Foxc2 induces Wnt4 and Bmp4 expression during muscle regeneration and osteogenesis - PubMed (original) (raw)

Foxc2 induces Wnt4 and Bmp4 expression during muscle regeneration and osteogenesis

M C Gozo et al. Cell Death Differ. 2013 Aug.

Abstract

Proliferation and fusion of myoblasts is a well-orchestrated process occurring during muscle development and regeneration. Although myoblasts are known to originate from muscle satellite cells, the molecular mechanisms that coordinate their commitment toward differentiation are poorly understood. Here, we present a novel role for the transcription factor Forkhead box protein C2 (Foxc2) in regulating proliferation and preventing premature differentiation of activated muscle satellite cells. We demonstrate that Foxc2 expression is upregulated early in activated mouse muscle satellite cells and then diminishes during myogenesis. In undifferentiated C2C12 myoblasts, downregulation of endogenous Foxc2 expression leads to a decrease in proliferation, whereas forced expression of FOXC2 sustains proliferation and prevents differentiation into myotubes. We also show that FOXC2 induces Wnt signaling by direct interaction with the Wnt4 (wingless-type MMTV integration site family member-4) promoter region. The resulting elevated expression of bone morphogenetic protein-4 (Bmp4) and RhoA-GTP proteins inhibits the proper myoblast alignment and fusion required for myotube formation. Interestingly, continuous forced expression of FOXC2 alters the commitment of C2C12 myoblasts toward osteogenic differentiation, which is consistent with FOXC2 expression observed in patients with myositis ossificans, an abnormal bone growth within muscle tissue. In summary, our results suggest that (a) Foxc2 regulates the proliferation of multipotent muscle satellite cells; (b) downregulation of Foxc2 is critical for myogenesis to progress; and (c) sustained Foxc2 expression in myoblast cells suppresses myogenesis and alters their lineage commitment toward osteogenesis by inducing the Wnt4 and Bmp4 signaling pathways.

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Figures

Figure 1

Figure 1

Foxc2 is expressed in activated muscle satellite cells during adult muscle regeneration. (a) Schematic diagram of PBS or CTX injection into the leg muscle of 10-week-old mice. Muscle was harvested 4, 6 and 14 days post injection. BrdU was injected intraperitoneally 24 h before tissue harvesting. (b) Immunohistochemistry was performed to assess the expression levels of BrdU, MyoD and Foxc2 at day 4 in the CTX- and PBS-injected mice. Images were taken with an Olympus DP25 camera with an Olympus BX43 light microscope. Scale bar, 50 _μ_m. (c) A cell suspension was prepared from the harvested tissue and fixed with permeabilization/fixation buffer to measure Foxc2 expression levels in BrdU+ and MyoD+ cells using FACS analysis. (d) Foxc2 expression levels were measured in Pax7+ and MyoD+ cells

Figure 2

Figure 2

Endogenous Foxc2 expression diminishes during myogenic differentiation and forced expression of FOXC2 inhibits myogenic differentiation. (a) Schematic representation of the differentiation assay and the time points at which C2C12 cells were analyzed. At 75% confluency, the cell culture mainly consists of proliferating myoblasts (PM). Cells were induced to undergo myogenic differentiation at 100% confluency (D0) and maintained in differentiation media up to 5 days post confluency (D1–D5). (b) qRT-PCR was performed at different time points to assess endogenous Foxc2 mRNA levels during differentiation. (c) Western blot analysis was performed to determine Foxc2 endogenous protein levels during differentiation. (d) C2C12-EV and C2C12-FOXC2 cells were generated as stable cell lines. Cells at different stages of myogenic differentiation were analyzed by qRT-PCR using primers specific for human FOXC2. (e) Forced expression of FOXC2 during different stages of differentiation was confirmed by western blot analysis using a FOXC2 antibody. (f) Bright-field images of C2C12-EV and C2C12-FOXC2 cells were taken at different stages of differentiation. Cells were visualized at × 10 magnification using an Olympus IX51 microscope

