Aberrant cell adhesion molecule expression in human bronchopulmonary sequestration and congenital cystic adenomatoid malformation

Abstract

In many organs, integrins and cadherins are partly regulated by Hox genes, but their interactions in airway morphogenesis and congenital lung diseases are unknown. We previously showed that the Hox protein HoxB5 is abnormally increased in bronchopulmonary sequestration (BPS) and congenital cystic adenomatoid malformation (CCAM), congenital lung lesions with abnormal airway branching. We now report on alpha(2)-, alpha(3)-, and beta(1)-integrin and E-cadherin expression in normal human lung and in BPS and CCAM tissue previously shown to have abnormal HoxB5 expression and on the relationship of cell adhesion molecule expression to Hoxb5 regulation. alpha(2)-, alpha(3)-, and beta(1)-integrins and E-cadherin expression in normal human lung and BPS and CCAM were evaluated using Western blot and immunohistochemistry. Fetal mouse lung fibroblasts with Hoxb5-specific siRNA downregulation were evaluated for alpha(2)-integrin protein levels by Western blot. Compared with normal human lung, a previously undetected alpha(2)-integrin isoform potentially lacking essential cytoplasmic sequences was significantly increased in BPS and CCAM, and alpha(2)-integrin spatial and cellular expression was more intense. E-cadherin protein levels were also significantly increased, whereas alpha(3) increased in CCAM compared with canalicular, but not with alveolar, stage lung. beta(1)-integrin levels were unchanged. We conclude that in BPS and CCAM, altered alpha(2)-integrin cytoplasmic signaling contributes to abnormal cellular behavior in these lung lesions. Aberrant cell adhesion molecule and Hox protein regulation are likely part of the mechanism involved in the development of BPS and CCAM.

Fig. 1.

Schematic of primary antibody binding sites to α₂-integrin transmembrane protein. Antibody 1 (Chemicon, Temecula, CA) detects essential regulatory sequences in the cytoplasmic domain of α₂-integrin of mouse and human origin. Antibody 2 (Santa Cruz Biotechnology, Santa Cruz, CA) detects sequences in the extracellular domain of α₂-integrin of human origin.

Fig. 2.

α₂-integrin representative Western blots and summary densitometry. A: Western blots performed with antibody 1 (detects cytoplasmic sequences of α₂-integrin) did not demonstrate any difference in protein levels of the 150-kDa protein isoform of α₂-integrin between normal human lung from the canalicular (Can) and alveolar (Alv) stage of lung development and bronchopulmonary sequestration (BPS) and congenital cystic adenomatoid malformation (CCAM) tissue. B: Western blots using antibody 2 (detects extracellular sequences of α₂-integrin) detected a significantly upregulated 130-kDa protein isoform of α₂-integrin in BPS and CCAM compared with normal alveolar or canalicular stage lung tissue, but there were no differences in the 150-kDa α₂-integrin isoform (*P = 0.005, means ± SE; BPS and CCAM vs. Alv or Can). A and B: n = 6, Can; n = 3, Alv; n = 4, BPS; n = 7, CCAM. C: representative Western blot of one CCAM sample in absence (−) or presence (+) of blocking peptide for antibody 2 showed that when antibody 2 activity was blocked with α₂-specific blocking peptide against antibody 2, neither the 150- nor the 130-kDa protein isoform was detected. D: mouse fetal lung fibroblasts treated with Hoxb5-specific siRNA to downregulate Hoxb5 protein had significantly decreased α₂-integrin protein levels compared with scramble-treated cells or control (no treatment) (*P = 0.04, means ± SE, n = 5).

Fig. 3.

α₂-integrin immunostaining in CCAM and BPS. A–F: representative CCAM tissue sections stained for α₂-integrin with blue alkaline phosphatase immunohistochemistry and nuclear Fast Red counterstain. G–I: α₂-integrin stained with purple VIP-HRP immunolocalization and Hoxb5 protein (H, I) immunolocalized with brown DAB-HRP immunolocalization and counterstained with methyl green. Bar = 100 μm in D, 50 μm in A and E, and 25 μm in B, C, F, G, H, and I. A: α₂-integrin antibody 1, immunolocalized α₂-integrin protein to mesenchymal and epithelial cells of CCAM cysts (* in A and B) and adjacent compressed lung (arrow in A and C) with similar intensity. D, E, F: in contrast, α₂-integrin antibody 2 immunostaining of adjacent section of CCAM shown in A–C revealed different regional localization of α₂-integrin. Compared with adjacent compressed lung regions (# in D, E, F), increased α₂-integrin staining in mesenchyme and epithelial cells was seen around CCAM cyst (* in D, E, F). Using antibody 2, BPS tissue (G) showed intense purple epithelial (arrow in G) and mesenchymal localization (* in G) of α₂-integrin compared with human fetal lung (17-wk gestation) (H), which had strong but less intense purple α₂-integrin localization in epithelial cells of branching airway tips (long arrows in H). These α₂-positive purple epithelial cells were surrounded by closely adjacent subepithelial fibroblasts with intense brown nuclear staining for HoxB5 protein (arrowheads in H). Regions of airways with less purple α₂-integrin staining (< in H) are not surrounded by closely adjacent HoxB5-positive brown cells. α₂-integrin was also localized diffusely in mesenchyme, especially in fibroblasts with brown nuclear Hoxb5 staining (short arrow in H). In absence of α₂-integrin antibody, there is no purple α₂-integrin immunostaining (I) but continued Hoxb5 brown nuclear staining in presence of Hoxb5 antibody.

Fig. 4.

α₃-integrin Western blots and immunohistochemistry. A: representative α₃-integrin Western blot and summary densitometry showed similar levels of α₃-integrin in BPS and CCAM tissue compared with alveolar stage lung tissue. CCAM tissue, however, had significantly increased α₃-integrin protein levels compared with canalicular period lung tissue samples (*P = 0.03, CCAM vs. Can, means ± SE; n = 6, Can; n = 3, Alv; n = 4, BPS; n = 7, CCAM). B: human fetal lung tissue (17-wk human gestation) showed α₃-integrin purple VIP staining at basal surface of columnar epithelial cells that had less intense subepithelial brown nuclear staining for Hoxb5 protein in mesenchymal cells (arrowhead). In contrast, airways (*) with less intense purple epithelial α₂-integrin staining had more intense brown Hoxb5 staining in adjacent subepithelial fibroblasts (arrow). α₃-integrin (purple) was also seen diffusely in mesenchyme but to a lesser degree than in airway epithelial cells and subepithelial fibroblasts. C: CCAM tissue had diffuse purple staining for α₃-integrin (arrowhead) and scattered brown nuclear staining for Hoxb5 protein (arrow). D: immunostaining control showed that in absence of Hoxb5 primary antibody, no brown DAB staining was seen with only purple VIP staining for α₃-integrin in presence of α₃-integrin primary antibody.

Fig. 5.

β₁-integrin representative Western blot and summary densitometry. β₁-integrin protein levels were not altered in BPS and CCAM compared with alveolar or canalicular stage human lung tissue. N = 6, Can; n = 3, Alv; n = 4, BPS; n = 7, CCAM.

Fig. 6.

E-cadherin representative Western Blot and summary densitometry. Compared with alveolar stage human lung, E-cadherin protein levels were increased significantly in CCAM and showed a trend towards increased levels in BPS. Levels of E-cadherin in CCAM and BPS were more similar to canalicular stage lung tissue. *P = 0.04, CCAM vs. Alv, means ± SE, n = 6, Can; n = 3, Alv; n = 4, BPS; n = 7, CCAM.