Clinical Science (2009) 116, 27–35 (Printed in Great Britain) doi:10.1042/CS20080068
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Thyroid transcription factor-1 (TTF-1/Nkx2.1/
TITF1) gene 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.
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TTF-1 [thyroid transcription factor-1; also known as Nkx2.1, T/EBP (thyroid-specific-enhancerbinding protein) 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 genes in the thyroid, lung and the central nervous system. In the lung,
TTF-1 controls the expression of surfactant proteins that are essential for lung stability and lung
host defence. Human TTF-1 is encoded by a single gene located on chromosome 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 cooperative actions of HNF-3 [hepatocyte nuclear factor-3; also known as FOXA (forkhead box A)],
Sp (specificity protein) 1, Sp3, GATA-6 and HOXB3 (homeobox B3) transcription factors. There
is limited information on the regulation of TTF-1 gene expression 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
TTF-1 [thyroid transcription factor-1; also known as
Nkx2.1, T/EBP (thyroid-specific-enhancer-binding protein) or TITF1], a member of the homeodomain-containing transcription factor family, activates the expression of
select genes in the thyroid, lung and restricted regions
of the brain [1]. Homeodomain-containing transcriptional
factors play key roles in the control of embryonic development and differentiation [2,3]. A homeobox is a
180 bp DNA sequence motif encoding a protein domain
that can bind the DNA in a sequence-specific manner.
Homeodomain-containing proteins control the transcriptional 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, microphthalmiaassociated 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
was first identified in the HOM and HOX (homeobox) genes that control body axis patterning in the fruit
fly Drosophila melanogaster. TTF-1 was first purified
from rat thyroid FRTL-5 cells as a transcription factor
activating the rat thyroglobulin promoter in a thyroidcell-specific manner [4]. cDNA cloning experiments
determined that the TTF-1 mRNA encodes a protein
of approx. 38 kDa, and that the TTF-1 DNA-binding
domain is a novel mammalian homeodomain that displays significant homology with the Drosophila NK-2
homeodomain [5]. These studies also demonstrated the
presence of TTF-1 mRNA in the lung and TTF-1 DNAbinding activity in lung nuclear extracts. Soon after the
first report on the cloning of TTF-1, independent studies
reported the cloning of T/EBP from FRTL-5 cells that
had sequence identity with TTF-1 [6].
TTF-1 controls the expression of several important
thyroid-specific and lung-specific genes. In the thyroid,
TTF-1 controls the expression of the thyroglobulin
[4], thyroperoxidase [6–9], thyrotropin receptor [10,11]
and sodium iodide symporter [12] genes and, in the lung,
TTF-1 is essential for the expression of SP (surfactant
protein)-A [13,14], SP-B [15,16], SP-C [17,18], CCSP
(Clara cell secretory protein) [19,20] and ABCA3 (ATPbinding-cassette transporter A3) [21] genes (Figure 1).
Recent studies have determined that, in lung type II cells,
TTF-1 is required for the induction of a subset of genes
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that include LAMP3 (lysosomal-associated membrane
protein 3) and CEACAM6 (carcinoembryonic antigenrelated cell adhesion molecule 6) [22]. Ectopic expression
of TTF-1 in NIH 3T3 cells activates the expression of
nestin, an intermediate filament protein expressed in
neuroepithelial stem cells, identifying nestin as a TTF-1regulated gene in the central nervous system [23]. TTF1 appears to be important for the expression of the T1α
promoter [24]. The expression of T1α is restricted to alveolar type I, but not type II, epithelial cells. It is not known
whether TTF-1 is expressed in alveolar type I cells, but
the occurrence of a TTF-1 DNA-binding site in the T1α
promoter and activation of the T1α promoter by TTF-1
suggest that TTF-1 might be expressed in type I cells.
