Clinical Science (2009) 116, 27–35 (Printed in Great Britain) doi:10.1042/CS20080068 27

REVIEW Thyroid -1 (TTF-1/Nkx2.1/ TITF1) regulation in the lung

Vijay BOGGARAM Department of Molecular Biology, The University of Texas Health Science Center at Tyler, 11937 US Highway 271, Tyler, TX 75708-3154, U.S.A.

ABSTRACT

TTF-1 [thyroid transcription factor-1; also known as Nkx2.1, T/EBP (thyroid-specific-enhancer- binding ) or TITF1] is a homeodomain-containing transcription factor essential for the morphogenesis and differentiation of the thyroid, lung and ventral forebrain. TTF-1 controls the expression of select in the thyroid, lung and the central nervous system. In the lung, TTF-1 controls the expression of surfactant that are essential for lung stability and lung host defence. Human TTF-1 is encoded by a single gene located on 14 and is organized into two/three exons and one/two introns. Multiple transcription start sites and alternative splicing produce mRNAs with heterogeneity at the 5 end. The 3 end of the TTF-1 mRNA is characterized by a rather long untranslated region. The amino acid sequences of TTF-1 from human, rat, mouse and other species are very similar, indicating a high degree of sequence conservation. TTF-1 promoter activity is maintained by the combinatorial or co- operative actions of HNF-3 [hepatocyte nuclear factor-3; also known as FOXA (forkhead box A)], Sp (specificity protein) 1, Sp3, GATA-6 and HOXB3 ( B3) transcription factors. There is limited information on the regulation of TTF-1 by hormones, cytokines and other biological agents. Glucocorticoids, cAMP and TGF-β (transforming growth factor-β)have stimulatory effects on TTF-1 expression, whereas TNF-α (tumour necrosis factor-α) and ceramide have inhibitory effects on TTF-1 DNA-binding activity in lung cells. Haplo-insufficiency of TTF-1 in humans causes hypothyroidism, respiratory dysfunction and recurring pulmonary infections, underlining the importance of optimal TTF-1 levels for the maintenance of thyroid and lung function. Recent studies have implicated TTF-1 as a lineage-specific proto-oncogene for lung cancer.

INTRODUCTION ofthebrain[1].Homeodomain-containingtranscriptional factors play key roles in the control of embryonic devel- TTF-1 [thyroid transcription factor-1; also known as opment and differentiation [2,3]. A homeobox is a Nkx2.1, T/EBP (thyroid-specific-enhancer-binding pro- 180 bp DNA sequence motif encoding a protein domain tein) or TITF1], a member of the homeodomain-contain- that can bind the DNA in a sequence-specific manner. ing transcription factor family, activates the expression of Homeodomain-containing proteins control the tran- select genes in the thyroid, lung and restricted regions scriptional activation of target genes by binding to specific

Key words: inflammation, lung cancer, morphogenesis, promoter regulation, surfactant, thyroid transcription factor (TTF). Abbreviations: CCSP, Clara cell secretory protein; DEE, diesel engine emissions; EMT, epithelial–mesenchymal transition; FOX, forkhead box; HNF, hepatocyte nuclear factor; HOX, homeobox; IPF, idiopathic pulmonary fibrosis; MITF, microphthalmia- associated transcription factor; NSCLC, non-SCLC; PKA, protein kinase A; RNAi, RNA interference; SCC, squamous cell carcinoma; SCLC, small-cell lung carcinoma; SP, surfactant protein; T/EBP, thyroid-specific-enhancer-binding protein; TGF-β, transforming growth factor-β;TNF-α, tumour necrosis factor-α; TSH, thyroid-stimulating hormone; TTF-1, thyroid transcription factor-1. Correspondence: Dr Vijay Boggaram (email [email protected]).

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Figure 1 Expression of TTF-1 and TTF-1-responsive genes in the lung A schematic diagram of an alveolus showing the locations of alveolar type I and type II cells and Clara epithelial cells. TTF-1 is expressed by alveolar type II and Clara epithelial cells and regulates the expression of SP and CCSP by these cells.

