(Received for publication, December 14, 1994; and in revised form, January 19, 1995)
From the
The human gene encoding thyroid transcription factor-1 (TTF-1), a homeodomain-containing nuclear transcription protein of the Nkx2 gene family, was isolated and characterized. Human TTF-1 was encoded by a single gene locus spanning approximately 3.3 kilobases and consisted of two exons and a single intron. The TTF-1 cDNA and polypeptide of 371 amino acids have been highly conserved, sharing 98% identity with the rat TTF-1 polypeptide. Human TTF-1 mRNA and polypeptide were selectively expressed in human and mouse pulmonary adenocarcinoma cell lines. In addition to its presence in thyroid gland epithelium, the human TTF-1 protein was detected by immunohistochemistry in human fetal lung as early as 11 weeks of gestation, being localized in the nuclei of epithelial cells of the developing airways. After birth, TTF-1 was selectively expressed in Type II epithelial cells in the alveoli and in subsets of bronchiolar epithelial cells in the conducting regions of the lung. The 5`-flanking region of the human TTF-1 gene directed transcription of luciferase cDNA in a lung epithelial cell-selective manner. The conservation and distribution of TTF-1 in the human respiratory tract support its role in the regulation of lung development and surfactant homeostasis.
Thyroid transcription factor-1 (TTF-1) ()is a 38-kDa
nuclear protein initially identified as a mediator of thyroid-specific
gene transcription(1, 2) . TTF-1 contains a highly
conserved homeobox domain and a 17-amino acid domain characterizing it
as a member of the Nkx2 family of homeodomain transcription
factors. TTF-1 activates thyroglobulin and thyroperoxidase gene
transcription in thyroid adenocarcinoma cells and is expressed in
epithelial cells of the developing and mature thyroid gland in the rat,
consistent with its role in thyroid epithelial cell gene
expression(2, 3, 4) . A potential role for
TTF-1 in lung epithelial cell gene expression was recently demonstrated
by the finding that TTF-1 activates the transcription of human
surfactant protein B gene(5) . TTF-1 binds to two closely
apposed TTF-1 DNA binding sites located -80 to -100 base
pairs from the start of SP-B gene transcription. TTF-1 is also
expressed in the embryonic forebrain and in the respiratory epithelium
of the rat, appearing in fetal lung buds of the embryonic rat lung on
postconceptional day 9 or 10(4) .
Like the thyroid gland, the lung develops as an outpouching of epithelial cells from the foregut endoderm. The fetal lung then undergoes branching morphogenesis and cellular differentiation to form the complex respiratory epithelium characteristic of the mature lung. Morphologic and biochemical criteria distinguish subsets of respiratory epithelial cells that line the proximal and distal respiratory tract in mammals. A number of proteins are selectively expressed by respiratory epithelial cells, including pulmonary surfactant proteins A, B, and C and the Clara cell secretory protein (CCSP) (for review, see (6) ). These cellular markers distinguish subsets of cells in the conducting and alveolar region of the lung in nonciliated respiratory epithelial and alveolar Type II epithelial cells. The surfactant proteins play important roles in surfactant homeostasis, enhancing the spreading of alveolar phospholipids and contributing to the regulation of the uptake and recycling of surfactant by Type II epithelial cells. The similarity of the temporal-spatial distribution of TTF-1 and surfactant protein B in the developing lung provides further support for the potential role of TTF-1 in the regulation of surfactant function of lung epithelial cell gene expression. In the work of Bohinski et al.(5) , the promoter elements for surfactant proteins B and C as well as the CCSP gene were transactivated by TTF-1 in vitro, suggesting that TTF-1 may also play a more general role in lung epithelial cell gene expression.
While the cDNAs encoding rat (1) and canine
TTF-1 ()have been isolated and characterized, there is less
information regarding the structure and function of the TTF-1 gene and
its distribution in the human lung. Because of the potential importance
of TTF-1 in the differentiation and function of the respiratory
epithelium, we have assessed the immunohistochemical localization of
this homeodomain protein in the human respiratory epithelium. The human
TTF-1 gene was isolated, and the promoter elements that contribute to
the regulation of expression of TTF-1 mRNA in lung cells were
identified.
