Temporal-spatial expression and transcriptional regulation of alpha7 nicotinic acetylcholine receptor by thyroid transcription factor-1 and early growth response factor-1 during murine lung development.

Nicotinic acetylcholine receptors are ligand-gated ion channels formed by five homologous subunits that are involved in processes including signal transduction, proliferation, and apoptosis. The developmental role of these receptors, however, is unclear. In the present investigation, alpha(7) nicotinic acetylcholine receptor expression was assessed by immunohistochemistry in mouse lungs from embryonic day (E)13.5 to postnatal day (PN)20. Transcriptional mechanisms that regulate alpha(7) were assessed by the transfection of murine bronchiolar cells with a reporter containing 1.1 kb of the mouse alpha(7) promoter, TTF-1, and Egr-1. alpha(7) was initially detected at E13.5 in pulmonary mesenchymal cells and in the epithelium of the primitive tubules at E15.5. From E18.5 to PN1, alpha(7) was expressed in conducting airway and saccule epithelial cells. By PN10, expression was observed in the peripheral epithelium and on luminal membranes of bronchiolar epithelial cells in the proximal lung, a pattern that continued through PN20. From E15.5 to PN20, type II alveolar cells expressed both prosurfactant protein C and alpha(7). From E18.5 to PN20, Clara cells in the bronchiolar epithelium co-expressed Clara cell secretory protein and alpha(7). TTF-1 dose-dependently activated alpha(7) transcription in vitro by binding specific TTF-1 regulatory elements in the mouse alpha(7) promoter. Furthermore, alpha(7) was not detected in TTF-1-null mice and markedly increased in TTF-1-overexpressing mice. Conversely, Egr-1 inhibited alpha(7) expression. Temporal-spatial alpha(7) expression supports the concept that these receptors function during normal pulmonary morphogenesis. A model is also supported whereby alpha(7) is induced by the essential pulmonary transcription factor TTF-1 and suppressed by Egr-1 during pulmonary development.

Neuronal and non-neuronal nicotinic acetylcholine receptors (nAChRs) 2 are members of a family of ligand-gated ion channels, which also includes glycine, GABA A , and 5HT3 receptors (1). nAChRs are pentameric oligomers composed of five related subunits arranged around a central ion channel that allows ion flow (usually calcium or sodium) following ligand binding. Several receptor subunits have been identified and are classified as either agonist-binding (␣ 2 -␣ 9 ) or structural (␤ 2 -␤ 4 ) subunits (2,3).
One particular nAChR composed entirely of ␣ 7 subunits is specifically characterized as having ␣-bungarotoxin sensitivity. In addition to synaptically associated characteristics, such as high Ca 2ϩ permeability and rapid desensitization (3), ␣ 7 homomeric nAChRs have been implicated in several important biological activities, including neurite outgrowth (4 -7), cell growth (8), neural development and cell death (9 -11), presynaptic control of neurotransmitter release (12), and modulation of proliferative responses in lung tumor cells (13,14). The expression of nicotinic receptors in the developing lung of monkeys following nicotine exposure has recently been identified in cells of the developing alveoli, airway epithelium, submucous glands, airway-associated nerve fibers, airway and arterial smooth muscle cells, airway fibroblasts, and free alveolar macrophages (15), but animal studies aimed at understanding the roles of these receptors during normal pulmonary development have been limited. Given the numerous roles of ␣ 7 nAChRs and the multiple cell types in which they are expressed, it is clear that precise mechanisms function to control temporal and spatial patterns of ␣ 7 expression.
Thyroid transcription factor (TTF)-1 is a member of the homeodomain-containing Nkx2 transcription factor family. TTF-1 is expressed in the lung, thyroid, ventral forebrain, and the pituitary (16 -18). TTF-1 mRNA is detected in the mouse at E10 (19) and is located predominantly in the lung periphery during pulmonary development (17). TTF-1 activates the expression of genes critical to lung development and function such as surfactant proteins (SPs), Clara cell secretory protein (CCSP), essential growth factors, and those associated with host defense and vasculogenesis (19,20). Inactivation of TTF-1 causes tracheo-esophageal fistulae and impairment of pulmonary branching, causing severe lung hypoplasia (21). In concert with other transcription factors, TTF-1 binds TTF-1 regulatory elements (TREs) in the promoters of genes selectively expressed in respiratory epithelial cells to influence cell differentiation and gene expression during lung morphogenesis. Although studies show that ␣ 7 is detected in cells known to express TTF-1 (15), no regulatory mechanism has been proposed linking the two in the lung to date.
