RESUMO
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.
Assuntos
Antiportadores de Cloreto-Bicarbonato/fisiologia , Regulação da Expressão Gênica , Proteínas de Membrana Transportadoras/fisiologia , Proteínas Nucleares/fisiologia , Neoplasias da Glândula Tireoide/metabolismo , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Northern Blotting , Western Blotting , Diferenciação Celular , Antiportadores de Cloreto-Bicarbonato/metabolismo , Cromatina , Imunoprecipitação da Cromatina , DNA/química , Regulação para Baixo , Genes Dominantes , Células HeLa , Humanos , Imunoprecipitação , Íntrons , Íons , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/química , Oligonucleotídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Interferência de RNA , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transportadores de Sulfato , Glândula Tireoide/metabolismo , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/química , TransfecçãoRESUMO
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.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Homeodomínio/metabolismo , Simportadores/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Tumoral , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Expressão Gênica , Células HeLa , Proteína Homeobox Nkx-2.5 , Humanos , Lactação/genética , Lactação/metabolismo , Mutagênese Sítio-Dirigida , Gravidez , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Ratos , Glândula Tireoide/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transfecção , Tretinoína/farmacologiaRESUMO
The Sonic hedgehog (Shh) pathway plays a critical role in hair follicle physiology and is constitutively active in basal cell carcinomas (BCCs), the most common human malignancy. Type 3 iodothyronine deiodinase (D3), the thyroid hormone-inactivating enzyme, is frequently expressed in proliferating and neoplastic cells, but its role in this context is unknown. Here we show that Shh, through Gli2, directly induces D3 in proliferating keratinocytes and in mouse and human BCCs. We demonstrate that Gli-induced D3 reduces intracellular active thyroid hormone, thus resulting in increased cyclin D1 and keratinocyte proliferation. D3 knockdown caused a 5-fold reduction in the growth of BCC xenografts in nude mice. Shh-induced thyroid hormone degradation via D3 synergizes with the Shh-mediated reduction of the type 2 deiodinase, the thyroxine-activating enzyme, and both effects are reversed by cAMP. This previously unrecognized functional cross-talk between Shh/Gli2 and thyroid hormone in keratinocytes is a pathway by which Shh produces its proliferative effects and offers a potential therapeutic approach to BCC.