Review Article Vitamin D Signaling in Myogenesis: Potential for Treatment of...

Review ArticleVitamin D Signaling in Myogenesis Potential forTreatment of Sarcopenia

Akira Wagatsuma1 and Kunihiro Sakuma2

1 Graduate School of Information Science and Technology The University of Tokyo 7-3-1 Hongo Bunkyo-ku Tokyo 113-8656 Japan2 Research Center for Physical Fitness Sports and Health Toyohashi University of Technology 1-1 HibarigaokaTempaku-cho Toyohashi 441-8580 Japan

Correspondence should be addressed to Akira Wagatsuma wagatsuma1969yahoocojp

Received 25 April 2014 Accepted 3 June 2014 Published 30 June 2014

Academic Editor Giuseppe DrsquoAntona

Copyright copy 2014 A Wagatsuma and K Sakuma This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Muscle mass and strength progressively decrease with age which results in a condition known as sarcopenia Sarcopenia wouldlead to physical disability poor quality of life and death Therefore much is expected of an effective intervention for sarcopeniaEpidemiologic clinical and laboratory evidence suggest an effect of vitamin D onmuscle function However the precise molecularand cellular mechanisms remain to be elucidated Recent studies suggest that vitamin D receptor (VDR) might be expressedin muscle fibers and vitamin D signaling via VDR plays a role in the regulation of myoblast proliferation and differentiationUnderstanding how vitamin D signaling contributes to myogenesis will provide a valuable insight into an effective nutritionalstrategy to moderate sarcopenia Here we will summarize the current knowledge about the effect of vitamin D on skeletal muscleand myogenic cells and discuss the potential for treatment of sarcopenia

1 Introduction

Muscle wasting is observed in various disease states in con-ditions of reduced neuromuscular activity with ageing Age-related muscle wasting is referred to as ldquosarcopeniardquo coinedby Irwin H Rosenberg from the Greek words sarx (meaningflesh) and penia (meaning loss) [1 2] There has been noconsensus about definition of sarcopenia suitable for use inresearch and clinical practice [3] Therefore some studies[4 5] suggest a working definition of sarcopenia sarcopeniais a syndrome characterized by progressive and generalizedloss of skeletal muscle mass and strength with a risk ofadverse outcomes such as physical disability poor quality oflife and death Sarcopenia is characterized by the fact thatit progresses very slowly throughout several decades Musclemass fairly consistently decreases at a rate of approximately05ndash1year beginning at 40 years of age [6 7] and the ratedramatically accelerates after the age of 65 years [8] Musclestrength appears to decline more rapidly than muscle massMuscle strength declines at a rate of 3-4 per year in menand 25ndash3 per year in women aged 75 years [9] Although

the precise molecular and cellular mechanisms underlyingage-related loss of muscle mass and strength have remainedunknown [10 11] multiple contributing factors have beenproposed The development and progress of sarcopenia havebeen thought to be mediated by the combination of thesecontributing factors

Based on large-scale studies [12ndash16] on average it isestimated that the prevalence of sarcopenia reaches 5ndash13 inthose aged 60ndash70 years and ranges from 11 to 50 in thoseaged over 80 years [17] In USA in 2000 it was estimated thatdirect healthcare costs related to sarcopenia were $185 billion($108 billion in men $77 billion in women) which repre-sented approximately 15 of total healthcare expendituresfor that year [18] Globally the number of people aged over 60years is 600 million in the year 2000 [19] It is predicted thatpeople aged over 65 years will double by 2020 and will tripleby 2050 [20]Therefore sarcopenia is being recognized as notonly a serious healthcare problem but also a social problemMuch is expected of an effective intervention for sarcopeniaNutritional interventions would be a promising candidate incombating sarcopenia

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 121254 13 pageshttpdxdoiorg1011552014121254

2 BioMed Research International

Epidemiologic clinical and laboratory evidence providean effect of vitamin D onmuscle function Numerous studieshave investigated the effect of vitamin D supplementationon muscle strength and physical performance in elderlypeople However the precise molecular and cellular mecha-nisms remain to be elucidated Immunohistochemical studieshave demonstrated that vitamin D receptor (VDR) mightbe localized in human muscle fibers [21ndash23] with somecontradictions [24 25] In addition recent studies havereported that vitamin D signaling via VDR plays a role in theregulation of myoblast proliferation and differentiation [26ndash32] Understanding how vitamin D signaling contributes tomyogenesis will provide a valuable insight into an effectivenutritional strategy to moderate sarcopenia Here we willsummarize the current knowledge about effect of vitaminD on skeletal muscle and myogenic cells and discuss thepotential for treatment of sarcopenia

2 Vitamin D Signaling Pathways Genomicand Nongenomic Pathways

Vitamin D signaling has been extensively investigated in avariety of cell types During the past two decades consid-erable progress has been made in understanding the actionof 112057225-dihydroxyvitamin D

3[112057225(OH)

2D3] on myogenic

cellsThebiological effect of 112057225(OH)2D3is exerted through

genomic or nongenomic mechanisms (for reviews see [33ndash35]) Better understanding of the molecular and cellularmechanisms of vitamin D action on skeletal muscle willenable us to develop an effective intervention for sarcope-nia We will focus on 112057225(OH)

2D3signaling via VDR in

genomic and nongenomic mechanism related to myogeniccells although rapid alteration in intracellular calcium whichis nongenomically regulated by 112057225(OH)

2D3 has been well

demonstrated both in vivo and in vitro for details excellentreview article is already available on this subject [36]

An active form 112057225(OH)2D3 acts by binding to VDR

[33] The binding affinity of 25(OH)D3for human vitamin

D receptor (VDR) is approximately 500 times less than thatof 112057225(OH)

2D3but the circulating level of 25(OH)D

3is

approximately 1000 times higher than that of 112057225(OH)2D3

[37 38] In genomic mechanism 112057225(OH)2D3binds to

VDR and is transported to the nucleus [35] VDR is het-erodimerized with 9-cis-retinoic acid receptor (RXR) andVDRRXR complex modulates gene expression via bindingto specific target gene promoter regions known as vitaminD response elements (VDREs) to activate or suppress theirexpression [35] In general VDREs possess either a directrepeat of two hexanucleotide half-elements with a spacer ofthree nucleotides (DR3) or an everted repeat of two half-elements with a spacer of six nucleotides (ER6) motif withDR3s being the most common [39] Wang et al [40] investi-gated direct 112057225(OH)

2D3-target genes on a large scale by

using a combined approach of microarray analysis and insilico genome-wide screens for DR3 and ER6-type VDREsMicroarray analyses performed with RNA from humanSCC25 cells treated with 112057225(OH)

2D3and cycloheximide

an inhibitor of protein synthesis revealed 913 regulated

genes [40] Of the 913 genes 734 genes were induced and179 genes were repressed by treatment of 112057225(OH)

2D3

[40] In addition a screening of the mouse genome iden-tified more than 3000 conserved VDREs and 158 humangenes containing conserved elements were 112057225(OH)

2D3-

regulated on microarrays [40] These results support theirbroad physiological actions of 112057225(OH)

2D3in a variety of

cell typesWith respect to several genes related to myogene-

sis we will describe them in more detail For example112057225(OH)

2D3induced expression of the gene encoding

Foxo1 [40] which is a member of the FOXO subfamilyof forkheadwinged helix family of transcription factorsgoverns muscle growth metabolism and myoblast differen-tiation When transfected C2C12 cells with adenoviral vectorencoded a constitutively active Foxo1 mutant they effectivelyblocked myoblast differentiation [47] This was partly res-cued by inhibition of Notch signaling [47] which inhibitsmyoblast differentiation [48] In addition loss of Foxo1function precluded Notch signaling-mediated inhibition ofmyoblast differentiation [47] To elucidate the possible roleof Notch signaling in Foxo1-mediated inhibition of myoblastdifferentiation by combining coculture system transfec-tion assay chromatin immunoprecipitation assay and shortinterfering RNA (siRNA) technology authors showed thatFoxo1 physically and functionally interacted with Notch bypromoting corepressor clearance fromDNA binding proteinCSL [CBF1RBPjkSu(H)Lag-1] leading to inhibition ofmyoblast differentiation through activation of Notch targetgenes [47] Another gene Id (inhibitor of differentiation)gene is also known target of 112057225(OH)

2D3[49] Id mRNA

was constitutively expressed in rat osteoblastic osteosarcomaROS1728 cells and its level was transcriptionally suppressedby 112057225(OH)

2D3[50] 112057225(OH)

2D3exerted its negative

effect on Id1 gene transcription via the 57 bp upstreamresponse sequence (minus1146minus1090) [49] Id proteins (Id1 Id2Id3 and Id4) dimerize and neutralize the transcriptionalactivity of basic helix-loop-helix (bHLH) proteins [51] Ithas been shown that Id inhibits MyoD activity either byforming transcriptionally inactive complexes of MyoD-Idor by forming heterodimers with E-proteins and effectivelyblocking the formation of active MyoDE-protein complexes[52] At this time there are only limited data available onId expression and vitamin D during muscle developmentFor example in VDR knockout mice with abnormal muscledevelopment there were no differences in expression levelsof Id1 and Id2 [46] Therefore we cannot conclude whethervitaminD regulatesmyogenesis bymodulating Id expression

A nongenomic response to 112057225(OH)2D3is character-

ized by a rapid (the seconds to minutes range) activationof signaling cascades and an insensitivity to inhibitors oftranscription and protein synthesis [34] The rapid responseto 112057225(OH)

2D3has been hypothesized to elicit the classic

VDR translocation to the plasma membrane When treatingchick myoblasts with 112057225(OH)

2D3 translocation of VDR

from the nucleus to the plasma membrane rapidly occurredwithin 5min after the addition of 112057225(OH)

2D3[53] This

translocation was blocked by colchicine suggesting the pos-sible role of the intracellular microtubular transport system

BioMed Research International 3

in the distribution of VDR [53] The VDR translocationappears to depend on intact caveolae that are specializedplasmalemmal microdomains originally studied in numer-ous cell types for their involvement in the transcytosis ofmacromolecules [54] Confocal microscopy revealed that112057225(OH)

2D3-induced VDR translocation to the plasma

membrane was abolished by methyl-beta-cyclodextrin areagent to disrupt the caveolae structure [55] Both disrup-tion of caveolae and siRNA-mediated silencing of caveolin-1 suppressed 112057225(OH)

2D3-dependent activation of pro-

tooncogene c-Src (cellular Src) with tyrosine-specific proteinkinase activity [55] Immunocytochemical analysis providedevidence that caveolin-1 colocalized with c-Src near theplasmamembrane under basal conditions [55]When treatedwith 112057225(OH)

2D3 the colocalization of caveolin-1 and c-Src

was disrupted and theywere redistributed into cytoplasm andnucleus [55] On the basis of these results it can be hypoth-esized that (1) interaction caveolin-1c-Src inactivates thekinase under basal conditions and (2) when 112057225(OH)

2D3

stimulates VDR translocation to the plasma membraneit dissociates the caveolin-1c-Src complex allowing c-Srcactivation [55] Non-genomic action of 112057225(OH)

2D3might

be required for a reciprocal interaction between c-Src andcaveoline-1 Besides the classical VDR it has been identifiedas a potential candidate as an alternate membrane-associatedreceptor for 112057225(OH)

