Figure 3

The MEK-ERK pathway negatively regulates bim expression through the bim 3'UTR. (A) Structure of the bim-LUC and bim-LUC+3'UTR reporter constructs. Bim-LUC+3'UTR was constructed by cloning and inserting the 4.2 kb rat bim 3'UTR into the bim-LUC construct [8], upstream of the SV40 late poly (A) signal and downstream of the luciferase reporter gene. The locations of two conserved FOXO binding sites and an inverted CCAAT box (ICB) are shown. (B) Sympathetic neurons were microinjected with bim-LUC at 10 ng/μl or bim-LUC+3'UTR at 14 ng/μl, along with pRL-TK to control for injection efficiency (5 ng/μl). The cells were maintained in medium containing NGF (+NGF) or withdrawn from NGF (-NGF) for 16 hours, after which time luciferase activity was measured. Relative luciferase activity was determined by normalisation of Firefly luciferase activity to Renilla luciferase activity (pRL-TK refers to Renilla luciferase under the control of the thymidine kinase promoter). The data is presented as the mean ± S.E., n = 5. Bim-LUC is activated significantly following NGF withdrawal (p = 0.001). Addition of the bim 3' UTR significantly decreases the basal promoter level of bim-LUC (p = 0.003). (C) The basal levels of bim-LUC and bim-LUC+3'UTR were normalised to 1 and the induction factors of the two constructs were compared. Addition of the bim 3' UTR significantly increases the induction of bim-LUC following NGF withdrawal (p = 0.018). (D) Sympathetic neurons were microinjected with bim-LUC at 10 ng/μl or bim-LUC+3'UTR at 14 ng/μl, along with pRL-TK (5 ng/μl). The cells were maintained in medium containing NGF and either left untreated (-U0126) or treated with U0126 at 10 μM (+U0126) for 16 hours, after which time luciferase activity was measured. The data is presented as the mean ± S.E., n = 6. Bim-LUC+3'UTR is activated significantly following inhibition of the MEK-ERK pathway by treatment with U0126 (p = 0.015). Bim-LUC is not activated significantly following treatment with U0126. (E) The basal levels of bim-LUC and bim-LUC+3'UTR were normalised to 1 and the induction factors of the two constructs were compared. Addition of the bim 3' UTR significantly increases the induction of bim-LUC upon inhibition of the MEK-ERK pathway (p = 0.022).