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Mariastella Zannini - One of the best experts on this subject based on the ideXlab platform.
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Candidate genes and pathways downstream of PAX8 involved in ovarian high-grade serous carcinoma
Oncotarget, 2016Co-Authors: Tiziana De Cristofaro, Tina Di Palma, Amata Amy Soriano, A Monticelli, Ornella Affinito, Sergio Cocozza, Mariastella ZanniniAbstract:// Tiziana de Cristofaro 1, * , Tina Di Palma 1, * , Amata Amy Soriano 1, 2 , Antonella Monticelli 1 , Ornella Affinito 1, 2 , Sergio Cocozza 2 , Mariastella Zannini 1 1 IEOS, Institute of Experimental Endocrinology and Oncology “G. Salvatore”, National Research Council, Naples, Italy 2 Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy * These authors contributed equally to this work Correspondence to: Mariastella Zannini, email: s.zannini@ieos.cnr.it Keywords: PAX8, ovarian cancer, fallopian tube secretory epithelial cells, RNA-seq, Transcriptional networks Received: March 24, 2016 Accepted: May 16, 2016 Published: May 31, 2016 ABSTRACT Understanding the biology and molecular pathogenesis of ovarian epithelial cancer (EOC) is key to developing improved diagnostic and prognostic indicators and effective therapies. Although research has traditionally focused on the hypothesis that high-grade serous carcinoma (HGSC) arises from the ovarian surface epithelium (OSE), recent studies suggest that additional sites of origin exist and a substantial proportion of cases may arise from precursor lesions located in the Fallopian tubal epithelium (FTE). In FTE cells, the Transcription Factor PAX8 is a marker of the secretory cell lineage and its expression is retained in 96% of EOC. We have recently reported that PAX8 is involved in the tumorigenic phenotype of ovarian cancer cells. In this study, to uncover genes and pathways downstream of PAX8 involved in ovarian carcinoma we have determined the molecular profiles of ovarian cancer cells and in parallel of Fallopian tube epithelial cells by means of a silencing approach followed by an RNA-seq analysis. Interestingly, we highlighted the involvement of pathways like WNT signaling, epithelial-mesenchymal transition, p53 and apoptosis. We believe that our analysis has led to the identification of candidate genes and pathways regulated by PAX8 that could be additional targets for the therapy of ovarian carcinoma.
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Neuropilin-2 Is a Newly Identified Target of PAX8 in Thyroid Cells
PloS one, 2015Co-Authors: Valeria Lucci, Tina Di Palma, Mariastella ZanniniAbstract:PAX8 is a Transcription Factor essential for thyroid gland development, as well as for the maintenance of the thyroid differentiated state in the adult. In particular, PAX8 has been comprehensively shown to regulate genes that are considered markers of thyroid differentiation. However, a better knowledge of genes Transcriptionally regulated by PAX8 is desirable to clarify its role in endocrine syndromes and cancer susceptibility. In order to further investigate PAX8 downstream targets, we recently performed a genome-wide expression analysis following PAX8 knockdown in FRTL-5 thyroid cells and Neuropilin-2 was identified as a potential Transcriptional target of PAX8. In this study, we determined the role of the Transcription Factor PAX8 in the regulation of Neuropilin-2 expression. Indeed, in thyroid cells PAX8 directly binds the Neuropilin-2 promoter leading to its Transcriptional repression. Interestingly, we observed an inverse correlation between the expression of PAX8 and Neuropilin-2 in thyroid carcinoma tissues and cell lines compared to non-tumor counterparts, suggesting a critical role of PAX8 in regulating Neuropilin-2 expression in vivo. Notably, ectopic overexpression of PAX8 in FB-2 thyroid cancer cells promotes Neuropilin-2 downregulation producing a significant reduction in cell proliferation, migration ability, and invasion activity and reverting the cell phenotype from mesenchymal to a more epithelial one. These findings uncover the novel interplay between PAX8 and Neuropilin-2, which is likely to be important in the pathogenesis of thyroid diseases.
