1 wnt/β-catenin signaling regulates multiple steps of myogenesis
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WNT/β-catenin signaling regulates multiple steps of myogenesis by regulating step-specific 1
targets 2
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Akiko Suzuki1,2, Richard C. Pelikan2, and Junichi Iwata1,2,3,* 4
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1Department of Diagnostic & Biomedical Sciences, and 2Center for Craniofacial Research, the 6
University of Texas Health Science Center at Houston School of Dentistry; 3The University of 7
Texas Graduate School of Biomedical Sciences at Houston, USA 8
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Competing financial interests 10
The authors declare no competing financial interests 11
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*Address correspondence to Junichi Iwata, Junichi.Iwata@uth.tmc.edu. 13
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Running title: WNT/β-catenin signaling regulates muscle development 15
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Keywords: WNT/β-catenin signaling, cell cycle, myoblast fusion, muscle homeostasis 17
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The word count for the Materials and Methods section: 1,177 words (7,328 characters excluding 19
spaces) 20
The combined word count for the Introduction, Results, and Discussion sections: 4,156 words 21
(24,976 characters excluding spaces) 22
MCB Accepted Manuscript Posted Online 9 March 2015Mol. Cell. Biol. doi:10.1128/MCB.01180-14Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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ABSTRACT 23
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Molecules involved in WNT/β-catenin signaling show specific spatiotemporal expression and 25
play vital roles in myogenesis; however, it is still largely unknown how WNT/β-catenin 26
signaling regulates each step of myogenesis. Here, we show that WNT/β-catenin signaling can 27
control diverse biological processes of myogenesis by regulating step-specific molecules. In 28
order to identify the temporally specific roles of WNT/β-catenin signaling molecules in muscle 29
development and homeostasis, we used in vitro culture systems for both primary mouse 30
myoblasts and C2C12 cells, which can differentiate into myofibers. We found that a blockade of 31
WNT/β-catenin signaling in the proliferating cells decreases proliferation activity, but does not 32
induce cell death, through the regulation of genes cyclin A2 (Ccna2) and cell division cycle 25C 33
(Cdc25c). During muscle differentiation, the inhibition of WNT/β-catenin signaling blocks 34
myoblast fusion through the inhibition of the Fermitin family homolog 2 (Fermt2) gene. 35
Blocking WNT/β-catenin signaling in the well-differentiated myofibers results in the failure of 36
maintenance of their structure by disruption of cadherin/β-catenin/actin complex formation, 37
which plays a crucial role in connecting a myofiber’s cytoskeleton to the surrounding 38
extracellular matrix. Thus, our results indicate that WNT/β-catenin signaling can regulate 39
multiple steps of myogenesis, including cell proliferation, myoblast fusion, and homeostasis, by 40
targeting step-specific molecules. 41
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INTRODUCTION 42
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Skeletal muscle plays highly specialized roles in the generation of force and is capable of 44
extensive metabolic and functional plasticity. Skeletal muscle also exhibits robust regenerative 45
capacity, and its formation is composed of complex and highly regulated processes among 46
embryonic development and regeneration in mature skeletal musculature (1). Muscle precursor 47
cells (aka satellite cells in the adult) lie under, or are embedded in, the basal lamina of the 48
myofiber in skeletal muscles and are the major source of regeneration and growth of skeletal 49
muscles (2). During development and regeneration in response to injury or recovery from 50
atrophy, muscle precursor cells start to proliferate, at which stage they are referred to as 51
myoblasts, and subsequently differentiate to form new myofibers or fuse with existing myofibers 52
(2). These processes require fine-tuned regulations of proliferation and differentiation that are 53
likely regulated by signal transduction pathways. 54
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The WNT family (wingless-type MMTV integration site family) of signaling molecules 56
consists of 21 secreted glycoprotein ligands. WNT ligands are essential to activate downstream 57
pathways (canonical β-catenin-dependent and/or non-canonical β-catenin independent pathways) 58
in physiological and pathological conditions. WNT/β-catenin signaling can regulate a variety of 59
genes in a specific spatiotemporal manner (3). In the absence of WNT ligands, β-catenin is 60
incorporated into a protein complex containing AXIN, adenomatous polyposis coli (APC) and 61
the serine-threonine kinase glycogen synthase kinase-3 (GSK3β), which is hereafter termed the 62
“destruction complex”. The destruction complex phosphorylates β-catenin and leads it to a 63
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degradation process by the ubiquitin-proteasome (4). In the presence of WNT ligands, WNT 64
ligands bind to a Frizzled receptor (FZD) and the low-density lipoprotein receptor-related protein 65
5/6 (LRP5/6), followed by the recruitment of AXIN to LRP5/6 (5). Subsequently, the destruction 66
complex is inactivated, and β-catenin can escape from its incorporation into the destruction 67
complex, resulting in its stabilization and translocation into the nucleus (4). Increased nuclear 68
concentrations of β-catenin result in induction of target genes by interacting with transcriptional 69
co-activators such as members of the T-cell factor/Lymphocyte-enhancement factor-1 70
(TCF/LEF-1) family (6). In addition, cytoplasmic accumulated β-catenin is involved in cell-cell 71
interactions in combination with cadherin and actin (7). 72
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WNT signaling is essential for a variety of developmental and regenerative processes 74
including embryonic muscle development and maintenance of skeletal muscle homeostasis in the 75
adult (8-10). For example, WNT ligands regulate the specification of skeletal myoblasts in the 76
paraxial mesoderm and induce location-specific expression of muscle regulatory factors (11). 77
Treatment with secreted Frizzled-related protein 3 (sFRP3), a soluble WNT antagonist, reduces 78
skeletal myogenesis in a dose-dependent fashion (12). Indeed, mice with deficiency of WNT/β-79
catenin signaling (Ctnnb1-/- mice) are embryonic lethal by E8.5 with increased cell death (13). 80
Mice with conditional depletion of β-catenin in the muscle precursor Pax7+ cell lineage 81
(Ctnnb1F/F;Pax7-Cre mice) show reduced muscle mass and slow myofibers (14). Mice with 82
expression of a constitutively active stabilized form of β-catenin in the Pax7+ lineage 83
(CtnnbΔex3;Pax7-Cre mice) exhibit reduced myogenesis but increased slow myofibers (14, 15). 84
Thus, dysregulation of WNT/β-catenin signaling can lead to severe developmental defects and 85
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perturbation of muscle homeostasis. Nevertheless, the temporally specific roles of WNT/β-86
catenin signaling during myogenesis remain unknown. 87
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The multiple steps of muscle development and regeneration, beginning with muscle 89
progenitor cell activation and ending with myofiber formation, are all subject to separate levels 90
of regulation and are affected by a variety of muscle disorders and atrophy (2, 14). In this study, 91
we investigated the role of WNT/β-catenin signaling in muscle biology, including cell 92
proliferation, differentiation, and homeostasis of muscle cells. We used both primary myoblasts 93
