AJP - Lung AJP citation statistics
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 287: L752-L763, 2004. First published June 11, 2004; doi:10.1152/ajplung.00026.2004
1040-0605/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/4/L752    most recent
00026.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Driskell, R. R.
Right arrow Articles by Engelhardt, J. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Driskell, R. R.
Right arrow Articles by Engelhardt, J. F.

Wnt-responsive element controls Lef-1 promoter expression during submucosal gland morphogenesis

Ryan R. Driskell,1 Xiaoming Liu,1 Meihui Luo,1 Mohammed Filali,1 Weihong Zhou,1 Duane Abbott,1 Ningli Cheng,1 Chris Moothart,1 Curt D. Sigmund,2,3 and John F. Engelhardt1,2,3

1Departments of Anatomy and Cell Biology and 2Internal Medicine, and 3Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, University of Iowa College of Medicine, Iowa City, Iowa 52242

Submitted 29 January 2004 ; accepted in final form 4 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Regulated expression of lymphoid enhancer factor 1 (Lef-1) plays an obligatory role in the transcriptional control of epithelial bud formation during airway submucosal gland and mammary gland development. However, regions of the Lef-1 promoter required for spatial and temporal regulation during glandular development have yet to be defined. We hypothesized that a previously reported 110-bp Wnt-responsive element (WRE) in the Lef-1 promoter, which can be induced by Wnt-3a/{beta}-catenin signals, may also play a role in regulating Lef-1 expression during airway and mammary gland development. Here we show that the Lef-1 promoter is also responsive to Wnt-1 signals in both airway and mammary epithelial cell lines. To better understand the importance of the WRE in dynamically regulating Lef-1 promoter activation in these two types of epithelia in vivo, we utilized LacZ reporter transgenic mice to evaluate the significance of Wnt-responsive sequences in the Lef-1 promoter during glandular bud formation. A 2.5-kb Lef-1 promoter fragment partially reproduced endogenous Lef-1 expression patterns in a subset of cell types involved in both mammary gland and submucosal glandular bud development. Interestingly, removal of the 110-bp WRE from the Lef-1 promoter ablated expression in nasal and tracheal submucosal glandular buds while having no significant effect on developmental expression in mammary glandular buds. These findings suggest that Wnt regulation of the Lef-1 promoter at the WRE may play an important role during airway submucosal glandular bud formation.

development; transgenic mice; gene regulation; airway; lymphoid enhancer factor 1

SUBMUCOSAL GLANDS IN THE LUNG play an important role in many hypersecretory diseases, such as asthma, chronic bronchitis, and cystic fibrosis (12, 19). In this context, submucosal gland hypertrophy and hyperplasia lead to excessive mucous secretion into the airways. Furthermore, in cystic fibrosis, submucosal glands may play an important role in innate immunity defects in the lung that result from defective cystic fibrosis transmembrane conductance regulatory expression in the serous tubule of submucosal glands (10, 33). Recently, submucosal glands have also been implicated as a potential stem cell niche in the proximal airway (5). Despite the potential importance of submucosal glands in airway disease and stem cell biology, relatively little is known about the processes that control submucosal gland development and growth in vivo.

In the mouse, airway submucosal glands exist in the nasal mucosa and proximal trachea. Whereas nasal submucosal glands develop after embryonic day 15 (E15), tracheal gland development initiates during the first postnatal weeks of life. Although relatively little is known about the factors that regulate the initial stages of submucosal gland development in the airway, the known developmental biology of other bud-forming organs has presented some recurring themes. The development of bud-forming organs is controlled by highly regulated interactions between bud-forming epithelia and the underlying mesenchymal layers that stimulate signal transduction cascades and the transcription of genes important to epithelial cell movement and differentiation. The Wnt/{beta}-catenin/lymphoid enhancer factor 1 (Lef-1) signaling pathway is one of the most extensively studied transcriptional cascades involved in various types of organogenesis (8, 14–16, 37). In this context, Wnt proteins bind to transmembrane Frizzled receptors and activate signaling cascades by stabilizing {beta}-catenin and its interactions with transcriptional factors of the T-cell factor (TCF)/Lef-1 family. After translocation to the nucleus, {beta}-catenin/Lef-1 and {beta}-catenin/TCF complexes modulate the transcription of target genes through the assembly of multiprotein complexes (3, 6, 18, 25).

The TCF/LEF family is composed of Lef-1, Tcf-1, Tcf-3, and Tcf-4. Studies using Lef-1 knockout models have demonstrated that expression of the Lef-1 gene is required for a wide range of inductive epithelial/mesenchymal interactions involved in mammary gland, tooth, vibrissa, hair, and airway submucosal gland development (8, 31). The importance of Lef-1 in glandular development is seen in Lef-1-deficient mice, which lack mammary glands and airway submucosal glands. In the context of airway submucosal gland development, Lef-1 mRNA expression is induced within the epithelial bud at the earliest stages of glandular development (7) and is functionally required for bud formation (8). Because Lef-1 is thus far the earliest factor known to be induced during submucosal glandular bud formation, knowledge of its transcriptional regulation could provide significant insight into the biology of gland development.

Several studies have begun to address the signals responsible for regulating Lef-1 gene expression in cell lines. For example, in vitro studies from our laboratory, focusing on the human Lef-1 promoter, have demonstrated transcriptional responsiveness to Wnt-3a and {beta}-catenin (11). In this case, dissection of the upstream region of the endogenous Lef-1 gene in HEK-293 kidney cells defined a minimal promoter fragment from –2700 to –200 bp relative to the ATG start codon (as +1 bp). This region of the promoter contains four transcriptional start sites, TSS1 (–1329 bp), TSS2 (–1195 bp), TSS3 (–499 bp), and TSS4 (–376 bp), two of which are induced by Wnt-3a (TSS1 and TSS3; Fig. 1A). In addition, these studies also identified a 110-bp promoter segment that is required for responsiveness to Wnt-3a/{beta}-catenin. When this Wnt-responsive element (WRE) was deleted from the Lef-1 promoter, basal levels of transcription were significantly increased, and Wnt responsiveness was lost. Furthermore, this WRE was able to convey Wnt-3a/{beta}-catenin responsiveness to a heterologous minimal promoter in a context-independent fashion, suggesting it is an enhancer capable of responding to Wnt signals (11). Other groups have also demonstrated that isolated segments of the Lef-1 promoter are responsive to specific TCF isoforms in a {beta}-catenin-dependent manner (1). Because there are no consensus TCF binding sites within the WRE, these findings suggest that unknown transcription factors may bind at the WRE and coordinate Wnt signals with upstream TCF elements. Given the apparent complexities of Lef-1 promoter regulation by the Wnt/{beta}-catenin/TCF cascade and its diverse roles in the developmental processes of multiple organ systems, in vivo studies of the Lef-1 promoter are required to more clearly dissect its regulatory components.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 1. Schematic representation of lymphoid enhancer factor 1 (Lef-1) promoter/reporter constructs used for cell line and transgenic analyses. A: genomic fragment of the human Lef-1 gene containing its first 2 exons (black), ATG start codon at +1 bp, 4 transcriptional start sites (TSS), a Wnt-responsive element (WRE) localized at –876 to –769 bp, and a T-cell factor (TCF) binding site at –921 bp (11). B: genomic fragment contains a previously identified promoter segment (–2,700 to –200 bp) that was used to generate the LF–2700/–200-LacZ reporter construct containing 2.5 kb of the promoter. C: WRE-deleted promoter/reporter construct [LF–2700(ID)/–200-LacZ] contains an internal 110-bp deletion but is otherwise identical to LF–2700/–200-LacZ.

