Reduced Smad3 protein expression and altered transforming growth factor-beta1-mediated signaling in cystic fibrosis epithelial cells.

Cystic fibrosis (CF) is a disease characterized by an aggressive inflammatory response in the airways. Given the antiinflammatory properties of transforming growth factor (TGF)-beta1, it was our goal to examine components of TGF-beta1-mediated signaling in both a cultured cell model and a mouse model of CF. A CF-related reduction of protein levels of the TGF-beta1 signaling molecule Smad3 was found in both of these model systems, whereas Smad4 levels were unchanged. Functional effects of reduced Smad3 expression are manifest in our cultured cell model, as reduced basal and TGF-beta1-stimulated levels of luciferase expression using the TGF-beta1-responsive reporter construct 3TP-Lux in the CF-phenotype cells compared with control cells. However, TGF-beta1-stimulated responses using the A3-Luc reporter construct were normal in both cell lines. These results suggest that select TGF-beta1-mediated signaling pathways are impaired in CF epithelial cells. This selective loss of Smad3 protein expression in CF epithelium may also influence inflammatory responses. Our data demonstrate that both CF-phenotype cells lacking Smad3 expression, and A549 cells expressing a dominant-negative Smad3, are unable to support TGF-beta1-mediated inhibition of either the interleukin (IL)-8 or the NOS2 promoter. We conclude that a CF-related reduction in Smad3 protein expression selectively alters TGF- beta1-mediated signaling in CF epithelium, potentially contributing to aggressive inflammatory responses.

In vivo alterations of IFN regulatory factor-1 and PIAS1 protein levels in cystic fibrosis epithelium.

Inducible nitric oxide synthase-2 (NOS2) expression has been shown to be reduced in cystic fibrosis (CF) epithelial cells. Reduced NOS2 expression is unexpected, given the inflammatory nature of CF airway disease, and is an indication that cell-signaling mechanisms necessary for proper NOS2 regulation are probably altered in CF epithelium. Therefore, we examined the expression levels of regulatory factors necessary for NOS2 expression in CF epithelium and showed that IFN regulatory factor-1 (IRF-1) is necessary for full NOS2 expression. Mice lacking IRF-1 expression have diminished epithelial NOS2 expression, as well as reduced NO-dependent chloride transport across the nasal epithelia. Furthermore, IRF-1 protein expression is reduced in nasal and intestinal epithelial cells from CF mice, suggesting a possible mechanism for the CF-related reduction of epithelial NOS2 expression. Active signal transducer and activator of transcription-1 (Stat1) is necessary for both NOS2 and IRF-1 expression. We found that protein levels of Stat1 were increased in CF cells, but that the active phosphorylated form of Stat1 was bound to the protein inhibitor of activated Stat1 (PIAS1). We propose that increased levels of PIAS1 diminish certain cell-signaling pathways, resulting in reduced IRF-1 and NOS2 expression in CF epithelial cells.

Cystic fibrosis transmembrane conductance regulator-dependent regulation of epithelial inducible nitric oxide synthase expression.

Recent evidence has shown that the inducible form of nitric oxide (NO) synthase (NOS2) has reduced expression in airway epithelia of patients with cystic fibrosis (CF) despite the presence of chronic inflammation. The goal of this paper is to determine whether NOS2 expression is regulated by the presence of functional CF transmembrane conductance regulator (CFTR). Using a human trachea epithelial cell line in which CFTR activity is blocked by the overexpression of the CFTR regulatory domain, we found that loss of CFTR activity reduces NOS2 messenger RNA expression as determined by reverse transcriptase/polymerase chain reaction and reduces overall NO production compared with mock-transfected controls. An in vivo model using mice lacking CFTR expression (cftr -/-), wild-type mice (cftr +/+), and cftr -/- mice that have had human CFTR introduced to the intestinal epithelium using the fatty acid binding protein (FABP) promoter (FABP-hcftr) was also examined. Electrical characterization confirmed that FABP-hcftr mice had corrected electrophysiologic properties compared with cftr -/- mice in the ileum, but FABP-hcftr nasal transepithelial potential difference measurements were identical to cftr -/- values showing specific intestinal correction. NOS2-specific immunostaining revealed that NOS2 expression is evident in sections of ileum and nasal epithelium of cftr +/+ mice but is absent in both tissues in cftr -/- mice. FABP-hcftr mice, however, show strong NOS2 staining in epithelial cells of the ileum but reduced staining in the nasal epithelium, suggesting a CFTR-related influence in the regulation of NOS2 expression in epithelial cells.

Nitric oxide-mediated regulation of transepithelial sodium and chloride transport in murine nasal epithelium.

Transepithelial ion transport is regulated by a variety of cellular factors. In light of recent evidence that nitric oxide (NO) production is decreased in cystic fibrosis airways, we examined the role of NO in regulating sodium and chloride transport in murine nasal epithelium. Acute intervention with the inducible NO synthase (iNOS)-selective inhibitor S-methylisothiourea resulted in an increase of amiloride-sensitive sodium absorption observed as a hyperpolarization of nasal transepithelial potential difference. Inhibition of iNOS expression with dexamethasone also hyperpolarized transepithelial potential difference, but only a portion of this increase proved to be amiloride sensitive. Chloride secretion was significantly inhibited in C57BL/6J mice by the addition of both S-methylisothiourea and dexamethasone. Mice lacking iNOS expression [NOS2(-/-)] also had a decreased chloride-secretory response compared with control mice. These data suggest that constitutive NO production likely plays some role in the downregulation of sodium absorption and leads to an increase in transepithelial chloride secretion.