TTF-1 is expressed at the earliest stages of thyroid,
lung and brain development and well before the expression of its target genes [25–27], suggesting that TTF-1
may have important roles in the development and differentiation of these organs. Consistent with a role for
TTF-1 in the organogenesis of the thyroid, lung and
brain, blocking TTF-1 expression in fetal lung explants
in culture resulted in an inhibition of branching morphogenesis [28]. Deletion of T/EBP (TTF-1) in mice resulted
in pups that are born dead, due to the complete lack of
lung parenchyma and thyroid, and to extensive defects
in the ventral region of the forebrain [29]. In the
adult mouse lung, TTF-1 is expressed predominantly
in alveolar type II epithelial cells, epithelial cells lining
Thyroid transcription factor-1 gene regulation in the lung
Figure 2 TTF-1 protein organization
A schematic diagram of the functional domains of TTF-1 protein. The N- and
C-terminal activation domains (AD), the homeodomain (HD) and the inhibitor
domain (ID) are shown. The locations of redox-sensitive cysteine residues (C)
and the serine-phosphorylation sites (S) in the TTF-1 protein are indicated. The
domain organization of TTF-1 protein shown in this diagram is based on the work
described by De Felice et al. [35]. The identification of redox-sensitive cysteine
residues and serine-phosporylation sites was described by Arnone et al. [37] and
Zannini et al. [40] respectively.
bronchi and bronchioles, and with a weaker expression
in the epithelial cells lining the trachea in a pattern similar
to the expression of SPs and CCSP [27]. TTF-1 expression remained constant during fetal human lung development and in the adult lung, indicating that TTF-1 levels
may not be subject to developmental regulation [30].
TTF-1 PROTEIN AND GENE STRUCTURE
TTF-1 from calf [5], rat [6], mouse [31] and human [32]
comprises a single polypeptide chain of 371–378 amino
acids with a molecular mass in the range of 38–42 kDa
(Figure 2). The amino acid sequences of human [32], rat
[6] and mouse [31] TTF-1 have 98 % sequence similarity,
with the complete conservation of the 60-amino-acid
homeodomain. A minor variant form of the human
TTF-1 transcript encoding a 30-amino-acid extension
at the N-terminal has been identified and was found to
give rise to a 44 kDa in-vitro-translated protein product
[33]. The 30-amino-acid extension of TTF-1 is highly
conserved in various mammalian species. The two TTF-1
transcripts are differentially expressed during mouse fetal
lung development, with the onset of accumulation of
the longer transcript occurring at a later stage than that
of the shorter transcript [34]. Both TTF-1 transcripts are
expressed in baboon lung and H441 lung epithelial cells
[33]. Two forms of TTF-1 protein with similar molecular
masses as the in-vitro-translated protein products of
the two TTF-1 transcripts are detected in MLE-15 lung
epithelial cells [34]. The two TTF-1 transcripts have differences in their capacity to activate the SP-C promoter
in co-transfection experiments, indicating functional
differences [34]. It is not clear whether the 44 kDa variant
form of TTF-1 is expressed in the lung; human lung
Clara H441 cells and primary type II cells express only
the 42 kDa form of TTF-1 and the expression of the
44 kDa form of TTF-1 was not detected [22].
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
protein that are involved in the transcriptional activation
of TTF-1 [35]. Either of the two activation domains was
found to activate a target promoter, indicating probable
functional redundancy [35]. The TTF-1 transcriptional
activation domains do not have any sequence similarity
with the activation domains of transcription factors characterized previously [36]. Despite this lack of similarity
with the activation domains of other transcription factors,
the N-terminal TTF-1 activation domain activates transcription by mechanisms similar to those used by acidic
activation domains [36]. Interestingly a glutamine-rich
region located C-terminal to the homeodomain functions as an inhibitory domain [35].
In vitro experiments have demonstrated that treatment
of TTF-1 with oxidizing agents, such as oxidized glutathione 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
to be due to the oxidation of two specific cysteine
residues located outside the TTF-1 homeodomain that
lead to the formation of higher-order oligomers [37].
Specifically, oxidation of Cys87 that resides in the Nterminal activation domain was implicated in the reduced
DNA-binding activity of the TTF-1 homeodomain [38].
It was suggested that oxidation of Cys87 interferes with
the correct folding of the homeodomain, resulting in its
decreased DNA-binding activity. Ref-1 (redox effector
factor-1) was shown to mediate the redox effects on the
TTF-1 homeodomain, thereby modulating TTF-1 transcriptional activity [38]. Redox regulation of TTF-1
DNA-binding activity could play important roles in the
control of target gene expression. In thyroid FRTL-5
cells, TSH (thyroid-stimulating hormone) up-regulates
thyroglobulin gene expression via increased binding of
Pax-8 and TTF-1 transcription factors to the thyroglobulin promoter [39]. Redox regulation by thioredoxin
was suggested to be responsible for the increased binding
of Pax-8 and TTF-1 to the thyroglobulin promoter in
TSH-treated FRTL-5 cells. TTF-1 DNA-binding activity
appears to be highly sensitive to oxidation, as shown by
its loss during the preparation of nuclear extracts in the
absence of reducing agents and the restoration of binding
activity by reducing agents ([37], and V. Boggaram,
unpublished work). TTF-1 is phosphorylated on seven
serine residues in thyroid FRTL-5 cells and in heterologous cells such as HeLa cells [40]. PKA (protein
kinase A) [14,41], PKC (protein kinase C) [40] and MST2
[mammalian Ste20 (sterile 20)-like protein kinase 2]
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TTF-1 PROMOTER REGULATION
IN THE LUNG
Figure 3 TTF-1 gene organization
A schematic diagram of the structure of human TTF-1 gene. Exons are represented
by rectangles and introns and 5 and 3 flanking regions are represented by lines.