Total RNA
(20 µg) was electrophoresed through a 1.0% agarose, 7% formaldehyde
gel, transferred to Hybond (Amersham) or Nytran (Schleicher &
Schuell), and bound to the filter by UV cross-linking. Filters were
hybridized overnight at 42 °C in 50% formaldehyde plus standard
sodium phosphate-EDTA solution as recommended, using P-random primer-labeled rat TTF-1 cDNA as probe. Filters
were washed to a final stringency of 0.2
saline/sodium/phosphate/EDTA, 0.1% SDS at 60 °C and exposed to
Kodak XAR-2 film.
Figure 2: Organization of the human TTF-1 gene and TTF-1 promoter-luciferase constructs. A, restriction map of the human TTF-1 gene showing position of exons (black), intron (hatched), translation start (ATG) and stop (TGA), and the homeodomain. B, representation of the luciferase constructs used in transfection assays.
Human NCI-H441-4 (H441) and mouse
MLE-15 cells were maintained as described
previously(5, 9) . NIH-3T3 cells (3T3) were maintained
in Dulbecco's modified Eagle's medium containing 10%
heat-inactivated bovine serum. Transfections were performed by the
calcium phosphate co-precipitation method as described by Rosenthal (10) except that glycerol shock was not used. Luciferase
reporter plasmid (5 pmol) and 2.5 pmol of the internal control plasmid,
pCMV-gal(11) , were co-transfected. Cells were incubated
for approximately 18 h, washed once with Hanks' balanced salt
solution (Life Technologies, Inc.), and returned to culture in original
media for an additional 24 h for MLE-15 cells, 72 h for H441 cells, and
48 h for 3T3 cells. Cells were harvested with reporter lysis buffer
(Promega) followed by a rapid single freeze-thaw cycle. The lysates
were prepared, and aliquots were assayed for
-galactosidase
activity (5) and for luciferase activity using a luminometer
(Analytical Luminescence Laboratory, San Diego, CA). To correct for
variations in transfection efficiency, assays were normalized to
-galactosidase activity.
Figure 1: Southern blot analysis of the human TTF-1 gene. 20 µg of DNA from the cosmid clone (A) or from H441-4 cells (B) was digested with BamHI (lane1), EcoRI (lane2), HindIII (lane3), or KpnI (lane4) and subjected to Southern analysis, using the rat TTF-1 cDNA as probe.
Figure 3: Nucleotide sequence and predicted amino acid sequence of human TTF-1 gene. The major start of transcription is marked (+1), and the polyadenylation signal (AATAAA) is underlined.
Figure 4:
Northern blot analysis of TTF-1 mRNA in
mouse lung and human and mouse pulmonary adenocarcinoma cells. A, Northern analysis of 20 µg of total RNA from MLE-15 (lane1), MLE F6 (lane 2), 3T3 (lane 3), and H441
cells (lane4). The probe used was the rat TTF-1
cDNA. B, Northern analysis of 15 µg of total RNA from
human cell lines HeLa (lane 1), H441 (lane2), H345 (lane3), H446 (lane4), BEAS-2B (lane5),
9/HTEo (lane6), and A549 (lane7). The probe used was a SacII-Sau3AI
fragment of rat TTF-1 cDNA, which did not contain any Nkx or homeobox
domain homology.
Figure 5:
Activity of TTF-1-luciferase constructs in
pulmonary adenocarcinoma cells and 3T3 fibroblasts. Luciferase assays
were performed after transfecting H441, MLE-15, and 3T3 cells using a
promoterless plasmid, pGL2-Basic, a plasmid containing 0.55 kb of human
TTF-1 promoter, and another containing 2.7 kb of human TTF-1 promoter.
The cells were co-transfected with a CMV--Gal construct as
described under ``Materials and Methods.'' Results are
plotted as units of luciferase activity per unit of
-galactosidase
and represent at least three separate experiments performed in
quadruplicate. Values are means ± S.E. (n = 3
separate experiments).
