The functional interaction between the paired domain transcription factor Pax8 and Smad3 is involved in transforming growth factor-beta repression of the sodium/iodide symporter gene.

Transforming growth factor-beta (TGF-beta) 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-beta on the regulation of sodium/iodide symporter (NIS) gene expression in PC Cl3 thyroid cells. TGF-beta 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-beta 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-beta was partially overcome by inhibitory Smad7 and mimicked by overexpression of either Smad3 or a constitutively activated mutant of TGF-beta 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-beta/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-beta treatment. The results suggest that, through activation of Smad3, TGF-beta decreases Pax8 DNA binding activity as well as Pax8 mRNA and protein levels, which are at least partially involved in TGF-beta-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.