 
To better understand the potential role of Wnt signaling in controlling the transcriptional activation of the Lef-1 gene during epithelial glandular bud formation, we compared regulation of the Lef-1 promoter between two organs (mammary glands and airway submucosal glands) that share a requirement for Lef-1 expression during glandular bud formation (8, 31). We first evaluated the importance of the WRE in regulating Wnt-1 induction of the Lef-1 promoter in mammary and lung epithelial cell lines. Wnt-1 was chosen for in vitro induction studies since it has been shown to induce mammary gland hyperplasia in mice and expansion of stem cell pools in this organ (22). In addition, we analyzed in vivo expression patterns of the intact and WRE-deleted Lef-1 promoter in transgenic mice. Results from these studies demonstrated that the WRE most dramatically influenced Wnt-1 responsiveness of the Lef-1 promoter in a subset of less-invasive tumor cell lines. Similarly, the WRE also appeared to influence Lef-1 promoter expression in a cell type-dependent fashion during mammary gland and airway submucosal gland development in vivo. Transgenic mice harboring a full-length, but not a WRE-deleted, 2.5-kb Lef-1 promoter fragment driving expression of a {beta}-galactosidase reporter gene demonstrated expression in a subset of nasal and tracheal submucosal glandular bud epithelial cells during the initial stages of gland development. In contrast, the WRE was not required for temporal regulation of the Lef-1 promoter in epithelial buds and mesenchymal cells of developing mammary glands. These studies have begun to shed light on the transcriptional control of the Lef-1 promoter during gland development and the importance of Wnt-responsive sequences in these processes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In vitro analysis of the Lef-1 promoter. Two mammary epithelial adenocarcinoma cell lines [MCF-7 and MDA-MB-231, obtained from American Type Culture Collection (ATCC)] and two transformed human airway epithelial cell lines (IB3 and A549; IB3 cells were a gift from William Guggino, Johns Hopkins Univ., and A549 cells were obtained from ATCC) were transfected using calcium phosphate/DNA precipitates according to standard protocols. Two previously described expression constructs [LF–2700/–200-LacZ and LF–2700(ID)/–200-LacZ] containing the human Lef-1 promoter upstream of the {beta}-galactosidase reporter gene were used to study the Wnt-1 responsiveness of the Lef-1 promoter (11). LF–2700/–200-LacZ contains sequences in the promoter that span from –2,700 to –200 bp (relative to the ATG start codon at +1). LF–2700(ID)/–200-LacZ has an internal deletion of 110 bp, which was shown to be responsive to Wnt signals (11). The culture conditions for all cell lines used DMEM with 10% FBS and 1% penicillin/streptomycin (GIBCO). Cells were grown to 50–70% confluence on 60-mm dishes before cotransfection with 3.5 µg of a given Lef-1 promoter/LacZ reporter construct and 0.5 µg of pGL3 plasmid, which encoded the luciferase gene under the control of the simian virus 40 promoter. Cells were harvested for {beta}-galactosidase and luciferase assays at 48 h posttransfection. Harvesting of cells was performed by washing in PBS, followed by lysis in 1x reporter buffer (Luciferase Assay System kit, Promega). Protein concentrations were determined by the Bradford method, and all lysates were normalized to the same protein concentration. Lysate (25 µg) was used for luciferase assays (Invitrogen), and 50 µg of lysate were used for {beta}-galactosidase assays using O-nitrophenyl-{beta}-D-galactopyranoside as a substrate (Sigma Chemical). Normalization of transfection efficiencies for each experimental point was performed by dividing the {beta}-galactosidase activity units by the relative light units of luciferase activity. The resulting value was then used to compare the expression level of a given construct to the promoterless LacZ plasmid backbone for the same experiment. For Wnt-1 induction assays, 5 µg of expression plasmid encoding Wnt-1 was transfected at the same time with the Lef-1 promoter-reporter and luciferase constructs, as described above. The total amount of DNA transfected in all experimental comparisons was always normalized by the inclusion of an empty vector plasmid control (pcDNA). Western blotting for Lef-1 protein was performed using an anti-Lef-1 antibody that recognizes the NH2 terminus of Lef-1 (Exalpha, Boston, MA).

Lef-1 promoter-reporter constructs and the generation of transgenic mice. Transgene-containing fragments from LF–2700/–200-LacZ and LF–2700(ID)/–200-LacZ, which lacked plasmid backbone sequences, were excised from the parental plasmid and gel purified using the Qiagen gel extraction kit (Qiagen, Valencia, CA). Transgenic founders were generated by injecting the linear transgene cassette into the pronucleus of fertilized F2 oocytes from (C57BL/6J x SJL/J) F1 parents (Jackson Lab, Bar Harbor, ME). Embryos were subsequently transferred into ICR pseudopregnant females (Harlan, Indianapolis, IN). Transgenic founders and progeny were screened by PCR and/or Southern blotting (BamHI digest) using DNA prepared from tail biopsies. Primer sets EL905 5'-CAAACTTCAGCTTCCCTTCTGCTG-3' and EL906 5'-GACGAGGAAGAAGGAACTGAAGAC-3' were derived from a {beta}-galactosidase transgene and used for PCR screening. These primers identified a 379-bp {beta}-galactosidase transgene band in positive transgenic mice. The number of integration sites was determined for each founder using Southern blotting of the F1 progeny's tail DNA and a single cutter enzyme in the {beta}-galactosidase transgene. When F1 progeny from a single founder had more than one integration site, each integration event was bred onto a C57BL/6J background and evaluated as an independent founder. All animal experiments were monitored regularly and maintained in accordance with the principles and procedures outlined in the National Institutes of Health guidelines for the care and use of experimental animals.

Histochemical detection of {beta}-galactosidase activity in transgenic mice. Timed pregnancies were established between C57BL/6J males and heterozygous, transgene-positive females. The appearance of a vaginal plug in the morning following breeding was designated as E0.5 days. The genotype of the embryos was determined by the PCR of DNA harvested from the yolk sac or part of the embryo. Embryos and/or tissue samples were dissected in cold PBS at designated ages and fixed [0.2% glutaraldehyde, 5 mM EGTA, pH 7.3, 2 mM MgCl2, 0.02% Nonidet P-40 (NP-40), 0.01% deoxycholate, 2% paraformaldehyde, 0.1 M sodium phosphate, pH 8.8] for 15–60 min at room temperature with rocking. After being washed in detergent solution (0.02% NP-40, 0.01% deoxycholate, 2 mM MgCl2, 0.1 M sodium phosphate, pH 8.0) three times for 10 min at room temperature, the embryos were stained in 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (X-gal) solution (1 mg/ml X-gal, 5 mM potassium ferricyanide, and 5 mM potassium ferrocyanide in the wash buffer) in the dark at 37°C for 3 h, followed by room temperature incubation overnight. The stained embryos were postfixed in 0.5% glutaraldehyde/10% formalin after being washed three times in PBS. When histological sections were evaluated, the embryos were decalcified (CalRite) and equilibrated in 30% sucrose and embedded in an optimum cutting temperature compound. For X-gal staining of the cryosection (12 µm), sections were fixed in 2% paraformaldehyde for 1–2 min, washed five times for 5 min, and X-gal stained at 37°C overnight, as described above. Sections were counterstained with propidium iodide before being photographed. Whole mount X-gal staining analysis for mammary and nasal glands was performed on at least three litters from each line with a total of at least 30 embryos stained for each line, and time points were evaluated (E12.5-E16.5 at 1-day intervals). Because heterozygous mice were used for all breedings, negative internal control embryos were included in each litter. At least three embryos were sectioned and histologically evaluated for each of the time points and tissues described. Tracheal X-gal staining from newborn 3-day-old pups was performed on microdissected tracheas. Genotyping was performed subsequent to staining, and no false-positive staining patterns were observed.

