Figure 4

Sp1-dependent regulation of hTLR2 promoter activity and TLR2 gene expression. (A) Assessment of the effect of mithramycin A treatment on SP1-induced TLR2 promoter activation after co-transfecting HeLa cells with the pGL3-T2P (-120) or pGL3-null and with pcDNA3.1 empty vector the SP1 or SP3 plasmids into HeLa cells. Cells were either untreated or treated 1 hr after transfection with 50 nM mithramycin A for 24 hr. Luciferase activity is expressed as the percent activity of the pGL3-T2P (-120) plasmid in pcDNA3.1-transfected, mitA-untreated cells. (B) Assessment of the effect of mutating the SP1 binding site within TLR2 -120/+110 promoter region. The pGL3-T2P (-120) or the pGL3-T2P (-120) SPm construct was co-transfected into HeLa cells with pcDNA3.1 or SP1 plasmid. The HeLa cells were also co-transfected with the pGL3-null and pcDNA3.1 plasmids as negative control. Luciferase activity is expressed as the percent activity of the pGL3-T2P (-120) construct in pcDNA3.1-co-transfected cells. Data are presented as the mean ± S.E.M. from 3 independent experiments performed in triplicate. *, p < 0.05; **, p < 0.0001 as assessed by ANOVA with Tukey-Kramer method. (C) SP1-dependent TLR2 mRNA expression in human epithelial cells. CFBE41o- cells were either treated with mitA (0.5, 1, 5 μM) for 24 hr or untreated. Semi-quantitative RT-PCR analysis was performed using the total RNA extracted from either CFBE41o- or 16HBE14o- cells. TLR2 expression was quantified by normalizing to GAPDH (control) and assayed using Image Gauge (top of the gel images). Results are representative of three independent experiments.