and C2C12 cells (a myoblast cell line) that have the ability to differentiate into myofibers in the 94
culture with differentiation inducers. This process provides an opportunity to investigate the 95
temporally specific role of WNT/β-catenin signaling during myogenesis. We found that WNT/β-96
catenin signaling can regulate multiple steps of muscle development, ranging from cell 97
proliferation to homeostasis, through the regulation of step-specific targets. Knowledge of the 98
temporally specific regulatory mechanism may be applied to therapeutic approaches to stimulate 99
effective skeletal muscle regeneration following muscle trauma or atrophy. 100
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MATERIALS AND METHODS 103
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Cell culture. C2C12 cells, a murine skeletal muscle cell line, were obtained from the American 105
Type Culture Collection (ATCC; CRL-1772). Primary myoblasts were isolated from the limb 106
and tongue of C57B6/J mice, as described previously (16). Briefly, for preparation of primary 107
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myoblasts, hind limb muscle and tongue were dissected out from E15.5 C57B6/J mouse embryos 108
and digested by a 1.8 U/ml dispase solution (Gibco) for one hour at 37 °C and 5 % CO2. 109
Digested tissues were then suspended with growth medium [Dulbecco’s Modified Eagle’s 110
Medium (DMEM) supplemented with 10% fetal bovine serum, penicillin, streptomycin, 2 mM 111
L-glutamate, 1 mM sodium pyruvate, and nonessential amino acids], and cells were collected by 112
centrifugation. Resuspended cells in growth medium were placed into a cell culture dish coated 113
with human fibronectin (BD BiocoatTM, BD Falcon) and cultured at 37 °C and 5 % CO2 in a 114
humidified incubator. Cell proliferation assays were performed using a cell counting kit (Dojindo 115
Molecular Technologies). Cells were treated with IWR1-endo (Tocris Bioscience, Bristol, UK) 116
at indicated concentration (0–80 µM) for 24 to 48 hours. BrdU incorporation assays (BrdU 117
incorporation for the last 1 hour) were performed using cells treated with vehicle or 80 µM 118
IWR1-endo for 36 hours. Incorporated BrdU was stained with a rat polyclonal antibody against 119
BrdU (Abcam), as described previously (17, 18). Myogenic differentiation was induced under 120
muscle differentiation medium [DMEM supplemented with 2% horse serum, 2 mM L-glutamate, 121
penicillin, streptomycin, and insulin (100 ng/ml)] for indicated days. To examine the effect of 122
IWR1-endo on myogenic differentiation, cells were treated with IWR1-endo for three to five 123
days at indicated concentration (0–10 µM). To investigate the effect of IWR1-endo on 124
maintenance of myofibers, well-differentiated C2C12 cells were cultured with vehicle or 1 μM 125
IWR1-endo under differentiation medium for another three to five days. The siRNA knockdown 126
for Ctnnb1 (Invitrogen), Ccna2, Cdc25c, and Fermt2 (Sigma-Aldrich) was performed as 127
described previously (17, 19). The overexpression of Fermt2 (OriGene Technologies, Inc., 128
Rockville, MD) was also performed as described previously (17). 129
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Tongue organ culture. Timed-pregnant C57B6/J mice were sacrificed at E14.5. Each 131
tongue was micro-dissected out and cultured in serum-free chemically defined BGJb medium 132
supplemented with penicillin, streptomycin, and 100 µg/ml ascorbic acid (16, 17). Tongue 133
explants were treated with vehicle or 80 µM IWR1-endo for 24 hours for BrdU incorporation 134
assay or for 72 hours for differentiation assay and then harvested, fixed in 4% 135
paraformaldehyde/0.1 M phosphate buffer (pH 7.4), and processed to analyze histology. BrdU 136
staining was performed as described previously (17). Immunohistological staining for myosin 137
heavy chain (MYH) was also performed as described previously (16, 20). 138
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Evaluation of myoblast fusion. After a five-day culture under the differentiation 140
medium with vehicle or IWR1-endo (1 µM), C2C12 cells were stained with MYH and 141
counterstained with DAPI for nuclei. The extent of fusion was calculated using the fusion index 142
(21, 22): Fusion % = (the number of nuclei in multiple nuclei myotubes) per (total number of 143
nuclei in MYH positive cells and myotubes) x 100. 144
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Immunoprecipitation. Extracts in cell lysis buffer containing 10 mM Tris (pH 7.5), 100 146
mM NaCl, 1% Triton X-100, and CompleteTM protease inhibitor (Roche Diagnostics) were lysed. 147
Immunoprecipitation assays were performed as described previously (17). A mouse monoclonal 148
antibody against pan-cadherin (Abcam) and a rabbit polyclonal antibody against actin (Abcam) 149
were used for immunoprecipitation assays. 150
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Immunoblotting. Immunoblots were performed, as described previously (18), using 152
rabbit polyclonal antibodies against cleaved caspase 3, active β-catenin, phosphorylated β-153
catenin (Cell Signaling Technology), and actin (Abcam) and mouse monoclonal antibodies 154
against pan-cadherin (Abcam) and GAPDH (Millipore). 155
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Immunofluorescent analysis. Immunofluorescent analysis was performed, as described 157
previously (20), using antibodies as follows: a rabbit polyclonal antibody against active β-catenin 158
(Cell Signaling Technology) and mouse monoclonal antibodies against MYH (Sigma-Aldrich) 159
and pan-cadherin (Abcam). DyLightTM 554-conjugated Phalloidin was used for staining F-actin 160
(Cell signaling Technology). Fluorescent images were taken by an inverted fluorescent 161
microscope (IX71, Olympus). Confocal images were taken by a laser confocal scanning 162
microscope (IX81 Fluoveiw FV1000, Olympus). 163
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Quantitative RT-PCR. For cell proliferation assays, total RNAs isolated from C2C12 165
cells treated with 80 µM IWR1-endo, or control vehicle, for 36 hours (n=6 per group), were 166
dissected with a QIAshredder and RNeasy mini extraction kit (Qiagen), as described previously 167
(19, 23). Gene expression profiling for cell cycle regulators was performed by PCR arrays 168
(PrimePCR assay, BioRad laboratories). The PCR primers were purchased from BioRad. For 169
myoblast fusion assays, total RNAs isolated from C2C12 cells cultured under differentiation 170
medium with 1 µM IWR1-endo or control vehicle for 12 hours to five days (n=6 per group) were 171
dissected with the QIAshredder and RNeasy mini extraction kit (Qiagen), as described 172
previously (16). For time-course experiments, C2C12 cells were treated with 0.1 μg/ml WNT3A 173
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for the indicated time with 80 µM IWR1-endo or control vehicle for cell proliferation and with 1 174
µM IWR1-endo or control vehicle for differentiation. The following PCR primers were used for 175
further specific analysis: Ccna2, 5’-TTGCTCCTCTTAAGGACCTTCC-3’ and 5’-176
CATTTAACCTCCATTTCCCTAAGGT-3’; Cdc25c, 5’-TGACGTCTATAGCCCCACC-3’ and 177
5’-TAAGCGGAGAGGCAGACATC-3’; Fermt2, 5’-GATCACTTTGGAAGGCGGGA-3’ and 178
5’-GCGCGTACTGCTTCTCGTTA-3’; and Gapdh, 5’-AACTTTGGCATTTGGAAGG-3’ and 179
5’-ACACATTGGGGGTAGGAACA-3’. The PCR primers for Ctnnb1 were purchased from 180
Santa Cruz Biotechnology, Inc. 181
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Reporter assay. Cignal TCF/LEF reporter construct (Qiagen) was transfected in C2C12 183
cells using Lipofectamine 3000 (Invitrogen). C12C12 cells were cultured under growth medium 184
or differentiation medium with/without 0.1 μg/ml WNT3A (R&D) and IWR1-endo (80 µM for 185
cell proliferation and 1 µM for differentiation) for 24 hours following the manufacturer’s 186
protocol. Luciferase assays were performed by the Dual-Glo® Luciferase assay system 187
(Promega). 188
