Cytokines Stimulate GTP Cyclohydrolase I Gene Expression in Cultured Human Umbilical Vein Endothelial Cells

From the Departments of Anesthesiology and Pharmacology, Mayo Clinic, Rochester, Minn (Z.S.K., A.S.); and the Laboratory of Cell Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, Md (S.M.).

Correspondence to Zvonimir S. Katusic, MD, PhD, Department of Anesthesiology, Mayo Clinic, 200 First St SW, Rochester, MN 55905.

Abstract—In vascular endothelial cells, tetrahydrobiopterin serves as an essential cofactor required for enzymatic activity of nitric oxide synthase. GTP cyclohydrolase I is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Previous studies have demonstrated that proinflammatory cytokines stimulate production of tetrahydrobiopterin in endothelial cells. Long-term regulation of GTP cyclohydrolase I gene expression in endothelium has not been studied. The present study was designed to determine whether the cytokines tumor necrosis factor- (TNF-), interferon- (INF-), and interleukin-1ß (IL-1ß) stimulate tetrahydrobiopterin synthesis by increasing expression of GTP cyclohydrolase I mRNA in endothelial cells. The relative reverse transcription polymerase chain reaction was used to quantify expression of GTP cyclohydrolase I mRNA in cultured human umbilical vein endothelial cells. Nuclear run-on assay was performed to determine the transcription rate of GTP cyclohydrolase I gene. GTP cyclohydrolase I enzymatic activity and production of tetrahydrobiopterin were measured in cell extracts. After incubation with TNF- (2 µg/mL), INF- (200 U/mL), and IL-1ß (5 U/mL) for 24 hours, significantly increased expression of GTP cyclohydrolase I mRNA was detected. Cytokines increased the transcription rate of GTP cyclohydrolase I 3.6-fold. This increase was associated with increased GTP cyclohydrolase I enzymatic activity and elevation of intracellular levels of tetrahydrobiopterin. An RNA synthesis inhibitor, actinomycin D (2 µg/mL), inhibited cytokine-induced expression of GTP cyclohydrolase I gene. A protein synthesis inhibitor, cycloheximide (0.5 µg/mL), did not affect expression of GTP cyclohydrolase I mRNA but blocked the increase in enzyme activity, as well as production of tetrahydrobiopterin. Incubation of endothelial cells for 24 hours in the presence of 8-bromoadenosine 3':5'-cyclic monophosphate (10^-3 mol/L) did not affect expression of GTP cyclohydrolase I mRNA. These results demonstrate that in vascular endothelial cells, cytokines increase production of tetrahydrobiopterin by stimulating expression of GTP cyclohydrolase I gene. This effect is apparently due to increased transcription rather than stabilization of mRNA. Regulation of GTP cyclohydrolase I gene expression by cytokines may play an important role in control of endothelial nitric oxide synthesis.

Key Words: nitric oxide • tetrahydrobiopterin • tumor necrosis factor- • interferon- • interleukin-1ß

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