Function of the homeo and paired domain proteins TTF-1 and Pax-8 in thyroid cell proliferation.

The thyroid transcription factors TTF-1 and Pax-8 are homeobox- and paired box-containing genes, respectively, that are responsible for thyroid-specific gene expression, thyroid development, and thyroid cell differentiation. However, it is not clear if such factors play a role in thyroid cell proliferation. The antisense oligonucleotide strategy was used in order to clarify this point. Treatment of quiescent FRTL-5 thyroid cells with TTF-1 or Pax-8 antisense oligonucleotides caused a significant reduction in thyroid-stimulating hormone and insulin-like growth factor-I-stimulated cell proliferation, measured by DNA synthesis and cell counting. The same results were obtained with forskolin indicating that the TTF-1 or Pax-8 role in mediating the thyroid-stimulating hormone growth effect occurred via the cAMP pathway. The effect was higher with TTF-1 as the blockage by this factor caused a 65% decrease in cell proliferation compared to the control. Pax-8 blocking lead only to a 30% decrease. The blocking of both thyroid transcription factors together did not result in an additive effect. These data provide direct evidence that both homeo and paired box gene expression is essential for FRTL-5 thyroid cell proliferation, with each one possibly playing a different regulatory role.

The thyroid transcription factors TTF-1 and Pax-8 are homeobox-and paired box-containing genes, respectively, that are responsible for thyroid-specific gene expression, thyroid development, and thyroid cell differentiation. However, it is not clear if such factors play a role in thyroid cell proliferation. The antisense oligonucleotide strategy was used in order to clarify this point. Treatment of quiescent FRTL-5 thyroid cells with TTF-1 or Pax-8 antisense oligonucleotides caused a significant reduction in thyroid-stimulating hormone and insulinlike growth factor-I-stimulated cell proliferation, measured by DNA synthesis and cell counting. The same results were obtained with forskolin indicating that the TTF-1 or Pax-8 role in mediating the thyroid-stimulating hormone growth effect occurred via the cAMP pathway. The effect was higher with TTF-1 as the blockage by this factor caused a 65% decrease in cell proliferation compared to the control. Pax-8 blocking lead only to a 30% decrease. The blocking of both thyroid transcription factors together did not result in an additive effect. These data provide direct evidence that both homeo and paired box gene expression is essential for FRTL-5 thyroid cell proliferation, with each one possibly playing a different regulatory role.
Homeo-and paired domain-containing proteins are transcriptional regulators that participate in cell growth and differentiation processes (1,2). The thyroid-specific transcription factors TTF-1 and Pax-8 are homeo and paired box genes, respectively, that are responsible for thyroglobulin and thyroperoxidase gene expression (3)(4)(5)(6). In addition, TTF-1 binds to the TSH 1 receptor (TSH-R) gene promoter (7,8). Both transcription factors are responsible for thyroid development, demonstrated by the fact that their expression precedes the onset of their target genes tyroglobulin, thyroperoxidase, and TSH-R by 5 days (9,10). Clear evidence for the major role of TTF-1 and Pax-8 in thyroid cell differentiation is that transformed FRTL-5 thyroid cells, where TTF-1 and/or Pax-8 are not expressed (11), loose their thyroid phenotype. It has also been reported that undifferentiated thyroid carcinomas do not express either TTF-1 or Pax-8 (12). However, it has not yet been demonstrated the role of both transcription factors in thyroid cell growth.
