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.
Furuta et al. BMC Molecular Biology 2008 9:39 doi:10.1186/1471-2199-9-39