The transcription factor NF-kB mediates thyrotropin-stimulated expression of thyroid differentiation markers

GEYSELS, RC; PEYRET, V; MARTI, M; NAZAR, M; REALE, C; BERNAL BARQUERO, CE; MIRANDA, L; MARTI, MA; VITO, P; MASINI-REPISO AM; NICOLA, JP

THYROID

MARY ANN LIEBERT INC

Lugar: New York; Año: 2021 vol. 31 p. 299 - 314

1050-7256

Background:The Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB)transcription factors are key regulators of cell survival, proliferation andgene expression. Although activation of NF-κB signaling in thyroid follicularcells following thyrotropin (TSH) receptor (TSHR) engagement has been reported,the downstream signaling leading to NF-κB activation remains unexplored. Here, wesought to elucidate the mechanism that regulates NF-κB signaling activation inresponse to TSH stimulation. Methods:Fisher rat-derived thyroid cell lines and primary cultures of NF-κB essential modulator (NEMO)-deficientmice thyrocytes were used as models. Signaling pathways leading to theactivation of NF-κB were investigated using chemical inhibitors andphospho-specific antibodies. Luciferase reporter gene assays and site-directedmutagenesis were used to monitor NF-κB-dependent gene transcriptional activity.Expression of thyroid differentiation markers was assessed by RT/qPCR andWestern blot. Chromatin immunoprecipitation was carried out to investigate NF-κBsubunit p65 DNA binding, and siRNA-mediated gene-knock-down approaches wereused for studying gene function.Results: Usingthyroid cell lines, we observed that TSH treatment leads to PKC-mediatedcanonical NF-κB p65 subunit nuclear translocation. Moreover, TSH stimulation phosphorylatesthe kinase TAK-1, and its knock-down abolished TSH-induced NF-κBtranscriptional activity. TSH induces the transcriptional activity of the NF-κBsubunit p65 in a PKA-dependent phosphorylation at Ser-276. Additionally, p65phosphorylation at Ser-276 induced acetyl transferase p300 recruitment leadingto its acetylation on Lys-310, thus enhancing its transcriptional activity. Evaluationof the role played by NF-κB in thyroid physiology demonstrated that the canonicalNF-κB inhibitor BAY11-7082 reduced TSH-induced expression of thyroiddifferentiation markers. Of note, the involvement of NF-κB signaling in thyroidphysiology was confirmed assessing TSH-induced gene expression in primarycultures of NEMO-deficient mice thyrocytes. Moreover, chromatinimmunoprecipitation and knock-down experiments revealed that p65 is a nucleareffector of TSH actions inducing the transcripcional expression of thyroiddifferentiation markers.Conclusions: Altogether our results point to NF-κB as a pivotalmediator in the TSH-induced thyroid follicular cell differentiation, a relevantfinding of potential physiological and pathophysiological implications.