The transition from quiescent to proliferating FRTL-5 thyroid cells requires the action of both TSH and IGF-I (13)(14)(15)(16). A significant amount of evidence suggests that TSH and IGF-I, could in some way, regulate TTF-1 and Pax-8 activity since mutations in the TTF-1/Pax-8 binding sites of thyroglobulin and thyroperoxidase promoters reduce their response to TSH and IGF-I (17,18). The aim of the present work was to study whether TTF-1 and Pax-8 play a role in thyroid cell growth in response to TSH and IGF-I. We used antisense oligonucleotides to inhibit TTF-1 or Pax-8 expression in FRTL-5 thyroid cells. This treatment led to a marked decrease in the rate of DNA synthesis and cell number in FRTL-5 cells in response to TSH and/or IGF-I. The inhibitory effect was higher when TTF-1 was blocked than that observed for Pax-8 blockage. Blocking both transcription factors simultaneously did not result in an additive effect. These data provide direct evidence that both TTF-1 and Pax-8 gene expression is required not only for the establishment and maintenance of the differentiated phenotype, but also is essential for thyroid cell proliferation.

EXPERIMENTAL PROCEDURES
Materials-Tissue culture medium, bovine TSH, bovine insulin, and TPA were purchased from Sigma. Rat IGF-I was from Amgen Biological (Thousand Oaks, CA) and forskolin was from Calbiochem. Donor calf serum was from Life Technologies, Inc., and Nytran and nitrocellulose filters from Schleicher & Schuell. The ECL kit, streptavidin-horseradish peroxidase conjugate, prestained protein marker, [ 3 H]thymidine, [␥-32 P]ATP and [␣-32 P]dCTP were from Amersham Corp.
Cell Culture-The FRTL-5 cells (ATCC CRL 8305; American Type Culture Collection, Rockville, MD) used in this study are a continuous strain of functioning epithelial cells derived from normal Fisher rat thyroid. The cells were cultured as described elsewhere (19) in Coon's modified Ham's F-12 medium supplemented with 5% donor calf serum and a six-hormone mixture including 0.5 milliunit/ml TSH and 10 g/ml insulin (6H medium). Cells were used, unless otherwise noted, when they approached confluence. Quiescent FRTL-5 cells were obtained after deprivation for 4 days of TSH and insulin by maintaining in 0.2% calf serum (4H medium).
Northern Blot Analysis-Total RNA was isolated by the guanidinium-thyocyanate-phenol procedure (20) from FRTL-5 cells after different treatments. Samples of total RNA were electrophoresed in 1% agarose gels containing 2.2 M formaldehyde. RNA was blotted onto nylon filters as suggested by the manufacturer. Methylene blue staining of the blots revealed the integrity of the RNA and the presence of equal amounts in each lane. Hybridization and washing (21) were carried out with specific probes for TTF-1 (4), Pax-8 (6), and c-fos (22) labeled by random oligo priming.
Western Blotting-Nuclear extracts were prepared (23) from FRTL-5 cells maintained under the different study conditions. Protein concentration was determined with the Bio-Rad protein assay kit with bovine * This work was supported by Grants BIO2-CT93-0454 (CE) and DGICYT PB94-0092, CAM 182/92 (Spain). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed: Instituto de Investigaciones Biomédicas, C/Arturo Duperier No. 4, Madrid 28029, Spain. Tel.: 34-1-5854644; Fax: 34-1-5854587. 1 The abbreviations use are: TSH, thyroid-stimulating hormone; serum albumin as the standard. Nuclear proteins (20 g of each sample) were separated by 8% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes using a Bio-Rad transfer apparatus (10 mA/gel, overnight) with 20% methanol, 25 mM Tris base, and 192 mM glycine as the transfer buffer. Membranes were blocked for 1-2 h at room temperature in TBS-T buffer (20 mM Tris-HCl, 137 mM NaCl, 0.1% Tween-20, pH 7.5) containing 10% bovine serum albumin. Membranes were incubated at room temperature for 1 h in TBS-T containing 10% bovine serum albumin and ␣-TTF-1 antibody (1 g/ml) and then washed three times, 10 min each, with 10 ml of TBS-T buffer with 10% bovine serum albumin. Membranes were then incubated for 60 min in TBS-T buffer plus 10% bovine serum albumin with a 1:1000 dilution of ␣-TTF-1 antibody or a 1:5000 dilution of streptavidin-horseradish peroxidase conjugate as appropriate followed by two 30-min washes with TBS-T buffer. Inmunoreactive bands were visualized using an ECL kit as described by the manufacturer. Electrophoretic Mobility Shift Assays-Nuclear proteins (5 g) from FRTL-5 prepared as above were incubated with 50 pg of 32 P-labeled double-stranded DNA oligonucleotide P (5Ј-CTA AGC TTG AGT GGG CAT CAG AGC ATG GAG TC-3Ј) the site at which Pax-8 binds to thyroperoxidase promoter. The binding reactions were performed as described elsewhere (17) except that the salt concentration was reduced to 75 mM KCl, optimal binding conditions for Pax-8 (5,6). In competition experiments, the unlabeled competitor was added in a 100-fold excess and was always preincubated with the extract for 10 min on ice prior to addition of the labeled probe. The resulting DNA-protein complexes were separated from free DNA on a 5% polyacrylamide gel (17).
