WNT-5A and WNT-5B modulate calcium homeostasis in airway smooth muscle

Rationale Airway hyperresponsiveness is a common feature of asthma explained in part by an excessive contractile response of the airway smooth muscle (ASM). The underlying mechanisms are complex and in need of study. WNT-5A and WNT-5B, two members of the WNT signaling pathway, may be of significance, as we have previously shown an active role for these ligands in cultured ASM and an increased WNT-5A expression in ASM isolated from asthmatics compared to healthy controls. Methods Using immortalized human bronchial smooth muscle cell lines, we investigated the role of WNT-5A and WNT-5B in ASM. Adopting a gain-of-function approach, we treated cells with recombinant WNT-5A or WNT-5B (100-1000 ng/ml) up to 96 hours. Furthermore, bovine tracheal smooth muscle (BTSM) strips were isolated and treated with recombinant WNT-5A or WNT-5B to determine isometric contraction. Results An initial microarray study indicated a modulatory role for WNT-5A, highlighting a wide subset of genes involved in mitochondrial function. Subsequent studies confirmed this role, indicating that treatment with recombinant WNT-5A and WNT-5B induced a strong depolarization of the mitochondrial membrane (Δψm), as was assessed with the fluorescent probe JC-1. Depolarization occurred dose dependently where the strongest effect was observed 48 hours following stimulation (32% and 22% for WNT-5A and WNT-5B respectively). Changes in Δψm were not due to initiation of apoptosis, as we could not find any difference in intracellular ATP levels nor in expression of mitochondrial mediated caspase-9 initiation. In addition, an Alamar Blue conversion assay indicated increased cellular viability following WNT treatment. Due to their crucial role in calcium buffering, we hypothesized that the observed mitochondrial membrane depolarization was calcium dependent. Using the calcium-sensitive ratiometric fluorescent dye fura-2, the involvement of calcium was corroborated as we observed a strong increase in resting cytosolic calcium levels following WNT-5A or WNT-5B 48 hour pre-treatment. Interestingly, direct WNT stimulation did not induce intracellular calcium release. To extent our findings in a functional setting, we pre-treated isolated BTSM strips with WNT-5A or WNT-5B for 48 hours. WNT-5A increased maximal contractility (25%) and sensitivity to histamine in isolated BTSM strips, whereas WNT-5B had no effect. Conclusions Our findings indicate a role for WNT-5A and WNT-5B in the regulation of calcium levels in ASM cells that likely includes a previously unidentified mitochondrial component. These findings may be of relevance to increased ASM contractility in asthma.