Early growth response gene 1 (Egr-1), also known as TIS-8, Krox24, Zif268, and NGFI-A (22), is a member of an emerging group of zinc finger transcription factors that modulate responsive genes by binding GC-rich regions of promoter sequences that customarily lack TATA boxes. As terminal differentiation proceeds, several cell types up-regulate Egr-1 expression (23). Members of this zinc finger-containing transcription factor family include Egr-1-4 and WT1 (Wilms tumor sup-pressor gene), and they are categorized as both inhibitory and stimulatory. Although Egr-1 can both transactivate and suppress targeted gene expression, it usually transcriptionally inhibits expression (24). The mouse ␣ 7 promoter contains three closely related GC-rich regions that have Egr-1 binding activity (25). Furthermore, mutation of a GC-rich sequence at 172 base pairs upstream of the rat ␣ 7 start site leads to an increase in promoter activity, indicating a potentially repressive element (26). Although Egr-1 potentially interacts with several transcription factors to regulate targeted genes, how it interacts in concert with such factors during lung development is unclear.
In this paper, we report that ␣ 7 nAChRs are expressed in subsets of pulmonary epithelial cells during stages of lung morphogenesis and that these receptors are up-regulated by TTF-1 and down-regulated by Egr-1. Furthermore, because TTF-1 and Egr-1 regulatory elements are conserved in the promoters of ␣ 7 nAChRs in several species, we report that these two factors may function in a common mechanism to temporally and spatially regulate the expression of ␣ 7 nAChRs in the lung.

MATERIALS AND METHODS
Antibodies-A mouse ␣ 7 monoclonal antibody (kindly gifted by Drs. Scott Rogers and Lorise Gahring) was generated at the University of Utah Health Sciences Center. This antibody recognizes the cytoplasmic domain (NH 2 -GFLRMKRPGEDKVRPAC-COOH) and has been demonstrated to interact with tissues embedded in paraffin (27). The ␣ 7 monoclonal antibody was used at a dilution of 1:100. A Clara cell-specific rabbit polyclonal antibody generated by the Pulmonary Biology Division at the Cincinnati Children's Hospital Medical Center (CCHMC) against recombinant mouse CCSP was used at a dilution of 1:500. A second rabbit polyclonal antibody to proSP-C was also generated by CCHMC against the first 35 residues of the amino terminus of the human type II epithelial cell-specific proSP-C and used at a dilution of 1:200. Specificity of the CCSP and proSP-C antibodies was determined using Western blotting analysis (not shown).
Immunohistochemistry-Immunohistochemical staining for ␣ 7 was performed using a "mouse-on-mouse" monoclonal antibody kit (Vector Laboratories, Burlingame, CA). Briefly, 5-m paraffin sections were deparaffinized and rehydrated. The sections were treated with 3% hydrogen peroxide in methanol for 15 min to quench endogenous peroxidase. Development in diaminobenzidine was followed by incubation in Tris-cobalt, which enhanced antigen localization, and by counterstaining with nuclear fast red. The sections were then dehydrated in a series of ethanols, washed in three changes of xylene, and mounted under coverslips with Permount. Control sections were incubated in blocking serum alone.