DNA sequences via the homeodomain. The homeobox that include LAMP3 (lysosomal-associated membrane was first identified in the HOM and HOX (homeo- protein 3) and CEACAM6 (carcinoembryonic antigen- box) genes that control body axis patterning in the fruit related cell adhesion molecule 6) [22]. Ectopic expression fly Drosophila melanogaster. TTF-1 was first purified of TTF-1 in NIH 3T3 cells activates the expression of from rat thyroid FRTL-5 cells as a transcription factor nestin, an intermediate filament protein expressed in activating the rat thyroglobulin promoter in a thyroid- neuroepithelial stem cells, identifying nestin as a TTF-1- cell-specific manner [4]. cDNA cloning experiments regulated gene in the central nervous system [23]. TTF- determined that the TTF-1 mRNA encodes a protein 1 appears to be important for the expression of the T1α of approx. 38 kDa, and that the TTF-1 DNA-binding promoter [24]. The expression of T1α is restricted to alve- domain is a novel mammalian homeodomain that dis- olar type I, but not type II, epithelial cells. It is not known plays significant homology with the Drosophila NK-2 whether TTF-1 is expressed in alveolar type I cells, but homeodomain [5]. These studies also demonstrated the the occurrence of a TTF-1 DNA-binding site in the T1α presence of TTF-1 mRNA in the lung and TTF-1 DNA- promoter and activation of the T1α promoter by TTF-1 binding activity in lung nuclear extracts. Soon after the suggest that TTF-1 might be expressed in type I cells. first report on the cloning of TTF-1, independent studies TTF-1 is expressed at the earliest stages of thyroid, reported the cloning of T/EBP from FRTL-5 cells that lung and brain development and well before the express- had sequence identity with TTF-1 [6]. ion of its target genes [25–27], suggesting that TTF-1 TTF-1 controls the expression of several important may have important roles in the development and dif- thyroid-specific and lung-specific genes. In the thyroid, ferentiation of these organs. Consistent with a role for TTF-1 controls the expression of the thyroglobulin TTF-1 in the organogenesis of the thyroid, lung and [4], thyroperoxidase [6–9], thyrotropin [10,11] brain, blocking TTF-1 expression in fetal lung explants and sodium iodide symporter [12] genes and, in the lung, in culture resulted in an inhibition of branching morpho- TTF-1 is essential for the expression of SP (surfactant genesis [28]. Deletion of T/EBP (TTF-1) in mice resulted protein)-A [13,14], SP-B [15,16], SP-C [17,18], CCSP in pups that are born dead, due to the complete lack of (Clara cell secretory protein) [19,20] and ABCA3 (ATP- lung parenchyma and thyroid, and to extensive defects binding-cassette transporter A3) [21] genes (Figure 1). in the ventral region of the forebrain [29]. In the Recent studies have determined that, in lung type II cells, adult mouse lung, TTF-1 is expressed predominantly TTF-1 is required for the induction of a subset of genes in alveolar type II epithelial cells, epithelial cells lining

