TTF-1 Protein Research Articles

Nestin Is a Neuroepithelial Target Gene of Thyroid Transcription Factor-1, a Homeoprotein Required for Forebrain Organogenesis

Thyroid transcription factor-1 (TTF-1, also known as NKX2.1 and T/EBP), a transcription factor belonging to the NKX-2 family of homeodomain-containing genes, plays an essential role in the organogenesis of the thyroid gland, lung, and ventral forebrain. Nestin is an intermediate filament protein strongly expressed in multipotential neuroepithelial stem cells and rapidly down-regulated during postnatal life. Here we show that stable fibroblastic clones expressing TTF-1 acquire a phenotype reminiscent of neuroepithelial cells in culture and up-regulate the endogenous nestin gene. TTF-1 transactivates in HeLa and NIH3T3 cells a reporter gene driven by a central nervous system-specific enhancer element from the second intron of the rat nestin gene, where it recognizes a DNA-binding site (NestBS) whose sequence resembles a nuclear hormone/cAMP-responsive element very different from canonical TTF-1 binding sites. Nuclear extracts from the head of mouse embryos form a retarded complex with NestBS of the same mobility of the extracts obtained from TTF1-expressing clones, which is either abolished or supershifted in the presence of two different antibodies recognizing the TTF-1 protein. Thus, the neuroepithelial marker nestin is a direct central nervous system-specific target gene of TTF-1, leading to the hypothesis that it might be the effector through which TTF-1 plays its role in the organogenesis of the forebrain.

Down-regulation of thyroid transcription factor-1 gene expression in fetal lung hypoplasia is restored by glucocorticoids.

The thyroid transcription factor (TTF)-1 has an essential role in lung morphogenesis and development. It is involved in the transcription of surfactant proteins (SP), which are critical in respiratory function. Neonates with congenital diaphragmatic hernia die of respiratory failure caused by pulmonary hypoplasia with associated biochemical immaturity. To gain new insights into the causes of this disorder and the effect of prenatal hormonal treatment on reducing mortality in these infants, we evaluated the expression of TTF-1 as marker of lung morphogenesis and SP-B as marker of lung maturity. Using a rat model of lung immaturity, we show that TTF-1 and SP-B messenger RNA (mRNA) levels are drastically reduced in congenital lung hypoplasia. Interestingly, prenatal dexamethasone (Dex) treatment increased both TTF-1 and SP-B mRNAs over control levels when administered to rats with lung hypoplasia, but it had no effect on TTF-1 or a moderate effect on SP-B mRNA when administered to control rats. TRH alone also increases TTF-1 and SP-B mRNA levels but to a lesser extent than Dex. When administered together with Dex, TRH counteracts the induction observed with the glucocorticoid. The decrease in TTF-1 mRNA levels in lung hypoplasia is paralleled by a down-regulation of TTF-1 protein levels, as well as by a decrease in the TTF-1/DNA complex when the TTF-1-binding site of the SP-B promoter was used as a probe. Both parameters were reestablished after glucocorticoid treatment. Moreover, the regulation of TTF-1 gene expression described in this report is accompanied by the same regulation in its promoter activity, as demonstrated in transfection experiments performed in H-441 human lung-derived adenocarcinoma cells. In conclusion, our data demonstrate, for the first time, that lung hypoplasia and the associated respiratory dysfunction caused by SP-B deficiency are caused, in part, by down-regulation of TTF-1 gene expression. The observations that prenatal glucocorticoid treatment induces the expression of TTF-1 supports routine in utero glucocorticoid treatment of patients expected to have lung hypoplasia.

The mitotically phosphorylated form of the transcription termination factor TTF-1 is associated with the repressed rDNA transcription machinery.