Filled and open areas represent untranslated and translated regions of the exons
respectively. The arrows indicate the locations of transcription start sites. The
locations of translation initiation (ATG) and translation termination (TGA) codons
and of functionally important transcription-factor-binding sites are also shown.
The organization of the TTF-1 gene shown is based on the structure of the TTF-1
gene described by Hamdan et al. [33].
kinase [42] are able to phosphorylate TTF-1. Increased
TTF-1 phosphorylation by PKA activated gene transcription of SP-B [41] and SP-A [14] in H441 lung epithelial cells and primary alveolar type II cells respectively,
suggesting that alterations in TTF-1 phosphorylation
status can modulate target gene expression. Abnormalities in lung morphogenesis and decreased expression of
SPs in homozygous mice expressing a TTF-1 mutant
in which all of the seven serine phosphorylation sites
had been mutated underlines the importance of TTF-1
phosphorylation in the control of target gene expression
[43].
TTF-1 is encoded by a single-copy gene that is
located on chromosome 14 in humans and chromosome
12 in the mouse [5]. Initial studies concluded that rat
[44,45], mouse [31] and human [32,33] TTF-1 genes were
organized into two exons and one intron. Other studies
have determined that the rat TTF-1 gene is organized
into three exons and two introns [46] (Figure 3). Previous
studies on the cloning and sequencing of a large number
of TTF-1 cDNAs from a fetal human lung cDNA library
have concluded that the human TTF-1 gene, similar to
the rat TTF-1 gene, is also organized into three exons and
two introns [33]. The newly identified exon I contains
an ATG codon that falls in-frame with the initiator ATG
identified previously. In MLE-15 and H441 human lung
epithelial cells, the start site of transcription for the TTF-1
gene was mapped to −196 bp relative to the initiator ATG
codon [32]. In rat FRTL-5 thyroid cells and human lung
H441 cells, multiple T/EBP (TTF-1) transcripts having
variable lengths of 5 untranslated regions were identified
by cDNA cloning and sequencing, indicating the use
of multiple promoters and the occurrence of alternative
splicing [33,45,46]. Determination of transcription start
sites for TTF-1 transcription in thyroid and lung showed
multiple start sites of transcription in agreement with the
existence of multiple TTF-1 mRNAs [45,46].
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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
presence of cis-DNA elements necessary for lung-specific
expression [32,33,47]. The identification of a new exon I
in the TTF-1 gene and analysis of genomic regions indicated the presence of two promoter regions which support reporter gene expression in lung cells in vitro [33].
One lies within the first intron (proximal promoter) and
the other upstream of the first exon (distal promoter).
A TTF-1 genomic fragment that included both the
upstream and the intronic promoter regions expressed
the reporter gene at a significantly higher level than a
fragment containing the intronic promoter alone [32].
These findings suggest interactions between the two promoters. The proximal promoter does not contain a traditional TATA sequence, but instead contains a sequence
TAAAA that has some degree of similarity to the TATA
element, whereas the distal promoter lacks a TATA-like
sequence altogether. The TTF-1 proximal promoter
contains two closely located DNA elements that bind
HNF-3α [hepatocyte nuclear factor-3α; also known as
FOXA1 (forkhead box A1)] and HNF-3β (also known
as FOXA2) factors in MLE-15 lung cells and activate
promoter activity [32]. The distal promoter contains a
GC-rich sequence that binds Sp1 and Sp3 (specificity
protein 1 and 3 respectively) transcription factors that
regulate TTF-1 promoter activity [48]. The TTF-1
promoter is activated by co-expression of TTF-1 in
FRTL-5 thyroid cells and HepG2 cells, indicating that
the TTF-1 promoter is subject to positive autoregulation
[45,49]. The TTF-1-binding site(s) that controls positive
autoregulation of the TTF-1 promoter has not been identified or characterized. A DNA element that binds the
zinc finger protein GATA-6 was identified in the mouse
proximal TTF-1 promoter and found to be important for
promoter activity in MLE-15 lung epithelial cells [50].
