatal type MHC (Fig. 4BGo) in the cytoplasm of small muscle fibers of quadriceps femoris muscle from 3-wk-old VDR -/- mice, whereas type II (adult fast) MHC expression in VDR -/- muscle was the same as VDR +/+ controls (Fig. 4Go, C and F). At the age of 8 wk, although the embryonic MHC had disappeared, persistent expression of neonatal MHC was still detectable in VDR -/- mice (data not shown). There were no differences in expression levels of Id1 and Id2, known targets of vitamin D (15) in either 3- or 8-wk-old mice (data not shown). All the above findings were confirmed at the mRNA level as shown in Fig. 5Go: expression of myf5, myogenin, E12 and E47, both of which are produced from the same E2A gene, and neonatal MHC mRNA was higher in VDR -/- mice than that of VDR +/+ mice at 3 and 8 wk. VDR mRNA was only detectable in 3-wk-old wild-type muscle under our experimental conditions. These results are consistent with the notion that the absence of VDR disturbs the coordinate pattern of expression of myogenic transcription factors during myocyte development, causing altered levels of differentiation-associated, lineage-specific gene expression and thereby morphological abnormalities. The possibility that these alterations were secondary effects of systemic changes appeared unlikely, because abnormalities in both morphology and MyoD family expression were observed already in 3-wk-old VDR -/- mice and also in mice fed with high-calcium diet to rescue the phenotype of rickets that showed normal circulating levels of calcium, phosphate, and PTH (Ref. 11 and data not shown).



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  		FIG. 3. Immunohistochemical analysis of MyoD family transcription factors in skeletal muscle tissues of 3-wk-old mice. Quadriceps femoris muscle tissue sections of 3-wk-old VDR -/- (A–E) and VDR +/+ (F–J) mice were analyzed by immunohistochemistry for expression of Myf5 (A and F), E2A (B and G), myogenin (C and H), MyoD (D and I), and MRF4 (E and J) as described in Materials and Methods. Expression of myf5, E2A, and myogenin was only detectable in VDR -/- muscles, which was mostly localized in nuclear and perinuclear regions. Virtually the same results were obtained in biceps femoris, medial gastrocnemius, anterior tibial, and soleus muscles (data not shown).

 


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  		FIG. 4. Immunohistochemical analysis of MHC isoforms in skeletal muscle tissues of 3-wk-old mice. Quadriceps femoris muscle tissue sections of 3-wk-old VDR -/- (A–C) and VDR +/+ (D–F) mice were analyzed by immunohistochemistry for expression of MHC of embryonic type (A and D), neonatal type (B and E), and adult fast type (type II) (C and F), as described in Materials and Methods. In the lower half of panels A and D, nonspecific staining of the intercellular space without specific primary antibodies (2° only) is shown. Also note that in panels C and F, some type I fibers scattered in the field are devoid of staining in contrast to the diffuse cytoplasmic staining of surrounding type II fibers. Scale bar, 40 µm.

 


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  		FIG. 5. Expression of MyoD family and MHC mRNA in skeletal muscle from VDR -/- and +/+ mice. Expression of myf5, myogenin, E2A, neonatal type MHC, and VDR mRNA in hindlimb skeletal muscle was analyzed by RT-PCR in 3- and 8-wk-old VDR -/- and +/+ mice as described in Materials and Methods. For each PCR, a negative control without reverse transcriptase is also shown.

