Integrin αvβ6 mediates epithelial-mesenchymal transition in human bronchial epithelial cells induced by lipopolysaccharides of Pseudomonas aeruginosa via TGF-β1-Smad2/3 signaling pathway

Abstract

Lower respiratory tract infection due to Pseudomonas aeruginosa has become increasingly challenging, resulting in a worse morbidity and mortality. Airway remodeling is a common phenomenon in this process, to which epithelial-mesenchymal transition (EMT) may contribute as an important promoter. Previous studies showed that epithelium-specific integrin αvβ6-mediated EMT was involved in pulmonary fibrosis via transforming growth factor-β1 (TGF-β1) signaling, but whether integrin αvβ6 plays a role in the P. aeruginosa-associated airway remodeling remains unknown. BEAS-2B cells were incubated with lipopolysaccharide (LPS) from P. aeruginosa in the presence or the absence of integrin αvβ6-blocking antibodies. Morphologic changes were observed by an inverted microscopy. The EMT markers were detected using Western blotting and immunofluorescence. The activation of TGF-β1-Smad2/3 signaling pathway was assessed. Furthermore, matrix metalloproteinase (MMP)-2 and -9 in the medium were measured using ELISA. P. aeruginosa's LPS decreased the expression of the epithelial marker E-cadherin and promoted the mesenchymal markers, vimentin and α-smooth muscle actin in BEAS-2B cells. The expression of integrin αvβ6 was significantly increased during EMT process. Blocking integrin αvβ6 could attenuate P. aeruginosa's LPS-induced EMT markers' expression via TGF-β1-Smad2/3 signaling pathway. Furthermore, blocking integrin αvβ6 could prevent morphologic changes and oversecretion of MMP-2 and -9. Integrin αvβ6 mediates epithelial-mesenchymal transition in human bronchial epithelial cells induced by lipopolysaccharides of P. aeruginosa via TGF-β1-Smad2/3 signaling pathway and might be a promising therapeutic target for P. aeruginosa-associated airway remodeling.

Fig. 1

P. aeruginosa’s LPS-induced EMT in BEAS-2B cells. BEAS-2B cells were incubated with 2 μg/mL P. aeruginosa’s LPS for 24, 48, or 72 h. Western blotting revealed that LPS decreased the expression of E-Cad and upregulated the expression of Vi and α-SMA in a time-dependent manner (a). Immunofluorescence showed similar results of the upregulated mesenchymal marker Vi and downregulation of the epithelial marker, E-Cad, in a time-dependent manner (b). Compared with the basal condition, a fibroblast-like, spindle-shaped morphology was induced by P. aeruginosa’s LPS for 72 h in BEAS-2B cells (c). The data represent the mean ± SEM, n = 3. *p < 0.05 versus the control

Fig. 2

The expression of integrin αvβ6 in BEAS-2B cells was significantly upregulated by P. aeruginosa’s LPS in a time-dependent manner. BEAS-2B cells were exposed to 2 μg/mL P. aeruginosa’s LPS for 24, 48, or 72 h. Western blotting revealed that LPS increased the expression of integrin β6 in a time-dependent manner. The data represent the mean ± SEM, n = 3. *p < 0.05 versus the control

Fig. 3

Blocking integrin αvβ6 could abrogate EMT in BEAS-2B cells induced by P. aeruginosa’s LPS. BEAS-2B cells were incubated with integrin αvβ6–blocking antibody 10D5 (30 μg/mL) or specific inhibitor of TGF-β1-Smad2/3 signaling, SB431542 (10 μM), for 2 h prior to incubation with 2 μg/mL P. aeruginosa’s LPS for 72 h. Western blotting showed that the decrease of the epithelial marker, E-Cad expression, and the increase of mesenchymal markers, Vi and α-SMA, induced by P. aeruginosa’s LPS were reversed by integrin αvβ6–blocking antibody 10D5 as well as SB431542 (a). Immunofluorescence showed similar results of E-Cad and Vi expressions (b). The data represent the mean ± SEM, n = 3. *p < 0.05 versus the control (Con), #p < 0.05 versus LPS

Fig. 4

Integrin αvβ6 mediated P. aeruginosa’s LPS-induced EMT in BEAS-2B cells via TGF-β1-Smad2/3 signaling pathway. BEAS-2B cells were pretreated with integrin αvβ6–blocking antibody 10D5 (30 μg/mL) or TGF-β1 inhibitor, SB431542 (10 μM), for 2 h prior to incubation with 2 μg/mL P. aeruginosa’s LPS for 72 h. Total-Smad2/3 and p-Smad2/3 were detected using Western blotting (a). The secretion of active TGF-β1 in the culture medium was measured by ELISA (b). The results showed that the phosphorylation of Smad2/3 was increased by P. aeruginosa’s LPS, which were ameliorated by integrin αvβ6–blocking antibody 10D5 as well as SB431542. In addition, elevated secretion of active TGF-β1 was induced by P. aeruginosa’s LPS, which could be inhibited by integrin αvβ6–blocking antibody 10D5. The data represent the mean ± SEM, n = 3. *p < 0.05 versus Con, #p < 0.05 versus LPS

Fig. 5

Blocking integrin αvβ6 could prevent morphologic changes and the increase of MMP-2 and -9 secretion induced by P. aeruginosa’s LPS. Cell morphologic change was ameliorated by 10D5 and SB431542 (a). ELISA showed that the secretion of active MMP-2 and -9 was significantly increased by P. aeruginosa’s LPS which could be attenuated by integrin αvβ6–blocking antibody 10D5 as well as specific inhibitor of TGF-β1-Smad2/3 signaling, inhibitor SB431542 (b). The data represent the mean ± SEM, n = 3. *p < 0.05 versus Con, #p < 0.05 versus LPS

Fig. 6

Summary. Integrin αvβ6 could regulate EMT in BEAS-2B cells induced by P. aeruginosa’s LPS via activation of TGF-β1-Smad2/3 signaling pathway. Compared with globally blocking TGF-β1, integrin αvβ6 blocking might be a more promising option to prevent airway remodeling induced by P. aeruginosa