TGF-β potentiates airway smooth muscle responsiveness to bradykinin

Abstract

The molecular mechanisms by which bradykinin induces excessive airway obstruction in asthmatics remain unknown. Transforming growth factor (TGF)-β has been involved in regulating airway inflammation and remodeling in asthma, although it is unknown whether TGF-β can modulate bradykinin-associated bronchial hyperresponsiveness. To test whether TGF-β directly modulates airway smooth muscle (ASM) responsiveness to bradykinin, isolated murine tracheal rings were used to assess whether TGF-β alters ASM contractile responsiveness to bradykinin. Interestingly, we found TGF-β-treated murine rings (12.5 ng/ml, 18 h) exhibited increased expression of bradykinin 2 (B2) receptors and became hyperreactive to bradykinin, as shown by increases in maximal contractile responses and receptor distribution. We investigated the effect of TGF-β on bradykinin-evoked calcium signals since calcium is a key molecule regulating ASM excitation-contraction coupling. We reported that TGF-β, in a dose- (0.5–10 ng/ml) and time- (2–24 h) dependent manner, increased mRNA and protein expression of the B2 receptor in cultured human ASM cells. Maximal B2 receptor protein expression that colocalized with CD44, a marker of membrane cell surface, occurred after 18 h of TGF-β treatment and was further confirmed using fluorescence microscopy. TGF-β (2.5 ng/ml, 18 h) also increased bradykinin-induced intracellular calcium mobilization in fura-2-loaded ASM cells. TGF-β-mediated enhancement of calcium mobilization was not attenuated with indomethacin, a cyclooxygenase inhibitor. These data demonstrate for the first time that TGF-β may play a role in mediating airway hyperresponsiveness to bradykinin seen in asthmatics by enhancing ASM contractile responsiveness to bradykinin, possibly as a result of increased B2 receptor expression and signaling.