TGF-β responsive co-modulators, Smad2 and 4, increase the activities of both GAL4–MEF2A and GAL4–MEF2C. (A and B) C2C12 cells were co-transfected with pCMV5B–TβRI(T204D), pCMV5B–Smad2, pCMV5B–Smad4, 5×GAL4-luc, GAL4(DBD), GAL4–MEF2A, GAL4–MEF2C and pSV-βgal as indicated and treated with (+) or without (–) 2 ng/ml of TGF-β in 1% FBS. The transcriptional activation domains of both MEF2 isoforms are fused to the DNA binding domain (DBD) of GAL4 to form GAL4–MEF2A (91–507 amino acids) and GAL4–MEF2C (87–442 amino acids). GAL4(DBD) contains the GAL4 DBD only. The reporter plasmid, 5×GAL4-luc, contains five copies of the GAL4 binding site linked to the adenovirus EIB promoter and the firefly luciferase gene. After 72 h, β-galactosidase activities were measured and used to normalise luciferase activity values. Two or more sets of assays were performed in both COS and C2C12 cells with comparable results. Each data point is a mean of triplicate samples from single experiments and the error bars represent the standard error of the mean (SEM).

In C2C12 cells, the activities of both GAL4–MEF2A and GAL4–MEF2C are enhanced by the over-expression of Smad2 and 4 with wild-type TGF-β receptors (TβR-I and -II), but not in the presence of a TGF-β dominant negative receptor, TβRII(K277R). (A–D) C2C12 cells were co-transfected with pCMV5B–TβRII(K277R), pCMV5B–TβR-I, pCMV5B–TβR-II, pCMV5B–Smad2, pCMV5B–Smad3, pCMV5B–Smad4, 5×GAL4-luc, GAL4(DBD), GAL4–MEF2A(91–507), GAL4–MEF2C(87–442) and pSV-βgal as indicated. Twenty-four hours after transfection, growth medium was replaced with 1% FBS ± 2 ng/ml of TGF-β. Cells were harvested 72 h after transfection for luciferase assays. β-galactosidase activities were used to normalise for transfection efficiency. Two or more sets of experiments were performed with comparable results. Each data point is the mean of triplicate samples from single experiments and the error bars represent the SEM.

A physical interaction between endogenous Smad2 and MEF2A in C2C12 mt was detected through co-immunoprecipitation. (A and B) COS cells were transiently transfected with pCMV5B–TβRI(T204D), pCMV5B–Smad2, pCMV5B–Smad4 and pMT2–MEF2A as indicated. Cell lysates were immunoprecipitated with antibodies raised against either MEF2A (α-MEF2A), or Smad2 (α-Smad2). (A) An immunoblot probed by an α-Smad2 antibody contains: 15 µl of anti-MEF2A immunoprecipitate (α-MEF2A ip) in lanes 1 and 3; 15 µl of anti-Smad2 immunoprecipitate (α-Smad2 ip) in lanes 5 and 7; and 30 µg of C2C12 cell extract in lane 9. The arrow indicates Smad2 protein. (B) An immunoblot probed by an α-MEF2A antibody contains: 15 µl of α-Smad2 ip in lane 1; 1 µl of α-Smad2 ip in lane 2; 20 µg of C2C12 cell extract in lane 4. Lane 3 was empty (C) An immunoblot probed by an α-MEF2A antibody contains: a molecular weight marker in lane 1; 1 µl of α-MEF2A ip from myoblast lysate (mb) in lane 2; 1 µl of α-MEF2A ip from myotube lysate (mt) in lane 3; 20 µl of α-Smad2 ip from mb in lane 5; and 20 µl of α-Smad2 ip from mt in lane 6. (D) A negative control for the co-immunoprecipitation assays was performed. An immunoblot probed with an α-MEF2A antibody contains: 20 µl of α-mouse IgG ip from mt lysate in lane 1; 50 µg of mt cellular extract (mt) in lane 3; 1 µl of mt lysate in lane 5 (used for α-Smad2 ip in lane 7); 20 µl of α-Smad2 ip from mt in lane 7; 15 µl of pre-cleared supernatant from α-Smad2 ip (lane 7) in lane 9; and a molecular weight marker in lane 10. Lanes 2, 4, 6 and 8 were empty. (B–D) The arrow indicates MEF2A protein. (A–D) The arrowheads indicate IgG recognised by the goat anti-rabbit secondary antibody. The bands below the IgG are non-specific. (E) C2C12 mb were plated at 25% confluence and 24 h later the medium was replaced with differentiation medium with (+) or without (–) 2 ng/ml TGF-β. After 2, 4 and 6 days (d), protein expression was analysed by loading 20 µg of each extract on a SDS–polyacrylamide gel and western blotting using an anti-Smad2 antibody. Jurkats cell extract (JC) was used as a control.

