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TTF-1 and PAX-8 are tissue-specific transcription factors expressed in the thyroid follicular cells, contributing to the maintenance of the differentiated phenotype. In fact, it has been demonstrated that TTF-1 and PAX-8 are able to activate transcription from thyroglobulin and thyroperoxidase (TPO) promoters, the transcriptional activity of which is in vivo restricted only to the thyroid follicular cell. In order to gain insight into how these transcription factors control in vivo the differentiation of the thyroid cell and to have a better molecular characterization of human thyroid tumors, TTF-1, PAX-8, thyroglobulin, and TPO mRNA levels were measured in nonmalignant and malignant human thyroid tissues. Results indicate that the expression of TTF-1 and PAX-8 is not sufficient per se for the expression of the thyroid-differentiated phenotype. Furthermore, in follicular adenomas, PAX-8 mRNA levels are strictly related to TPO mRNA levels, suggesting that the amount of PAX-8 could play a role in the modulation of TPO gene expression. TTF-1 mRNA is always well detectable in papillary carcinomas and, in contrast, always absent in anaplastic carcinomas. Identical results were obtained when the expression of TTF-1 protein was investigated using immunohistochemistry. Thus, TTF-1 gene expression could be a molecular marker in order to distinguish these two types of thyroid neoplasms.

Activating point mutations in the Ras oncogene occur in a large number of human tumors, especially of epithelial origin. In thyroid follicular cells, ectopic expression of oncogenic H-Ras results in growth factor-independent proliferation, loss of differentiation and tumor formation in nude mice. In fibroblasts concomitant activation of the MAP kinase cascade and the small GTPase Rac-1 leads to full malignant transformation. We have tested the effects of these key downstream mediators of Ras in thyroid epithelial cells, by stably expressing either a constitutively active form of MEK-1 (MEK(deltaN3/S218E/S222D)), a constitutively active form of Rac-1 (Val12-Rac), or both. While the activation of one molecule or the other results in a weak phenotype, concomitant activation of both MEK-1 and Rac-1 in thyroid cells leads to growth factor-independent proliferation, morphological transformation and anchorage-independent growth. However, in contrast to Ras-transformed thyroid cells, the ones expressing the constitutively active forms of MEK-1 and Rac-1 maintain their differentiate phenotype and fail to form tumors when injected into nude mice. Thus, in thyroid epithelial cells, concomitant activation of MEK-1 and Rac-1 can reproduce only a subset of the Ras-induced effects and is not sufficient to cause full malignant transformation. Significantly, Ras-mediated increased proliferation and loss of differentiation can be dissociated in these cells.

The conformational stability of TTF-1HD has been determined by CD-monitored thermal denaturation and isothermal urea unfolding studies. The Gibbs free energy of stabilization found are 1.44 and 1.26 kcal.mol-1, respectively. TTF-1HD exhibits a Tm of 42 degrees C and a delta Cp of 80 cal.mol-1.K-1 indicating that TTF-1HD, when free in solution, is a mobile flexible segment folded into loose helices. Such a flexibility would be relevant for the DNA-binding function of this homeodomain. In fact, a small reduction of the alpha-helical content of TTF-1HD significantly modifies its DNA-binding activity.

Background The molecular mechanisms leading to a fully differentiated thyrocite are still object of intense study even if it is well known that thyroglobulin, thyroperoxidase, NIS and TSHr are the marker genes of thyroid differentiation. It is also well known that Pax8, TTF-1, Foxe1 and Hhex are the thyroid-enriched transcription factors responsible for the expression of the above genes, thus are responsible for the differentiated thyroid phenotype. In particular, the role of Pax8 in the fully developed thyroid gland was studied in depth and it was established that it plays a key role in thyroid development and differentiation. However, to date the bases for the thyroid-enriched expression of this transcription factor have not been unraveled yet. Here, we report the identification and characterization of a functional thyroid-specific enhancer element located far upstream of the Pax8 gene. Results We hypothesized that regulatory cis-acting elements are conserved among mammalian genes. Comparison of a genomic region extending for about 100 kb at the 5'-flanking region of the mouse and human Pax8 gene revealed several conserved regions that were tested for enhancer activity in thyroid and non-thyroid cells. Using this approach we identified one putative thyroid-specific regulatory element located 84.6 kb upstream of the Pax8 transcription start site. The in silico data were verified by promoter-reporter assays in thyroid and non-thyroid cells. Interestingly, the identified far upstream element manifested a very high transcriptional activity in the thyroid cell line PC Cl3, but showed no activity in HeLa cells. In addition, the data here reported indicate that the thyroid-enriched transcription factor TTF-1 is able to bind in vitro and in vivo the Pax8 far upstream element, and is capable to activate transcription from it. Conclusions Results of this study reveal the presence of a thyroid-specific regulatory element in the 5' upstream region of the Pax8 gene. The identification of this regulatory element represents the first step in the investigation of upstream regulatory mechanisms that control Pax8 transcription during thyroid differentiation and are relevant to further studies on Pax8 as a candidate gene for thyroid dysgenesis.