Fluorescent co-localization experiments included deparaffinization and rehydration followed by blocking in 2% normal donkey serum. Sections were then incubated in a combination of mouse anti-␣ 7 monoclonal antibody (1:10) and rabbit anti-CCSP (1:500) or mouse anti-␣ 7 FIGURE 1. Immunohistochemical staining reveals ␣ 7 nAChR expression in the lung during pulmonary morphogenesis. A, ␣ 7 was expressed in pulmonary mesenchyme (arrow) during the early pseudoglandular period of lung development (E13.5). B, during the late pseudoglandular period (E15.5), ␣ 7 was detected in both the pulmonary mesenchyme (arrowhead) and in the apical membranes of pulmonary epithelial cells of the primitive tubules (arrows). C, during the saccular stage of development (E18.5), ␣ 7 expression persisted in the apical regions of most (but not all) epithelial cells. Staining of the respiratory epithelium was also observed at PN1 (D, arrows), a period in which faint staining of the bronchiolar epithelium was initially detected (not shown in this image). E, as alveolarization proceeds, immunostaining of PN10 lung revealed continued ␣ 7 expression in the thinning respiratory epithelium (arrows) and in the proximal lung (inset). F, by PN20, distinct and punctate staining of ␣ 7 was observed in a subset of bronchiolar cells (arrows), although respiratory epithelial cell expression persisted (arrowhead). No staining was observed in sections without primary antibodies. Images are 100ϫ magnification. monoclonal antibody (1:10) and rabbit anti-proSP-C (1:200) for 1 h. After two rinses in phosphate-buffered saline, the sections were incubated with Alexa488-conjugated donkey anti-mouse IgG (1:200) for 30 min. After two more rinses in phosphate-buffered saline, the sections were incubated in Alexa568-conjugated donkey anti-rabbit IgG (1:200) for 30 min. After further rinsing, the sections were mounted with 4Ј,6diamidino-2-phenylindole-containing Vectashield (Vector Laboratories) and examined using an Olympus Fuoview TM laser-scanning confocal microscope.
RNA Isolation and Reverse Transcription (RT)-PCR Analysis-Total RNA was isolated from whole mouse lung using TRIzol reagent (Invitrogen) and from cells grown in culture by using the Absolutely RNA RT-PCR Miniprep kit (Stratagene, La Jolla, CA). 2-g aliquots of total RNA were treated with 1 l each of RNasin (Promega, Madison, WI) and DNase I (Invitrogen) at room temperature for 15 min. The DNase I was heat-denatured at 90°C for 10 min, and reverse transcription was performed with SuperScript TM II-RT (Invitrogen) according to the manufacturer's protocol. PCR was performed using 2-l aliquots of the generated cDNA using Taq polymerase (Roche Applied Science). Products were electrophoresed on a 1.5% agarose gel with appropriate molecular weight standards. Bands were quantified using NIH Image software available on the internet at rsb.info.nih.gov/nih-image. Gene expression was assessed in three replicate pools, and representative data  Co-localization of ␣ 7 with a mouse monoclonal antibody (green) and CCSP with a rabbit polyclonal antibody (red) were determined in PN10 mouse lungs (A-C). Co-localization of ␣ 7 (green) and proSP-C with a rabbit polyclonal antibody (red) were determined at PN1 (D-F) and PN20 (G-I). Co-localization of ␣ 7 and CCSP (C) or proSP-C (F, I) was observed in many (arrows) but not all cells in the bronchiolar or respiratory epithelium (arrowheads). Negative control sections not exposed to primary antibodies revealed only background fluorescence. Bars represent 50 m (A-F) and 20 m (G-I). Whole lung RNA was isolated, 1.0 g was reverse transcribed, and the resulting cDNA was subjected to PCR analysis. ␣ 7 nAChR mRNA was initially detected at E15.5, and its expression persisted through E18.5. ␣ 7 expression increased with elevated TTF-1 expression and decreasing Egr-1 expression. Depicted values represent band densities relative to GAPDH. TGG TAG GTG TTA TT-3Ј; and GAPDH forward, 5Ј-TGG GGC CAA AAG GGT CAT CAT CTC-3Ј and reverse, 5Ј-GCC GCC TGC TTC ACC ACC TTC TT-3Ј. PCR parameters included an initial heating at 95°C for 2 min. ␣ 7 was amplified by 35 cycles at 95°C for 30 s, 50°C for 30 s, and 72°C for 1 min. TTF-1 was amplified by 25 cycles at 94°C for 45 s, 60°C for 45 s, and 72°C for 2 min. Egr-1 was amplified by 30 cycles at 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min. GAPDH was amplified by 15 cycles at 94°C for 30 s, 62°C for 30 s, and 72°C for 1 min. All amplifications were followed by a 7-min extension at 72°C.