In situ hybridization for Lef-1 mRNA. The proline-rich region (450 bp) of the mouse Lef-1 cDNA (GenBank GI:6754531) was cloned into a pBlueScript KS plasmid (Stratagene, La Jolla, CA) using KpnI and EcoRV restriction sites. This region did not overlap with conserved high mobility group DNA-binding domains. Mouse Lef-1 sense and antisense digoxigenin-labeled RNA probes were prepared by in vitro transcription using a Dig-RNA labeling kit with T3 and T7 RNA polymerase, respectively, per the manufacturer's instructions (Roche Molecular Biochemicals, Mannheim, Germany). Whole mount hybridization was performed essentially as previously described (17, 34); however, a higher temperature (70°C) was employed during hybridization and washing steps. The proteinase K incubation times (5–60 min) were also adjusted for different stages of embryos. Embryos were photographed after dehydration in a series of 25, 50, 75, and 100% methanol, after which they were cleared in 80% glycerol. In situ hybridization for Lef-1 RNA in nasal submucosal glands used cryosections with digoxigenin-labeled RNA probes and were performed as previously described (7–9).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Wnt-1 responsiveness of the human Lef-1 promoter in airway and mammary epithelial cell lines. The highly regulated spatial and temporal expression patterns of Lef-1 mRNA during embryogenesis suggest that cell type-specific regulation of the Lef-1 promoter plays an important role in organogenesis (31). The identification of Lef-1 promoter elements responsible for the inductive expression of Lef-1 will help clarify the transcriptional regulatory cascades important in developmental processes controlled by Lef-1. In this context, previous studies in HEK-293 kidney cells have demonstrated that Wnt-3a can induce the human Lef-1 promoter in a {beta}-catenin-dependent fashion (11). Given the importance of Wnt pathways during development of many organs, we sought to characterize whether Wnt induction of the Lef-1 promoter might play a role during airway submucosal gland and mammary gland development. The rationale for comparing developmental regulation of the Lef-1 promoter in these two types of glandular epithelia stems from the fact that Lef-1 expression is essential for the induction of glandular bud formation in both these organs (7, 8, 31).

Since Wnt-3a pathways have not been associated with mammary gland or submucosal gland organogenesis, we first sought to clarify whether alternative Wnts known to play a functional role in gland development might similarly induce the Lef-1 promoter. In this context, Wnt-1 has been previously shown to play a role in mammary gland carcinogenesis (4, 26, 29, 38). Furthermore, overexpression of Wnt-1 in mammary glands has been shown to increase expansion of stem/progenitor cell pools (as measured by a Hoechst-negative side population) in the adult mammary gland and results in mammary hyperplasia and malignancy (22). Given the shared requirement for Lef-1 expression during mammary and airway submucosal gland development, we first sought to evaluate whether Wnt-1 could also induce the Lef-1 promoter in several airway and mammary epithelial transformed cell lines. Two airway epithelial cell lines (A549 and IB3) and two mammary epithelial cell lines (MCF-7 and MBD-MA-231) were chosen for analysis in transient transfection assays of Lef-1 promoter function. Two Lef-1 promoter/LacZ reporter constructs were chosen for analysis of Wnt-1 induction based on previous studies characterizing the Lef-1 promoter in HEK-293 cells (11). The first of these reporter constructs (LF–2700/–200-LacZ) harbored a 2.5-kb fragment of the human Lef-1 promoter (–2,700 to –200 bp) containing all four known TSS (Fig. 1B). This region of the promoter was previously shown to maintain the highest level of Wnt-3a induction in 293 cells (11). The second construct [LF–2700(ID)/–200-LacZ] contained this same promoter fragment with an internal 110-bp deletion of a WRE located at –876 to 769 bp (Fig. 1C).

Lef-1 promoter expression profiles in IB3, A549, MCF-7, and MDA-MB-231 cells were examined by transient transfection of the promoter/reporter plasmids with either a negative control plasmid (pcDNA) or a plasmid coding for Wnt-1. Our analysis revealed that Wnt-1 was able to induce LF–2700/–200-LacZ expression in all cell lines tested to varying degrees (Fig. 2, A–D). In airway epithelial cell lines, Wnt-1 responsiveness was highest in IB3 cells (224-fold induction), which demonstrated the lowest level of baseline endogenous Lef-1 protein (Fig. 2E). In contrast, Lef-1 promoter induction by Wnt-1 was significantly less in A549 cells (6-fold induction), which had much higher baseline levels of endogenous Lef-1 protein (Fig. 2E). Interestingly, A549 cells have been demonstrated to have a highly invasive phenotype (28). Hence, the higher level of baseline Lef-1 activation and reduced Lef-1 promoter responsiveness to Wnt-1 in A549 cells may reflect constitutively activated Wnt signaling pathways. Lef-1 promoter induction by Wnt-1 was slightly higher in the more invasive mammary epithelial cell line MDA-MB-231 cells (17-fold) compared with the less invasive MCF-7 cells (7-fold). However, in contrast to the two airway epithelial cell lines analyzed, the endogenous baseline levels of Lef-1 protein were similar between these two mammary epithelial cells. These studies demonstrate for the first time that the Lef-1 promoter is also responsive to Wnt-1 signals and substantiate previous reports demonstrating Wnt-3a, {beta}-catenin, and/or TCF responsiveness of the human Lef-1 promoter in HEK-293 and COS-7 cells (1, 11).



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 2. Wnt-1 induction of the Lef-1 promoter in airway and mammary epithelial cell lines and its dependence on the WRE. A–D: Wnt-1-mediated induction of the indicated Lef-1/LacZ reporters was evaluated in IB3, MDA-MB-231, A549, and MCF-7 cells. Methods utilized cotransfection of a Lef-1/LacZ reporter with a Wnt-1 expression plasmid or a control plasmid, pcDNA. Results depict the mean (± SE, n = 3) relative expression over the promoterless LacZ vector backbone at 48 h posttransfection. Fold induction in the presence of Wnt-1 is given above each data set. E: Western blot analysis of total cellular lysate (75 µg) from IB3, A549, MCF-7, and MBD-MA-231 cell lines using an NH2-terminal anti-Lef-1 antibody to detect endogenous Lef-1 levels and an anti-I{kappa}B{alpha} antibody as an internal control for protein loading. Various elements and restriction sites used for cloning are denoted with the bp locations. {beta}-gal, {beta}-galactosidase.

