Abstract 3685: Role Of The GTP Cyclohydrolase Regulatory Protein In Modulating Human Endothelial Cell Nitric Oxide Production
GTP cyclohydrolase-I (GTPCHI) is the rate-limiting enzyme involved in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase. We have previously shown that laminar shear stress increases BH4 production in human aortic endothelial cells (HAECs) by enhancing GTPCHI activity via Casein Kinase 2 (CK2)-dependent phosphorylation on serine 81. CK2 is usually constitutively active and phosphorylates an array of substrates when their phosphorylation sites are exposed upon release from binding partners. We therefore sought to determine if GTPCHI binding proteins might modulate its activity. One such protein is Activator of Heat Shock 90 kDa Protein (Aha1) which was found to interact with GTPCHI in a yeast two-hybrid system. We found that HAECs express Aha1 by western blotting; however, downregulation of Aha1 by siRNA transfection did not affect HAEC BH4 levels. In non-endothelial cells, another binding partner of GTPCHI is the GTP cyclohydrolase I feedback regulatory protein (GFRP). We found that GFRP is abundantly expressed in HAECs by real time PCR. Transfection with siRNA against GFRP caused a striking increase of BH4 levels in HAECs (from near undetectable values to 46.4 ± 5.0 pmol/mg protein, p<0.0001) and non-silencing siRNA had no effect on BH4 levels. GFRP siRNA treatment of HAECs also increased phosphorylation of GTPCHI on serine 81 and augmented its enzymatic activity (from near undetectable values to 1.65 ± 0.12 μUnits/mg protein, p<0.0001) without changing its protein level. Exposure of HAECs transfected with GFRP siRNA to laminar shear stress did not further increase BH4 levels (48.7 ± 2.9 pmol/mg protein, p<0.71). Finally, we found that HAEC NO production is dramatically increased following downregulation of GFRP compared to non-silencing siRNA (3891.5 ± 969.0 pmol/mg protein vs. 683.5 ± 141.4 pmol/mg protein, p=0.02). Thus, GFRP plays a major role in regulation of human endothelial cell GTPCHI enzyme activity, BH4 levels and NO production. While Aha1 has been reported to bind GTPCHI, our data indicate that it has little role in modulating its activity in endothelial cells.