Bacterial LPS is a potent biological activator with multiple actions. It has been proposed as an etiopathogenic agent in TG-induced thyroid autoimmune disease models through an immunostimulatory effect. We previously reported that LPS increased TG level by activation of TG gene expression at transcriptional level in FRTL-5 cells, revealing for the first time the ability of LPS to directly affect self-protein expression in the thyroid cell. Here we analyze the molecular mechanism by which LPS modulates TG gene expression in FRTL-5. Our previous data indicated that LPS (0.01-1µg/ml) increased TSH-induced TG level (immunofluorescence/Western blot; maximum 0.1µg/ml, 48h, 1.78/1.82-fold, p<0.001/p<0.01). Here we demonstrated that LPS increased TG mRNA expression at 3-6h (Northern blot;maximum 3h, 1.8-fold, p<0.01). Transfections assays evidenced that LPS increased the activity of minimal TG promoter containing TTF-1, TTF-2 and Pax8/TTF-1 (C site) binding sites (-168 to +36 bp; pTG Luc; maximum 24h, 2-fold, p<0.01 ). LPS induced a stimulation of a construct containing 5 C sites in tandem linked to TATA-box/CAT gene similarly at 24-48h (1.6-fold, p<0.01). Transfection of pTG Luc mutated at the Pax8-binding site evidenced no stimulation under LPS treatment. In EMSAs it was observed that LPS increased the binding of proteins to C site (oligo C; 3-48h; maximum 3h, 1.8-fold, p<0.001). Evidence that LPS was able to increase the formation of Pax8 and TTF-1/C site complexes was obtained by supershifts, EMSAs with mutated oligo C able to bind only Pax8 (Cp) or TTF-1 (Ct), or EMSAs with oligo C, Cp or Ct as cold competitors. Expression of Pax8 and TTF-1 mRNA and protein was also increased by LPS.

Conclusions of the Abstract

These findings provide insight into the LPS ability to stimulate antigenic and hormonogenic TG protein, a fact of possible pathophysiological implicance. The LPS-induced transcriptional activation of TG gene could be exerted through an enhanced C site-mediated transactivation by Pax8 and TTF-1. A novel property of LPS to increase Pax8 and TTF-1 expression was evidenced.