Figure 3

Effect of BMP7 on survivin expression and binding of Runx2 to survivin promoter in serum-starved C4-2B cells. (a) The effect of BMP7 on survivin expression. C4-2B cells were pretreated with medium containing 0.1% serum for 24 h followed by supplementation of BMP7 with or without transfection of pAML1-ETO. The expression levels of survivin protein in the cells at 48 h post transfection were compared with that of the corresponding controls. Equal loading of the cells lysates was determined using actin levels. Semi-quantitative RT-PCR was used to determine the expression of the transfected AML1-ETO gene. (b) Suppression of the antiapoptotic effect of BMP7 by AML1-ETO. C4-2B cells cultured in 0.1% serum with or without BMP7 treatment were transfected with the indicated amount of pAML1-ETO and subjected to TUNEL assay. BMP7 treatment significantly reduced cell apoptosis (P<0.05). However, cells treated with 1 μg of pAML1-ETO showed significantly higher apoptotic rate (P<0.01) compared with control cells or cells transfected with 0.5 μg pAML1-ETO, indicating that AML-ETO had a counteracting effect and the effect was dose-dependent. (c) Increased occupancy of region-II of the human survivin promoter by Runx2 in BMP7-treated cells. Serum-starved C4-2B cells were cultured with or without BMP7 and cross-linking ChIP was performed to examine the Runx2 binding to region-I (upper panel) and region-II (lower panel) of the survivin promoter, respectively. Runx2 binding was only evident for region-II of the promoter in cells stimulated with BMP7. (d) Luciferase reporter constructs that contained various lengths of survivin promoter (pLuc1430, pLuc1270, and pLuc649) are shown on the upper panel. The promoter pLuc1430 retained the Runx2-binding sites of the region-II as marked. Effect of BMP7 on the activity of the promoter constructs in serum-starved C4-2B cells is illustrated in the lower panel. The activity of each construct was analyzed in serum-starved cells with or without supplementation of BMP7. The pAML1-ETO plasmids were also transfected as indicated to examine the effect of Runx2 binding on the promoter activity. The cells were cotransfected with β-galactosidase and all activities are presented relative to the activity of pLuc1430 after normalization with respect to β-galactosidase activity. For pLuc1430 plasmid, P-values determined were as follows: ±BMP7 (<0.01); +BMP7, ±0.5 μg AML1-ETO (<0.01); and +BMP7, ±1 μg AML1-ETO (<0.001). For the pLuc 1270 plasmid, the corresponding values were <0.01, not significant, and <0.05, and for pLuc649, not significant. Transfection with ptkLuc (a construct with a minimal promoter) was used as a negative control in the assays.