Adipocyte signaling affects thyroid-specific gene expression via down-regulation of TTF-2/FOXE1.

Obesity affects thyroid gland function. Hypothyroidism, thyroid nodules, goiter, and thyroid cancer are more frequent in patients with higher BMI values. Although these data are supported by many clinical and epidemiological studies, our knowledge is very scarce at the molecular level. In this study, we present the first experimental evidence that adipocyte signaling downregulates the expression of thyroid-specific transcription factor 2 (TTF-2/FoxE1). It plays a crucial role in thyroid development and thyroid homeostasis and it is strictly connected to thyroid cancer as well. We provide in vivo and in vitro evidence that inhibition of TTF-2/FoxE1 gene expression is mediated by adipocyte signaling.

TTF-1/Nkx2.1 functional connection with mutated EGFR relies on LRIG1 and ß-catenin pathways in lung cancer cells.

In non-small lung cancer, the expression of the transcription factor TTF-1/Nkx2.1 correlates with the presence of EGFR mutations, therefore TTF-1/Nkx2.1 expression is used to optimize an EGFR testing strategy and to guide clinical treatment. We investigate the molecular mechanisms underlying the functional connection between EGFR and TTF-1/Nkx2.1 gene expression in lung adenocarcinoma. Using the H1975 cell line as a non-small cell lung cancer model system and short hairpin RNA, we have selected clones with TTF-1/Nkx2.1 silenced expression. We have found that Leucine-rich immunoglobulin repeats-1 (LRIG1) gene is a direct target of TTF-1/Nkx2.1 and the transcription factor binding to the LRIG1 genomic sequence inhibits its gene expression. In TTF-1/Nkx2.1 depleted clones, we have found high levels of LRIG1 and decreased presence of EGFR protein. Furthermore, in TTF-1/Nkx2.1 depleted clones we detected a reduced ß-catenin level and we provide experimental evidence indicating that TTF-1/Nkx2.1 gene expression is regulated by ß-catenin. Published studies indicate that LRIG1 triggers EGFR degradation and that mutated EGFR induces ß-catenin activity. Hence, with the present study we show that mutated EGFR, enhancing ß-catenin, stimulates TTF-1/Nkx2.1 gene expression and, at the same time, TTF-1/Nkx2.1, down-regulating LRIG1, sustains EGFR pathway. Therefore, LRIG1 and ß-catenin mediate the functional connection between TTF-1/Nkx2.1 and mutated EGFR.

Functional characterization of two novel mutations in TTF-1/NKX2.1 homeodomain in patients with benign hereditary chorea.

The thyroid transcription factor 1 (TTF-1) is encoded, on chromosome 14q13, by the gene termed TITF-1/NKX2.1. Mutations in this gene have been associated with chorea, hypothyroidism, and lung disease, all included in the "brain-thyroid-lung syndrome." We here describe two cases of novel missense mutations [NM_003317.3:c.516G>T and c.623G>C resulting in p.(Gln172His) and p.(Trp208Ser), respectively] in TITF-1/NKX2-1 in non-consanguineous patients. We provide a functional study of the role of the two mutations on the TTF-1 ability to bind DNA and to trans-activate both thyroid and lung specific gene promoters. Our results confirm the difficulty to correlate the TTF-1 activity with the clinical phenotype of affected patients and highlight the need to increase the limited knowledge we have on the activity of TTF-1 in neuronal cells.

Large scale analysis of transcription factor TTF-1/NKX2.1 target genes in GnRH secreting cell line GT1-7.

TTF-1/Nkx2.1 is a homeodomain-containing transcription factor required for the proper development of ventral forebrain, including some structures of the hypothalamus. TTF-1/Nkx2.1 remains expressed in the hypothalamus after birth and it plays a crucial role during sexual development. To identify putative TTF-1/Nkx2.1 target genes in GnRH neurons, we have studied the gene expression profile of the GT1-7 cells exogenously expressing TTF-1/Nkx2.1 coding gene. Our transcriptome analysis confirms that TTF-1/Nkx2.1 is involved in neuron morphogenesis and differentiation. Many of the newly identified TTF-1/Nkx2.1 target genes have a direct involvement with the central regulation of sexual maturity. In particular, we have identified Sparc as a gene directly regulated by TTF-1/Nkx2.1 at the promoter level. To further support the role of TTF-1 in GnRH neurons, we show that Sparc is involved in the regulation of the GnRH secretion in GT1-7 cells.

