Conditional knockout of GCH1 in leukocytes and endothelial cells: A novel model to study tetrahydrobiopterin-dependent NOS functions in vivo

Abstract

Tetrahydrobiopterin (BH4) is an essential co-factor for NO production from NOS enzymes. When BH4 levels become limiting these enzymes can become ‘un-coupled’, leading to superoxide production. GTP cyclohydrolase I (GTPCH), encoded by GCH1 , is an essential enzyme in the biosynthesis of BH4. We designed mice harbouring a ‘floxed’ portion of the GCH1 locus within the active site of the enzyme (GCHfl/fl mice). We crossed these with mice expressing the cre enzyme under control of the Tie2 promoter (GCHfl/fl Tie2cre). Cre expression causes efficient excision of the floxed allele in all leukocytes and endothelial cells, as detected by PCR for the floxed or knockout allele. Leukocyte or endothelial cell rich tissues such as aorta and spleen showed a significant decrease in BH4 content whereas other tissues such as liver showed no change in biopterin levels. Isolation of primary endothelial cells or leukocytes showed these cells to be deficient in BH4. Bone Marrow Derived Macrophages cultured from GCHfl/fl mice show GTPCH protein expression and BH4 production, but have no iNOS expression. Stimulation with Lipopolysaccharide and Interferon- γ increases BH4 production and also induces iNOS protein expression and NO-derived nitrite accumulation. GCHfl/fl Tie2cre cells show no GTPCH protein expression and barely detectable levels of BH4 and, despite normal iNOS protein levels following stimulation, exhibit no nitrite accumulation. Furthermore GCHfl/fl Tie2cre BMDM show enhanced ROS production, as measured by DHE-HPLC.