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The TTF-1 homeodomain shares 82% identity with the Drosophila NK-2 homeodomain and only 27– 40% identity with other homeodomains, identifying it as a member of the NK2 family of homeodomain proteins [5]. Deletion mapping studies have shown that most of the DNA-binding activity of TTF-1 is contributed by the homeodomain [5]. Mapping studies have identified two activation domains located at the N- and C-termini of the Figure 2 TTF-1 protein organization protein that are involved in the transcriptional activation A schematic diagram of the functional domains of TTF-1 protein. The N- and of TTF-1 [35]. Either of the two activation domains was C-terminal activation domains (AD), the homeodomain (HD) and the inhibitor found to activate a target promoter, indicating probable domain (ID) are shown. The locations of redox-sensitive cysteine residues (C) functional redundancy [35]. The TTF-1 transcriptional and the serine-phosphorylation sites (S) in the TTF-1 protein are indicated. The activation domains do not have any sequence similarity domain organization of TTF-1 protein shown in this diagram is based on the work with the activation domains of transcription factors char- described by De Felice et al. [35]. The identification of redox-sensitive cysteine acterized previously [36]. Despite this lack of similarity residues and serine-phosporylation sites was described by Arnone et al. [37] and with the activation domains of other transcription factors, Zannini et al. [40] respectively. the N-terminal TTF-1 activation domain activates tran- scription by mechanisms similar to those used by acidic bronchi and bronchioles, and with a weaker expression activation domains [36]. Interestingly a glutamine-rich in the epithelial cells lining the trachea in a pattern similar region located C-terminal to the homeodomain func- to the expression of SPs and CCSP [27]. TTF-1 express- tions as an inhibitory domain [35]. ion remained constant during fetal human lung develop- In vitro experiments have demonstrated that treatment ment and in the adult lung, indicating that TTF-1 levels of TTF-1 with oxidizing agents, such as oxidized gluta- may not be subject to developmental regulation [30]. thione and diamide, inactivated TTF-1 DNA-binding activity, which was reversed by dithiothreitol [37]. The decrease in the TTF-1 DNA-binding activity was found TTF-1 PROTEIN AND GENE STRUCTURE to be due to the oxidation of two specific cysteine residues located outside the TTF-1 homeodomain that TTF-1 from calf [5], rat [6], mouse [31] and human [32] lead to the formation of higher-order oligomers [37]. comprises a single polypeptide chain of 371–378 amino Specifically, oxidation of Cys87 that resides in the N- acids with a molecular mass in the range of 38–42 kDa terminal activation domain was implicated in the reduced (Figure 2). The amino acid sequences of human [32], rat DNA-binding activity of the TTF-1 homeodomain [38]. [6] and mouse [31] TTF-1 have 98% sequence similarity, It was suggested that oxidation of Cys87 interferes with with the complete conservation of the 60-amino-acid the correct folding of the homeodomain, resulting in its homeodomain. A minor variant form of the human decreased DNA-binding activity. Ref-1 (redox effector TTF-1 transcript encoding a 30-amino-acid extension factor-1) was shown to mediate the redox effects on the at the N-terminal has been identified and was found to TTF-1 homeodomain, thereby modulating TTF-1 tran- give rise to a 44 kDa in-vitro-translated protein product scriptional activity [38]. Redox regulation of TTF-1 [33]. The 30-amino-acid extension of TTF-1 is highly DNA-binding activity could play important roles in the conserved in various mammalian species. The two TTF-1 control of target gene expression. In thyroid FRTL-5 transcripts are differentially expressed during mouse fetal cells, TSH (thyroid-stimulating hormone) up-regulates lung development, with the onset of accumulation of thyroglobulin gene expression via increased binding of the longer transcript occurring at a later stage than that Pax-8 and TTF-1 transcription factors to the thyro- of the shorter transcript [34]. Both TTF-1 transcripts are globulin promoter [39]. Redox regulation by thioredoxin expressed in baboon lung and H441 lung epithelial cells was suggested to be responsible for the increased binding [33]. Two forms of TTF-1 protein with similar molecular of Pax-8 and TTF-1 to the thyroglobulin promoter in massesasthein-vitro-translated protein products of TSH-treated FRTL-5 cells. TTF-1 DNA-binding activity the two TTF-1 transcripts are detected in MLE-15 lung appears to be highly sensitive to oxidation, as shown by epithelial cells [34]. The two TTF-1 transcripts have dif- its loss during the preparation of nuclear extracts in the ferences in their capacity to activate the SP-C promoter absence of reducing agents and the restoration of binding in co-transfection experiments, indicating functional activity by reducing agents ([37], and V. Boggaram, differences [34]. It is not clear whether the 44 kDa variant unpublished work). TTF-1 is phosphorylated on seven form of TTF-1 is expressed in the lung; human lung serine residues in thyroid FRTL-5 cells and in hetero- Clara H441 cells and primary type II cells express only logous cells such as HeLa cells [40]. PKA (protein the 42 kDa form of TTF-1 and the expression of the kinase A) [14,41], PKC (protein kinase C) [40] and MST2 44 kDa form of TTF-1 was not detected [22]. [mammalian Ste20 (sterile 20)-like protein kinase 2]

