NF-κB p65 Subunit Mediates Lipopolysaccharide-Induced Na⁺/I⁻Symporter Gene Expression by Involving Functional Interaction with the Paired Domain Transcription Factor Pax8

Abstract

The Gram-negative bacterial endotoxin lipopolysaccharide (LPS) elicits a variety of biological responses. Na(+)/I(-) symporter (NIS)-mediated iodide uptake is the main rate-limiting step in thyroid hormonogenesis. We have recently reported that LPS stimulates TSH-induced iodide uptake. Here, we further analyzed the molecular mechanism involved in the LPS-induced NIS expression in Fisher rat thyroid cell line 5 (FRTL-5) thyroid cells. We observed an increase in TSH-induced NIS mRNA expression in a dose-dependent manner upon LPS treatment. LPS enhanced the TSH-stimulated NIS promoter activity denoting the NIS-upstream enhancer region (NUE) as responsible for the stimulatory effects. We characterized a novel putative conserved kappaB site for the transcription factor nuclear factor-kappaB (NF-kappaB) within the NUE region. NUE contains two binding sites for the transcription factor paired box 8 (Pax8), main regulator of NIS transcription. A physical interaction was observed between the NF-kappaB p65 subunit and paired box 8 (Pax8), which appears to be responsible for the synergic effect displayed by these transcription factors on NIS gene transcription. Moreover, functional blockage of NF-kappaB signaling and site-directed mutagenesis of the kappaB cis-acting element abrogated LPS stimulation. Silencing expression of p65 confirmed its participation as an effector of LPS-induced NIS stimulation. Furthermore, chromatin immunoprecipitation corroborated that NIS is a novel target gene for p65 transactivation in response to LPS. Moreover, we were able to corroborate the LPS-stimulatory effect on thyroid cells in vivo in LPS-treated rats, supporting that thyrocytes are capable of responding to systemic infections. In conclusion, our results reveal a new mechanism involving p65 in the LPS-induced NIS expression, denoting a novel aspect in thyroid cell differentiation.

Demonstration of Innate Immune Responses in the Thyroid Gland: Potential to Sense Danger and a Possible Trigger for Autoimmune Reactions

“…dsRNA and LPS increased NFjB activity (Fig. 2B), which is consistent with the previous reports showing the functional expression of TLR3 and TLR4 in FRTL-5 cells (19)(20)(21)(22). dsDNA also stimulated these two reporter genes as previously described (26).…”

“…Although mRNA from human thyroid tissue could contain mRNAs from other types of cells (fibroblasts, endothelial cells, and infiltrated leukocytes), FRTL-5 cells are pure thyroid cells without contamination by other cell types. Therefore, the results indicate that thyroid cells actually express TLRs as previously demonstrated for TLR3 and TLR4 expression in human and rat thyroid cells (19)(20)(21)(22). …”

“…It was demonstrated that rat and human thyroid cells express functional TLR3, which recognizes dsRNA (19,20). Also, it was shown that TLR4, which mediates responses to LPS, is expressed in FRTL-5 cells (21,22). Moreover, the expression of TLR4 and accessory molecules with a basolateral localization has been described in normal rat thyroid cells (22).…”

NF-κB Essential Modulator (NEMO) Is Critical for Thyroid Function

“…Although an involvement of NF-B in the regulation of Nis expression has been already reported (34), we now show that, in addition to NIS, NF-B controls the expression of a panel of thyroid markers, including TTF1, PAX8, TPO, and thyroglobulin. In this context, our work provides a molecular explanation for some phenotypical aspects of human disorders associated with mutations in NEMO.…”

The iodide transport defect-causing mutation R124H: a δ-amino group at position 124 is critical for maturation and trafficking of the Na+/I− symporter (NIS)

“…Mutagenesis of R111A, R124A and H, R127A, E119A, E122A in human NIS cloned into pcDNA3.1, or R124Q, C440A, S and T in hemagglutinin-tagged human NIS cloned into pcDNA3.1 were introduced using QuikChange TM Site-Directed Mutagenesis Kit (Strategene, La Jolla, CA, USA) as reported (Nicola et al, 2010). All constructs were sequenced to verify specific nucleotide substitutions.…”