 
Interestingly, different regions of the Lef-1 promoter appear to facilitate Wnt-1 induction in IB3 cells compared with A549, MCF-7, and MDA-MB-231 cells. In IB3 airway cells, deletion of the previously characterized WRE (–879 to –769 bp) from the Lef-1 promoter [LF–2700(ID)/–200-LacZ] completely abolished Wnt-1 induction (Fig. 2A). Similarly, deletion of the WRE decreased Wnt-1 induction of MCF-7 cells from 7.2- to 2.5-fold (Fig. 2D). In contrast, Wnt-1 responsiveness in mammary MDA-MB-231 and A549 cells was not significantly affected by the loss of the 110-bp WRE (Fig. 2, B and C). Interestingly, there was a trend for the more highly invasive cell lines (A549 and MDA-MB-231) to retain Wnt responsiveness of the Lef-1 promoter in the absence of the WRE. This is most clearly evident when comparing MDA-MB-231 (high invasive) and MCF-7 (low invasive) cells (2, 27). In summary, these findings suggest that the WRE imparts Wnt-1 responsiveness to the Lef-1 promoter in a cell type-specific fashion not previously appreciated. However, the cell type specificity of this responsiveness is likely a feature of the transformed phenotype rather than the tissue type of origin.

Generation of transgenic mouse lines with the human Lef-1 promoter controlling {beta}-galactosidase reporter gene expression. To better understand the in vivo significance of the WRE in the Lef-1 promoter, we generated two different transgenic lines harboring the Lef-1 promoter. Eight independent founder lines were created for each of the two transgene cassettes evaluated in the above cell lines [LF–2700/–200-LacZ and LF–2700(ID)/–200-LacZ; Table 1]. All lines contained a single integration event except for line 19298/2, which had two integration events that were subsequently bred out to single integration founders.


View this table:
[in this window]
[in a new window]
  		Table 1. Expression patterns of X-gal staining in LF–2700/–200-LacZ and LF–2700(ID)/–200-LacZ mice

 
Three of the eight transgenic founder lines from the LF–2700/–200-LacZ construct (24191/2, 11434/4, and 11439/1) exhibited positive {beta}-galactosidase staining at various stages of embryo development from E12.5 to E16.5 (Fig. 3, A–E). The remaining five lines did not express transgene at any stage of embryo development and were presumed to have integrated the transgene cassette into an inactive region of the genome. The whole mount embryo staining patterns seen in these three transgene-expressing founders were very similar in vibrissa/hair follicles, ear, and brain, but did differ with respect to limb bud and mammary gland expression (Table 1). No X-gal staining was observed in transgene-negative littermates (Fig. 3, K–O). In contrast to the LF–2700/–200-LacZ lines, a higher percentage of transgene-expressing embryos was obtained from founders generated with the LF–2700(ID)/–200-LacZ construct. Six of the eight founder lines derived from the LF–2700(ID)/–200-LacZ construct (18959/3, 19351/1, 19350/1, 19298/2, 19304/2, and 19298/2.2) demonstrated detectable {beta}-galactosidase transgene expression at various stages of embryogenesis (Fig. 3, F–J). However, these six lines demonstrated much more diversity in whole mount staining patterns at visible tissue sites, including the mammary glands, vibrissa/hair follicles, limb buds, brain, and ear (Table 1). Consequently, differences in Lef-1 promoter expression caused by deletion of the WRE could not be singled out by whole mount X-gal staining of embryos. To this end, we focused our analysis to histologically compare X-gal staining in airway submucosal glands and mammary glands at the initial stages of glandular bud formation.



View larger version (52K):
[in this window]
[in a new window]
  		Fig. 3. Whole mount 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (X-gal) staining analysis of LF–2700/–200-LacZ and LF–2700(ID)/–200-LacZ transgenic mice. Whole mount X-gal staining of embryos was performed at embryonic days E12.5-E16.5 for all LF–2700/–200-LacZ and LF–2700(ID)/–200-LacZ transgenic lines (Table 1). Representative lines are shown for the 11434/4 founder line containing the LF–2700/–200-LacZ reporter construct (A–E), the 18959/3 founder line containing the LF–2700(ID)/–200-LacZ reporter construct (F–J), and transgene-negative embryos (K–O).

 
Regulated expression of the Lef-1 promoter during the initial stages of mammary gland development is independent of the WRE. The formation of mammary glands during embryogenesis initiates at ~E11.5. This process involves epithelial and mesenchymal interactions that result in an invading epithelial sprout or cord protruding into the mesenchyme fat pad precursor that opens at the nipple (32). Lef-1, a key transcription factor involved in bud-forming epithelial/mesenchymal interaction, is the first identifiable transcriptional factor found to be expressed in initiating mammary placode, and in the absence of Lef-1, mice do not develop mammary glands (31). As such, Lef-1 mRNA is significantly upregulated during the initial stage of mammary glandular bud formation in both the epithelial buds and surrounding mesenchyme (31). Previously reported analyses have revealed that Lef-1 is expressed in the basal cells of the developing epidermis and in the epithelial cells of the mammary bud at E11.5-E12.5. By E13.5-E15.5, Lef-1 expression transitions to the mammary mesenchyme and is turned off in the mammary epithelium (Fig. 4A) (13, 24).



View larger version (116K):
[in this window]
[in a new window]
  		Fig. 4. Lef-1 promoter activity during the early stages of mammary gland development. A: illustration depicting previously characterized endogenous Lef-1 mRNA expression patterns during mammary glandular bud formation (13, 24, 31). During the initial stages of mammary glandular bud formation (E12.5), Lef-1 is expressed in the invading epithelium bud and at sites in the surface dermal epithelium in the proximity of the bud. At E13.5, expression of Lef-1 mRNA moves from the epithelium to the mesenchymal cells surrounding the bud. B and C: whole mount in situ hybridization for Lef-1 mRNA using an antisense (cRNA) probe (B) or a sense RNA (sRNA) probe (C) in E12.5 embryos. Lef-1 mRNA was detected in vibrissa follicles (vf) and mammary gland buds (mg). No staining was observed in whole mount in situ hybridization using an sRNA Lef-1 probe. D–I: whole mount X-gal staining of mammary glands in E12.5 and E13.5 transgenic embryos demonstrating positive LacZ staining only in the mammary gland buds of transgene-positive animals. Transgenic-negative control littermates at E12.5 and E13.5 did not show X-gal staining. J–M: histological analysis of X-gal staining in sections from E12.5 and E13.5 transgenic embryos demonstrating predominant Lef-1-LacZ expression in mammary epithelia (me) and mammary mesenchyme (mm) of early-stage mammary gland buds. The construct of each transgenic line and the embryonic stage is given in each panel.

 
As previously described (31), the induction of Lef-1 mRNA was observed by whole mount in situ hybridization of developing mammary glands at the E12.5 bud stage (Fig. 4, B and C). Of the eight LF–2700/–200-LacZ transgenic founder lines harboring a full-length 2.5-kb Lef-1 promoter, three expressed {beta}-galactosidase in E12.5-E16.5 stage embryos (11434/4, 11439/1, 24191/2). Two of the lines (11434/4 and 24191/2) demonstrated mammary expression in all three thoracic and two inguinal mammary buds from E12.5 to E15.5 embryos (Fig. 4, F and G). Interestingly, expression was limited predominantly to the mesenchymal cells surrounding the mammary bud in both lines (Fig. 4, J and K). This pattern of expression overlapped with that previously reported for Lef-1 mRNA, with the notable exclusion of expression in the epithelium of the mammary bud at E12.5. Furthermore, mesenchymal expression in these two lines appeared to be activated slightly earlier than previously described for mRNA.

