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

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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

	Introduction

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Tetrahydrobiopterin serves as the natural cofactor for enzymatic activity of nitric oxide synthases.¹ Although this cofactor plays an important role in the cardiovascular system, little is known about the regulatory mechanisms that control its biosynthesis in vascular endothelial cells. Previous studies on cultured HUVECs demonstrated that the cytokines TNF-, INF-, and IL-1ß stimulate production of tetrahydrobiopterin, with subsequent elevation of endothelial nitric oxide synthase activity.^{2 3} This stimulatory effect is apparently mediated by increased enzymatic activity of GTP cyclohydrolase I, which is the rate-limiting enzyme for synthesis of tetrahydrobiopterin.² Long-term regulation of GTP cyclohydrolase I mRNA levels in endothelial cells has not been studied previously. Therefore, the present study was designed to determine whether cytokines increase tetrahydrobiopterin levels by stimulating expression of the GTP cyclohydrolase I gene in cultured HUVECs.

	Methods

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Endothelial Cells
Experiments were performed on HUVECs purchased from Clonetics. Cryopreserved cells were obtained in primary passage (500 000 cells per ampuole). They were grown in gelatin-coated flasks in optimized endothelial growth medium supplied by Clonetics. The cells were passaged by exposure to trypsin/EDTA for about 120 seconds in HEPES-buffered saline and reseeded in T-175 flasks. Cells grew to confluence after about 5 days in 95% humidified air/5% CO₂ at 37°C and were used up to passage level six.

Quantification of mRNA Levels
Total RNA was isolated by RNA STAT-60 kit (Tele-Test "B"/Inc). Integrity of the isolated RNA was evaluated by visualizing the ethidium bromide–stained nucleic acids after electrophoresis through 1% agarose gel. RNA concentrations were calculated from the optical density at 260 nm, and the purity was determined by A₂₆₀/A₂₈₀.

Cellular RNA (1, 2, and 4 µg) was reverse transcribed in a 20-µL reaction mixture containing 200 U reverse transcriptase (Superscript GIBCO-BRL, Life Technologies, Inc). For PCR amplification, each 25-µL reaction mixture contained 1 µL of cDNA derived from 1 µg of total RNA, 1x10⁶ cpm of labeled 5'-primer, 10 pmol of each unlabeled primer, 2 U of Taq DNA polymerase, 2.5 µL of 10x buffer provided with Taq DNA polymerase, and optimal concentration of MgCl₂ (3.5 mmol/L). The PCR conditions were as follows: denaturation for 30 seconds at 94°C, annealing for 30 seconds at 59°C, and extension for 30 seconds at 72°C. Oligo 4.0 software was used to design primers and determine optimal conditions for PCR reaction⁴ (Table 1).

Incorporation of ³²P–end-labeled primers of known specific radioactivity into PCR product was measured as a function of PCR cycle number. The total amount of radioactivity incorporated from end-labeled primer is proportional to product copy number and is independent of product size. Sense primers were labeled with ³²P using T4 polynucleotide kinase. Unincorporated isotope was removed by using Nick columns (Pharmacia), and percent incorporation was determined, permitting calculation of the specific radioactivity of the radiolabeled primers.

The PCR product was separated from unincorporated primer by electrophoresis through 1.5% agarose gels. The band migrating at the predicted size was excised, and radioactivity was determined by Cerenkov counting. The point in the exponential amplification range for comparison was selected by inspection of semilogarithmic plots of counts per minute versus cycle number.

Subcloning and DNA Sequencing
DNA obtained with PCR was extracted using Geneclean (Bio 101) and subcloned in pCR-Script Sk(+) cloning vector (Stratagene), which is based on pBluescript II Sk(+). Phagemid DNA was isolated, and double-stranded DNA was sequenced using Sequenase 2.0 (United States Biochemical) with primers complementary to the T3 and KS promoters in pBluescript.⁵

Estimates of Transcription Rate
Nuclear run-on assays were performed using a modified method described by Greenberg and Bender.⁶ Cells and solutions were maintained on ice throughout the procedure. Briefly, endothelial cells were trypsinized and centrifuged at 1500 rpm for 5 minutes. Cells were washed twice in phosphate-buffered saline followed by a single wash in resuspension buffer containing 10 mmol/L Tris, 10 mmol/L NaCl, and 3 mmol/L MgCl₂. Cell pellets were resuspended in a small volume of resuspension buffer. Cells were lysed by the addition of 4 vol resuspension buffer containing 0.125% NP-40. Nuclei were isolated by centrifugation as above. Nuclear pellets were suspended in storage buffer containing 50 mmol/L Tris, 5 mmol/L MgCl₂, 100 mmol/L EDTA, and 40% glycerol and stored under liquid nitrogen until further analysis.

