Epithelial N-cadherin and nuclear β-catenin are up-regulated during early development of human lung

Kaarteenaho, Riitta; Lappi-Blanco, Elisa; Lehtonen, Siri

doi:10.1186/1471-213X-10-113

Research article
Open access
Published: 16 November 2010

Epithelial N-cadherin and nuclear β-catenin are up-regulated during early development of human lung

Riitta Kaarteenaho^1,2,3,
Elisa Lappi-Blanco^2,3 &
Siri Lehtonen^4,5,6

BMC Developmental Biology volume 10, Article number: 113 (2010) Cite this article

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Abstract

Background

The aim of this study was to analyze the cell-specific expression of E- and N-cadherin and β-catenin in developing human lung tissues from 12 to 40 weeks of gestation.

Methods

Fortyseven cases of developing human lung including pseudoglandular, canalicular, saccular and alveolar periods were analyzed by immunohistochemisty for E- and N-cadherin and β-catenin and twentyone cases were also investigated by RT-PCR for E- and N-cadherin and β-catenin. For identifying the lung cells, the sections were also stained with antibodies against thyroid transcription factor-1 (TTF-1) and caveolin-1. Normal adult lung tissue was used as a control.

E-cadherin was strongly expressed in epithelium of bronchi and large bronchioles from week 12 onwards and it was also positive in alveoli in pretype II cells and type II cells. N-cadherin was present in most of the epithelial cells of bronchi and the largest bronchioles during the pseudo-glandular and canalicular periods. N-cadherin was not detected in epithelium of developing alveoli. β-catenin was strongly membrane-bound and positively expressed in bronchial epithelium from week 12 to week 40; it showed nuclear positivity in both developing airway epithelium and in the cells underneath the epithelium during pseudo-glandular period and to a lesser degree also in the canalicular period. β-catenin was positive in pretype II cells as well as in type I and type II pneumocytes within alveoli.

RT-PCR analyses revealed detectable amounts of RNAs of E- and N-cadherin and β-catenin in all cases studied. The amounts of RNAs were higher in early stages of gestation.

Conclusions

E-cadherin is widely expressed in bronchial and alveolar epithelial cells. N-cadherin exhibit extensive epithelial positivity in bronchial epithelial cells during early lung development. The presence of β-catenin was observed in several cell types with a distinct location in tissue and cells in various gestational stages, indicating that it possesses several roles during lung development. The expressions of protein and mRNAs of E- and N-cadherin and β-catenin were higher in early gestation compared to of the end. Moreover, the expressions of these factors were higher during the lung development than in the adult human lung.

Background

There is still little detailed knowledge of how cells differentiate during ontogenesis in the lung as well as in pulmonary diseases. It has been assumed that signalling processes occuring during pulmonary fibrosis and lung cancer may share similarities with the various stages of human lung development, thus by studying lung development it might be possible to acquire valuable information that may be useful when researching fibrotic and neoplastic lung diseases [1]. The epithelial changes are regulated by the extracellular matrix (ECM), which is an essential element in the process of branching morphogenesis, and also participating in the control of cell phenotype expression. In our previous studies, we have observed that the levels of several ECM proteins e.g. tenascin-C and precursors of collagen I and III are increased in certain localizations during human lung development as well as in fibrotic lung disorders when compared to the healthy adult normal lung [2–5].

Cells are connected with each other or the ECM in different ways but it is clear that adhesion molecules are extremely important in cellular junctions. The cadherins represent an important subclass of adhesion molecules [6]. The cytoplasmic domain of cadherins interacts with catenins, and this complex associates with actin filaments [7]. The cadherin superfamily consists of several members and one of these, E-cadherin is expressed in epithelial cells and it is nowadays a commonly used marker of cell epithelial phenotype in studies focusing on the epithelial-mesenchymal transition (EMT) [8]. N-cadherin was originally found to be expressed in neural and muscle cells, but subsequently it is observed be an element of mesenchymal cells [9]. In studies investigating the EMT, N-cadherin has been used as a marker of mesenchymal differentiation [8]. Specific cadherins have been shown to directly stimulate cellular differentiation into certain types of tissue [10]. Recently, expression of N-cadherin has been observed to be present in epithelial lung tumors in addition of E-cadherin and β-catenin [11].