To determine whether the WRE was critical for the observed mesenchymal expression of the Lef-1 promoter during mammary bud formation, we evaluated mammary gland expression in six of the eight LF–2700(ID)/–200-LacZ transgenic lines that expressed any level of {beta}-galactosidase during embryo development (Table 1). Two of these six lines evaluated (19298/2 and 18959/3) expressed {beta}-galactosidase in the mammary glands (Fig. 4, H and I). Mesenchymal expression was observed in both of these lines; however, the temporal regulation of expression differed (Fig. 4, L and M). Mesenchymal expression was initiated in the 18959/3 line at E12.5 and was subsequently extinguished by E13.5. In contrast, mesenchymal expression was initiated at E13.5 in the 19298/2 line. Although the onset of mesenchymal expression was different in the two WRE-deleted lines, the spatial patterns of expression overlapped with that seen in the LF–2700/–200-LacZ lines. The most notable difference between the LF–2700(ID)/–200-LacZ and the LF–2700/–200-LacZ lines was the appearance of {beta}-galactosidase expression in the mammary epithelium in only the WRE-deleted promoter. As shown in Fig. 4L, expression of {beta}-galactosidase in the 19298/2 line was observed in the mammary epithelial bud at E12.5, which switched to a mesenchymal expression pattern by E13.5. This pattern of temporally and spatially restricted expression was very similar to that previously reported for Lef-1 mRNA (13, 24, 31). However, this was the only founder that reproduced endogenous Lef-1 expression patterns in mammary glands. The second LF–2700(ID)/–200-LacZ line (18959/3) that expressed in mammary gland epithelium demonstrated a delayed, weaker level of epithelial expression at E13.5 that followed the mesenchymal expression at E12.5 (Fig. 4M). This pattern of expression demonstrated an incorrect regulatory switch from epithelial to mesenchymal expression. Given the apparent slight differences (1 day) in the onset and shutoff of mesenchymal and/or epithelial expression seen in both the LF–2700/–200-LacZ and LF–2700(ID)/–200-LacZ lines, we conclude that variation in the integration site of the transgene cassette leads to subtle difference in the temporal regulation of the promoter. This implies that sequences are missing from in the Lef-1 promoter transgene cassette necessary for insulating transcription from variations in chromatin structure.

The WRE is required for epithelial-specific regulation of the Lef-1 promoter during nasal submucosal gland development. Nasal submucosal gland development initiates from the nasal epithelium between E15 and E16, where stage 1 buds emerge from the simple cuboidal airway epithelium. Although Lef-1 expression is required for nasal submucosal gland development in the mouse (8), Lef-1 mRNA expression has not been previously characterized in the mouse nasal glandular epithelium. To this end, we sought to confirm endogenous Lef-1 mRNA expression patterns in the mouse nasal epithelium at stages of glandular bud formation. Findings from these studies demonstrated widespread Lef-1 mRNA expression in both the surface nasal epithelium and glandular buds (Fig. 5, A and B), similar to that seen during the initial stages of mammary gland development (31). To determine whether the 2.5-kb promoter could regulate expression in nasal submucosal glands, we evaluated the {beta}-galactosidase staining patterns in the three LF–2700/–200-LacZ transgenic founder lines (24191/2, 11434/4, and 11439/1) that demonstrated transcriptionally active transgenes during embryo development (see Table 1). All of these founder lines demonstrated LacZ transgene expression in a subset of cells contained within E16.5 nasal submucosal gland epithelial buds (Fig. 5, C–H), which was never observed in transgene-negative littermates (Fig. 5I). Interestingly, such staining was not observed in any of the eight LF–2700(ID)/–200-LacZ WRE-deleted lines (Fig. 5J). Given that Lef-1 mRNA expression was seen in nasal surface airway epithelium (Fig. 5A) but was not observed in LF–2700/–200-LacZ transgenic mice, we conclude that Lef-1 promoter segments outside the –2,700/–200 bp control expression in surface airway cell types. Furthermore, these data also demonstrate that the WRE is required for epithelial cell expression of the Lef-1 promoter in nasal glandular buds. This finding is in stark contrast to observations in mammary gland (Fig. 4) and vibrissa/hair follicles (23).



View larger version (123K):
[in this window]
[in a new window]
  		Fig. 5. Lef-1 promoter activity during the early stages of nasal submucosal gland development is dependent on the WRE. A and B: in situ hybridization for Lef-1 mRNA using an antisense (cRNA) probe (A) or a sense (sRNA) probe (B). Only the Lef-1 cRNA probe demonstrated Lef-1-mRNA expression in the nasal surface airway epithelium (SAE) and invaginating nasal gland buds of E16.5 embryos. C–H: histological analysis of X-gal staining in sections from E16.5 LF–2700/–200-LacZ embryos demonstrating predominant LacZ expression in nasal gland buds (gb) of transgene-positive (Trans+) embryos. Three separate LF–2700/–200-LacZ lines, 11439/1 (C), 11434/4 (D, E, and H), and 24191/2 (F and G), are shown. Transgene expression was not seen in the nasal SAE. Transgenic-negative (Trans–) littermate controls did not show staining (I). J and K: histological analysis of X-gal staining in sections from E16.5 LF–2700(ID)/–200-LacZ 19298/2 transgenic embryos demonstrated no LacZ staining in nasal gland buds or SAE in either the transgenic-positive or the transgenic-negative embryos.

 
Expression of the Lef-1 promoter in proximal tracheal glandular buds and surrounding surface airway epithelium is dependent on the WRE. Tracheal gland development in the mouse initiates during the first postnatal week of life. Therefore, we evaluated Lef-1 promoter expression patterns in 3-day-old neonatal excised tracheas (Fig. 6, A–D). En face X-gal staining patterns demonstrated a surprisingly widespread expression of transgene in the proximal end of the trachea in two of the eight LF–2700/–200-LacZ lines (24191/2 and 11434/4), which was never observed in transgene-negative littermates (Fig. 6, A–D) or in any of the eight LF–2700(ID)/–200-LacZ lines (data not shown). Histological analysis of cryosections revealed a peculiar staining pattern in developing tracheal submucosal glandular buds and the surrounding surface airway epithelium characterized by punctate staining in the surface airway epithelium surrounding a more diffusely staining glandular bud (Fig. 6, E and F). In contrast, this staining was never seen in transgene-negative littermates from these two lines (Fig. 6, G and H) or any of the eight LF–2700(ID)/–200-LacZ lines (Fig. 6, I–L). In addition to the typical number of embryos screened for each founder line (described in MATERIALS AND METHODS), we performed blinded staining and genotyping experiments on four independent litters derived from a C57BL/6 x 11434/4 heterozygous transgenic line cross to confirm that the staining pattern observed was genuine. From a total of 31 embryos, 16 were transgene positive and 15 were transgene negative. Fifteen of the 16 transgene-positive animals demonstrate the type of staining presented in Fig. 6, whereas none of the 15 transgene-negative animals demonstrated positive staining. The single negative staining transgene-positive embryo may represent a false-negative condition due to air bubbles in the trachea limiting penetration of the X-gal substrate. Hence, we do not attribute this type of staining to variability in the transgenic line background.



