RESEARCH ARTICLE Mechanisms of Cigarette Smoke Effects on Human Airway Smooth Muscle

Cigarette smoke contributes to or exacerbates airway diseases such as asthma and COPD, where airway hyperresponsiveness and airway smooth muscle (ASM) proliferation are key features. While factors such as inflammation contribute to asthma in part by enhanc-ing agonist-induced intracellular Ca2+ ([Ca2+]i) responses of ASM, the mechanisms by which cigarette smoke affect ASM are still under investigation. In the present study, we test-ed the hypothesis that cigarette smoke enhances the expression and function of Ca2+ regu-latory proteins leading to increased store operated Ca2+ entry (SOCE) and cell proliferation. Using isolated human ASM (hASM) cells, incubated in the presence and absence cigarette smoke extract (CSE) we determined ([Ca2+]i) responses and expression of relevant pro-teins as well as ASM proliferation, reactive oxidant species (ROS) and cytokine generation. CSE enhanced [Ca2+]i responses to agonist and SOCE: effects mediated by increased ex-pression of TRPC3, CD38, STIM1, and/or Orai1, evident by attenuation of CSE effects when siRNAs against these proteins were used, particularly Orai1. CSE also increased hASM ROS generation and cytokine secretion. In addition, we found in the airways of pa-tients with long-term smoking history, TRPC3 and CD38 expression were significantly in-creased compared to life-long never-smokers, supporting the role of these proteins in smoking effects. Finally, CSE enhanced hASM proliferation, an effect confirmed by upregu-lation of PCNA and Cyclin E. These results support a critical role for Ca2+ regulatory pro-teins and enhanced SOCE to alter airway structure and function in smoking-related airway disease