2D3 125D

3-MARRS (membrane-

associated rapid response steroid binding) also known asERp57 GRp58 ERp60 and Pdia3 [56] Since 125D

3-MARRS

has been shown to function in various cell types [57] it alsomay potentially mediate vitamin D signaling in myogeniccells

The c-Src tyrosine kinase induced by 112057225(OH)2D3is

required for activation of mitogen-activated protein kinases(MAPKs) ERK12 (extracellular signal-regulated kinase12) [58] and p38 [59] 112057225(OH)

2D3rapidly promoted

phosphorylation of ERK12 through c-Src activation [58]Raf-1RasMEK (MAPKERK kinase) and PKC120572 (proteinkinase C alpha) [60] In addition to ERK12 activation112057225(OH)

2D3rapidly stimulated MKK3MKK6 (mitogen-

activated protein kinase kinases 36)p38 MAPK through c-Src activation [59] Although another MAPK family mem-ber JNK12 (c-Jun NH2-terminal kinase 12) was alsoactivated by 112057225(OH)

2D3[59] an upstream mediator of

112057225(OH)2D3-dependent JNK12 activation was character-

ized less than that of ERK12 and p38 The molecular linksbetween JNK and c-Src have been shown in Drosophilamelanogaster The JNK homolog Basket (Bsk) is requiredfor epidermal closure [61] Src42A a Drosophila c-Src pro-tooncogene homolog functions in epidermal closure duringboth embryogenesis and metamorphosis [61] The severity ofthe epidermal closure defect in the Src42A mutant dependedon the Bsk activity These results suggest the possibility thatJNK activation in mammals may also be required for Srctyrosine kinase activity These MAPK signaling pathwayshave been shown to contribute to myogenesis [62ndash65] Forexample inactivation of the Raf-1MEK12ERK12 pathwayin MM14 cells through the overexpression of dominant neg-ative mutants of Raf-1 blocked ERK12 activity and preventedmyoblast proliferation [62] Pharmacological blockade of

p38120572120573 kinases by SB203580 inhibited myoblast differen-tiation [63ndash65] JNK was involved in regulating myostatinsignaling [66] which is known as a member of tumor growthfactor120573 family and functions as a negative regulator ofmusclegrowth [67] MAPK signaling pathways function at differentstages of myogenesis

Apart from MAPKs PI3K (phosphatidyl inositol 3-kinase)Akt signaling pathway which is essential for ini-tiation of myoblast differentiation [68] also seems to beactivated by 112057225(OH)

2D3 After exposure to 112057225(OH)

2D3

Akt phosphorylation was enhanced through PI3K in C2C12cells [68] Intriguingly suppression of c-Src activity by PP2a specific inhibitor for all members of the Src family andknockdown of c-Src expression by siRNA decreased Aktphosphorylation in 112057225(OH)

2D3-treated C2C12 cells [28]

In addition when treating C2C12 cells with 112057225(OH)2D3

in the presence of U0126 or SB203580 to inhibit ERK12and p38 MAPK respectively SB203580 but not U0126markedly blocked both basal and 112057225(OH)

2D3-inducedAkt

phosphorylation These results suggest that 112057225(OH)2D3-

induced Akt phosphorylation may occur through c-Src andp38 MAPK [28] Taken together 112057225(OH)

2D3can simul-

taneously activate multiple signaling pathways in myogeniccells but their relative contribution to myogenesis remains tobe established

3 Effects of Ageing on SerumConcentration of Vitamin D MuscleMorphology and Muscle Fiber Type

VitaminDstatusvarieswithage[69] Serumlevelsof 25(OH)D3

are qualitatively categorized as deficiency (lt20 ngL orlt50 nM) insufficiency (21ndash29 ngL or 50ndash75 nM) and nor-mal (30 ngL or gt75 nM) [70] van der Wielen et al [69]measured wintertime serum 25(OH)D

3concentrations in

824 elderly people from 11 European countries [69] Theyreported that 36 of men and 47 of women had 25(OH)D

3

concentrations below 30 nM [69] Vitamin D deficiency inelderly is thought to occur mainly due to restricted sunlightexposure reduced dietary vitamin D intake and decreasedcapacity of the skin to produce vitamin D [69] MacLaughlinand Holick [71] examined the effects of ageing on thecapacity of the skin to produce previtamin D3 in the skinby comparing young subjects (8 and 18 years old) with agedsubjects (77 and 82 years old) They showed that ageingdecreased the capacity less than half of young subjects [71]suggesting that elderly people are potentially at risk forvitamin D insufficiencydeficiency

Vitamin D deficiency appears to be associated withchanges in muscle morphology For example patients withosteomalacic myopathy associated with vitamin D deficiencyshow degenerative changes such as opaque fibers ghost-like necrotic fibers regenerating fibers enlarged interfibrillarspaces infiltration of fat fibrosis glycogen granules andtype II muscle fiber atrophy [72] As is the case withvitamin D-deficient patients it is well known that elderlypeople show aberrant muscle morphology Scelsi et al [73]performed histochemical and ultrastructure analysis using


biopsies taken from the vastus lateralis of healthy sedentarymen and women aged 65ndash89 years They observed myofib-rillar disorganization streaming of Z-line rod formationintracellular lipid droplets lysosomes and type II musclefiber atrophy [73] The very elderly people had ldquoflattenedrdquo orldquocrushedrdquo shaped muscle fibers whereas the young peoplehad mature-appearing polygonal muscle fibers [74] Theseaberrant changes were much more pronounced in the typeII muscle fibers than in type I muscle fibers [74] Althoughthe precise mechanisms remain to be elucidated it can bespeculated that specific type II muscle fiber atrophy withageing may be associated with a muscle fiber type-specificreduction in satellite cell content Satellite cells are essentialfor normal muscle growth [75] Verdijk et al [76] examinedwhether satellite cells could specifically decrease in type IImuscle fibers in the elderly people Biopsies were taken fromthe vastus lateralis of elderly (average age 76 years) andyoung (average age 20 years) healthy males [76] They foundsignificant reduction in the proportion and mean cross-sectional area of the type II muscle fibers and the numberof satellite cells per type II muscle fiber in elderly subjectscompared to young subjects [76] This study is the firstto show type II muscle fiber atrophy in elderly people tobe associated with a muscle fiber type-specific decline insatellite cell content It remains unknown whether vitaminD supplementation specifically attenuates atrophy of typeII muscle fibers with recruitment of satellite cell Whethervitamin D has positive effects on myoblast proliferation anddifferentiation is currently under debate Recent studies [27]suggest that vitaminD treatment enhances fast type (type IIa)MyHC expression in fully differentiated C2C12 myotubesType II muscle fibers contain type IIa MyHC [77] Thereforevitamin D could potentially contribute to the changes inphenotype of existing muscle fibers andor the maintenanceof type II muscle fibers

4 Effects of Ageing on Expression of VDR

Bischoff-Ferrari et al [22] investigated the effect of ageingon VDR expression in human skeletal muscle Biopsies weretaken from the gluteus medius of 20 female patients under-going total hip arthroplasty (average age 716 years) andfrom the transversospinalis muscle of 12 female patients withspinal operations (average age 552 years) Immunohisto-chemical analysis revealed that the number of VDR-positivemyonuclei decreased with ageing [22] Importantly VDRexpression was not affected by 25(OH)D

3or 112057225(OH)

2D3

levels [22] Buitrago et al [78] showed that silencing ofVDR expression in C2C12 myoblasts suppressed p38 MAPKphosphorylation and decreased ERK12 activation inducedby 112057225(OH)

2D3 Tanaka et al [31] demonstrated that

knockdown of VDR expression resulted in downregulationof MyHC mRNA in differentiating C2C12 myoblasts whentreated with 112057225(OH)

2D3 Therefore it is possible that

decreased expression of VDR observed in elderly peoplemight reduce the functional response of the muscle fibers to112057225(OH)

2D3

5 Effects of Vitamin D Supplementation onMuscle Injury

The regenerative potential of skeletal muscle decreases withage [79ndash81] Satellite cells are absolutely required for muscleregeneration [82] Satellite cells are defined anatomicallyby their position beneath the basal lamina and adhered tomuscle fibers [83] They traditionally considered as a pop-ulation of skeletal muscle-specific committed progenitorsplay a crucial role in the postnatal maintenance repair andregeneration [75] Under normal physiological conditionsthey remain in a quiescent and undifferentiated state [7584] However when skeletal muscle is damaged by unaccus-tomed exercise or mechanical trauma they are activated toproliferate differentiate and fuse with the already existingmuscle fibers or fuse to form new muscle fibers [75 84] Fewstudies have examined the effects of vitamin D treatment onmuscle injury Stratos et al [85] investigated whether system-ically applied vitamin D could restore muscle function andmorphology after trauma Rats were injected subcutaneouslywith 7-dehydrocholesterol (332000 IUkg) immediately aftercrush injury and muscle samples were collected at days 1 414 and 42 after injury [85] Vitamin D treatment increasedcell proliferation and inhibited occurrence of apoptosis atday 4 compared to control rats [85] In addition a fasterrecovery of contraction forces was observed at day 42 invitamin D-treatment group compared to control group [85]Notably the number of satellite cells was not influenced byvitamin D [85] suggesting the possibility that vitamin Dsupplementation has relatively little effect on satellite cellfunction in vivo It is necessary to scrutinize thoroughlyefficacy duration optimal dose and side effects in relationto vitamin D treatment Srikuea et al [29] demonstratedthat VDR was highly expressed in the nuclei of regeneratingmuscle fibers indicating a potential role for vitamin D inmuscle regeneration following injury Relationship of vitaminD signaling and myogenesis will be discussed below inSection 10

6 Vitamin D and Type 2 Diabetes MellitusAlthough the incidence of type 2 diabetes mellitus increaseswith age [86] the precise underlyingmechanisms are still notfully understood Skeletal muscle is the primary target forinsulin action and glucose disposal Therefore elderly peoplewith excessive loss of muscle mass are at risk for developmentof type 2 diabetes mellitus [87] Meta-analysis reveals thatvitamin D supplementation has beneficial effects amongpatients with glucose intolerance or insulin resistance atbaseline [88] However an explanation for the beneficial roleof vitamin D supplementation in the lowering of glycemia indiabetes mellitus remains to be determined Skeletal musclecan increase glucose uptake through insulin-dependent andmuscle contraction-dependentmechanisms [89] Insulin andmuscle contractions stimulate glucose transport in skeletalmuscle via translocation of intracellular glucose transportertype 4 (GLUT4) to the cell surface Manna and Jain [90]examined the mechanism by which vitamin D supplemen-tation regulates glucose metabolism in 3T3L1 adipocytes


When 3T3L1 adipocytes were treated with high glucosein the presence of 112057225(OH)

2D3 it increased expression

of GLUT4 and its translocation to cell surface glucoseuptake and glucose utilization [90] 112057225(OH)

2D3also

enhanced cystathionine-120574-lyase (CSE) activation and H2S

formation [90] which is an important signaling moleculeproduced mainly by CSE in the cardiovascular system [91]Furthermore the effect of 112057225(OH)

2D3on GLUT4 translo-

cation and glucose utilization was prevented by chemicalinhibition or silencing of CSE [90] In muscle cells itis currently not known whether CSE may be associatedwith 112057225(OH)