Plasmid Construction and Mutagenesis-Primers were designed to retrieve 1.1 kb of the mouse ␣ 7 promoter by high fidelity PCR using the expand long template PCR system (Roche Applied Science). Promoterbased studies that demonstrate sufficient transcriptional regulation (28) were reviewed and TTF-1 regulatory elements were identified to determine the length of the promoter (1.1 kb) used in these experiments. The amplified ␣ 7 promoter fragment was directionally cloned into the pGL3-basic luciferase reporter plasmid (Promega) and verified by sequencing analysis. Site-directed mutagenesis of potential TTF-1 and Egr-1 binding sites were performed using the reporter construct (pGL3-1.1␣ 7 ) and the QuikChange TM site-directed mutagenesis kit (Stratagene). Briefly, synthetic oligonucleotides containing the desired mutation for TTF-1 (CAAG3 CCCC) and Egr-1 (GCGGGGGCG3 G-CGAAAGCG) were extended during a PCR reaction. The products were digested with DpnI to remove the wild-type DNA. The nicked vector DNA was then transformed into XL1-blue supercompetent cells and repaired. All constructs were verified by nucleotide sequencing.
Transfection and Reporter Gene Assays-Functional assays of reporter gene constructs were performed by transient transfection of Beas2B cells using FuGENE 6 reagent (Roche Applied Science). Beas2B is a transformed human bronchiolar epithelial cell line. Cells in 35-mm dishes at 40 -50% confluence were transfected with three plasmids at the following concentrations: 500 ng of pRSV-␤-gal, 100 ng of pGL3-1.1␣ 7 , 100 -500 ng of pCMV-TTF-1 and/or pCMV-Egr-1, and pCDNA control vector to bring the total DNA concentration to 1.1 g. The cells were allowed to grow to confluence (48 h) and were washed with cold phosphate-buffered saline, lysed, and snap frozen for several hours. The plates were scraped and centrifuged, and the cleared supernatant was used for both ␤-gal and luciferase assays. ␤-Gal assays were performed as previously described (20). Reporter assays were normalized for transfection efficiency based on the ␤-gal activity. Luciferase activity was determined in 10 l of extract at room temperature with 100 l of luciferase reagent (Promega) for 10 s after a 2-s delay in a MicroLumat LB 96P luminometer.
In Vitro Assessment of ␣ 7 Inducibility-Expression of ␣ 7 was assessed in a pulmonary adenocarcinoma cell line (A-549) before and after transfection with either pCMV-TTF-1 or pCMV-Egr-1. A-549 cells were selected because they endogenously express ␣ 7 . As outlined previously, FuGENE 6 was used to transfect A-549 cells with a total of 1.0 g of DNA that included the expression vectors (100 or 500 ng), 500 ng of Rous sarcoma virus-␤-gal, and empty vector as needed.
In Vivo Assessment of ␣ 7 Inducibility-Using the immunohistochemical techniques previously described, ␣ 7 expression was assessed in E16.5 and E18.5 lungs from mice with genetically altered TTF-1 activity. Dr. Jeffrey Whitsett at the Cincinnati Children's Hospital Medical Center generously gifted TTF-1-null mice and SP-C-mediated lung-specific TTF-1-overexpressing mice. Both transgenic mouse lines were bred and maintained as previously described (21,30).
Statistical Analysis-Results are presented as the means Ϯ S.D. of six replicate pools/treatment group. Means were compared using the Student's t test, with values of p Յ 0.05 considered to be statistically significant.

RESULTS
␣ 7 nAChR Expression during Lung Development-The distribution of ␣ 7 was assessed in the mouse lung by immunohistochemistry from E13.5 to PN20. At E13.5, ␣ 7 was predominantly observed in the pulmonary mesenchyme but only faintly detected in the epithelium of the primitive tubules (Fig. 1A). Although mesenchymal staining diminished at E15.5, ␣ 7 expression in the epithelium increased and was restricted to luminal cell surfaces (Fig. 1B). ␣ 7 expression persisted in the peripheral epithelial cells perinatally. Although frequent staining was observed in the respiratory epithelial cells at E18.5 (Fig. 1C) and PN1 (Fig. 1D), ␣ 7 expression may not have reached a threshold required for detection in all respiratory epithelial cells. ␣ 7 expression was also initially observed in the proximal bronchiolar epithelial cells at PN1 (not shown). During the period of alveologenesis (PN5-30), luminal surfaces of thinning respiratory epithelial cells stained positively for ␣ 7 (Fig. 1, E and F). Detection of ␣ 7 at PN10 (Fig. 1E) and PN20 (Fig. 1F) was also observed in distinct subsets of bronchiolar epithelial cells in the proximal lung. No staining was observed in sections stained without primary antibodies.
