The Transcriptional Regulatory Effects Of C/EBP?on Amyloid Precursor Protein Gene Expression

Objective Alzheimer's disease is one of the most common neurodegenerative diseases. One of the pathological features of Alzheimer's disease is the presence of fibrillary amyloid-? deposits, which result from cleavage of the amyloid precursor protein. The CCAAT/enhancer binding protein ?(c/EBP ?) is a member of the transcription factor family consisting of six functionally and structurally related basic leucine-zipper DNA-binding proteins. As a complex transcription factor, c/EBP ? plays an important role in the regulation of gene expression. This study was designed to investigate the effects of transcription factor c/ EBP ? on APP gene promoter activities and the possible mechanism in regulation of APP gene expression, to further explore the role of c/EBP ? in AD pathogenesis and hope to provide a new idea for prevention and treatment of AD.Methods 1. The c/EBP ? lentivirus expression vector and Sp1 eukaryotic expression vector were constructed with molecular cloning and PCR techniques; 2. We co-transfected the c/EBP ? eukaryotic expression vector, p CDNA3-c/EBP ?, together with luciferase reporter constructs of APP promoter p GL3-APP-170/147 into the U87 cells to verify the effects of c/EBP ? on APP promoter activities by luciferase assay. 3. To extract total RNA from the c/EBP ? lentivirus infected HT22 cells with Trizol and then to do the reverse transcription, use the transcripts as templates to synthesize the first strand of c DNA and then perform the Real time PCR to evaluate the effects of c/EBP ? on amyloid precursor protein(APP) and Sp1 gene expression. 4. To extract total proteins from the c/EBP ? lentivirus infected HT22 cells and the effects of c/EBP ? on amyloid precursor protein(APP), Sp1 and P65 gene expression were estimated with Western blot. 5. Forty-eight hours after HT22 cells were co-transfected with c/EBP ? and P300 eukaryotic expression plasmids, the cells were harvested, and the cellular proteins were extracted for Co IP.Results 1. The c/EBP ? lentivirus expression vector and SP1 eukaryotic expression vector were successfully constructed. 2. The results of luciferase assay demonstrated that c/EBP ? could up-regulate APP promoter activities. 3. The results of Q-PCR confirmed that c/EBP ? could increase APP and Sp1 gene expressions in transcriptional level. 4. The results of Western blot confirmed that c/EBP ? could increase APP, Sp1 and P65 gene expressions in translational levels. 5. The results of Co IP showed that the c/EBP ? and P300 proteins had physical interaction.Conclusion The c/EBP ? can upregulate APP and Sp1 gene expression. The possible mechanism for c/EBP ? to upregulate APP gene expression maybe through the following pathways: 1. The c/EBP ? facilitate the endogenous Sp1 gene expression and the increasing expression of Sp1 would promote the APP gene expression since Sp1 is known as a positive regulator TF for APP gene expression, which could bind to the GC boxes located in the APP promoter 5'UTR. 2. The c/EBP ? could increase P65 gene expressions in translational levels. We hypothesized that c/EBP ? is likely to activate NF-?B pathway to regulate APP gene expression. 3. The c/EBP ? and P300 proteins had physical interaction. We hypothesized that c/EBP ? and P300 proteins could form the transcription complex in this way to regulate APP gene expression.