β-catenin serves many roles in the maintenance of cell architecture, for example it can bind to the cytoplasmic tail of E-cadherin. The WNT/β-catenin signal transduction pathway has been shown to control embryonic patterning [12]. The cell specific expression profile of E- and N-cadherin during developing human lung is still unclear, though the role of β-catenin in lung organogenesis has been evaluated more extensively [13]. A previous study indicated that β-catenin signalling was required for the formation of the distal, but not the proximal, airways, and that the excision of β-catenin in epithelial cells caused respiratory failure and death [14].

The aim of the immunohistochemical study was to examine the cell-specific expression of E- and N-cadherin and β-catenin in normal human developing lung at different gestational ages i.e. from week 12 to week 40 during the pseudoglandular, canalicular, saccular and alveolar periods. We hypothesized that the expression and localization of these factors could vary during the different developmental periods. In addition to protein localization by immunohistochemistry, the amounts of RNAs were evaluated in some cases by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR).

Methods

Patients and handling of specimens

Samples of lung tissue were retrieved from the files of the Department of Pathology, Oulu University Hospital. The study protocol was approved by the Ethical committee of the Northern Ostrobothnia Hospital District (Stament 64/2001, amedment 68/2005). The National Supervisory Authority for Welfare and Health (Valvira) has given a license for this study for the use of lung tissues of abortive foetuses for research purposes (Reg. nr. 7323/05.01.00.06/2009). Since this is a retrospective material, the informed consent permission has been given by the National Supervisory Authority for Welfare and Health, which is the national licensing authority for social welfare and health care (please see http://www.valvira.fi/en/licensing).

The study material for developing lung consisted of 47 cases of spontaneous abortion, stillbirth, and autopsied infants who had died for different reasons without lung disorders within 1-2 days after birth in Oulu University Hospital between 1990 and 2002. Autopsies had been performed within one day in most cases and within two days in four cases. Causes of death of the infants were abortion (n = 19), abruption of placenta (n = 9), rupture of fetal membranes (n = 2), feto-fetal transfusion (n = 2), sacral teratoma (n = 2), prolapse or aplasia of the umbilical artery (n = 3), placentitis or chorioamnionitis (n = 4), hemochromatosis (n = 1), hydrocephalus (n = 1), meningomyelocele (n = 1), encephalocele (n = 1), holoprosencephaly (n = 1) and hemorrhage of caput (n = 1). Infants with pneumonia, cardiac abnormalities or features of maceration were excluded. The gestational ages of infants ranged from 12 to 40 weeks, corresponding to the pseudoglandular (day 52 to week 16, 13 cases), canalicular (weeks 16-28, 17 cases), saccular (weeks 28-36, 9 cases) and alveolar (weeks 36-40, 8 cases) periods. Uninvolved peripheral lung tissue from adults, used as a control, was obtained from six patients operated on for benign lung tumour. The clinical information was obtained from the patient records.

Lung samples, which had been taken from different parts of the left or right lung were fixed in 10% formalin and then dehydrated and embedded in paraffin. Sections, which were 5 μm thick, were stained with hematoxylin-eosin. All material was re-evaluated, and one representative tissue block from each case was selected for the immunohistochemical studies. In order to identify the phenotype of the various lung cells, the sections were also stained with antibodies against thyroid transcription factor (TTF-1) (a staining for type II pneumocytes) and caveolin-1 (a staining for type I pneumocytes) and some selected cases also with vimentin (a staining for fibroblast and myofibroblast).

