Abstract
Ca2+ entry into airway epithelia is important for activation of the NFAT family of transcription factors and expression of genes including epidermal growth factor that help orchestrate local inflammatory responses. However, the identity of epithelial Ca2+ channel that activates these transcriptional responses is unclear. In many other non-excitable cells, store-operated Ca2+ entry is a major route for Ca2+ influx and is mediated by STIM1 and Orai1 proteins. This study was performed to determine if store-operated Ca2+ channels were expressed in human bronchial epithelial cells and, if so, whether they coupled Ca2+ entry to gene expression. Cytoplasmic Ca2+ measurements, patch clamp recordings, RNAi knockdown and functional assays were used to identify and then investigate the role of these Ca2+ channels in activating the NFAT and c-fos pathways and EGF expression. STIM1 and Orai1 mRNA transcripts as well as proteins were robustly in epithelial cells and formed functional Ca2+ channels. Ca2+ entry through the channels activated expression of c-fos and EGF as well as an NFAT-dependent reporter gene. Store-operated Ca2+ entry was also important for epithelial cell migration in a scrape wound assay. These findings indicate that store-operated Ca2+ channels play an important role in stimulating airway epithelial cell gene expression and therefore comprise a novel potential therapeutic target for the treatment of chronic asthma and related airway disorders.
Highlights
A common theme in chronic asthma is significant remodelling of the airway wall [1]
Store-operated Ca2+ influx is present in 16HBE cells We tested for the presence of store-operated Ca2+ entry in the human bronchial epithelial cell line (16HBE) by stimulating cells with the sarcoplasmic/endoplasmic reticulum Ca2+ATPase inhibitor thapsigargin (2 mM) in Ca2+-free external solution [28,29]
We found that the extent of fast inactivation over a broad voltage range was indistinguishable between RBL-1 and 16HBE cells (Figure 3E), consistent with the presence of Ca2+ release-activated Ca2+ (CRAC) channels in the epithelial cell line
Summary
A common theme in chronic asthma is significant remodelling of the airway wall [1]. Changes include an increase in both smooth muscle mass and sensitivity to contractile triggers, accumulation of extracellular matrix below the epithelial basement membrane, appearance of gaps between epithelia and an increase in the number of mucus-producing goblet cells within the epithelial cell layer [2].Airway epithelia lie at the interface between a host and its environment and thereby comprise a first line of defence against air-borne allergens. We show that store-operated CRAC channels are present and functional in human airway epithelial cells. We found that the extent of fast inactivation over a broad voltage range was indistinguishable between RBL-1 and 16HBE cells (Figure 3E), consistent with the presence of CRAC channels in the epithelial cell line.
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