Proceedings of The Physiological Society
University College Dublin (2009) Proc Physiol Soc 15, C103
Knockdown of SERCA-2 in human airway smooth muscle from healthy subjects recapitulates a phenotype associated with asthma
O. O. Ojo1, K. Mahn1, M. R. Holt3, S. J. Hirst2, T. H. Lee1, J. P. Ward3
1. MRC and Asthma UK Centre in Allergic Mechanisms of Asthma, King's College London, London, United Kingdom. 2. Department of Physiology, Monash University, Melbourne, Victoria, Australia. 3. King's College London, London, United Kingdom.
Cytosolic [Ca2+] plays a critical role in the function of airway smooth muscle (ASM) and the sarco/endoplasmic reticulum [Ca2+] ATPase (SERCA) is an important mechanism for regulating cytosolic [Ca2+]. ASM from asthmatics is characterised by increased proliferative and migratory responses, and we have reported that ASM cells obtained from asthmatic volunteers exhibit abnormal Ca2+ homeostasis arising from reduced SERCA-2 expression (Mahn et al, 2007). The aim of this study was to determine whether knockdown of SERCA-2 in ASM cells from healthy subjects could reproduce features of the pro-asthmatic phenotype. METHODS: The study was approved by the local ethical committee and informed consent obtained. ASM cells from subjects with and without mild asthma were cultured from endobronchial biopsies (n=10). ASM cells from healthy donors were transfected with siRNA against SERCA-2 (sASM) or a scrambled siRNA (cASM). Knockdown efficiency was confirmed by Western blot. Changes in cytosolic [Ca2+] were estimated using Fura PE3; proliferation was assessed by 3[H]-thymidine incorporation with migration determined by cell spreading. RESULTS: sASM exhibited a 30% reduction in SERCA-2 protein expression, comparable with that found in ASM from asthmatics. Bradykinin (1µM)-evoked [Ca2+] release was reduced by 47±24% (mean±SEM) in sASM compared with cASM (p<0.05; n=4), consistent with reduced store capacity. Recovery of cytosolic [Ca2+] to baseline, an index of SERCA-mediated Ca2+ uptake, was increased from 109±27 sec in cASM to 274±78 sec in sASM (p<0.05; n=4), similar again to that found with ASM from asthmatics (Mahn et al, 2007). SERCA-2 knockdown also increased 10% fetal bovine serum (FBS)-dependent proliferation from 285±10% (versus FBS-free medium) in cASM, to 417±26% in sASM (p<0.02; n=3). The latter was comparable with responses in ASM from asthmatics (450±90%; n=3). Cell spreading, taken as a functional readout of cell motility, showed increased spreading or early laminapodia and filapodia formation (expressed as pixels/3min) in sASM (387±118) compared with cASM (90±28; n=7; p<0.05). Conclusion: Knockdown of SERCA-2 in healthy ASM recapitulates key changes in cellular function that have been previously observed in ASM from asthmatics. This suggests that reduced SERCA-2 expression, with consequent changes in cytosolic Ca2+ homeostasis, may play an important role in the development of ASM changes in airway wall remodeling in asthma
Where applicable, experiments conform with Society ethical requirements