In vitro transcription was performed with 25 to 44x10⁶ nuclei. The reaction was performed in 25 mmol/L Tris, 12.5 mmol/L MgCl₂, 750 mmol/L KCl, 1.25 mmol/L each ATP, CTP, and GTP, and 30 µL of [³²P]UTP (3000 Ci/mmol/L) at 30°C for 30 minutes. Identical numbers of nuclei were used from control and cytokine-treated cells. The reaction was stopped by the addition of RNase-free DNase I in HSB buffer (0.5 mol/L NaCl, 50 mmol/L MgCl₂, 2 mmol/L CaCl₂, and 10 mmol/L Tris) for 10 minutes at 30°C. The nuclei were lysed in 10% SDS, 50 mmol/L EDTA, and 100 mmol/L Tris and treated with 200 µg/mL proteinase K for 45 minutes at 42°C. RNA was phenol/chloroform extracted and precipitated with 3 mol/L NH₄OAC and equal volume of isopropanol. Centrifuged RNA pellets were suspended in TE buffer (10 mmol/L Tris and 1 mmol/L EDTA) and separated from unincorporated nucleotides on a Micron-100 column (Amicon, Inc). RNA was denatured in 0.2 mol/L NaOH, neutralized with 0.24 mol/L HEPES, and precipitated in ethanol overnight at -20°C. RNA pellets were suspended in TES solution (10 mmol/L TES, 10 mmol/L EDTA, and 0.2% [wt/vol] SDS), and 5 µL was used to determine counts per minute.

Linearized cDNA (5 µg each) for human GTP cyclohydrolase I (a generous gift of Dr T. Nagatsu), GAPDH (500 bp cloned into pCRII, Invitrogen, a gift from Dr Emanuel Strehler), and plasmid pRc/CMV (Invitrogen) were immobilized onto supported nitrocellulose (GIBCO-BRL, Life Technologies, Inc) by a slot-blot apparatus (Schleicher & Schuell). Membranes were prehybridized overnight at 65°C in 10 mmol/L TES, 10 mmol/L EDTA, 0.2% (wt/vol) SDS, 0.3 mol/L NaCl, 2x Denhardt's solution, and 0.25 mg/mL yeast tRNA. Equal counts (5 to 10x10⁶ cpm/mL) of radiolabeled transcripts from control and cytokine-treated cells were added to the membranes in fresh hybridization solution and hybridized for 48 to 72 hours at 65°C. The membranes were washed three times in 2x SSC and 0.1% SDS and twice in 0.1x SSC and 0.1% SDS for 30 minutes at 65°C. The membranes were exposed to autoradiography at -80°C. Autoradiograms were scanned on a UMAX UC1260 flatbed scanner (UMAX Data Systems, Inc), using Adobe Photoshop 3.0 software. Densitometric analysis was performed using NIH Image 1.61 software.

Determination of Tetrahydrobiopterin and GTP Cyclohydrolase I Activity in Cell Extracts
After incubation with different drugs, cells were harvested with trypsin, washed with PBS, pelleted by centrifugation, and stored frozen at -80°C. Measurements of tetrahydrobiopterin were performed with high-performance liquid chromatography analysis after oxidation by MnO₂ or iodine as previously described.⁷ GTP cyclohydrolase I activity was determined by high-performance liquid chromatography measurements of neopterin released from dihydroneopterin triphosphate after oxidation and phosphatase treatment by minor modification of previously described methods.²

Drugs
The following drugs were used: actinomycin D, 8-bromo-cAMP, cycloheximide (Sigma Chemical Co), INF-, IL-1ß, and TNF- (Collaborative Biomedical Products).

