Proc Natl Acad Sci U S A 97(24): 13144-13149

Pax8 has a key role in thyroid cell differentiation

^{Stazione Zoologica “A. Dohrn,” Villa Comunale, 80121 Naples, Italy; and}^{Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli, Federico II, via Pansini 5, 80131 Naples, Italy}

^{Present address: Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030, Vienna, Austria.}

^{To whom reprint requests should be addressed. E-mail:}ti.nzs.ahpla@allets.

Edited by Michael G. Rosenfeld, University of California at San Diego, La Jolla, CA, and approved September 20, 2000

Received 2000 Jul 19

Abstract

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.

Keywords: Pax genes, gene expression

Abstract

Terminally differentiated cells express genes whose products are undetectable in other cell types. The isolation of regulatory DNA sequences responsible for such cell-type-specific expression and the cloning of the cDNAs encoding cognate transcription factors revealed, in the majority of cases, that a combination of transcription factors is unique to a cell type. Thus, transcription of cell-type-specific genes appears to depend on synergy between transcription factors (1, 2). Frequently, transcription factors controlling the expression of genes specific to the terminal differentiation state of a given cell type play an additional role earlier in the development of the same cell type. As a consequence, the identification of factors controlling cell-type-specific gene expression also provides relevant information on earlier developmental stages in many organs and cell lineages. However, a direct assessment of the role of many transcription factors in cell-type-specific gene expression has proven difficult, given that mice lacking the relevant transcription factors often show complete absence of the implicated cell type.

To elucidate the mechanism(s) operating in the establishment and maintenance of cell-type-specific expression, we have been studying a model system represented by the thyroid follicular cells, the most abundant cell population of the thyroid gland. These cells are responsible for thyroid hormone synthesis and are characterized by the expression of thyroid-specific genes such as thyroglobulin (Tg) and thyroperoxidase (TPO), which are exclusively expressed in this organ, and by the expression of genes that have been detected only in a few tissues other than the thyroid, such as the TSH receptor (TSHr) and the sodium/iodide symporter (NIS) (2, 3). Tg and TPO promoters have been extensively studied, and three transcription factors have been cloned that specifically bind and activate these two promoters (3). The three factors are TTF-1 (Thyroid Transcription Factor-1), TTF-2 (Thyroid Transcription Factor-2), and Pax8. TTF-1 is a homeodomain-containing protein expressed in embryonic diencepahlon, thyroid, and lung (4); TTF-2 is a forkhead domain-containing protein expressed in pituitary and thyroid glands (5), and Pax8 is a member of the murine Pax family of paired domain-containing genes, expressed in the developing kidney, the neural tube, and the developing and adult thyroid (6). Thus, none of these transcription factors is expressed only in the thyroid, but their combination is unique to this organ and is likely to be responsible for early commitment and differentiation of thyrocytes.

In this study, we focused on the transcription factor Pax8. Pax8 binds to a single site on the promoters of the Tg and TPO genes and, in both promoters, the Pax8-binding site overlaps with that of TTF-1 (7). In the case of the Tg promoter, all mutations that abolish Pax8 binding also greatly reduce that of TTF-1, making it difficult to conclude which of the two factors is playing an important role for promoter expression at this site. Conversely, in the TPO promoter it has been possible to find a mutation that interferes only with Pax8 and that greatly reduces promoter activity, thus providing strong evidence for an important role of Pax8 in thyroid-specific gene expression (7). In addition, Pax8 was shown, in transient transfection assays, to activate transcription from the TPO and, albeit at a much lower level, the Tg promoters in nonthyroid cells. These data prompted us to propose these two thyroid-specific genes as putative targets for Pax8 action (7). Very recently, Pax8 has also been involved in thyroid-specific expression of the rat gene encoding for the sodium/iodide symporter (8). However, given both the exceedingly elevated concentration of transcription factors and of the cognate target promoters that are achieved in transient transfection assays, and given that Pax8-dependent transcriptional activation of the endogenous Tg, TPO, and NIS genes had not yet been reported, there was a need for direct evidence for Pax8-dependent transcription from the chromosomal Tg, TPO, and NIS genes. The finding that Pax8 knockout mice exhibit a total absence of the thyroid follicular cells (9), while indicating an essential role for this gene in thyroid organogenesis, could not contribute to a definitive assessment of the role of Pax8 in controlling the expression of the thyroid-differentiated phenotype, because the thyroid cells precursors disappear before the onset of the Tg, TPO, and NIS gene expression. Also, the association between mutations in PAX8 with human thyroid dysgenesis (10) underlines an important role for this protein in organogenesis but could not contribute to a further understanding of its involvement on the expression of thyroid-specific genes.

Several thyroid-derived cell lines have been established, which maintain in vitro the expression of the differentiation markers. Among these cell lines, we have used the PC Cl3 cells (PC cells) (11) that express the Tg, TPO, NIS, and TSHr genes and depend on TSH for proliferation and for the expression of the differentiated phenotype (12). These cells also express the transcription factors TTF-1, TTF-2, and Pax8.

Transformed cell lines have been obtained by the expression of different oncogenes in PC cells (11, 13). In most cases, transformation causes loss of the differentiated phenotype, TSH-independent proliferation, and tumor formation on injection in nude mice. When the thyroid-specific transcription factors have been analyzed in these cell lines, often an impairment of their expression or activity has been found. However, more than one factor is usually affected, thus suggesting that complex changes in the transcriptional program have occurred. As a consequence of the complexity of the phenotype, a detailed study of the individual role of each factor in differentiation is often difficult.

In contrast, we report in this paper that transformation of PC cells with the polyoma middle T antigen (PCPy), resulting in loss of the differentiated down-regulation of Tg, TPO, and NIS expression and TSH-independent proliferation, is associated with specific loss of Pax8 expression. In contrast, TTF-1 is detected at wild-type levels, and TTF-2 is moderately reduced. We demonstrate in this paper that the introduction of Pax8 expression vectors in PCPy cells, followed by the synthesis of a functional Pax8 protein, is sufficient to activate expression of the endogenous Tg, TPO, and NIS, thus providing direct evidence that this transcription factor plays a key role in thyroid-specific expression of many genes. However, PCPy cells expressing Pax8 still show an altered growth potential, indicating that transformation affects the gene expression program and the proliferation of these cells by using two distinct mechanisms, only one of which involves Pax8.

We have investigated the mechanisms through which Pax8 reactivates Tg transcription. We obtained strong evidence that Pax8 acts directly on the Tg promoter and that the interaction with another thyroid-specific protein is required. Experiments aiming at the localization of the functional domains in Pax8 have been performed.

Acknowledgments

We thank Mario De Felice, Caterina Missero, and Lucio Nitsch for reviewing the manuscript and for helpful discussions. This work was supported by grants from the Consiglio Nazionale delle Ricerche “Target Project on Biotechnology,” the Ministero per l'Università e la Ricerca Scientifica Project “Molecular mechanisms responsible for differentiation of thyroid cells: diagnostic and therapeutic applications,” the Consiglio Nazionale delle Ricerche Progetto Strategico “Modifiche posttrascrizionali dell'espressione genica,” and the Programma Biotecnologie legge 95/95 (Ministero dell'Università e della Ricerca Scientifica e Tecnologica). We further acknowledge support from the Associazione Italiana per la Ricerca sul Cancro and from the Telethon Foundation (Grant D.67)

Acknowledgments

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.240336397.

Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.240336397

Footnotes

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