Figure 3

Inhibition of TLR8 signaling by simvastatin is independent of the prenylation pathway. (A) Schematic showing the principle of the bioluminescence reporter for detection of inhibition of protein prenylation in cells. HEK Blue TLR8 cells were transfected with a plasmid coding for a Gal4-VP16-GFP transcription factor bearing a prenylation (geranylgeranylation) site from Cdc42 at its C-terminus (Gal4-VP16-GFP-Cdc42 tail). This drives transcription of firefly luciferase (F Luc) from a second transfected plasmid under the control of a synthetic promoter containing 5 Gal4 DNA-binding sites. When prenylated, the Gal4-VP16-GFPCdc42 tail associates with membranes, reducing its ability to drive firefly luciferase (F Luc) expression. (B) Inhibition of prenylation by 10 µg/mL simvastatin for 30 min prevents association of Gal4-VP16-GFP-Cdc42 tail with membranes, allowing the transactivator to accumulate in the nucleoplasm and augment reporter expression. The presence of 10 µmol/L GGPP fully reverses the effect of simvastatin. (C) Prenylation is inhibited by 10 µg/mL simvastatin and reversed by 10 µmol/L GGPP in HEK Blue TLR8 cells treated with 5 µg/mL R-848 for 16 h. (D) NF-κB/AP-1 activation is inhibited in HEK-Blue TLR8 cells by simvastatin, but is unaffected by 10 µmol/L GGPP.