C The Authors Journal compilation C 2009 Biochemical Society 30 V. Boggaram

TTF-1 PROMOTER REGULATION IN THE LUNG

The 5 flanking region of the TTF-1 gene supports high level reporter gene expression in H441 and MLE-15 lung cells compared with non-lung cells, such as NIH 3T3 cells, and to A549 lung cells that do not display differentiated characteristics of type II cells, indicating the Figure 3 TTF-1 gene organization presence of cis-DNA elements necessary for lung-specific A schematic diagram of the structure of human TTF-1 gene. Exons are represented expression [32,33,47]. The identification of a new exon I by rectangles and introns and 5 and 3 flanking regions are represented by lines. in the TTF-1 gene and analysis of genomic regions indi- Filled and open areas represent untranslated and translated regions of the exons cated the presence of two promoter regions which sup- respectively. The arrows indicate the locations of transcription start sites. The port reporter gene expression in lung cells in vitro [33]. locations of translation initiation (ATG) and translation termination (TGA) codons One lies within the first intron (proximal promoter) and and of functionally important transcription-factor-binding sites are also shown. the other upstream of the first exon (distal promoter). The organization of the TTF-1 gene shown is based on the structure of the TTF-1 A TTF-1 genomic fragment that included both the gene described by Hamdan et al. [33]. upstream and the intronic promoter regions expressed the reporter gene at a significantly higher level than a fragment containing the intronic promoter alone [32]. kinase [42] are able to phosphorylate TTF-1. Increased These findings suggest interactions between the two pro- TTF-1 phosphorylation by PKA activated gene tran- moters. The proximal promoter does not contain a tradi- scription of SP-B [41] and SP-A [14] in H441 lung epi- tional TATA sequence, but instead contains a sequence thelial cells and primary alveolar type II cells respectively, TAAAA that has some degree of similarity to the TATA suggesting that alterations in TTF-1 phosphorylation element, whereas the distal promoter lacks a TATA-like status can modulate target gene expression. Abnormal- sequence altogether. The TTF-1 proximal promoter ities in lung morphogenesis and decreased expression of contains two closely located DNA elements that bind SPs in homozygous mice expressing a TTF-1 mutant HNF-3α [hepatocyte nuclear factor-3α; also known as in which all of the seven serine phosphorylation sites FOXA1 (forkhead box A1)] and HNF-3β (also known had been mutated underlines the importance of TTF-1 as FOXA2) factors in MLE-15 lung cells and activate phosphorylation in the control of target gene expression promoter activity [32]. The distal promoter contains a [43]. GC-rich sequence that binds Sp1 and Sp3 (specificity TTF-1 is encoded by a single-copy gene that is protein 1 and 3 respectively) transcription factors that located on chromosome 14 in humans and chromosome regulate TTF-1 promoter activity [48]. The TTF-1 12 in the mouse [5]. Initial studies concluded that rat promoter is activated by co-expression of TTF-1 in [44,45], mouse [31] and human [32,33] TTF-1 genes were FRTL-5 thyroid cells and HepG2 cells, indicating that organized into two exons and one intron. Other studies the TTF-1 promoter is subject to positive autoregulation have determined that the rat TTF-1 gene is organized [45,49]. The TTF-1-binding site(s) that controls positive into three exons and two introns [46] (Figure 3). Previous autoregulation of the TTF-1 promoter has not been iden- studies on the cloning and sequencing of a large number tified or characterized. A DNA element that binds the of TTF-1 cDNAs from a fetal human lung cDNA library zinc finger protein GATA-6 was identified in the mouse have concluded that the human TTF-1 gene, similar to proximal TTF-1 promoter and found to be important for the rat TTF-1 gene, is also organized into three exons and promoter activity in MLE-15 lung epithelial cells [50]. two introns [33]. The newly identified exon I contains However, mice deficient in GATA-6 expressed TTF-1 an ATG codon that falls in-frame with the initiator ATG in bronchiolar epithelial cells, suggesting that GATA-6 identified previously. In MLE-15 and H441 human lung may not be required for TTF-1 expression in bronchiolar epithelial cells, the start site of transcription for the TTF-1 epithelial cells [51]. The homeobox protein HOX3B gene was mapped to −196 bp relative to the initiator ATG binds to a highly conserved DNA element in the TTF-1 codon [32]. In rat FRTL-5 thyroid cells and human lung proximal promoter and activates promoter activity in co- H441 cells, multiple T/EBP (TTF-1) transcripts having transfection experiments in HeLa cells [52]. The signi- variable lengths of 5 untranslated regions were identified ficance of HOX3B regulation of TTF-1 promoter activity by cDNA cloning and sequencing, indicating the use in lung epithelial cells is not clear because HOX3B of multiple promoters and the occurrence of alternative expression appears to be restricted to the mesenchyme splicing [33,45,46]. Determination of transcription start in the developing lung [53]. The occurrence of multiple sites for TTF-1 transcription in thyroid and lung showed functionally important binding sites in the TTF-1 multiple start sites of transcription in agreement with the promoter suggests that the promoter activity is controlled existence of multiple TTF-1 mRNAs [45,46]. via combinatorial or co-operative interactions between

