Sp1 plays an important role in nicotine-mediated LDLR expression.

<p>(A) Ca9-22 cells were pre-treated with or without mithramycin for 1 h. After washes, the cells were stimulated with 100 μM of nicotine for 3 h and luciferase activity was measured. (B) Ca9-22 cells were transfected with various concentrations of siRNA against Sp1 or control siRNA (con) for 3 h. After transfection, the cells were stimulated with or without 100 μM of nicotine for 3 h. The expression level of LDLR mRNA was examined by real-time PCR. (C) The silencing effect of siRNA transfection on the expression of Sp1 was assessed by real-time PCR. Sp1 expression level of control siRNA-transfected Ca9-22 cell was set as 1. The data are presented as mean ± SD of at least 3 separate experiments. *p < 0.05.</p

Nicotine stimulation leads to Sp1 binding to R3.

<p>(A) Left panel: Ca9-22 cells were stimulated with (+) or without (-) nicotine for 3 h. After stimulation, the cells were harvested and the nuclear extracts were prepared. Twenty μg of nuclear extract was incubated with biotinylated R3 probe with (+) or without (-) cold R3 probe. The samples were separated by native PAGE and transferred to a nylon membrane. The retarded band was detected by incubating the membrane with streptavidine-HRP followed by ECL. The representative of 3 separate experiments was shown. Right panel: Nicotine-stimulated nuclear extract was incubated with or without R2 or R3 probe and the retarded bands were detected as described above. (B) Nicotine-stimulated nuclear extracts were prepared from Ca9-22 cells and subjected to Stre-Av precipitation assays. Upper panel: lane 1: Stre-Av precipitation assay performed without nuclear extract. Lane 2: Stre-Av precipitation assay with nuclear extract. Lane 3: Stre-Av precipitation assay with biotinylated and non-labeled R3 probes. Lower panel: lane 1: Stre-Av precipitation assay performed without probe. Lane 2: Stre-Av precipitation assay with R2 probe. Lane 3: Stre-Av precipitation assay with R3 probe.</p

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

<p>(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.</p

Increased expression of LDLR in the gingival epithelium from smoking patients.

<p>(A) The gingival tissues were excised from smoking (lower panel) or non-smoking patients (upper panel) and fixed with 5% acetic acid-ethanol. The tissues were embedded in paraffin and 4 μm sections were prepared. The specimens were incubated with anti-LDLR Ab followed by FITC-conjugated goat anti-rabbit IgG Ab. Images were viewed and photographed using a LSM510 confocal laser microscope (Carl Zeiss, Heidelberg, Germany). Green, LDLR; red, nuclei with monomeric cyanine nucleic acid stain. Scale bar (white line): 50 μm.</p

Nicotine-mediated LDLR up-regulation is dependent on nAChR.

<p>(A) The expression of nAChR subunits was assessed by real-time PCR for Ca9-22 (upper panel) and primary cultured human gingival epithelial cells (lower panel). The expression level of GAPDH was set as 1. Relative expression level of each nAChR subunit to GAPDH was shown. (B) Ca9-22 cells were pre-treated with graded concentrations of αBtx for 1 h. The cells were washed and further incubated with 100 μM of nicotine for 3 h. LDLR expression was examined by real-time PCR. Data from at least 3 separate experiments are shown (mean ± SD). *p < 0.05.</p