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Heterologous Cftr Expression is Antagonized By Nitric Oxide (No) ♦ 1955

Cystic fibrosis is the most frequent autosomal recessive inherited disease among Caucasians. CF is caused by mutations of the CFTR gene. Novel therapies for CF currently under trial include pharmacological modulators of epithelial ion transport, CFTR expression and/or processing, anti-inflammatory therapy and gene therapy. To improve the efficiency of gene therapy, host responses limiting CFTR transgene expression need to be identified, and strategies to overcome these limitations have to be designed. We studied the mechanisms of NO-dependent inhibition of heterologous CFTR expression in a model epithelium. LLCPK1 cells stably transduced with CFTR (LLCPKWTCFTR) were subjected to treatment with NO derived either from a chemical NO-donor (diethyl triamine; DETA) or produced by 3T3 cells stably transduced with the inducible NO-synthase (3T3iNOS). CFTR expression was quantified using immunoprecipitation followed by in vitro phosphorylation, SDS-PAGE analysis and phosphor imaging densitometry. Exposure to NO produced either by DETA, or by 3T3iNOS cells inhibited heterologous CFTR expression in LLCPKWTCFTR cells.The inclusion of freshly isolated red blood cells, i.e., NO scavengers, rescued CFTR expression in DETA treated cells and treatment with L-NG-monomethyl arginine, i.e., a NOS inhibitor, abrogated the inhibitory effect of the co-cultured 3T3iNOS cells on CFTR expression in LLCPKWTCFTR cells. None of the treatment conditions were cytotoxic for LLCPKWTCFTR cells as determined by LDH release and/or apoptosis assays. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylyl cyclase, had no effect on NO-inhibition of CFTR expression, and 8Br-cGMP had no effect on heterologous CFTR expression. These data indicate that heterologous CFTR expression is inhibited by NO in a model epithelium, and that this inhibition is not mediated by cGMP-dependent signaling. These findings caution that inflammation and consequent NO generation should be considered as potential stumbling blocks for CF gene therapy.