To identify the types of epithelial cells expressing ␣ 7, dual immunofluorescent labeling was performed at various time points (Fig. 2). ␣ 7 staining was observed in the non-ciliated Clara cells in the conducting airways during alveologenesis (Fig. 2A). These cells slightly protrude into the luminal space and stain for CCSP, a Clara cell-specific marker (Fig. 2B). When the images were merged, ␣ 7 co-localized with many CCSP-expressing Clara cells, but not all (Fig. 2C). ␣ 7 also co-localized with proSP-C in type II respiratory epithelial cells in the lung periphery (Fig. 2). Discriminate staining of ␣ 7 was observed throughout the distal lung from PN1 to PN20 (Fig. 2, D and G). ␣ 7 expression patterns correlated well with proSP-C immunolocalization (Fig. 2, E and H). Merged images reveal that ␣ 7 is expressed in many type II respiratory epithelial . TTF-1 activates and Egr-1 represses ␣ 7 promoter activity. A, genomic DNA of mouse ␣ 7 promoter (Ϫ1100 to ϩ50). Squares represent putative TTF-1 regulatory elements (TREs) and rectangles represent Egr-1 regulatory elements (EREs). The promoter sequence was ligated into a luciferase-reporting vector, pGL3 (pGL3-1.1␣ 7 ). Murine bronchiolar epithelial cells (Beas2B) were transfected with 500 ng of pRVS-␤-gal, 100 ng of pGL3-1.1␣ 7 , and a total of 500 ng of pCMV-TTF-1, pCMV-Egr-1, and/or empty vector. A total of 1.1 g of DNA was transfected. Luciferase assays were performed in triplicate 2 days after transfection. Significant differences in luciferase levels from reporter alone are noted at p Յ 0.05 (*) and p Յ 0.01 (**). B, co-transfection of pGL3-1.1␣ 7 with 100 -500 ng of pCMV-TTF-1 expression vector activated the transcription of luciferase in a dose-dependent manner. C, co-transfection with at least 100 ng of pCMV-Egr-1 significantly inhibited promoter activation. D, although the ␣ 7 promoter was activated by TTF-1 and inhibited by Egr-1, an equal quantity of Egr-1 was sufficient to eliminate the stimulatory effects of TTF-1.
cells, but the co-localization is not complete (Fig. 2, F and I). Only background fluorescence was observed in negative control sections not exposed to primary antibodies. ␣ 7 RNA Expression Patterns-␣ 7 mRNA expression was assessed by RT-PCR of whole lung RNA from E12.5 to E18.5 (Fig. 3). It is widely accepted that nAChRs are not robustly expressed on the membranes of specific cells; therefore, nAChR detection at both the protein and mes-sage levels has been enigmatic. Nevertheless, ␣ 7 mRNA was initially detected by RT-PCR at E15.5, and its expression persisted until just before birth (E18.5) (Fig. 3). Detection of ␣ 7 mRNA correlated with increased ␣ 7 immunostaining in the pulmonary epithelium. Interestingly, RT-PCR demonstrated that the onset of ␣ 7 mRNA expression in the lung is consistent with increased pulmonary TTF-1 expression. Furthermore, the detection of ␣ 7 correlated with a decrease in the transcriptional inhibitor Egr-1 (Fig. 3).
To identify potential TREs and EREs that function to regulate ␣ 7 expression, site-directed mutagenesis was performed to alter putative TTF-1 and Egr-1 binding sites (Fig. 5). Although transactivation by FIGURE 5. TTF-1 and Egr-1 sensitivity in mouse ␣ 7 promoter constructs. A, a schematic representing the DNA sequence of the mouse ␣ 7 promoter (Ϫ1100 to ϩ50) in which 5 TREs (CAAG) and 3 EREs (GCGGGGGCG) are located. Constructs were generated by ligating the promoter sequence into the pGL3-basic vector (pGL3-1.1␣ 7 ). ␣ 7 promoter activity was assessed during transfection experiments conducted in the bronchiolar epithelial cell line Beas2B, as shown in B-D. B, the ␣ 7 promoter was activated by TTF-1 and inhibited by Egr-1. Equal quantities of Egr-1 eliminated the stimulatory effects of TTF-1. C, mutagenesis of TREs significantly reduced luciferase transcription. The two proximal TREs were demonstrated to be the most important sites in TTF-1-mediated ␣ 7 activation. D, although Egr-1 interaction with any of the three EREs inhibited luciferase transcription, the two proximal EREs had the most inhibitory effects on the ␣ 7 promoter. Luciferase assays were performed in triplicate 2 days after transfection. Significant differences in luciferase levels from reporter alone are noted at p Յ 0.05 (*) and p Յ 0.01 (**).