MEF2 proteins associate with GST–Smad2 in vitro. (Top) In a GST pull-down assay, in vitro translated [³⁵S]methionine-labelled MEF2A (2 µl) or MEF2C (2.5 µl) was mixed with 5 µg of GST (lanes 3 and 6) or GST–Smad2 (lanes 4 and 7) immobilised on glutathione–agarose beads. After washing, ³⁵S-labelled bound proteins were analysed by SDS–PAGE and autoradiography. Aliquots of 0.4 µl MEF2A (lane 2) and 0.5 µl MEF2C (lane 5) in vitro translation reactions were included as references. The arrows indicate the position of MEF2A or MEF2C. (Bottom) Coomassie blue staining of the gel shows that comparable amount of the GST or GST–Smad2 proteins were loaded. The open arrows (lanes 3, 4, 6 and 7) indicate the position of the corresponding GST or GST–Smad2 fusion proteins.

Localisation of MEF2A, Smad1, Smad2, phosphorylated-Smad2 and Smad3 in cultured muscle cells. C2C12 mb were grown in differentiation media in the absence of exogenous TGF-β (–TGF-β) until myotubes formed after 4 days (left and middle panels). At this point, myotubes were either fixed for immunofluorescence or exposed to differentiation medium + 2 ng/ml TGF-β (+TGF-β) for 24 h (right panels). Mt were fixed and immunostained using specific antibodies raised against MEF2A, Smad1, Smad2, phospho-Smad2 or Smad3.

MEF2A is not retained in the cytoplasm by TGF-β signalling. (A and B) C2C12 mb were co-transfected with Flag–pCMVB–MADR2(3SA) and cytomegalovirus–eGFP using lipofectamine. The expression of GFP was used to determine which cells had been transfected. Cells were fixed with methanol and immunostained using either an anti-Flag (αFlag) antibody or an anti-MEF2A antibody (αMEF2A).

The Smad2–MEF2 interaction is dependent on p38 MAPK phosphorylation sites in MEF2C and in mt, the endogenous MEF2 activity is increased by the addition of TGF-β. (A) COS cells were co-transfected with pSV-βgal, pCMV5B–Smad2, pCMV5B–Smad3, pCMV5B–Smad4, pCMV5B–TβRI(T204D), GAL4(DBD), GAL4–MEF2C(87–442), GAL4–MEF2C(S387A), GAL4–MEF2C(T293,300A) and 5×GAL4-luc as indicated. The construct GAL4–MEF2C(S387A) contains a replacement of serine 387 for alanine and GAL4–MEF2C(T293,300A) contains the exchange of two threonines for alanines at positions 293 and 300. Cells were harvested 72 h after transfection for luciferase assays. (B and C) C2C12 cells were co-transfected with pSV-βgal, pMT2, pMT2-MEF2A, pMEF2-luc, GAL4–MEF2A, GAL4–MEF2C, GAL4(DBD) and 5×GAL4-luc as indicated. Twenty-four hours later the medium was replaced with growth medium. One day later, growth medium was replaced with differentiation medium, which was replaced every 2 days. Mt formed after 4 days, at which point the medium was replaced with differentiation medium and either 2 ng/ml TGF-β (+) or an equal volume of TGF-β diluent (–). Cells were harvested after 24 h for luciferase assays. (A–C) β-galactosidase activities were used to normalise for transfection efficiency. Two or more sets of experiments were performed with comparable results. Each data point is a mean of triplicate samples from single experiments and the error bars represent the SEM.