A novel role of the NF-kappaB transcription factor in the thyrotropin-regulated thyroid differentiation gene expression

NICOLA JP ; NAZAR M; MASINI - REPISO AM

Paris

Congreso; 14th International thyroid Congress (ITC 2010); 2010

EUROPEAN THYROID ASSOCIATION

Thyrotropin (TSH), is a prime regulator of thyroid gland 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-êB) is an ubiquitously expressed transcription factor activated in response to different signals involved in immune response, cell survival and proliferation. NF-êB transcription factors function as dimers of five different subunits including p65(Rel A), RelB, c-Rel, p50 and p52. The 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 thyroid physiology has not been explored. It was demonstrated that TSH increased the p65/p50 heterodimer in association with activation of NF-êB-driven promoter and IL-6 expression whereas only p50-containing NF-êB was obtained in the absence of TSH in the rat thyroid cell line FRTL-5. Therefore, we sought to investigate a possible role for NF-êB in the TSH-induced gene expression in FRTL-5 cells.

We observed a degradation of the cytosolic êB inhibitor (IêB-á) in response to TSH resulting in nuclear translocation of the NF-êB p65 subunit which suggests NF-êB activation. Inhibition of PKA activity by H89 blocked the TSH-stimulated p65 nuclear recruitment. TSH induced the phosphorylation of p65 at Ser-276. By contrast, no induction was observed in the presence of H89. Moreover, TSH activated the NF-êB reporter gene which did not occur under blockage of p65 phosphorylation.

To explore the participation of NF-êB in the TSH-induced gene expression, we used Bay 11-7082, a specific NF-êB inhibitor. Bay 11-7082 significantly reduced the TSH-induced expression of the proteins involved in thyroid hormonogenesis Na⁺/I^- symporter, thyroperoxidase, DUOX2 and thyroglobulin. In agreement, similar results were obtained upon inhibition of NF-êB activation by an IêB-á dominant-negative mutant. The blockage of NF-êB 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 NF-êB consensus site in the promoter regions of the four marker genes of thyroid differentiation. Morover, disruption of the êB 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-êB p65 subunit to the promoters 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-êB p65 subunit constitutes a novel mediator of crucial importance in the TSH-induced gene expression. 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-kB is involved in the pathogenesis of various diseases, these observations could have potential implication in thyroid pathophysiology.