Sherif E. Gabriel

Transmembrane Protein 16A (TMEM16A) Is a Ca2+-regulated Cl– Secretory Channel in Mouse Airways

For almost two decades, it has been postulated that calcium-activated Cl– channels (CaCCs) play a role in airway epithelial Cl– secretion, but until recently, the molecular identity of the airway CaCC(s) was unknown. Recent studies have unequivocally identified TMEM16A as a glandular epithelial CaCC. We have studied the airway bioelectrics of neonatal mice homozygous for a null allele of Tmem16a (Tmem16a–/–) to investigate the role of this channel in Cl– secretion in airway surface epithelium. When compared with wild-type tracheas, the Tmem16a–/– tracheas exhibited a >60% reduction in purinoceptor (UTP)-regulated CaCC activity. Other members of the Tmem16 gene family, including Tmem16f and Tmem16k, were also detected by reverse transcription-PCR in neonatal tracheal epithelium, suggesting that other family members could be considered as contributing to the small residual UTP response. TMEM16A, however, appeared to contribute little to unstimulated Cl– secretion, whereas studies with cystic fibrosis transmembrane conductance regulator (CFTR)-deficient mice and wild-type littermates revealed that unstimulated Cl– secretion reflected ∼50% CFTR activity and ∼50% non-Tmem16a activity. Interestingly, the tracheas of both the Tmem16a–/– and the CFTR–/– mice exhibited similar congenital cartilaginous defects that may reflect a common Cl– secretory defect mediated by the molecularly distinct Cl– channels. Importantly, the residual CaCC activity in Tmem16a–/– mice appeared inadequate for normal airway hydration because Tmem16a–/– tracheas exhibited significant, neonatal, lumenal mucus accumulation. Our data suggest that TMEM16A CaCC-mediated Cl– secretion appears to be necessary for normal airway surface liquid homeostasis.

POLARIZED DISTRIBUTION OF KEY MEMBRANE TRANSPORT PROTEINS IN THE RAT SUBMANDIBULAR GLAND

Abstract Immunofluorescence labelling and confocal microscopy were employed to examine the polarized distribution of several membrane transport proteins believed to be essential for salivary secretion in the rat submandibular gland. The Na+/K+-ATPase, Na+/H+ exchanger isoform 1 (NHE1), and the secretory Na+/K+/2Cl–cotransporter isoform were all found in the basolateral membranes of acinar and intralobular duct cells. Anion exchanger isoform 2 (AE2) was found only in the basolateral membranes of acinar cells, while AE1 was absent from glandular epithelial cells. Aquaporin 5 was detected in the apical membranes of acinar cells, while the cystic fibrosis transmembrane conductance regulator was found only in apical membranes of intralobular duct cells. NHEs 2 and 3 were found in the apical membranes of both acinar and intralobular duct cells. Our results are generally consistent with the expected distribution of most transporters based on previous physiological and pharmacological experiments. However, the apical localization of NHEs 2 and 3, and the presence of the secretory isoform of the Na+/K+/2Cl–cotransporter in intralobular duct cells were not predicted.

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Delivering CFTR to ciliated cells of cystic fibrosis (CF) patients fully restores ion and fluid transport to the lumenal surface of airway epithelium and returns mucus transport rates to those of non-CF airways.

Coordinated release of nucleotides and mucin from human airway epithelial Calu-3 cells

The efficiency of the mucociliary clearance (MCC) process that removes noxious materials from airway surfaces depends on the balance between mucin secretion, airway surface liquid (ASL) volume, and ciliary beating. Effective mucin dispersion into ASL requires salt and water secretion onto the mucosal surface, but how mucin secretion rate is coordinated with ion and, ultimately, water transport rates is poorly understood. Several components of MCC, including electrolyte and water transport, are regulated by nucleotides in the ASL interacting with purinergic receptors. Using polarized monolayers of airway epithelial Calu‐3 cells, we investigated whether mucin secretion was accompanied by nucleotide release. Electron microscopic analyses of Calu‐3 cells identified subapical granules that resembled goblet cell mucin granules. Real‐time confocal microscopic analyses revealed that subapical granules, labelled with FM 1‐43 or quinacrine, were competent for Ca2+‐regulated exocytosis. Granules containing MUC5AC were apically secreted via Ca2+‐regulated exocytosis as demonstrated by combined immunolocalization and slot blot analyses. In addition, Calu‐3 cells exhibited Ca2+‐regulated apical release of ATP and UDP‐glucose, a substrate of glycosylation reactions within the secretory pathway. Neither mucin secretion nor ATP release from Calu‐3 cells were affected by activation or inhibition of the cystic fibrosis transmembrane conductance regulator. In SPOC1 cells, an airway goblet cell model, purinergic P2Y2 receptor‐stimulated increase of cytosolic Ca2+ concentration resulted in secretion of both mucins and nucleotides. Our data suggest that nucleotide release is a mechanism by which mucin‐secreting goblet cells produce paracrine signals for mucin hydration within the ASL.