C The Authors Journal compilation C 2009 Biochemical Society Thyroid transcription factor-1 gene regulation in the lung 31 the transcription factors. Although TTF-1 5 flanking primary and metastatic lung cancer, in particular for the genomic DNA fragments from the baboon that included identification of the lung as the primary site of metastatic both the distal and proximal promoters or only the distal adenocarcinoma [66–68]. TTF-1 is also useful in distin- promoter failed to direct LacZ reporter gene expression guishing malignant pleural mesotheliomas from adeno- in the lung or thyroid, they expressed the LacZ gene in the carcinomas [69]. SCLCs (small-cell lung carcinomas) also trachea and subsets of cells localized in the hypothalamus have TTF-1 immunoreactivity. Although SCLCs display in transgenic mice [54]. These intriguing findings suggest a positive reaction for TTF-1, TTF-1 may not be a specific that additional TTF-1 regulatory sequences may be marker for SCLCs, as small cell carcinomas from tissues required for lung- and thyroid-specific expression. other than lung have positive reactions [70]. It has been suggested that TTF-1 in combination with cytokeratin TTF-1 EXPRESSION IN LUNG DISEASES 20 would be very useful as a marker for distinguishing SCLCs from small cell carcinomas of other primary sites TTF-1 is expressed at the onset of lung morphogenesis in [70]. In primary NSCLC (non-SCLC), TTF-1 immuno- epithelial cells and throughout fetal lung development. reactivity was significantly correlated with adenocar- In the postnatal lung, TTF-1 expression is restricted pri- cinomas, rather than SCCs (squamous cell carcinomas) marily to alveolar type II cells and subsets of non-ciliated [71,72]. There appears to be considerable variation in bronchiolar epithelial cells. There is limited information TTF-1 immunoreactivity in NSCLC. Although TTF-1 on the regulation of TTF-1 levels in lung diseases. TTF-1 immunoreactivity is not frequently encountered in SCCs, immunostaining was reduced or absent in type II cells in reverse transcriptase–PCR analysis of lung SCC cell regions of acute inflammation, oedema, haemorrhage and lines detected TTF-1 expression in three out of four atelectasis in lungs of infants with HMD (hyaline mem- cell lines [73], indicating that the lack of or low TTF-1 brane disease) and BPD (bronchopulmonary dysplasia) immunoreactivity in SCCs is due to low TTF-1 ex- [25]. In contrast, TTF-1 immunostaining was prominent pression. in cells in regions of the lung that were undergoing regen- Several studies have investigated whether TTF-1 eration [25]. Partial deficiency or haplo-insufficiency of expression can serve as a prognostic indicator for survival TTF-1 in humans heterozygous for TTF-1 mutations is in lung cancer patients [71,72,74–76]. There appears to associated with hypothyroidism, choreoathetosis, mus- be a strong correlation between TTF-1 expression and cular hypotonia, respiratory dysfunction and recurrent median survival