The transcription termination factor TTF-1 exerts two functions in ribosomal gene (rDNA) transcription: facilitating initiation and mediating termination of transcription. Using HeLa cells, we show that TTF-1 protein is colocalized with the active transcription machinery in the nucleolus and also with the inactive machinery present in certain mitotic nucleolar organizer regions (NORs) when rDNA transcription is repressed. We also show that TTF-1 is specifically phosphorylated during mitosis in a manner dependent on the cdc2-cyclin B kinase pathway and on an okadaic acid-sensitive phosphatase. Interestingly, the mitotically phosphorylated form of TTF-1 appearing at the G(2)/M transition phase was more easily solubilized than was the interphase form. This indicates that the chromatin-binding affinity of TTF-1 appears to be different in mitotic chromosomes compared to the interphase nucleolus. Correlated with this, the other DNA-binding factor, UBF, which interferes with chromatin conformation in the rDNA promoter, was more strongly bound to rDNA during mitosis than at interphase. The reorganization of the mitotic rDNA promoter might be induced by phosphorylation of certain components of the rDNA transcription machinery and participate in silencing of rDNA during mitosis.

TTF-1 protein expression in pleural malignant mesotheliomas and adenocarcinomas of the lung

TTF-1 is a tissue-specific transcription factor expressed in the epithelial cells of thyroid and lung. This study investigates the immunohistochemical expression of TTF-1 in pleural malignant mesotheliomas (MM) and adenocarcinomas (AC) of the lung, respectively. For this purpose, 33 biopsy specimens of pulmonary AC and 24 specimens of MM were studied. TTF-1 immunoreactivity was identified in 19 of 33 cases of AC (57.5%) and in none of the 24 cases of MM. Positivity for TTF-1 was 100% specific and 57.5% sensitive for lung AC. Alternatively, negativity for TTF-1 was 57.5% specific and 100% sensitive for MM. These results suggest that TTF-1 can be favourably added to the immunohistochemical diagnostic panel for distinction between AC of the lung involving the pleura and pleural MM.

Transfection of TTF-1 gene induces thyroglobulin gene expression in undifferentiated FRT cells

The thyroglobulin gene, the substrate for thyroid hormone biosynthesis, is not expressed in the FRT cell line, which, even though it manifests the polarised epithelial phenotype, does not express any of the thyroid functional properties. Two transcription factors, TTF-1 and Pax-8, have been implicated in thyroid specific expression of the thyroglobulin gene. FRT cells contain Pax-8 but they lack TTF-1. In this paper, we show that transfection of TTF-1 expression vectors in FRT cells results in activation of thyroglobulin gene expression. If the expression vector encoded for TTF-1–ER, a fusion gene coding for the entire TTF-1 protein fused to the hormone-binding domain of the steroid receptor, under the control of the RSV promoter, thyroglobulin gene expression was controlled by estrogen. These data provide a direct demonstration that TTF-1 activates the chromosomal thyroglobulin promoter. Since transfection of TTF-1 expression vectors in non-thyroid cell types did not result in thyroglobulin gene expression, it is suggested that Pax-8, in addition, perhaps, to a specific cellular environment, might be required for thyroid specific expression of the thyroglobulin gene.

SPATIOTEMPORAL CHANGES IN EXPRESSION OF TTF-1 IN AN ANIMAL MODEL OF BPD.† 1369

TTF-1 is a homeodomain transcriptional regulator expressed in thyroid, lung& parts of brain. In vitro, TTF-1 binds to and activates transcription from pulmonary and thyroid specific gene promoters. We have shown that suppression of TTF-1 leads to abnormal pulmonary epithelial morphogenesis and structural defects (Minoo et al., Dev. Biol. 172:694-698). Because Bronchopulmonary dysplasia, BPD, affects the structural & functional organization of the lung, we proposed that alternative morphogenetic signals released by tissue injury &/or repair subvert normal lung organogenesis by alternations in spatiotemporal regulation of TTF-1. To test this hypothesis, we used immunological and molecular probes that we have recently developed in our laboratory to determine the pattern of expression of TTF-1 protein and mRNA during late lung development and in chronic lung disease in a baboon model of BPD. mRNA for TTF-1 increased as a function of gestational age which correlated with expansion of the epithelial cell population. Nuclear staining for TTF-1 protein was localized to some but not all epithelial cells lining the airways & presumptive pre-alveolar spaces during late lung development. Relationship between expression of TTF-1 & surfactant gene expression in vivo is not known and is currently underway. Expression of TTF-1 was reduced in the lungs of baboons delivered at 140 day gestational age & ventilated with prolonged (6 to 10 days) hyperoxia. More specifically however, areas of collapsed airways and pre-alveolar airspaces were marked by the absence of TTF-1 protein as assayed by immunostaining. In contrast, epithelial cells in the lungs of control (PRN) animals was positive for TTF-1. Therefore, chronic lung injury which leads to structural aberrations characteristic of BPD, is accompanied by suppression of TTF-1 in pulmonary epithelial cells. These observations have significant implications regarding etiology of, & design of novel therapies for BPD. Supported by HL56221. The BPD Resource Center is supported by HL52636.

