Gregory E. Conner

Pannexin 1 Contributes to ATP Release in Airway Epithelia

ATP is a paracrine regulator of critical airway epithelial cell functions, but the mechanism of its release is poorly understood. Pannexin (Panx) proteins, related to invertebrate innexins, form channels (called pannexons) that are able to release ATP from several cell types. Thus, ATP release via pannexons was examined in airway epithelial cells. Quantitative RT-PCR showed Panx1 expression in normal human airway epithelial cells during redifferentiation at the air-liquid interface (ALI), at a level comparable to that of alveolar macrophages; Panx3 was not expressed. Immunohistochemistry showed Panx1 expression at the apical pole of airway epithelia. ALI cultures exposed to hypotonic stress released ATP to an estimated maximum of 255 (+/-64) nM within 1 minute after challenge (n = 6 cultures from three different lungs) or to approximately 1.5 (+/-0.4) microM, recalculated to a normal airway surface liquid volume. Using date- and culture-matched cells (each n > or = 16 from 4 different lungs), the pannexon inhibitors carbenoxolone (10 microM) and probenecid (1 mM), but not the connexon inhibitor flufenamic acid (100 microM), inhibited ATP release by approximately 60%. The drugs affected Panx1 currents in Xenopus oocytes expressing exogenous Panx1 correspondingly. In addition, suppression of Panx1 expression using lentivirus-mediated production of shRNA in differentiated airway epithelial cells inhibited ATP release upon hypotonic stress by approximately 60% as well. These data not only show that Panx1 is expressed apically in differentiated airway epithelial cells but also that it contributes to ATP release in these cells.

Hyaluronan serves a novel role in airway mucosal host defense

Enzymes secreted onto epithelial surfaces play a vital role in innate mucosal defense, but are believed to be steadily removed from the surface by mechanical actions. Thus, the amount and availability of enzymes on the surface are thought to be maintained by secretion. In contrast to this paradigm, we show here that enzymes are retained at the apical surface of the airway epithelium by binding to surface‐associated hya‐luronan, providing an apical enzyme pool ‘ready for use’ and protected from ciliary clearance. We have studied lactoperoxidase, which prevents bacterial colonization of the airway, and kallikrein, which mediates allergic bronchoconstriction that limits the inhalation of noxious substances. Binding to hyaluronan inhibits kallikrein, which is needed only in certain situations, whereas lactoperoxidase, useful at all times, does not change its activity. Hyaluronan itself interacts with the receptor for hyaluronic acid‐mediated motility (RHAMM or CD168) that is expressed at the apex of ciliated airway epithelial cells. Functionally, hyaluronan binding to RHAMM stimulates ciliary beating. Thus, hyaluronan plays a previouslyunrecognized pivotal role in mucosal host defense by stimulating ciliaryclearance of foreign material while simultaneously retaining enzymes important for homeostasis at the apical surface so that they cannot be removed by ciliary action.—Forteza, R., Lieb, T., Aoki, T., Savani, R. C., Conner, G. E., Salathe, M. Hyaluronan serves a novel role in airway mucosal host defense. FASEB J. 15, 2179–2186 (2001)

Soluble adenylyl cyclase is localized to cilia and contributes to ciliary beat frequency regulation via production of cAMP

Ciliated airway epithelial cells are subject to sustained changes in intracellular CO2/HCO3 − during exacerbations of airway diseases, but the role of CO2/HCO3 −-sensitive soluble adenylyl cyclase (sAC) in ciliary beat regulation is unknown. We now show not only sAC expression in human airway epithelia (by RT-PCR, Western blotting, and immunofluorescence) but also its specific localization to the axoneme (Western blotting and immunofluorescence). Real time estimations of [cAMP] changes in ciliated cells, using FRET between fluorescently tagged PKA subunits (expressed under the foxj1 promoter solely in ciliated cells), revealed CO2/HCO3 −-mediated cAMP production. This cAMP production was specifically blocked by sAC inhibitors but not by transmembrane adenylyl cyclase (tmAC) inhibitors. In addition, this cAMP production stimulated ciliary beat frequency (CBF) independently of intracellular pH because PKA and sAC inhibitors were uniquely able to block CO2/HCO3 −-mediated changes in CBF (while tmAC inhibitors had no effect). Thus, sAC is localized to motile airway cilia and it contributes to the regulation of human airway CBF. In addition, CO2/HCO3 − increases indeed reversibly stimulate intracellular cAMP production by sAC in intact cells.