rates in patients with lung cancer. Patients pulmonary infections [55–59]. TTF-1 mutations in these with tumours having strong TTF-1 expression had patients were characterized as complete gene deletions, statistically significant better median survival rates than missense mutations or nonsense mutations. Respiratory those with tumours having no or weak expression (>57.3 dysfunction and recurrent pulmonary infections in these compared with 39.4 −+ 5.2 months respectively; P = 0.006 patients could be the result of reduced expression of SPs according to a long-rank test) [71]. Indeed a meta-analysis as a consequence of TTF-1 deficiency. TTF-1 expression concluded that TTF-1 expression is a good prognostic was significantly reduced in nitrofen-induced lung hypo- factor for survival in NSCLC [77]. The reasons for the plasia in rats, and glucocorticoid treatment prevented the close association between TTF-1 expression and survival reduction in TTF-1 levels [60]. Similarly, nitrofen de- rates in patients with NSCLC are not well understood. creased TTF-1 promoter activity in H441 lung epithelial Although one study found no correlation between TTF-1 cells, indicating that the decreased TTF-1 expression is expression levels and tumour differentiation [75], another due to reduced gene transcription [60]. Other studies study found that the TTF-1 expression level was posit- have found that TTF-1 expression is unaltered [61] or ively associated with tumour differentiation in NSCLCs increased [62,63] in hypoplastic lungs of nitrofen-treated [71]. rats. In contrast with the alterations in TTF-1 levels in There exists an intimate association between cell lin- hypoplastic lungs of nitrofen-treated rats, no changes eage and tumorigenesis, indicating common mechanisms in lung TTF-1 expression were observed in an ovine for cell lineage and tumour progression and survival. In- model of surgically created congenital diaphragmatic deed results from several studies have supported a lineage- hernia [64] or in human fetuses with diaphragmatic hernia dependency model for tumorigenesis ([78], but see [78a]). [65]. These findings suggest that the alterations in TTF-1 It has been suggested that aberrant functioning of lineage- levels in nitrofen-induced diaphragmatic hernia may be survival pathways might underlie carcinogenesis and the result of the effects of nitrofen on TTF-1 expression tumour progression. For example, lineage-dependency in and not due to diaphragmatic hernia itself. tumorigenesis might rely on the deregulated expression of TTF-1 AS A LINEAGE-SPECIFIC ONCOGENE a master gene(s) that controls developmental and essential IN LUNG CANCER functions. It has been suggested that transcription factors and signalling proteins might fill the role of lineage- Owing to its cell/tissue-restricted expression, TTF-1 survival oncogenes. The master transcriptional regulator has been used widely as a marker for the diagnosis of MITF (microphthalmia-associated transcription factor)