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Comparative analysis of transcription factor binding site. The UCSC genome 190
browser was used to obtain the genomic sequences of the following murine genes including 5 191
kbp upstream of the respective transcription start site: Ccna2 (NM_009828.2), Ccnb1 192
(NM_172301.3), Cdc25c (NM_009860.2), Ccnd2 (NM_009829.3), Cdk1 (NM_007659.3), Cdk4 193
(NM_009870.3), Cdkn1a (NM_007669.4), Dmd (NM_007868.5), E2f5 (NM_007892.2), and 194
Fermt2 (NM_146054.2). These sequences were then mapped to seven additional mammalian 195
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genomes [human (Build 38), chimpanzee (Build 2.1.4), orangutan (Build 2.0.2), rhesus macaque 196
(Build 1.0), rat (Build 5), dog (Build 3.1), and horse (Build equCab2)] through the BLAST tool, 197
as described previously (16, 19). Multiple alignments for these sequences were obtained using 198
the Clustal Omega tool with default parameters and settings (24). Binding motifs of LEF1 199
(minimal core sites: 5’-CTTTG-3’or 5’-CAAAG-3’; optimal sites: 5’-CTTTGWW-3’ or 5’-200
WWCAAAG-3’, W=A/T) (25-27) were searched in the aligned DNA sequences. 201
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Chromatin immunoprecipitation (ChIP) assay. Cell extracts were incubated with β-203
catenin antibody (Abcam) overnight at 4°C followed by precipitation with magnetic beads. 204
Washing and elution of the immune complexes, as well as precipitation of DNA, were performed 205
according to standard procedures, as described previously (16, 19). The putative LEF1 target 206
sites of the genes in the immune complexes were detected by PCR using the following primers: 207
Ccna2 gene, 5’-TGGTGTTGCAGATCTACCGT-3’ and 5’-208
TCTGCTAACAAAATGGCAATGC-3’ (-2364 to -2154); Cdc25c gene, 5’-209
TTCATCGGTCTCAGCTTCCC-3’ and 5’-AGTCACCACTGAGCCTTGTC-3’ (-3164 to -210
3029); and Fermt2 gene, 5’-TAGAGGTTCTAGCGGGGGTT-3’ and 5’-211
TTCAGGCCTTGGCTTTGAGT-3’ (-3138 to -2949). Positions of PCR fragments correspond to 212
NCBI mouse genome Build 38 (mm10). 213
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Statistical analysis. Two-tailed student’s t tests were applied for statistical analysis. A p 215
value ≤ 0.05 was considered statistically significant. For all graphs, data are represented as mean 216
± standard deviation (SD). 217
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RESULTS 219
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WNT/β-catenin signaling regulates cell proliferation activity through Ccna2 and Cdc25c 221
expression. 222
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We have recently reported that upregulation of Dkk1/4, WNT/β-catenin antagonists, 224
inhibits muscle development (20), suggesting that WNT/β-catenin signaling plays a role in 225
muscle development. To explore how WNT/β-catenin signaling regulates myogenesis, we used a 226
cell culture system of C2C12 cells, which are an immortalized mouse myoblast cell line 227
originally established from normal adult C3H mouse hind limb muscle. C2C12 cells express 228
WNT ligands during cell proliferation and differentiation (28). We performed cell proliferation 229
assays using C2C12 cells treated with or without WNT/β-catenin inhibitor IWR1-endo (0-80 230
μM) that can promote AXIN stabilization and thereby inhibit WNT signaling (29). Cell 231
proliferation activity was decreased by IWR1-endo treatment in the dose-dependent manner (Fig. 232
1A and B). To validate cell proliferation defects after treatment with IWR1-endo (80 μM) for 36 233
hours, we performed BrdU incorporation assay. Cells incorporated BrdU were reduced by the 234
treatment with IWR1-endo (Fig. 1C and D). We further confirmed that IWR1-endo had no 235
toxicity on C2C12 cells at the concentrations we tested. We detected no apoptosis with treatment 236
of 80 μM IWR1-endo (Fig. 1E). IWR1-endo specifically inhibits WNT/β-catenin signaling by 237
induction of the β-catenin destruction complex formation (29). As expected, non-phosphorylated 238
(active) β-catenin was decreased, and phosphorylated (inactive) β-catenin was increased at 12 239
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and 24 hours after the treatment with 80 μM IWR1-endo (Fig. 1F). To test whether IWR1-endo 240
specifically blocks WNT/β-catenin signaling cascade through transcriptional response elements 241
of LEF/TCF, which is a well-known β-catenin co-transcription factor, we performed LEF/TCF 242
luciferase reporter assays after treatment with WNT3A (0.1 μg/ml) and IWR1-endo (80 μM) for 243
24 hours using proliferating cells. WNT3A induced LEF/TCF-mediated promoter activity 244
approximately six times, and IWR1-endo (80 μM) completely inhibited the induction of 245
LEF/TCF-mediated promoter activity (Fig. 1G). These results indicate that IWR1-endo inhibits 246
canonical WNT/β-catenin signaling. 247
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Next, to identify downstream targets of WNT/β-catenin signaling, we performed PCR 249
array analyses of C2C12 cells treated with or without 80 μM IWR1-endo for 36 hours (n=5 per 250
group). In this analysis, we uncovered 7 genes that were differentially expressed (≥1.5-fold, 251
p<0.05), 2 genes that were more abundant in the treated group, and 5 genes that were more 252
abundant in the control group (Table 1). The genes altered in the treated group were consistent 253
with cell proliferation defects, with significant reductions in the expression levels of transcripts 254
related to cell cycle transition. We analyzed the promoter sequence (including 5-kbp upstream of 255
the transcription start site) of the candidate target genes. We found that both Ccna2 and Cdc25c 256
genomic regions have a phylogenetically conserved LEF1 recognition sequence (Tables 2 and 3). 257
To test whether β-catenin could bind to the promoter regions of Ccna2 and Cdc25c, we 258
performed chromatin immunoprecipitation (ChIP) assays. The binding site for LEF1 in each 259
gene was immunoprecipitated with β-catenin antibody but not with control IgG. Furthermore, the 260
immunoprecipitated amounts were decreased after the treatment with 80 μM IWR1-endo for 24 261
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hours in both Ccna2 and Cdc25c, indicating that the binding site for LEF1 in each Ccna2 and 262
Cdc25c gene is functional and responsive to WNT/β-catenin signaling activity (Fig. 1H and I). 263
Importantly, gene expression of Ccna2 and Cdc25c was induced by WNT3A ligands (0.1 μg/ml) 264
in the time dependent manner (Fig. 1J and K). Additionally, we analyzed cell proliferation 265
activity after treatment with siRNA knockdown for Ccna2, Cdc25c, and Ccna2/Cdc25c 266
combination. Gene expression of Ccna2 and Cdc25c was significantly reduced after the siRNA 267
knockdown by 80 % and 60 % reduction, respectively (Fig. 1L). Cell proliferation activity was 268
significantly reduced after the knockdown of Ccna2, Cdc25c, or Ccna2/Cdc25c combination 269
(Fig. 1M). Furthermore, we analyzed gene expression of Ccna2 and Cdc25c after siRNA 270
knockdown for Ctnnb1 (Fig. 1N and O). Cell proliferation activity was significantly reduced 271
after the knockdown for Ctnnb1 (Fig. 1P and Q). Taken together, our results indicate that gene 272
expression of Ccna2 and Cdc25c is regulated by the WNT/β-catenin pathway, and CCNA2 and 273
CDC25C play a crucial role during myoblast proliferation. 274
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WNT/β-catenin signaling regulates muscle differentiation through Fermt2 expression. 276
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Next, we investigated myogenic differentiation of C2C12 cells under a differentiation 278
medium with (at both 1 and 10 μM) and without IWR1-endo. Without the IWR1-endo treatment, 279
we could detect myofibers after a three-day culture in the differentiation medium. After a five-280
day culture in the differentiation medium without IWR1-endo, C2C12 cells were well-developed 281
into multinucleated myofibers. In contrast, C2C12 cells cultured in the differentiation medium 282