Calculations and Statistics
All experiments were conducted with cells obtained from at least three different donors. In each set, n represents the number of experiments. Statistical evaluation was done by paired Student's t test. Means were considered significantly different at P<.05.

	Results

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Using specific primers, mRNA for GTP cyclohydrolase I and GAPDH were detected in HUVECs by PCR. Both PCR products were found to be of predicted size on 1.5% agarose gels. Sequencing of human endothelial GTP cyclohydrolase I PCR product revealed 100% homology with reported human liver sequence⁸ (data not shown). This finding, together with restriction enzyme analysis of the GAPDH PCR product, confirmed specificity of amplified sequences.

Quantitative analysis of electrophoretically isolated GTP cyclohydrolase I and GAPDH PCR products showed exponential amplification of the semilogarithmic plot of counts per minute incorporated versus cycle number (Fig 1). Thirty and 25 cycles (middle portion of exponential amplification) were chosen for further studies of GTP cyclohydrolase I and GAPDH gene expression, respectively.

View larger version (13K): [in this window] [in a new window]   Figure 1. Exponential amplification of GTP cyclohydrolase I (left) and GAPDH (right) cDNA from control HUVECs by PCR. Values are mean±SEM, n=4. Note a logarithmic increase in the amount of amplified cDNA as a function of cycle number.

Incubation of HUVECs for 24 hours in the presence of TNF- (2 µg/mL), IFN- (200 U/mL), and IL-1ß (5 U/mL) significantly increased expression of GTP cyclohydrolase I mRNA (Fig 2). Induction of GTP cyclohydrolase I mRNA was apparent by 3 hours, peaked at 8 hours, and was sustained at high levels for at least 24 hours (Fig 3). In contrast, the same cocktail of cytokines did not affect expression of the GAPDH gene (Figs 2 and 3). Cytokines also stimulated GTP cyclohydrolase I enzymatic activity and production of tetrahydrobiopterin (Fig 4).

View larger version (10K): [in this window] [in a new window]   Figure 2. Stimulatory effect of cytokines IL-1ß (5 U/mL), TNF- (2 µg/mL), and INF- (200 U/mL) on expression of GTP cyclohydrolase I (GTPCH-I) mRNA from HUVECs. Cytokines did not affect expression of GAPDH mRNA. Values are mean±SEM, n=5 to 13. *P<.05; significantly different from corresponding control.

View larger version (12K): [in this window] [in a new window]   Figure 3. Time course of the cytokine (IL-1ß, 5 U/mL; TNF-, 2 µg/mL; INF, 200 U/mL) effect on expression of GTP cyclohydrolase I (GTPCH-I; left) mRNA and GAPDH (right) mRNA. Values are mean±SEM, n=3.

View larger version (10K): [in this window] [in a new window]   Figure 4. Stimulatory effect of cytokines IL-1ß (5 U/mL), TNF- (2 µg/mL), and INF- (200 U/mL) on GTP cyclohydrolase I (GTPCH-1) enzymatic activity and production of tetrahydrobiopterin in HUVECs. Values are mean±SEM, n=8 to 13. *P<.05; significantly different from corresponding control.

Actinomycin D (2 µg/mL) inhibited cytokine-induced expression of GTP cyclohydrolase I in HUVECs (Fig 4). In contrast, cycloheximide (0.5 µg/mL) did not block the stimulatory effect of cytokines (Fig 5). Actinomycin D and cycloheximide abolished and significantly reduced cytokine-induced production of tetrahydrobiopterin, respectively (Fig 6).

View larger version (24K): [in this window] [in a new window]   Figure 5. Effects of actinomycin D (2 µg/mL) and cycloheximide (0.5 µg/mL) on GTP cyclohydrolase I mRNA induction by cytokines in HUVECs. Cells were treated with cytokines or cytokines plus actinomycin D or cycloheximide for 24 hours. Values are mean±SEM, n=6. *P<.05; significantly different from control.