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PAX8 modulates the expression of Wnt4 that is necessary for the maintenance of the epithelial phenotype of thyroid cells
BMC molecular biology, 2014Co-Authors: Maria Grazia Filippone, Tina Di Palma, Valeria Lucci, Mariastella ZanniniAbstract:Background The Transcription Factor PAX8 is expressed during thyroid development and is involved in the morphogenesis of the thyroid gland and maintenance of the differentiated phenotype. In particular, PAX8 has been shown to regulate genes that are considered markers of thyroid differentiation. Recently, the analysis of the gene expression profile of FRTL-5 differentiated thyroid cells after the silencing of PAX8 identified Wnt4 as a novel target. Like the other members of the Wnt family, Wnt4 has been implicated in several developmental processes including regulation of cell fate and patterning during embryogenesis. To date, the only evidence on Wnt4 in thyroid concerns its down-regulation necessary for the progression of thyroid epithelial tumors.
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PAX8 modulates the expression of Wnt4 that is necessary for the maintenance of the epithelial phenotype of thyroid cells
BMC Molecular Biology, 2014Co-Authors: Maria Grazia Filippone, Tina Di Palma, Valeria Lucci, Mariastella ZanniniAbstract:Background The Transcription Factor PAX8 is expressed during thyroid development and is involved in the morphogenesis of the thyroid gland and maintenance of the differentiated phenotype. In particular , PAX8 has been shown to regulate genes that are considered markers of thyroid differentiation. Recently, the analysis of the gene expression profile of FRTL-5 differentiated thyroid cells after the silencing of PAX8 identified Wnt4 as a novel target. Like the other members of the Wnt family, Wnt4 has been implicated in several developmental processes including regulation of cell fate and patterning during embryogenesis. To date, the only evidence on Wnt4 in thyroid concerns its down-regulation necessary for the progression of thyroid epithelial tumors. Results Here we demonstrate that PAX8 is involved in the Transcriptional modulation of Wnt4 gene expression directly binding to its 5’-flanking region, and that Wnt4 expression in FRTL-5 cells is TSH-dependent. Interestingly, we also show that in thyroid cells a reduced expression of Wnt4 correlates with the alteration of the epithelial phenotype and that the overexpression of Wnt4 in thyroid cancer cells is able to inhibit cellular migration. Conclusions We have identified and characterized a functional PAX8 binding site in the 5’-flanking region of the Wnt4 gene and we show that PAX8 modulates the expression of Wnt4 in thyroid cells. Taken together, our results suggest that in thyroid cells Wnt4 expression correlates with the integrity of the epithelial phenotype and is reduced when this integrity is perturbed. In the end, we would like to suggest that the overexpression of Wnt4 in thyroid cancer cells is able to revert the mesenchymal phenotype.
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An essential role for PAX8 in the Transcriptional regulation of cadherin-16 in thyroid cells.
Molecular endocrinology (Baltimore Md.), 2011Co-Authors: Tiziana De Cristofaro, Tina Di Palma, Mario De Felice, Imma Fichera, Valeria Lucci, Luca Parrillo, Mariastella ZanniniAbstract: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.
Pilar Santisteban - One of the best experts on this subject based on the ideXlab platform.
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PAX8 controls thyroid follicular polarity through cadherin 16
Journal of Cell Science, 2017Co-Authors: Petrina Koumarianou, Gonzalo Gomezlopez, Pilar SantistebanAbstract:Organization of epithelial cells during follicular lumen formation is crucial for thyroid morphogenesis and function of the thyroid gland; however, the molecular mechanisms underlying this are poorly understood. To investigate this process, we established three-dimensional (3D) epithelial culture model systems using Fischer rat thyroid (FRT) cells or murine primary thyrocytes that developed polarized spherical structures with a central lumen, mimicking thyroid follicles. Using microarray-based differential expression analysis of FRT cells grown under 2D or 3D conditions, followed by RNA-mediated interference (RNAi) and morphogenetic analysis, we identified a key role for the thyroid Transcription Factor PAX8 and its target cadherin-16 (Cdh16) in the generation of polarized follicle-like structures. Silencing PAX8 expression inhibited the acquisition of apical-basal membrane polarity and impaired lumen formation. Both laminin and β1-integrin (Itgb1) expression was reduced, and cell cytoskeleton polarized distribution was altered. Silencing Cdh16 expression also led to the formation of defective structures characterized by very low laminin expression at the follicle-matrix interface, downregulation of Itgb1, and unpolarized distribution of cell cytoskeleton. Our results demonstrate that PAX8 controls apical-basal follicular polarization and follicle formation through Cdh16.
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Wnt-independent role of β-catenin in thyroid cell proliferation and differentiation.
