Transforming growth factor-β-induced transcription of the Alzheimer β-amyloid precursor protein gene involves interaction between the CTCF-complex and Smads

Abstract

Transforming growth factor-β-1 (TGF-β), a key regulator of the brain responses to injury and inflammation, has been implicated in upregulating the expression of the Alzheimer amyloid precursor protein (APP) and Alzheimer's disease (AD) pathogenesis. However, little is known about the mechanisms underlying the effects of TGF-β on APP expression. Analysis of APP promoter activity upstream of the chloramphenicol acetyltransferase reporter gene in normal human astrocytes (NHAs), revealed that the APP promoter binding β (APBβ) site (−93/−82) is responsive to TGF-β. This site interacts with the zinc finger nuclear factor CTCF, involved in APP transcriptional activity. As determined by gel shift assay, there was no significant difference in the CTCF–APBβ complex binding activity in the presence or absence of TGF-β treatment of NHAs. To further investigate the contributions of the CTCF-complex and Smad proteins to the TGF-β induced APP promoter activity, we examined the distribution of these factors and their DNA binding activity. Interestingly, upon TGF-β treatment both Smads 3 and 4 were translocated to the nuclei in contrast to Smad 2, which was cytoplasmic. However, CTCF was predominantly localized in the nuclei irrespective of TGF-β treatment. Gel super shift assay coupled with Western blot analysis showed that Smads 3 and 4 specifically associated with the CTCF–APBβ complex. In addition, AD brain sections showed increased expression and nuclear localization of Smad 4, which correlated with higher levels of APP and TGF-β. However, over expression of Smad 4 on its own was not sufficient to affect APP expression. These results demonstrate that TGF-β activation of Smad protein complexes promotes transcription of the APP gene. Increased synthesis of APP may in part determine Aβ production and deposition in affected AD brain.