Figure 4. GSK3β regulation of BACE1 transcription is dependent on NF-κB p65 expression.

(A) pBACE1-4NF-κB plasmid contains the 4 NF-κB cis-elements from the human BACE1 promoter upstream of the firefly luciferase reporter gene. N2a cells were co-transfected with pBACE1-4NF-κB and pCMV-RLuc. Transfected cells were treated with vehicle solution (control) or 10 ng/ml TNF-α with/without 5 μM ARA for 24 hours. (B) N2a cells were co-transfected with pBACE1-4NF-κB plasmid and pMTF-p65 or a vector control. Transfected cells were then treated with a vehicle solution or 5 μM ARA for 24 hours. (C) pNF-κB-Luc was co-transfected with pMTF-p65 or a vector control and treated with a vehicle solution or 5 μM ARA for 24 hours. Renilla luciferase was used to normalize for transfection efficiency. Values are expressed as mean ± SEM. n = 4; *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test. (D) Wild-type MEFs and RelA-KO MEFs which are dysfunctional for NF-κB activity, were co-transfected with a 3.5-kb human BACE1 promoter and S9A-GSK3β or a control vector. S9A-GSK3β overexpression in MEFs. significantly increased luciferase activity (*P < 0.05, Student’s t test), whereas RelA-KO MEFs did not have any significant effect. Luciferase activity is indicative of BACE1 promoter activity. All promoter data shown represent an average of at least 4 independent experiments, with each condition performed in triplicate. (E) N2a cells were treated with 5 μM ARA for 24 hours, followed by cell fractionation. Cytosolic and nuclear fractions were subjected to SDS-PAGE. ARA treatment significantly reduced NF-κB p65 levels in the (F) nuclear fraction (n = 6; **P < 0.001, Student’s t test) and (G) cytosolic fraction. n = 6; ***P < 0.001, Student’s t test. Values are expressed as mean ± SEM.