Inhibition of myoblast differentiation by Sfrp1 and Sfrp2 - Springer Link

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Mar 6, 2008 - Abstract Secreted Frizzled-related proteins (Sfrps) are extracellular ... of mouse Sfrp1 and Sfrp2 was cloned into the pcDNA3.1/ myc-His ...
Cell Tissue Res (2008) 332:299–306 DOI 10.1007/s00441-008-0574-z

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Inhibition of myoblast differentiation by Sfrp1 and Sfrp2 Simon Descamps & Hayat Arzouk & Francis Bacou & Henri Bernardi & Yann Fedon & Stéphanie Gay & Yves Reyne & Bernadette Rossano & Jonathan Levin

Received: 5 September 2007 / Accepted: 9 January 2008 / Published online: 6 March 2008 # Springer-Verlag 2008

Abstract Secreted Frizzled-related proteins (Sfrps) are extracellular regulators of Wnt signalling and play important roles in developmental and oncogenic processes. They are known to be upregulated in regenerating muscle and in myoblast cultures but their function is unknown. Here, we show that the addition of recombinant Sfrp1 or Sfrp2 to C2C12 cell line cultures or to primary cultures of satellite cells results in the inhibition of myotube formation with no significant effect on the cell cycle or apoptosis. Even though at confluence, treated and untreated cultures are identical in appearance, analyses have shown that, for maximum effect, the cells have to be treated while they are proliferating. Furthermore, removal of Sfrp from the culture medium during differentiation restores normal myotube formation. We conclude that Sfrp1 and Sfrp2 act to prevent myoblasts from entering the terminal differentiation process. Keywords Sfrp1 . Sfrp2 . Myoblast . Proliferation . Differentiation . Cell culture . Mouse . Rabbit

Introduction Satellite cells are skeletal muscle stem cells situated between the basal lamina and sarcolemma of mature myofibres. In S. Descamps and J. Levin contributed equally to this work. S. Descamps : H. Arzouk : F. Bacou : H. Bernardi : Y. Fedon : S. Gay : Y. Reyne : B. Rossano : J. Levin (*) INRA, UMR 866 Différenciation cellulaire et croissance, 2 place Viala, 34060 Montpellier, France e-mail: [email protected] S. Descamps Université Montpellier 2, place Eugène Bataillon, 34095 Montpellier, France

the adult, they are mitotically and metabolically quiescent (Mauro 1961). Satellite cell activation is dramatically enhanced in response to exercise, muscle damage or degenerative muscle diseases (Charge and Rudnicki 2004). Once activated, satellite cells proliferate and their progeny progress down the myogenic lineage pathway to become fusioncompetent myoblasts (Hawke and Garry 2001; Schultz and McCormick 1994). The various signalling factors responsible for the initiation and control of satellite cell proliferation and differentiation are not, as yet, completely defined. Among the factors implicated in this process are the secreted Frizzled-related proteins (Sfrps; Levin et al. 2001) whose function is considered to be that of modulating the Wnt signalling pathway. The Wnt-secreted proteins are a family of 19 glycosylated lipid-modified proteins that are powerful regulators of embryonic development, proliferation, cell differentiation and migration (Logan and Nusse 2004). Signal transduction takes place after Wnt binding to the extracellular domain of a Frizzled receptor (Bhanot et al. 1996). The five Sfrps in the human and mouse genome are composed of two domains: a netrin-like domain of unknown function and a cysteine-rich domain (CRD) that possesses a high degree of structural and sequence similarity to the CRD of Frizzled receptors (Finch et al. 1997; Hoang et al. 1996; Leyns et al. 1997; Melkonyan et al. 1997; Rattner et al. 1997; Salic et al. 1997; Wang et al. 1997). The Frizzled CRD mediates binding to Wnt ligands (He et al. 1997; Wu and Nusse 2002). The homologous CRD of Sfrp has also been shown to be sufficient and necessary for interaction with Wnt proteins (Bafico et al. 1999; Lescher et al. 1998; Lin et al. 1997). In order to shed more light on the function of Sfrps in the muscle, we have examined the effect of recombinant Sfrp1 and Sfrp2 on the proliferation and differentiation of

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the C2C12 mouse myoblast cell line and on satellite cell primary cultures. Our data indicate that treatment with Sfrp1 and Sfrp2 inhibits myotube formation of both C2C12 cultures and satellite cell primary cultures. We also show that addition of Sfrp2 results in a 50% diminution of myogenin and Mrf4 mRNA expression, whereas MyoD and Myf5 mRNA expression is not affected.

Materials and methods Expression and purification of recombinant mouse Sfrp1 and Sfrp2 proteins Recombinant proteins were prepared as described by Uren et al. (2000) with slight modifications. The coding sequence of mouse Sfrp1 and Sfrp2 was cloned into the pcDNA3.1/ myc-His eukaryotic expression vector (Invitrogen). Subconfluent Madin-Darby-canine kidney cells, grown in Dulbecco_s modified Eagle_s medium (Sigma) supplemented with 5% fetal calf serum (Dominique Dutscher), were transfected with Sfrp1 and Sfrp2 expression plasmids by using the jetPEI transfection system (Qbiogene). Transfected cells were selected with Geneticine (100 mg/ml). Individual G418-resistant clones were isolated and the expression of the recombinant proteins was determined by Western blot with an anti-myc antibody (clone 9E10; Upstate). Conditioned medium was collected from heparintreated cells (100 μg/ml) and the myc/his-Sfrps were purified by using a Ni-NTA agarose resin (Qiagen) and elution with 250 mM imidazole. Purified protein was subjected to analysis by SDS-polyacrylamide gel electrophoresis. Gels were stained with Coomassie brilliant blue to verify purity and concentration. The protein was also subjected to Western blot analysis with anti-myc antibodies to confirm its identity. Table 1 Effect of the absence (Control) or presence (Sfrp2) of 5 μg/ ml Sfrp2 on the cell cycle or cell death in C2C12 cultures. P>0.05 was considered non-significant (NS). Cell cycle Cells were harvested 48 h after seeding. The percentage±SD of cells in G0/G1, S and G2/M phases and the statistical significance of the differences in each phase Stage of cell cycle or cell type Cell cycle G0/G1 S G2/M Cell type Viable cells Apoptotic cells Necrotic cells Total cell death