2D3-induced glucose metabolism Therefore

further studies are required to elucidate the physiologicalrole of CSE in regulation of glucose metabolism in skeletalmuscle Tamilselvan et al [92] examined the effect of cal-citriol (125 dihydroxycholecalciferol) on the expression ofVDR insulin receptor (IR) and GLUT4 in L6 cells whenexposed to high glucose and high insulin which mimicstype 2 diabetic model [92] Calcitriol partially restored VDRIR and GLUT4 expression in type 2 diabetic model [92]raising the possibility that vitamin D could contribute toimproving insulin signaling in type 2 diabetes mellitus In arecent study the effect of 112057225(OH)

2D3on glucose uptake

in rat skeletal muscle is investigated [93] 112057225(OH)2D3

stimulated glucose uptake with increased expression ofGLUT4 protein and enhanced translocation of GLUT4 to theplasmamembrane not through PI3K-signaling pathway [93]which is essential for insulin-stimulated GLUT4 transloca-tion and glucose transport [94] In addition 112057225(OH)

2D3-

stimulated glucose uptake was suppressed concomitantlywith downregulation of GLUT4 protein by treatment withcycloheximide [93] suggesting that it may be mediated bygenomic signaling of vitamin D Taken together vitaminD may improve glucose metabolism in skeletal muscle bymodulating GLUT4 expression and translocation throughinsulin-dependent andor insulin-independent mechanisms

7 Vitamin D Receptor Expression in SkeletalMuscle and Myogenic Cells

VDR is known to be expressed in a wide variety of tissuesincluding bone bronchus intestine kidneymammary glandpancreas parathyroid pituitary gland prostate gland spleentestis and thymus [25] However there has been still somecontroversy as to whether VDR is expressed in skeletalmuscle [24 29 32] For example some studies have failed todetect VDR in skeletal muscle [24 25 95ndash97] other studieshave shown that VDR protein andor mRNA are detectablein skeletal muscle [21ndash23 29 46 98] and myogenic cells[26 27 29 31 32 42 45 46 53 55 98ndash104] In briefWang et al [105] call into question the specificity of variouscommercially available VDR antibodies They systematicallycharacterized these antibodies in terms of their specificityand immunosensitivity using negative control samples fromVDR knockout mice [105] They demonstrated that themouse monoclonal VDR antibody against the C-terminusof human VDR D-6 (Santa Cruz Biotechnology) possesseshigh specificity high sensitivity and versatility [105] They

showed that VDR protein was not detected in skeletal muscleby immunohistochemical analysis using this antibody andthat VDR mRNA was detectable only at extremely low levelsby quantitative RT-PCR assay [24] By contrast Kislingeret al [106] used large scale gel-free tandem mass spec-trometry to monitor global proteome alterations throughoutthe myogenic differentiation program in C2C12 cells Theyobserved upregulation of VDR protein during early stage ofmyoblast differentiation [106] Srikuea et al [29] providedstrong evidence for the presence of VDR in myogenic cellsby combining immunoblot assay immunocytochemical anal-ysis PCR-based cloning and DNA sequencing to validatethe expression of VDR in C2C12 cells They showed that thefull-length VDR mRNA transcript could be isolated frommyoblasts and myotubes and VDR protein was primarilylocalized in the nucleus of myoblasts and in the cytoplasmof myotubes [29] In addition they examined the localizationof VDR protein using a model of myogenesis in vivo BaCl

2

treatment was used to induce regeneration and immuno-histochemical analysis was performed on sections fromcontrol and regeneratingmuscle In controlmuscle VDRwasdetected in muscle fibers but levels were very low whereasin regenerating muscle VDR expression was detected in thecentral nuclei of newly regenerating muscle fibers [29] Morerecently Girgis et al [32] demonstrated that VDR proteinwas detectable in C2C12 myoblasts by immunoblot assayusing VDR antibody (D-6) The discrepancy among studiesmay be explained at least in part by the difference in theexpression of VDR during the stages of muscle developmentFor example Endo et al [46] reported that VDR mRNAwas detected in skeletal muscle from 3-week-old wild-typemice but not 8-week-old wild-type mice Wang and DeLuca[24] showed that VDR protein was undetectable in skeletalmuscle from 6- to 7-week-old C57BL6 mice A similar resultwas also reported by Srikuea et al [29] using 12-week-oldC57BL6miceTherefore VDR expressionmay be dependenton the context of muscle development It requires furtherclarification whether VDR is expressed in muscle fibers

8 The Conversion of25 (OH)D3 to 112057225(OH)2D3 MightOccur in Myogenic Cells

Vitamin D in the form of vitamin D3 is synthesized from

7-dehydrocholesterol in the skin through the action of ultra-violet irradiation [33] Alternatively vitamin D in the formof either vitamin D

2or vitamin D

3 can also be taken in the

diet [33] An active form 112057225(OH)2D3 is synthesized from

vitamin D3through two hydroxylation steps [33] Vitamin

D3is converted to 25-hydroxyvitamin D

3[25(OH)D

3] in

the liver by 25-hydroxylases (encoded by the gene CYP27A1)[33] The generated 25(OH)D

3is further hydroxylated to

112057225(OH)2D3

by 25-hydroxyvitamin D3

1120572-hydroxylase(encoded by the gene CYP27B1) in the kidney [33] HoweverCYP27B1 has been detected in various extrarenal tissues[107 108] raising the possibility that 112057225(OH)

2D3might be

locally synthesized and activate VDR in myogenic cells [2932] Inactive formof vitaminD

3 25(OH)D

3 could inhibit cell


proliferation in a similar manner to 112057225(OH)2D3[29 32]

indicating that the conversion of 25(OH)D3to 112057225(OH)

2D3

by CYP27B1 occurs in myogenic cells Girgis et al [32] con-firmed this possibility using luciferase reporter assay systemthat luciferase activity results from 112057225(OH)

2D3binding

to GAL-4-VDR and subsequent activation of the UASTKluciferase gene via its GAL4 promoter They transfectedC2C12 cells with GAL4-VDR (switch) and UASTK luciferasereporter with treatment of 25(OH)D

3and showed that

luciferase activity increased in a dose-dependent mannersuggesting the conversion of 25(OH)D

3to 112057225(OH)

2D3

by CYP27B1 and the subsequent activation of luciferaseexpression via 125(OH)

2D-bound GAL4-VDR [32] Srikuea

et al [29] confirmed that C2C12 cells express the full-lengthCYP27B1 mRNA transcript and CYP27B1 protein could bedetected in the cytoplasm of myoblasts exhibiting partiallyoverlapping with the mitochondria to which CYP27B1 hasbeen reported to be typically localized [109] Furthermorethey showed that siRNA-mediated knockdown of CYP27B1could alleviate inhibitory effects of 25(OH)D

3on cell pro-

liferation [29] These observations provide direct evidencethat CYP27B1 is biologically active in myogenic cells andmediates to convert 25(OH)D

3to 112057225(OH)

2D3 However

it should be noted that the agonistic action of 25(OH)D3has

been demonstrated in cells derived from CYP27B1 knockoutmice [110] Although further studies are needed to elucidatethe basic mechanisms locally synthesized 112057225(OH)

2D3

in myogenic cells might act through autocrineparacrinemechanisms via VDR

9 The Role of VDR in Muscle Development

Since the process of myogenesis has been extensively studiedboth in vivo and in vitro substantial progress has been madein understanding the molecular and cellular mechanismsThe myogenic regulatory factors a group of basic helix-loop-helix transcription factors consisting of MyoD Myf5myogenin and MRF4 play critical roles in myogenesis [111]MyoD and Myf5 have redundant functions in myoblastspecification [112 113] whereasmyogenin [114 115] and eitherMyoD or MRF4 [116] are required for differentiation Thesemyogenic factors can formheterodimers in combinationwithless specific factors such as members of E12E47 [117] whichare generated by alternative splicing of the E2A gene [118]leading to activation of muscle-specific gene transcription[117]

VDR knockout mice model has provided insight intothe possible physiological roles of vitamin D signaling viaits receptor in muscle development [46] VDR null micerecapitulate a human disease of vitamin D resistance vitaminD-dependent rickets type II [119] VDR null mice grow nor-mally until weaning and thereafter develop variousmetabolicabnormalities including hypocalcemia hypophosphatemiasecondary hyperparathyroidism and bone deformity [46119] Muscle fiber diameter of VDR null mice was approx-imately 20 smaller and fiber size was more variable thanthat of the wild-type mice at 3 weeks of age (before weaning)By 8 weeks of age these morphological changes were more

prominent in the VDR null mice compared to the wild-type mice suggesting either a progressive nature of theabnormalities caused by the absence of VDR or additiveeffects of systemic metabolic changes already present atthis age [46] Although there are neither degenerative nornecrotic changes in VDR null mice the aberrant myofiberswere observed diffusely without any preference to type I ortype II fibers [46] Based on these results they suggest thatthe absence of VDR induces these abnormalities probablyin late stages of fiber maturation andor in metabolism ofmature muscle fibers Tanaka et al [31] showed that siRNA-mediated knockdown of VDR inhibited myotube formationconcomitantly with downregulation of MyoD and myogeninusing C2C12 and G8 cells These results demonstrate that asubstantial level of signaling via VDR is required for normalmuscle development and myogenesis in vitro

Furthermore Myf5 myogenin and E2A but not MyoDand MRF4 were aberrantly and persistently upregulated atthe protein andor mRNA levels in VDR null mice at 3 weeksof age [46] Consistent with the deregulated expression ofMRFs that controlmuscle phenotype VDR null mice showedaberrantly increased expression of embryonic and neonatalMyHC isoforms but not type II (adult fast twitch) MyHCisoform [46]These findings observed in VDR null mice mayreflect compensatory response to a reduction in muscle fibersize For example it can be hypothesized that in VDR nullmice satellite cells may be anomalously activated proliferateand differentiate to form new myonuclei that fuse with exist-ing fibers to restore normal fiber size Finally they examinedwhether 112057225(OH)

2D3could directly downregulate MRFs

and neonatal MyHC gene expression in C2C12 myoblasts112057225(OH)

2D3(10 nM) decreased the steady-state expression

levels of these genes [46] Overall these results support arole of VDR in the regulation of muscle development butthe precise mechanisms remain to be elucidated and theinterpretation is further complicated since negative vitaminD response elements [120ndash122] in the promoter region ofgenes encodingMyf5 andmyogenin have not been identified

10 Effects of 112057225(OH)2D3 on MyoblastProliferation and Differentiation

As referred to above decline of intrinsic regenerative poten-tial of skeletal muscle is a hallmark of ageing [79ndash81] andmay be due to age-related changes in satellite cell func-tion If vitamin D treatment does lead to improvements inmuscle function in elderly people more attention should bedirected to the effect of vitamin D

3on myoblast proliferation

and differentiation Research on effect of 112057225(OH)2D3

on myogenesis has been performed using an in vitro cellculture system The effects of 112057225(OH)

2D3on myoblast

proliferation and differentiation are summarized in Table 1Early studies [41 43] have reported that 112057225(OH)

2D3stim-

ulates proliferation of myogenic cells Giuliani and Boland[41] reported that 112057225(OH)

2D3(013 nM) increased cell

density of chick myoblasts Drittanti et al [43] showed that112057225(OH)

