Distinct contributions of R1, R2, and R3 of LDLR gene regulatory regions to nicotine-mediated LDLR expression.

(A) Schematic illustration of the 271 bp of the 5ʹ-UTR of the LDLR gene. Nucleotide numbering is relative to the translation initiation site AUG where A is +1. The positions of R1 (-103), R2, and R3 (-68) were indicated as boxes. WT indicates the wild type structure. R1, R2, and R3 represent the mutant constructs lacking each sequence. Each fragment was subcloned in the pGL4-basic vector and used for luciferase assays. Ca9-22 cells were transfected with WT (B) or with R1, R2, or R3 (C) along with normalized pRL-CMV vector for 3 h. After transfection, the cells were stimulated with or without nicotine for 3 h and luciferase activity was measured. At least 3 independent experiments were performed. The data are presented as mean ± SD. *p < 0.05.