The Lactoperoxidase System Links Anion Transport To Host Defense in Cystic Fibrosis

Chronic respiratory infections in cystic fibrosis result from CFTR channel mutations but how these impair antibacterial defense is less clear. Airway host defense depends on lactoperoxidase (LPO) that requires thiocyanate (SCN−) to function and epithelia use CFTR to concentrate SCN− at the apical surface. To test whether CFTR mutations result in impaired LPO‐mediated host defense, CF epithelial SCN− transport was measured. CF epithelia had significantly lower transport rates and did not accumulate SCN− in the apical compartment. The lower CF [SCN−] did not support LPO antibacterial activity. Modeling of airway LPO activity suggested that reduced transport impairs LPO‐mediated defense and cannot be compensated by LPO or H2O2 upregulation.

Transcellular thiocyanate transport by human airway epithelia

Human airway mucosa synthesizes and secretes lactoperoxidase (LPO). As H2O2 and thiocyanate (SCN−) are also present, a functional LPO antibacterial defence system exists in the airways. SCN− concentrations in several epithelial secretions are higher than in serum, although the mechanisms of transepithelial transport and accumulation in these secretions are unknown. To examine SCN− accumulation in secretions, human airway epithelial cells, re‐differentiated at the air–liquid interface, were used in open‐circuit conditions. [14C]SCN−, in the basolateral medium, was transported across the epithelium and concentrated tenfold at the apical surface. Measurement of the transepithelial potential showed that the basolateral compartment was positive relative to the apical surface (13.7 ± 1.8 mV) and therefore unfavourable for passive movement of SCN−. Transport was dependent on basolateral [SCN−] and saturable (Km,app= 69 ± 25 μm); was inhibited by increased apical [SCN−]; and was dependent on the presence of basolateral Na+. Perchlorate (Ki,app= 0.6 ± 0.05 μm) and iodide (Ki,app= 9 ± 8 μm) in the basolateral medium reversibly inhibited transport, but furosemide did not. Iodide was also transported (Km,app= 111 ± 69 μm). RT‐PCR and immunohistochemistry confirmed expression of Na+−I− symporter (NIS) in the airways. SCN− transport was insensitive to apical disulphonic acid Cl− channel blockers, but sensitive to apical glibenclamide and arylaminobenzoates. Forskolin and dibutyryl cAMP increased transport. These data suggest SCN− transport may occur through basolateral NIS‐mediated SCN− concentration inside cells, followed by release through an apical channel, perhaps cystic fibrosis transmembrane conductance regulator.

Oxidative epithelial host defense is regulated by infectious and inflammatory stimuli

Epithelia express oxidative antimicrobial protection that uses lactoperoxidase (LPO), hydrogen peroxide (H(2)O(2)), and thiocyanate to generate the reactive hypothiocyanite. Duox1 and Duox2, found in epithelia, are hypothesized to provide H(2)O(2) for use by LPO. To investigate the regulation of oxidative LPO-mediated host defense by bacterial and inflammatory stimuli, LPO and Duox mRNA were followed in differentiated primary human airway epithelial cells challenged with Pseudomonas aeruginosa flagellin or IFN-gamma. Flagellin upregulated Duox2 mRNA 20-fold, but upregulated LPO mRNA only 2.5-fold. IFN-gamma increased Duox2 mRNA 127-fold and upregulated LPO mRNA 10-fold. DuoxA2, needed for Duox2 activity, was also upregulated by flagellin and IFN-gamma. Both stimuli increased H(2)O(2) synthesis and LPO-dependent killing of P. aeruginosa. Reduction of Duox1 by siRNA showed little effect on basal H(2)O(2) production, whereas Duox2 siRNA markedly reduced basal H(2)O(2) production and resulted in an 8-fold increase in Nox4 mRNA. In conclusion, large increases in Duox2-mediated H(2)O(2) production seem to be coordinated with increases in LPO mRNA and, without increased LPO, H(2)O(2) levels in airway secretion are expected to increase substantially. The data suggest that Duox2 is the major contributor to basal H(2)O(2) synthesis despite the presence of greater amounts of Duox1.

Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells

Airway ciliary beat frequency regulation is complex but in part influenced by cyclic adenosine monophosphate (cAMP)-mediated changes in cAMP-dependent kinase activity, yet the cAMP concentration required for increases in ciliary beat frequency and the temporal relationship between ciliary beat frequency and cAMP changes are unknown. A lentiviral gene transfer system was developed to express a fluorescence resonance energy transfer (FRET)-based cAMP sensor in ciliated cells. Expression of fluorescently tagged cAMP-dependent kinase subunits from the ciliated-cell-specific foxj1 promoter enhanced expression in fully differentiated ciliated human airway epithelial cells, and permitted simultaneous measurements of ciliary beat frequency and cAMP (represented by the FRET ratio). Apical application of forskolin (1 μM, 10 μM, 20 μM) and, in permeabilized cells, basolateral cAMP (20 μM, 50 μM, 100 μM) caused dose-dependent, albeit similar and simultaneous–increases in cAMP and ciliary beat frequency. However, decreases in cAMP preceded decreases in ciliary beat frequency, suggesting that either cellular cAMP decreases before ciliary cAMP or the dephosphorylation of target proteins by phosphatases occur at a rate slower than the rate of cAMP hydrolysis.

