Over-expression of GTP-cyclohydrolase 1 feedback regulatory protein attenuates LPS and cytokine-stimulated nitric oxide production

GTP-cyclohydrolase 1 (GTP-CH1) catalyses the first and rate-limiting step for the de novo production of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase (NOS). The GTP-CH1-BH4 pathway is emerging as an important regulator in a number of pathologies associated with over-production of nitric oxide (NO) and hence a more detailed understanding of this pathway may lead to novel therapeutic targets for the treatment of certain vascular diseases. GTP-CH1 activity can be inhibited by BH4 through its protein-protein interactions with GTP-CH1 regula- tory protein (GFRP), and transcriptional and post-translational modification of both GTP-CH1 and GFRP have been reported in response to proinflammatory stimuli. However, the functional significance of GFRP/GTP-CH1 interactions on NO pathways has not yet been demonstrated. We aimed to investigate whether over-expression of GFRP could affect NO production in living cells. Over-expression of N-terminally Myc-tagged recombinant human GFRP in the murine endothelial cell line sEnd 1 resulted in no significant effect on basal BH4 nor NO levels but significantly attenu- ated the rise in BH4 and NO observed following lipopolysaccharide and cytokine stim- ulation of cells. This study demonstrates that GFRP can play a direct regulatory role in iNOS-mediated NO synthesis and suggests that the allosteric regulation of GTP-CH1 activity by GFRP may be an important mechanism regulating BH4 and NO levels in vivo.