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 reported the ability of LPS to stimulate TSH-induced NIS expression. The aim of this work was to analyze the molecular mechanism involved in the LPS action in the thyroid cell line FRTL-5. LPS increased NIS mRNA and protein expression. TSH-induced transcription of the transfected rat NIS promoter was activated by LPS. Testing internal deletions of the promoter, we defined the NIS upstream enhancer (NUE) as responsible for the LPS stimulatory effect. NUE contains two Pax8 binding sites. Site-directed mutagenesis showed a critical role of Pax8 C site in LPS action. Bioinformatics analysis showed a novel conserved site for NFkB in NUE. Functional blockage of NFkB signaling antagonized LPS effect. Mutagenesis of NFkB site abrogates LPS stimulation. Co-expression of Pax8 and p65 in HeLa or Cos-7 cells synergistically activates NIS promoter transcription. A physical interaction between Pax8 and p65 was evidenced. Furthermore, ChIP experiments confirmed that NIS is a novel target gene for p65 transactivation in response to LPS. Our results thus reveal a new mechanism of p65 in regulating thyroid cell differentiation by a functional interaction with Pax8.
