Canonical Wnt signaling is involved in switching from cell proliferation to myogenic differentiation of mouse myoblast cells
© Tanaka et al; licensee BioMed Central Ltd. 2011
Received: 20 June 2011
Accepted: 5 October 2011
Published: 5 October 2011
Wnt/β-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and β-catenin are required for muscle-specific gene transcription in embryonic carcinoma cells and satellite-cell proliferation during adult skeletal muscle regeneration. Downstream targets of canonical Wnt signaling are cyclin D1 and c-myc. However both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Underlying molecular mechanisms of β-catenin signaling during myogenic differentiation remain unknown.
Using C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. We confirmed that several Wnt signaling components, including Wnt9a, Sfrp2 and porcupine, were consistently upregulated in differentiating C2C12 cells. Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4. TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts. FH535, a small-molecule inhibitor of β-catenin/Tcf complex formation, reduced basal β-catenin in the cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased both cytosolic and membrane-bound β-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated β-catenin (Tyr654) during myogenic differentiation.
These results suggest that various Wnt ligands control subcellular β-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via β-catenin likely acts as a molecular switch that regulates the transition from cell proliferation to myogenic differentiation.
Wnt signaling plays key roles in stem cell maintenance and adult tissue homeostasis [1, 2]. In addition, Wnt signaling controls cell proliferation and differentiation, as well as organized cell movements and tissue polarity establishment. Wnt signaling dysregulation can induce degenerative and cancerous disorders. The Wnt signaling pathway has gained attention as a potential therapeutic target for cancer treatment, as well as research interest in regenerative medicine and stem cell biology.
Members of the Wnt family are involved in various stages of skeletal muscle development and regeneration . Wnt1 and Wnt3a expression in the developing neural tube initiate myogenic differentiation in dorsal and medial somites [4, 5]. Wnt3a overexpression significantly decreases terminally differentiated myogenic cells and causes chick limb malformation by inhibiting chondrogenesis [6, 7]. In chick embryos, Wnt4 is expressed in developing limbs, particularly in the central elbow region and joint interzones of the wrist-forming region . Wnt4 overexpression induces muscle satellite cell markers Pax7 and MyoD, and increases skeletal muscle mass in chick embryos . Wnt5a and Wnt11 have been implicated in varying the number of fast and/or slow myofiber types; Wnt5a increases and decreases, the number of slow and fast myofibers, respectively, whereas Wnt11 has a reversion activity on myofiber specification . Compared to the characterization of these Wnt ligands, intracellular Wnt signaling cooperation during skeletal muscle development and homeostasis is not fully understood.
Wnt family proteins consist of two subfamilies based on downstream intracellular signaling. The canonical Wnt pathway stabilizes β-catenin and activates target genes via TCF/Lef transcription factors. Other Wnt pathways are independent of β-catenin signaling and known as non-canonical Wnt pathways that include stimulation of intracellular Ca2+ release and activation of phospholipase C and protein kinase C. Non-canonical signaling pathways also activate G proteins, RhoGTPases and c-Jun N-terminal kinase (JNK). A recent studies showed that the β-catenin pathway is inhibited by Ror that contains extracellular immunoglobulin (Ig)-like, frizzled-like cysteine-rich, kringle, cytoplasmic tyrosine kinase and proline-rich domains . Ror2 negatively regulates the β-catenin pathway at the TCF-mediated transcription level and activates JNK . The Wnt/Ror pathway is considered to be involved in non-canonical pathways.
Previously, we demonstrated that Wnt4 overexpression increases skeletal muscle mass in chick embryos . Wnt4 signaling pathway involvement in skeletal muscle development has been debated, although the level of involvement is dependent on the cell type and context of other regulatory influences. Indeed, Wnt4 can function via the canonical Wnt/β-catenin signaling pathway , whereas Wnt4 is mediated by JNK in frog eye and human kidney development [13–15]. While Wnt4 functions are well defined, the underlying mechanisms that regulate expression remain largely unknown.
