Bacterial lipopolysaccharide (LPS), an endotoxin from Gram-negative bacteria, exerts a variety of biological responses. The Na+/I- symporter (NIS)-mediated iodide uptake is the main rate-limiting step in thyroid hormonogenesis. We have recently reported the ability of LPS to stimulate the TSH-induced NIS expression. The aim of this work was to analyze the molecular mechanism involved in the LPS-induced NIS expression in the thyroid cell line FRTL-5. LPS increased the TSH-induced NIS mRNA expression and the functional activity of the transfected rat NIS promoter. Testing internal deletions of the promoter, we could define the NIS upstream enhancer (NUE) as responsible for the LPS stimulatory effect. Site-directed mutagenesis showed a critical role of a Pax8 binding site (named C) in LPS action. Bioinformatics analysis over NUE region looking for conserved sites of LPS canonical effectors, showed a novel conserved site for the transcription factor NFkB (region kB). Functional blockage of NFkB signaling by pharmacological agents antagonized LPS effect. Site directed mutagenesis of the kB site abrogates LPS stimulation. Co-expression of Pax8 and the main NFkB-subunit effector p65, but not p50 synergistically activated NIS promoter transcription in Cos-7 cells. Silencing of endogenous p65 levels by siRNA confirmed its participation as effector of LPS action on NIS expression. Furthermore, ChIP experiments corroborated that NIS is a novel target gene for p65 transactivation in response to LPS. In conclusion, our results thus reveal a new mechanism involving p65 in the LPS-induced NIS regulation, denoting a novel transcription factor related to NIS expression.
