Cytokines stimulate GTP cyclohydrolase I gene expression in cultured human umbilical vein endothelial cells.
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-alpha (TNF-alpha), interferon-gamma (INF-gamma), and interleukin-1beta (IL-1beta) 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-alpha (2 microg/mL), INF-gamma (200 U/mL), and IL-1beta (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 microg/mL), inhibited cytokine-induced expression of GTP cyclohydrolase I gene. A protein synthesis inhibitor, cycloheximide (0.5 microg/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.
Katusic
Z S
ZS
Department of Anesthesiology, Mayo Clinic, Rochester, Minn. 55905, USA.
Stelter
A
A
Milstien
S
S
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D016207:Cytokines / Q000494:pharmacology*; D003609:Dactinomycin / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006136:GTP Cyclohydrolase / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011622:Pterins / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins / Q000187:drug effects / Q000378:metabolism
D016207:Cytokines; D007375:Interleukin-1; D011622:Pterins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C011552:tetrahydropterin; D003609:Dactinomycin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D007371:Interferon-gamma; D003513:Cycloheximide; D006136:GTP Cyclohydrolase