In the present study, we demonstrate a direct role for d-aspartate in regulating hippocampal synaptic plasticity. These evidences were obtained using two different experimental strategies which enabled a non-physiological increase of endogenous d-aspartate levels in the mouse hippocampus: a genetic approach based on the targeted deletion of d-aspartate oxidase gene and another based on the oral administration of d-aspartate. Overall, our results indicate that increased d-aspartate content does not affect basal properties of synaptic transmission but enhances long-term potentiation in hippocampal slices from both genetic and pharmacological animal models. Besides electrophysiological data, behavioral analysis suggests that altered levels of d-aspartate in the hippocampus do not perturb basal spatial learning and memory abilities, but may selectively interfere with the dynamic NMDAR-dependent processes underlying cognitive flexibility. In the present study, we demonstrate a direct role for d-aspartate in regulating hippocampal synaptic plasticity. These evidences were obtained using two different experimental strategies which enabled a non-physiological increase of endogenous d-aspartate levels in the mouse hippocampus: a genetic approach based on the targeted deletion of d-aspartate oxidase gene and another based on the oral administration of d-aspartate. Overall, our results indicate that increased d-aspartate content does not affect basal properties of synaptic transmission but enhances long-term potentiation in hippocampal slices from both genetic and pharmacological animal models. Besides electrophysiological data, behavioral analysis suggests that altered levels of d-aspartate in the hippocampus do not perturb basal spatial learning and memory abilities, but may selectively interfere with the dynamic NMDAR-dependent processes underlying cognitive flexibility. © 2007 Elsevier Inc. All rights reserved.

We have conditionally inactivated the E-cadherin gene in the thyroid follicular cells of mouse embryo to unravel its role in thyroid development. We used the Cre-loxP system in which the Cre-recombinase was expressed under the control of the tissue-specific thyroglobulin promoter that becomes active at embryonic d 15. At postnatal d 7, thyroid follicle lumens in the knockout mice were about 30% smaller with respect to control mice and had an irregular shape. E-cadherin was almost completely absent in thyrocytes, beta-catenin was significantly reduced, whereas no change in gamma-catenin was detected. alpha-Catenin was also reduced on the cell plasma membrane. Despite the dramatic loss of E-cadherin and beta-catenin, cell-cell junctions were not affected, the distribution of tight junction proteins was unaltered, and no increase of thyroglobulin circulating in the blood was observed. In addition, we found that other members of the cadherin family, the R-cadherin and the Ksp-cadherin, were expressed in thyrocytes and that their membrane distribution was not altered in the E-cadherin conditional knockout mouse. Our results indicate that E-cadherin has a role in the development of the thyroid gland and in the expression of beta-catenin, but it is not essential for the maintenance of follicular cell adhesion.

Ras proteins are highly related GTPases that have key roles in regulating growth, differentiation and tumorigenesis. Gene-targeting experiments have shown that, out of the three mammalian ras genes, only K-ras is essential for normal mouse embryogenesis, and that mice deprived of H-ras and/or N-ras show no major phenotype. We generated mice (HrasKI) in which the K-ras gene had been modified to encode H-Ras protein. HrasKI mice produce undetectable amounts of K-Ras but - in contrast to mice homozygous for a null K-ras allele - they are born at the expected mendelian frequency, indicating that H-Ras can be substituted for K-Ras in embryonic development. However, adult HrasKI mice show dilated cardiomyopathy associated with arterial hypertension. Our results show that K-Ras can be replaced by H-Ras in its essential function in embryogenesis, and indicate that K-Ras has a unique role in cardiovascular homeostasis. © 2005 European Molecular Biology Organization.

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.

The transcription factor Pax8 plays an important role in the expression of the differentiated phenotype of thyroid follicular cells. It has recently been shown that Pax8 is necessary for thyroglobulin (Tg) gene expression in the fully differentiated rat thyroid cell line PC. We have used the PC model system to investigate the role of Pax8 as a mediator of TSH regulation of Tg gene expression. We have demonstrated that Pax8 expression, as well as Tg expression, is severely reduced in cells grown in the absence of hormones and serum. The re-addition of TSH or forskolin to the culture medium is able to restore to wild-type levels the expression of both Pax8 and Tg. We have determined that the action of TSH/forskolin on Pax8 is at the transcriptional level. However, the re-expression of Pax8 can be observed several hours before that of Tg, suggesting that either another factor is needed or that Pax8 itself must be post-translationally modified by a newly synthesized protein to become active. To distinguish between these two possibilities we have stably transfected into PC cells an exogenous Pax8 that is expressed independently of TSH. Our results indicate that in these cells the Tg promoter is still dependent on TSH despite the constitutive presence of Pax8. Furthermore, we also show that in this condition Tg gene transcription requires de novo protein synthesis. In conclusion, TSH regulates the expression of Pax8 at a transcriptional level and also regulates the activity of Pax8 by controlling the expression of one or more as yet unknown factors.

