The sodium/iodide symporter (NIS) is a plasma membrane protein that mediates active iodide transport in thyroid and mammary cells. It is a prerequisite for radioiodide treatment of thyroid cancer and a promising diagnostic and therapeutic tool for breast cancer. We investigated the molecular mechanisms governing NIS expression in mammary cells. Here we report that Nkx-2.5, a cardiac homeobox transcription factor that is also expressed in the thyroid primordium, is a potent inducer of the NIS promoter. By binding to two specific promoter sites (N2 and W), Nkx-2.5 induced the rNIS promoter (about 50-fold over the basal level). Interestingly, coincident with NIS expression, Nkx-2.5 mRNA and protein were present in lactating, but not virgin, mammary glands in two human breast cancer samples and in all-trans retinoic acid (tRA)-stimulated MCF-7 breast cancer cells. A cotransfected dominant-negative Nkx-2.5 mutant abolished tRA-induced endogenous NIS induction, which shows that Nkx-2.5 activity is critical for this process. Remarkably, in MCF-7 cells, Nkx-2.5 overexpression alone was sufficient to induce NIS and iodide uptake. In conclusion, Nkx-2.5 is a novel relevant transcriptional regulator of mammary NIS and could thus be exploited to manipulate NIS expression in breast cancer treatment strategies.

By producing T3 from T4, type 2 iodothyronine deiodinase (D2) catalyzes the first step in the cascade underlying the effect exerted by thyroid hormone. Type 2 iodothyronine deiodinase mRNA is expressed at high levels in human heart but is barely detectable in the corresponding rodent tissue. Although the heart is a major target of thyroid hormone, the role of cardiac D2 and the factors that regulate its expression are unknown. Here we report that the human Dio2 promoter is very sensitive to the cardiac transcription factors Nkx-2.5 and GATA-4. Nkx-2.5 transactivates a 6.5-kb human (h)Dio2-chloramphenicol acetyltransferase construct, with maximal induction reached with a 633-bp proximal promoter region. Interestingly, despite 73% identity with the corresponding human region, the rat Dio2 promoter is much less responsive to Nkx-2.5 induction. Using EMSA, we found that two sites in the human promoter (C and D) specifically bind Nkx-2.5. In coexpression studies, GATA-4 alone was a poor inducer of the hDio2 promoter; however in synergy with Nkx-2.5, it activated D2 reporter gene expression in the human, but not the rat promoter. Functional analysis showed that both C and D sites are required for the complete Nkx-2.5 response and for the Nkx-2.5/GATA-4 synergistic effect. In neonatal rat primary myocardiocytes, most of the hDio2-chloramphenicol acetyltransferase activity was suppressed by mutation of the Nkx-2.5 binding sites. Finally, a mutant Nkx-2.5 protein (N188K), which causes, in heterozygosity, congenital heart diseases, did not transactivate the Dio2 promoter and interfered with its activity in cardiomyocytes, possibly by titrating endogenous Nkx-2.5 protein away from the promoter. In conclusion, this study shows that Nkx-2.5 and GATA-4 play prime roles in Dio2 gene regulation in the human heart and suggests that it is their synergistic action in humans that causes the differential expression of the cardiac Dio2 gene between humans and rats.

Thyroid transcription factor gene 1 (TTF-1) is a homeobox-containing gene involved in thyroid organogenesis. During early thyroid development, the homeobox gene Nkx-2.5 is expressed in thyroid precursor cells coincident with the appearance of TTF-1. The aim of this study was to investigate the molecular mechanisms underlying thyroid-specific gene expression. We show that the Nkx-2.5 C terminus interacts with the TTF-1 homeodomain and, moreover, that the expression of a dominant-negative Nkx-2.5 isoform (N188K) in thyroid cells reduces TTF-1-driven transcription by titrating TTF-1 away from its target DNA. This process reduced the expression of several thyroid-specific genes, including pendrin and thyroglobulin. Similarly, down-regulation of TTF-1 by RNA interference reduced the expression of both genes, whose promoters are sensitive to and directly associate with TTF-1 in the chromatin context. In conclusion, we demonstrate that pendrin and thyroglobulin are downstream targets in vivo of TTF-1, whose action is a prime factor in controlling thyroid differentiation in vivo.

A New Method for the Experimental Assessment of Finger Haemodynamic Effects Induced by a Hydraulic Breaker in OperativeConditions: Matteo VALENTINO, et al.

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