Rho Signaling Regulates Pannexin 1-mediated ATP Release from Airway Epithelia

Abstract

ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia.The mucociliary clearance process that removes foreign particles and pathogens from the airways is the primary innate defense mechanism in the lung (1). Nucleotides and nucleosides within the airway surface liquid (ASL) 3 regulate key components of mucociliary clearance via activation of epithelial cell surface purinergic receptors (2, 3). ATP activates the G q -coupled P2Y 2 receptor that promotes mucin secretion and ciliary beat frequency and regulates electrolyte transport and ASL volume production by inhibiting sodium absorption (4 -10) and promoting cystic fibrosis transmembrane conductance regulator and Ca 2ϩ -activated Cl Ϫ channel activity (11-16). Adenosine, generated from the hydrolysis of ATP, activates the G scoupled A 2b receptor that promotes cyclic AMP-regulated cystic fibrosis transmembrane conductance regulator Cl Ϫ channel activity (17) and increases cilia beat frequency (5). Although ATP and adenosine are naturally occurring signaling molecules in ASL (18 -22), the mechanisms of airway epithelial ATP release into the ASL are poorly understood.The lung epithelia exhibit a complex cellular composition, and thus, several mechanisms and pathways are likely involved in the release of nucleotides onto airway surfaces. Our recent studies with goblet-like cell models indicate that ATP and other nucleotides are released concomitantly with MUC5AC, a secretory mucin...

Pannexin 1, an ATP Release Channel, Is Activated by Caspase Cleavage of Its Pore-associated C-terminal Autoinhibitory Region

“…In other cases of ball-and-chain inhibition, there has been no physiological mechanism described for cutting the chain to permanently remove channel inhibition, as we have found for caspase-mediated cleavage and activation of hPANX1. Although this cleavage-based mechanism seems appropriate for a terminal process such as apoptosis, we expect that more subtle mechanisms will account for other forms of PANX1 activation associated with different physiological conditions (10,26,27). Nevertheless, because C-terminal inhibition in the intact channel depends on its association with the pore, our data suggest that these other PANX1 activation mechanisms will also require some disruption of the C terminus from its inhibitory interaction site within the channel pore.…”

The cellular life of pannexins

“…15,16,35,52 Surprisingly, the overall viability and health of Panx1 null mice is relatively unaltered, which seems counterintuitive when we consider the ubiquitous expression of Panx1 in mouse organs and tissues. These findings raise three essential questions: is Panx1 not that important after all?…”

“…Some options include preadsorption with cognate peptides as shown in Figures 6 and 7, or the use of tissues and cells from Panx null mouse models as has been reported for the Panx1 CT-395 antibody. 15,35 Although Panx3 was found within intracellular compartments in rat cochlear bone, 46 it was also found at the cell surface and within intracellular compartments in murine chondrocytes and chondrogenic cell lines (ATDC5). 21 Panx2, on the other hand, has been localized primarily to intracellular compartments 9,38,39 and displays only limited cell surface distribution that is increased when coexpressed with Panx1.…”

“…16 In another case, a significant reduction in the hypotonicity-induced ATP release was observed in bronchial epithelial cells and excised tracheas from Panx1 null mice. 15 It is likely that further phenotypes will be revealed by placing Panx1 null mice under different sources of stress. It is possible though that the other two members of the pannexin family could be compensating for the lack of Panx1 as either Panx2 or Panx3 is often coexpressed with Panx1 in many cells and tissues.…”

Function and Regulation of Pannexin 1 Channels in the Vascular Endothelium

“…These new peptides have shed light on the gating of Cx43 hemichannels which appears to be tightly regulated by interactions between the intracellular domains of Cx43. Due to the increasing success of mimetic peptides in targeting Cx hemichannels, a mimetic peptide of the first extracellular loop of Panx1 termed 10 panx1 was developed and has subsequently been shown to significantly inhibits channel currents, dye uptake and ATP release mediated by Panx1 in a number of cell types [164,196,197]. The Panx mimetic peptide has not escaped the issue of cross-inhibition, however, as it was recently shown that the 10 panx1 peptide also blocks Cx46 hemichannel currents [193].…”