Airway epithelial cytoglobin regulates nitric oxide synthase and development of primary ciliary dyskinesia and situs inversus

PROJECT SUMMARY One of the major causes of sino-pulmonary disease is primary ciliary dyskinesia (PCD), also called immotile cilia syndrome. PCD is an inherited disease affecting 1 in ~20,000 individuals. More than 50 genetic variants have been identified that are associated with immotile, dyskinetic, or aplastic cilia, and produce a similar clinical syndrome of chronic sinusitis, bronchitis and bronchiectasis, recurrent pneumonia, infertility, and often situs inversus (Kartagener syndrome). Exhaled nitric oxide (NO) derived from the epithelial NO synthases (NOSs) is low in PCD patients and serves as a sensitive and specific diagnostic test for the disease. Cystic fibrosis (CF) and acute viral infections can also lower exhaled NO levels, but the mechanism that underlies the low NO and how this might contribute to the disease remains unknown. In our preliminary data, using knock-down methodologies in zebrafish and mice, we report on a novel and unexpected finding: we have identified cytoglobin as a conserved protein that is essential for developmental stage-specific NO signaling and ciliary motility, required for the establishment of left-right patterning in the developing embryo and normal airway ciliary motility. In this proposal we hypothesize that airway epithelial cytoglobin co-localizes with cilia and NOS as a metabolon to regulate NOS-NO signaling and ciliary assembly and function. We further propose that cytoglobin modulation of NOS-NO influences organ laterality development, potentially explaining the link between airway inflammation/oxidative stress, NO production and signaling, ciliary function in PCD, and organ laterality determination, with relevance to acute viral infection, smoking, and CF. Finally, we propose to exploit this new discovery therapeutically, by testing drugs that activate the NOS-NO-soluble guanylate cyclase (sGC) pathway to correct ciliary function in our zebrafish, mouse, and human airway epithelial models. Aim 1 will explore the mechanisms of cytoglobin-dependent regulation of zebrafish, mouse, and human NOS and cilia function. We have developed multiple mouse models, including CYGB knock-out, gain-of- function (H81Q), and loss-of-function (K116E) mutations that affect heme ligand binding and oxidation-reduction, and will evaluate interactions with NOS isoforms in zebrafish and mice and in human airway epithelial cell lines from patients with PCD. Aim 2 will test the hypothesis that therapeutic activation of the CYGB-NOS-NO-sGC signaling pathway can restore airway epithelial ciliary function and developmental abnormalities in zebrafish, mouse, and human models. Translational evaluation of genomic gain and loss of function and drug treatments will be performed in human PCD airway epithelial cell cultures to identify new therapeutic approaches to improve muco- ciliary clearance in airway disease.