NO is a free radical that mediates a wide array of cellular functions in many tissues. It is generated from L-arginine by NO-synthases (NOS), with additional production of L-citrulline. NOS isoforms expression has been demonstrated in thyroid cells. Previous reports indicate that NO donors induce thyroid dedifferentiation. However, the functional significance of thyrocyte-produced endogenous NO has not been examined. We previously reported an increase of the TSH-stimulated iodide uptake and thyroglobulin (TG) mRNA expression by the NOS inhibitor, L-NAME (1mM, 48 h) in FRTL-5 cells. This work aimed to explore the role of endogenously produced NO on expression of thyroid-specific genes and proliferation in FRTL-5 cells. Treatment with TSH for 24-48 h increased NO (14C-citrulline; maximum TSH 200µUI/ml, 24 h, 2-fold, p<0.05) indicating a TSH-dependent NO production. To analyse the NO action on TG gene expression at transcriptional level, minimal TG promoter region (-168 to +36 bp) was transfected. An increment of TSH (200µUI/ml)-stimulated activity of TG promoter after incubation with two NOS inhibitors, L-NAME and L-NMMA (0.05mM) for 12-24 h was evidenced (maximum 24h, 1.8-fold, p<0.05). The presence of L-NAME also induced an increase of TSH-stimulated NIS mRNA expression (Northern-Blot; 48h, 2-fold, p<0.001). A significant increase of TSH-induced transcriptional activity of a NIS promoter region (-2841 to +13 bp) was obtained in presence of L-NMMA (24h, 2-fold, p<0.05). We also analyzed the action of endogenous NO on cell proliferation. The presence of L-NMMA (0.1mM) induced an increase of the TSH (100-300 µUI/ml)-stimulated cell proliferation (³H-thymidine incorporation) in a concentration-dependent manner at 24-48 h (maximum 24h, 2-fold, p<0.001). In conclusion, our data provide strong evidence that endogenous NO could act as a negative signal for TSH-stimulated expression of specific genes and proliferation in thyroid cell.
Since TSH induced NO production, a novel physiological role of NO to regulate the stimulatory action of TSH could be proposed.