In this study, we investigate Wnt signaling during differentiation of C2C12 cells that can differentiate into contractile myotubes under a low-serum condition . We use microarray analysis to identify the expression profile of Wnt signaling components during myoblast differentiation. In addition, several Wnt members were overexpressed in C2C12 cells to assess their roles in myoblast differentiation. We also examined small-molecule inhibitor effects on Wnt signaling to evaluate β-catenin/TCF complex and membrane-bound β-catenin involvement in regulating cell proliferation and myoblast differentiation.
Expression of Wnt signaling molecules during myogenic differentiation of C2C12 cells
Mouse mesenchymal C2C12 cells can differentiate into muscle cells under a low-serum condition . Using real-time PCR, we investigated the expression of endogenous Wnt signaling components before and after C2C12 cell differentiation. Expression profiles of cells cultured in proliferation medium were used as controls for comparisons to the second and fourth days of culture in differentiation medium.
Wnt-4 enhances myogenic differentiation of C2C12 cells and antagonizes the canonical Wnt signaling pathway
Next, we evaluated myogenic differentiation induced by Wnt family members. Expression plasmids containing Wnt3a, Wnt4, Wnt5a, Wnt6, Wnt7a, Wnt9a and Wnt10a cDNAs were transfected into C2C12 cells. Differentiation was determined by troponin T immunostaining. Although transfection efficiency as indicated by HA-tag immunostaining was ~10%, troponin T expression in Wnt4-transfected cells was 3.5-fold higher compared with that of the control (Additional file 4). Troponin T expression was also significantly increased with Wnt6, Wnt7a and Wnt9a overexpression, but not Wnt3a, Wnt5a and Wnt10a. Similar results were also obtained with immunostaining, when skeletal muscle myosin heavy chain was used as a marker protein in place of troponin T (data not shown).
β-Catenin is a molecular switch between C2C12 cell proliferation or differentiation
C2C12 cells overexpressing Wnt3a, Wnt4 and Wnt5a were stained with the same monoclonal antibody to determine cellular phospho-β-catenin in differentiation medium. Consistent with troponin T expression, decreased vesicles containing phospho-β-catenin (Y654) were observed in Wnt3a-overexpressing cells cultured in differentiation medium, whereas Wnt4- and Wnt5a-overexpressing cells contained increased vesicles compared with that of the control cells expressing eGFP (Figure 10F, H, J, L).
Lastly, we analyzed localization of β-catenin within cells overexpressing Wnt3a, Wnt4 and Wnt5a. Wnt3a overexpression resulted in an elevated cytoplasmic β-catenin level in both proliferation and differentiation mediums (Figure 10O, P). In contrast, Wnt4 and Wnt5a overexpression showed an increase in nuclear and membrane-bound β-catenin levels (Figure 10Q, R, S, T). Collectively, our results suggested that Wnt family members differentially regulate β-catenin phosphorylation and subcellular localization. Moreover, β-catenin is required to maintain cell proliferation and acts as a molecular switch that regulates myogenic differentiation.
Wnt/β-catenin signaling is essential for skeletal muscle development and regeneration. Wnt signals via β-catenin are necessary to induce muscle-specific gene transcription in P19 embryonic carcinoma cells . Canonical Wnt signaling also promotes muscle satellite-cell proliferation in response to skeletal muscle injury . The β-catenin/TCF complex binds cyclin D1 and c-myc promoters, and increases expression in carcinoma cells. However, β-catenin/TCF target gene expression is decreased in differentiating C2C12 myoblast cells. Cyclin D1, c-myc and fosl1 down-regulation as well as a compensating cyclin D3 elevation suggest pathway switching from β-catenin dominant signaling to other pathways. We demonstrated that Wnt family members, including Wnt2b, Wnt4, Wnt6, Wnt9a, and Wnt10a, are significantly elevated following myogenic differentiation of C2C12 cells. Within the Wnt family, Wnt4, Wnt6, Wnt7a, and Wnt9a induce myogenic differentiation following transient overexpression in C2C12 cells, whereas Wnt3a, Wnt5a, and Wnt10a do not affect troponin T expression. Wnt4 overexpression optimally promotes myogenesis and antagonizes transcription mediated by β-catenin/TCF. We therefore selected Wnt3a, Wnt4 and Wnt5a to analyze canonical and/or non-canonical signaling during myogenic differentiation of C2C12 cells.
