Search

BACKGROUND: TAZ (Transcriptional co-Activator with PDZ-binding motif), is a biologically potent transcriptional coactivator and functions by binding to the PPXY motif present in several transcription factors. Notably, TAZ behaves as a transducer linking cytoplasmic signaling events to transcriptional regulation in the nucleus. Several different factors regulate TAZ expression and/or function. In particular, a major regulation of TAZ activity occurs through the Hippo pathway by a phosphorylation-mediated mechanism that causes its cytoplasmic sequestration or degradation. RESULTS: Here we demonstrate that AMOTL2 robustly co-immunoprecipitates with TAZ, and their interaction is dependent on the WW domain of TAZ and the PPXY motif in the N-terminus of AMOTL2. Furthermore, we show that AMOTL2 colocalizes with TAZ in the cytoplasm of H441 human lung cells and regulates TAZ cytoplasm-to-nucleus translocation through direct protein-protein interaction. Interestingly, the overexpression of AMOTL2 inhibits the functional cooperation between the transcription factor TTF-1 and TAZ on the Surfactant C gene promoter, as well as the expression of other known target genes of these regulatory factors. CONCLUSIONS: Taken together, our results suggest an inhibitory role of AMOTL2 on TAZ ability to co-activate transcription and describe a different mechanism, Hippo pathway-independent, that modulates the activity of TAZ in lung cells through the interaction with Angiomotin-like 2 (AMOTL2).

Cadherin-16 was originally identified as a tissue-specific cadherin present exclusively in kidney. Only recently, Cadherin-16 has been detected also on the plasma membrane of mouse thyrocytes. This last finding prompted us to note that the expression profile of Cadherin-16 resembles that of the transcription factor Pax8, a member of the Pax (paired-box) gene family, predominantly expressed in the developing and adult kidney and thyroid. Pax8 has been extensively characterized in the thyroid and shown to be a master gene for thyroid development and differentiation. In this study, we determined the role of the transcription factor Pax8 in the regulation of Cadherin-16 expression. We demonstrate that the Cadherin-16 minimal promoter is transcriptionally active in thyroid cells as well as in kidney cells, that Pax8 is able to activate transcription from a Cadherin-16 promoter reporter construct, and more importantly, that indeed Pax8 is able to bind in vivo the Cadherin-16 promoter region. In addition, by means of Pax8 RNA interference in thyroid cells and by analyzing Pax8 null mice, we demonstrate that Pax8 regulates also in vivo the expression of Cadherin-16. Finally, we reveal that the expression of Cadherin-16 is TSH dependent in FRTL-5 thyroid cells and significantly reduced in mouse thyroid carcinomas. Therefore, we conclude that Cadherin-16 is a novel downstream target of the transcription factor Pax8, likely since the early steps of thyroid development, and that its expression is associated with the fully differentiated state of the thyroid cell.

BACKGROUND: The differentiation program of thyroid follicular cells (TFCs), by far the most abundant cell population of the thyroid gland, relies on the interplay between sequence-specific transcription factors and transcriptional coregulators with the basal transcriptional machinery of the cell. However, the molecular mechanisms leading to the fully differentiated thyrocyte are still the object of intense study. The transcription factor Pax8, a member of the Paired-box gene family, has been demonstrated to be a critical regulator required for proper development and differentiation of thyroid follicular cells. Despite being Pax8 well-characterized with respect to its role in regulating genes involved in thyroid differentiation, genomics approaches aiming at the identification of additional Pax8 targets are lacking and the biological pathways controlled by this transcription factor are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: To identify unique downstream targets of Pax8, we investigated the genome-wide effect of Pax8 silencing comparing the transcriptome of silenced versus normal differentiated FRTL-5 thyroid cells. In total, 2815 genes were found modulated 72 h after Pax8 RNAi, induced or repressed. Genes previously reported to be regulated by Pax8 in FRTL-5 cells were confirmed. In addition, novel targets genes involved in functional processes such as DNA replication, anion transport, kinase activity, apoptosis and cellular processes were newly identified. Transcriptome analysis highlighted that Pax8 is a key molecule for thyroid morphogenesis and differentiation. CONCLUSIONS/SIGNIFICANCE: This is the first large-scale study aimed at the identification of new genes regulated by Pax8, a master regulator of thyroid development and differentiation. The biological pathways and target genes controlled by Pax8 will have considerable importance to understand thyroid disease progression as well as to set up novel therapeutic strategies.

