MyoD Over-Expression Rescues GST-bFGF Repressed Myogenesis

Abstract

During embryogenesis, basic fibroblast growth factor (bFGF) is released from neural tube and myotome to promote myogenic fate in the somite, and is routinely used for the culture of adult skeletal muscle (SKM) stem cells (MuSC, called satellite cells). However, the mechanism employed by bFGF to promote SKM lineage and MuSC proliferation has not been analyzed in detail. Furthermore, the question of if the post-translational modification (PTM) of bFGF is important to its stemness-promoting effect has not been answered. In this study, GST-bFGF was expressed and purified from E.coli, which lacks the PTM system in eukaryotes. We found that both GST-bFGF and commercially available bFGF activated the Akt-Erk pathway and had strong cell proliferation effect on C2C12 myoblasts and MuSC. GST-bFGF reversibly compromised the myogenesis of C2C12 myoblasts and MuSC, and it increased the expression of Myf5, Pax3/7, and Cyclin D1 but strongly repressed that of MyoD, suggesting the maintenance of myogenic stemness amid repressed MyoD expression. The proliferation effect of GST-bFGF was conserved in C2C12 over-expressed with MyoD (C2C12-tTA-MyoD), implying its independence of the down-regulation of MyoD. In addition, the repressive effect of GST-bFGF on myogenic differentiation was almost totally rescued by the over-expression of MyoD. Together, these evidences suggest that (1) GST-bFGF and bFGF have similar effects on myogenic cell proliferation and differentiation, and (2) GST-bFGF can promote MuSC stemness and proliferation by differentially regulating MRFs and Pax3/7, (3) MyoD repression by GST-bFGF is reversible and independent of the proliferation effect, and (4) GST-bFGF can be a good substitute for bFGF in sustaining MuSC stemness and proliferation.

Keywords: MyoD; bFGF; cell cycle; differentiation; muscle; myogenesis.

Figure 1

Recombinant GST-bFGF activates Akt–Erk pathways. (A,B) Recombinant GST and GST-bFGF proteins were induced and expressed in E. coli (A) and purified by glutathione beads, before being sterilized by passing through a filter with 0.45 μm pore size (B). Bovine serum albumin (BSA) was used to block non-specific adsorption of the filter and also as a protein level (12.5~100 μg/mL) reference. (C) C2C12 myoblasts in both growth medium (GM, 10% FCS) and differentiation medium (DM, 5% HS) were treated with GST (100 ng/mL) or GST-bFGF (50 and 100 ng/mL) for 24 h. The signaling proteins in total lysate were examined by Western blot, and the signal of Gapdh served as an input control. (D) The FGF reporter FIRE-luc was transfected into C2C12 and kept in GM with/without GST-bFGF and bFGF (50 ng/mL) for 48 h before being harvested for luciferase activity assay. The activity in GST treated cells was set as one-fold activation. **: p < 0.01 vs. GST. #: p < 0.05 vs. GST-bFGF. All experiments in this manuscript were repeated for >three times, unless otherwise indicated. The FIRE-luc treatment by bFGF was repeated twice, with triplicate in each assay.

Figure 2

Recombinant GST-bFGF activates myoblast proliferation in GM. C2C12 myoblasts in GM were treated with GST or GST-bFGF protein for 48 h and their cell number (A) and cell cycle stages (B) were examined. Various doses (as indicated) of GST-bFGF were used in (A) and the morphology of cells (bright field under phase-contrast) after treatment (as indicated in ng/mL) is also shown in the right panel. Scale bar: 0.4 mm. (C) Primary satellite cells were treated as in (A), and the effect of GST-bFGF and bFGF was compared in (D). Cells in (B) were treated with 100 ng/mL GST-bFGF, but those in (C,D) were treated with 50 ng/mL GST-bFGF. Cell cycle stages were determined by flowcytometry after cells had been stained with propidium iodide (PI). * and **, respectively denoted p < 0.05 and p < 0.01 vs. GST treated cell. ns: non-significant. #: cell number.

Figure 3

GST-bFGF reversibly represses myogenic differentiation. (A) The proliferation of C2C12 cells were seeded at various densities and their numbers after GST-bFGF treatment for 48 h were counted. (B) C2C12 myoblasts in DM were treated with GST or GST-bFGF protein for 48 h and their cell number were examined. (C) Schematics showing the GST-bFGF treatment. Myoblasts were treated with GST-bFGF at the proliferation stage, when kept in growth medium (GM only) or in both proliferation and differentiation stages (GM + DM4). Cells of both treatments were harvested on DM4 and were stained for MHC to examine their morphology (D) and fusion index (E). * and **: p < 0.05 and p < 0.01 vs. GST-treated cells. Scale bar: 0.25 mm.

Figure 4

GST-bFGF signaling targets myogenic and cell cycle genes. The effect of GST-bFGF (50 ng/mL) on the expression of MyoD and cell cycle genes in C2C12 myoblasts kept in growth GM (A,D,F) or DM (B,C), and in satellite cells (E), and was examined by qRT-PCR. Myoblasts in (B) were treated with various doses of GST-bFGF in DM, as indicated. (D) The effect of GST-bFGF on MyoD and Cyclin D1 protein levels in C2C12 cells kept in GM was examined with Western blot. The effect of GST–FGF and FGF on MyoD expression was compared in (E). The level of each gene in GST-treated cells was arbitrarily set as 1-old. * and **: p < 0.05 and p < 0.01 vs. GST treated cells.

Figure 5

Establishment of MyoD over-expressed C2C12 stable clones. (A) Schematics showing the TET-off system, in which tTA binding to TRE is inhibited by doxycycline (Dox, 25 ng/mL). C2C12 stable clones (C2-tTA-MyoD) carrying MyoD over-expression system regulated by the TET-off system was established, and its morphology (B) and the expression of MyoD and GFP at mRNA (C) and protein (D) levels are shown. Scale bar: 0. 25 mm. **: p < 0.01 vs. Dox treated cells (Dox (+)).

Figure 6

MyoD over-expression rescues GST-bFGF-repressed myogenesis. The effect of various doses of GST-bFGF on C2-tTA-MyoD proliferation when MyoD was over-expressed (Dox (−)) was examined (A), and their morphology is shown in (B). C2-tTA-MyoD myoblasts were treated with various doses of GST-bFGF during differentiation and their MHC-stained morphology (C), fusion index (D), and mRNA levels of MyoD and Cyclin D1 (E) are shown here. Dox (+)/(−): with/without Doxycycline (25 ng/mL). * and **: p < 0.05 and p < 0.01 vs. GST treated cells. Scale bars in (B,C) are 0.4 mm and 0.2 mm, respectively.

Figure 7

GST-bFGF signaling enhances the stemness of satellite cells. (A) Schematics showing the GST-bFGF treatment. C2C12 and primary satellite cells were treated with GST-bFGF at the proliferation stage and kept in growth medium (GM), or in both proliferation and differentiation stages (GM + DM2/DM4). Some cells treated in the GM were allowed to differentiate in DM for 2–4 days, without GST-bFGF (GM + ΔDM2/DM4). Cells were harvested after 2 days in GM or 2–4 days in DM, as indicated by the last arrow. (B,C) The expression of MRFs and Pax3/7 in C2C12 (B) and satellite cells (C) under different treatment patterns of GST-bFGF was detected with qRT-PCR, and compared with GST-treated control cells in pairs (relative to those in GST treated cells). The level of each gene in GST-treated cells was set as one-fold (not shown in the figure). * and **: p < 0.05 and p < 0.01 vs. GST treated cells.