with 1 μM IWR1-endo were mostly mononucleated and failed to differentiate into myotubes. 283
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This suggests that WNT/β-catenin signaling is crucial for myoblast fusion, a critical process for 284
the terminal differentiation of skeletal muscle. We further tested higher concentrations of IWR1-285
endo. We found that treatment with IWR1-endo at 10 μM and higher blocked myoblast 286
differentiation of C2C12 cells (Fig. 2A). To quantify the fusion defect with treatment of 1 μM 287
IWR1-endo, we counted the number of nuclei in the cells stained with a myosin heavy chain 288
(MYH), a differentiation marker for myofibers. In the control, almost 100% of muscle cells had 289
more than 3 nuclei [as shown by fusion index (ratio of muscle cells with multiple nuclei per all 290
muscle cells)]. In contrast, approximately 90% of the cells treated with 1 μM IWR1-endo had 291
single nuclei in each cell (Fig. 2B and C). Consistent with the IWR1-endo induced fusion defect, 292
the length of muscle fibers in the treated group was significantly shorter than those in the control 293
group (approximately 1.4 mm in control vs. approximately 0.1 mm in the treated group) (Fig. 294
2D). Furthermore, we confirmed that IWR1-endo treatment decreased non-phosphorylated 295
(active) β-catenin and increased phosphorylated (inactive) β-catenin at 1 and 10 μM (Fig. 2E). 296
Importantly, the treatment with either 1 or 10 μM IWR1-endo induced no apoptosis in the 297
process of muscle differentiation (Fig. 2F). To validate further that IWR1-endo treatment had no 298
toxic effects on myoblasts, we cultured C2C12 cells with 1 μM IWR1-endo under the 299
differentiation medium for five days (fusion defects at Day 5 treated with IWR1-endo) and then 300
switched the medium with or without 1 μM IWR1-endo for another five days (Fig. 2G). The 301
inhibited myoblast fusion at Day 5 was completely restored at Day 10 without IWR1-endo 302
(IWR1-endo was removed from the culture medium after five days of the culture), indicating that 303
WNT/β-catenin signaling specifically inhibits myoblast fusion but does not induce apoptosis. 304
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After this, we explored target molecules which were involved in myoblast fusion and 306
altered by the 1 μM IWR1-endo treatment at Day 5. Among 11 candidate molecules (Nphs1, 307
Ptk2, Fermt2, Adam12, Itga3, Itgb1, Cdh2, Cdh15, Myof, Dysf, and Tmem8c), gene expression of 308
Fermitin family homolog 2 (Fermt2, aka Kindlin2) was significantly decreased by the treatment 309
of 1 μM IWR1-endo (1.8-fold reduction, p<0.01) (Fig. 3A). Decreased Fermt2 expression at Day 310
2 was restored at Day 7 after the depletion of 1 μM IWR1-endo, indicating that Fermt2 311
expression is regulated by WNT/β-catenin signaling (Fig. 3B). To test whether IWR1-endo 312
specifically blocks WNT/β-catenin signaling cascade through transcriptional response elements 313
of LEF/TCF, we performed LEF/TCF luciferase reporter assays after treatment with WNT3A 314
(0.1 μg/ml) and IWR1-endo (1 μM) at the muscle differentiation stage. WNT3A induced 315
LEF/TCF-mediated promoter activity approximately six times, and IWR1-endo (1 μM) 316
completely inhibited the induction of LEF/TCF-mediated promoter activity (Fig. 3C). These 317
results indicate that IWR1-endo inhibits canonical WNT/β-catenin signaling. FERMT2, a focal 318
adhesion protein, can regulate integrin activation and mediate cell-cell or cell-matrix adhesion 319
(30, 31). We analyzed the promoter sequence of the Fermt2 gene (including 5-kbp upstream of 320
the transcription start site). We found that the murine Fermt2 genomic region has a conserved 321
LEF1 recognition sequence (Tables 1 and 2). To test whether β-catenin could bind to the 322
promoter region of Fermt2, we performed ChIP assays. The binding site for LEF1 in the 323
promoter region of Fermt2 was immunoprecipitated with β-catenin antibody but not with control 324
IgG. In addition, the immunoprecipitated amounts were decreased by treatment with 1 μM 325
IWR1-endo for 48 hours, indicating that the binding site for LEF1 in the Fermt2 promoter region 326
is functional and responsible for WNT/β-catenin signaling activity (Fig. 3D and E). Gene 327
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expression of Fermt2 was induced by WNT3A ligands (0.1 μg/ml) in the time dependent manner 328
(Fig. 3F). Furthermore, gene expression of Fermt2 was significantly reduced after siRNA 329
knockdown for Ctnnb1 (Fig. 3G and H). Myoblast fusion was disrupted by the knockdown of 330
Ctnnb1 (Fig. 3I). Taken together, our results indicate that gene expression of Fermt2 is regulated 331
by the WNT/β-catenin pathway. 332
333
To validate the function of Fermt2 during muscle differentiation, we cultured C2C12 334
cells under the differentiation medium with treatment of siRNA knockdown for Fermt2. Gene 335
expression of Fermt2 was significantly reduced after the siRNA knockdown at 80 % reduction 336
(Fig. 3J). Myoblast fusion was disrupted by the knockdown of Fermt2 (Fig. 3K). In addition, 337
overexpression of Fermt2 in the condition of 1 μM IWR1-endo treatment restored the myofiber 338
fusion defect (Fig. 3L and M). Altogether, our results indicate that gene expression of Fermt2 is 339
regulated by WNT/β-catenin pathway, and Fermt2 plays a crucial role in myoblast fusion. 340
341
The cadherin/β-catenin/actin complex formation mediated by WNT/β-catenin signaling is 342
crucial for muscle homeostasis. 343
344
To test if WNT/β-catenin signaling is involved in muscle homeostasis, we treated well-345
differentiated C2C12 cells with or without 1 μM IWR1-endo (Fig. 4A and B). The long and 346
straightened myofibers were disrupted and changed their form to a round, multinucleated shape 347
after the IWR1-endo treatment (Fig. 4B). Next, we analyzed gene expression of the molecules 348
related to cytoskeletal structure formation (MYH1/4, caveolin 3, and desmin); however, we 349
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failed to identify genes altered by the treatment with 1 μM IWR1-endo (Fig. 4C), which suggests 350
that the WNT/β-catenin pathway may regulate cytoskeletal structure formation at the 351
posttranscriptional level. Previous studies have demonstrated that β-catenin can bind to both 352
cadherin and actin (32, 33). An extracellular matrix niche is crucial for regulating the movement 353
and morphology of skeletal muscle cells (34). We hypothesized that the cadherin/β-catenin/actin 354
complex plays a crucial role in myofiber morphogenesis by anchoring skeletal structure 355
molecules to the connective tissue (niche) composed of an extracellular matrix. To test the 356
molecular interaction among them, we performed immunoprecipitation for actin, cadherin, and 357
β-catenin (Fig. 4D). Our results indicate that cadherin/β-catenin/actin complex existed in 358
myofibers and that it was disrupted by the treatment with 1 μM IWR1-endo. Thus, β-catenin is 359
crucial for forming the cadherin/β-catenin/actin complex. In addition, we confirmed that 1 μM 360
IWR1-endo induced no apoptosis at the maturation/maintenance stage of muscle cells (Fig. 4E). 361
To further validate our findings of the complex formation regulated by WNT/β-catenin signaling, 362
we performed immunofluorescent analyses of these complex molecules with or without 363
treatment of 1 μM IWR1-endo. Actin stained with phalloidin was straightened and paralleled 364
each other in control. In contrast, actin formation was disrupted and accumulated after the 365
treatment with 1 μM IWR1-endo (Fig. 4F). Without IWR1-endo treatment, cadherin and β-366
catenin were co-localized in the myofibers. In contrast, there was poor co-localization of 367
cadherin and β-catenin after the treatment with 1 μM IWR1-endo (Fig. 4G and H). Thus, our 368
data indicate that β-catenin is an essential binding partner for both the cytoplasmic tail of 369