 
1,25(OH)2D3 down-regulates myf5 and myogenin expression by myoblasts in vitro
Our results suggest that temporally strict down-regulation of myogenic differentiation factors requires the presence of VDR. Therefore, we finally examined whether or not 1,25(OH)2D3 was able to directly down-regulate MyoD family gene expression in myocyte-lineage cells in vitro. C2C12 myoblasts were grown to 80% confluence and then treated with 10 nM 1,25(OH)2D3 or a vehicle alone in the presence of 10% charcoal-treated fetal bovine serum for 48–96 h. During this phase of initial differentiation, less than 5% of myotubes appeared (data not shown). There were no apparent differences in the overall differentiational process between vehicle-treated and 1,25(OH)2D3-treated cells. As shown in Fig. 6AGo, VDR mRNA was found to be expressed at constant levels throughout the experimental period. In control cells, myf5 was already expressed in growing cells, and the level of expression stayed the same. In contrast, expression of myogenin and neonatal type MHC showed a gradual increase in a time-dependent manner. Treatment with 10 nM 1,25(OH)2D3 for 48 h caused a decrease in the steady-state levels of myf5, myogenin, and neonatal MHC, and the 1,25(OH)2D3 effects lasted up to 96 h (Fig. 6AGo). The effects on myogenin and myf5 expression were also confirmed quantitatively by Northern blot analysis (Fig. 6Go, B and C). Thus, these in vitro results using myoblast cultures have in large part recapitulated our in vivo findings and are in agreement with our hypothesis that 1,25(OH)2D participates in physiological regulation of muscle development, particularly playing a role in temporally strict down-regulation of some myogenic differentiation factors through VDR.



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  		FIG. 6. Down-regulation of myf5, myogenin, and MHC neonatal type mRNA by 1,25D3 in C2C12 myoblastic cell line. A, 80% confluent C2C12 cells were treated with a vehicle alone or 10 nM 1,25D3 for indicated times and analyzed for mRNA expression of myf5, myogenin, MHC neonatal type, VDR and glyceraldehyde-3-phosphate dehydrogenase by RT-PCR as described in Materials and Methods. For each PCR, a negative control without reverse transcriptase is also shown. B, The same RNA samples as panel A were analyzed for myogenin and actin mRNA expression by Northern blot analysis as described under Materials and Methods to show quantitative difference. C, Poly A+ RNA was purified as described under Materials and Methods and analyzed for expression of myf5, myogenin, and actin mRNA by Northern blot analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Defects in VDR-dependent vitamin D actions cause rickets or osteomalacia. Although rachitic or osteomalacic myopathy has long been described, the molecular pathogenesis remains elusive. One of the central questions to be answered is whether the myopathy is caused by impairment of direct VDR-dependent actions of active vitamin D on muscle or by secondary metabolic changes including hypocalcemia, hypophosphatemia, and hyperparathyroidism. Some clinical studies have indicated that the extent of hypocalcemia and/or hypophosphatemia does not correlate well with the severity of myopathy and that correction of hypocalcemia does not lead to a cure of the muscle symptoms (16), which supports an involvement of direct VDR actions. However, others have demonstrated that PTH excess leads to similar muscle atrophy and weakness causing increased intracellular calcium (17) and impaired production of contractile proteins (18). Efforts to obtain conclusive results have been hampered by an inability to test direct effects of VDR in human muscles and absence of appropriate animal models of rickets for this purpose.

We have recently generated VDR gene deleted mice as an animal model of type II vitamin D-dependent rickets. VDR -/- mice almost completely recapitulated the human disease and showed most of the characteristic abnormalities including hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, increased serum levels of 1,25D and alkaline phosphatase, decreased 24,25-dihydroxyvitamin D, and osteopathy (11). A unique feature in these model mice is that they grow normally and show no bone or metabolic abnormalities until they are weaned, presumably due to high calcium content or other critical nutrients in the breast milk. In the present study, we took advantage of this feature and were able to demonstrate that the absence of VDR causes muscle abnormality independently of secondary effects of systemic metabolic changes. Three lines of evidence from the present study support physiological roles of direct VDR actions on skeletal muscle: firstly, VDR -/- mice developed apparent morphological abnormalities in skeletal muscle and a deregulated pattern of muscle gene expression before weaning; secondly, the same changes were still observed in older rescued VDR -/- mice fed with high calcium diet; and thirdly, direct negative regulatory effects of 1,25(OH)2D on muscle gene expression were at least in part reproduced in cultured myoblasts in vitro. Thus, our results suggest that the skeletal muscle is a direct physiological target of VDR actions and that the absence of VDR in situ caused muscle abnormalities in VDR -/- mice, although secondary changes such as hypocalcemia, hypophosphatemia, and hyperparathyroidism may contribute in an additive and/or modulatory manner.

