Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC284

Poster Communications

The asthma associated cytokines IL-13 and TGFβ synergistically increase expression of TRPC6 in human airway smooth muscle

Y. Shaifta1, C. Ribeiro1, C. Karner1, J. P. Ward1

1. Asthma Allergy and Respiratory Science, King''s College London, London, United Kingdom.


Asthma has been associated with a defect in intracellular calcium homeostasis of airway smooth muscle (ASM) (Mahn et al., 2010), and we have shown that at least part of this is related to decreased expression of the sarcoendoplasmic reticulum calcium ATPase type 2 isoform (SERCA-2) (Mahn et al., 2009), although increased influx of calcium through channels has also been implicated (Trian et al., 2007). There is little indication that these defects in calcium handling are related to any genetic defect, and evidence from our laboratory and others suggests that at least the reduced expression of SERCA-2 is instead related to the action of asthma-associated inflammatory cytokines such as transforming growth factor β (TGFβ) and interleukin-13 (IL-13). In terms of calcium entry pathways, it has been suggested that channels formed of TRPC6 may make a major contribution to agonist-induced calcium entry in ASM (Mahn et al., 2010). We therefore examined the effects of treatment with TGFβ and IL-13 on human bronchial ASM cells in primary culture, to determine whether these conditions also affected expression of TRPC6. Human ASM cells were cultured from endobronchial biopsies taken from healthy subjects (n=4-6), with informed consent and local ethical permission.. Following growth-arrest, cells were stimulated with 10 ng/ml IL-13 and/or 10 ng/ml TGFβ1. TRPC6 mRNA expression was analysed at 6hrs using real-time qPCR and protein expression at 24hrs using Western blot. Antibodies for TRPC6 are notoriously poor, and we therefore first validated a new antibody using a TRPC6-GFP construct expressed in HEK293 cells, using both the TRPC6 antibody (Sigma) and a high quality GFP antibody. In ASM cells, IL-13 had no significant effect on expression of either TRPC6 message or protein. TGFβ1 however significantly increased mRNA expression by a factor of 3 +/- 0.6 (SEM, p<0.05), and in combination with IL-13 there showed a synergistic effect in that expression was increased by a factor of 7.5 +/- 1.1 (P<0.01). Similarly, Western blot revealed no change in TRCP6 protein expression at the predicted MW of ~100kD (114 +/- 14% control, n=4) following treatment with IL-13, whereas TGFβ1 alone caused an significant increase to 181 +/- 22% (P<0.01). Again, TGFβ1 and IL-13 in combination caused a further synergistic increase in TRPC6 protein to 248 +/- 27% of control (P<0.001). These data suggest that the inflammatory milieu associated with asthma causes alterations to both ASM calcium sequestration mechanisms and calcium influx mechanisms, which together could be responsible for the exaggerated response to agonists that is associated with airway hyperresponsiveness in asthma. The apparent synergistic effect of IL-13 and TGFβ1 may be of particular importance, but the underlying mechanisms currently remain obscure.

Where applicable, experiments conform with Society ethical requirements