View larger version (112K):
[in this window]
[in a new window]
  		Fig. 6. Lef-1 promoter activity in newborn proximal tracheal gland buds and surrounding SAE is dependent on the WRE. A–D: whole mount X-gal staining of dissected tracheas from transgene-positive and -negative 3-day-old neonates of 2 LF–2700/–200-LacZ lines. X-gal staining can be seen at the very proximal end of the trachea only in transgenic-positive pups (A and B). No staining was observed in tracheas from transgenic-negative neonates (C and D). E–H: histological analysis of X-gal-stained whole mount tracheas from 2 LF–2700/–200-LacZ lines demonstrated staining in the SAE and in the developing submucosal gland buds of transgenic-positive neonates (E and F). No staining was seen in transgene-negative littermate control tracheas (G and H). I–L: histological analysis of LF–2700(ID)/–200-LacZ X-gal-stained trachea showed no staining in either the transgenic-positive (I and J) or -negative (K and L) littermates in the SAE or the submucosal gland buds.

 
These findings suggest several intriguing possibilities regarding the Lef-1 promoter in the proximal trachea. First, the WRE in the Lef-1 promoter appears to be necessary for expression in tracheal submucosal glandular buds and the surrounding surface epithelium. This is similar to what was observed in nasal submucosal glandular buds. However, one of the three LF–2700/–200-LacZ lines (11439/1) that expressed in nasal submucosal glandular buds did not express in the proximal trachea. This finding suggests that regulation of the Lef-1 promoter in these two glandular regions may be slightly different, despite a similar dependence on the WRE. Given the very different spatial distribution of staining in the proximal trachea compared with the nasal glandular buds, this hypothesis seems reasonable.

Other sites of Lef-1 promoter expression. Although the focus of this report was to evaluate Lef-1 promoter regulation during nasal submucosal glandular and mammary glandular bud formation, both the LF–2700/–200-LacZ and LF–2700(ID)/–200-LacZ lines expressed at other sites, including brain, limb buds, vibrissa (whisker) follicles, and hair follicles (Table 1 and Fig. 3). Findings evaluating Lef-1 promoter regulation in vibrissa and hair follicles have demonstrated that epithelial- and mesenchymal-restricted patterns of Lef-1 expression are uniquely regulated by the Wnt/{beta}-catenin-responsive sequences (WRE) in the Lef-1 promoter during follicle formation (23). In this context, the WRE was necessary for mesenchymal expression within mesenchymal condensates and dermal papillae of developing hair/vibrissa follicles (Table 1). Hence, expression in these regions was only observed in the LF–2700/–200-LacZ and not the LF–2700(ID)/–200-LacZ lines. In summary, these results suggest that Wnt/{beta}-catenin-responsive sequences in the Lef-1 promoter play an important role in coordinating epithelial and mesenchymal expression during vibrissa/hair follicle development through unique transcriptional pathways.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Despite the wealth of knowledge on developmental biology and branching morphogenesis of the lung, relatively little is known about the molecular cues that control submucosal gland development following formation of the airways. Lef-1-deficient mice develop normal lungs and airways, with the exception of absent submucosal glands. This selective involvement of Lef-1 in airway gland development, but not lung development, supports the notion that transcription cascades controlling Lef-1 gene expression are uniquely regulated in epithelial progenitors responsible for submucosal gland development. Despite the seemingly selective role of Lef-1 in airway gland development, this transcription factor is also very important in regulating epithelial-mesenchymal interactions in many other bud-forming organs. In this regard, comparative analyses of Lef-1 regulatory mechanisms may shed important insights into the transcriptional control of epithelial cell fates in general.

Wnt signaling plays an important role in regulating epithelial-mesenchymal interaction during the development of many organs, including the lung (30). The transcriptional programs activated by Wnts often involve the mobilization of {beta}-catenin to the nucleus and the {beta}-catenin-dependent activation of TCF/Lef-1 transcriptional regulators (36). The finding that the Lef-1 promoter is also responsive to Wnt-3a/{beta}-catenin pathways in HEK-293 cells (11) suggests that Lef-1 protein is not only transcriptionally activated by Wnt/{beta}-catenin signaling but also transcriptionally regulated by these same signals. Our studies in a number of mammary and airway epithelial cell lines demonstrate that Wnt-1 can also transcriptionally activate the Lef-1 promoter. Interestingly, regulatory elements in the Lef-1 promoter responsible for Wnt-1 induction appear to have a variable dependence on the previously identified 110-bp WRE, which is solely responsible for Wnt-3a induction of the promoter in HEK-293 cells. These findings suggest that Wnt-1 activation of the Lef-1 promoter can be controlled by sequences both within and outside the WRE, depending on the cell phenotype. This finding may reflect a capacity of the Lef-1 promoter to be temporally regulated in vivo (i.e., transcriptionally turned on and off) in both airway and mammary gland epithelial cells during the process of gland formation. Although the exact signaling networks responsible for Wnt activation of the Lef-1 promoter remain to be determined, differences in endogenous Wnt expression profiles of the four transformed cell lines analyzed may account for some of the variability in both the level and WRE dependence of Wnt-1 induction. For example, Wnt-3 and Wnt-7 are highly expressed in MDA-MB-231 and A549 cells, whereas Wnt-3 is highly expressed in MCF-7 cells (see ATCC datasheet) (20, 21). Hence, Wnt inducibility of the Lef-1 promoter in each cell line analyzed may represent the potential of each cell type to induce the Lef-1 promoter under a given environmental condition or cell phenotype found during development.

The ability of Wnts to induce the Lef-1 promoter and the dependence of this induction on the WRE in certain cell phenotypes provide an opportunity to better understand the potential roles of Wnt induction in regulating Lef-1-dependent developmental processes such as glandular bud formation. Our results evaluating the dependence of the Lef-1 promoter on its WRE for spatial and temporal regulation during the early stages of mammary gland and submucosal gland formation have suggested that although these two organs share a similar dependence on Lef-1 during development, the regulatory processes that control Lef-1 gene transcription are quite distinct. During nasal and tracheal submucosal gland development, the presence of the WRE in the Lef-1 promoter was absolutely required for expression. These findings suggest that Wnt induction of the Lef-1 promoter may play an important role in the transcriptional activation of Lef-1 in airway epithelial cells. However, based on Lef-1 mRNA localization studies in nasal submucosal glands, the 2.5-kb region of the Lef-1 promoter evaluated only partially reconstituted expression in epithelial cells of nasal glandular buds and not in the surface airway epithelium. These findings suggest that the transcriptional programs that regulate the Lef-1 promoter in surface airway epithelial cells are distinct from that in the glandular bud. Interestingly, expression patterns of the Lef-1 promoter in proximal tracheal submucosal glandular buds and surrounding surface airway epithelium appear to also be slightly different than that for nasal submucosal glands. Although expression of the Lef-1 promoter at both sites was dependent on the WRE, the finding of discordant nasal and tracheal gland expression in a single founder (i.e., nasal glandular bud expression was seen in the 11439/1 line, which did not demonstrate tracheal gland expression) suggests the transcriptional programs regulating the promoter in these two regions may also be distinct. Given the fact that Lef-1 promoter expression in proximal trachea also demonstrated widespread staining in the surface airway epithelium surrounding gland ducts, this hypothesis appears reasonable. Interestingly, mouse tracheal submucosal gland ducts have been proposed as a niche for the expansion of bromodeoxyuridine label-retaining airway epithelial cells following SO2 injury (5). This finding suggests the intriguing possibility that Lef-1 expression in this region is regulated by Wnts in a stem cell pool occupying this unique niche. Given the dependence of this expression on the WRE and the fact that Wnt-1 and Wnt-3a can induce self-renewal of hematopoietic and mammary gland stem cells (22, 35), such a potential link is worthy of further investigation.

