Thyrotropin Modifies Activation of Nuclear Factor KappaB by Tumour Necrosis Factor Alpha in Rat Thyroid Cell Line

We have recently demonstrated that nuclear factor kappaB (NF-kappaB) mediates the tumour necrosis factor alpha (TNF-alpha)-dependent expression of the gene encoding interleukin 6 (IL-6) in rat thyroid FRTL-5 cells cultured in the presence of thyrotropin (TSH). In the present study we investigated how TSH is involved in the activation of NF-kappaB by TNF-alpha in the cells. Electrophoretic mobility-shift assay revealed that, in the absence of TSH, TNF-alpha activated a single protein-DNA complex containing the p50 subunit but not other NF-kappaB subunits such as p65. In contrast, two distinct protein-DNA complexes were activated in the presence of TSH: the faster-migrating complex contained only p50 subunit; the slower-migrating complex consisted of p65-p50 heterodimer. This TSH effect was mimicked by forskolin and thyroid-stimulating antibodies obtained from patients with Graves's disease, suggesting that an increase in intracellular cAMP is responsible for the induction of different NF-kappaBs by TNF-alpha. A transient transfection study with a luciferase reporter gene driven by multimerized NF-kappaB sites demonstrated that TNF-alpha increased the luciferase activities only in the presence of TSH, and that this increase was inhibited by the co-transfection of mutant p65, which prevented the function of wild-type p65 in a dominant-negative manner. Accordingly, TNF-alpha activated the expression of the IL-6 gene in the presence of TSH but not in its absence. Although the expression of the p105 gene, another known target for NF-kappaB, was increased by TNF-alpha in the absence of TSH, the presence of TSH further increased the mRNA level. Taken together, these observations indicate that the presence of TSH is crucial for the NF-kappaB-mediated actions of TNF-alpha on thyroid follicular cells.