We found that Wnt4 suppresses canonical Wnt signaling mediated by the β-catenin/TCF complex and promotes myogenic differentiation. However, numerous reports indicate that activation of the Wnt/β-catenin signaling pathway increases myogenic differentiation directly or indirectly [24–30]. One possible explanation may be that canonical Wnt signaling maintains proliferation of myoblast and progenitors, such as satellite cells and reserve cells, and subsequently induces myogenic differentiation. Indeed, canonical Wnt signaling downstream targets cyclin D1 and c-myc are suppressed during myoblast differentiation  (Figure 1). This observation is consistent with c-myc overexpression in C2C12 cells resulting in inhibition of myoblast fusion and myotube formation . Moreover, increased canonical Wnt signaling is related to skeletal muscle aging [32, 33]. Further analyses of the cell cycle and β-catenin are required to characterize canonical Wnt signaling during myogenic differentiation.
Recent studies have shown that Wnt4 activates the canonical β-catenin pathway in C2C12 cells . This difference of results may be partly due to cell culture conditions and C2C12 cell characteristics. First, the C2C12 cell cycle is not synchronized with medium replacement and various cell cycle phases are observed, as shown in Figure 6. Second, serum components in the medium may affect the results, because proliferation and differentiation of C2C12 cells is controlled by lysophosphatidic acid and cholesterol [34, 35]. Third, C2C12 cell characteristics may substantially change during serial passaging. Therefore, Wnt4 could mediate either canonical or non-canonical signaling pathways and overexpression may promote myogenic differentiation of C2C12 cells. During the transition from cell proliferation to differentiation in C2C12 cells, β-catenin expression is down-regulated and troponin T expression is predominantly observed in β-catenin-negative cells (Figure 6). Therefore, β-catenin signaling may be required during early myogenic differentiation stages and unnecessary or inhibitory toward myotube formation after myogenic determination.
Functional switching from cell proliferation to myogenic differentiation is accompanied by changes in cell migration, as revealed by a scratch test analysis (Figure 4). Maintenance of an undifferentiated state with Wnt3a overexpression results in decreased migration, while transition toward myogenic differentiation further reduces migration possibly via non-canonical pathways. Wnt3a activity via the canonical pathway affects cell survival and proliferation. Additionally, Wnt3a is required for maintenance of the myoblast undifferentiated state as well as differentiation induced by Wnt4 signaling.
Our results suggest that β-catenin acts as a molecular switch between proliferation and differentiation of C2C12 cells. The β-catenin/Tcf complex is essential for C2C12 cell proliferation, and inhibition of complex formation by FH535 promotes cell death, although the detailed mechanism of FH535 inhibition is unknown at present. β-Catenin is phosphorylated by various kinases, which causes β-catenin degradation or signaling activity. K252a inhibition of Y654 phosphorylation shifts β-catenin localization from the cytoplasm to the cell membrane, which stimulates myotube formation (Figure 10). Wnt4 induces β-catenin shuttling from the nucleus to the cytoplasm and the regulatory mechanism is not known.
We found that various Wnt ligands regulate differentially subcellular β-catenin localization as well as myoblast proliferation and myotube formation. These results implicate β-catenin in functional switching from cell proliferation to myogenic differentiation.