Cell Proliferation-Quiescent FRTL-5 cells in 24 well plates were incubated for 3 days with sense or antisense oligonucleotides at a final concentration of 5 M. To measure DNA synthesis, 1 Ci/well of [ 3 H]thymidine was added, and half of the groups were stimulated with different ligands during a further 24 h, in the continued presence of antisense or sense oligonucleotides, respectively. Cells were then harvested, and radioactivity was measured using standard scintillation counting. The same experimental approach was followed in the experiments in which the cells were counted. In this case no [ 3 H]thymidine was added, and the cells were harvested by trypsinization.

Treatment with TTF-1 or Pax-8 Antisense Oligonucleotides Decreases mRNA and Protein Levels of Both Genes in FRTL-5
Cells-In order to demonstrate that the thyroid-specific transcription factors TTF-1 and Pax-8 play a role in thyroid cell proliferation, we used the antisense oligonucleotide approach to FRTL-5 cells, whose transition from quiescence to proliferation requires the action of TSH and IGF-I. Due to the relative stability of TTF-1 and Pax-8 proteins, 2 we found it necessary to maintain the cells in the presence of antisense oligonucleotides (5 M) for a minimum of 4 days. In order to demonstrate the success of the antisense strategy, we first examined the effect of antisense oligonucleotides on TTF-1 and Pax-8 mRNA levels. Confluent FRTL-5 cells growing in 6H medium were treated with either TTF-1 (Fig. 1A) or Pax-8 (Fig. 1B) sense or antisense oligonucleotides. Northern blots containing 20 g of total RNA from the above-mentioned cells were hybridized with labeled TTF-1 and Pax-8 cDNA probes. The results obtained (top panel) showed that the treatment with TTF-1 or Pax-8 antisense oligomers led to a marked reduction, to almost undetectable levels, of both TTF -1 (lane 3, A) and Pax-8 (lane 3, B) mRNAs. There was no change in the mRNA levels of either transcription factor from sense oligomer treated cells (lanes 2,  A and B). These showed the same levels as the ones detected in untreated cells (lanes 1, A and B). Another control for antisense specificity, besides the sense oligonucleotides, was performed hybridizing the above Northern blots with the other transcription factor probe. The results obtained shown that the level of the alternative transcription factor did not change, indicating that the effect observed is specific (middle panel). The integrity of 28 S and 18 S ribosomal RNAs from the blots after staining with methylene blue is shown in the bottom panel.
Since the half-life of TTF-1 and Pax-8 mRNAs is short compared to the long half-life of the protein we decided to study whether the antisense treatment also reduced the protein levels. For this purpose we detected the presence of TTF-1 and Pax-8 proteins by Western blotting and electrophoretic mobility shift assay, respectively. Nuclear proteins (20 g) extracted from confluent FRTL-5 cells or from the same cells treated for 4 days with sense or antisense TTF-1 oligonucleotide were separated in a 8% SDS-polyacrylamide gel electrophoresis, together with a prestained protein marker. Nuclear extracts from RAT-1 fibroblasts were used as a negative control. After protein transfer to nitrocellulose, the Western blot was probed with anti-TTF-1 antibodies. Inmunoreactive bands were visualized using the ECL kit. As shown in Fig. 2A, a clear and specific decrease is detected in TTF-1 protein in antisensetreated cells (lane 3) in comparison to cells treated with a control TTF-1 sense oligonucleotide (lane 1). TTF-1 protein, as expected, was not present in RAT-1 fibroblasts (lane 2).