cadherin and actin and is crucial for the maintenance of myofiber morphology. 370
371
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Molecular mechanisms of myogenesis mediated by WNT/β-catenin signaling are conserved 372
in primary muscle cells. 373
374
We tested whether the mechanisms we found in C2C12 cells were conserved in primary 375
muscle cells from C57B6/J mice. We isolated primary muscle cells from the limb and tongue of 376
mice. We performed BrdU incorporation assays to test cell proliferation activity with or without 377
treatment of 80 μM IWR1-endo. In primary myoblasts from both the limb and tongue, IWR1-378
endo reduced cell proliferation activity (Fig. 5A-C). Gene expression of Ccna2 and Cdc25c was 379
significantly decreased by treatment of IWR1-endo in both primary limb and tongue myoblasts 380
(Fig. 5D). Then, we investigated muscle differentiation of primary myoblasts from both the limb 381
and tongue. The treatment with 1 μM IWR1-endo strikingly inhibited myoblast fusion in both 382
primary limb and tongue myoblasts. Gene expression of Fermt2 was significantly reduced by the 383
treatment with IWR1-endo (Fig. 5F). Myoblasts differentiated into myofibers within seven days 384
in a culture under the differentiation medium in the control. In contrast, treatment with 1 μM 385
IWR1-endo blocked myotube formation by inhibiting myoblast fusion (Fig. 5G). Next, we 386
investigated the maintenance ability of muscle cells. The well-differentiated and straightened 387
myofibers changed their shape after treatment with 1 μM IWR1-endo, as seen in C2C12 cells 388
(Fig. 5H). These results indicate that WNT/β-catenin-mediated signaling mechanisms are well 389
conserved in primary muscle cells. Furthermore, to test if the mechanisms were conserved in a 390
muscular tissue, we cultured tongue explants from C57B6/J mice with or without IWR1-endo. In 391
the tongue explants, IWR1-endo treatment (80 μM, 24 hours) resulted in decreased cell 392
proliferation, as seen in our cell culture system (Fig. 6A and B). In addition, IWR1-endo 393
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treatment (80 μM, 72 hours) resulted in defects in the elongation of muscle fibers (Fig. 6C). 394
Collectively, these results suggest that WNT/β-catenin signaling can regulate multiple steps of 395
muscle development, including proliferation, differentiation, and structural homeostasis, by 396
regulating step-specific targets (Fig. 6D). 397
398
DISCUSSION 399
400
Mice with deficiency in WNT ligands and their receptors FZDs show early embryonic lethality, 401
often affecting multiple tissues (3, 10). WNT/β-catenin signaling is active within myogenic cells, 402
as shown by BAT-gal mice, in which seven TCF/LEF-binding sites drive nuclear LacZ 403
expression when bound by transcriptionally active TCF/LEFs (35). During axial myogenesis, 404
WNT/β-catenin signaling is crucial for dermomyotome and myotome formation (14, 36-38). 405
WNT ligands can positively regulate the number of dermomyotomal Pax3+ and Pax7+ 406
progenitors (39-41). During fetal myogenesis, β-catenin affects myofiber number and positively 407
regulates the number of slow fibers in mice with conditional deletion of Ctnnb1 in muscle 408
progenitor cells (Pax7iCre/+;Ctnnb1Δ/fl2-6 mice) (14). Subsequently, WNT ligands are necessary 409
and sufficient to promote normal Myf5 and MyoD expression (42-45). WNT/β-catenin activation 410
also induces satellite cell proliferation during skeletal muscle regeneration (46). Compared with 411
studies on early muscle specification as described above, relatively little is known about prenatal 412
and postnatal myogenesis. 413
414
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In this study, we investigated how WNT/β-catenin signaling regulates muscle biology 415
using both primary myoblasts and C2C12 cells. We found that WNT/β-catenin signaling can 416
regulate specific targets crucial for each myoblast proliferation, fusion, and homeostasis of 417
myofibers. We also found that during myoblast proliferation, gene expression of Ccna2 and 418
Cdc25c was specifically downregulated by the inhibition of WNT/β-catenin signaling by IWR1-419
endo. Ccna2 is ubiquitously expressed and is essential for cell-cycle progression of embryonic 420
stem cells (47). Mice with loss of Ccna2 exhibit early embryonic lethality (48). In cancer, Ccna2 421
is often aberrantly expressed and impacts cell proliferation (49). The CDC25 protein 422
phosphatases (CDC25A, -B, and -C) drive cell cycle transitions by activating key components of 423
the cell cycle engine (50). Mice with loss of Cdc25c exhibit normal development, suggesting that 424
the other CDC25 protein phosphatases can compensate for its function during embryogenesis 425
(51). Indeed, mice lacking Cdc25a or all three Cdc25 genes are early embryonic lethal with 426
growth retardation by E7.5 (50). Although WNT/β-catenin signaling targets gene expression of 427
both Ccna2 and Cdc25c, gene expression of Ccna2 mediated by WNT/β-catenin signaling may 428
be more important for myoblast proliferation in vivo. 429
430
A recent study suggests a potential role of WNT/β-catenin signaling in myoblast fusion 431
(52). Members of the R-spondin family (RSPO) of secreted cysteine-rich proteins (RSPO1, -2, -3, 432
and -4) activate the WNT/β-catenin signaling pathway at the receptor level (53, 54). RSPO2 433
promotes myogenic differentiation and myocyte fusion via activation of WNT/β-catenin 434
signaling in C2C12 cells (55). RSPOs can regulate WNT receptor expression and control 435
myogenesis through gene expression of the TGFβ antagonist Fst (56). Thus, WNT signaling is 436
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activated through WNT ligands, RSPO, and non-canonical (β-catenin-independent) WNT 437
signaling activators during muscle development. During myoblast fusion, the cell signaling 438
pathway is tightly regulated and controls downstream target molecules involved in membrane 439
fusion and cytoskeletal remodeling (57). For example, a recent study indicates that Tmem8c (aka 440
Myomaker) specifically expresses in skeletal muscles and plays a crucial role in myoblast fusion 441
during muscle development (58). In our study, the bioinformatics analysis of Tmem8c promoter 442
region (up to 5-kb upstream of transcription start site of Tmem8c gene) detected no TCF/LEF 443
optimal binding site (WNT/ β-catenin response element). In addition, gene expression of 444
Tmem8c was not altered by IWR1-endo treatment. These data suggest that gene expression of 445
Tmem8c is regulated by the other signaling pathways such as transforming growth factor beta 446
(TGFβ), fibroblast growth factor (FGF), and hedgehog (HH). Among 11 candidate molecules, 447
only Fermt2 expression was significantly (> 1.5-fold change, p < 0.05) decreased by IWR1-endo 448
treatment. The other molecules involved in myoblast fusion tested in this study (Nphs1, Ptk2, 449
Adam12, Itga3, Itgb1, Cdh2, Cdh15, Myof, and Dysf) may be also controlled by the other 450
signaling pathways. 451
452
A previous study shows that RNAi knockdown for Fermt2 in C2C12 cells results in 453
significant abnormalities during the early stages of myogenesis (31). In addition, targeted 454
disruption of the murine Fermt2 gene results in early embryonic lethality with musculature 455
developmental defects (59). These findings, coupled with our results, indicate that WNT/β-456
catenin-mediated Fermt2 expression is crucial for myogenic fusion. Although Fermt2 null mice 457
are lethal by the stage of muscle fusion, the future study using mice with conditional deletion of 458
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Fermt2 will allow us to investigate the role of Fermt2 during muscle fusion. Moreover, Fermt2 459
may play additional roles aside from myoblast fusion during muscle development. Interestingly, 460
different steps of muscle differentiation are also regulated by WNT/β-catenin signaling. For 461
example, inhibition of GSK3β results in activated WNT/β-catenin signaling and leads to 462
