Upregulation of MUC5AC by VEGF in human primary bronchial epithelial cells: implications for asthma

Abstract

Background: Airway mucus hypersecretion is an important pathophysiological feature in asthma. Mucins are glycoproteins that are mainly responsible for the viscoelastic property of mucus, and MUC5AC is a major mucin glycoprotein that is overproduced in asthma. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthmatics. Therefore, we sought to investigate the effect of VEGF on MUC5AC expression and study the underlying mechanisms.

Methods: In order to elucidate the precise mechanism underlying the effect of VEGF on MUC5AC expression, we tested the effects of VEGF on RhoA activation and the association of caveolin-1 and VEGFR2 in Primary Bronchial Epithelial Cells.

Results: VEGF up-regulated MUC5AC mRNA and protein levels in a dose- and time-dependent manner, and activated RhoA. Additionally, VEGF-induced MUC5AC expression and RhoA activation were enhanced by disrupting caveolae with cholesterol depletion and reversed by cholesterol repletion, and inhibited by a selective VEGF receptor 2 (VEGFR2) inhibitor SU1498. Furthermore, phospho-VEGFR2 expression was decreased via overexpression of caveolin-1. VEGF treatment reduced the association of caveolin-1 and VEGFR2.

Conclusion: Collectively, our findings suggest that VEGF up-regulates MUC5AC expression and RhoA activation by interaction with VEGFR2, and this phenomenon was related with the association of caveolin-1 and VEGFR2. Further studies on these mechanisms are needed to facilitate the development of treatments for asthma.

Keywords: Asthma; Caveolin-1; MUC5AC; Vascular endothelial growth factor.

Fig. 1

VEGF promotes MUC5AC expression at both mRNA and protein level. PBECs were incubated with indicated doses of VEGF for 9 h, and then real-time PCR performed. The values are normalized relative to the GAPDH standard (A). PBECs were incubated at indicated times of VEGF (50 ng/ml), and then real-time PCR performed (B). PBECs were incubated with indicated doses of VEGF for 24 h (C). PBECs were incubated at indicated times of VEGF (50 ng/ml), and then western blotting analysis for MUC5AC was performed. β-actin was used as a loading control (D). All data are representative of three independent experiments. The blots were quantified by densitometry. Values represent the means ± SEM. ^*P < 0.05, ^**P < 0.005 vs. control; n = 3

Fig. 2

VEGF-induced MUC5AC upregulation depends on RhoA/Rho kinase activation. PBECs were incubated with indicated doses of Rho kinase inhibitor HA-1077 for 2 h before treatment with VEGF (50 ng/ml) for 24 h, and then western blotting analysis for MUC5AC was performed. β-actin was used as a loading control (A). PBECs were transfected with Control siRNA or RhoA siRNA, and then western blotting analysis for RhoA-GTP, Total RhoA, and MUC5AC was performed. β-actin was used as a loading control (B). All data are representative of three independent experiments. The blots were quantified by densitometry. Values represent the means ± SEM. ^*P < 0.05 vs. control; ^# P < 0.05 vs. VEGF alone; n = 3

Fig. 3

Caveolar disruption enhanced the VEGF-induced RhoA activation and MUC5AC up-regulation. PBECs were treated with VEGF (50 ng/ml) in the presence or absence of pretreatment with the caveolar-disrupting agent cyclodextrin (CD; 5 mM, 30 min) or filipin (2.5 g/ml, 30 min). Reversal of drug effects was sought with simultaneous cholesterol repletion (Chol; 15 g/ml) given at the time of cyclodextrin administration. RhoA activity was assessed by pull-down assay of GTP-bound RhoA (24 kDa) as described in Method (A). MUC5AC protein levels were assessed by Western blot (B). All data are representative of three independent experiments. The blots were quantified by densitometry. Values represent the means ± SEM. ^*P < 0.05 vs. control; ^# P < 0.05 vs. VEGF alone; n = 3

Fig. 4

Effect of SU1498 on VEGF-induced RhoA activation and MUC5AC expression in PBECs. PBECs were incubated with indicated doses of SU1498 for 2 h before treatment with VEGF (50 ng/ml) for 24 h, and then western blotting analysis for RhoA activation was performed. Total RhoA was used as a loading control (A). PBECs were incubated with indicated doses of SU1498 for 2 h before treatment with VEGF (50 ng/ml) for 24 h, western blotting analysis for MUC5AC activation was performed. β-actin was used as a loading control (B). All data are representative of three independent experiments. The blots were quantified by densitometry. Values represent the means ± SEM. ^*P < 0.05 vs. control; ^# P < 0.05 vs. VEGF alone; n = 3

Fig. 5

Transfection of caveolin-1 in PBECs inhibits VEGFR2 phosphorylation. PBECs were transiently transfected with caveolin-1 cDNAs. Forty-eight hours posttransfection, cells were serum-starved and lysed. VEGFR-2 was immune- precipitated from cell extracts and phosphorylation was monitored by Western blotting with an anti-phospho-VEGFR2 antibody. Actin in the supernatant was probed to ensure equal immunoprecipitation across conditions Data was representative of three independent experiments

Fig. 6

VEGFR2 association with caveolin-1 is markedly decreased by treatment with VEGF, cyclodextrin, or filipin and is partially rescued by treatment with cholesterol repletion. VEGFR2 was immunopricipitated from PBECs lysates, and its association with caveolin-1 was assessed by Western blot. Actin in the supernatant was probed to ensure equal immunoprecipitation across conditions (A). PBECs were treated with VEGF (50 ng/ml) in the presence or absence of pretreatment with the caveolar-disrupting agent cyclodextrin (CD; 5 mM, 30 min) or filipin (2.5 g/ml, 30 min). Reversal of drug effects was sought with simultaneous cholesterol repletion (Chol; 15 g/ml) given at the time of cyclodextrin administration. Caveolin-1was immunopricipitated from PBECs lysates, and its association with VEGFR2 was assessed by Western blot. Actin in the supernatant was probed to ensure equal immunoprecipitation across conditions (B). All data are representative of three independent experiments