Molecular endocrinology (Baltimore Md.), 2014Co-Authors: Ana Sastre-perona, Pilar SantistebanAbstract:The Wnt/β-catenin pathway has been associated with thyroid cell growth and tumorigenesis. However, little is known regarding its involvement in the response to the key regulators of thyroid cell proliferation and differentiation. Here we show that TSH and IGF-1 increase β-catenin nuclear accumulation and its Transcriptional activity in differentiated thyroid cells. This effect takes place in a Wnt-independent manner because TSH and IGF-1, through the activation of protein kinase A and protein kinase B/Akt, phosphorylate β-catenin at S552 and S675, which results in β-catenin release from E-cadherin at the adherens junctions. Nuclear β-catenin regulates thyroid cell proliferation, because its silencing or the overexpression of a dominant-negative form of T-cell Factor 4 resulted in reduced levels of cyclin D1 and DNA synthesis. Furthermore, the β-catenin silencing markedly reduced the expression of PAX8, the main Transcription Factor involved in epithelial thyroid cell differentiation. Finally, we observed that β-catenin physically interacts with the Transcription Factor PAX8, increasing its Transcriptional activity on the sodium iodide symporter (NIS) gene, a critical gene required for thyroid cell physiology. Taken together, our findings show that β-catenin plays a not yet described role in thyroid function including a functional interaction with PAX8.
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Genome-wide analysis of PAX8 binding provides new insights into thyroid functions
BMC genomics, 2012Co-Authors: Sergio Ruiz-llorente, Ana Sastre-perona, Enrique Carrillo Santa De Pau, Cristina Montero-conde, Gonzalo Gómez-lópez, James A. Fagin, Alfonso Valencia, David G. Pisano, Pilar SantistebanAbstract:The Transcription Factor PAX8 is essential for the differentiation of thyroid cells. However, there are few data on genes Transcriptionally regulated by PAX8 other than thyroid-related genes. To better understand the role of PAX8 in the biology of thyroid cells, we obtained Transcriptional profiles of PAX8-silenced PCCl3 thyroid cells using whole genome expression arrays and integrated these signals with global cis-regulatory sequencing studies performed by ChIP-Seq analysis Exhaustive analysis of PAX8 immunoprecipitated peaks demonstrated preferential binding to intragenic regions and CpG-enriched islands, which suggests a role of PAX8 in Transcriptional regulation of orphan CpG regions. In addition, ChIP-Seq allowed us to identify PAX8 partners, including proteins involved in tertiary DNA structure (CTCF) and chromatin remodeling (Sp1), and these direct Transcriptional interactions were confirmed in vivo. Moreover, both Factors modulate PAX8-dependent Transcriptional activation of the sodium iodide symporter (Nis) gene promoter. We ultimately combined putative and novel PAX8 binding sites with actual target gene expression regulation to define PAX8-dependent genes. Functional classification suggests that PAX8-regulated genes may be directly involved in important processes of thyroid cell function such as cell proliferation and differentiation, apoptosis, cell polarity, motion and adhesion, and a plethora of DNA/protein-related processes. Our study provides novel insights into the role of PAX8 in thyroid biology, exerted through Transcriptional regulation of important genes involved in critical thyrocyte processes. In addition, we found new Transcriptional partners of PAX8, which functionally cooperate with PAX8 in the regulation of thyroid gene Transcription. Besides, our data demonstrate preferential location of PAX8 in non-promoter CpG regions. These data point to an orphan CpG island-mediated mechanism that represents a novel role of PAX8 in the Transcriptional output of the thyrocyte.