enhanced differentiation of C2 myogenic cells (60). The RSPO family of proteins, WNT/β-463
catenin signaling agonists, can promote myogenic differentiation in cell culture (55). 464
Pharmacological activations of WNT/β-catenin signaling can also facilitate the differentiation of 465
satellite cells and myoblasts (60-62). Thus, WNT/β-catenin signaling may play a pivotal role for 466
the volume and size of skeletal muscles by regulating various steps of myoblast differentiation 467
and fusion. 468
469
In this study, we found that during muscle maturation/maintenance, a blockade of 470
WNT/β-catenin signaling resulted in disrupted cell adhesion via loss of cadherin/β-catenin/actin 471
complex formation. Previous studies indicate that β-catenin plays dual roles in WNT/β-catenin 472
signaling and in cadherin-mediated cell adhesion (32, 63). Beta-catenin co-localized with 473
cadherin in membranes may be essential for proper cell structural formation. Cadherin family 474
molecules exhibit distinctly regulated tissue distribution and control morphogenesis. Skeletal 475
muscle cells express both N-cadherin (encoded by Cdh2) and M-cadherin (encoded by Cdh15) 476
throughout myogenesis, from progenitors to myofibers, in both development and regeneration 477
(64, 65). Mice with loss of Cdh2 are embryonic lethal by E10 with disorganized somites (66). 478
The in vitro studies of Cdh2-/- cells indicate that N-cadherin is not essential for myogenesis, but 479
other cadherins may compensate for the lack of N-cadherin (67). M-cadherin is a classical 480
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calcium-dependent cell adhesion molecule that is highly expressed in developing skeletal 481
muscle, satellite cells, and cerebellum (65). Mice with loss of M-cadherin exhibit no gross 482
developmental defects (68). These results strongly suggest that loss of the combination of N- and 483
M-cadherins or other cadherins, or both, is essential for myogenic defects. The actin family is 484
categorized into four muscle-specific isoforms: α-skeletal; α-cardiac; α-smooth; and γ-smooth. It 485
is further categorized into two ubiquitously expressed isoforms: β-cytoplasmic and γ-cytoplasmic 486
(69). Skeletal myoblasts almost exclusively express β- and γ-cytoplasmic actins, but on 487
differentiation, these cytoplasmic isoforms are drastically downregulated and sequentially 488
replaced by α-cardiac and α-smooth actins, and ultimately α-skeletal actin (69). Mice with 489
deficiency of α-skeletal actin are indistinguishable from their littermates at birth but die in the 490
early neonatal period (postnatal day 1 to 9) with skeletal muscle defects (70). Recent studies 491
indicate that γ-cytoplasmic actin is required for cytoskeletal maintenance but not development 492
(71, 72). Thus, actin isoforms may also largely compensate for the lack of an individual actin. 493
Compared with cadherin and actin, β-catenin may play the most critical role in the cadherin/β-494
catenin/actin complex formation. 495
496
Among skeletal muscles, tongue muscles are the most developed muscle at birth for 497
suckling, compared with the other muscles (73, 74). Interestingly, mice lacking Myf5 and Pax3 498
do not develop skeletal muscle in the trunk or limb, yet head muscles form normally (75). In 499
chick embryo, WNT signals, which promote trunk myogenesis, have been shown to block 500
myogenesis in branchial arches (76). These findings suggest that craniofacial muscle 501
development may be differently regulated compared to that of trunk and limb muscles. In this 502
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study, we investigated primary myoblasts from the tongue compared with that of the limb. We 503
found that requirement of WNT/β-catenin signaling was similar among tongue and limb 504
myogenesis. 505
506
Our findings may be relevant in human disease, given that altered WNT/β-catenin 507
signaling is implicated in multiple malformations and syndromes including muscle disorders in 508
humans (77-79). For example, in patients with muscular defects, such as myopathies and 509
atrophy, WNT/β-catenin signaling is most likely altered due to genetic and/or epigenetic cause(s) 510
(80). Our results highlight that WNT/β-catenin signaling is a widely used mechanism in skeletal 511
muscles which regulate step-specific targets during myogenesis. Our findings on the mechanisms 512
of action of WNT/β-catenin signaling offer several intriguing possibilities into the potential for 513
therapeutic interventions. 514
515
516
ACKOWLEDGMENTS 517
518
We thank Dr. Yoshihiro Komatsu for discussion. This study was supported by the UT Houston 519
School of Dentistry faculty start-up fund to Junichi Iwata. 520
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62. Brack AS, Conboy IM, Conboy MJ, Shen J, Rando TA. 2008. A temporal switch 699
from notch to Wnt signaling in muscle stem cells is necessary for normal adult 700
myogenesis. Cell stem cell 2:50-59. 701
63. Gates J, Peifer M. 2005. Can 1000 reviews be wrong? Actin, alpha-Catenin, and 702
adherens junctions. Cell 123:769-772. 703
64. Krauss RS, Cole F, Gaio U, Takaesu G, Zhang W, Kang JS. 2005. Close encounters: 704
regulation of vertebrate skeletal myogenesis by cell-cell contact. Journal of cell science 705
118:2355-2362. 706
65. Krauss RS. 2010. Regulation of promyogenic signal transduction by cell-cell contact and 707
adhesion. Experimental cell research 316:3042-3049. 708
66. Radice GL, Rayburn H, Matsunami H, Knudsen KA, Takeichi M, Hynes RO. 1997. 709
Developmental defects in mouse embryos lacking N-cadherin. Developmental biology 710
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67. Charlton CA, Mohler WA, Radice GL, Hynes RO, Blau HM. 1997. Fusion 712
competence of myoblasts rendered genetically null for N-cadherin in culture. The Journal 713
of cell biology 138:331-336. 714
68. Hollnagel A, Grund C, Franke WW, Arnold HH. 2002. The cell adhesion molecule 715
M-cadherin is not essential for muscle development and regeneration. Molecular and 716
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69. Jaeger MA, Sonnemann KJ, Fitzsimons DP, Prins KW, Ervasti JM. 2009. Context-718
dependent functional substitution of alpha-skeletal actin by gamma-cytoplasmic actin. 719
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70. Crawford K, Flick R, Close L, Shelly D, Paul R, Bove K, Kumar A, Lessard J. 2002. 722
Mice lacking skeletal muscle actin show reduced muscle strength and growth deficits and 723
die during the neonatal period. Molecular and cellular biology 22:5887-5896. 724
71. Belyantseva IA, Perrin BJ, Sonnemann KJ, Zhu M, Stepanyan R, McGee J, 725
Frolenkov GI, Walsh EJ, Friderici KH, Friedman TB, Ervasti JM. 2009. Gamma-726
actin is required for cytoskeletal maintenance but not development. Proceedings of the 727
National Academy of Sciences of the United States of America 106:9703-9708. 728
72. Sonnemann KJ, Fitzsimons DP, Patel JR, Liu Y, Schneider MF, Moss RL, Ervasti 729
JM. 2006. Cytoplasmic gamma-actin is not required for skeletal muscle development but 730
its absence leads to a progressive myopathy. Developmental cell 11:387-397. 731
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73. Noden DM, Francis-West P. 2006. The differentiation and morphogenesis of 732
craniofacial muscles. Developmental dynamics : an official publication of the American 733
Association of Anatomists 235:1194-1218. 734
74. Yamane A. 2005. Embryonic and postnatal development of masticatory and tongue 735
muscles. Cell and tissue research 322:183-189. 736
75. Tajbakhsh S, Rocancourt D, Cossu G, Buckingham M. 1997. Redefining the genetic 737
hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD. 738
Cell 89:127-138. 739
76. Tzahor E, Kempf H, Mootoosamy RC, Poon AC, Abzhanov A, Tabin CJ, Dietrich S, 740
Lassar AB. 2003. Antagonists of Wnt and BMP signaling promote the formation of 741
vertebrate head muscle. Genes & development 17:3087-3099. 742