Study of TTF-1 gene expression in dog thyrocytes in primary culture

TTF-1 is a homeodomain-containing transcription factor mainly expressed in the thyroid where it controls the tissue-specific expression of the thyrogobulin, thyroperoxidase and TSH receptor genes. It is therefore potentially implicated in the hormonal control exerted by thyrotropin via the second messenger cyclic AMP on the transcription of these genes in thyrocytes. In order to investigate whether there exists a relationship between the stimulation of the cAMP pathway and TTF-1 gene expression in these cells, we have compared the amounts of TTF-1 protein, its state of phosphorylation and its subcellular distribution in control and cAMP-stimulated dog thyrocytes in primary culture. Dog TTF-1 was expressed in bacteria as a fusion protein and antibodies were raised against the dog TTF-1 moiety. Stimulation of the thyrocytes by cyclic AMP agonist only marginally increased TTF-1 gene expression as shown for the mRNA by RNase protection assay and for the protein by immunoblotting and immunoprecipitation of extracts from 35S-methionine labelled cells. The phosphorylation state of TTF-1 was investigated by immunoprecipitation of extracts from 32P-labelled thyrocytes. Phosphorylation level appeared to be essentially unaffected by forskolin treatment of the cells. We also looked for differences in the use of phosphorylation sites by partial proteolytic digestion of immunoprecipitated 32P-labelled TTF-1 with Glu-C and Asp-N endoproteases. Comparison of radioactivity distribution amongst the generated fragements did not reveal any difference in the pattern of TTF-1 phosphorylation in control and forskolin conditions. Lastly, in situ detection of TTF-1 by immunofluorescence demonstrated that the protein was localized in the nucleus of the cells, irrespective of the culture conditions. No major change in TTF-1 gene expression upon stimulation of the thyrocyte with a cAMP agonist could thus be detected in this study. The absence of an obvious modification of the TTF-1 protein itself in response to cAMP stimulation may indicate that other transcription factor(s) or co-factor(s) are involved in the control exerted by cAMP on the expression of thyroid-specific genes.

The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain

TTF-1, a homeodomain-containing transcription factor, which is required for the specific expression of the thyroglobulin and thyroperoxidase gene promoters in differentiated thyroid cell lines, is expressed at the very beginning of rat thyroid differentiation. TTF-1 mRNA is detected in the endodermal cells of the thyroid rudiment in the rat embryo and precedes the expression of the two known target genes by 5 days. No delay is observed between the appearance of TTF-1 mRNA and protein, which shows a clear nuclear localization. In the adult thyroid, TTF-1 is present only in the endoderm-derived follicular cells. Two additional domains of expression of TTF-1 have been observed, the lung and restricted areas of the brain. In the lung, TTF-1 mRNA and protein are also present at the earliest stages of differentiation and are later confined to the bronchial epithelium. In the brain, TTF-1 appears to be restricted to structures of diencephalic origin, including the developing neurohypophysis. The early detection of TTF-1 in the endodermal cells of the thyroid and lung anlage and in restricted neuroblast populations indicates that TTF-1 may have a role in cell determination in these three systems and that additional mechanisms may be involved in the activation of thyroid-specific gene expression.