Nitric oxide (NO) is a ubiquitous signaling moleculeinvolved in a wide variety of cellular physiological processes. In thyroidcells, NO-synthase III-endogenously produced NO reduces thyrotropin(TSH)-stimulated thyroid specific gene expression, suggesting a potentialautocrine role of NO in modulating thyroid function. Further studies indicatethat NO induces thyroid dedifferentiation, since NO donors repressTSH-stimulated I^- uptake.

To investigate the molecular mechanism underlying theNO-inhibited Na⁺/I^- Symporter (NIS)-mediated I^-uptake in thyroid cells.

FRTL-5 cells, a line of highly differentiated ratthyroid-derived cells, were incubated with different NO donors (sodium nitroprusside,S-nitrosoglutathione, Spermine NONOate). NIS function was measured using ¹²⁵I^-transport assays, and NIS expression was evaluated through western blot,RT/qPCR, and gene reporter assays. NIS post-transcriptional modifications wereevaluated in FRTL-5 cells stably expressing N-terminal HA-tagged NIS.

NO donors reduced I^- uptake in aconcentration-dependent manner, which correlated with decreased NIS proteinexpression. NO-reduced I^- uptake resulted from transcriptionalrepression of NIS gene rather than post-transcriptional modifications impairingfunctional NIS expression at the plasma membrane. We observed that NO donorsrepress TSH-induced NIS gene expression by reducing the NF-κB subunit p65-dependenttranscriptional activity rather than affecting Pax8 expression ortranscriptional activity. NO-promoted Cys-38 p65 S-nitrosylation reducesp65-mediated transactivation of the NIS promoter in response to TSHstimulation.

We demonstrated that exogenous NO-induced p65 S-nitrosylationrepressed TSH-stimulated NIS gene transcription, thus leading to a subsequentreduction of NIS-mediated I^- uptake in rat thyrocytes. Thesefindings support the participation of NO as an inhibitory signal molecule tocounterbalance TSH-stimulated NF-κB activation, thus modulating TSH-inducedthyroid specific gene expression.