77. Al-Qattan MM. 2011. WNT pathways and upper limb anomalies. The Journal of hand 743
surgery, European volume 36:9-22. 744
78. Kim N, Vu TH. 2006. Parabronchial smooth muscle cells and alveolar myofibroblasts in 745
lung development. Birth defects research. Part C, Embryo today : reviews 78:80-89. 746
79. He F, Chen Y. 2012. Wnt signaling in lip and palate development. Frontiers of oral 747
biology 16:81-90. 748
80. Alexander MS, Kawahara G, Motohashi N, Casar JC, Eisenberg I, Myers JA, 749
Gasperini MJ, Estrella EA, Kho AT, Mitsuhashi S, Shapiro F, Kang PB, Kunkel 750
LM. 2013. MicroRNA-199a is induced in dystrophic muscle and affects WNT signaling, 751
cell proliferation, and myogenic differentiation. Cell death and differentiation 20:1194-752
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Abbreviations 754
755
APC, adenomatous polyposis coli; ChIP, chromatin immunoprecipitation; FZD, Frizzled 756
receptor; GSK3β, serine-threonine kinase glycogen synthase kinase-3; H&E, Hematoxylin and 757
Eosin; IP, immunoprecipitation; LEF-1, Lymphocyte-enhancement factor-1; LRP5/6, low-758
density lipoprotein receptor-related protein 5/6; MyHC, myosin heavy chain; NCBI, National 759
Center for Biotechnology Information; sFRP3, secreted Frizzled-related protein 3; TCF, T-cell 760
factor. 761
762
763
FIGURE LEGENDS 764
765
FIG 1. WNT/β-catenin signaling regulates myoblast cell proliferation via Ccna2 and Cdc25c 766
expression. (A) Cell proliferation assay of C2C12 cells with IWR1-endo treatment for 0-48 hours 767
at each indicated concentration (0, 1, 5, 10, 20, 40, 60, and 80 μM). N=6 per group. ***, p<0.001. 768
(B) Cell proliferation ratio at 48 hours after treatment with IWR1-endo at indicated 769
concentrations (0-80 μM). N=6 per group. ***, p<0.001. (C) BrdU staining (brown) in C2C12 770
cells after treatment with vehicle control or 80 μM IWR1-endo for 24 hours. Nuclei are 771
counterstained with Hematoxylin. Arrows indicate BrdU positive cells. Scale bar, 50 μm. (D) 772
Quantitation of the percentage of BrdU-labeled nuclei in C2C12 cells treated with 80 μM IWR1-773
endo or vehicle control for 24 hours. N=12 per each group. ***, p<0.001. (E) Immunoblotting 774
analysis of caspase 3 in C2C12 cells treated with vehicle control (lane 1), 80 μM IWR1-endo 775
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(lane 2), or positive control (tissue sample from adult wild-type mouse small intestine, lane 3) for 776
24 hours. (F) Immunoblotting analysis of time course (0, 6, 12, and 24 hours) for activated β-777
catenin (ABC), phosphorylated β-catenin (p-β-catenin), and GAPDH in C2C12 cells after 778
treatment with vehicle control or 80 μM IWR1-endo for 24 hours. (G) Reporter assay for 779
WNT/β-catenin signaling activity after treatment with/without (+/-) WNT3A (0.1 μg/ml) and 780
IWR1-endo (80 μM) in C2C12 cells cultured in growth medium. (H) ChIP analysis of DNA 781
fragments immunoprecipitated with a β-catenin-specific antibody or with an isotype-specific 782
(IgG) control antibody after treatment with/without 80 μM IWR1-endo (IWR1) for 24 hours. 783
Immunoprecipitated products were PCR amplified with primers flanking the putative LEF1-784
binding region of indicated genes. 2% input lane shows PCR amplification of the sonicated 785
chromatin before immunoprecipitation. (I) ChIP-qPCR analysis of β-catenin binding on the 786
Ccna2 and Cdc25c promoter regions using β-catenin antibody following treatment with/without 787
80 μM IWR1-endo treatment for 24 hours. ChIP with IgG without IWR1-endo treatment 788
(orange), IgG with IWR1-endo treatment (green), α-β-catenin antibody without IWR1-endo 789
treatment (red), and α-β-catenin antibody with IWR1-endo treatment (blue). (J, K) Quantitative 790
RT-PCR analysis of gene expression of Ccna2 (J) and Cdc25c (K) after treatment with WNT3A 791
(0.1 μg/ml) for 0, 1, 2, and 3 hours (h) with/without 80 μM IWR1-endo. **, p<0.01; ***, 792
p<0.001. (L) Quantitative RT-PCR analysis after treatment with siRNA for control (orange), 793
Ccna2 (red), Ccna2/Cdc25c combination (green), and Cdc25c (blue). ***, p<0.001. (M) Cell 794
proliferation assay of C2C12 cells treated with siRNA for control (orange), Ccna2 (red), Cdc25c 795
(blue), and Ccna2/Cdc25c combination (green). ***, p<0.001. (N) Ctnnb1 expression after 796
treatment with siRNA for control (opened column) and Ctnnb1 (closed column). ***, p<0.001. 797
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(O) Quantitative RT-PCR analysis of Ccna2 and Cdc25c expression after treatment with siRNA 798
for control (opened column) and Ctnnb1 (closed column). ***, p<0.001. **, p<0.01. (P) BrdU 799
staining (brown) in C2C12 cells after treatment with control or Ctnnb1 siRNA knockdown for 24 800
hours. Nuclei are counterstained with Hematoxylin. Arrows indicate BrdU positive cells. Scale 801
bar, 50 μm. (Q) Quantitation of the percentage of BrdU-labeled nuclei in C2C12 cells after 802
treatment with control or Ctnnb1 siRNA knockdown for 24 hours. N=12 per each group. ***, 803
p<0.001. 804
805
FIG 2. WNT/β-catenin signaling regulates myoblast fusion during muscle development. (A) 806
Immunohistochemical analysis of myosin heavy chain (green) in C2C12 cells during muscle 807
differentiation (Day 3 and Day 5 after induction for muscle differentiation) with IWR1-endo at 0, 808
1, or 10 μM. Negative control was continued to culture C2C12 cells in growth medium. Cells are 809
counterstained with DAPI (blue). Scale bars 100 μm. (B) The number of nuclei (x-axis of graph) 810
was measured in MYH positive cells. Vehicle control (blue bars), 1 μM IWR1-endo treatment 811
(red bars). (C) Fusion index in cells treated with vehicle control (blue bar) or 1 μM IWR1-endo 812
(red bar). ***, p<0.001. (D) Length of muscle fibers. Vehicle control (blue bar), 1 μM IWR1-813
endo (red bar). ***, p<0.001. (E) Immunoblotting analysis of activated β-catenin (ABC) and 814
phosphorylated β-catenin (P-β-catenin) after treatment with vehicle (Control) or IWR1-endo (1 815
or 10 μM) for three days. (F) Immunoblotting analysis of caspase 3 after treatment with vehicle 816
(Control) or IWR1-endo (1 or 10 μM) for three days. PC, positive control (tissue sample from 817
adult wild-type mouse small intestine). (G) C2C12 cells were cultured under differentiation 818
medium with vehicle (control) or 1 μM IWR1-endo for five days (Day 5). The cells treated with 819
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1 μM IWR1-endo for 5 days (Day 5) were continued to culture with or without 1 μM IWR1-endo 820
for another five days (Day 10). The fusion defect by the treatment with 1 μM IWR1-endo at Day 821
5 was rescued by the removal of IWR1-endo at Day 10. 822
823
FIG 3. WNT/β-catenin signaling regulates Fermt2 expression during myoblast fusion. (A) PCR 824
array analysis after treatment with vehicle control (blue bars) or 1 μM IWR1-endo (red bars) for 825
48 hours. N=5 per treatment. **, p<0.01. (B) Quantitative RT-PCR analysis of Fermt2 after 826
treatment with vehicle control (blue) or 1 μM IWR1-endo (red) for two days (Day 2) or after 827
another two-days culture after the removal of 1 μM IWR1-endo at Day 5 of muscle 828
differentiation (Day 7, green). **, p<0.01. (C) Reporter assay for WNT/β-catenin signaling 829
activity after treatment with/without (+/-) WNT3A (0.1 μg/ml) and IWR1-endo (1 μM) in C2C12 830
cells cultured under differentiation medium. (D) ChIP analysis of DNA fragments 831
immunoprecipitated with a β-catenin-specific antibody or with an isotype-specific (IgG) control 832
antibody after treatment with/without 1 μM IWR1-endo (IWR1) for 48 hours under the 833
differentiation medium. Immunoprecipitated products were PCR amplified with primers flanking 834
the putative LEF1-binding region of Fermt2. 2% input lane shows PCR amplification of the 835
sonicated chromatin before immunoprecipitation. (E) ChIP-qPCR analysis of β-catenin binding 836
on the Fermt2 promoter region using β-catenin antibody following treatment with/without 1 μM 837