2D3(01 nM) had biphasic effects on DNA synthe-

sis 112057225(OH)2D3exhibited a mitogenic effect in proliferat-

ing chick myoblasts followed by an inhibitory effect during


Table 1 Effects of 112057225(OH)2D3 on proliferation and differentiation in myogenic cells

Muscle cell type Concentration[112057225(OH)2D3]

Proliferation Differentiation Method of VDR detection Reference

Myoblast(chick) 013 nM uarr uarr NI Giuliani and Boland

1984 [41]

G8 3ndash300 nM darr NI Equilibrium binding assaychromatography Simpson et al 1985 [42]

Myoblast(chick) 01 nM uarr uarr NI Drittanti et al 1989 [43]

Myoblast(chick) 1 nM uarr uarr NI Capiati et al 1999 [44]

C2C12 1 nM ND NI Immunoblot Stio et al 2002 [45]

C2C12 10 nM NI ND RT-PCR Endo et al 2003 [46]

C2C12 100 nM darr uarr

RT-PCR immunoblot andimmunocytochemistry Garcia et al 2011 [26]

C2C12 1ndash100 nM darr darr RT-PCR Okuno et al 2012 [27]

C2C12 1 nM uarr uarr NI Buitrago et al 2012 [28]

C2C12 20 nM2 120583M [25(OH)D3]

darr darr

RT-PCR PCR cloning DNAsequencing immunocytochemistry

and immunoblotSrikuea et al 2012 [29]

C2C12 01 pMndash10 120583M NI darr NI Ryan et al 2013 [30]

C2C12 G8 1ndash100 nM NI darr RT-PCR Tanaka et al 2013 [31]

C2C121ndash100 nM1ndash100 nM[25(OH)D3]

darr darr RT-PCR immunoblot Girgis et al 2014 [32]

Promote (uarr) inhibit (darr) no difference between vehicle and treatment (ND) not investigated (NI)

the subsequent stage of myoblast differentiation Capiati et al[44] showed that 112057225(OH)

2D3(1 nM) increases the rate of

[3H] thymidine incorporation into DNA in chick myoblastsIn addition they investigated the role of PKC in mediatingthe effect of 112057225(OH)

2D3using a PKC inhibitor PKC

activity increased after treatment of 112057225(OH)2D3[44] The

specific PKC inhibitor calphostin suppressed 112057225(OH)2D3

stimulation of DNA synthesis in proliferatingmyoblasts [44]Finally they examined 112057225(OH)

2D3-dependent changes

in the expression of PKC isoforms 120572 120573 120575 120576 and 120577 [44]They identified PKC120572 as main isoform correlated with theearly stimulation of myoblast proliferation by 112057225(OH)

2D3

[44] By contrast several studies suggest that overall112057225(OH)

2D3or 25(OH)D

3appears to have antiproliferative

effect on myogenic cells [26 27 29 32 42] 112057225(OH)2D3

(1ndash100 nM) inhibited proliferation of C2C12 myoblasts in adose-dependent manner [27 32] without inducing necroticand apoptotic cell death [32] Okuno et al [27] showedthat 112057225(OH)

2D3arrested the cells in the G0G1 phase

concomitantly with induction of cyclin-dependent kinase(CDK) inhibitors p21WAF1CIP1 that facilitates cell cycle with-drawal [123] and p27Kip1 that inhibits a wide range of CDKsessential for cell cycle progression [124] Girgis et al [32]

also reported the increased expression of genes involved inG0G1 arrest includingRb (retinoblastomaprotein) andATM(ataxia telangiectasia mutated) and decreased expression ofgenes involved in G1S transition including c-myc (cellularmyc) and cyclin-D1 In addition they found reduced c-mycprotein and hypophosphorylated Rb protein [32] The activeform hypophosphorylated Rb blocks entry into S-phase byinhibiting the E2F transcriptional program [125 126] Insummary the effects of 112057225(OH)

2D3on myoblast prolif-

eration remain inconclusive The discrepancy may be dueto the differences in the experimental settings For exampledifferent cell type (primary cells or immortalized cell lines)112057225(OH)

2D3concentration serum concentration duration

of cell culture and duration of treatment are employedFurther studies are needed to clarify the role of 112057225(OH)

2D3

on myoblast proliferationSome studies [43 44] reported that 112057225(OH)

2D3(01 or

1 nM) had inhibitory effects on DNA synthesis in differenti-ating chick myoblasts with an increased MyHC expressionincreasedmyofibrillar andmicrosomal protein synthesis andan elevation of creatine kinase activity Garcia et al [26]reported that prolonged treatment of C2C12 myoblasts with112057225(OH)

2D3(100 nM) enhanced myoblast differentiation


by inhibiting cell proliferation andmodulating the expressionof promyogenic and antimyogenic growth factors using a cul-ture system without reducing serum concentration to initiatecell differentiation They showed that 112057225(OH)

2D3down-

regulated insulin-like growth factor-I (IGF-I) and myostatinexpression and upregulates IGF-II and follistatin expression[26] Follistatin antagonizes myostatin-mediated inhibitionof myogenesis [127] Intriguingly inhibition of myostatinis characterized by increased expression of IGF-1 and IGF-II [128ndash133] which are known to be potent stimulus ofmyogenesis [134 135] Therefore it can be hypothesizedthat 112057225(OH)

2D3may contribute to myogenesis by induc-

ing IGF-II expression through modulation of myostatin-follistatin system It should be noted however that in theseculture conditions only small thin myotubes with few nucleiwere observed on day 10 [26] This may not recapitulate nor-mal C2C12 myoblast differentiation as previously reported[136]

In general C2C12 myoblasts normally proliferate and aremononucleated when kept subconfluently in high-mitogenmedium (eg 10ndash20 fetal bovine serum) To initiate cellcycle exit and myogenic differentiation by switching fromhigh-mitogen medium to low-mitogen medium (eg 2horse serum) they fuse and differentiate into postmi-totic elongated and multinucleated myotubes Using thisC2C12 myoblast differentiation system Buitrago et al [28]showed that 112057225(OH)

2D3(1 nM) enhanced the expression

of MyHC and myogenin at 72 h after treatment By contrastOkuno et al [27] investigated the effects of 112057225(OH)

2D3

(1ndash100 nM) on differentiating and differentiated stage ofC2C12 myoblasts In differentiating phase 112057225(OH)

2D3

treatment downregulated the expression of neonatal myosinheavy chain and myogenin and inhibited myotube forma-tion in a dose-dependent manner (1ndash100 nM) [27] Theyshowed that the expression of fast MyHC isoform increasedwhen fully differentiated myotubes were treated with 1 and10 nM 112057225(OH)

2D3[27] Girgis et al [32] investigated the

prolonged treatment of 112057225(OH)2D3(100 nM) on C2C12

myoblast differentiation When myoblast was treated with112057225(OH)

2D3throughout myogenesis including prolifera-

tive differentiating and differentiated stages myotube for-mation was delayed by day 10 concomitantly with downreg-ulation of Myf5 and myogenin [32] However intriguinglymyotubes treated with 112057225(OH)

2D3exhibit larger cell

size than nontreated myotubes [32] These results suggestthat 112057225(OH)

2D3may biphasically act in the process of

early and late myoblast differentiation Furthermore theyshowed that the hypertrophic effect of 112057225(OH)

2D3on

myotubes is accompanied with downregulation of myostatin[32] Several studies have provided evidence that myostatinacts as a negative regulator of the Aktmammalian targetof rapamycin (mTOR) signaling pathway [137ndash140] whichplays a key role in the regulation of protein synthesis [141]For example Trendelenburg et al [139] show that myostatinreduces AktmTOR signaling complex 1 (TORC1)p70 S6kinase (p70S6K) signaling inhibiting myoblast differentia-tion and reducing myotube size In addition 112057225(OH)

2D3

induced Akt phosphorylation in differentiating C2C12 cells[28] Intriguingly 112057225(OH)

2D3sensitizes the AktmTOR

signaling pathway to the stimulating effect of leucine andinsulin resulting in a further activation of protein synthesisin C2C12 myotubes [104] Taken together 112057225(OH)

2D3

may have an anabolic effect on myotubes by modulatingAktmTOR signaling probably through genomic and nonge-nomic mechanisms

11 Conclusions

The randomized-controlled studies and meta-analysis sup-port a role of vitamin D in improving the age-relateddecline in muscle function However the effect remainsinconclusive Girgis et al [32] emphasize that large studiesemploying standardized reproducible assessments of musclestrength and double-blinded treatment regimens are requiredto identify the effect of vitamin D supplementation onmuscle function and guide the recommended level of vitaminD intake Although it remains intensely debated whetherVDR is expressed in skeletal muscle research on VDR nullmice provides insight into the physiological roles of vitaminD in muscle development and suggests that a substantiallevel of signaling via VDR is required for normal musclegrowth VDR expression seems to be affected by ageingsuggesting that this might reduce the functional responseof the muscle fibers to vitamin D Vitamin D appears tofunction in primary myoblasts and established myoblast celllines Despite limited evidence available at the time vitaminD might have an anabolic effect on myotubes by modulatingmultiple intracellular signaling pathways probably throughgenomic and nongenomic mechanisms However not allstudies support this result Further studies on the potentialimpact of vitamin D onmuscle morphology and function arerequired to develop the effective intervention for sarcopenia

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This research was supported by MEXT (the Ministry ofEducation Culture Sports Science and Technology) (Grantin Aid for Scientific Research (C) 25350882) Japan

References

[1] I H Rosenberg ldquoSummary comments epidemiological andmethodological problems in determining nutritional status ofolder personsrdquo The American Journal of Clinical Nutrition vol50 no 5 pp 1231ndash1233 1989

[2] I H Rosenberg ldquoSarcopenia origins and clinical relevancerdquoJournal of Nutrition vol 127 no 5 supplement pp 990Sndash991S1997

[3] A J Cruz-Jentoft J P Baeyens J M Bauer et al ldquoSarcopeniaEuropean consensus on definition and diagnosis report of theEuropean Working Group on Sarcopenia in Older Peoplerdquo AgeAgeing vol 39 no 4 pp 412ndash423 2010


[4] B H Goodpaster S W Park T B Harris et al ldquoThe loss ofskeletal muscle strength mass and quality in older adults TheHealth Aging and Body Composition Studyrdquo The Journals ofGerontology A Biological Sciences and Medical Sciences vol 61no 10 pp 1059ndash1064 2006

[5] M J Delmonico T B Harris J-S Lee et al ldquoAlternativedefinitions of sarcopenia lower extremity performance andfunctional impairment with aging in older men and womenrdquoJournal of the AmericanGeriatrics Society vol 55 no 5 pp 769ndash774 2007

[6] J F Aloia D M McGowan A N Vaswani P Ross and S HCohn ldquoRelationship ofmenopause to skeletal andmusclemassrdquoThe American Journal of Clinical Nutrition vol 53 no 6 pp1378ndash1383 1991

[7] V A Hughes W R Frontera R RoubenoffW J Evans andMA Fiatarone Singh ldquoLongitudinal changes in body compositionin older men and women role of body weight change andphysical activityrdquo The American Journal of Clinical Nutritionvol 76 no 2 pp 473ndash481 2002

[8] K S Nair ldquoAging musclerdquo The American Journal of ClinicalNutrition vol 81 no 5 pp 953ndash963 2005