Figure 6:
Immunohistochemistry of TTF-1 in fetal,
newborn, and adult lung. Immunoperoxidase staining was utilized to
stain human lung samples from 12 weeks of gestation (A and B), 37 weeks of gestation (C and D), and
adult (E and F). PanelF represents
the control panel of adult lung without primary antibody. Nuclear
staining was detected in airway epithelial cells. Slides were
counterstained with hematoxylin (A, B, C,
and D) or nuclear fast red (E and F). A, B, and C, magnification is 530; D, E, and F, magnification is
425.
Human TTF-1 was detected in respiratory epithelial cells from as early as 11 weeks of gestation to adulthood, marking distinct subsets of nonciliated respiratory epithelial cells in the conducting airway and Type II epithelial cells within the alveoli postnatally. Transcriptional elements from the 5`-flanking region of the human TTF-1 gene provide, at least in part, for the cell-selective expression of TTF-1 in the respiratory epithelium. The temporal-spatial pattern of human TTF-1 polypeptide is consistent with its importance in the regulation of surfactant protein synthesis and perhaps with a more general role in the early differentiation of the foregut epithelium during fetal lung development. The structure and function of TTF-1 have been highly conserved during evolution, providing further support for its potential role in mammalian lung development and in surfactant homeostasis.
The polypeptide sequences of human TTF-1 have been
strongly conserved among mammalian species. TTF-1 is a member of the Nkx2 class of homeodomain-containing proteins, initially
isolated by screening of thyroid cDNA library with oligonucleotides
based on a partial amino acid sequence of the bovine TTF-1
protein(1) . TTF-1 contains a highly conserved homeodomain DNA
binding domain distinct from the Antenapedia class of transcriptional
proteins and is also distinguished by the presence of a highly
conserved 17-amino acid peptide domain characteristic of genes of the Nkx2 gene family(1, 16) . The predicted TTF-1
polypeptide shares 98-99% identity with that predicted from the
TTF-1 cDNA from canine and rat(1) , with
conservation of the homeodomain and the 17-amino acid Nkx2 domain. While the gene structures of other mammalian TTF-1 genes
have not been reported, the 5`-flanking region of the TTF-1 gene has
also been highly conserved between the rat (17) and the human
TTF-1 genes, supporting the likelihood of shared regulatory
characteristics of the TTF-1 genes.
The temporal-spatial pattern of TTF-1 expression in the developing human lung recapitulates the findings in the developing rat lung previously described by Lazzaro et al.(4) . In the rat, TTF-1 is expressed in the primordial thyroid epithelium between postconceptional days 10 and 11 and at a similar time in the primordial lung buds, being expressed at high levels in the distal tips of the developing airways. In the human fetal lung, the distribution of TTF-1 staining is similar to the pattern of SP-C mRNA in the distal respiratory epithelium(14) , a finding consistent with the observation that TTF-1 strongly activated lung cell-specific SP-C gene transcription(5) . However, SP-C was nearly completely excluded from the conducting airway epithelium in the more mature human (14) and rodent lung(18) , while TTF-1 was detected in both nonciliated respiratory epithelial cells in the conducting airways and in alveolar Type II cells. The distribution of TTF-1 in the conducting airway in human lung included regions of the respiratory epithelium expressing SP-A, SP-B, and CCSP genes. The promoters of each of these genes are transactivated by TTF-1 in vitro(5) . Thus TTF-1 expression includes distinct subsets of epithelial cells that express the lung epithelial cell-specific markers; however, the presence of TTF-1 alone is not sufficient to explain the heterogeneous distribution of these markers in the developing fetal and mature lung.
