Terminal sialylation is altered in airway cells with impaired CFTR-mediated chloride transport.
ABSTRACT Reduced terminal sialylation at the surface of airway epithelial cells from patients with cystic fibrosis may predispose them to bacterial infection. To determine whether a lack of chloride transport or misprocessing of mutant cystic fibrosis transmembrane conductance regulator (CFTR) is critical for the alterations in glycosylation, we studied a normal human tracheal epithelial cell line (9/HTEo(-)) transfected with the regulatory (R) domain of CFTR, which blocks CFTR-mediated chloride transport; DeltaF508 CFTR, which is misprocessed, wild-type CFTR; or empty vector. Reduced cAMP-stimulated chloride transport is seen in the R domain and DeltaF508 transfectants. These two cell lines had consistent, significantly reduced binding of elderberry bark lectin, which recognizes terminal sialic acid in the alpha-2,6 configuration. Binding of other lectins, including Maakia amurensis lectin, which recognizes sialic acid in the alpha-2,3 configuration, was comparable in all cell lines. Because the cell surface change occurred in R domain-transfected cells, which continue to express wild-type CFTR, it cannot be related entirely to misprocessed or overexpressed CFTR. It is associated most closely with reduced CFTR activity.
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ABSTRACT: Chronic lung colonization with Pseudomonas aeruginosa is anticipated in cystic fibrosis (CF). Abnormal terminal glycosylation has been implicated as a candidate for this condition. We previously reported a down-regulation of mannose-6-phosphate isomerase (MPI) for core N-glycan production in the CFTR-defective human cell line (IB3). We found a 40% decrease in N-glycosylation of IB3 cells compared with CFTR-corrected human cell line (S9), along with a threefold-lower surface attachment of P. aeruginosa strain, PAO1. There was a twofold increase in intracellular bacteria in S9 cells compared with IB3 cells. After a 4-hour clearance period, intracellular bacteria in IB3 cells increased twofold. Comparatively, a twofold decrease in intracellular bacteria occurred in S9 cells. Gene augmentation in IB3 cells with hMPI or hCFTR reversed these IB3 deficiencies. Mannose-6-phosphate can be produced from external mannose independent of MPI, and correction in the IB3 clearance deficiencies was observed when cultured in mannose-rich medium. An in vivo model for P. aeruginosa colonization in the upper airways revealed an increased bacterial burden in the trachea and oropharynx of nontherapeutic CF mice compared with mice treated either with an intratracheal delivery adeno-associated viral vector 5 expressing murine MPI, or a hypermannose water diet. Finally, a modest lung inflammatory response was observed in CF mice, and was partially corrected by both treatments. Augmenting N-glycosylation to attenuate colonization of P. aeruginosa in CF airways reveals a new therapeutic avenue for a hallmark disease condition in CF.American Journal of Respiratory Cell and Molecular Biology 06/2011; 44(6):824-30. · 4.15 Impact Factor
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ABSTRACT: Malfunction of the cell surface glycoprotein, cystic fibrosis transmembrane conductance regulator, is the molecular hallmark of cystic fibrosis (CF), causing salt imbalance across the lung epithelium and biochemical and biophysical alterations of the mucous secretion and airway surfaces. Abnormal glycosylation of both secreted and membrane-tethered airway mucins in CF hosts are reported by a substantial body of literature and correlates with bacterial infection and inflammation in CF airways, features that are linked to the CF pathology. It is established that Pseudomonas aeruginosa and other CF-typic bacteria use the altered host mucin glycosylation as receptors for adhesion by dedicated lectins and adhesins recognizing an array of the aberrantly expressed glycan determinants. This review aims to describe the aberrant mucin glycosylation phenotype observed in CF airways relative to the non-CF equivalent by summarizing the wealth of literature on this topic. The possible causes and effects of altered glycosylation in the respiratory system are discussed. Specific attention is given to the adhesion mechanisms of the opportunistic P. aeruginosa, which utilizes the molecular alterations of the lung to gain access to the normally sterile airways. Finally, the emerging glycosylation-based therapeutics that show promising potential for reducing bacterial infection in individuals with CF by molecular mimicry mechanisms are discussed.Expert Review of Respiratory Medicine 10/2013; 7(5):553-76.
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ABSTRACT: The pathophysiology of cystic fibrosis (CF) inflammatory lung disease is not well understood. CF airway epithelial cells respond to inflammatory stimuli with increased production of proinflammatory cytokines as a result of increased NF-kappaB activation. Peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibits NF-kappaB activity and is reported to be reduced in CF. If PPARgamma participates in regulatory dysfunction in the CF lung, perhaps PPARgamma ligands might be useful therapeutically. Cell models of CF airway epithelium were used to evaluate PPARgamma expression and binding to NF-kappaB at basal and under conditions of inflammatory stimulation by Pseudomonas aeruginosa or TNFalpha/IL-1beta. An animal model of CF was used to evaluate the potential of PPARgamma agonists as therapeutic agents in vivo. In vitro, PPARgamma agonists reduced IL-8 and MMP-9 release from airway epithelial cells in response to PAO1 or TNFalpha/IL-1beta stimulation. Less NF-kappaB bound to PPARgamma in CF than normal cells, in two different assays; PPARgamma agonists abrogated this reduction. PPARgamma bound less to its target DNA sequence in CF cells. To test the importance of the reported PPARgamma inactivation by phosphorylation, we observed that inhibitors of ERK, but not JNK, were synergistic with PPARgamma agonists in reducing IL-8 secretion. In vivo, administration of PPARgamma agonists reduced airway inflammation in response to acute infection with P. aeruginosa in CF, but not wild-type, mice. In summary, PPARgamma inhibits the inflammatory response in CF, at least in part by interaction with NF-kappaB in airway epithelial cells. PPARgamma agonists may be therapeutic in CF.AJP Lung Cellular and Molecular Physiology 07/2008; 295(2):L303-13. · 3.52 Impact Factor