Regulation of murine airway surface liquid volume by CFTR and Ca2+-activated Cl- conductances

Two Cl− conductances have been described in the apical membrane of both human and murine proximal airway epithelia that are thought to play predominant roles in airway hydration: (1) CFTR, which is cAMP regulated and (2) the Ca2+-activated Cl− conductance (CaCC) whose molecular identity is uncertain. In addition to second messenger regulation, cross talk between these two channels may also exist and, whereas CFTR is absent or defective in cystic fibrosis (CF) airways, CaCC is preserved, and may even be up-regulated. Increased CaCC activity in CF airways is controversial. Hence, we have investigated the effects of CFTR on CaCC activity and have also assessed the relative contributions of these two conductances to airway surface liquid (ASL) height (volume) in murine tracheal epithelia. We find that CaCC is up-regulated in intact murine CF tracheal epithelia, which leads to an increase in UTP-mediated Cl−/volume secretion. This up-regulation is dependent on cell polarity and is lost in nonpolarized epithelia. We find no role for an increased electrical driving force in CaCC up-regulation but do find an increased Ca2+ signal in response to mucosal nucleotides that may contribute to the increased Cl−/volume secretion seen in intact epithelia. CFTR plays a critical role in maintaining ASL height under basal conditions and accordingly, ASL height is reduced in CF epithelia. In contrast, CaCC does not appear to significantly affect basal ASL height, but does appear to be important in regulating ASL height in response to released agonists (e.g., mucosal nucleotides). We conclude that both CaCC and the Ca2+ signal are increased in CF airway epithelia, and that they contribute to acute but not basal regulation of ASL height.

Characterization of the oligomeric structure of the Ca(2+)-activated Cl- channel Ano1/TMEM16A.

Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca2+-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl− secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca2+, suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.

A novel plant-derived inhibitor of cAMP-mediated fluid and chloride secretion

We have identified an agent (SP-303) that shows efficacy against in vivo cholera toxin-induced fluid secretion and in vitro cAMP-mediated Cl-secretion. Administration of cholera toxin to adult mice results in an increase in fluid accumulation (FA) in the small intestine (FA ratio = 0.63 vs. 1.86 in control vs. cholera toxin-treated animals, respectively). This elevation in FA induced by cholera toxin was significantly reduced (FA ratio = 0.70) in animals treated with a 100 mg/kg dose of SP-303 at the same time as the cholera treatment. Moreover, when SP-303 was administered 3 h after cholera toxin, a dose-dependent inhibition of FA levels was observed with a half-maximal inhibitory dose of 10 mg/kg. In Ussing chamber studies of Caco-2 or T84 monolayer preparations, SP-303 had a significant effect on both basal current and forskolin-stimulated Cl- current. SP-303 also induced an increase in resistance that paralleled the observed decrease in current. These data suggest that SP-303 has an inhibitory effect on cAMP-mediated Cl- and fluid secretion. Thus SP-303 may prove to be a useful broad-spectrum antidiarrheal agent.

Bestrophin-2 mediates bicarbonate transport by goblet cells in mouse colon

Anion transport by the colonic mucosa maintains the hydration and pH of the colonic lumen, and its disruption causes a variety of diarrheal diseases. Cholinergic agonists raise cytosolic Ca2+ levels and stimulate anion secretion, but the mechanisms underlying this effect remain unclear. Cholinergic stimulation of anion secretion may occur via activation of Ca2+-activated Cl- channels (CaCCs) or an increase in the Cl- driving force through CFTR after activation of Ca2+-dependent K+ channels. Here we investigated the role of a candidate CaCC protein, bestrophin-2 (Best2), using Best2-/- mice. Cholinergic stimulation of anion current was greatly reduced in Best2-/- mice, consistent with our proposed role for Best2 as a CaCC. However, immunostaining revealed Best2 localized to the basolateral membrane of mucin-secreting colonic goblet cells, not the apical membrane of Cl--secreting enterocytes. In addition, in the absence of HCO3-, cholinergic-activated current was identical in control and Best2-/- tissue preparations, which suggests that most of the Best2 current was carried by HCO3-. These data delineate an alternative model of cholinergic regulation of colonic anion secretion in which goblet cells play a critical role in HCO3- homeostasis. We therefore propose that Best2 is a HCO3- channel that works in concert with a Cl:HCO3- exchanger in the apical membrane to affect transcellular HCO3- transport. Furthermore, previous models implicating CFTR in cholinergic Cl- secretion may be explained by substantial downregulation of Best2 in Cftr-/- mice.

Novel human bronchial epithelial cell lines for cystic fibrosis research.

Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three DeltaF508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 Omega.cm(2). In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1beta, TNF-alpha, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development.

Enhanced T cell cytokine gene expression in mouse airway obliterative bronchiolitis.

BACKGROUND Obliterative bronchiolitis (OB), chronic allograft rejection of the lung, is a major cause of morbidity and mortality after lung transplantation. Previous studies using the heterotopic mouse trachea model of chronic airway rejection have shown a T cell infiltrate composed of CD4+ and CD8+ T cells. The goal of these experiments was to characterize the pattern of T lymphocyte cytokines during chronic airway rejection using the heterotopic mouse trachea model. METHODS Isografts (BALB/c into BALB/c) and allografts (BALB/c into C57BL/6) were implanted into cyclosporin-treated animals and harvested 2, 4, 6, and 10 weeks posttransplant. Cytokine mRNA expression in these grafts was determined using reverse transcription polymerase chain reactions. Expression of Th1 cytokines, interleukin- (IL) 2 and gamma-interferon, and Th2 cytokines, IL-4, and IL-10 were analyzed, as well as the cytotoxic lymphocyte product granzyme B and expressed relative to beta-actin gene expression. RESULTS In allografts, expression of IL-2 (P=0.002), gamma-interferon (P=2x10(-6)), granzyme B (P=0.003), IL-4 (P=0.06), and IL-10 (P=8x10(-6)) were 2- to 10-fold higher compared to isografts throughout the time-course of graft injury. Th1 and cytotoxic lymphocyte gene expression were increased to a greater extent than Th2 cytokines in allografts compared with isografts, and both Th1 and Th2 cytokine gene expression persisted at 6-10 weeks. CONCLUSIONS These data suggest that Th1, Th2, and cytotoxic lymphocyte subtypes all contribute to the development of obliterative bronchiolitis in the heterotopic mouse trachea model. Efforts to reduce the development of obliterative bronchiolitis may require the antagonism of multiple T cell pathways.