Thyrotropin (TSH) is a prime regulator of thyroid growth and function. Physiological actions of TSH are largely mediated by activation of adenylate cyclase, followed by cAMP elevation and protein kinase A (PKA) activation. The Nuclear Factor-kappa B (NF-κB) is an ubiquitous transcription factor activated in response to different signals rolled in immune response, cell survival and proliferation. NF-κB transcription factors function as dimers of five different subunits including p65, RelB, c-Rel, p50 and p52. The p65 constitutes the main effector and the most studied subunit. Activation of NF-κB in thyroid cells by TSH or TSH receptor stimulating antibodies has been reported, although the function of NF-κB in thyroid physiology has not been explored. Moreover, the composition of NF-κB subunits activated by TNF-α in thyroid cells is altered by the presence TSH, increasing the p65/p50 heterodimer in association with activation of NF-κB-driven promoter and IL-6 expression. Therefore, we sought to investigate a potential role for NF-κB in the TSH-induced gene expression in FRTL-5 thyroid cell line.
We observed a degradation of the cytosolic κB inhibitor (IκB-α) in response to TSH resulting in nuclear translocation of the NF-κB p65 subunit which suggests NF-κB activation. Inhibition of PKA activity by H89 blocked the TSH-stimulated p65 nuclear recruitment. TSH induced the phosphorylation of p65 at Ser-276 in dependency of PKA activity. Moreover, TSH activated an artificial NF-κB reporter gene which did not occur under blockage of p65 phosphorylation.
To explore the participation of NF-κB in the TSH-induced gene expression, we used Bay 11-7082, a specific NF-κB inhibitor. Bay 11-7082 significantly reduced the TSH-induced expression of the proteins involved in thyroid hormonogenesis Na+/I- symporter, thyroperoxidase, dual oxidase-2 and thyroglobulin. In agreement, similar results were obtained upon inhibition of NF-κB activation by an IκB-α dominant-negative mutant. The blockage of NF-κB signaling also reduced the TSH-stimulated mRNA expression and promoter activity of these genes, which seems to indicate a transcriptional event. Interestingly, bioinformatical analysis revealed the presence of a NF-κB consensus site in the promoter regions of the four marker genes of thyroid differentiation. Moreover, disruption of the κB site in the NIS-enhancer region diminished the TSH-induced NIS increase. Silencing of p65 by siRNA corroborated its original role in TSH-induced gene expression. Furthermore, chromatin immunoprecipitation assay confirmed the TSH-stimulated binding of the NF-κB p65 subunit to the promoter region of these genes. In addition, we established a link between p65 phosphorylation and trans-activation of the TSH-responsive gene NIS by inactivating the PKA phosphorylation site Ser-276 on p65 which substantially reduced the TSH-induced NIS expression.
Concluding, these findings provide evidence that the NF-κB p65 subunit constitutes a novel mediator of crucial importance in the TSH-induced gene expression. Thus, our study demonstrates that PKA plays a pivotal role in p65 activation induced by TSH. The regulation of both transcriptional and binding activities of p65 by PKA seems likely to occur via phosphorylation of Ser-276. Since NF-κB is involved in the pathogenesis of various diseases, these observations could have potential implication in thyroid pathophysiology.