[9] W K Mitchell J Williams P Atherton M Larvin J Lund andMNarici ldquoSarcopenia dynapenia and the impact of advancingage on human skeletal muscle size and strength a quantitativereviewrdquo Frontiers in Physiology vol 3 article 260 2012

[10] H Augustin and L Partridge ldquoInvertebrate models of age-related muscle degenerationrdquo Biochimica et Biophysica ActamdashGeneral Subjects vol 1790 no 10 pp 1084ndash1094 2009

[11] F Demontis R Piccirillo A L Goldberg and N Perri-mon ldquoMechanisms of skeletal muscle aging insights fromDrosophila and mammalian modelsrdquo Disease Models amp Mech-anisms vol 6 no 6 pp 1339ndash1352 2013

[12] R N Baumgartner K M Koehler and D Gallagher ldquoEpi-demiology of sarcopenia among the elderly in New MexicordquoAmerican Journal of Epidemiology vol 147 no 8 pp 755ndash7631998

[13] I Janssen S B Heymsfield and R Ross ldquoLow relative skeletalmuscle mass (sarcopenia) in older persons is associated withfunctional impairment and physical disabilityrdquo Journal of theAmerican Geriatrics Society vol 50 no 5 pp 889ndash896 2002

[14] F Lauretani C R Russo S Bandinelli et al ldquoAge-associatedchanges in skeletalmuscles and their effect onmobility an oper-ational diagnosis of sarcopeniardquo Journal of Applied Physiologyvol 95 no 5 pp 1851ndash1860 2003

[15] Y Rolland V Lauwers-Cances M Cournot et al ldquoSarcopeniacalf circumference and physical function of elderly womena cross-sectional studyrdquo Journal of the American GeriatricsSociety vol 51 no 8 pp 1120ndash1124 2003

[16] I Janssen ldquoInfluence of sarcopenia on the development ofphysical disability the cardiovascular health studyrdquo Journal ofthe American Geriatrics Society vol 54 no 1 pp 56ndash62 2006

[17] S von Haehling J E Morley and S D Anker ldquoAn overviewof sarcopenia facts and numbers on prevalence and clinicalimpactrdquo Journal of Cachexia Sarcopenia and Muscle vol 1 no2 pp 129ndash133 2010

[18] I Janssen D S Shepard P T Katzmarzyk and R RoubenoffldquoThehealthcare costs of sarcopenia in theUnited Statesrdquo Journalof the American Geriatrics Society vol 52 no 1 pp 80ndash85 2004

[19] ldquoWhat Works Healing the Healthcare Staffing ShortagerdquoPricewaterhouse-Coopers Health Research Institute 2007

[20] World Population Ageing 1950ndash2050 Department of Economicand Social Affairs United Nations 2001 httpwwwunorgesapopulationpublicationsworldageing19502050

[21] H A Bischoff M Borchers F Gudat et al ldquoIn situ detectionof 125-dihydroxyvitaminD

3receptor in human skeletal muscle

tissuerdquoTheHistochemical Journal vol 33 no 1 pp 19ndash24 2001[22] H A Bischoff-Ferrari M Borchers F Gudat U Durmuller

andW Dick ldquoVitamin D receptor expression in human muscletissue decreases with agerdquo Journal of Bone andMineral Researchvol 19 no 2 pp 265ndash269 2004

[23] L Ceglia M da Silva Morais L K Park et al ldquoMulti-stepimmunofluorescent analysis of vitamin D receptor loci andmyosin heavy chain isoforms in human skeletalmusclerdquo Journalof Molecular Histology vol 41 no 2-3 pp 137ndash142 2010

[24] Y Wang and H F DeLuca ldquoIs the vitamin D receptor found inmusclerdquo Endocrinology vol 152 no 2 pp 354ndash363 2011

[25] Y Wang J Zhu and H F DeLuca ldquoWhere is the vitamin Dreceptorrdquo Archives of Biochemistry and Biophysics vol 523 no1 pp 123ndash133 2012

[26] L A Garcia K K King M G Ferrini K C Norris andJ N Artaza ldquo125(OH)

2vitamin D

3stimulates myogenic

differentiation by inhibiting cell proliferation and modulatingthe expression of promyogenic growth factors and myostatin inC2C12skeletal muscle cellsrdquo Endocrinology vol 152 no 8 pp

2976ndash2986 2011[27] H Okuno K N Kishimoto M Hatori and E Itoi ldquo112057225-dihy-

droxyvitamin D3enhances fast-myosin heavy chain expres-

sion in differentiated C2C12

myoblastsrdquo Cell Biology Interna-tional vol 36 no 5 pp 441ndash447 2012

[28] C G Buitrago N S Arango and R L Boland ldquo112057225(OH)2D3-

dependent modulation of Akt in proliferating and differentiat-ingC2C12skeletalmuscle cellsrdquo Journal of Cellular Biochemistry

vol 113 no 4 pp 1170ndash1181 2012[29] R Srikuea X Zhang O-K Park-Sarge and K A Esser ldquoVDR

and CYP27B1 are expressed in C2C12

cells and regeneratingskeletal muscle potential role in suppression of myoblastproliferationrdquo American Journal of PhysiologymdashCell Physiologyvol 303 no 4 pp C396ndashC405 2012

[30] K J Ryan Z C Daniel L J Craggs T Parr and J M BrameldldquoDose-dependent effects of vitaminD on transdifferentiation ofskeletal muscle cells to adipose cellsrdquo Journal of Endocrinologyvol 217 no 1 pp 45ndash58 2013

[31] M Tanaka K N Kishimoto H Okuno H Saito and E ItoildquoVitamin D receptor gene silencing effects on differentiation ofmyogenic cell linesrdquoMuscle ampNerve vol 49 no 5 pp 700ndash7082014

[32] C M Girgis R J Clifton-Bligh N Mokbel K Cheng andJ E Gunton ldquoVitamin D signaling regulates proliferationdifferentiation andmyotube size in C

2C12skeletal muscle cellsrdquo

Endocrinology vol 155 no 2 pp 347ndash357 2014[33] G Jones S A Strugnell and H F DeLuca ldquoCurrent under-

standing of the molecular actions of vitamin Drdquo PhysiologicalReviews vol 78 no 4 pp 1193ndash1231 1998

[34] R Losel and M Wehling ldquoNongenomic actions of steroidhormonesrdquo Nature Reviews Molecular Cell Biology vol 4 no1 pp 46ndash56 2003

[35] K K Deeb D L Trump and C S Johnson ldquoVitamin Dsignalling pathways in cancer potential for anticancer thera-peuticsrdquoNature Reviews Cancer vol 7 no 9 pp 684ndash700 2007

[36] C M Girgis R J Clifton-Bligh M W Hamrick M F Holickand J E Gunton ldquoThe roles of vitamin D in skeletal muscle


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[37] R Bouillon W H Okamura and A W Norman ldquoStructure-function relationships in the vitamin D endocrine systemrdquoEndocrine Reviews vol 16 no 2 pp 200ndash257 1995

[38] Y-R Lou S Qiao R Talonpoika H Syvala and P TuohimaaldquoThe role of vitamin D

3metabolism in prostate cancerrdquo The

Journal of Steroid Biochemistry and Molecular Biology vol 92no 4 pp 317ndash325 2004

[39] M R Haussler P W Jurutka M Mizwicki and A W NormanldquoVitamin D receptor (VDR)-mediated actions of 112057225(OH)

2

vitamin D3 genomic and non-genomic mechanismsrdquo Best

PracticeampResearch Clinical Endocrinology andMetabolism vol25 no 4 pp 543ndash559 2011

[40] T-T Wang L E Tavera-Mendoza D Laperriere et al ldquoLarge-scale in silico andmicroarray-based identification of direct 125-dihydroxyvitamin D

3target genesrdquo Molecular Endocrinology

vol 19 no 11 pp 2685ndash2695 2005[41] D L Giuliani and R L Boland ldquoEffects of vitamin D

3

metabolites on calcium fluxes in intact chicken skeletal muscleand myoblasts cultured in vitrordquo Calcified Tissue Internationalvol 36 no 2 pp 200ndash205 1984

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3receptors and activities inmusclerdquo

The Journal of Biological Chemistry vol 260 no 15 pp 8882ndash8891 1985

[43] L Drittanti A R de Boland and R Boland ldquoModulation ofDNA synthesis in cultured muscle cells by 125-dihydroxyvi-tamin D-3rdquo Biochimica et Biophysica ActamdashMolecular CellResearch vol 1014 no 2 pp 112ndash119 1989

[44] D A Capiati M T Tellez-Inon and R L Boland ldquoPartic-ipation of protein kinase C 120572 in 125-dihydroxy-vitamin D

3

regulation of chick myoblast proliferation and differentiationrdquoMolecular and Cellular Endocrinology vol 153 no 1-2 pp 39ndash45 1999

[45] M Stio A Celli and C Treves ldquoSynergistic effect of vitaminD derivatives and retinoids on C

2C12

skeletal muscle cellsrdquoIUBMB Life vol 53 no 3 pp 175ndash181 2002

[46] I Endo D Inoue T Mitsui et al ldquoDeletion of vitamin Dreceptor gene in mice results in abnormal skeletal muscledevelopment with deregulated expression of myoregulatorytranscription factorsrdquo Endocrinology vol 144 no 12 pp 5138ndash5144 2003

[47] T Kitamura Y I Kitamura Y Funahashi et al ldquoA FoxoNotchpathway controls myogenic differentiation and fiber type spec-ificationrdquoThe Journal of Clinical Investigation vol 117 no 9 pp2477ndash2485 2007

[48] M F Buas and T Kadesch ldquoRegulation of skeletal myogenesisby Notchrdquo Experimental Cell Research vol 316 no 18 pp 3028ndash3033 2010

[49] Y Ezura O Tournay A Nifuji and M Noda ldquoIdentificationof a novel suppressive vitamin D response sequence in the51015840-Flanking region of the murine Id1 generdquo The Journal ofBiological Chemistry vol 272 no 47 pp 29865ndash29872 1997

[50] N Kawaguchi H F Deluca and M Noda ldquoId gene expres-sion and its suppression by 125-dihydroxyvitamin D

3in rat

osteoblastic osteosarcoma cellsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 89 no10 pp 4569ndash4572 1992

[51] A Lasorella R Benezra and A Iavarone ldquoThe ID proteinsmaster regulators of cancer stem cells and tumour aggressive-nessrdquo Nature Reviews Cancer vol 14 no 2 pp 77ndash91 2014

[52] Y Jen H Weintraub and R Benezra ldquoOverexpression of Idprotein inhibits the muscle differentiation program in vivoassociation of Id with E2A proteinsrdquoGenes amp Development vol6 no 8 pp 1466ndash1479 1992

[53] D Capiati S Benassati and R L Boland ldquo125(OH)2-vitamin

D3induces translocation of the vitaminD receptor (VDR) to the

plasma membrane in skeletal muscle cellsrdquo Journal of CellularBiochemistry vol 86 no 1 pp 128ndash135 2002

[54] P W Shaul and R G Anderson ldquoRole of plasmalemmal cave-olae in signal transductionrdquo American Journal of PhysiologymdashLung Cellular and Molecular Physiology vol 275 no 5 ppL843ndashL851 1998