Nuclear extracts (5 g) from FRTL-5 cells, previously incubated with sense or antisense Pax-8 oligonucleotides, were tested for their ability to bind a 32-base pair oligonucleotide derived from the Ϫ30 to Ϫ61 region within the thyroperoxidase promoter which has been shown to recognize the transcription factor Pax-8 (5, 6). A retarded band was detected in the gel shift assay when nuclear extracts from both control or sense treated FRTL-5 cells were used (Fig. 2B, lanes 2 and 3). The absence of Pax-8 was demonstrated when nuclear extracts from cells treated with antisense oligonucleotides were used, since no retarded band corresponding to Pax-8⅐DNA complex was observed (lane 4). The specificity of the complex was demonstrated by competition experiments. The complex was competed by a 100-fold excess of unlabeled oligonucleotide P (lanes 5-7).
TTF-1 and Pax-8 Antisense Oligonucleotides Inhibit TSHand IGF-I-stimulated Thyroid Cell Proliferation-In order to study whether TTF-1 and Pax-8 play a role in FRTL-5 cell proliferation in response to TSH and IGF-I, the following experiments were carried out. Quiescent FRTL-5 cells were incubated with 5 M TTF-1 and/or Pax-8 sense or antisense oligonucleotides in 24-well plates for 3 days. For the DNA synthesis  Fig. 3A, FRTL-5 cells treated with TTF-1 antisense oligonucleotide exhibited a 65% decrease in the DNA synthesis in response to 1 nM TSH, 100 ng/ml of IGF-I, or both ligands together. When the cells were treated with Pax-8 antisense oligonucleotides, a 30% decrease was found in DNA synthesis in response to the same ligands (Fig. 3B). The TSH effect was mimicked in both cases, by 5 M forskolin but not by 1 M TPA (not shown). Since proliferation is a complex process involving many cellular functions, we ruled out any toxic or other nonspecific effect of the antisense oligomer by treatment of the FRTL-5 cells with TTF-1 or Pax-8 sense oligonucleotide. We found this inhibition of thyroid cell proliferation to be specific, since addition of 5 M TTF-1 or Pax-8 sense oligonucleotide to the cells had no effect (Fig. 3, A and B). In this case, the DNA synthesis in response to growth factors was as expected and has been published previously (13,16). To exclude the possibility that the bases from the oligonucleotides could decrease the specific activity of the [ 3 H]thymidine, we confirmed the effect of antisense TTF-1 or Pax-8 treatment on FRTL-5 thyroid cell proliferation by cell counting 36 h after stimulation (Fig. 3, C and D). The effect of TTF-1 and Pax-8 on DNA synthesis or cell number was not additive, since the result obtained when the cells were incubated with both antisense oligonucleotides together was the same as that observed with the TTF-1 antisense oligonucleotide alone. DISCUSSION Different evidences suggest that the thyroid-specific transcription factors TTF-1 and Pax-8, homeo and paired box genes, respectively, are necessary for thyroid phenotype determination, thyroid development (9, 10), as well as for differentiation (11,12). Since this kind of genes are also involved in cell proliferation (1)(2), the aim of this work was to demonstrate that both TTF-1 and Pax-8 are necessary in processes of thyroid cell growth. We have used an effective and simple antisense approach for the specific depletion of TTF-1 and Pax-8 from FRTL-5 cells. The cells treated with sense or antisense oligonucleotides were viable and exhibited apparently normal cellular phenotype. Sense oligonucleotides or scrambled sequences were used as non-blocking controls. The oligonucleo-tides used in this work were changed every 24 h and appeared to be effective. The controls used for measuring the antisense effect were those recommended previously (24). The target RNAs and proteins levels after antisense oligonucleotide treatment were clearly reduced compared to that of control sense treatment. We consider that the approach used up to this point was appropriate to demonstrate our aim.
