Asthma associated cytokines IL-13 and TGF-β affect expression of calcium handling proteins in human airway smooth muscle (hASM) cells. We have previously shown an increase in TRPC6 expression and have now investigated the pathways involved and any change in calcium influx using Flufenamic acid (FFA). The hASM cells were derived from endobronchial biopsies of healthy volunteers, as approved by the local ethical committee and with informed consent. These were grown and then serum starved for 72h before treatment with cytokines. Sequence specific SMAD2 and SMAD3 siRNAs were cloned and transfected using the AmaxaTM nucleofector device (Lonza). Protein expression was measured via western blots. Intracellular calcium was measured as a ratio of 340/380nm excitation after loading with Fura3-PE/AM. Stimulating hASM cells with TGF-β alone and in combination with IL-13 caused a significant increase in TRPC6 protein (n=9 p<0.001 and n=17 p<0.001). TRPC6 splice variants were non-uniformly affected by the combination of IL-13 and TGF-β at one week; 75kDa variant increased by 720 ± 196 % compared to control (p=0.02) whereas the ≈100kDa variants increased by 185 ± 20 % (p=0.003). SMAD2/3 regulates this increase (see figure 1). The increase in TRPC6 expression by TGFβ is not altered by a scramble siRNA, but both SMAD2 and SMAD3 siRNAs, alone and in combination, inhibit this affect. It has also been found that the combination of IL-13 and TGF-β and TGF-β alone also reduced SMAD3 protein expression significantly (p<0.001, n =7). Flufenamic acid (FFA) has been shown to act as a TRPC6 agonist (Foster et al., 2009). Stimulating hASM cells treated with IL-13 and TGF-β with FFA decreased the peak calcium response at 72 hours (p=0.02, n=7-8) and 1 week (p=0.002, n=4-5). This decrease in peak calcium may be due to the high increase in expression of the 75kDa TRPC6 variant, which could be displacing the functional ≈100kDa variants. TGF-β but not IL-13 results in a non-uniform increase in TRPC6 splice variants in hASM cells which is regulated partly by SMAD2 and significantly by SMAD3. An alteration in the calcium handling properties of the hASM results, which as previously described in the literature, could play a key role in the pathophysiology of asthma.