Fluorescence-activated cell sorting analysis and apoptosis assays For fluorescence-activated cell sorting (FACS) assays, cells were trypsinized and harvested, washed with cold phosphatebuffered saline (PBS) several times, gently fixed with 85% cold ethanol and incubated at 4°C for 30 min. Fixed cells were spun down, resuspended in DNA staining solution (1 mg/ml RNase A, 0.5 mg/ml propidium iodide, 0.05% Triton X-100, 250 mg/ml TriNaCitrate) and subjected to flow cytometry by using FACS can (Becton Dickinson). For apoptosis assays, the Annexin V assay with propidium iodide labeling was performed on cells according to the manufacturer’s instructions (Pharmingen) to monitor the fraction of necrotic (propidium-iodide-positive and annexin-V-negative cells) or apoptotic cells (annexin-V-positive). Cell cultures The murine skeletal muscle cell line C2C12 was obtained from the American Type Culture Collection (ATCC CRL1772). C2C12 myoblasts were cultured according to Levin et al. (2001). Primary cultures were prepared from mice and New Zealand white rabbit muscles, both from our own breeding stocks. All animals were treated in accordance with institutional and national guidelines. Mice satellite cells were isolated from the whole muscles of the paw. Cells were plated at a density of 2×104 cells/cm2 on Matrigel-coated Petri dishes (BD Biosciences), in 80% Ham’s F12, 20% horse serum, containing glutamine, penicillin and amphotericin B (Invitrogen). They were maintained at 37°C in a water-saturated atmosphere containing 5% CO2 in air. After 2 days, cells were washed with Ham’s F12 and placed in complete medium supplemented with 5 ng/ml basic fibroblast growth factor. Rabbits were sacrificed by an overdose of sodium pentobarbitone. Satellite

of treated cells versus control cells is presented. Cell death Cells were harvested at confluence. The percentage±SD of viable, necrotic and apoptotic cells and of total cell death (necrotic+apoptotic cell percentages) and the statistical significance of differences in each phase of treated cells versus control cells is presented

Control

Sfrp2

P-value

39.96±1.72 34.47±2.49 25.57±1.25

40.43±2.53 36.01±1.92 23.56±3.08

0.569, NS 0.941, NS 0.494, NS

92.30±1.50 3.99±0.80 2.07±1.93 7.21±2.22

87.89±1.47 5.95±0.36 2.97±1.79 11.05±1.68

0.122, NS 0.098, NS 0.446, NS 0.115, NS

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cells were isolated from the semimembranosus accessorius muscle as previously described (Levin et al. 2001). Immunofluorescence Cell cultures were fixed for 20 min at room temperature in 3.7% formaldehyde buffered in PBS, permeabilized in PBS containing 0.2% Tween 20 and blocked for 30 min in PBS that contained 0.5% bovine serum albumin (Sigma). Monoclonal antibody against troponin T (clone JLT-12 Sigma) was diluted 1:100 in blocking solution and incubated with the cells for 1 h at 37°C. After undergoing three washing steps in PBS, cells were incubated for 30 min

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Fig. 1 a–f Effect of Sfrp1 and Sfrp2 on myotube formation. Fluorescent images of C2C12 myoblasts at day 8 after immunostaining with a monoclonal anti-troponin T antibody. Cells were either left untreated (a, b) or were treated with either 0.5 or 5 μg/ml Sfrp1 (c, d) or 0.5 or 5 μg/ml Sfrp2 (e, f), 1 day after plating. At confluence (2 days later), growth medium (GM) was replaced by differentiation medium (DM) and new protein was added. Medium was then changed every 2 days and fresh protein was added with each change. Bar 100 μm. g–i Effects of the different concentrations of Sfrp1 on the differentiation of C2C12 cells. Bar charts showing the variation in the percentage of total nuclei

No. of nuclei

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% surface myotube

% nuclei in myotube

with fluorescein-conjugated secondary antibodies (Jackson Immunoresearch Laboratory). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Sigma). Images were aquired with Kappa Imagebase software (Claravision) by using a Kappa DX20 digital charge-coupled device camera connected to a Zeiss Axiophot fluorescent microscope. The number of nuclei, the percentage surface stained by troponin T (a muscle differentiation marker) and the fusion index (percentage of nuclei present in a myotube, i.e. a troponin-T-positive structure having at least two nuclei) were determined by averaging eight different fields randomly chosen from at least three dishes from each culture and condition. Each culture and condition was

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in myotubes (g), the percentage of the photomicrograph occupied by myotubes, i.e. cell surfaces expressing troponin T (h) and the total number of nuclei as a function of Sfrp1 concentration (i). Recombinant Sfrp1 was added at 0.5 μg/ml, 2.5 μg/ml or 5 μg/ml at 1 day after plating. Two days later (at confluence), GM was replaced by DM and fresh protein added. DM and protein were renewed 2 days later. After 5 days in DM, cells were stained with troponin T and with DAPI. Photomicrographs were then analysed with Perfect Image. The bar charts show the means±SEM of eight different fields. They were analysed by ANOVA followed by Tukey HSD. *P