Plasmid construction, cell culture and transfection assay
eGFP cDNA was purchased from Wako Chemicals as GFP pQBI-polII. Wnt4 and eGFP cDNAs were subcloned into pcDNA3.2DEST (Invitrogen, Carlsbad, CA) for overexpression in C2C12 cells. A HA-tag was added to the Wnt4 C-terminal using mouse Wnt3a cDNA in pUSEamp (Upstate Biotechnology Inc., Temecula, CA) that contained the HA-tag sequence at the C-terminal end, after subcloning to replace the Wnt3a sequence with Wnt4. All Wnt cDNAs used in the present studies contained HA-tag sequence at the C-terminal end. Wnt3aHA, Wnt4HA, Wnt5aHA, Wnt6HA, Wnt7aHA, Wnt9aHA, Wnt10aHA and eGFP (Wako Chemicals, Osaka, Japan) cDNAs were subcloned into pcDNA3.2 (Invitrogen) for transfection and transient expression.
The C2C12 cell line (myoblast-like cell line from the C3H mouse) was purchased from the RIKEN Cell Bank (RIKEN, Wako, Japan) [16, 36] and cultured in Dulbecco's modified Eagle's medium (D-MEM) supplemented with 10% fetal bovine serum (FBS). At 12-24 h after subculture, transfection was performed using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Transfected C2C12 cells were cultured in differentiation medium consisting of 2% horse serum in D-MEM to induce myogenesis.
Real-time quantitative PCR
C2C12 cells were harvested at days 2 (n = 3) and 4 (n = 3) after replacement of proliferation medium with differentiation medium. Total RNA was extracted from C2C12 cells using ISOGEN (NIPPON GENE CO., Tokyo, Japan) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA (RT2 First Strand Kit, SABiosciences) and the cDNA used for quantitative PCR analysis. Wnt signaling component expression in C2C12 cells was analyzed by quantitative Real-Time RT-PCR using a SA Biosciences RT2Profiler™ PCR Array (http://www.sabiosciences.com/rt_pcr_product/HTML/PAMM-043A.html) and RT2 SYBR Green Master Mixes. The PCR condition was 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Analyses were performed on an ABI Real-Time PCR System 7500 (Applied Biosystems, Foster City, CA). Data were evaluated by the ΔΔCt method against control cells cultured in proliferation medium (n = 4), using an on-line template (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php). ΔCt values were calculated by subtracting average cycle threshold (Ct) values of housekeeping genes (glucuronidase, beta, Gusb; Heat shock protein 90 alpha (cytosolic), class B member 1, Hsp90ab1; glyceraldehyde-3-phosphate dehydrogenase, Gapdh; actin, beta, Actb; and hypoxanthine guanine phosphoribosyl transferase 1, Hprt1). MIAME (Minimum Information About a Microarray Experiment) compliant array data including raw data is deposited in GEO (Gene Expression Omnibus) at NCBI [GSE30077].
Viral vector production and treatment
Adenoviruses carrying Wnt cDNAs were prepared using a ViraPower adenovirus expression system (Invitrogen). Human Wnt3a, Wnt4 and Wnt5a cDNAs (Origene, Rockville, MD) were PCR-amplified and subcloned into a pAd/CMV/V5-DEST vector (Invitrogen) using a Gateway system with LR clonase (Invitrogen). eGFP cDNA was used as a control after subcloning into a pAd/CMV/V5-DEST vector. Plasmids were purified and digested with PacI (New England Biolabs Japan Inc., Tokyo, Japan). Linearized plasmids (1-2 μg) were then mixed with 3 μl Lipofectamine 2000 in 200 μl Opti-MEM medium (Invitrogen) and transfected into subconfluent 293A cells (Invitrogen) cultured in 1 ml Opti-MEM in 35 mm plates (Nunc, Thermo Fisher Scientific, Yokohama, Japan). 293A cells were cultured in 100 mm plates for 2 weeks in proliferation medium that was exchanged every 3 days. Cells and medium were harvested upon cell detachment from culture plates, freeze-thawed four times and then centrifuged to obtain adenovirus-enriched supernatants. Aliquots of supernatant were added to fresh 293A cells and cultured for 2-3 days to propagate the adenovirus. After 2-fold amplification, adenovirus-containing medium was used as the virus stock. Viral titers were determined by a plaque-forming assay using 293A cells.