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that is involved in the differentiation and survival of silicon carbide whiskers, an asbestos substitute, into rats melanocytes is a case in point [79]. Deregulation of MITF produced fibrotic changes and decreased the expression of has been linked to metastatic melanoma. SP-A and TTF-1 [83]. Changes in SP-A and TTF-1 levels The characteristics of TTF-1, namely its role in lung were apparent as early as 3 days after the exposure and development, cell-restricted expression in the lung and reduced expression was maintained for up to 6 months high-level expression in lung adenocarcinomas, strongly following the single exposure. In studies to assess the point to TTF-1 as a potential lineage-survival oncogene impact of inhaled DEE (diesel engine emissions) on in lung cancer [80]. Analysis of large numbers of lung tu- the clearance of Pseudomonas aeruginosa infection in mours and lung cancer cell lines has revealed amplification mice, it was found that prior exposure of mice to DEE of the TTF-1 gene in lung adenocarcinoma. Although one significantly reduced the expression of TTF-1 as well study reported amplification of the TTF-1 gene in 2% of as of CCSP [84]. Exposure to DEE decreased lung the tumours analysed [80], other studies have reported clearance of P. aeruginosa and exacerbated lung inflam- amplification rates of 12% [81] and 11% [82]. Interest- mation and injury. Pro-inflammatory agents such as ingly the occurrence of higher TTF-1 gene amplifications TNF-α (tumour necrosis factor-α) [85] and ceramide [86] at metastatic sites than primary sites was observed [82]. inhibited SP-B promoter activity in H441 lung cells by Amplification of the TTF-1 gene in lung adenocarcinoma reducing TTF-1 DNA-binding activity. The molecular cell lines is associated with increased expression of TTF- mechanisms responsible for the TNF-α and ceramide in- 1 [80,82]. Sequencing of the TTF-1 open reading frame hibition of TTF-1 DNA-binding activity are not known. from four NSCLC cell lines exhibiting amplification did Glucocorticoid- and cAMP-dependent differentiation of not reveal any DNA mutations, indicating that TTF-1 alveolar type II epithelial cells in vitro is associated levels would be of importance [82]. Reduction in endo- with the up-regulation of TTF-1 and SP expression genous TTF-1 levels by RNAi (RNA interference)- [87]. It is not known whether the inductive effects of mediated knockdown in a variety of lung cancer cell lines glucocorticoid and cAMP on TTF-1 levels are due to their with TTF-1 amplification decreased cell proliferation, direct effects on TTF-1 gene expression or secondary to indicating the importance of elevated TTF-1 levels for the their effects on the differentiation of alveolar type II cells. growth of cells [80–82]. Lung cancer cell lines without Chronic exposure of primary rat alveolar epithelial cells amplification of the TTF-1 gene but with detectable to a combination of TGF-β (transforming growth factor- expression of TTF-1 (H1155) [82] and those without de- β)andTNF-α resulted in decreased TTF-1 immunoreact- tectable expression of TTF-1 (NCI-H23, A549) [80] were ivity with a concomitant increase in immunoreactivity for not affected by RNAi-mediated knockdown of TTF-1, α-SMA (α-smooth muscle actin) and other mesenchymal underlining further the functional importance of elevated markers, indicating induction of an EMT (epithelial– TTF-1 levels for cell survival and proliferation. A mesenchymal transition) [88]. It was suggested that EMT reduction in endogenous TTF-1 levels by RNAi- could be responsible for the accumulation of fibroblasts mediated knockdown resulted in decreased cell-cycle and myofibroblasts in IPF (idiopathic pulmonary progression and increased apoptosis [80–82]. These fibrosis). Although TGF-β1 increases TTF-1 expression findings have demonstrated that sustained expression of in non-tumorigenic E10 mouse lung epithelial cells, it had TTF-1 is necessary for the growth and survival of a subset no effect on TTF-1 expression in tumorigenic E9 mouse lung adenocarcinoma and implicate TTF-1 as lineage- lung epithelial cells [89]. In E10 cells, TGF-β1 induction specific proto-oncogene for lung cancer. Apart from gene of TTF-1 expression occurs via the TGF-βII receptor and amplification, TTF-1 levels in lung adenocarcinoma cell Smad signalling with the involvement of Sp1 and Sp3 tran- lines and tumours can be up-regulated by transcriptional scription factors. Sp1 and Sp3 exert opposing effects on and post-transcriptional mechanisms, and by the control the TTF-1 promoter; although Sp1 increases TTF-1 pro- of protein stability. It remains to be determined whether moter activity, Sp3 negated the inductive effects of Sp1. any of these mechanisms operate along with TTF-1 gene amplification to elevate TTF-1 levels in lung adenocar- cinomas. It is not known whether TTF-1 directly con- CONCLUSIONS trols the survival of cancer cells or via another downstream effector molecule(s), whose identity remains TTF-1 plays key roles in the control of cell/tissue-specific to be determined. gene expression and in the morphogenesis and differenti- ation of the thyroid, lung and brain. Recent studies have suggested that TTF-1 plays the role of a proto-oncogene REGULATION OF TTF-1 GENE EXPRESSION in lung cancer. Considering the important roles that IN THE LUNG TTF-1 plays, abnormal expression of TTF-1 probably contributes to the pathogenesis of lung, thyroid and other There is limited information on the regulation of TTF-1 diseases. Molecular mechanisms that control cell/tissue- expression in the lung. Intratracheal instillation of specific expression of TTF-1 and TTF-1 transcriptional

