Gene Expression Profiling in Cells with Enhanced ?-Secretase Activity

Magold, Alexandra I.; Cacquevel, Matthias; Fraering, Patrick C.

Importantly, we found several key players of the three Wnt pathways to be transcriptionally altered in response to enhanced csecretase. We confirmed one of these, Wnt3a, to be increased by 2.8-fold in S-1 cells (Fig. 3 ). Aoyama et al. have reported that Wnt3a can influence Notch protein levels and increase Notch1 activation [37] , which increases the effect of enhanced c-secretase even further through substrate enhancement. Thus, enhanced csecretase activity may lead to increased WNT3A transcription, which in turn can increase the protein levels of the NICD-carrying c-secretase substrate. This proposed enhancement of the canonical Wnt pathway is further supported by the recent observation that b-catenin (the central protein that also ties PS1 to the pathway) modulates the level and transcriptional activity of Notch1/NICD through their direct interaction [38] . Several proteins, including Frizzled and Disheveled (Dvl), relay the Wnt signal along the canonical pathway between Wnt and b-catenin. We found that they both show increased gene transcription in our microarray analysis. DVL3 was confirmed by qPCR to increase in mRNA copy numbers by 3-fold (Fig. 3) . Taken together with the microarray data showing differential transcription of PROC, DKK, LRP5/6, GBP, AXIN, b-catenin, C-JUN and CYC D (all part of the canonical Wnt pathway-for further details see 'Discussion'), our data suggest a strong transcriptional effect on this pathway by c-secretase activity and resulting alterations in gene expression. CYC D has also been reported to be downregulated by Protein tyrosine phosphatase receptor type G (PTPRG) [39] . We confirmed by real time PCR that PTPRG transcript level is reduced by 515-fold (Fig. 3) . AMN1 (levels down 978-fold) has also been connected with cell cycle regulation in yeast, but its role in mammals is not well known. TERA, a gene of unknown function (levels down 24-fold), has also been associated with Wnt antagonism (see Fig. 3 and results of human cortex analysis). b-actin served as housekeeping gene.