IWR1-endo treatment for 48 hours under the differentiation medium. ChIP with IgG without 838
IWR1-endo treatment (orange), IgG with 1 μM IWR1-endo treatment (green), α-β-catenin 839
antibody without IWR1-endo treatment (red), and α-β-catenin antibody with 1 μM IWR1-endo 840
treatment (blue). (F) Quantitative RT-PCR analysis of gene expression of Fermt2 after treatment 841
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with WNT3A (0.1 μg/ml) for 0, 1, 2, and 3 hours (h) with/without 1 μM IWR1-endo. ***, 842
p<0.001. (G) Ctnnb1 expression after treatment with siRNA for control (opened column) and 843
Ctnnb1 (closed column). ***, p<0.001. (H) Quantitative RT-PCR analysis of Fermt2 expression 844
after treatment with siRNA for control (opened column) and Ctnnb1 (closed column). ***, 845
p<0.001. (I) Immunofluorescent images for MHC in C2C12 cells treated with siRNA for control 846
and Ctnnb1. Cells are counterstained with DAPI (blue). Scale bars 100 μm. (J) Quantitative RT-847
PCR analysis after treatment with siRNA for control (opened column) and Fermt2 (closed 848
column). ***, p<0.001. (K) Immunofluorescent images for MHC in C2C12 cells treated with 849
siRNA for control and Fermt2. Cells are counterstained with DAPI (blue). Scale bars 100 μm. 850
(L) Quantitative RT-PCR analysis after treatment with overexpression vector for control (opened 851
column) and Fermt2 (closed column). ***, p<0.001. (M) Immunofluorescent images for MHC in 852
C2C12 cells treated with overexpression vector for control and Fermt2. Cells are counterstained 853
with DAPI (blue). Scale bars 100 μm. 854
855
FIG 4. WNT/β-catenin signaling regulates homeostasis. (A) Schematic diagram of time 856
schedule. After the completion of myofiber formation (Day 0), myofibers were cultured under 857
differentiation medium with vehicle (Control) or IWR1-endo for indicated time. (B) 858
Immunohistochemical analysis of MYH (green) in the differentiated C2C12 cells treated with 859
vehicle (Control) or 1 μM IWR1-endo for indicated days (Day 0, 3, and 5). Cells are 860
counterstained with DAPI (blue). Scale bars 100 μm. (C) Quantitative RT-PCR analysis of 861
indicated molecules in the differentiated C2C12 cells treated with vehicle (Control) or 1 μM 862
IWR1-endo at Day 3. NS, not significant. (D) Immunoblotting (IB) analysis with indicated 863
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antibodies after immunoprecipitation (IP) by beads with indicated antibody of extracts from 864
C2C12 cells treated with vehicle (Control) or 1 μM IWR1-endo at Day 5. Lane 1 and 2: 2% input 865
from vehicle control (lane 1) and IWR1-endo (lane 2) treatment groups. Lane 3 and 4: IgG 866
control for vehicle control (lane 3) and IWR1-endo (lane 4) treatment group. (E) Immunoblotting 867
analysis of caspase 3 treated with vehicle control or IWR1-endo. PC, positive control (tissue 868
sample from adult wild-type small intestine). (F) Immunofluorescent analysis of Phalloidin (red) 869
or cadherin (green)/β-catenin (red) in C2C12 cells at Day 5. Cells are counterstained with DAPI 870
(blue). Top images, scale bar 25 μm; lower images, scale bar 20 μm. (G) Confocal microscopic 871
images for cadherin (green) and β-catenin (red) in C2C12 cells at Day 5. Cells are counterstained 872
with DAPI (blue). Scale bar 25 μm. (H) Quantification of colocalized dots. Colocalized dots 873
(yellow) per total dots (green or red) on the plasma membrane. ***, p<0.001. 874
875
FIG 5. WNT/β-catenin signaling mechanism in primary myoblasts. (A) BrdU staining (brown) 876
in primary myoblasts from the limb after treatment with vehicle (control) or IWR1-endo for 24 877
hours. Nuclei are counterstained with Hematoxylin. Arrows indicate BrdU positive nuclei. Scale 878
bar, 50 μm. (B) BrdU staining (brown) in primary myoblasts from the tongue after treatment 879
with vehicle (control) or IWR1-endo for 24 hours. Nuclei are counterstained with Hematoxylin. 880
Arrows indicate BrdU positive nuclei. Scale bar, 50 μm. (C) Quantitation of the percentage of 881
BrdU-labeled nuclei in primary myoblasts derived from the limb or the tongue treated with 882
vehicle (blue bars) or IWR1-endo (red bars) for 24 hours. N=6 per each treatment. ***, p<0.001. 883
(D) Quantitative RT-PCR analysis of Ccna2 and Cdc25c in primary myoblasts from the limb (L) 884
or the tongue (T) after treatment with vehicle (blue bars) or IWR1-endo (red bars) for 24 hours. 885
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N=6 per each treatment. *, p<0.05; **, p<0.01. (E) Fusion index of primary muscle cells from 886
the limb or the tongue after myogenic induction with vehicle (blue bars) or IWR1-endo (red 887
bars) for three days. ***, p<0.001. (F) Quantitative RT-PCR analysis of Fermt2 in primary cells 888
from the limb or the tongue after treatment with vehicle (blue bars) or IWR1-endo (red bars) for 889
three days. ***, p<0.001. (G) Immunofluorescent analysis of MYH (green) in primary muscle 890
cells from the limb (upper panels) or the tongue (lower panels) cultured under the differentiation 891
medium with vehicle or IWR1-endo for five days. Cells are counterstained with DAPI (blue). 892
Scale bars 100 μm. (H) Immunofluorescent analysis of MYH (green) in primary muscle cells 893
from the tongue after well-differentiated into myofibers. The well-differentiated cells (Day 0) 894
were treated with vehicle (control) or 1 μM IWR1-endo for seven days (Day 7) under the 895
differentiation medium. Cells are counterstained with DAPI (blue). Scale bars 100 μm. 896
897
FIG 6. Conserved WNT/β-catenin signaling mechanisms in tongue explants. (A) BrdU staining 898
(brown) in tongue explants from C57B6/J mice after treatment with vehicle (control) or 1 μM 899
IWR1-endo for 24 hours. Nuclei are counterstained with Methylene Blue. Arrows indicate BrdU 900
positive nuclei. Scale bar, 50 μm. (B) Quantitation of the percentage of BrdU-labeled nuclei in 901
tongue explants treated with vehicle (blue bars) or 1 μM IWR1-endo (red bars) for 24 hours. 902
N=6 per each treatment. ***, p<0.001. (C) Immunohistological analysis of MYH (green) in 903
tongue explants cultured with vehicle or 10 μM IWR1-endo for five days. Cells are 904
counterstained with DAPI (blue). Scale bar, 50 μm. (D) Model of WNT/β-catenin signaling 905
during myogenesis. The schematic diagram depicts our model of the mechanism of WNT/β-906
catenin signaling pathway in muscle cells. WNT/β-catenin signaling regulates gene expression of 907
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Ccna2 and Cdc25c in the cell proliferation stage. WNT/β-catenin signaling regulates gene 908
expression of Fermt2 in the differentiation stage. Βeta-catenin forms a complex with cadherin 909
and actin to elongate the myofiber. Disruption of the complex formation results in disorganized 910
myofiber morphology. 911
912
Table 1. Differentially expressed genes related to cell proliferation based on PCR array analysis 913
in C2C12 cells treated with vehicle control or IWR1-endo for 24 hours. Table lists cell cycle 914
regulators altered with 1 μM IWR1-endo treatment compared to controls expressed as a ratio. *, 915
p<0.05; **, p<0.01; ***, p<0.001. Ratio based on the geometric means of expression scores 916
derived from control and treatment groups. All relevant transcripts with a ≥1.5-fold change with 917
p<0.05 are shown. 918
919
Table 2. Distribution of potential LEF1 recognition sequences in promoters of selected genes of 920
interest. Sequences 5-kbp upstream of transcription start sites of indicated genes were analyzed. 921
The number of minimal (CTTTG) and optimal (CTTTGWW) binding sites of indicated genes 922
are given. Note that the optimal binding sites are a subset of the minimal core sites. The number 923
of phylogenetically conserved sites in at least six mammals is given in the right column. 924
925
Table 3. Evolutionary conserved LEF1 recognition sites within the promoters of Ccna2, Cdc25c, 926
and Fermt2. Binding site motif is underlined in all cases. 927
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