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activity are not fully understood. Likewise cellular path- 12 Endo, T., Kaneshige, M., Nakazato, M., Ohmori, M., ways and agents that regulate TTF-1 expression remain to Harii, N. and Onaya, T. (1997) Thyroid transcription factor-1 activates the promoter activity of rat thyroid + − be explored. Because TTF-1 is essential for the expression Na /I symporter gene. Mol. Endocrinol. 11, 1747–1755 of SP and other genes important for lung function, abnor- 13 Bruno, M. D., Bohinski, R. J., Huelsman, K. M., Whitsett, mal expression and/or activity of TTF-1 may underlie the J. A. and Korfhagen, T. R. (1995) Lung cell-specific expression of the murine surfactant protein A (SP-A) gene pathogenesis of a variety of lung inflammatory diseases. is mediated by interactions between the SP-A promoter The recent discovery of TTF-1 as a proto-oncogene for and thyroid transcription factor-1. J. Biol. Chem. 270, 6531–6536 lung cancer has opened new avenues of research that 14 Li, J., Gao, E. and Mendelson, C. R. (1998) Cyclic might lead to novel therapies for lung cancer. A better AMP-responsive expression of the surfactant protein-A understanding of agents and pathways and molecular gene is mediated by increased DNA binding and transcriptional activity of thyroid transcription factor-1. mechanisms that control TTF-1 expression in the lung J. Biol. Chem. 273, 4592–4600 will be useful for the development of novel treatment 15 Bohinski, R. J., Di Lauro, R. and Whitsett, J. A. (1994) The lung-specific surfactant protein B gene promoter is a strategies for lung diseases such as ARDS (acute respirat- target for thyroid transcription factor 1 and hepatocyte ory distress syndrome), IPF, lung cancer and others. nuclear factor 3, indicating common factors for organ-specific gene expression along the foregut axis. Mol. Cell. Biol. 14, 5671–5681 16 Margana, R. K. and Boggaram, V. (1997) Functional ACKNOWLEDGMENTS analysis of surfactant protein B (SP-B) promoter. Sp1, Sp3, TTF-1, and HNF-3α transcription factors are necessary for lung cell-specific activation of SP-B gene Research in my laboratory is supported by the National transcription. J. Biol. Chem. 272, 3083–3090 Institutes of Health Grant HL-48048. I thank Dr Mark 17 Kelly, S. E., Bachurski, C. J., Burhans, M. S. and Glasser, A.L. Atkinson for reading the manuscript prior to S. W. (1996) Transcription of the lung-specific surfactant protein C gene is mediated by thyroid transcription factor submission, and Dr Dorota Stankowska for preparing 1. J. Biol. Chem. 271, 6881–6888 Figure 1. 18 Liu, C., Glasser, S. W., Wan, H. and Whitsett, J. A. (2002) GATA-6 and thyroid transcription factor-1 directly interact and regulate surfactant protein-C gene expression. J. Biol. Chem. 277, 4519–4525 REFERENCES 19 Toonen, R. F., Gowan, S. and Bingle, C. D. 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Received 29 February 2008/1 May 2008; accepted 19 May 2008 Published on the Internet 28 November 2008, doi:10.1042/CS20080068

C The Authors Journal compilation C 2009 Biochemical Society