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Genome-wide analysis of PAX8 binding provides new insights into thyroid functions
BMC Genomics, 2012Co-Authors: Sergio Ruiz-llorente, Ana Sastre-perona, Cristina Montero-conde, Gonzalo Gómez-lópez, James A. Fagin, Alfonso Valencia, David G. Pisano, Enrique Carrillo Santa De Pau, Pilar SantistebanAbstract:Background The Transcription Factor PAX8 is essential for the differentiation of thyroid cells. However, there are few data on genes Transcriptionally regulated by PAX8 other than thyroid-related genes. To better understand the role of PAX8 in the biology of thyroid cells, we obtained Transcriptional profiles of PAX8-silenced PCCl3 thyroid cells using whole genome expression arrays and integrated these signals with global cis-regulatory sequencing studies performed by ChIP-Seq analysis Results Exhaustive analysis of PAX8 immunoprecipitated peaks demonstrated preferential binding to intragenic regions and CpG-enriched islands, which suggests a role of PAX8 in Transcriptional regulation of orphan CpG regions. In addition, ChIP-Seq allowed us to identify PAX8 partners, including proteins involved in tertiary DNA structure (CTCF) and chromatin remodeling (Sp1), and these direct Transcriptional interactions were confirmed in vivo . Moreover, both Factors modulate PAX8-dependent Transcriptional activation of the sodium iodide symporter ( Nis ) gene promoter. We ultimately combined putative and novel PAX8 binding sites with actual target gene expression regulation to define PAX8-dependent genes. Functional classification suggests that PAX8-regulated genes may be directly involved in important processes of thyroid cell function such as cell proliferation and differentiation, apoptosis, cell polarity, motion and adhesion, and a plethora of DNA/protein-related processes. Conclusion Our study provides novel insights into the role of PAX8 in thyroid biology, exerted through Transcriptional regulation of important genes involved in critical thyrocyte processes. In addition, we found new Transcriptional partners of PAX8, which functionally cooperate with PAX8 in the regulation of thyroid gene Transcription. Besides, our data demonstrate preferential location of PAX8 in non-promoter CpG regions. These data point to an orphan CpG island-mediated mechanism that represents a novel role of PAX8 in the Transcriptional output of the thyrocyte.
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the functional interaction between the paired domain Transcription Factor PAX8 and smad3 is involved in transforming growth Factor β repression of the sodium iodide symporter gene
Journal of Biological Chemistry, 2004Co-Authors: Eugenia Costamagna, Bibian Garcia, Pilar SantistebanAbstract:Abstract Transforming growth Factor-β (TGF-β) is a secreted protein that regulates proliferation, differentiation, and death in various cell types, including thyroid cells, although few details are known about its mechanisms of action in this cell type. Here, we studied the role of TGF-β on the regulation of sodium/iodide symporter (NIS) gene expression in PC Cl3 thyroid cells. TGF-β inhibits thyroid-stimulated hormone (TSH)-induced NIS mRNA and protein levels in a dose-dependent manner. This effect takes place at the Transcriptional level, as TGF-β inhibits TSH-induced Transcription of a luciferase reporter construct containing a 2.8-kb DNA fragment of the rat NIS promoter. The inhibitory effect of TGF-β was partially overcome by inhibitory Smad7 and mimicked by overexpression of either Smad3 or a constitutively activated mutant of TGF-β receptor I (acALK-5). Using internal deletions of the promoter, we defined a region between –2,841 to –1,941, which includes the NIS upstream enhancer (NUE), as responsible for the TGF-β/Smad inhibitory effect. NUE contains two binding sites for the paired domain Transcription Factor PAX8, the main Factor controlling NIS Transcription. The physical interaction observed between PAX8 and Smad3 appears to be responsible for the decrease in PAX8 binding to DNA. Expression of PAX8 mRNA and protein was also decreased by TGF-β treatment. The results suggest that, through activation of Smad3, TGF-β decreases PAX8 DNA binding activity as well as PAX8 mRNA and protein levels, which are at least partially involved in TGF-β-induced down-regulation of NIS gene expression in thyroid follicular cells. Our results thus demonstrate a novel mechanism of Smad3 function in regulating thyroid cell differentiation by functionally antagonizing the action of the paired domain Transcription Factor PAX8.
Roberto Di Lauro - One of the best experts on this subject based on the ideXlab platform.
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The paired box Transcription Factor PAX8 is essential for function and survival of adult thyroid cells.
Molecular and cellular endocrinology, 2014Co-Authors: Pina Marotta, Elena Amendola, Marzia Scarfò, Pasquale De Luca, Pietro Zoppoli, Angela Amoresano, Mario De Felice, Roberto Di LauroAbstract:The Transcription Factor PAX8 is already known to be essential at very early stages of mouse thyroid gland development, before the onset of thyroid hormone production. In this paper we show, using a conditional inactivation strategy, that the removal of the PAX8 protein late in gland development results in severe hypothyroidism, consequent to a reduced gland size and a deranged differentiation. These results demonstrate that PAX8 is also an essential player in controlling survival and differentiation of adult thyroid follicular cells.