Thyroid follicular cells are regulated by a variety of growthstimulating factors, including TSH and IGF-I. Both growth factors stimulate proliferation and differentiation (13-16, 25, 26). It is well accepted that TSH works through the cAMP pathway in regulating thyroid growth (27)(28)(29). Experiments measuring [ 3 H]thymidine incorporation and cell number in FRTL-5 cells treated with either sense or antisense oligonucleotides for TTF-1 or Pax-8 indicated the importance of both transcription factors in thyroid cell proliferation. The TTF-1and Pax-8-mediated growth are via the cAMP pathway, since the TSH effect was mimicked by forskolin but not by TPA. It is not yet clear how IGF-I regulates thyroid cell growth but an explanation may be that of the tyrosine kinase signaling pathway. The signaling pathways initiated by TSH and IGF-I are different in the early cytoplasmic steps but they converge at a later point in the signaling cascade since they both regulate the same thyroid genes (13,30,31) and DNA synthesis (15,26,32). Moreover, the fact that the TSH and IGF-I have an additive effect on growth suggests that the pathways through which both factors work are different. The precise mechanism by which these mitogens transmit their signals from the cell surface to the nucleus is unknown, as are the number of points where these signaling pathways interact with each other. One of these interactions is demonstrated in our study by the fact that the thyroid transcription factors TTF-1 and Pax-8 mediate the thyroid growth response elicited by TSH and IGF-I.
What roles do these factors play in thyroid cell growth? There are two possible theories. First, the simpler one, that both transcription factors are necessary for maintaining the thyroid phenotype. When the cells are not totally differentiated, by blocking TTF-1 and Pax-8, they do not respond to growth factors such as TSH and IGF-I. The other possibility, more complex but to us a very interesting explanation, is that both factors play a functional role in the regulation of thyroid cell cycle. This would result in TTF-1 or Pax-8 mRNA being regulated by TSH and IGF-I. Although other authors (8) have reported evidence that TTF-1 mRNA is down-regulated by TSH, in our conditions its mRNA is not regulated either by TSH, forskolin (data not shown) or by IGF-I (17). However, both growth factors could regulate TTF-1 by some other mechanism. One of the possible postranslational regulation mechanisms is protein phosphorylation. TTF-1 has been shown to be a phosphoprotein (33). Thus, our hypothesis is that at least for this transcription factor, TSH and IGF-I could regulate its phosphorylation state through the pathways used by such factors to induce thyroid cell growth.
The transition from quiescence to proliferation requires the presence of mitogenic stimulation. It is well accepted that different kinases are activated in many cell types in response to growth factors during the G 0 to G 1 transition in the cell cycle (34,35). These kinases could phosphorylate either TTF-1 or a cell cycle kinase, such as cdc2, that will be involved in TTF-1 activation. Phosphorylated TTF-1 could regulate one still unknown protein of the cell cycle. We can postulate the same theory in the case of Pax-8. It has been suggested (2) that Pax genes might be involved in growth regulation because cell cycle proteins, such as cdc2, whose steady state levels are growth regulated would be good candidates for common targets of developmental control (36,37).
To determine if the mechanism by which blocking TTF-1 mRNA interferes with DNA synthesis could implicate the regulation of genes involved in the proliferation, we analyzed the mRNA levels of c-fos. The results obtained (not shown) indicated that c-fos, a gene involved in FRTL-5 cell proliferation, remained unaffected in TTF-1 antisense-treated cells after TSH or IGF-I stimulation. At present, we can only speculate about the precise function of both thyroid transcription factors in the cell cycle control but it seems reasonable that it might regulate another set of genes expressed later in the growth response. The demonstration of this hypothesis will require a more careful study of possible targets of TTF-1 or Pax-8 action.