BACKGROUND: Congenital hypothyroidism (CH) is a common endocrine disease that occurs in about 1:3000 newborns. In 80-85% of the cases, CH is presumably secondary to thyroid dysgenesis (TD), a defect in the organogenesis of the gland leading to an ectopic (30-45%), absent (agenesis, 35-40%) or hypoplastic (5%) thyroid gland. The pathogenesis of TD is still largely unknown. Most cases of TD are sporadic, although familial occurrences have occasionally been described. Recently, mutations in the PAX8 transcription factor have been identified in patients with TD. OBJECTIVE: Our aim was to identify and functionally characterize novel PAX8 mutations with autosomal dominant transmission responsible for TD. DESIGN: The PAX8 gene was sequenced in a mother and child both suffering from congenital hypothyroidism (CH) because of thyroid hypoplasia. Subsequently, expression vectors encoding the mutated PAX8 were generated, and the effects of the mutation on both the DNA-binding capability and the transcriptional activity were evaluated. RESULTS: PAX8 gene sequencing revealed a heterozygous mutation that consists of the substitution of a histidine residue with a glutamine at position 55 of the PAX8 protein (H55Q). When tested in cotransfection experiments with a thyroglobulin promoter reporter construct, the mutant protein turned out to be still able to bind DNA in Electrophoretic Mobility Shift Assay assays but transcriptionally inactive. CONCLUSIONS: Our findings confirm the important role of PAX8 in normal thyroid development and support the evidence that in humans haploinsufficiency of PAX8 is associated with TD.

Pax8 is a transcription factor that plays an important role in the regulation of genes that are exclusively expressed in differentiated thyroid cells. In the thyroid cell environment, evidence exists that Pax8 is part of a multiprotein complex in which its transcriptional activity may be modulated by specific co-factors. In an attempt to identify proteins that interact with Pax8, we performed pull-down experiments challenging the GST-Pax8 fusion protein with protein extracts prepared from the thyroid differentiated cell line PC Cl3. By this approach, we isolated a 113-kDa protein that is able to associate with Pax8, which was further identified by mass fingerprint experiments as poly(ADP-ribose) polymerase 1 (PARP1). To further confirm this interaction, we also showed that PARP1 can be co-immunoprecipitated with Pax8 in vivo from a thyroid cell extract. Gel shifts experiments demonstrated that PARP1 binding to Pax8 significantly inhibits Pax8 binding to DNA. Accordingly, we provide evidence that the functional outcome of such an interaction is a significant downregulation of Pax8 transcriptional activity. In the context of thyroid-specific gene transcription, our results suggest that PARP1 behaves as an important negative co-factor involved in the regulation of Pax8-dependent gene expression.

To study the transforming activity of the dbl oncogene and its effect on normal development in vivo, we linked dbl cDNA to promoters with different cell type specificities and used the constructs to generate transgenic mice. The promoters included the mouse alpha A-crystallin promoter, the rat insulin II promoter, and a mouse metallothionein promoter. We also generated transgenic mice carrying a recombinant cosmid clone that contains the entire dbl gene. Mice with the crystallin promoter construct developed cataracts and expressed the dbl protein in their lenses. The architecture of the lenses suggested a block to the normal pattern of differentiation and elongation of the secondary fiber cells. Mice with the insulin II promoter expressed dbl protein in the pancreas but showed no evidence of diabetes and no apparent pancreatic beta-cell defects. Similarly, mice with the metallothionein promoter expressed dbl protein in heart and testes, but showed no pathologic abnormalities in these tissues even after treatment with heavy metals. However, one family of mice carrying the metallothionein promoter construct showed cataracts and a dramatic fibroblastic dysplasia of the lens. One family with the cosmid-dbl gene showed a nearly identical lenticular dysplasia, but with a slower developmental time course. Thus, although the dbl oncogene did not induce neoplasia in any of the mice studied, it is apparently capable of interfering with the ability of the lens epithelial cells to differentiate into lens fiber cells, and of inducing metaplasia of the epithelial cells into fibroblastic cells.

