The Na+/I- Symporter (NIS)-mediated iodide uptake is the main rate-limiting step in thyroid hormonogenesis. Thyrotropin (TSH), a prime regulator of thyroid gland growth and hormone synthesis, constitutes the central physiological control of NIS expression. The Nuclear Factor-kB (NF-êB) is an ubiquitously expressed transcription factor activated in response to different signals roled in cell survival, proliferation and gene expression. Among the five members composing the NF-êB family, p65 constitutes the main effector and the most studied subunit. Activation of NF-êB in thyroid cells by TSH or TSH receptor stimulating antibodies has been reported, although the function of NF-êB in normal thyroid physiology has not been explored. Therefore, we sought to investigate the role of NF-êB in the TSH-induced NIS gene expression in thyrocytes.
We observed a nuclear recruitment of the p65 NF-êB subunit in response to TSH in FRTL-5 cells, suggesting NF-êB activation. Inhibition of protein kinase A by H89 blocked the TSH-induced p65 nuclear recruitment. Phosphorylation of p65 at Ser-276 was induced following treatment with TSH. By contrast, no induction was observed in the presence of H89. TSH induced activation of NF-êB reporter gene. Such effect did not occur under conditions that blocked p65 phosphorylation. To uncover the participation of NF-êB in the TSH-induced NIS gene expression, we performed TSH treatment in the presence of Bay 11-7082, a specific NF-êB inhibitor. Bay 11-7082 significantly reduced the TSH-induced NIS expression. The blockage of NF-êB signaling also reduced the TSH-stimulated NIS promoter activity, thus indicating a transcriptional event. Bioinformatical analysis revealed the presence of a NF-êB consensus site in the NIS-upstream enhancer region. Disruption of the êB site diminished TSH effect. Silencing p65 expression corroborated its role in the TSH-induced gene expression. Furthermore, chromatin immunoprecipitation assay confirmed the binding of the p65 NF-êB subunit to the NIS promoter induced by TSH. We established a link between p65 phosphorylation and trans-activation of the TSH-responsive gene NIS by inactivating the PKA phosphorylation site Ser-276 which substantially reduced the TSH-induced NIS expression.
Concluding, these findings provide evidence that NF-êB constitutes a novel mediator of crucial importance in the TSH-induced NIS gene expression, a relevant finding of potential physiological and pathophysiological implication.
