Abstract
One significant pathophysiological mechanism underlying Cystic Fibrosis (CF) is the hyperabsorption of sodium through ENaC. Further understanding of this misregulation could help develop novel therapeutic targets for treating CF. Channel Activating Proteases (CAPs) have been shown to increase ENaC activity but it is not clear whether CAP activity is physiologically regulated, or whether this regulation is defective in CF airways. We have previously shown that normal primary human bronchial epithelial cultures (HBECs) exhibit a trypsin-sensitive transepithelial voltage (Vt) when airway surface liquie (ASL) has accumulated for 48 hours. Conversely, freshly washed normal and CF HBECs, as well as CF HBECs with accumulated ASLs, remained trypsin-insensitive indicating that ENaC is maximally activated in these cultures. This implies that the CAP inhibitor is soluble and accumulates in the ASL. Additionally, accumulation of this inhibitor is unable to inhibit ENaC conductance in CF cultures. (Tarran et al., 2006). We then searched for soluble CAP inhibitors with trypsin-coated beads, which were added to the ASL. Albumin-coated beads were used as a control for non-specific binding. Using mass spectrometry, we identified sPLUNC1. This protein is secreted, in vivo and in vitro, in the ASL although its function is unknown. To understand of the role of sPLUNC1 in airway physiology, we examined the capacity for ASL regulation in HBECs treated with either anti-sPLUNC1 or anti-luciferase shRNA as a control. qPCR verified a >90% knockdown efficiency, which resulted in significant ASL volume depletion in the sPLUNC1 knockdown HBECs as compared to the controls (ctrl, 9.0 ?1.8 uM; anti-sPLUNC1, 5 ?0.9uM; n=5). This was accompanied by a persistent trypsin-sensitive Vt in sPLUNC1-defecient cultures, indicating that they could no longer regulate ENaC. Our next aim was to examine the effect of sPLUNC1 on ENaC current. To do this, xenopus oocytes were injected with sPLUNC1 and ?,?,? ENaC subunits. sPLUNC1 was detected in the media of these oocyte by Western blot and showed a 43% (? 4%, n=40) inhibition of ENaC current when compared to ENaC injected oocytes alone. Western blot analysis revealed that this reduction in current was accompanied by a reduction in cleavage of ?, ? and ? ENaC subunits suggesting that SPLUNC1 prevents cleavage of ENaC by serine proteases. These findings indicate an important role for sPLUNC1 in airway physiology as it regulates ENaC current and consequently, ASL volume. Understanding the mechanism of sPLUNC1 may provide insight into novel therapies for regulating hyperactive ENaC in CF.