Figure 3

Figure 3

Markers of myogenic differentiation diminish upon forced continuous expression of FOXC2. (a and b) qRT-PCR of the myogenic regulatory factors MyoD (a) and myogenin (b) was performed on mRNA isolated from C2C12-EV and C2C12-FOXC2 cells during the proliferation stage (PM), at 100% confluency (D0) and 3 days post induction of myogenesis (D3). (c and d) Protein expression of MyoD (c) and myogenin (d) during myogenesis at D0 and D3 was assessed by immunofluorescence staining. Cells were visualized at × 10 magnification using an Olympus IX51 fluorescence microscope. Images are representative of three independent experiments. *represents statistically significance with P<0.005 between two groups

Figure 4

Figure 4

Forced expression of FOXC2 in C2C12 cells enhances myoblast proliferation, and delays cell-cycle exit and apoptosis upon myogenic induction. (a) The rate of proliferation of C2C12-EV and C2C12-FOXC2 cells was measured using Celltiterglo at 24, 72 and 120 h post seeding. Differentiation was induced at 72 h. (b) C2C12-EV and C2C12-FOXC2 cells were analyzed for cell-cycle progression by PI staining in undifferentiated proliferating myoblasts (PM), 100% confluent cells (D0) and in cells 24 h post myogenic induction (D1). The data are representative of three independent experiments. (c) C2C12-EV and C2C12-FOXC2 cells were harvested at D0 and D1 for protein expression analysis of the cell-cycle regulators p27 and Cdc25c by western blotting. GAPDH was used as a loading control. (d) The rate of apoptosis was analyzed by Annexin-V staining at the D0 and D1 time points. The data are representative of three or more independent experiments

Figure 5

Figure 5

FOXC2 upregulates Wnt4 expression resulting in the activation of Wnt/_β_C and RhoGTP signaling pathways. (a) Wnt4 mRNA levels were analyzed by qRT-PCR in C2C12-EV and C2C12-FOXC2 cells at 75% confluency (PM), 100% confluency (D0), and at days 1 and 3 post myogenic differentiation. (b) ChIP using anti-Pol-II and anti-human FOXC2 antibodies was performed in C2C12-EV and C2C12-FOXC2 cells at D1 and analyzed for interaction of ectopically expressed FOXC2 with the Wnt4 promoter region containing the putative FOXC2 DNA-binding domain. (c) A TOPflash and FOPflash dual luciferase assays were performed in C2C12-EV and C2C12-FOXC2 cells at D1 to assess Wnt/_β_C signaling activity. (d) Expression levels of RhoGTP were analyzed in C2C12-EV and C2C12-FOXC2 cells at D0 and D1 by pull-down using Rhottekin RBD agarose that binds to active RhoA and western blot analysis using an anti-RhoA monoclonal antibody. (e) C2C12-EV and C2C12-FOXC2 cells were seeded at equal densities and transfected with a dominant-negative RhoA (RhoA T19N-GFP). The cells were analyzed with a fluorescence microscope for myotube formation 5 days post myogenic induction. Images were taken with a × 10 objective and are representative of three independent experiments. (f) The number of multi-nucleated myotubes in C2C12-FOXC2 cells transfected with RhoA T19N-GFP or a control GFP vector was quantified and plotted to show the average number of myotubes per field (_n_=8). (g) Expression of the myogenic differentiation marker myogenin was analyzed by qRT-PCR in C2C12-FOXC2 cells transfected with RhoA T19N-GFP or a control GFP vector

