Transforming growth factor-␤ (TGF-␤) is a secreted protein that regulates proliferation, differentiation, and death in various cell types, including thyroid cells, although few details are known about its mechanisms of action in this cell type. Here, we studied the role of TGF-␤ on the regulation of sodium/iodide symporter (NIS) gene expression in PC Cl3 thyroid cells. TGF-␤ inhibits thyroid-stimulated hormone (TSH)-induced NIS mRNA and protein levels in a dose-dependent manner. This effect takes place at the transcriptional level, as TGF-␤ inhibits TSH-induced transcription of a luciferase reporter construct containing a 2.8-kb DNA fragment of the rat NIS promoter. The inhibitory effect of TGF-␤ was partially overcome by inhibitory Smad7 and mimicked by overexpression of either Smad3 or a constitutively activated mutant of TGF-␤ receptor I (acALK-5). Using internal deletions of the promoter, we defined a region between ؊2,841 to ؊1,941, which includes the NIS upstream enhancer (NUE), as responsible for the TGF-␤/Smad inhibitory effect. NUE contains two binding sites for the paired domain transcription factor Pax8, the main factor controlling NIS transcription. The physical interaction observed between Pax8 and Smad3 appears to be responsible for the decrease in Pax8 binding to DNA. Expression of Pax8 mRNA and protein was also decreased by TGF-␤ treatment. The results suggest that, through activation of Smad3, TGF-␤ decreases Pax8 DNA binding activity as well as Pax8 mRNA and protein levels, which are at least partially involved in TGF-␤-induced down-regulation of NIS gene expression in thyroid follicular cells. Our results thus demonstrate a novel mechanism of Smad3 function in regulating thyroid cell differentiation by functionally antagonizing the action of the paired domain transcription factor Pax8.Iodide is an essential element in thyroid physiology as a critical component of thyroxine and triiodothyronine molecules and a key regulator of thyroid gland function. The first step in iodide metabolism is represented by thyroid trapping, which is achieved by an active, energy-dependent transport process across the basolateral plasma membrane of the thyrocytes. The protein responsible for this process, the sodium/iodide symporter (NIS), 1 is an intrinsic plasma membrane protein that mediates active transport of I Ϫ in the thyroid, lactating mammary gland, stomach, and salivary glands (1-4). NIS plays key role in thyroid pathophysiology as the route by which iodide reaches the gland for thyroid hormone biosynthesis and as a means for diagnostic scintigraphic imaging and radioiodide therapy in hyperthyroidism and thyroid cancer (2, 4).The molecular characterization of NIS started with the cloning of the cDNA encoding rat NIS in 1996 (5). The rNIS gene has a minimal promoter between Ϫ199 and Ϫ110 bp (3, 6 -8) and an upstream enhancer, NUE (NIS upstream enhancer), between Ϫ2495 and Ϫ2264 bp (9). The NUE stimulates transcription in a thyroid-specific, cAMP-dependent manner and involves the most relevant aspect of NIS...

Signaling by TSH through its receptor leads to the dissociation of trimeric G proteins into Galpha and Gbetagamma. Galphas activates adenylyl cyclase, which increases cAMP levels that induce several effects in the thyroid cell, including transcription of the sodium-iodide symporter (NIS) gene through a mechanism involving Pax8 binding to the NIS promoter. Much less is known about the function of Gbetagamma in thyroid differentiation, and therefore we studied their role in TSH signaling. Gbetagamma overexpression inhibits NIS promoter activation and reduces NIS protein accumulation in response to TSH and forskolin. Conversely, inhibition of Gbetagamma-dependent pathways increases NIS promoter activity elicited by TSH but does not modify forskolin-induced activation. Gbetagamma dimers are being released from the Gs subfamily of proteins, because cholera toxin mimics the effects elicited by TSH, whereas pertussis toxin has no effect on NIS promoter activity. We also found that TSH stimulates Akt phosphorylation in a phosphoinositide 3-kinase (PI3K)-dependent and cAMP-independent manner. This is mediated by Gbetagamma, because its overexpression or specific sequestration, respectively, increased or reduced phosphorylated Akt levels upon TSH stimulation. Gbetagamma sequestration increases NIS protein levels induced by TSH and Pax8 binding to the NIS promoter, which is also increased by PI3K inhibition. This is, at least in part, caused by Gbetagamma-mediated Pax8 exclusion from the nucleus that is attenuated when PI3K activity is blocked. These data unequivocally demonstrate that Gbetagamma released by TSH action stimulate PI3K, inhibiting NIS gene expression in a cAMP-independent manner due to a decrease in Pax8 binding to the NIS promoter.