Oxidative stress is implicated in the pathogenesis of many inflammatory pulmonary diseases, including cystic fibrosis (CF). Delineating how oxidative stress stimulates CF transmembrane conductance regulator (CFTR) in airway epithelial cells is useful, both to increase the understanding of airways host defense and suggest therapeutic approaches to reduce the oxidant stress burden in the CF lung. Using the airway epithelial cell line Calu-3, we investigated the hypothesis that hydrogen peroxide (H 2 O 2 ), which stimulates anion efflux through CFTR, does so via the production of prostaglandin E 2 (PGE 2 ). Using iodide efflux as a biochemical marker of CFTR activity and short circuit current (I sc ) recordings, we found that the H 2 O 2 -stimulated efflux was abolished by cyclooxygenase-1 inhibition and potentially also involves microsomal prostaglandin E synthase-1 activity, implicating a role for PGE 2 production. Furthermore, H 2 O 2 application resulted in a rapid release of PGE 2 from Calu-3 cells. We additionally hypothesized that the PGE 2 subtype 4 (EP 4 ) receptor was involved in mediating this response. In the presence of (4Z)-7-[(rel-1S,2S,5R)-5-((1,1Ј-biphenyl-4-yl)methoxy)-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid (AH23848) (which blocks the EP 4 receptor), the H 2 O 2 -stimulated response was abolished. To investigate this finding in a polarized system, we measured the increase in I sc induced by H 2 O 2 addition in the presence and absence of AH23848. H 2 O 2 induced a robust increase in I sc , which was significantly attenuated in the presence of AH23848, suggesting some role for the EP 4 receptor. In conclusion, with H 2 O 2 as a model oxidant stress, stimulation of CFTR seems to involve PGE 2 production and likely EP 4 receptor activation in Calu-3 airway epithelial cells. This mechanism would be compromised in the CF airways.
Cells regulate their volume in response to changes in the osmolarity of both their extracellular and their intracellular environments. We investigated the ability of the human airway epithelial cell line Calu-3 to respond to changes in extracellular osmolarity. Although switching Calu-3 cells from an isosmotic to a hyperosmotic environment resulted in cell shrinkage, there was no compensatory mechanism for the cells to return to their original volume. In contrast, switching to a hyposmotic environment resulted in an initial cell swelling response, followed by a regulatory volume decrease (RVD). Pharmacologic studies demonstrate that the voltage-activated K+ channels Kv4.1 and (or) Kv4.3 play a crucial role in mediating this RVD response, and we demonstrated expression of these channel types at the mRNA and protein levels. Furthermore, inhibition of the large- and intermediate-conductance Ca2+-activated K+ channels KCa1.1 (maxi-K) and KCa3.1 (hIK) also implicated these channels as playing a role in volume recovery in Calu-3 cells. This report describes the nature of volume regulation in the widely used model cell line Calu-3.
Estrogen deficiency may contribute to extracellular matrix turnover in skin. This has led previous authors to postulate that aged skin heals less efficiently when compared to younger skin. Also, cigarette smokers have been shown to heal less efficiently than nonsmokers. Matrix metalloproteinase (MMP)-13, an enzyme that participates in the degradation of the extracellular matrix, has been implicated in physiologic aging and wound healing. This study investigates the effects of smoke exposure and estrogen deficiency on MMP-13 in young and aged female mouse skin. Young and aged female C57Bl/6J mice were ovariectomized. They were then randomly administered either 17beta-estradiol (E2) or placebo pellets. Half the animals in each age group were further randomized to exposure to cigarette smoke for a period of 6 months. Smoking and estrogen deficiency increased MMP-13 protein and activity in aged skin. The tissue inhibitors of metalloproteinases, which inhibit MMPs, activity was unchanged across all groups. E2 replacement decreased the actual level of MMP-13 protein and activity. We also found an increased collagen content and decreased ER receptor protein level in aged, smoke-exposed female mice. Our experimental data show that tobacco smoke exposure and estrogen deficiency are additive risk factors for promoting increased activity of MMP-13 in aged skin. These findings suggest that MMP-13 functions as a mediator of smoke-induced skin injury in susceptible, aged experimental female mice.
Interleukin (IL)‐22 is a pleiotropic cytokine which predominately targets epithelial cells. IL‐22 has been implicated in increasing epithelial host defence via a number of different mechanisms and we hypothesized its IL‐22 application would stimulate several protective mechanisms in airway epithelial cells. Using the model human airway bronchial epithelial cell line 16HBEo− we investigated the ability of IL‐22 to increase CFTR expression, and determined that treatment for 24 hours increased CFTR gene and protein expression, resulting in increased CFTR activity measured via iodide efflux assays. Furthermore, IL‐22 increased mRNA expression for the anti‐microbial peptides human beta‐defensin‐2 (hDB‐2) and neutrophil gelatinase‐associated lipocalin (NGAL), but had no effect on expression of the cathelicidin LL‐37. Additionally, we investigated the ability of IL‐22 to enhance epithelial repair using a mechanical wounding model, and determined that its presence significantly enhanced would healing in this model. IL‐22 application was associated with increased STAT‐3 phosphorylation. This is the first report of IL‐22 acting to increase CFTR expression and functionality, which together with its ability to increase anti‐microbial peptide expression and would healing ability, confirm its role as a potentially important factor in the airway epithelial host defence and repair. CFCanada and NSERC.
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