The Transcription Factor NF-κB Mediates Thyrotropin-Stimulated Expression of Thyroid Differentiation Markers

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor is a key regulator of cell survival, proliferation, and gene expression. Although activation of NF-κB signaling in thyroid follicular cells after thyrotropin (TSH) receptor (TSHR) engagement has been reported, the downstream signaling leading to NF-κB activation remains unexplored. Here, we sought to elucidate the mechanisms that regulate NF-κB signaling activation in response to TSH stimulation. Fisher rat-derived thyroid cell lines and primary cultures of NF-κB essential modulator (NEMO)-deficient mice thyrocytes were used as models. Signaling pathways leading to the activation of NF-κB were investigated by using chemical inhibitors and phospho-specific antibodies. Luciferase reporter gene assays and site-directed mutagenesis were used to monitor NF-κB-dependent gene transcriptional activity and the expression of thyroid differentiation markers was assessed by reverse transcription quantitative polymerase chain reaction and Western blot, respectively. Chromatin immunoprecipitation (ChIP) was carried out to investigate NF-κB subunit p65 DNA binding, and small interfering RNA (siRNA)-mediated gene knockdown approaches were used for studying gene function. Using thyroid cell lines, we observed that TSH treatment leads to protein kinase C (PKC)-mediated canonical NF-κB p65 subunit nuclear expression. Moreover, TSH stimulation phosphorylated the kinase TAK-1, and its knockdown abolished TSH-induced NF-κB transcriptional activity. TSH induced the transcriptional activity of the NF-κB subunit p65 in a protein kinase A (PKA)-dependent phosphorylation at Ser-276. In addition, p65 phosphorylation at Ser-276 induced acetyl transferase p300 recruitment, leading to its acetylation on Lys-310 and thereby enhancing its transcriptional activity. Evaluation of the role played by NF-κB in thyroid physiology demonstrated that the canonical NF-κB inhibitor BAY 11-7082 reduced TSH-induced expression of thyroid differentiation markers. The involvement of NF-κB signaling in thyroid physiology was confirmed by assessing the TSH-induced gene expression in primary cultures of NEMO-deficient mice thyrocytes. ChIP and the knockdown experiments revealed that p65 is a nuclear effector of TSH actions, inducing the transcripcional expression of thyroid differentiation markers. Taken together, our results point to NF-κB being a pivotal mediator in the TSH-induced thyroid follicular cell differentiation, a relevant finding with potential physiological and pathophysiological implications.