Antibodies and immunohistochemical staining

E-cadherin antibody (clone HECD-1) and N-cadherin antibody (clone 3B9) were purchased from Zymed Laboratories Inc (South San Francisco, CA, USA). β-catenin antibody (clone 5) was purchased from Transduction Laboratories (Lexington, KY, USA), TTF-1 (clone 8G7G31) from Dako (Carpinteria, CA), caveolin-1 (clone E249) from Abcam (Cambridge, UK) and vimentin (clone V9) from DakoCytomatin (Glostrup, Denmark). The sections were incubated overnight with antibodies to E-cadherin (diluted as supplied 1: 300), N-cadherin (diluted as supplied 1: 200), β-catenin (diluted as supplied 1:200), vimentin (diluted as supplied 1:1500), TTF-1 (diluted as supplied 1:100) and caveolin-1 (diluted as supplied 1:250). Immunohistochemistry was performed using paraffin-embedded biopsies. Tissue samples were sectioned and deparaffinized followed by an antigen retrieval procedure suitable for the antibody in question. Dako Envision Kit (Dako) was used in the immunohistochemistry with a 30 min incubation for the primary antibody. Negative controls were obtained by substituting non-immune rabbit or mouse serum and PBS for the primary antibodies.

Scoring of the immunoreactivity

The extent and intensity of E- and N-cadherin and β-catenin were evaluated semiquantitatively as negative (0), weak (+), moderate (++) or strong (+++) in different types of pulmonary cells, such as epithelial cells of bronchioles and bronchus, alveolar epithelium including pretype II cells, type I and II pneumocytes, endothelial cells, interstitial cells such as fibroblasts and myofibroblasts, and mesothelial cells. In the evaluation, membrane-bound positivity, cytoplasmic and nuclear positivity were quantified.

Quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR)

In 21 cases representing all developmental periods (pseudoglandular, n = 4; canalicular, n = 8; saccular, n = 7; alveolar, n = 4) one tissue block was selected for E-cadherin, N-cadherin and β-catenin analysis at the transcriptional level by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) as previously described [15]. Additionally five paraffin embedded samples of normal adult human lung were used as controls. As it has been shown previously RNA can be isolated from paraffin embedded tissue material for expression profiling [16, 17]. The total RNA was isolated from five 10 μm thick slices from each sample with Purelink FFPE total RNA isolation kit according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The isolated RNA was quantified and qualified spectrophotometrically and 0.5 μg of isolated RNA was converted in duplicate to cDNA by RevertAid first strand cDNA synthesis kit (Fermentas, EU) by using random hexamers as primers. Quantitative RT-PCR was performed by using the iQ5 Optical system (Bio-Rad Laboratories, Hercules, CA) with SYBR Green I technology (iQ Custom SYBR green supermix, Bio-Rad Laboratories). PCR was performed in duplicate from each cDNA by using 66°C as the annealing temperature. The primers were 5'-aaggtgacagagcctctggat-3'(forward, f) and 5'-cgtctgtggctgtgacct-3'(reverse, r) for E cadherin; 5'-cgagccgcctgcgctgccac-3 (f) and 5'-cgctgctctccgctccccgc-3 (r) for N-cadherin; 5'-tggatgggctgcctccaggtgac-3' (f) and 5'-accagcccacccctcgagccc-3'(r) for β-catenin; and 5'-gagtcaacggatttggtcgt-3' (f) and 5'-gacaagcttcccgttctcag-3'(r) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that was considered as a house-keeping gene in the data analysis. The results were counted by the Livak ΔΔC_t method using a sample of normal adult lung as a reference [18]. Differences between the groups were analysed by PASW Statistics software version 17 (SPSS, Chicago, IL, USA) by using Kruskal-Wallis test.

Results

Immunohistochemistry for E- and N-cadherin and β-catenin

Pseudoglandular period (weeks 12 to 16)

E-cadherin was strongly expressed in all the epithelial cells of developing bronchi and bronchioles of all sizes including also the smallest structures (Figure 1A, 1B). N-cadherin was also moderately or strongly expressed in the epithelium of bronchi and largest bronchioles whereas it was negative in the smallest developing airways (Figure 1C, 1D). β-catenin displayed strong membrane-bound positivity in bronchi and larger bronchioles, whereas its expression in the small developing airways was mainly strongly nuclear (Figure 1E, 1F, 1G). The cells underneath the epithelium, especially in smallest airways, showed also nuclear positivity for β-catenin (Figure 1G), and in contrast, these non-epithelial cells at the same localizations seemed to be negative for E-and N-cadherin.