However, mice deficient in GATA-6 expressed TTF-1
in bronchiolar epithelial cells, suggesting that GATA-6
may not be required for TTF-1 expression in bronchiolar
epithelial cells [51]. The homeobox protein HOX3B
binds to a highly conserved DNA element in the TTF-1
proximal promoter and activates promoter activity in cotransfection experiments in HeLa cells [52]. The significance of HOX3B regulation of TTF-1 promoter activity
in lung epithelial cells is not clear because HOX3B
expression appears to be restricted to the mesenchyme
in the developing lung [53]. The occurrence of multiple
functionally important binding sites in the TTF-1
promoter suggests that the promoter activity is controlled
via combinatorial or co-operative interactions between
Thyroid transcription factor-1 gene regulation in the lung
the transcription factors. Although TTF-1 5 flanking
genomic DNA fragments from the baboon that included
both the distal and proximal promoters or only the distal
promoter failed to direct LacZ reporter gene expression
in the lung or thyroid, they expressed the LacZ gene in the
trachea and subsets of cells localized in the hypothalamus
in transgenic mice [54]. These intriguing findings suggest
that additional TTF-1 regulatory sequences may be
required for lung- and thyroid-specific expression.
TTF-1 EXPRESSION IN LUNG DISEASES
TTF-1 is expressed at the onset of lung morphogenesis in
epithelial cells and throughout fetal lung development.
In the postnatal lung, TTF-1 expression is restricted primarily to alveolar type II cells and subsets of non-ciliated
bronchiolar epithelial cells. There is limited information
on the regulation of TTF-1 levels in lung diseases. TTF-1
immunostaining was reduced or absent in type II cells in
regions of acute inflammation, oedema, haemorrhage and
atelectasis in lungs of infants with HMD (hyaline membrane disease) and BPD (bronchopulmonary dysplasia)
[25]. In contrast, TTF-1 immunostaining was prominent
in cells in regions of the lung that were undergoing regeneration [25]. Partial deficiency or haplo-insufficiency of
TTF-1 in humans heterozygous for TTF-1 mutations is
associated with hypothyroidism, choreoathetosis, muscular hypotonia, respiratory dysfunction and recurrent
pulmonary infections [55–59]. TTF-1 mutations in these
patients were characterized as complete gene deletions,
missense mutations or nonsense mutations. Respiratory
dysfunction and recurrent pulmonary infections in these
patients could be the result of reduced expression of SPs
as a consequence of TTF-1 deficiency. TTF-1 expression
was significantly reduced in nitrofen-induced lung hypoplasia in rats, and glucocorticoid treatment prevented the
reduction in TTF-1 levels [60]. Similarly, nitrofen decreased TTF-1 promoter activity in H441 lung epithelial
cells, indicating that the decreased TTF-1 expression is
due to reduced gene transcription [60]. Other studies
have found that TTF-1 expression is unaltered [61] or
increased [62,63] in hypoplastic lungs of nitrofen-treated
rats. In contrast with the alterations in TTF-1 levels in
hypoplastic lungs of nitrofen-treated rats, no changes
in lung TTF-1 expression were observed in an ovine
model of surgically created congenital diaphragmatic
hernia [64] or in human fetuses with diaphragmatic hernia
[65]. These findings suggest that the alterations in TTF-1
levels in nitrofen-induced diaphragmatic hernia may be
the result of the effects of nitrofen on TTF-1 expression
and not due to diaphragmatic hernia itself.
TTF-1 AS A LINEAGE-SPECIFIC ONCOGENE
IN LUNG CANCER
Owing to its cell/tissue-restricted expression, TTF-1
has been used widely as a marker for the diagnosis of
primary and metastatic lung cancer, in particular for the
identification of the lung as the primary site of metastatic
adenocarcinoma [66–68]. TTF-1 is also useful in distinguishing malignant pleural mesotheliomas from adenocarcinomas [69]. SCLCs (small-cell lung carcinomas) also
have TTF-1 immunoreactivity. Although SCLCs display
a positive reaction for TTF-1, TTF-1 may not be a specific
marker for SCLCs, as small cell carcinomas from tissues
other than lung have positive reactions [70]. It has been
suggested that TTF-1 in combination with cytokeratin
20 would be very useful as a marker for distinguishing
SCLCs from small cell carcinomas of other primary sites
[70]. In primary NSCLC (non-SCLC), TTF-1 immunoreactivity was significantly correlated with adenocarcinomas, rather than SCCs (squamous cell carcinomas)
[71,72]. There appears to be considerable variation in
TTF-1 immunoreactivity in NSCLC. Although TTF-1
immunoreactivity is not frequently encountered in SCCs,
reverse transcriptase–PCR analysis of lung SCC cell
lines detected TTF-1 expression in three out of four
cell lines [73], indicating that the lack of or low TTF-1
immunoreactivity in SCCs is due to low TTF-1 expression.