FIGURE 6. ␣ 7 expression is induced by TTF-1 and eliminated by Egr-1 in A-549 cells, a pulmonary adenocarcinoma cell line.
A-549 cells were grown to ϳ40% confluence and transfected with either TTF-1 or Egr-1. Two days after transfection, whole RNA was isolated, reverse transcribed, and the resulting cDNA was subjected to PCR. By RT-PCR, A-549 cells endogenously express ␣ 7. ␣ 7 expression was markedly up-regulated in A-549 cells with 100 and 500 ng of TTF-1. ␣ 7 expression was reduced to below basal levels when transfected with 100 ng of Egr-1, and ␣ 7 was eliminated when 500 ng of Egr-1 was transfected. Depicted values represent band densities relative to GAPDH.
TTF-1 was inhibited by mutating each of the five TREs in the ␣ 7 promoter (Fig. 5C), the two proximal TREs were shown to be most significant in TTF-1-mediated activation. Stepwise mutation of the three EREs revealed that each functions to inhibit ␣ 7 transcription (Fig. 5D); however, mutation of the two proximal EREs completely prevented Egr-1-mediated suppression of ␣ 7 transcription.
TTF-1-mediated Effects on ␣ 7 Expression in Vivo-As previously described, ␣ 7 is detected in pulmonary epithelial cells at E18.5 (Figs. 1  and 7B). TTF-1-null mice die at birth because of impaired branching morphogenesis and severe lung hypoplasia. Similar to wild-type control sections that were not exposed to primary antibodies (Fig. 7A), ␣ 7 was not detected in pulmonary epithelial cells of TTF-1-null mice at E18.5 (Fig. 7C,  arrow). Conversely, transgenic mice with increased TTF-1 expression in SP-C-secreting type II alveolar cells revealed a marked increase in ␣ 7 staining (Fig. 7D). These disparate levels of ␣ 7 expression were also observed in the lungs of these genetically modified mice at E16.5 (Fig. 7, E and F).

DISCUSSION
The temporal-spatial distribution of ␣ 7, a highly expressed member of the nicotinic acetylcholine receptor subunit family, was determined during embryonic and postnatal development of the mouse lung. ␣ 7 protein was detected in subsets of epithelial cells in conducting and peripheral air spaces, with the sites and intensity of expression varying during development. It is likely that immunohistochemical detection is achieved after a threshold is met; an event possibly accomplished by the detection of numerous receptors in close proximity. ␣ 7 was primarily expressed in respiratory epithelial cells during the embryonic, pseudoglandular, cannalicular, and saccular stages of lung development. In addition to expression in the peripheral lung, ␣ 7 was also detected perinatally in distinct populations of bronchiolar epithelial cells. Conducting airway epithelial cell expression persisted throughout the period of remodeling associated with alveologenesis. In our studies, immunolabeling of ␣ 7 in the fetal lung was observed primarily on luminal epithelial cell membranes. Expression on the luminal membrane suggests that: 1) ␣ 7 may be localized on apical cell surfaces to accumulate the protein for receptor assembly and functionality in the postnatal lung or 2) ␣ 7 subunits may combine in utero to form functional nAChRs that bind ligand in the amniotic fluid and signal events that are essential during development. Several groups have shown that nAChRs are expressed in airway bronchiolar epithelium and that they form functional receptors, as demonstrated by electrophysiological analysis (31)(32)(33). Co-localization of ␣ 7 with CCSP, proSP-C, and sites of TTF-1 expression suggests that ␣ 7 is regulated by TTF-1 and, therefore, may play a role in the mediation of paracrine signaling between respiratory epithelial cells during pulmonary morphogenesis.
