(Received for publication, April 24, 1995; and in revised form, July 13, 1995)
From the
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.
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 ()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.
Figure 1: Effect of TTF-1 and Pax-8 antisense oligonucleotides on TTF-1 and Pax-8 mRNA levels. Total RNA from FRTL-5 thyroid cells cultured in control 6H medium or treated 4 days with 5 µM of TTF-1 (A) or Pax-8 (B) sense or antisense oligonucleotides was isolated. Northern blots of total RNA (20 µg) were hybridized with TTF-1 or Pax-8 cDNA probes (top panel). The specificity of the reduction in mRNA levels after each treatment is demonstrated by hybridization with the probe of the alternative transcription factor (middle panel). The sizes of TTF-1 and Pax-8 mRNAs are indicated. The bottom panel is the result of methylene blue staining of the membranes after transfer.
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 µg) 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 antisense-treated 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).
Figure 2:
Effect of TTF-1 and Pax-8 antisense
oligonucleotides on TTF-1 and Pax-8 protein levels. Nuclear extracts
from the different treatments were isolated. A, Western blot
analysis with nuclear proteins (20 µg) from FRTL-5 cells treated
with TTF-1 sense or antisense oligonucleotides and from RAT-1
fibroblasts were probed with a-TTF-1 antibodies. The size of the TTF-1
protein is shown. B, electrophoretic mobility shift assay from
nuclear extracts (4 µg) of control, Pax-8 sense, or antisense
treated FRTL-5 cells incubated with the labeled oligonucleotide P
(Pax-8-binding site). The specificity of the retarded complex was
established by competition with a 100-fold excess of unlabeled
oligonucleotide P. The migration of the Pax-8DNA complex is
shown.
Nuclear extracts (5 µg) 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 -30 to -61 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-8DNA 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).
Figure 3:
Effect of TTF-1 and Pax-8 antisense
oligonucleotides on FRTL-5 cells proliferation. Quiescent FRTL-5 cells
were incubated in 24-well plates with 5 µM of TTF-1 (A and C) or Pax-8 (B and D) sense or
antisense oligonucleotides for 4 days. Then half of the groups were
stimulated with different ligands for another 24 h. A and B, cells were pulse-labeled with 1 µCi of
[H]thymidine as described under
``Experimntal Procedures,'' and DNA synthesis was determined
as thymidine incorporation (C and D). Cell counts
were performed after trypsinization in the absence of
[
H]thymidine as described above. Data are average
values ± S.D. of three independent experiments performed in
triplicate. &cjs2108;, sense;
,
antisense.
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 oligonucleotides 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 growth-stimulating factors,
including TSH and IGF-I. Both growth factors stimulate proliferation
and
differentiation(13, 14, 15, 16, 25, 26) .
It is well accepted that TSH works through the cAMP pathway in
regulating thyroid growth(27, 28, 29) .
Experiments measuring [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-1- and
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 to G
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.