Identification of the minimal melanocyte-specific promoter in the melanocortin receptor 1 gene.

BACKGROUND: The understanding of cutaneous pigmentation biology is relevant from the biologic and clinical point of view. The binding of alpha-melanocortin and its specific receptor, on the plasma membrane of melanin synthesising cells, plays a crucial role in melanins biosynthesis. Furthermore, loss of MC1R function is associated with an increased incidence of melanoma and non-melanoma skin cancer. The expression of the alpha-melanocortin receptor gene is highly controlled but, at the present, region responsible for tissue-specific activity of the gene promoter has not been identified. METHODS: We have cloned the genomic sequences upstream the human MC1R coding gene. A DNA fragment of 5 kilobases upstream the human MC1R encoding sequence was placed in front of a reporter gene and several deletion mutants of such fragment have been prepared. These constructs have been tested for the ability to drive the melanocyte-specific gene expression of the reporter gene using transfection experiments in melanocyte and non-melanocyte cell lines. From these experiments we identified a DNA fragment with the ability to drive the gene transcription in a tissue-specific way and we used this small DNA fragment in DNA-protein interaction assays. RESULTS: We show that the 150 base pairs upstream the MC1R gene initiation codon are able to drive the melanocyte-specific gene transcription. Furthermore, we provide experimental evidences suggesting that on such minimal melanocyte-specific gene promoter can assemble tissue-specific complexes. CONCLUSION: The present results strongly imply that the transcriptional regulation of the melanocyte-specific MC1R gene requires an internal promoter located in the 150 base pairs upstream the initiation codon.

Functional characterization of a novel mutation in TITF-1 in a patient with benign hereditary chorea.

Benign hereditary chorea (BHC) is an autosomal dominant disorder of early onset characterised by non progressive choreic movements with normal cognitive function occasionally associated with hypothyroidism and respiratory problems. Numerous pieces of evidence link BHC with TITF-1/NKX2.1 gene mutations. We studied a patient with a familial benign hereditary chorea and normal thyroid and respiratory function. Sequence analysis of TITF-1 revealed the presence of a heterozygous C>T substitution at nucleotide 532, predicted to change an arginine (CGA) with a stop codon (TGA) at position 178 (R178X). A functional analysis shows that the mutated TTF-1 is not binding DNA, nor activating the canonical thyroid target gene promoter or interfering with the ability of wild type TTF-1 to activate transcription. In addition, the mutated protein is predominantly cytoplasmic, rather than nuclear as in the case of the wild type TTF-1. Thus, we have identified a new mutation in the TTF-1 coding gene in a patient with benign hereditary chorea. The results show that the mutation leads to a haploinsufficiency of TITF-1 and opens the question of genotype/phenotype correlation.

Retinoblastoma protein acts as Pax 8 transcriptional coactivator.

Control of cell proliferation and differentiation by the retinoblastoma protein (pRb) depends on its interactions with key cellular substrates. Available data indicate that pRb and the transcription factor Pax 8 play a crucial role in the differentiation of thyroid follicular cells. In this study, we show that pRb takes part in the complex assembled on the thyroperoxidase gene promoter acting as a transcriptional coactivator of Pax 8. Accordingly, pRb interacts with and potentiates Pax 8 transcriptional activity. In addition, we show that the downregulation of pRb gene expression, in thyrocytes, through RNA interference results in a reduction of the thyroperoxidase gene promoter activity mediated by the Pax 8-binding site. In agreement with these results and with the ability of the adenoviral protein E1A to bind pRb, we show that E1A downregulates Pax 8 activity and that such inhibition requires the E1A-Rb interaction. Furthermore, we show that the Pax 8/pRb synergy plays a role on the sodium/iodide symporter gene expression as well.

The synergistic activity of thyroid transcription factor 1 and Pax 8 relies on the promoter/enhancer interplay.

The transcription factors, thyroid transcription factor 1 (TTF-1) and Pax 8, play a pivotal role in the transcriptional regulation of the thyroid differentiation marker genes and in the differentiation of the thyroid follicular cells. They have a very restricted tissue distribution, and the thyrocyte is the only cell type with the simultaneous expression of these factors. Here we show that TTF-1 and Pax 8 cooperatively activate their target genes and that their synergistic activity requires the cross-talk between enhancer and gene promoter. We have characterized the cis and trans requirements of the TTF1/Pax 8 synergistic activity on the thyroperoxidase gene. We show that their synergy is also important for thyroglobulin gene transcription.