Supporting the notion that ␣ 7 forms functional receptors in utero is the concept that acetylcholine (ACh) is not only a neurotransmitter but also a local signaling molecule synthesized by many non-neuronal cells (34). In order for ACh to function as a hormone signal in the lung, ACh must be synthesized, secreted, and released locally. Proskocil et al. (34) show that choline is incorporated into pulmonary bronchiolar cells by a choline high affinity transporter, synthesized into ACh by choline acetyl transferase, and packaged into transport vesicles by a vesicular ACh transporter (34). Prior to choline acetyl transferase-mediated acetylation, however, choline has also been demonstrated to be an agonist for ␣ 7 nAChR (35) and therefore may be an additional ligand for ␣ 7 receptors. This is significant in the lung because choline can be derived from the degradation and recycling of lungspecific surfactant proteins and the recycling of various membranes (36). Like choline, locally synthesized and secreted ACh may act in a paracrine manner by binding ␣ 7 homomeric receptors. Following ligand binding, receptor depolarization may subsequently induce signaling events in utero that are required for normal lung organogenesis.
TTF-1 regulated ␣ 7 expression in Beas2B cells in vitro. Furthermore, ␣ 7 expression was ablated in TTF-1-null mouse lung and markedly increased in the lungs of transgenic mice that overexpress TTF-1.
Although TTF-1 directly induced ␣ 7 expression in pulmonary cells, the temporal-spatial expression patterns of TTF-1 and ␣ 7 during lung development were not identical. At E13.5, ␣ 7 protein was expressed in both the epithelium and mesenchyme, whereas TTF-1 is an epitheliumspecific transcription factor. Therefore, the expression of ␣ 7 is likely regulated by the activity of a combination of transcription factors. Deletion constructs and site-directed mutagenesis of putative proximal TTF-1 binding sites demonstrated the importance of TREs in the transcription of ␣ 7 . Because TTF-1 regulates target gene expression in concert with other regulatory factors, including NF-1, RAR, HNF-3, GATA-6, and AP-1, it is likely that the temporal-spatial distribution of ␣ 7 expression is influenced in a complex manner by other transcription factors. One such factor is Egr-1. Egr-1 ablated the stimulatory effects of TTF-1 in bronchiolar epithelial cells. Interestingly, TREs were observed in the promoters of ␣ 7 in several species (Fig. 8), and although less conserved, all screened mammals contained at least two EREs in the ␣ 7 promoter. Because binding sites for both factors are conserved and the opposing effects of TTF-1 and Egr-1 were demonstrated to directly influence ␣ 7 expression (Fig. 6), it is likely that TTF-1 and Egr-1 function in a complex mechanism that governs specific temporal and spatial patterns of ␣ 7 expression.
Nicotinic cholinergic signaling via ␣ 7 nAChRs in airway epithelial cells is likely affected by nicotine. Plasma nicotine levels in smokers peak during the day at 200 nM and drop to ϳ10 nM during periods of sleep (39,40). Epithelial cells directly exposed to smoke may experience nicotine levels that are 5-10-fold greater (40). Exposure to cigarette smoke during pregnancy adversely affects lung development as manifested by altered pulmonary function (41), increased respiratory illness (42), significantly reduced branching morphogenesis (43), and permanent airway obstruction in the proximal lung (44). Nicotine crosses the placenta and directly affects lung development in utero via an interaction with nAChRs in the developing and postnatal lung. Our studies demonstrated that these receptors are expressed in populations of epithelial cells in both the proximal and distal lung.
Prenatal nicotine exposure has been shown to dramatically increase ␣ 7 nAChR expression (21). Nicotine-induced up-regulation of ␣ 7 suggests that nicotine binds ␣ 7 nAChRs expressed in the fetal lung. Studies have shown that high levels of nicotine may activate (45) or inhibit (46) ␣ 7 nAChR expression in cells where airway nicotine is concentrated. Chronic nicotine exposure both up-regulates and permanently inhibits the function of many nAChRs (29,32); therefore, exposure to nicotine in utero may lead to high levels of inactivated ␣ 7 receptors. Studies aimed at clarifying the effects of nicotine on ␣ 7 expression and function during development are currently being conducted.
In summary, ␣ 7 nAChRs are expressed in specific epithelial cell types in the lung during development. ␣ 7 expression is regulated by several factors including TTF-1 and Egr-1, but expression patterns may be altered by nicotine exposure in the developing lung. Nicotine may directly influence normal cholinergic signaling by increasing ␣ 7 nAChR expression and indirectly affect signaling mechanisms by inactivating these receptors.