Figure 4. Transcriptional regulation by IGF-I of Survivin.

(A) NRP-152 cells were co-transfected with full length (FL) or truncations (Trunc-1 to -4) of rat Survivin promoter-Firefly luciferase reporter constructs, along with CMV-Renilla control reporter one day before a 24 h treatment with LR³-IGF-I or vehicle, and cells were then analyzed for dual luciferase reporter activity. (B) NRP-152 cells were co-transfected with Trunc-2 Survivin promoter-luciferase construct (rSur-pro-Luc#2) and CMV-Renilla as in A, and next day cells were treated with various kinase inhibitors (LY: 10 µM LY294004; Rap: 200 pM rapamycin; 10 µM of either SB431542, SB202190, SP600125 or U0126) or DMSO vehicle for 2 h before 24 h treatment with 2 nM LR³-IGF-I or vehicle. (C) NRP-152 cells were co-transfected with rSur-pro-Luc#2 (WT) or rSur-pro-Luc#2 mutated at CDE and CHR (CDE/CHR Mut) along with CMV-Renilla, next day cells were treated with 2 nM LR³-IGF-I or vehicle, and harvested for dual luciferase activity. Data shown are relative values of Firefly luciferase normalized to Renilla luciferase, and expressed as relative luciferase units (R.L.U.). Each bar represents the average of triplicate determinations ± S.E. Statistical significance (*p<0.01) was assessed by two-way Anova analysis of variance. D) IGF-I reverses the ability of TGF-β to suppress Survivin mRNA and such reversal is mitigated by rapamycin. NRP-152 cells plated in GM3 overnight were pre-treated for 2 h with either 200 nM rapamycin or vehicle, followed by overnight with 2 nM LR³-IGF-I or vehicle, and then treated with 10 ng/ml rhTGF-β1 for 24 h. RNA was extracted and processed for RT-PCR of Survivin, Osteopontin (Ost-1) and β-actin.