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.

The aberrant dysregulation of the inducible form of nitric oxide synthase (NOS2) is thought to play a role in many inflammatory disorders including cystic fibrosis (CF). The complex regulation of NOS2 expression is the subject of intense investigation, and one intriguing regulatory pathway known to influence NOS2 expression is the Rho GTPase cascade. We examined NOS2 regulation in response to inflammatory cytokines in a human alveolar epithelial cell line treated with inhibitors of different upstream and downstream components of the Rho GTPase pathway to better define potential signaling mechanisms. Statin-mediated 3-hydroxy-3-methylglutaryl-CoA reductase inhibition increased cytokine-dependent activation of the NOS2 promoter, reversible by the addition of geranylgeranyl pyrphosphate. However, inhibition of Rho-associated kinase (ROCK) with Y-27632 resulted in a decrease in NOS2 promoter activity, yet an increase in NOS2 mRNA and protein levels. Our results suggest that prenylation events influence NOS2 promoter activity independently of the Rho GTPase pathway and that Rho GTPase signaling mediated through ROCK suppresses NOS2 production downstream of promoter function at the message and protein level.