In contrast to submucosal glands, expression of the Lef-1 gene is activated in both epithelial and mesenchymal cells of the developing mammary gland. This process involves a temporal switch in epithelial to mesenchymal expression at ~E13.5. Findings from both the full-length and WRE-deleted Lef-1 promoter transgenic mice suggest that regulation of this temporal switch in expression requires additional sequences not currently contained within the promoter. Despite the fact that spatial expression patterns in mammary epithelium and mesenchyme closely mirrored that previously observed for Lef-1 protein and mRNA during the initial stages of mammary gland development (13, 24, 31), the temporal regulation of the promoter in these two cellular compartments was variable, often lagging or preceding previously reported Lef-1 mRNA expression patterns by one gestational day. Nonetheless, several notable differences and similarities can be seen with respect to expression of the Lef-1 promoter in other organs. First, expression of the Lef-1 promoter in mammary gland bud epithelial and mesenchymal cells was independent of the WRE. This is in contrast to the WRE dependence of epithelial Lef-1 expression submucosal glandular buds. Furthermore, the expression patterns seen in a single transgenic line did closely resemble temporal and spatial regulation of Lef-1 previously reported for mammary gland development (13, 24, 31). Hence, regulation at the WRE and potential Wnt signals that act on this site of the Lef-1 promoter appear to be unique to bud formation of airway but not mammary glands. Second, epithelial expression in mammary glandular buds was only seen in two of eight WRE-deleted lines, but not in any of the eight full-length WRE-inclusive promoter lines (Table 1). This finding suggests the intriguing possibility that the WRE may be inhibitory for epithelial expression in mammary glandular buds. In support of this hypothesis, analysis of vibrissa/hair follicle expression patterns in the LF–2700/–200-LacZ and LF–2700(ID)/–200-LacZ transgenic lines (23) has demonstrated similar findings as seen in mammary glands (see Table 1 for comparison). During vibrissa/hair follicle development, Lef-1 expression temporally shifts between epithelial and mesenchymal compartments of the developing follicle. As seen in the current studies on mammary glands, LF–2700/–200-LacZ lines expressed solely in the mesenchymal compartments of the follicle during development (Table 1). In contrast, a single line generated from the LF–2700(ID)/–200-LacZ construct (18959/3) demonstrated an epithelial-restricted pattern of expression in developing follicles. This same 18959/3 line and a second additional founder (19298/2) also demonstrated epithelial expression during mammary gland development. These findings support the notion that the WRE may play an inhibitory role in regulating epithelial expression within the developing hair follicle and mammary glands. Obviously, the fact that removal of the WRE promoted more native patterns of expression in epithelial cells of developing mammary glands and hair follicles suggests that additional Lef-1 promoter sequences are required to properly regulate the WRE.

