FIGURE 4.

PI 3-kinase/Akt signaling regulates BMP-2-induced NFATc1 protein and mRNA expression, promoter activation, and nuclear translocation. A–F, NFATc1 protein (A, C, and E) and mRNA (B, D, and F) expression was determined in C2C12 cells treated with BMP-2 with or without pretreatment with 12.5 μm Ly294002 (A and B) or in the presence or absence of adenoviral vectors expressing PTEN (Ad PTEN) (C and D) or dominant negative Akt (Ad DNAkt) (E and F). Immunoblotting with NFATc1 or actin antibody (A, C, and E) or qRT-PCR (B, D, and F) was performed. Immunoblotting with phosphorylated Akt (p-Akt) and Akt antibodies was performed in cell lysates treated with Ly294002 or infected with Ad PTEN (A and C). G–I, C2C12 cells were transfected with NFATc1-Luc plasmids with or without pretreatment with Ly294002 (G), Ad PTEN (H), or Ad DNAkt (I) with or without BMP-2 treatment. Luciferase activities were measured as described in Fig. 2D. For B, mean ± S.E. of quadruplicate measurements is shown. *, p < 0.001 versus control; **, p < 0.001 versus BMP-2-treated. For D and F, mean ± S.E. of quadruplicate measurements is shown. *, p < 0.01 versus control; **, p < 0.01 versus BMP-2-treated. For G, H, and I, mean ± S.E. of triplicate determinations is shown. *, p < 0.01 versus control; **, p < 0.001 versus BMP-2-treated. J–L, nuclear fractions were isolated from C2C12 cells treated with Ly294002 (J) or incubated with Ad PTEN (K) or Ad DNAkt (L). Immunoblotting was performed using antibody against NFATc1 (top panels) or lamin B (lower panels). Error bars represent S.E.