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Oncogenic Ras Blocks the cAMP Pathway and Dedifferentiates Thyroid Cells Via an Impairment of PAX8 Transcriptional Activity
Molecular endocrinology (Baltimore Md.), 2009Co-Authors: Maria Giuseppina Baratta, Immacolata Porreca, Roberto Di LauroAbstract:A deranged differentiation is often a landmark of transformed cells. We used a thyroid cell line expressing an inducible Ras oncoprotein in order to study the hierarchy of molecular events leading to suppression of thyroid-specific gene expression. We find that, upon Ras activation, there is an immediate global down-regulation of thyroid differentiation, which is associated with an inhibition of the cAMP signaling pathway. We demonstrate that an unusual negative cross talk between Ras oncogene and the cAMP pathway induces inactivation of the Transcription Factor PAX8 that we propose as a crucial event in Ras-induced dedifferentiation.
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Functional inactivation of the Transcription Factor PAX8 through oligomerization chain reaction.
Molecular endocrinology (Baltimore Md.), 2006Co-Authors: Barbara D'andrea, Roberto Iacone, Tina Di Palma, Roberto Nitsch, Maria Giuseppina Baratta, Lucio Nitsch, Roberto Di Lauro, Mariastella ZanniniAbstract:Among the approaches used to provide a functional inactivation of a target protein, we have chosen the recently described oligomerization chain reaction (OCR) strategy to functionally inactivate the Transcription Factor PAX8, a member of the Pax gene family expressed in thyroid cells. The OCR strategy is based on the fusion of the self-associating coiled-coil (CC) domain of the nuclear Factor promyelocytic leukemia (PML) to target proteins that are able to self-associate naturally or that form heterocomplexes. In the thyroid tissue, the Transcription Factor PAX8 is involved in the morphogenesis of the gland and in the Transcriptional regulation of thyroid-expressed genes. We have recently demonstrated that in thyroid cells PAX8 interacts biochemically and functionally with the Transcription Factor TTF-1 (thyroid Transcription Factor 1), and that such interaction leads to the synergistic activation of thyroglobulin (Tg) gene expression. Fusion of the CC domain to PAX8 leads to the formation of aberrant, nonfunctional high-molecular mass complexes to which TTF-1 is also recruited. The CC-PAX8 chimera inhibits the Transcriptional activity of PAX8 and of TTF-1 on both synthetic and physiological promoters and prevents the synergistic activation of the Tg promoter mediated by these two Transcription Factors. Furthermore, the expression of the CC-PAX8 chimera in differentiated thyroid cells leads to the down-regulation of the endogenous expression of several differentiation markers such as Tg, sodium/iodide symporter, Foxe1, TTF-1, and thyroid oxidase 2. These results demonstrate that the OCR is a useful tool to functionally inactivate a Transcription Factor. Moreover, by this approach, we identified Foxe1, TTF-1, and thyroid oxidase 2 as new direct targets of PAX8 or TTF-1.
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PAX8 has a key role in thyroid cell differentiation
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Marina Pasca Di Magliano, Roberto Di Lauro, M. ZanniniAbstract:Transformation of rat thyroid cells with polyoma virus middle T antigen results in loss of the thyroid-differentiated phenotype, measured as the expression of the thyroglobulin (Tg), thyroperoxidase (TPO), and sodium/iodide symporter (NIS) genes. Among the Transcription Factors involved in the regulation of these genes, TTF-1 and TTF-2 were still detected at nearly wild-type levels, while a specific loss of the paired domain Transcription Factor PAX8 was observed. In this study, we used the PCPy cell line as a model system to study the role of PAX8 in thyroid differentiation. We demonstrate that the reintroduction of PAX8 in PCPy cells is sufficient to activate expression of the endogenous genes encoding thyroglobulin, thyroperoxidase, and sodium/iodide symporter. Thus, this cell system provides direct evidence for the ability of PAX8 to activate Transcription of thyroid-specific genes at their chromosomal locus and strongly suggests a fundamental role of this Transcription Factor in the maintenance of functional differentiation in thyroid cells. Moreover, we show that PAX8 and TTF-1 cooperate in the activation of the thyroglobulin promoter and that additional thyroid-specific mechanism(s) are involved in such a cooperation. To identify the PAX8 domain able to mediate the specific activation of the thyroglobulin promoter, we transfected in PCPy cells three different PAX8 isoforms. The results of such experiments indicate that for the Transcriptional activation of thyroid-specific genes, PAX8 uses an as yet unidentified functional domain.