[55] C Buitrago and R Boland ldquoCaveolae and caveolin-1 areimplicated in 112057225(OH)

2-vitamin D

3-dependent modulation

of Src MAPK cascades and VDR localization in skeletal musclecellsrdquoThe Journal of Steroid Biochemistry andMolecular Biologyvol 121 no 1-2 pp 169ndash175 2010

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2D3membrane bind-

ing protein (125D3-MARRS) and phosphate uptake in intesti-

nal cellsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 19 pp 7392ndash7397 2004

[57] R C Khanal and I Nemere ldquoThe ERp57GRp58125D3-

MARRS receptor multiple functional roles in diverse cellsystemsrdquo Current Medicinal Chemistry vol 14 no 10 pp 1087ndash1093 2007

[58] C Buitrago R Boland and A R de Boland ldquoThe tyrosinekinase c-Src is required for 125(OH)

2-vitamin D

3signalling to

the nucleus in muscle cellsrdquo Biochimica et Biophysica ActamdashMolecular Cell Research vol 1541 no 3 pp 179ndash187 2001

[59] C G Buitrago A C Ronda A R de Boland and R BolandldquoMAP kinases p38 and JNK are activated by the steroidhormone 112057225(OH)

2-vitamin D

3in the C

2C12muscle cell linerdquo

Journal of Cellular Biochemistry vol 97 no 4 pp 698ndash7082006

[60] C G Buitrago V Gonzalez Pardo A R de Boland and RBoland ldquoActivation of Raf-1 through Ras and protein kinaseC120572mediates 112057225(OH)

2-vitaminD

3regulation of themitogen-

activated protein kinase pathway in muscle cellsrdquoThe Journal ofBiological Chemistry vol 278 no 4 pp 2199ndash2205 2003

[61] M Tateno Y Nishida and T Adachi-Yamada ldquoRegulation ofJNK by Src during Drosophila developmentrdquo Science vol 287no 5451 pp 324ndash327 2000

[62] N C Jones Y V Fedorov R S Rosenthal and B B OlwinldquoERK12 is required for myoblast proliferation but is dispens-able for muscle gene expression and cell fusionrdquo Journal ofCellular Physiology vol 186 no 1 pp 104ndash115 2001

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2C12

myogenesisrdquoThe Journal of Biological Chemistry vol 274 no 7pp 4341ndash4346 1999

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[65] Z Wu P J Woodring K S Bhakta et al ldquop38 and extracellularsignal-regulated kinases regulate the myogenic program atmultiple stepsrdquo Molecular and Cellular Biology vol 20 no 11pp 3951ndash3964 2000

[66] Z Huang D Chen K Zhang B Yu X Chen and J MengldquoRegulation of myostatin signaling by c-Jun N-terminal kinase


in C2C12cellsrdquo Cellular Signalling vol 19 no 11 pp 2286ndash2295

2007[67] A C McPherron A M Lawler and S-J Lee ldquoRegulation of

skeletal muscle mass in mice by a new TGF-120573 superfamilymemberrdquo Nature vol 387 no 6628 pp 83ndash90 1997

[68] E MWilson and P Rotwein ldquoSelective control of skeletal mus-cle differentiation by Akt1rdquoThe Journal of Biological Chemistryvol 282 no 8 pp 5106ndash5110 2007

[69] R P van derWielenM R LowikH van danBerg et al ldquoSerumvitamin D concentrations among elderly people in EuroperdquoTheLancet vol 346 no 8969 pp 207ndash210 1995

[70] M F Holick ldquoVitamin D deficiencyrdquoThe New England Journalof Medicine vol 357 no 3 pp 266ndash281 2007

[71] J MacLaughlin and M F Holick ldquoAging decreases the capacityof human skin to produce vitamin D

3rdquo The Journal of Clinical

Investigation vol 76 no 4 pp 1536ndash1538 1985[72] S Yoshikawa T Nakamura H Tanabe and T Imamura

ldquoOsteomalacic myopathyrdquo Endocrinologia Japonica vol 26supplement pp 65ndash72 1979

[73] R Scelsi C Marchetti and P Poggi ldquoHistochemical andultrastructural aspects of M vastus lateralis in sedentary oldpeople (age 65ndash89 years)rdquo Acta Neuropathologica vol 51 no 2pp 99ndash105 1980

[74] J L Andersen ldquoMuscle fibre type adaptation in the elderlyhuman musclerdquo Scandinavian Journal of Medicine amp Science inSports vol 13 no 1 pp 40ndash47 2003

[75] S B P Charge and M A Rudnicki ldquoCellular and molecularregulation of muscle regenerationrdquo Physiological Reviews vol84 no 1 pp 209ndash238 2004

[76] L B Verdijk R Koopman G Schaart K Meijer H H CM Savelberg and L J C Van Loon ldquoSatellite cell contentis specifically reduced in type II skeletal muscle fibers in theelderlyrdquo American Journal of PhysiologymdashEndocrinology andMetabolism vol 292 no 1 pp E151ndashE157 2007

[77] W Scott J Stevens and S A Binder-Macleod ldquoHuman skeletalmuscle fiber type classificationsrdquo Physical Therapy vol 81 no11 pp 1810ndash1816 2001

[78] C Buitrago V G Pardo and R Boland ldquoRole of VDR in 112057225-dihydroxyvitamin D

3-dependent non-genomic activation of

MAPKs Src and Akt in skeletal muscle cellsrdquo The Journal ofSteroid Biochemistry and Molecular Biology vol 136 no 1 pp125ndash130 2013

[79] A S Brack and T A Rando ldquoIntrinsic changes and extrinsicinfluences of myogenic stem cell function during agingrdquo StemCell Reviews vol 3 no 3 pp 226ndash237 2007

[80] S Carosio M G Berardinelli M Aucello and A MusaroldquoImpact of ageing onmuscle cell regenerationrdquo Ageing ResearchReviews vol 10 no 1 pp 35ndash42 2011

[81] Y C Jang M Sinha M Cerletti C Dallosso and A J WagersldquoSkeletal muscle stem cells effects of aging and metabolism onmuscle regenerative functionrdquo Cold Spring Harbor Symposia onQuantitative Biology vol 76 pp 101ndash111 2011

[82] F Relaix and P S Zammit ldquoSatellite cells are essential forskeletal muscle regeneration the cell on the edge returns centrestagerdquo Development vol 139 no 16 pp 2845ndash2856 2012

[83] A Mauro and W R Adams ldquoThe structure of the sarcolemmaof the frog skeletal muscle fiberrdquoThe Journal of Biophysical andBiochemical Cytology vol 10 no 4 supplement pp 177ndash1851961

[84] T J Hawke and D J Garry ldquoMyogenic satellite cells physiologyto molecular biologyrdquo Journal of Applied Physiology vol 91 no2 pp 534ndash551 2001

[85] I Stratos Z Li P Herlyn et al ldquoVitamin D increases cellularturnover and functionally restores the skeletal muscle aftercrush injury in ratsrdquoAmerican Journal of Pathology vol 182 no3 pp 895ndash904 2013

[86] P Zimmet K G Alberti and J Shaw ldquoGlobal and societalimplications of the diabetes epidemicrdquo Nature vol 414 no6865 pp 782ndash787 2001

[87] S W Park B H Goodpaster J S Lee et al ldquoExcessive lossof skeletal muscle mass in older adults with type 2 diabetesrdquoDiabetes Care vol 32 no 11 pp 1993ndash1997 2009

[88] J Mitri M D Muraru and A G Pittas ldquoVitamin D and type2 diabetes a systematic reviewrdquo European Journal of ClinicalNutrition vol 65 no 9 pp 1005ndash1015 2011

[89] N Jessen and L J Goodyear ldquoContraction signaling to glucosetransport in skeletal musclerdquo Journal of Applied Physiology vol99 no 1 pp 330ndash337 2005

[90] P Manna and S K Jain ldquoVitamin D up-regulates glucosetransporter 4 (GLUT4) translocation and glucose utilizationmediated by cystathionine-120574-lyase (CSE) activation and H

2S

formation in 3T3L1 adipocytesrdquoThe Journal of Biological Chem-istry vol 287 no 50 pp 42324ndash42332 2012

[91] O Kabil and R Banerjee ldquoRedox biochemistry of hydrogensulfiderdquoThe Journal of Biological Chemistry vol 285 no 29 pp21903ndash21907 2010

[92] B Tamilselvan K G Seshadri and G Venkatraman ldquoRoleof vitamin D on the expression of glucose transporters in L6myotubesrdquo Indian Journal of Endocrinology and Metabolismvol 17 supplement 1 pp S326ndashS328 2013

[93] A J Castro M J Frederico L H Cazarolli et al ldquoBetulinicacid and 1 25(OH)

2vitamin D

3share intracellular signal

transduction in glucose homeostasis in soleus musclerdquo TheInternational Journal of Biochemistry amp Cell Biology vol 48 pp18ndash27 2014

[94] S Lund G D Holman O Schmitz and O Pedersen ldquoCon-traction stimulates translocation of glucose transporter GLUT4in skeletal muscle through a mechanism distinct from that ofinsulinrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 92 no 13 pp 5817ndash5821 1995

[95] W E Stumpf M Sar F A Reid Y Tanaka and H F DeLucaldquoTarget cells for 125-dihydroxyvitamin D

3in intestinal tract

stomach kidney skin pituitary and parathyroidrdquo Science vol206 no 4423 pp 1188ndash1190 1979

[96] K Colston M Hirst and D Feldman ldquoOrgan distributionof the cytoplasmic 125-dihydroxycholecalciferol receptor invarious mouse tissuesrdquo Endocrinology vol 107 no 6 pp 1916ndash1922 1980

[97] M E Sandgren M Bronnegard and H F DeLuca ldquoTissue dis-tribution of the 1 25-dihydroxyvitamin D

3receptor in the male

ratrdquoBiochemical and Biophysical Research Communications vol181 no 2 pp 611ndash616 1991

[98] S B Zanello E D Collins M J Marinissen A W Normanand R L Boland ldquoVitamin d receptor expression in chickenmuscle tissue and cultured myoblastsrdquoHormone and MetabolicResearch vol 29 no 5 pp 231ndash236 1997

[99] E M Costa H M Blau and D Feldman ldquo125-dihydroxyvi-tamin D

3receptors and hormonal responses in cloned human

skeletal muscle cellsrdquo Endocrinology vol 119 no 5 pp 2214ndash2220 1986

[100] R Boland A Norman E Ritz andW Hasselbach ldquoPresence ofa 125-dihydroxy-vitamin D

3receptor in chick skeletal muscle

myoblastsrdquo Biochemical and Biophysical Research Communica-tions vol 128 no 1 pp 305ndash311 1985


[101] C Buitrago G Vazquez A R De Boland and R L BolandldquoActivation of Src kinase in skeletal muscle cells by 1 1 25-(OH2)-vitamin D

3correlates with tyrosine phosphorylation

of the vitamin D receptor (VDR) and VDR-Src interactionrdquoJournal of Cellular Biochemistry vol 79 no 2 pp 274ndash281 2000

[102] C Buitrago G Vazquez A R De Boland and R BolandldquoThe vitamin D receptor mediates rapid changes in muscleprotein tyrosine phosphorylation induced by 125(OH)

2D3rdquo

Biochemical and Biophysical Research Communications vol 289no 5 pp 1150ndash1156 2001

