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

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: Forty-seven cases of developing human lung including pseudoglandular, canalicular, saccular and alveolar periods were analyzed by immunohistochemistry for E- and N-cadherin and β-catenin and twenty-one 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.

Figure 1

Immunohistochemical stainings for E- and N-cadherin and β-catenin during pseudoglandular period of developing human lung. 1A and 1B. Epithelium of the developing airways is positive for E-cadherin (arrows). 1C and 1 D. Epithelium of some developing airways is positive for N-cadherin (arrows) whereas other developing airways are negative for N-cadherin (short arrows). 1E and 1F. Epithelial and mesenchymal cells of developing airways are positive for β-catenin. In most airways epithelial cells display nuclear positivity (arrows) whereas some airways exhibit membrane-bound positivity (short arrows). 1G. A high power field image showing nuclear positivity for β-catenin in epithelial cells of a small developing airway (arrows) surrounded by positively stained mesenchymal cells (short arrows). 1 H. A negative control in which the primary antibody has been substituted with non-immune serum. Arrows are indicating developing airways. Scale bars = 40 μm in 1A, 1C, 1F and 1H; scale bars = 20 μm in 1B, 1 D and 1G, and scale bar = 80 μm in 1E.

Figure 2

Immunohistochemical stainings for E- and N-cadherin and β-catenin during canalicular period of developing human lung. 2A and 2B. Epithelial cells of a developing bronchiolus (short arrows) and alveoli including pretype II pneumocytes (arrows) are positive for E-cadherin. 2C. Pretype II pneumocytes (short arrows) of alveoli are negative for N-cadherin while epithelial cells of a bronchiolus (arrow) are positive for N-cadherin. 2 D. In addition of the epithelial cells (arrows), some spindle shaped cells of a bronchiole showed also a positivity for N-cadherin (short arrow). 2E, 2F and 2G. Epithelial cells of developing airways including pretype II pneumocytes are positive for β-catenin showing mostly a nuclear positivity (arrows) and also a membrane-bound positivity in some cells (short arrows). 1 H. A negative control in which the primary antibody has been substituted with non-immune serum. Scale bars = 40 μm in 2A, 2C, 2E and 2 H; scale bars = 20 μm in 2B, 2D, 2F and 2G.

Figure 3

Immunohistochemical stainings for E- and N-cadherin and β-catenin during saccular period of developing human lung. 3A and 3B. A strong positivity for E-cadherin is seen in the epithelial cells of a bronchiole (arrows) and type II pneumocytes of alveoli are also positive for E-cadherin (short arrows). 3C. Alveolar epithelium is negative for N-cadherin (arrows). 3 D. A few epithelial cells of a bronchiole are positive for N-cadherin (arrows). 3E and 3F. Positive staining for β-catenin is seen in the epithelial cells of a bronchiole (arrows) and alveolar epithelial cells (short arrows). 3G. A high power field image showing a positive expression for β-catenin within alveoli. Some alveolar epithelial cells exhibit nuclear staining (arrows). 3 H. A negative control in which the primary antibody has been substituted with non-immune serum. Scale bars = 40 μm in 3A, 3C, 3E and 3H; scale bars = 20 μm in 3B, 3D, 3F and 3G.

Figure 4

Immunohistochemical stainings for E- and N-cadherin and β-catenin during alveolar period of developing human lung. 4A and 4B. Type II pneumocytes (short arrows) and bronchial epithelial cells (arrows) display a positive staining for E-cadherin. 4C. Alveoli do not show any positivity for N-cadherin. 4 D. Type II pneumocytes of alveoli (arrows) are strongly positive for β-catenin. 4F. An example of vimentin staining showing that alveoli are positive for it. 4G. A negative control in which the primary antibody has been substituted with non-immune serum. 4 H. A negative control in which the primary antibody has been substituted with PBS with haematoxylin counterstain. Scale bars = 40 μm in 4A, 4C and 4D; scale bars = 20 μm in 4B, 4E, 4F, 4G and 4 H.

Figure 5

Immunohistochemical stainings for E- and N-cadherin, β-catenin, TTF-1 and caveolin-1 in adult human lung. 5A. Type II pneumocytes of alveoli are positive for E-cadherin (arrows). 5B. Epithelial cells of a bronchiole are expressing E-cadherin (arrows). 5C. Cells of alveoli are negative for N-cadherin (arrows). 5 D. Type II pneumocytes (arrows) and to a lesser extent also type I pneumocytes of alveoli are positive for β-catenin. 5E. Epithelial cells of a bronchiole are positive for β-catenin. 5F. Type II pneumocytes exhibit a positive staining for TTF-1 (arrows). 5G. A staining of caveolin-1 is positive in alveolar epithelium (arrows). 5G. A negative control in which the primary antibody has been substituted with non-immune serum. Scale bars = 20 μm.

Figure 6

Immunohistochemical stainings for TTF-1 and caveolin-1 during pseudoglandular, canalicular, saccular and alveolar periods of developing human lung. 6A. Epithelial cells of small developing airways during pseudoglandular period are strongly positive for TTF-1 (arrows). 6B. Epithelial cells of a developing airway during pseudoglandular period are negative for caveolin-1 (arrow) while the cells surrounding the airway (short arrow) and obvious endothelial cells (arrowheads) are positive for it. 6C. Pretype II cells during the canalicular period are positive for TTF-1 (arrows). 6 D. Pretype II cells during the canalicular period do not show any positivity for caveolin-1 (arrows) while the cells rounding alveoli (short arrows) as well as the endothelial cells (arrowheads) are positively stained. 6E and 6G. Type II pneumocytes are positive for TTF-1 during saccular (6E) and alveolar (6G) periods (arrows). 6F and 6H. Caveolin-1 is positive during saccular (6F) and alveolar (6H) periods in alveolar epithelial cells (arrows) whereas bronchial epithelial cells do not exhibit any posivity (short arrows). Scale bars = 20 μm.

Figure 7

Quantitative RT-PCR analysis of E-cadherin (A), N-cadherin (B) and β-catenin (C). The RNA was isolated from paraffin embedded tissue samples of pseudoglandular (n = 3), canalicular (n = 8), saccular (n = 7) and alveolar (n = 3) periods and from adult lung (n = 5). The isolated RNA was reverse transcribed into cDNA before real-time PCR. The results were analysed against the GAPDH expression to give relative expression values and related to one adult lung sample. One asterisk represents the probability value below 0.05 and two asterisks values below 0.01 compared to healthy control group.