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the paired domain Transcription Factor PAX8 binds to the upstream enhancer of the rat sodium iodide symporter gene and participates in both thyroid specific and cyclic amp dependent Transcription
Molecular and Cellular Biology, 1999Co-Authors: Makoto Ohno, Mariastella Zannini, Orlie Levy, Nancy Carrasco, Roberto Di LauroAbstract:Cell type-specific gene Transcription is often dependent on a set of Transcription Factors whose combination is unique to that cell type. Three Transcription Factors, TTF-1, TTF-2, and PAX8, have been implicated in such a control in the case of thyroid-specific Transcription of the thyroglobulin and thyroperoxidase genes (13). TTF-1 is an homeodomain (HD)-containing protein, present in the developing thyroid, lung, and diencephalon (26). TTF-2, a forkhead protein, has been detected in the endoderm of the developing foregut, including the thyroid anlage, and in the anterior pituitary (41), while the paired-domain (PD) Factor PAX8 is present in both the thyroid and kidney (35). The unique combination of these Factors in the thyroid follicular cells strongly suggests that their interaction plays an important role in inducing a specific pattern of gene expression in these cells. Cyclic AMP (cAMP), whose intracellular level is elevated by the thyroid-stimulating hormone (TSH), is an important modulator of gene expression in thyroid cells (1, 2, 14, 18, 20, 22, 34, 37). Nevertheless, direct roles for the thyroid-restricted Factors TTF-1, TTF-2, and PAX8 in mediating the cAMP effects in thyroid cells have not yet been demonstrated. Interestingly, well-known mediators of Transcriptional regulation by cAMP, such as those acting through the cAMP responsive element (CRE) sequence (5) have been proposed to be involved only in the regulation of TSH receptor gene expression (22), but no conclusive evidence on their roles in the control exerted by TSH cAMP or on other thyroid-specific genes has been provided. To gain further insights into the mechanisms responsible for cAMP-dependent Transcription in thyroid cells and their relationships to thyroid-specific Transcriptional mechanisms, we decided to study the regulation of the gene encoding the Na/I symporter (NIS). Thyroid follicular cells accumulate iodide against a concentration gradient and utilize it for thyroid hormone biosynthesis (40). Iodide transport, which is catalyzed by NIS, is primarily regulated by TSH through cAMP (30, 36, 40) by at least two mechanisms. The first induces the activity of the NIS protein (27, 32), presumably by posttranslational modifications, while the second positively regulates NIS mRNA levels (24) in a cycloheximide-sensitive manner. In the present study, we isolated the rat NIS (rNIS) gene and searched for a regulatory element(s) capable of thyroid-specific and TSH-regulated Transcriptional activation. We identified in the upstream region of rNIS a short enhancer capable of increasing Transcription of its own or a heterologous promoter. This regulatory element shows four remarkable features. (i) It responds to cAMP only in differentiated thyroid cells, suggesting that a cell-type-specific mechanism is operating to mediate the Transcriptional response of NIS to cAMP. (ii) It can be activated by cAMP even when thyroid cells, as a consequence of chronic stimulation of the cAMP pathway, down-regulate their PKA levels and, hence, are unable to carry out Transcriptional activation of CRE-containing promoters (1). (iii) It requires the binding of PAX8, in addition to a degenerate CRE-like sequence, for Transcriptional activity. (iv) It can be activated in a cAMP-dependent manner in nonthyroid cells by cotransfection of a PAX8 expression vector. Taken together, these data suggest that the rNIS enhancer mediates thyroid-specific gene expression by the interaction of PAX8 with a novel cAMP-dependent pathway.
Michelle S Hirsch - One of the best experts on this subject based on the ideXlab platform.