The transcription factor thyroid transcription factor-1 (TTF-1) is a homeodomain-containing protein that belongs to the NK2 family of genes involved in organogenesis. TTF-1 is required for normal development of the forebrain, lung, and thyroid. In a search for factors that regulate TTF-1 transcriptional activity, we isolated three genes (T:G mismatch-specific thymine DNA glycosylase (TDG), homeodomain-interacting protein kinase 2 (HIPK2), and Ajuba), whose products can interact with TTF-1 in yeast and in mammalian cells. TDG is an enzyme involved in base excision repair. In the present paper, we show that TDG acts as a strong repressor of TTF-1 transcriptional activity in a dose-dependent manner, while HIPK2 and Ajuba display no effect on TTF-1 activity, at least under the tested conditions. TDG-mediated inhibition occurs specifically on TTF-1-responsive promoters in thyroid and non thyroid cells. TDG associates with TTF-1 in mammalian cells through the TTF-1 carboxyl-terminal activation domain and is independent of the homeodomain. These findings reveal a previously unsuspected role for the repair enzyme TDG as a transcriptional repressor and open new routes toward the understanding of the regulation of TTF-1 transcriptional activity.

Several homeobox-containing genes related to Drosophila Distal-less (Dll) have been isolated from a wide variety of organisms and have been shown to function as developmental regulators. While in Drosophila only one Dll gene has been described so far, in Vertebrates many components of the Dlx multigenic family have been characterized. This suggests that, during the evolution of the Chordate phylum, the Dlx genes arose from an ancestral Dll/Dlx gene via gene duplication. We have previously reported the isolation of two Dll-related homeoboxes from the protochordate Ciona intestinalis, and described their clustered arrangement (Gene 156 (1995) 253). Here we present the detailed genomic organization and spatial-temporal expression of these two genes, Ci-Dll-A and Ci-Dll-B, and describe the isolation and characterization of another member of the ascidian family of Dll-related genes, which we tentatively named Ci-Dll-C.

Expression of oncogenic v-H-Ras in the thyroid cell line FRTL-5 (FRTL-5(Ras)) results in uncontrolled proliferation, loss of thyroid-specific gene expression and tumorigenicity. Concomitant expression of constitutively activated MEK and Rac, two major H-Ras downstream effectors, in FRTL-5 (FRTL-5(MEK/Rac)) recapitulates H-Ras effects on proliferation and morphology. In contrast to FRTL-5(Ras), however, FRTL-5(MEK/Rac) cells remain differentiated and are not tumorigenic. To find H-Ras induced genes potentially responsible for tumorigenicity and loss of differentiation, we have used subtractive suppression hybridization (SSH), a PCR-based cDNA subtraction technique, between de-differentiated and tumorigenic FRTL-5(Ras) cells and differentiated and non-tumorigenic FRTL-5(MEK/Rac) cells. We examined 800 of the cDNA clones obtained after subtraction and verified their levels of expression in the two cell lines by reverse northern, identifying 337 H-Ras induced genes. By sequence analysis, we clustered 57 different genes. Among these, 39 were known genes (involved in diverse signal transduction processes regulating mitogenic activity, cell survival, cytoskeletal reorganization, stress response and invasion) while the remaining 18 clones were novel genes. Among the 57 H-Ras specific clones, we identified those genes whose expression is induced early by H-Ras. We suggest that these immediate-early genes may play a crucial role in H-Ras-mediated transformation in thyroid epithelial cells.

The thyroglobulin gene, the substrate for thyroid hormone biosynthesis, is not expressed in the FRT cell line, which, even though it manifests the polarised epithelial phenotype, does not express any of the thyroid functional properties. Two transcription factors, TTF-1 and Pax-8, have been implicated in thyroid specific expression of the thyroglobulin gene. FRT cells contain Pax-8 but they lack TTF-1. In this paper, we show that transfection of TTF-1 expression vectors in FRT cells results in activation of thyroglobulin gene expression. If the expression vector encoded for TTF-1-ER, a fusion gene coding for the entire TTF-1 protein fused to the hormone-binding domain of the steroid receptor, under the control of the RSV promoter, thyroglobulin gene expression was controlled by estrogen. These data provide a direct demonstration that TTF-1 activates the chromosomal thyroglobulin promoter. Since transfection of TTF-1 expression vectors in non-thyroid cell types did not result in thyroglobulin gene expression, it is suggested that Pax-8, in addition, perhaps, to a specific cellular environment, might be required for thyroid specific expression of the thyroglobulin gene. (C) 1997 Elsevier Science B.V.