MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF TOLL-LIKE RECEPTOR 4 AS LIPOPOLYSACCHARIDE RESPONSE MEDIATOR IN THE THYROID CELL.

NICOLA, JUAN PABLO; VÉLEZ, MARIA LAURA; LUCERO, ARIEL MAXIMILIANO; FOZZATTI, LAURA; GATTI, GERARDO; MACCIONI, MARIANA; MASINI-REPISO, ANA MARIA

Santiago de Chile

Congreso; XII Congreso de la Sociedad Latinoamericana de Tiroides (SLAT).; 2007

Sociedad Latinoamericana de Tiroides (SLAT).

Lipopolysaccharide (LPS), a glicolipid found in the outer membrane of Gram-negative bacteria, exerts a variety of biological effects mainly acting on immune cells. A previous study from our group reveal the ability of the endotoxin to exert a direct action on the thyroid cell up-regulating the TSH-stimulated thyroglobulin gene expression. We previously reported that LPS is also able to stimulate the TSH-induced iodide uptake, a key step in thyroid hormonogenesis, by increasing NIS expression. It has been well characterized in immune cells that LPS acts through Toll-like receptors (TLRs), specifically TLR4. These receptors are a family of proteins related to the interleukin-1 receptor, with many ligands and able to induce the LPS signal. These molecules include CD14, MD-2 and LBP. The present study aimed to elucidate the mechanism by which LPS is recognized in the thyroid cell and to extend our findings on LPS effect on thyroid function. We used a highly differentiated and characterized rat thyroid cell line, FRTL-5, which has the advantage of being a pure cel preparation with no contaminating cell that could potentially influence the LPS response. We used different approaches to study the expression of TLR4 receptor and accessories molecules, such as RT-PCR and Western blot in membrane fractions. We also studied localization and colocalization by Flow Cytometry. Surface Biotinylation and confocal Microscopy. FITC-LPS was used to analyze binding by Flow Cytometry. To obtain insight into TLR4 functionality, we performed treatments with different compounds to evaluate specificity and characterize the involvement of TLR4 in the effect of LPS on iodide uptake, NIS protein and mRNA expression. We used Lipid A and Polymixin as LPS agonist and antagonist respectively. TLR4 specific blocking antibody. We demonstrated for the first time, that the LPS receptor TLR4 and the molecule CD14 and MD-2 are expressed at the plasma membrane in the thyroid cell and are involved in the LPS recognition. We also evidenced that the TLR4-induced signaling is clearly involves in LPS effect on thyroid function.