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PAX8 reliably distinguishes ovarian serous tumors from malignant mesothelioma
The American Journal of Surgical Pathology, 2010Co-Authors: Anna R Laury, Joseph M. Corson, Jason L Hornick, Ruth Perets, Jeffrey F Krane, Ronny Drapkin, Michelle S HirschAbstract:Ovarian serous neoplasms can have morphologic overlap with malignant mesothelioma. The distinction is clinically important, yet most studies have failed to identify immunostains that reliably distinguish these 2 tumor types. Recently, Transcription Factor PAX8 was shown to be a sensitive and relatively specific marker for Mullerian tumors. In addition, some studies suggest that h-caldesmon is sensitive and specific for mesothelioma when compared with serous ovarian tumors. The goal of this study was to evaluate whether PAX8 and h-caldesmon expression can successfully distinguish mesothelioma from serous ovarian tumors. Immunohistochemistry was carried out using PAX8 and h-caldesmon antibodies on archival tissue from 254 ovarian serous tumors and 50 mesothelial tumors. Nuclear and cytoplasmic immunoreactivity were considered positive for PAX8 and h-caldesmon, respectively. PAX8 staining was present in 99% of high-grade serous ovarian carcinomas and all (100%) low-grade ovarian carcinomas and serous borderline tumors; however, only 74% of these cases (188/254) were diffusely positive in more than 50% of tumors cells, and intensity ranged from strong to weak. None of the pleural malignant mesotheliomas were reactive with PAX8. However, 2/23 (9%) peritoneal malignant mesotheliomas showed focal and/or weak staining for PAX8; the remaining cases were negative. Two well-differentiated papillary mesotheliomas and 1 multicystic mesothelioma each showed some staining for PAX8. h-caldesmon was negative in all serous neoplasms and all mesothelial neoplasms, except 1 pleural malignant mesothelioma which showed patchy immunoreactivity. Strong PAX8 staining is highly specific (P<0.00001) for ovarian serous tumors when compared with malignant mesotheliomas of the peritoneum and pleura. The presence of weak staining for PAX8 in the 3 "noninvasive" mesotheliomas questions the use for PAX8 in this differential diagnosis. On the basis of this study, h-caldesmon is not a useful marker for mesothelioma.
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PAX8 reliably distinguishes ovarian serous tumors from malignant mesothelioma.
The American journal of surgical pathology, 2010Co-Authors: Joseph M. Corson, Anna R Laury, Jason L Hornick, Ruth Perets, Jeffrey F Krane, Ronny Drapkin, Michelle S HirschAbstract:Ovarian serous neoplasms can have morphologic overlap with malignant mesothelioma. The distinction is clinically important, yet most studies have failed to identify immunostains that reliably distinguish these 2 tumor types. Recently, Transcription Factor PAX8 was shown to be a sensitive and relatively specific marker for Müllerian tumors. In addition, some studies suggest that h-caldesmon is sensitive and specific for mesothelioma when compared with serous ovarian tumors. The goal of this study was to evaluate whether PAX8 and h-caldesmon expression can successfully distinguish mesothelioma from serous ovarian tumors. Immunohistochemistry was carried out using PAX8 and h-caldesmon antibodies on archival tissue from 254 ovarian serous tumors and 50 mesothelial tumors. Nuclear and cytoplasmic immunoreactivity were considered positive for PAX8 and h-caldesmon, respectively. PAX8 staining was present in 99% of high-grade serous ovarian carcinomas and all (100%) low-grade ovarian carcinomas and serous borderline tumors; however, only 74% of these cases (188/254) were diffusely positive in more than 50% of tumors cells, and intensity ranged from strong to weak. None of the pleural malignant mesotheliomas were reactive with PAX8. However, 2/23 (9%) peritoneal malignant mesotheliomas showed focal and/or weak staining for PAX8; the remaining cases were negative. Two well-differentiated papillary mesotheliomas and 1 multicystic mesothelioma each showed some staining for PAX8. h-caldesmon was negative in all serous neoplasms and all mesothelial neoplasms, except 1 pleural malignant mesothelioma which showed patchy immunoreactivity. Strong PAX8 staining is highly specific (P
M. Zannini - One of the best experts on this subject based on the ideXlab platform.
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PAX8 has a critical role in epithelial cell survival and proliferation.