Several studies have investigated whether TTF-1
expression can serve as a prognostic indicator for survival
in lung cancer patients [71,72,74–76]. There appears to
be a strong correlation between TTF-1 expression and
median survival rates in patients with lung cancer. Patients
with tumours having strong TTF-1 expression had
statistically significant better median survival rates than
those with tumours having no or weak expression (> 57.3
compared with 39.4 +
− 5.2 months respectively; P = 0.006
according to a long-rank test) [71]. Indeed a meta-analysis
concluded that TTF-1 expression is a good prognostic
factor for survival in NSCLC [77]. The reasons for the
close association between TTF-1 expression and survival
rates in patients with NSCLC are not well understood.
Although one study found no correlation between TTF-1
expression levels and tumour differentiation [75], another
study found that the TTF-1 expression level was positively associated with tumour differentiation in NSCLCs
[71].
There exists an intimate association between cell lineage and tumorigenesis, indicating common mechanisms
for cell lineage and tumour progression and survival. Indeed results from several studies have supported a lineagedependency model for tumorigenesis ([78], but see [78a]).
It has been suggested that aberrant functioning of lineagesurvival pathways might underlie carcinogenesis and
tumour progression. For example, lineage-dependency in
tumorigenesis might rely on the deregulated expression of
a master gene(s) that controls developmental and essential
functions. It has been suggested that transcription factors
and signalling proteins might fill the role of lineagesurvival oncogenes. The master transcriptional regulator
MITF (microphthalmia-associated transcription factor)
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that is involved in the differentiation and survival of
melanocytes is a case in point [79]. Deregulation of MITF
has been linked to metastatic melanoma.
The characteristics of TTF-1, namely its role in lung
development, cell-restricted expression in the lung and
high-level expression in lung adenocarcinomas, strongly
point to TTF-1 as a potential lineage-survival oncogene
in lung cancer [80]. Analysis of large numbers of lung tumours and lung cancer cell lines has revealed amplification
of the TTF-1 gene in lung adenocarcinoma. Although one
study reported amplification of the TTF-1 gene in 2 % of
the tumours analysed [80], other studies have reported
amplification rates of 12 % [81] and 11 % [82]. Interestingly the occurrence of higher TTF-1 gene amplifications
at metastatic sites than primary sites was observed [82].
Amplification of the TTF-1 gene in lung adenocarcinoma
cell lines is associated with increased expression of TTF1 [80,82]. Sequencing of the TTF-1 open reading frame
from four NSCLC cell lines exhibiting amplification did
not reveal any DNA mutations, indicating that TTF-1
levels would be of importance [82]. Reduction in endogenous TTF-1 levels by RNAi (RNA interference)mediated knockdown in a variety of lung cancer cell lines
with TTF-1 amplification decreased cell proliferation,
indicating the importance of elevated TTF-1 levels for the
growth of cells [80–82]. Lung cancer cell lines without
amplification of the TTF-1 gene but with detectable
expression of TTF-1 (H1155) [82] and those without detectable expression of TTF-1 (NCI-H23, A549) [80] were
not affected by RNAi-mediated knockdown of TTF-1,
underlining further the functional importance of elevated
TTF-1 levels for cell survival and proliferation. A
reduction in endogenous TTF-1 levels by RNAimediated knockdown resulted in decreased cell-cycle
progression and increased apoptosis [80–82]. These
findings have demonstrated that sustained expression of
TTF-1 is necessary for the growth and survival of a subset
lung adenocarcinoma and implicate TTF-1 as lineagespecific proto-oncogene for lung cancer. Apart from gene
amplification, TTF-1 levels in lung adenocarcinoma cell
lines and tumours can be up-regulated by transcriptional
and post-transcriptional mechanisms, and by the control
of protein stability. It remains to be determined whether
any of these mechanisms operate along with TTF-1 gene
amplification to elevate TTF-1 levels in lung adenocarcinomas. It is not known whether TTF-1 directly controls the survival of cancer cells or via another
downstream effector molecule(s), whose identity remains
to be determined.