As a clue to the mechanism whereby the absence of VDR caused skeletal muscle abnormalities, we found prolonged up-regulation of a certain subset of myogenic regulatory factors: myf5, myogenin, and E2A. The transcription factors of the MyoD family play pivotal roles in muscle cell differentiation. During muscle differentiation, the four members of the family thus far identified, myf5, MyoD, myogenin, and MRF4, show a temporal and sequential pattern of expression that is subject to complex mutual regulation and exhibit distinct but overlapping functions that are not yet completely understood (12, 19). Expression of muscle-specific genes including MHC subtypes is under the control of the MyoD family members (20, 21, 22, 23). Although we currently have no evidence for a direct link between deregulated expression of myogenic transcription factors and the muscle phenotype observed in VDR -/- mice, it is plausible to assume that aberrant up-regulation of myf5, myogenin, and E2A leads to abnormal expression of MHC and muscle atrophy, because it appears that a strictly regulated, coordinate pattern of expression of the MyoD family defines the program of myocyte differentiation and maturation. Such an assumption is further supported by a previous report that transgenic myogenin overexpression in differentiated postmitotic muscle fibers in mice resulted in grossly normal muscle development but higher rates of neonatal mortality, probably due to mildly impaired muscle function (24).

The molecular mechanism by which myogenic transcription factors including myf5, myogenin, and E2A are aberrantly and persistently up-regulated is currently unknown. However, it is of note that, in the course of muscle differentiation, these genes are normally down-regulated during the stages in which VDR is expressed (25). We therefore assume that VDR is involved in transcriptional down-regulation of these genes during the process of physiological muscle differentiation. Our in vitro observations that 1,25(OH)2D was able to down-regulate myf5, myogenin, and neonatal MHC mRNA expression in C2C12 myoblasts further support this idea. However, we have not been able to identify known negative vitamin D response elements (26, 27, 28) in the promoter region of myf5 and myogenin genes. Further functional analysis of the promoters of MyoD family members may elucidate the down-regulatory mechanism of these genes through VDR.

Our findings may be clinically relevant to the musculoskeletal health in the aged, because vitamin D insufficiency has been shown to be associated with lower muscle strength and increased falling tendency in adults. Conversely, supplement of native vitamin D or treatment with active vitamin D has been reported to improve muscle functions and protect from falling events and falling-associated fractures (29, 30, 31, 32, 33). Whether the beneficial effects of vitamin D treatment occur via direct VDR actions on skeletal muscle cells or indirect mechanisms remains unclear. Interestingly, however, abnormal expression of MyoD family members and MHC isoforms has been reported in various models of immobilization and denervation (34, 35, 36, 37, 38). Considering the plasticity and highly adaptive nature of muscle fibers, it is conceivable that reprogramming and adaptations of muscle fibers may occur under various pathological conditions, particularly in elderly patients, and that these processes may be modulated by VDR-dependent vitamin D actions.

In summary, we have shown that VDR gene deleted mice exhibit abnormal skeletal muscle development. These abnormalities occur independently of secondary metabolic changes such as hypocalcemia and hypophosphatemia and are accompanied by deregulated expression of myogenic transcription factors and MHC isoforms. These effects appear to involve direct vitamin D actions on muscle through VDR, because similar effects were reproduced by treatment of VDR-positive myoblastic cells with 1,25(OH)2D in vitro. The present study can form a molecular basis of VDR actions on muscle and should help further establish the physiological roles of VDR in muscle development as well as pharmacological effects of vitamin D on muscle functions.


   Acknowledgments
 
We thank Kiyomi Yoshida for her helpful assistance in preparation of the manuscript.


   Footnotes
 
This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas 12137207 (to T.Ma.) and Grants-in-Aid for Scientific Research (B) (to T.Ma.) from the Ministry of Education, Science, Sports and Culture of Japan; and Fellowships from Japan Intractable Diseases Research Foundation (to D.I.).

Abbreviations: 1,25(OH)2D, 1{alpha},25-Dihydroxyvitamin D; MHC, myosin heavy chain; SSC, sodium chloride-sodium citrate; VDR, vitamin D receptor.

Received April 21, 2003.

Accepted for publication August 7, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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