Regulation of human airway ciliary beat frequency by intracellular pH.

pHi affects a number of cellular functions, but the influence of pHi on mammalian ciliary beat frequency (CBF) is not known. CBF and pHi of single human tracheobronchial epithelial cells in submerged culture were measured simultaneously using video microscopy (for CBF) and epifluorescence microscopy with the pH‐sensitive dye BCECF. Baseline CBF and pHi values in bicarbonate‐free medium were 7.2 ± 0.2 Hz and 7.49 ± 0.02, respectively (n= 63). Alkalization by ammonium pre‐pulse to pHi 7.78 ± 0.02 resulted in a 2.2 ± 0.1 Hz CBF increase (P < 0.05). Following removal of NH4Cl, pHi decreased to 7.24 ± 0.02 and CBF to 5.8 ± 0.1 Hz (P < 0.05). Removal of extracellular CO2 to change pHi resulted in similar CBF changes. Pre‐activation of cAMP‐dependent protein kinase (10 μm forskolin), broad inhibition of protein kinases (100 μm H‐7), inhibition of PKA (10 μm H‐89), nor inhibition of phosphatases (10 μm cyclosporin + 1.5 μm okadaic acid) changed pHi‐mediated changes in CBF, nor were they due to [Ca2+]i changes. CBF of basolaterally permeabilized human tracheobronchial cells, re‐differentiated at the air–liquid interface, was 3.9 ± 0.3, 5.7 ± 0.4, 7.0 ± 0.3 and 7.3 ± 0.3 Hz at basolateral i.e., intracellular pH of 6.8, 7.2, 7.6 and 8.0, respectively (n= 18). Thus, intracellular alkalization stimulates, while intracellular acidification attenuates human airway CBF. Since phosphorylation and [Ca2+]i changes did not seem to mediate pHi‐induced CBF changes, pHi may directly act on the ciliary motile machinery.

Functional Apical Large Conductance, Ca2+-activated, and Voltage-dependent K+ Channels Are Required for Maintenance of Airway Surface Liquid Volume

Large conductance, Ca2+-activated, and voltage-dependent K+ (BK) channels control a variety of physiological processes in nervous, muscular, and renal epithelial tissues. In bronchial airway epithelia, extracellular ATP-mediated, apical increases in intracellular Ca2+ are important signals for ion movement through the apical membrane and regulation of water secretion. Although other, mainly basolaterally expressed K+ channels are recognized as modulators of ion transport in airway epithelial cells, the role of BK in this process, especially as a regulator of airway surface liquid volume, has not been examined. Using patch clamp and Ussing chamber approaches, this study reveals that BK channels are present and functional at the apical membrane of airway epithelial cells. BK channels open in response to ATP stimulation at the apical membrane and allow K+ flux to the airway surface liquid, whereas no functional BK channels were found basolaterally. Ion transport modeling supports the notion that apically expressed BK channels are part of an apical loop current, favoring apical Cl− efflux. Importantly, apical BK channels were found to be critical for the maintenance of adequate airway surface liquid volume because continuous inhibition of BK channels or knockdown of KCNMA1, the gene coding for the BK α subunit (KCNMA1), lead to airway surface dehydration and thus periciliary fluid height collapse revealed by low ciliary beat frequency that could be fully rescued by addition of apical fluid. Thus, apical BK channels play an important, previously unrecognized role in maintaining adequate airway surface hydration.

Proteolytic Activation of Human Procathepsin D

Procathepsin D is a short-lived inactive precursor of the lysosomal aspartyl protease, cathepsin D. Pulse-chase analysis using radiolabeled amino acids demonstrated the existence of several biosynthetic intermediates during formation of mature cathepsin D (summarized in Figure 1). Procathepsin D is capable of autocatalytic cleavage to pseudocathepsin D. This was demonstrated using small quantities of procathepsin D isolated from cell culture media as well as using a non-glycosylated form of procathepsin D synthesized in a bacterial expression system. Complete conversion to the single-chain cathepsin D appears to require a second enzyme which is inhibited by leupeptin. This conclusion was drawn from the inability to produce single-chain enzyme from either procathepsin D or pseudocathepsin D in vitro as well as observations from addition of protease inhibitors to cell cultures. It appears that the conversion of procathepsin D to active single-chain enzyme falls between the paradigms of pepsinogen autoactivation and prorenin conversion by a separate enzyme.