Canalicular period (weeks 16 to 28)

E-cadherin was strongly expressed in the epithelial cells of bronchi and bronchioles and also in the pretype II cells of developing alveoli (Figure 2A, 2B). N-cadherin was faintly or moderately expressed in the cells of bronchi and bronchioles whereas the cells of developing alveoli were negative (Figure 2C, 2D). β-catenin was strongly membrane-bound positive in the cells of bronchi and bronchioles, whereas the cells of alveoli showed mainly nuclear-type of positivity especially at the beginning of the stage (Figure 2E, 2F, 2G). However, at the end of this period, the amount of membrane-bound positivity within alveolar epithelium increased, and correspondingly, the nuclear positivity decreased.

Saccular (weeks 28 to 36) period

E-cadherin was expressed strongly in the epithelial cells of bronchi and bronchioles with its expression being clearly positive within alveoli in type II cells (Figure 3A, 3B). N-cadherin was positive in some of the epithelial cells in bronchi and bronchioles, but its expression in alveoli remained negative (Figure 3C, 3D). β-catenin revealed membrane-bound positivity in the cells of bronchi and bronchioles, and the cells of alveolar epithelium were also mainly membrane-type positive, although some alveolar epithelial cells exhibited also nuclear-type positivity (Figure 3E, 3F, 3G).

Alveolar (weeks 36 to 40) period

E-cadherin was moderately positive in the epithelium of bronchi and bronchioles and in type II pneumocytes within alveoli (Figure 4A, 4B). N-cadherin was faintly expressed in only a few scattered cells of the epithelium of bronchi and bronchioles. Most of the bronchial epithelial cells were negative for N-cadherin, nor could it be detected in the alveoli (Figure 4C). β-catenin revealed strong membrane-bound positivity in the cells of bronchi and bronchioles, and the cells of alveolar epithelium were also mainly membrane-type positive, although some alveolar epithelial cells showed also nuclear-type positivity (Figure 4D, 4E).

Mesothelium, fibroblasts, myofibroblasts, smooth muscle cells and endothelium were positive for β-catenin, but negative for E- and N-cadherin. The neural cells displayed positivity for N-cadherin. In some cases during the pseudoglandular period, trachea was included within the tissue sample, and its epithelium was positive for E- and N-cadherin and β-catenin. Negative controls obtained by substituting non-immune rabbit or mouse serum and PBS for the primary antibodies showed no positive staining (Figure 1H, 2H, 3H, 4G, 4H).

The results of the scoring of the immunoreactivity for various proteins are shown in Table 1. One score shown for each protein in various periods is valid for every case analyzed. If the samples of the same developmental stage showed variable staining with each other, two scores are shown.

Table 1 Immunoreactivity scores for E- and N-cadherin, β-catenin, TTF-1 and caveolin-1 in different types of pulmonary cells in developing human lung during various gestational periods.

Full size table

Normal adult lung as a control

E-cadherin was expressed in most epithelial cells of normal adult human lung including type II pneumocytes and also in all bronchial epithelial cells whereas mesothelial cells remained negative (Figure 5A). Alveolar epithelial cells including both type I and II pneumocytes were negative for N-cadherin (Figure 5B), while mesothelial cells and some scattered basal cells of bronchioles were positive. β-catenin was expressed in type II pneumocytes and epithelium of bronchioles and also faintly in type I pneumocytes (Figure 5C, 5D). Endothelial cells, some mesenchymal cells, and mesothelium stained also positively for β-catenin. The staining pattern for β-catenin was mainly membranous, but some type II pneumocytes exhibited cytoplasmic expression pattern for β-catenin with nuclear enhancement. Type II pneumocytes and some epithelial cells of bronchioles showed a positive staining for TTF-1 (Figure 5E). Cells lining alveoli expressed positively for caveolin-1 (Figure 5F).