Cell death & disease, 2013Co-Authors: T.m. Di Palma, Maria Grazia Filippone, Giovanna Maria Pierantoni, Alfredo Fusco, Silvia Soddu, M. ZanniniAbstract:The Transcription Factor PAX8, a member of the Paired-box gene family, is 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 responsible for thyroid differentiation, its involvement in cell survival and proliferation has been hypothesized but remains unclear. Here, we show that PAX8 overexpression significantly increases proliferation and colony-forming efficiency of Fischer rat thyroid line 5 epithelial cells, although it is not sufficient to overcome their hormone dependence. More interestingly, we show that PAX8-specific silencing induces apoptosis through a p53-dependent pathway that involves caspase-3 activation and cleavage of poly(ADP)ribose polymerase. Our data indicate that tumor protein 53 induced nuclear protein 1 (tp53inp1), a positive regulator of p53-dependent cell cycle arrest and apoptosis, is a Transcriptional target of PAX8 and is upregulated by PAX8 knockdown. Remarkably, tp53inp1 silencing significantly abolishes PAX8-induced apoptosis thus suggesting that tp53inp1 may be the mediator of the observed effects. In conclusion, our data highlight that PAX8 is required for the survival of differentiated epithelial cells and its expression levels are able to modulate the proliferation rate of such cells.
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PAX8 protein stability is controlled by sumoylation.
Journal of molecular endocrinology, 2008Co-Authors: Tiziana De Cristofaro, Barbara D'andrea, Lucio Nitsch, Anna Mascia, Andrea Pappalardo, M. ZanniniAbstract:The Transcription Factor PAX8 is involved in the morphogenesis of the thyroid gland and in the maintenance of the differentiated thyroid phenotype. Despite the critical role played by PAX8 during thyroid development and differentiation, very little is known of its post-translational modifications and how these modifications may regulate its activity. We focused our attention on the study of a specific post-translational modification, i.e., sumoylation. Sumoylation is a dynamic and reversible process regulating gene expression by altering Transcription Factor stability, protein-protein interaction and subcellular localization of target proteins. The analysis of PAX8 protein sequence revealed the presence of one sumoylation consensus motif (psiKxE), strongly conserved among mammals, amphibians, and fish. We demonstrated that PAX8 is sumoylated by the addition of a single small ubiquitin-like modifier (SUMO) molecule on its lysine residue 309 and that PAX8(K309R), a substitution mutant in which the candidate lysine is replaced with an arginine, is no longer modified by SUMO. In addition, we analyzed whether protein inhibitor of activated signal transducers and activators of Transcription (PIASy), a member of the PIAS STAT family of proteins, could function as a SUMO ligase and we demonstrated that indeed PIASy is able to increase the fraction of sumoylated PAX8. Interestingly, we show that PAX8 is targeted in the SUMO nuclear bodies, which are structures that regulate the nucleoplasmic concentration of Transcription Factors by SUMO trapping. Finally, we report here that the steady-state protein level of PAX8 is controlled by sumoylation.
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Hormonal control of the Transcription Factor PAX8 and its role in the regulation of thyroglobulin gene expression in thyroid cells.
The Journal of endocrinology, 2002Co-Authors: Anna Mascia, Lucio Nitsch, R Di Lauro, M. ZanniniAbstract: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.
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PAX8 has a key role in thyroid cell differentiation
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Marina Pasca Di Magliano, Roberto Di Lauro, M. ZanniniAbstract:Transformation of rat thyroid cells with polyoma virus middle T antigen results in loss of the thyroid-differentiated phenotype, measured as the expression of the thyroglobulin (Tg), thyroperoxidase (TPO), and sodium/iodide symporter (NIS) genes. Among the Transcription Factors involved in the regulation of these genes, TTF-1 and TTF-2 were still detected at nearly wild-type levels, while a specific loss of the paired domain Transcription Factor PAX8 was observed. In this study, we used the PCPy cell line as a model system to study the role of PAX8 in thyroid differentiation. We demonstrate that the reintroduction of PAX8 in PCPy cells is sufficient to activate expression of the endogenous genes encoding thyroglobulin, thyroperoxidase, and sodium/iodide symporter. Thus, this cell system provides direct evidence for the ability of PAX8 to activate Transcription of thyroid-specific genes at their chromosomal locus and strongly suggests a fundamental role of this Transcription Factor in the maintenance of functional differentiation in thyroid cells. Moreover, we show that PAX8 and TTF-1 cooperate in the activation of the thyroglobulin promoter and that additional thyroid-specific mechanism(s) are involved in such a cooperation. To identify the PAX8 domain able to mediate the specific activation of the thyroglobulin promoter, we transfected in PCPy cells three different PAX8 isoforms. The results of such experiments indicate that for the Transcriptional activation of thyroid-specific genes, PAX8 uses an as yet unidentified functional domain.