Regulation of actin dynamics by WNT-5A: implications for human airway smooth muscle contraction

Abstract

A defining feature of asthma is airway hyperresponsiveness (AHR), which underlies the exaggerated bronchoconstriction response of asthmatics. The role of the airway smooth muscle (ASM) in AHR has garnered increasing interest over the years, but how asthmatic ASM differs from healthy ASM is still an active topic of debate. WNT-5A is increasingly expressed in asthmatic ASM and has been linked with Th2-high asthma. Due to its link with calcium and cytoskeletal remodelling, we propose that WNT-5A may modulate ASM contractility. We demonstrated that WNT-5A can increase maximum isometric tension in bovine tracheal smooth muscle strips. In addition, we show that WNT-5A is preferentially expressed in contractile human airway myocytes compared to proliferative cells, suggesting an active role in maintaining contractility. Furthermore, WNT-5A treatment drives actin polymerisation, but has no effect on intracellular calcium flux. Next, we demonstrated that WNT-5A directly regulates TGF-β1-induced expression of α-SMA via ROCK-mediated actin polymerization. These findings suggest that WNT-5A modulates fundamental mechanisms that affect ASM contraction and thus may be of relevance for AHR in asthma.

Figure 1. WNT-5A potentiates histamine-induced maximum isometric tension.

(a) Organ-cultured bovine tracheal smooth muscle strips were pre-incubated with WNT-5A (500 ng/mL) for 48 hours and a cumulative dose-response curve to histamine for the maximum isometric tension was constructed. Data represents five independent experiments, each performed in duplicate. (b) Alpha smooth muscle actin and smooth muscle myosin heavy chain immunoblot of lysates from bovine tracheal smooth muscle strips, normalised against GAPDH. Strips were pre-incubated with WNT-5A (500 ng/mL) for 48 hours. Data represents six independent experiments. Cropped images are shown. Full-length blots are presented in Supplementary Fig. 1. α-SMA and GAPDH blots were derived from the same gel; SMMHC was run on a separate gel. Data is expressed as the mean ± SEM.

Figure 2. Calcium handling is not affected by WNT-5A.

(a) Representative Fura-2 traces (left) and quantification (right) of intracellular calcium (Ca²⁺_i) changes with respect to time of immortalized human airway smooth muscle cells exposed to WNT-5A (500 ng/mL) or histamine (10⁻⁴ M). 30–40 cells were simultaneously measured in the presence of extracellular Ca²⁺ and collectively determined the response. Data represents four independent experiments. *vs pre-stim. (b) Airway smooth muscle cells were pre-incubated with WNT-5A (500 ng/mL) for 48 hours and a dose-response curve to histamine for the maximum Ca²⁺_i peak response was constructed. Data represents four independent experiments. (c) mRNA of airway smooth muscle cells pre-incubated with WNT-5A (500 ng/mL) for 24 hours was isolated and subjected to RT-qPCR. Data represents five independent experiments. Data is expressed as the mean ± SEM.

Figure 3. Involvement of WNT-5A in the regulation of alpha smooth muscle actin.

(a) Alpha smooth muscle actin, caveolin-1 and WNT-5A immunoblot of lysates from cultured human airway smooth muscle cells, normalised against GAPDH. Cell were grown to 50% confluence in serum-enriched (10% FBS) DMEM (serum-fed group) or grown to confluence and then serum-deprived in Ham’s F12 supplemented with ITS (serum-starved group) for 7 days. Cropped images are shown. Full-length blots are presented in Supplementary Fig. 1. α-SMA/GAPDH and caveolin-1/WNT-5A/GAPDH were derived from the same gel. Data represents four independent experiments. *vs serum-fed. (b) Airway smooth muscle actin immunoblot as performed in (a). Cells were treated with WNT-5A for 24 hours. Full-length blots are presented in Supplementary Fig. 1. Data represents three independent experiments. (c) Representative immunofluorescent images of a Phalloidin (F-actin, green) and DNAse I (G-actin, red) staining of airway smooth muscle cells exposed to WNT-5A (200 ng/mL) for 2 hours, and the corresponding quantification. White arrowhead points to filamentous actin. Dashed line represents a single cell boundary. Horizontal line represents the mean. Data is expressed as the mean ± SEM.

Figure 4. ROCK activation underlies WNT-5A-induced actin polymerisation.

(a) Representative immunofluorescent images of a Phalloidin (F-actin, green) and DNAse I (G-actin, red) staining of airway smooth muscle cells exposed to WNT-5A (200 ng/mL) for 2 hours in the presence or absence of Y27632 (1.0 μM), and the corresponding quantification. White arrowhead points to filamentous actin. Dashed line represents a single cell boundary. Horizontal line represents the mean. (b) Organ-cultured bovine tracheal smooth muscle strips were pre-incubated with WNT-5A (500 ng/mL) and/or Y27632 (1.0 μM) for 48 hours and a cumulative dose-response curve to histamine for the maximum isometric tension was constructed. *vs Control, $ vs WNT-5A + Y27632. Data represents five independent experiments, each performed in duplicate. Data is expressed as the mean ± SEM.

Figure 5. WNT-5A mediates TGF-β1 induced actin polymerization.

Representative immunofluorescent images of a Phalloidin (F-actin, green) and DNAse I (G-actin, red) staining of airway smooth muscle cells exposed to TGF-β1 (2 ng/mL) for 48 hours in the presence or absence of (a) latrunculin A (0.1 μM), or (b) WNT-5A-specific siRNA. White arrowhead points to filamentous actin. Dashed line represents a single cell boundary. Horizontal line represents the mean. (c) mRNA of airway smooth muscle cells pre-incubated with WNT-5A siRNA or control siRNA (30 pmol) for 36 hours was isolated and subjected to RT-qPCR. *vs control siRNA. Data represents three independent experiments. Data is expressed as the mean ± SEM.

Figure 6. TGF-β1-induced actin expression utilises WNT-5A.

(a) Alpha smooth muscle actin immunoblot of lysates from cultured human airway smooth muscle cells, normalised against GAPDH. Cells were treated with TGF-β1 (2 ng/mL) for 48 hours in the presence or absence of latrunculin A (0.1 μM). Cropped images are shown. Full-length blots are presented in Supplementary Fig. 1. α-SMA and GAPDH were derived from the same gel. Data represents four independent experiments. *vs control, $ vs TGF-β1. (b) Alpha smooth muscle actin immunoblot performed as in (a). Cells were transfected with a WNT-5A-specific siRNA construct or scrambled negative control siRNA and treated with TGF-β1 (2 ng/mL) for 48 hours. Full-length blots are presented in Supplementary Fig. 1. Data represents six independent experiments. *vs control siRNA, $ vs control siRNA + TGF-β1. (c) Alpha smooth muscle actin immunoblot performed as in (a). Cells were treated with TGF-β1 (2 ng/mL) for 48 hours in the presence or absence of WNT-5A (200 ng/mL). Full-length blots are presented in Supplementary Fig. 1. Data represents three independent experiments. *vs control, $ vs TGF-β1. Data is expressed as the mean ± SEM.