The Akt/protein kinase B serine/threonine kinase is a downstream effector of phosphoinositide 3-kinase (PI3K). Akt is an important component of mitogenic and antiapoptotic signaling pathways and is implicated in neoplastic transformation. Thyroid cells in culture retain a differentiated phenotype consisting of epithelial cell morphology and the expression of several tissue-specific genes. The survival and proliferation of these cells depend on thyrotropin and a mixture of five additional hormones that includes insulin. The regulation of proliferation and the expression of the thyroid differentiation program are intimately connected processes. As a result, oncogenes that induce hormone-independent proliferation invariably impair the expression of the thyroid-specific differentiation markers. Given that thyrotropin and insulin stimulate Akt activation in thyroid cells, we set out to determine the effects of Akt on thyroid cell proliferation, survival, and differentiation. To this end, we expressed constitutively active myristylated Akt (myrAkt) in PC Cl 3 thyroid cells. The myrAkt-expressing cells continued to proliferate, even in the absence of hormones, and they were resistant to programmed cell death induced by starvation. These effects were paralleled by the induction of the G1 cyclins D3 and E and by the inhibition of induction of the proapoptotic Fas, Fas ligand, and BAD genes in starved cells. However, in marked contrast with several other oncogenes, myrAkt did not interfere with the expression of thyroid differentiation functions. These results unveil the existence of an Akt-triggered thyroid cell pathway that modulates proliferation and survival without affecting the expression of the thyroid cell differentiated phenotype.

Expression of mutated versions of the p53 gene deranged the differentiation program of thyroid cells and resulted in deregulated growth. Specifically, p53 mutants in several residues of the DNA-binding region induced thyrotropin (TSH) -independent growth and inhibition of the expression of thyroid-specific genes. The loss of the differentiated phenotype invariably correlated with the blockage of the expression of the genes coding for the thyroid transcriptional factors PAX-8 and TTF2. Conversely, thyroid cells transfected with a p53 gene mutated at codon 392, located outside the DNA-binding region, stimulated the expression of differentiation genes in the absence of the TSH, and induced TSH-independent growth. cAMP intracellular levels were higher in thyroid cells transfected with the p53 gene mutated at the 392 site than in the untransfected thyroid cells, but lower in the cells transfected with the other mutated p53 genes. Fra-1 and c-jun were induced by p53, resulting in increased AP-1 levels. The results of this study suggest that p53 exerts effects on cAMP transduction pathway in thyroid cells, which are exquisitely sensitive to cAMP.

The Pax gene family encodes transcription factors that are essential in organogenesis and in the differentiation of various organs in higher eukaryotes. Pax proteins have a DNA binding domain at the N-terminus, and a transcriptional activation domain at the C-terminus. How these domains interact with the transcriptional machinery of the cell is still unclear. In the present paper, we describe the identification by means of immunological screening of the WW domain binding protein WBP-2 as a biochemical interactor of Pax8 (a WW domain is a protein-interaction domain containing two conserved tryptophan residues). Pax8 is required for the morphogenesis of the thyroid gland and for the maintenance of the thyroid differentiated cellular phenotype. WBP-2 was identified originally as a WW domain binding protein, and its function is still unknown. WBP-2 binds to Pax8 in vitro in pull-down assays, and in vivo in tissue culture cells in co-immunoprecipitation assays. Interestingly, Pax8 does not contain a WW domain. Our results point to the identification of a new protein-interacting domain that is present in the C-terminal portion of Pax8 and that is required for protein-protein interaction with WBP-2. Our results demonstrate that WBP-2 is not a transcriptional coactivator of Pax8, but rather behaves as an adaptor molecule, as suggested in other studies.

The most frequent genetic alterations described thus far in human papillary thyroid carcinomas are somatic rearrangements of the RET proto-oncogene, which generate the chimeric RET/PTC oncogenes. We recently found that the expression of the RET/PTC1 oncogene blocked the expression of the thyroid-differentiated phenotype in rat thyroid epithelial cell line PC CI 3 (PC). Here, we show that this block occurs at a transcriptional level; indeed, the thyroid-specific thyroglobulin and thyroperoxidase gene promoters were inactive in PC-PTC cells. Specific transcription factors, namely, TTF-1 and Pax-8, regulate the expression of differentiated functions in thyroid cells. Here, we show that Pax-8 is expressed at reduced levels in PC-PTC cells and that its adoptive overexpression is unable to restore the activity of target promoters. In contrast, TTF-1 expression is unaltered in PC-PTC cells; however, by using a synthetic promoter that contains its specific target sequence, we demonstrate that TTF-1 is inactive in PC-PTC cells. We conclude that the RET/PTC1 oncogene alters the expression of the thyroid-differentiated phenotype by at least two different mechanisms, ie., down-regulation of Pax-8 protein and mRNA expression and impaired function of TTF-1 and Pax-8, which occurs at a posttranslational level.