TI - LRP5Delta causes active beta-catenin signaling in MCF7 cells . AB - In order to study the consequences of LRP5Delta receptor expression we employed the widely used MCF7 mammary adenocarcinoma cell line which expressed the LRP5Delta receptor but not LRP5wt (Figure 2A) , and has been shown to accumulate beta-catenin in the nucleus [25] , [33] . We first chose to test the TOPFLASH/FOPFLASH and the pTOPGlow/pFOPGlow TCF luciferase reporters , although conflicting results regarding detection of active beta-catenin signaling using these reporters in breast cancer cell lines have been published [14] , [34] , [35] . Control siRNAs and three highly specific siRNAs directed against LRP5 mRNA were transfected to MCF7 cells . The specificity and silencing potential of the LRP5 siRNAs were ascertained at the mRNA and protein level in MCF7 cells (Figure 2B) , as we have shown previously in sHPT-1 parathyroid tumor cells [26] . Transfection of siLRP5Delta , specific for the internally truncated LRP5 receptor , as well as of siLRP5tot directed against exon 13 present in both LRP5 wt and LRP5Delta transcripts , resulted in markedly reduced non-PHOSphorylated active beta-catenin level , compared to control siRNAs and siLRP5wt (Figure 2C) . siLRP5wt was directed to exon 10 , not included in the LRP5Delta transcript .sibeta-catenin was included as positive control . Similarily , the endogenous beta-catenin activity in MCF7 ( Figure 2D , left panel ) , as measured by using the TOPFLASH/FOPFLASH or the pTOP/Glow/pFOPGlow TCF/beta-catenin luciferase reporters ( 6-8 fold ) , was dependent on maintained expression of LRP5Delta and beta-catenin ( Figure 2D , right panels ) . The various siRNAs displayed no effect on FOPFLASH [26] or pFOPGlow reporter activities ( data not shown ) , which contain mutated TCF binding elements in their promoters [34] . The experiments were done using our in house strain of MCF7 or fresh MCF7 cells from ATCC , which similarily expressed LRP5Delta and showed beta-catenin activity by the TOPFLASH and pTOPGlow assays . Next we employed the natural beta-catenin responsive DKK1 promoter [36] -[38] instead of the synthetic minimal promoters of TOPFLASH and pTOPGlow . Clearly , the DKK1 promoter activity in transfected MCF7 cells was dependent on the TCF binding elements ( TBEs ) , and also on maintained expression of LRP5Delta and beta-catenin (Figure 3A) . Thus , this confirmed the results obtained with the TOPFLASH and pTOPGlow reporters (Figure 2D) . Furthermore , we transfected the various siRNAs to MCF7 cells and determined the endogenous DKK1 mRNA expression level . In accordance with the above results , transfection of siLRP5Delta and siLRP5tot , but not of siLRP5wt or Control siRNA resulted in significantly reduced endogenous DKK1 expression (Figure 3B) . In an attempt to address possible explanations for conflicting results regarding detection of beta-catenin activity by the TOPFLASH and pTOPGlow assays [14] , [34] , [35] , we performed transfections in the presence of varying cell densities . We routinely plate cells rather sparsely ( 2x105 cells/35 mm dish , 104 cells/96-well microplates ) as compared to for example 80% confluency [34] . As expected , the transfection efficiency ( beta-galactosidase activity ) was reduced with increasing cell density ( not shown ) , and clearly the TOPFLASH/FOPFLASH as well as the pTOPGlow/pFOPGlow ratios decreased with increasing cell density (Figure 3C) . Thus , cell density seemed to be an important determinant of WNT/beta-catenin signaling constituting one possible explanation for published inconsistencies . In summary , the results showed that maintained expression of the internally truncated LRP5 receptor in MCF7 cells appeared necessary for accumulation of transcriptionally active beta-catenin .