Figure 6

Figure 6

Wnt4 knockdown in C2C12-FOXC2 cells results in partial rescue of myotube formation, and a decrease in Wnt/_β_C signaling and RhoGTPase activity. (a) C2C12-FOXC2 cell lines with stable Wnt4 knockdown were generated using two lentiviral Wnt4 shRNAs (sh1 and sh2). Wnt4 knockdown was confirmed by qRT-PCR. (b) C2C12-EV and C2C12-FOXC2 cells with a scrambled shRNA (scr) or an shRNA targeting Wnt4 (Wnt4 sh2) were induced to undergo myogenic differentiation. The cells were analyzed for myotube formation 5 days post induction. (c) A TOPflash and FOPflash dual luciferase reporter assay was performed in C2C12-EV and C2C12-FOXC2 cells with Wnt4 knockdown or the scrambled control to determine Wnt/_β_C activity 24 h post myogenic induction (D1). The cells were analyzed for luciferase activity 48 h post shRNA transfection. The fold changes between C2C12-FOXC2 scr and Wnt4-knockdown reporter activities were compared relative to the control C2C12-EV scr reporter activity. (d) The cells were analyzed for RhoGTPase activity at D1 by pull-down using Rhottekin beads and western blot analysis using an anti-RhoA monoclonal antibody. The lower panel shows the densitometry values of the western blot that was scanned with an Odyssey infrared imaging system and quantified using the Image Studio software

Figure 7

Figure 7

FOXC2 alters the lineage commitment of myoblasts toward osteogenesis by upregulation of Bmp4. (a) A representative image of a heterotopic bone in a muscle of a patient with myositis ossificans. Sections of formalin-fixed, paraffin-embedded tissue biopsies were stained using a FOXC2 antibody. The asterisk indicates the muscle area. The heterotopic bone area in the rectangle is shown at high magnification. Images were taken with an Olympus DP25 camera with an Olympus BX43 light microscope. Scale bar, 50 _μ_m. (b) C2C12-EV and C2C12-FOXC2 cells were induced to undergo osteogenesis using recombinant BMP2. The cells were stained for bone ALP at days 1, 2 and 5 post induction. (c) Expression levels of the osteogenic markers Runx2, Osterix and osteocalcin in C2C12-EV and C2C12-FOXC2 cells in the absence of BMP2 at 5 days post confluency were analyzed by qRT-PCR. (d) Analysis of Bmp4 mRNA expression by qRT-PCR in C2C12-EV and C2C12-FOXC2 cells during myogenic induction. (e) Western blot analysis of secreted Bmp4 in cell culture media of C2C12-EV and C2C12-FOXC2 cells at day 1 post myogenic induction. (f) Western blot analysis of phospho-Smad1/5 in C2C12-EV and C2C12-FOXC2 cells in the presence of PBS, a Bmp4-neutralizing antibody (anti-Bmp4) or the Bmp4 inhibitor Noggin. The lower panel shows the densitometry values of the western blot that was scanned with an Odyssey infrared imaging system and quantified using the Image Studio software. (g) Myogenic induction of C2C12-EV and C2C12-FOXC2 cells was performed in the presence of PBS or Noggin, and analyzed for myotube formation at day 5 post myogenic induction by bright-field microscopy (upper panels) and immunofluorescence visualization of cytoskeletal actin and nuclear MyoD (lower panels). The white arrowheads indicate partial myotube formation in C2C12-FOXC2 cells. (h and i) Bmp4 expression in C2C12-FOXC2 Wnt4-knockdown cells was analyzed for Bmp4 mRNA by qRT-PCR (h) and for secreted protein expression by western blot analysis using an antibody against Bmp4 (i)

Figure 8

Figure 8

Proposed model for FOXC2 regulation of myoblast proliferation and osteogenic differentiation via upregulation of the Wnt and Bmp signaling pathways. FOXC2 is present in mesenchymal progenitor cells, but its expression diminishes during myogenic differentiation. Forced continuous expression of FOXC2 in C2C12 myoblasts promotes the binding of FOXC2 to the promoter region of Wnt4, which in turn leads to upregulation of Wnt4 expression and activation of canonical (_β_C) and non-canonical (RhoA) Wnt signaling, as well as increased levels of RhoGTP and Bmp4. Failure to downregulate FOXC2 in muscle progenitors inhibits myogenesis and induces osteogenesis

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