Comparative analysis of Lef-1 promoter activity in several organs has suggested that the WRE may uniquely regulate epithelial-specific expression during glandular bud formation. Although the exact positive and/or negative regulatory pathways that act on the WRE remain to be determined, these findings imply that Wnt regulation at the Lef-1 promoter WRE may be a critical component of such regulation. The identity of the Wnt pathway(s) critical for regulated Lef-1 promoter expression at the WRE during gland development remains unknown. Studies in cell lines have demonstrated that both Wnt-3a and Wnt-1 induction of the Lef-1 promoter can be dependent on the WRE. However, as seen in both airway and mammary epithelial cells, Wnt-1 can also induce the Lef-1 promoter independently of the WRE. Such findings appear to reflect a complex context-specific involvement of the WRE in expression of the Lef-1 promoter in vivo during development. Hence, the mechanisms of Wnt regulation of the Lef-1 promoter may be considerably more complex than previously anticipated. Further delineation of such regulation will likely shed light on pathological conditions involving airway gland hyperplasia as seen in asthma, chronic bronchitis, and cystic fibrosis or mammary gland carcinogenesis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-47967.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the editorial assistance of Dr. Gregory Leno and Leah Williams.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. F. Engelhardt, Dept. of Anatomy and Cell Biology, Univ. of Iowa, Rm. 1-111 BSB, 51 Newton Road, Iowa City, IA 52242-1109 (E-mail: john-engelhardt{at}uiowa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Atcha FA, Munguia JE, Li TW, Hovanes K, and Waterman ML. A new {beta}-catenin-dependent activation domain in T cell factor. J Biol Chem 278: 16169–16175, 2003.[Abstract/Free Full Text]
  2. Bajou K, Lewalle JM, Martinez CR, Soria C, Lu H, Noel A, and Foidart JM. Human breast adenocarcinoma cell lines promote angiogenesis by providing cells with uPA-PAI-1 and by enhancing their expression. Int J Cancer 100: 501–506, 2002.[CrossRef][ISI][Medline]
  3. Billin AN, Thirlwell H, and Ayer DE. {beta}-Catenin-histone deacetylase interactions regulate the transition of LEF1 from a transcriptional repressor to an activator. Mol Cell Biol 20: 6882–6890, 2000.[Abstract/Free Full Text]
  4. Bocchinfuso WP, Hively WP, Couse JF, Varmus HE, and Korach KS. A mouse mammary tumor virus-Wnt-1 transgene induces mammary gland hyperplasia and tumorigenesis in mice lacking estrogen receptor-{alpha}. Cancer Res 59: 1869–1876, 1999.[Abstract/Free Full Text]
  5. Borthwick DW, Shahbazian M, Krantz QT, Dorin JR, and Randell SH. Evidence for stem-cell niches in the tracheal epithelium. Am J Respir Cell Mol Biol 24: 662–670, 2001.[Abstract/Free Full Text]
  6. Brantjes H, Roose J, van De Wetering M, and Clevers H. All Tcf HMG box transcription factors interact with Groucho-related co-repressors. Nucleic Acids Res 29: 1410–1419, 2001.[Abstract/Free Full Text]
  7. Duan D, Sehgal A, Yao J, and Engelhardt JF. Lef1 transcription factor expression defines airway progenitor cell targets for in utero gene therapy of submucosal gland in cystic fibrosis. Am J Respir Cell Mol Biol 18: 750–758, 1998.[Abstract/Free Full Text]
  8. Duan D, Yue Y, Zhou W, Labed B, Ritchie TC, Grosschedl R, and Engelhardt JF. Submucosal gland development in the airway is controlled by lymphoid enhancer binding factor 1 (LEF1). Development 126: 4441–4453, 1999.[Abstract]
  9. Engelhardt JF, Schlossberg H, Yankaskas JR, and Dudus L. Progenitor cells of the adult human airway involved in submucosal gland development. Development 121: 2031–2046, 1995.[Abstract]
  10. Engelhardt JF, Yankaskas JR, and Wilson JM. In vivo retroviral gene transfer into human bronchial epithelia of xenografts. J Clin Invest 90: 2598–2607, 1992.[ISI][Medline]
  11. Filali M, Cheng N, Abbott D, Leontiev V, and Engelhardt JF. Wnt-3A/{beta}-catenin signaling induces transcription from the LEF-1 promoter. J Biol Chem 277: 33398–33410, 2002.[Abstract/Free Full Text]
  12. Finkbeiner WE. Physiology and pathology of tracheobronchial glands. Respir Physiol 118: 77–83, 1999.[CrossRef][ISI][Medline]
  13. Foley J, Dann P, Hong J, Cosgrove J, Dreyer B, Rimm D, Dunbar M, Philbrick W, and Wysolmerski J. Parathyroid hormone-related protein maintains mammary epithelial fate and triggers nipple skin differentiation during embryonic breast development. Development 128: 513–525, 2001.[Abstract]
  14. Fuchs E, Merrill BJ, Jamora C, and DasGupta R. At the roots of a never-ending cycle. Dev Cell 1: 13–25, 2001.[CrossRef][ISI][Medline]
  15. Galceran J, Farinas I, Depew MJ, Clevers H, and Grosschedl R. Wnt3a–/–-like phenotype and limb deficiency in Lef1(–/–)Tcf1(–/–) mice. Genes Dev 13: 709–717, 1999.[Abstract/Free Full Text]
  16. Gat U, DasGupta R, Degenstein L, and Fuchs E. De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated {beta}-catenin in skin. Cell 95: 605–614, 1998.[CrossRef][ISI][Medline]
  17. Hogan B. Manipulating the Mouse Embryo: A Laboratory Manual. Plainview, NY: Cold Spring Harbor Laboratory Press, 1994.
  18. Hsu SC, Galceran J, and Grosschedl R. Modulation of transcriptional regulation by LEF-1 in response to Wnt-1 signaling and association with {beta}-catenin. Mol Cell Biol 18: 4807–4818, 1998.[Abstract/Free Full Text]
  19. Jeffery PK. Remodeling in asthma and chronic obstructive lung disease. Am J Respir Crit Care Med 164: S28–S38, 2001.[Abstract/Free Full Text]
  20. Katoh M. Molecular cloning and characterization of human WNT3. Int J Oncol 19: 977–982, 2001.[ISI][Medline]
  21. Kirikoshi H, Sekihara H, and Katoh M. Molecular cloning and characterization of human WNT7B. Int J Oncol 19: 779–783, 2001.[ISI][Medline]
  22. Liu BY, McDermott SP, Khwaja SS, and Alexander CM. The transforming activity of Wnt effectors correlates with their ability to induce the accumulation of mammary progenitor cells. Proc Natl Acad Sci USA 101: 4158–4163, 2004.[Abstract/Free Full Text]
  23. Liu X, Driskell RR, Luo M, Abbott D, Filali M, Cheng N, Sigmund CD, and Engelhardt JF. Characterization of Lef-1 promoter segments that facilitate inductive developmental expression in skin. J Inv Derm 123: 264–274, 2004.
  24. Michno K, Boras-Granic K, Mill P, Hui CC, and Hamel PA. Shh expression is required for embryonic hair follicle but not mammary gland development. Dev Biol 264: 153–165, 2003.[CrossRef][ISI][Medline]
  25. Novak A, Hsu SC, Leung-Hagesteijn C, Radeva G, Papkoff J, Montesano R, Roskelley C, Grosschedl R, and Dedhar S. Cell adhesion and the integrin-linked kinase regulate the LEF-1 and {beta}-catenin signaling pathways. Proc Natl Acad Sci USA 95: 4374–4379, 1998.[Abstract/Free Full Text]
  26. Papkoff J and Aikawa M. WNT-1 and HGF regulate GSK3 {beta} activity and {beta}-catenin signaling in mammary epithelial cells. Biochem Biophys Res Commun 247: 851–858, 1998.[CrossRef][ISI][Medline]
  27. Polette M, Gilles C, Marchand V, Seiki M, Tournier JM, and Birembaut P. Induction of membrane-type matrix metalloproteinase 1 (MT1-MMP) expression in human fibroblasts by breast adenocarcinoma cells. Clin Exp Metastasis 15: 157–163, 1997.[CrossRef][ISI][Medline]
  28. Ramesh R, Ito I, Gopalan B, Saito Y, Mhashilkar AM, and Chada S. Ectopic production of MDA-7/IL-24 inhibits invasion and migration of human lung cancer cells. Mol Ther 9: 510–518, 2004.[CrossRef][ISI][Medline]
  29. Shackleford GM, MacArthur CA, Kwan HC, and Varmus HE. Mouse mammary tumor virus infection accelerates mammary carcinogenesis in Wnt-1 transgenic mice by insertional activation of int-2/Fgf-3 and hst/Fgf-4. Proc Natl Acad Sci USA 90: 740–744, 1993.[Abstract/Free Full Text]
  30. Shannon JM and Hyatt BA. Epithelial-mesenchymal interactions in the developing lung. Annu Rev Physiol 66: 625–645, 2004.[CrossRef][ISI][Medline]
  31. Van Genderen C, Okamura RM, Farinas I, Quo RG, Parslow TG, Bruhn L, and Grosschedl R. Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice. Genes Dev 8: 2691–2703, 1994.[Abstract/Free Full Text]
  32. Veltmaat JM, Mailleux AA, Thiery JP, and Bellusci S. Mouse embryonic mammogenesis as a model for the molecular regulation of pattern formation. Differentiation 71: 1–17, 2003.[CrossRef][ISI][Medline]
  33. Wang X, Zhang Y, Amberson A, and Engelhardt JF. New models of the tracheal airway define the glandular contribution to airway surface fluid and electrolyte composition. Am J Respir Cell Mol Biol 24: 195–202, 2001.[Abstract/Free Full Text]
  34. Wassarman PM and DePamphilis ML. Guide To Techniques in Mouse Development. San Diego, CA: Academic, 1993.
  35. Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya T, Yates JR III, and Nusse R. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423: 448–452, 2003.[CrossRef][Medline]
  36. Wodarz A and Nusse R. Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol 14: 59–88, 1998.[CrossRef][ISI][Medline]
  37. Yasumoto K, Takeda K, Saito H, Watanabe K, Takahashi K, and Shibahara S. Microphthalmia-associated transcription factor interacts with LEF-1, a mediator of Wnt signaling. EMBO J 21: 2703–2714, 2002.[CrossRef][ISI][Medline]
  38. Ziemer LT, Pennica D, and Levine AJ. Identification of a mouse homolog of the human BTEB2 transcription factor as a {beta}-catenin-independent Wnt-1-responsive gene. Mol Cell Biol 21: 562–574, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Proc Am Thorac SocHome page
X. Liu and J. F. Engelhardt
The Glandular Stem/Progenitor Cell Niche in Airway Development and Repair
Proceedings of the ATS, August 15, 2008; 5(6): 682 - 688.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
M. Amen, X. Liu, U. Vadlamudi, G. Elizondo, E. Diamond, J. F. Engelhardt, and B. A. Amendt
PITX2 and {beta}-Catenin Interactions Regulate Lef-1 Isoform Expression
Mol. Cell. Biol., November 1, 2007; 27(21): 7560 - 7573.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
S. Kuure, A. Popsueva, M. Jakobson, K. Sainio, and H. Sariola
Glycogen Synthase Kinase-3 Inactivation and Stabilization of beta-Catenin Induce Nephron Differentiation in Isolated Mouse and Rat Kidney Mesenchymes
J. Am. Soc. Nephrol., April 1, 2007; 18(4): 1130 - 1139.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
T. W.-H. Li, J.-H. T. Ting, N. N. Yokoyama, A. Bernstein, M. van de Wetering, and M. L. Waterman
Wnt Activation and Alternative Promoter Repression of LEF1 in Colon Cancer.
Mol. Cell. Biol., July 1, 2006; 26(14): 5284 - 5299.
[Abstract] [Full Text] [PDF]

This Article
Right arrow