[103] R Boland A R De Boland C Buitrago et al ldquoNon-genomic stimulation of tyrosine phosphorylation cascades by125(OH)

2D3by VDR-dependent and -independent mecha-

nisms inmuscle cellsrdquo Steroids vol 67 no 6 pp 477ndash482 2002[104] J Salles A Chanet C Giraudet et al ldquo125(OH)

2-vitamin D

3

enhances the stimulating effect of leucine and insulin on proteinsynthesis rate through AktPKB andmTORmediated pathwaysin murine C

2C12

skeletal myotubesrdquo Molecular Nutrition ampFood Research vol 57 no 12 pp 2137ndash2146 2013

[105] Y Wang B R Becklund and H F DeLuca ldquoIdentification ofa highly specific and versatile vitamin D receptor antibodyrdquoArchives of Biochemistry and Biophysics vol 494 no 2 pp 166ndash177 2010

[106] T Kislinger A O Gramolini Y Pan K Rahman D HMacLennan and A Emili ldquoProteome dynamics during C

2C12

myoblast differentiationrdquo Molecular and Cellular Proteomicsvol 4 no 7 pp 887ndash901 2005

[107] D Zehnder R Bland M C Williams et al ldquoExtrarenalexpression of 25-hydroxyvitamin D3-1120572-hydroxylaserdquo Journalof Clinical Endocrinology amp Metabolism vol 86 no 2 pp 888ndash894 2001

[108] J S Adams and M Hewison ldquoExtrarenal expression of the 25-hydroxyvitamin D-1-hydroxylaserdquoArchives of Biochemistry andBiophysics vol 523 no 1 pp 95ndash102 2012

[109] Y Nakamura T-A Eto T Taniguchi et al ldquoPurificationand characterization of 25-hydroxyvitamin D

31120572- hydroxylase

from rat kidney mitochondriardquo FEBS Letters vol 419 no 1 pp45ndash48 1997

[110] Y R Lou F Molnar M Perakyla et al ldquo25-Hydroxyvitamin D3

is an agonistic vitamin D receptor ligandrdquoThe Journal of SteroidBiochemistry and Molecular Biology vol 118 no 3 pp 162ndash1702010

[111] K Bismuth and F Relaix ldquoGenetic regulation of skeletal muscledevelopmentrdquo Experimental Cell Research vol 316 no 18 pp3081ndash3086 2010

[112] T Braun M A Rudnicki H-H Arnold and R JaenischldquoTargeted inactivation of the muscle regulatory gene Myf-5results in abnormal rib development and perinatal deathrdquo Cellvol 71 no 3 pp 369ndash382 1992

[113] M A Rudnicki T Braun S Hinuma and R Jaenisch ldquoInacti-vation of MyoD in mice leads to up-regulation of the myogenicHLH gene Myf-5 and results in apparently normal muscledevelopmentrdquo Cell vol 71 no 3 pp 383ndash390 1992

[114] P Hasty A Bradley J H Morris et al ldquoMuscle deficiencyand neonatal death in mice with a targeted mutation in themyogenin generdquo Nature vol 364 no 6437 pp 501ndash506 1993

[115] Y Nabeshima K Hanaoka M Hayasaka et al ldquoMyogenin genedisruption results in perinatal lethality because of severemuscledefectrdquo Nature vol 364 no 6437 pp 532ndash535 1993

[116] A Rawls M R Valdez W Zhang J Richardson W H Kleinand ENOlson ldquoOverlapping functions of themyogenic bHLH

genes MRF4 and MyoD revealed in double mutant micerdquoDevelopment vol 125 no 13 pp 2349ndash2358 1998

[117] A B Lassar R L DavisW EWright et al ldquoFunctional activityof myogenic HLH proteins requires hetero-oligomerizationwith E12E47-like proteins in vivordquo Cell vol 66 no 2 pp 305ndash315 1991

[118] X-H Sun and D Baltimore ldquoAn inhibitory domain of E12transcription factor prevents DNA binding in E12 homodimersbut not in E12 heterodimersrdquo Cell vol 64 no 2 pp 459ndash4701991

[119] T Yoshizawa Y Handa Y Uematsu et al ldquoMice lacking thevitamin D receptor exhibit impaired bone formation uterinehypoplasia and growth retardation after weaningrdquo NatureGenetics vol 16 no 4 pp 391ndash396 1997

[120] K Sakoda M Fujiwara S Arai et al ldquoIsolation of a genomicDNA fragment having negative vitamin D response elementrdquoBiochemical and Biophysical Research Communications vol 219no 1 pp 31ndash35 1996

[121] T Nishishita T Okazaki T Ishikawa et al ldquoA negative vitaminD response DNA element in the human parathyroid hormone-related peptide gene binds to vitamin D receptor along with Kuantigen to mediate negative gene regulation by vitamin DrdquoTheJournal of Biological Chemistry vol 273 no 18 pp 10901ndash109071998

[122] S Seoane M Alonso C Segura and R Perez-FernandezldquoLocalization of a negative vitamin D response sequence inthe human growth hormone generdquo Biochemical and BiophysicalResearch Communications vol 292 no 1 pp 250ndash255 2002

[123] G P Dotto ldquop21(WAF1Cip1) more than a break to the cellcyclerdquo Biochimica et Biophysica ActamdashReviews on Cancer vol1471 no 1 pp M43ndashM56 2000

[124] C J Sherr and JM Roberts ldquoCDK inhibitors positive and neg-ative regulators ofG1-phase progressionrdquoGenesampDevelopmentvol 13 no 12 pp 1501ndash1512 1999

[125] J R Nevins ldquoE2F a link between the Rb tumor suppressorprotein and viral oncoproteinsrdquo Science vol 258 no 5081 pp424ndash429 1992

[126] C J Sherr ldquoCancer cell cyclesrdquo Science vol 274 no 5293 pp1672ndash1677 1996

[127] H Amthor G Nicholas I McKinnell et al ldquoFollistatin com-plexes Myostatin and antagonises Myostatin-mediated inhibi-tion of myogenesisrdquo Developmental Biology vol 270 no 1 pp19ndash30 2004

[128] H Kocamis S A Gahr L Batelli A F Hubbs and J KilleferldquoIGF-I IGF-II and IGF-receptor-1 transcript and IGF-II pro-tein expression in myostatin knockout mice tissuesrdquo Muscle ampNerve vol 26 no 1 pp 55ndash63 2002

[129] H Gilson O Schakman L Combaret et al ldquoMyostatingene deletion prevents glucocorticoid-inducedmuscle atrophyrdquoEndocrinology vol 148 no 1 pp 452ndash460 2007

[130] A Marshall M S Salerno MThomas et al ldquoMighty is a novelpromyogenic factor in skeletal myogenesisrdquo Experimental CellResearch vol 314 no 5 pp 1013ndash1029 2008

[131] H Gilson O Schakman S Kalista P Lause K Tsuchida andJ-P Thissen ldquoFollistatin induces muscle hypertrophy throughsatellite cell proliferation and inhibition of both myostatin andactivinrdquo American Journal of PhysiologymdashEndocrinology andMetabolism vol 297 no 1 pp E157ndashE164 2009

[132] M Miyake S Hayashi Y Taketa et al ldquoMyostatin down-regulates the IGF-2 expression via ALK-Smad signaling duringmyogenesis in cattlerdquo Animal Science Journal vol 81 no 2 pp223ndash229 2010


[133] N G Williams J P Interlichia M F Jackson D Hwang PCohen and B D Rodgers ldquoEndocrine actions of myostatinsystemic regulation of the igf and igf binding protein axisrdquoEndocrinology vol 152 no 1 pp 172ndash180 2011

[134] C Duan H Ren and S Gao ldquoInsulin-like growth factors(IGFs) IGF receptors and IGF-binding proteins roles inskeletal muscle growth and differentiationrdquo General and Com-parative Endocrinology vol 167 no 3 pp 344ndash351 2010

[135] N Zanou and P Gailly ldquoSkeletal muscle hypertrophy andregeneration interplay between the myogenic regulatory fac-tors (MRFs) and insulin-like growth factors (IGFs) pathwaysrdquoCellular and Molecular Life Sciences vol 70 no 21 pp 4117ndash4130 2013

[136] X Shen J M Collier M Hlaing et al ldquoGenome-wide exami-nation of myoblast cell cycle withdrawal during differentiationrdquoDevelopmental Dynamics vol 226 no 1 pp 128ndash138 2003

[137] A Amirouche A-C Durieux S Banzet et al ldquoDown-regulation of Aktmammalian target of rapamycin signalingpathway in response to myostatin overexpression in skeletalmusclerdquo Endocrinology vol 150 no 1 pp 286ndash294 2009

[138] R Sartori GMilanM Patron et al ldquoSmad2 and 3 transcriptionfactors control muscle mass in adulthoodrdquo American Journal ofPhysiologymdashCell Physiology vol 296 no 6 pp C1248ndashC12572009

[139] A U Trendelenburg A Meyer D Rohner J Boyle SHatakeyama and D J Glass ldquoMyostatin reduces AktTORC1p70S6K signaling inhibiting myoblast differentiation andmyotube sizerdquoAmerican Journal of PhysiologymdashCell Physiologyvol 296 no 6 pp C1258ndashC1270 2009

[140] C Lipina H Kendall A C McPherron P M Taylor andH S Hundal ldquoMechanisms involved in the enhancement ofmammalian target of rapamycin signalling and hypertrophy inskeletal muscle of myostatin-deficient micerdquo FEBS Letters vol584 no 11 pp 2403ndash2408 2010

[141] D D Sarbassov S M Ali and D M Sabatini ldquoGrowing rolesfor the mTOR pathwayrdquo Current Opinion in Cell Biology vol 17no 6 pp 596ndash603 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Epidemiologic clinical and laboratory evidence providean effect of vitamin D onmuscle function Numerous studieshave investigated the effect of vitamin D supplementationon muscle strength and physical performance in elderlypeople However the precise molecular and cellular mecha-nisms remain to be elucidated Immunohistochemical studieshave demonstrated that vitamin D receptor (VDR) mightbe localized in human muscle fibers [21ndash23] with somecontradictions [24 25] In addition recent studies havereported that vitamin D signaling via VDR plays a role in theregulation of myoblast proliferation and differentiation [26ndash32] Understanding how vitamin D signaling contributes tomyogenesis will provide a valuable insight into an effectivenutritional strategy to moderate sarcopenia Here we willsummarize the current knowledge about effect of vitaminD on skeletal muscle and myogenic cells and discuss thepotential for treatment of sarcopenia

2 Vitamin D Signaling Pathways Genomicand Nongenomic Pathways

Vitamin D signaling has been extensively investigated in avariety of cell types During the past two decades consid-erable progress has been made in understanding the actionof 112057225-dihydroxyvitamin D

3[112057225(OH)

2D3] on myogenic

cellsThebiological effect of 112057225(OH)2D3is exerted through

genomic or nongenomic mechanisms (for reviews see [33ndash35]) Better understanding of the molecular and cellularmechanisms of vitamin D action on skeletal muscle willenable us to develop an effective intervention for sarcope-nia We will focus on 112057225(OH)

2D3signaling via VDR in

genomic and nongenomic mechanism related to myogeniccells although rapid alteration in intracellular calcium whichis nongenomically regulated by 112057225(OH)