C2C12 cells were seeded into 24 well plates at 2.5 × 104 cells/well and cultured for 24 h. Cells were then cotransfected with 0.2 μg DNA constructs (Wnt3aHA + pcDNA3.2, Wnt4HA + pcDNA3.2, Wnt3aHA + Wnt4HA), 0.2 μg reporter plasmid (TOPFLASH, FOPFLASH) and 0.2 μg internal control pRG-TK, then cultured for a further 24 h. Luciferase activity was measured and normalized for transfection efficiency using a Dual-Glo luciferase assay system (Promega, Madison, WI). Graphs show the average of three independent experiments with normalized transfection efficiency using Rluc.
Scratch test analysis
Scratch testing was performed using C2C12 cells transfected with Wnt3a, Wnt4, Wnt5a and eGFP in pcDNA3.2, which were cultured in 35 mm dishes. Two days after transfection, the cell monolayer was scratched using a toothpick. Cell proliferation and migration were recorded at 5 min intervals and observed for 16 h using an AQUACOSMOS image acquisition and analysis system (Hamamatsu, Japan). Initial cells counts were subtracted from the 9 h counting.
Cells were fixed in a solution of ethanol: formalin: acetic acid: H2O (14:2:1:6, v/v) for 10 min. After three PBS washes, cells were treated with a blocking buffer consisting of 2% goat serum, 2% skim milk, 0.2% Tween20 in Tris-buffered saline (TBS; 50 mM Tris-HCl, pH 7.5, 150 mM NaCl) for 30 min. Then, cells were incubated with anti-troponin T (MAB1487, clone TT-98, Abnova, CA), anti-β-catenin (C2206, Sigma-Aldrich) and anti-phospho-β-catenin (Y654) (ab24925, Abcam, Tokyo, Japan) antibodies at 1:40, 1:200 and 1:50 dilutions, respectively, in blocking buffer at 4°C overnight. After three TBS washes, cells were incubated with a 1:200 dilution of secondary Alexa Fluor 594 or 488-conjugated goat anti-mouse or rabbit IgG antibodies (A11032, A11037, A11029, Molecular Probes, Invitrogen) for 1 h. After a TBS wash, cell nuclei were stained with 1 μg/ml 4', 6'-diamino-2-phenylindole solution (DAPI, DOJINDO, Kumamoto, Japan). Fluorescent images were taken using All-in-One Fluorescence Microscope BZ-9000 (Keyence Japan, Osaka, Japan).
C2C12 cells were seeded into 24 well plates at 2.5 × 104 cells/well and cultured for 24 h. Cells were incubated in proliferation or differentiation medium with or without chemical inhibitors FH535 (1 μM), GW9662 (1 μM) and K-252a (3.6 nM).
Western blot analysis
At 24 h post-subculture, C2C12 cell culture medium was changed to differentiation medium supplemented with FH535 (1 μM) or K-252a (3.6 nM). After 72 h, cells were harvested to prepare total protein extracts.
Cytosolic β-catenin accumulation was assayed as described elsewhere [37, 38]. Protein extracts were resolved by electrophoresis in 5-20% sodium dodecyl sulfate-polyacrylamide gels. Anti-β-catenin (BD Transduction Laboratory, San Diego, CA), anti-phospho-β-catenin (Y654) (ab24925, Abcam), and anti-GPR78 (Santa Cruz) antibodies were used at 1:2000, 1:500, and 1:5000 dilutions, respectively. Proteins were detected using an ECL Plus System (Amersham Pharmacia Biotech, Uppsala, Sweden) with a 1:10 reagent dilution. Band intensities were determined using Image J software (NIH USA).
Acknowledgements and Funding
We thank Dr. Shin-ichiro Nishimatsu for helpful discussion about results and the manuscript, Dr. Tomohiro Narita for technical support and helpful advice, and Keiko Fujioka and Satomi Misao for excellent technical assistance. This work was supported by a research grant for Comprehensive Research on Disability Health and Welfare from the Ministry of Health, Labour and Welfare of Japan, by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (23590227), and by Research Project grants from Kawasaki Medical School (21-102, 22-A17).
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