Regulation of actin dynamics by wnt-5a: Implications for human airway smooth muscle contraction
2016
An important pathophysiological feature of asthma is airway hyperresponsiveness (AHR), characterized by exaggerated bronchoconstriction in which the airway smooth muscle (ASM) is fundamentally involved. How the ASM in asthmatics differs from that in non-asthmatics is a current focus for research. We have shown previously that WNT-5A is increasingly expressed in ASM of mild to moderate asthmatics. WNT proteins have been implicated in the regulation of cytoskeletal filaments and microtubules, thus, we tested the hypothesis that WNT-5A is a determinant of the contractile response of ASM through modulation of the cytoskeleton. Using bovine tracheal smooth muscle strips, we found that following pre-incubation with recombinant WNT-5A (500 ng/mL, 48 h), maximum isometric tension induced by histamine was increased (Emax = 1,67 fold of control, p <0.01). We used immortalized human ASM cells to study the underlying mechanisms. In myocytes loaded with the ratiometric calcium indicator, Fura-2, we found that neither direct exposure to WNT-5A nor pre-incubation (48 h) had any effect on calcium flux handling. Moreover, WNT-5A treatment for 24 h did not yield any detectable change in mRNA expression of the major calcium channels or receptors (IP3R, SERCA2 and RyR1/2/3). However, while direct exposure to WNT-5A (10-200 ng/mL, 24 h) had no effect on total alpha smooth muscle actin (α-SMA) protein, it did lead to increased abundance of filamentous actin (labelled with fluorochrome-conjugated phalloidin). These changes, which were visible after 2 hours, could be completely prevented by treatment with the Rho kinase inhibitor Y27632 (1 uM). Interestingly, ASM cells serum-deprived for 7 days to adopt a contractile phenotype also showed greater WNT-5A protein expression (1,99 fold of control, p <0.05) compared to the proliferative phenotype (serum-fed cells). We next assessed whether TGF-β1 played an upstream role in the WNT-5A response. We found that WNT-5A siRNA not only blocked TGF-β1-induced actin polymerization, but could also abrogate TGF-β1-induced accumulation of α-SMA protein. In line with this contention, adding WNT-5A on top of TGF-β1 synergistically induced α-SMA protein abundance (2,21 fold of TGF-β, p <0.01). In summary, WNT-5A promotes increased agonist-induced airway smooth muscle contraction, most likely by increasing actin polymerization and actin expression in concerted action with TGF-β1. These results may be relevant in the understanding of AHR and warrant further investigation.
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