2D3 has been well

demonstrated both in vivo and in vitro for details excellentreview article is already available on this subject [36]

An active form 112057225(OH)2D3 acts by binding to VDR

[33] The binding affinity of 25(OH)D3for human vitamin

D receptor (VDR) is approximately 500 times less than thatof 112057225(OH)

2D3but the circulating level of 25(OH)D

3is

approximately 1000 times higher than that of 112057225(OH)2D3

[37 38] In genomic mechanism 112057225(OH)2D3binds to

VDR and is transported to the nucleus [35] VDR is het-erodimerized with 9-cis-retinoic acid receptor (RXR) andVDRRXR complex modulates gene expression via bindingto specific target gene promoter regions known as vitaminD response elements (VDREs) to activate or suppress theirexpression [35] In general VDREs possess either a directrepeat of two hexanucleotide half-elements with a spacer ofthree nucleotides (DR3) or an everted repeat of two half-elements with a spacer of six nucleotides (ER6) motif withDR3s being the most common [39] Wang et al [40] investi-gated direct 112057225(OH)

2D3-target genes on a large scale by

using a combined approach of microarray analysis and insilico genome-wide screens for DR3 and ER6-type VDREsMicroarray analyses performed with RNA from humanSCC25 cells treated with 112057225(OH)

2D3and cycloheximide

an inhibitor of protein synthesis revealed 913 regulated

genes [40] Of the 913 genes 734 genes were induced and179 genes were repressed by treatment of 112057225(OH)

2D3

[40] In addition a screening of the mouse genome iden-tified more than 3000 conserved VDREs and 158 humangenes containing conserved elements were 112057225(OH)

2D3-

regulated on microarrays [40] These results support theirbroad physiological actions of 112057225(OH)

2D3in a variety of

cell typesWith respect to several genes related to myogene-

sis we will describe them in more detail For example112057225(OH)

2D3induced expression of the gene encoding

Foxo1 [40] which is a member of the FOXO subfamilyof forkheadwinged helix family of transcription factorsgoverns muscle growth metabolism and myoblast differen-tiation When transfected C2C12 cells with adenoviral vectorencoded a constitutively active Foxo1 mutant they effectivelyblocked myoblast differentiation [47] This was partly res-cued by inhibition of Notch signaling [47] which inhibitsmyoblast differentiation [48] In addition loss of Foxo1function precluded Notch signaling-mediated inhibition ofmyoblast differentiation [47] To elucidate the possible roleof Notch signaling in Foxo1-mediated inhibition of myoblastdifferentiation by combining coculture system transfec-tion assay chromatin immunoprecipitation assay and shortinterfering RNA (siRNA) technology authors showed thatFoxo1 physically and functionally interacted with Notch bypromoting corepressor clearance fromDNA binding proteinCSL [CBF1RBPjkSu(H)Lag-1] leading to inhibition ofmyoblast differentiation through activation of Notch targetgenes [47] Another gene Id (inhibitor of differentiation)gene is also known target of 112057225(OH)

2D3[49] Id mRNA

was constitutively expressed in rat osteoblastic osteosarcomaROS1728 cells and its level was transcriptionally suppressedby 112057225(OH)

2D3[50] 112057225(OH)

2D3exerted its negative

effect on Id1 gene transcription via the 57 bp upstreamresponse sequence (minus1146minus1090) [49] Id proteins (Id1 Id2Id3 and Id4) dimerize and neutralize the transcriptionalactivity of basic helix-loop-helix (bHLH) proteins [51] Ithas been shown that Id inhibits MyoD activity either byforming transcriptionally inactive complexes of MyoD-Idor by forming heterodimers with E-proteins and effectivelyblocking the formation of active MyoDE-protein complexes[52] At this time there are only limited data available onId expression and vitamin D during muscle developmentFor example in VDR knockout mice with abnormal muscledevelopment there were no differences in expression levelsof Id1 and Id2 [46] Therefore we cannot conclude whethervitaminD regulatesmyogenesis bymodulating Id expression

A nongenomic response to 112057225(OH)2D3is character-

ized by a rapid (the seconds to minutes range) activationof signaling cascades and an insensitivity to inhibitors oftranscription and protein synthesis [34] The rapid responseto 112057225(OH)

2D3has been hypothesized to elicit the classic

VDR translocation to the plasma membrane When treatingchick myoblasts with 112057225(OH)

2D3 translocation of VDR

from the nucleus to the plasma membrane rapidly occurredwithin 5min after the addition of 112057225(OH)

2D3[53] This

translocation was blocked by colchicine suggesting the pos-sible role of the intracellular microtubular transport system









2D3


2D3might


2D3 125D

3-MARRS (membrane-


3-MARRS




2D3rapidly promoted










2D3





2D3-inducedAkt



2D3can simul-





3concentrations in


3







3or 112057225(OH)

2D3






2D3








2D3also










2D3-





2





2or vitamin D





3[25(OH)D

3] in



112057225(OH)2D3



2D3might be


3 25(OH)D





2D3


2D3binding


3and showed that


3to 112057225(OH)

2D3




3on cell pro-


3to 112057225(OH)

2D3 However



2D3
















2D3on myoblast


2D3stim-












1984 [41]










C2C12 20 nM2 120583M [25(OH)D3]

darr darr

















2D3


2D3or 25(OH)D











2D3


2D3(01 or





2D3down-







2D3


2D3











2D3on


2D3




2D3


11 Conclusions




Acknowledgment


References






























2vitamin D




























2






3







3



2C12







3in rat









2-vitamin D




2D3membrane bind-






2-vitamin D

3signalling to



2-vitamin D

3in the C




2-vitaminD






2C12




































2S





2vitamin D























2D3rdquo





2-vitamin D

3


2C12




2C12

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









2D3


2D3might


2D3 125D

3-MARRS (membrane-


3-MARRS




2D3rapidly promoted










2D3





2D3-inducedAkt



2D3can simul-





3concentrations in


3







3or 112057225(OH)

2D3






2D3








2D3also










2D3-





2





2or vitamin D





3[25(OH)D

3] in



112057225(OH)2D3



2D3might be


3 25(OH)D





2D3


2D3binding


3and showed that


3to 112057225(OH)

2D3




3on cell pro-


3to 112057225(OH)

2D3 However



2D3
















2D3on myoblast


2D3stim-












1984 [41]










C2C12 20 nM2 120583M [25(OH)D3]

darr darr

















2D3


2D3or 25(OH)D











2D3


2D3(01 or





2D3down-







2D3


2D3











2D3on


2D3




2D3


11 Conclusions




Acknowledgment


References






























2vitamin D




























2






3







3



2C12







3in rat









2-vitamin D




2D3membrane bind-






2-vitamin D

3signalling to



2-vitamin D

3in the C




2-vitaminD






2C12




































2S





2vitamin D























2D3rdquo





2-vitamin D

3


2C12




2C12

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3or 112057225(OH)

2D3






2D3








2D3also










2D3-





2





2or vitamin D





3[25(OH)D

3] in



112057225(OH)2D3



2D3might be


3 25(OH)D





2D3


2D3binding


3and showed that


3to 112057225(OH)

2D3




3on cell pro-


3to 112057225(OH)

2D3 However



2D3
















2D3on myoblast


2D3stim-












1984 [41]










C2C12 20 nM2 120583M [25(OH)D3]

darr darr

















2D3


2D3or 25(OH)D











2D3


2D3(01 or





2D3down-







2D3


2D3











2D3on


2D3




2D3


11 Conclusions




Acknowledgment


References






























2vitamin D




























2






3







3



2C12







3in rat









2-vitamin D




2D3membrane bind-






2-vitamin D

3signalling to



2-vitamin D

3in the C




2-vitaminD






2C12




































2S





2vitamin D























2D3rdquo





2-vitamin D

3


2C12




2C12

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





2D3also










2D3-





2





2or vitamin D





3[25(OH)D

3] in



112057225(OH)2D3



2D3might be


3 25(OH)D





2D3


2D3binding


3and showed that


3to 112057225(OH)

2D3




3on cell pro-


3to 112057225(OH)

2D3 However



2D3
















2D3on myoblast


2D3stim-












1984 [41]










C2C12 20 nM2 120583M [25(OH)D3]

darr darr

















2D3


2D3or 25(OH)D











2D3


2D3(01 or





2D3down-







2D3


2D3











2D3on


2D3




2D3


11 Conclusions




Acknowledgment


References






























2vitamin D




























2






3







3



2C12







3in rat









2-vitamin D




2D3membrane bind-






2-vitamin D

3signalling to



2-vitamin D

3in the C




2-vitaminD






2C12




































2S





2vitamin D























2D3rdquo





2-vitamin D

3


2C12




2C12

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2D3


2D3binding


3and showed that


3to 112057225(OH)

2D3




3on cell pro-


3to 112057225(OH)

2D3 However



2D3
















2D3on myoblast


2D3stim-












1984 [41]










C2C12 20 nM2 120583M [25(OH)D3]

darr darr

















2D3


2D3or 25(OH)D











2D3


2D3(01 or





2D3down-







2D3


2D3











2D3on


2D3




2D3


11 Conclusions




Acknowledgment


References






























2vitamin D




























2






3







3



2C12







3in rat









2-vitamin D




2D3membrane bind-






2-vitamin D

3signalling to



2-vitamin D

3in the C




2-vitaminD






2C12




































2S





2vitamin D























2D3rdquo





2-vitamin D

3


2C12




2C12

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






1984 [41]










C2C12 20 nM2 120583M [25(OH)D3]

darr darr

















2D3


2D3or 25(OH)D











2D3


2D3(01 or





2D3down-







2D3


2D3











2D3on


2D3




2D3


11 Conclusions




Acknowledgment


References






























2vitamin D




























2






3







3



2C12







3in rat









2-vitamin D




2D3membrane bind-






2-vitamin D

3signalling to



2-vitamin D

3in the C




2-vitaminD






2C12




































2S





2vitamin D























2D3rdquo





2-vitamin D

3


2C12




2C12

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



2D3down-







2D3


2D3











2D3on


2D3




2D3


11 Conclusions




Acknowledgment


References






























2vitamin D




























2






3







3



2C12







3in rat









2-vitamin D




2D3membrane bind-






2-vitamin D

3signalling to



2-vitamin D

3in the C




2-vitaminD






2C12




































2S





2vitamin D























2D3rdquo





2-vitamin D

3


2C12




2C12

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























2vitamin D




























2






3







3



2C12







3in rat









2-vitamin D




2D3membrane bind-






2-vitamin D

3signalling to



2-vitamin D

3in the C




2-vitaminD






2C12




































2S





2vitamin D























2D3rdquo





2-vitamin D

3


2C12




2C12

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








2






3







3



2C12







3in rat









2-vitamin D




2D3membrane bind-






2-vitamin D

3signalling to



2-vitamin D

3in the C




2-vitaminD






2C12




































2S





2vitamin D























2D3rdquo





2-vitamin D

3


2C12




2C12

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































2S





2vitamin D























2D3rdquo





2-vitamin D

3


2C12




2C12

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






2D3rdquo





2-vitamin D

3


2C12




2C12

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Review Article Vitamin D Signaling in Myogenesis: Potential for Treatment of...

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Transcript of Review Article Vitamin D Signaling in Myogenesis: Potential for Treatment of...