TY - JOUR T1 - TGF-β alters TRPC6 expression and calcium handling via smad2/3 in human airway smooth muscle JF - European Respiratory Journal JO - Eur Respir J VL - 42 IS - Suppl 57 SP - P556 AU - David Wright AU - Yasin Shaifta AU - Vladimir Snetkov AU - Jeremy Ward Y1 - 2013/09/01 UR - http://erj.ersjournals.com/content/42/Suppl_57/P556.abstract N2 - Rationale: IL-13 and TGF-β have been shown to affect expression of calcium handling proteins in airway smooth muscle (ASM). We hypothesise they also alter expression of TRPC6 channel proteins.Methods: Cultured ASM cells were serum starved for 72h before treatment. SMAD2 and SMAD3 siRNAs were transfected using AmaxaTM nucleofector (Lonza). Protein expression was measured via western blots, and intracellular calcium with Fura3-PE/AM.Results: Treatment of ASM cells for 1 week with TGF-β (10ng/ml) increased TRPC6 protein (285 ± 48% n=17 p<0.001); IL-13 (10ng/ml) had no additional effect (279 ± 37% n=17 p<0.001). TRPC6 splice variants were non-uniformly affected; a 75kDa variant increased by 720 ± 196 % (p=0.02) whereas the functional ∼100kDa variants increased by 185 ± 20 % (p=0.003). Knockdown of SMAD2 or 3 abolished the increase.TGF-β significantly reduced SMAD3 protein (p<0.001, n=7). Following treatment with TGF-β, elevation in calcium elicited by bradykinin (0.5μM) or the TRPC6 agonist flufenamic acid (10uM) was decreased at 72 hours (p=0.02, n=7-8) and 1 week (p=0.002, n=4-5).Conclusions: TGF-β but not IL-13 results in differential increases in TRPC6 splice variant expression, regulated by SMAD2/3. As this was accompanied by reduced calcium mobilisation, we speculate increased expression of non-functional TRPC6 variants may contribute to impaired calcium handling in asthma. ER -