REGULATION OF TTF-1 GENE EXPRESSION
IN THE LUNG
There is limited information on the regulation of TTF-1
expression in the lung. Intratracheal instillation of
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silicon carbide whiskers, an asbestos substitute, into rats
produced fibrotic changes and decreased the expression of
SP-A and TTF-1 [83]. Changes in SP-A and TTF-1 levels
were apparent as early as 3 days after the exposure and
reduced expression was maintained for up to 6 months
following the single exposure. In studies to assess the
impact of inhaled DEE (diesel engine emissions) on
the clearance of Pseudomonas aeruginosa infection in
mice, it was found that prior exposure of mice to DEE
significantly reduced the expression of TTF-1 as well
as of CCSP [84]. Exposure to DEE decreased lung
clearance of P. aeruginosa and exacerbated lung inflammation and injury. Pro-inflammatory agents such as
TNF-α (tumour necrosis factor-α) [85] and ceramide [86]
inhibited SP-B promoter activity in H441 lung cells by
reducing TTF-1 DNA-binding activity. The molecular
mechanisms responsible for the TNF-α and ceramide inhibition of TTF-1 DNA-binding activity are not known.
Glucocorticoid- and cAMP-dependent differentiation of
alveolar type II epithelial cells in vitro is associated
with the up-regulation of TTF-1 and SP expression
[87]. It is not known whether the inductive effects of
glucocorticoid and cAMP on TTF-1 levels are due to their
direct effects on TTF-1 gene expression or secondary to
their effects on the differentiation of alveolar type II cells.
Chronic exposure of primary rat alveolar epithelial cells
to a combination of TGF-β (transforming growth factorβ) and TNF-α resulted in decreased TTF-1 immunoreactivity with a concomitant increase in immunoreactivity for
α-SMA (α-smooth muscle actin) and other mesenchymal
markers, indicating induction of an EMT (epithelial–
mesenchymal transition) [88]. It was suggested that EMT
could be responsible for the accumulation of fibroblasts
and myofibroblasts in IPF (idiopathic pulmonary
fibrosis). Although TGF-β1 increases TTF-1 expression
in non-tumorigenic E10 mouse lung epithelial cells, it had
no effect on TTF-1 expression in tumorigenic E9 mouse
lung epithelial cells [89]. In E10 cells, TGF-β1 induction
of TTF-1 expression occurs via the TGF-βII receptor and
Smad signalling with the involvement of Sp1 and Sp3 transcription factors. Sp1 and Sp3 exert opposing effects on
the TTF-1 promoter; although Sp1 increases TTF-1 promoter activity, Sp3 negated the inductive effects of Sp1.
CONCLUSIONS
TTF-1 plays key roles in the control of cell/tissue-specific
gene expression and in the morphogenesis and differentiation of the thyroid, lung and brain. Recent studies have
suggested that TTF-1 plays the role of a proto-oncogene
in lung cancer. Considering the important roles that
TTF-1 plays, abnormal expression of TTF-1 probably
contributes to the pathogenesis of lung, thyroid and other
diseases. Molecular mechanisms that control cell/tissuespecific expression of TTF-1 and TTF-1 transcriptional
Thyroid transcription factor-1 gene regulation in the lung
activity are not fully understood. Likewise cellular pathways and agents that regulate TTF-1 expression remain to
be explored. Because TTF-1 is essential for the expression
of SP and other genes important for lung function, abnormal expression and/or activity of TTF-1 may underlie the
pathogenesis of a variety of lung inflammatory diseases.
The recent discovery of TTF-1 as a proto-oncogene for
lung cancer has opened new avenues of research that
might lead to novel therapies for lung cancer. A better
understanding of agents and pathways and molecular
mechanisms that control TTF-1 expression in the lung
will be useful for the development of novel treatment
strategies for lung diseases such as ARDS (acute respiratory distress syndrome), IPF, lung cancer and others.
ACKNOWLEDGMENTS
Research in my laboratory is supported by the National
Institutes of Health Grant HL-48048. I thank Dr Mark
A. L. Atkinson for reading the manuscript prior to
submission, and Dr Dorota Stankowska for preparing
Figure 1.
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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
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