In the human, TTF-1 was expressed
at the highest levels in the tips of the fetal lung buds and in the
alveolar Type II epithelial cells in the postnatal lung. TTF-1 staining
was excluded from the ciliated respiratory epithelial cells and from
terminally differentiated Type I epithelial cells, neither of which
contain detectable levels of SP-A, SP-B, SP-C, or CCSP. It is therefore
not surprising that TTF-1 mRNA was detected in several differentiated
cell lines including mouse MLE-15 and human H441-4 adenocarcinoma
cells that express surfactant proteins. Surprisingly, TTF-1 mRNA was
also detected in a small cell carcinoma cell line H345 and was detected
in 12 of 12 human small cell carcinomas in clinical pathology
specimens, ()a tumor type that generally does not express
surfactant proteins. TTF-1 mRNA was not present in HeLa, 3T3
fibroblasts, or human pulmonary adenocarcinoma cell line A549 that does
not express surfactant proteins. In the present study, TTF-1 was also
detected in the human fetal thyroid epithelium by immunohistochemistry
(data not shown), consistent with previous findings in the rat thyroid
and in thyroid carcinoma cells wherein TTF-1 is known to activate
thyroperoxidase and thyroglobulin synthesis (1, 2, 4) . Surfactant proteins are not
detected in the thyroid gland or in thyroid carcinoma cells, suggesting
that in addition to TTF-1, other factors must further distinguish lung
and thyroid-specific gene transcription. Whether thyroid and lung cell
specificity is mediated by differences of the binding sites for TTF-1
or by the interactions of TTF-1 with other transcriptional proteins
(for example, PAX-8 in the thyroid) is unclear at present.
The start of transcription and the proximal 5`-flanking region of the TTF-1 gene were highly conserved in the rat and human. Lung-selective expression of luciferase reporter gene was conferred by the 5`-flanking regions in the TTF-1-luciferase constructs when transfected into cells in vitro. The 5`-flanking region of the human TTF-1 gene contains a TATA-like element, located approximately -30 base pairs from the start of transcription. The sequence at the start of transcription located at -196 base pairs from the ATG is similar to that recently reported for the TTF-1 gene in the rat(17) . The coding region of the human TTF-1 gene predicts a polypeptide of 371 amino acids (38.6 kDa). The size of the major human TTF-1 mRNA (approximately 2.3 kb) is similar to that of rat lung, mouse, and human pulmonary adenocarcinoma cells. The 5`-flanking region of the human and rat TTF-1 genes is also strongly conserved, sharing 98% identity in the region -142 to +103 base pairs relative to the start of transcription(17) . These findings are consistent with the shared temporal-spatial distribution of TTF-1 gene expression in both human and rat thyroid and lung. TTF-1 is also expressed in the rat diencephalon; however, in the present study we could not assess its distribution in the fetal human brain.
While the 5`-flanking region of the human TTF-1 gene was expressed at much higher levels in pulmonary adenocarcinoma cells, significant expression, albeit at lower levels, was also detected in 3T3 cells. The finding that elements between 2.7 kb and the start of transcription conferred higher level expression in lung cells than in 3T3 cells supports the presence of elements stimulating activity in lung cells in that region. Binding and activation of TTF-1 expression by Hox 2.7 was recently described by Guazzi et al.(17) . This region of the rat TTF-1 gene has been nearly completely conserved in the human gene. Hox 2.7 is expressed in the developing lung(7) ; however, its precise colocalization and potential role in TTF-1 expression have not been discerned. The 5`-region of the TTF-1 gene also contained elements consistent with SP-1 binding as well as potential sites for binding of TTF-1 to its own promoter. Whether the TTF-1 interacts with these elements to modulate its own expression is unclear. The finding that TTF-1-luciferase constructs were expressed at low levels in a non-lung cell supports the concept that critical lung-specific elements may not be included in the 2.7-kb TTF-1 promoter constructs or that cell-specific function of the TTF-1 gene is further influenced by the context of the sites that distinguish the endogenous TTF-1 gene locus but are lacking from the TTF-luciferase constructs tested.
In conclusion, the strong conservation of the TTF-1 gene and polypeptide among species, its temporal and spatial distribution in the developing respiratory epithelium, and the lung cell-selective expression of TTF-1-luciferase constructs support the important role of this nuclear transcription factor in lung differentiation and gene expression. The abundance of TTF-1 in alveolar Type II cells is consistent with its role in the modulation of surfactant protein gene expression and surfactant homeostasis that is critical to postnatal adaptation to air breathing.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U19816[GenBank].