View larger version (12K): [in this window] [in a new window]   Figure 6. Inhibitory effects of actinomycin D (2 µg/mL) and cycloheximide (0.5 µg/mL) on cytokine-induced production of tetrahydrobiopterin. Cells were treated with cytokines or cytokines plus actinomycin D or cycloheximide for 24 hours. Values are mean±SEM, n=4 to 6. *P<.05; significantly different from values obtained in cells treated only with cytokines. N.D. indicates not detectable.

To determine whether the response of GTP cyclohydrolase I to cytokines involved change in mRNA transcription, nuclear run-on experiments were performed. Run-on transcription assays demonstrated that the transcriptional rate of GTP cyclohydrolase I was increased 3.6±0.6-fold (n=7; P<.05) after 24 hours exposure to cytokines (Fig 7). In contrast, only 1.6±0.2-fold increase in transcriptional rate of GAPDH was detected (n=7; P>.05). Nonspecific hybridization was ruled out by the absence of signals on nylon membranes slot blotted with a plasmid pRc/CMV.

View larger version (107K): [in this window] [in a new window]   Figure 7. Effect of cytokines on the rate of transcription of GTP cyclohydrolase I (GTPCH-I) mRNA in the endothelium. HUVECs were exposed to TNF- (2 µg/mL), INF- (200 U/mL), and IL-1ß (5 U/mL) for 24 hours. Nuclei were isolated and in vitro transcription was performed in the presence of [32P]UTP. Newly elongated 32P-labeled RNA transcripts were hybridized with a nylon membrane previously slot blotted with human GTP cyclohydrolase I and GAPDH cDNA, whereas pRc/CMV plasmid served as a control for nonspecific hybridization.

Incubation of endothelial cells for 24 hours with 8-bromo cAMP (10^-3 mol/L) did not affect expression of GTP cyclohydrolase I or GAPDH mRNA (Table 2).

	Discussion

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The major new finding of the present study is that in cultured HUVECs, cytokine-induced increase in GTP cyclohydrolase I activity is a result of increased levels of GTP cyclohydrolase I mRNA. This finding suggests that regulation of GTP cyclohydrolase I gene expression may play an important role in control of tetrahydrobiopterin biosynthesis in vascular endothelium. The results of our study also confirmed the previously reported ability of cytokines to increase endothelial GTP cyclohydrolase I enzymatic activity and production of tetrahydrobiopterin.^{2 3} Our findings are in agreement with recent results obtained by Simmons et al (1996),⁹ demonstrating the ability of cytokines to increase GTP cyclohydrolase I mRNA expression in cultured cardiac microvascular endothelial cells.

Two types of RT-PCR assays are currently in use. In the quantitative RT-PCR assay, known amounts of synthetic RNA (or cDNA) are coamplified in the same tube as a sample of cellular RNA. The quantitative RT-PCR assay allows the determination of the number of mRNA molecules per cell. However, in the relative RT-PCR assay (used in the present study), a housekeeping gene (GAPDH) and the experimental gene product (GTP cyclohydrolase I) are amplified in separate tubes. The relative RT-PCR method allows the determination of differences in mRNA levels between different samples.¹⁰ Extensive analysis of parameters involved in the quantitative RT-PCR assay clearly demonstrated that the relative and quantitative RT-PCR assays yielded comparable results.¹¹ To use the relative RT-PCR technique to measure GTP cyclohydrolase I cDNA, we determined the number of PCR cycles that were in the exponential range of amplification. Most importantly, the major advantage of this approach was that it enabled us to study expression of low abundant GTP cyclohydrolase I mRNA in endothelial cells.

Cytokines significantly increased transcription rate of GTP cyclohydrolase I by 3.6-fold. This finding is in contrast to the levels of GTP-cyclohydrolase I mRNA, which were increased fourfold to fivefold by cytokines. Although it is very difficult to compare semiquantitative data obtained with PCR and run-on assay, our findings raise the possibility that cytokines may have an additional effect on GTP cyclohydrolase I mRNA stability. Further studies are needed to characterize contribution of this mechanism to cytokine-induced increase in expression of GTP cyclohydrolase I gene in endothelial cells.

The ability of the RNA synthesis inhibitor actinomycin D¹² to inhibit cytokine-induced increase in expression of GTP cyclohydrolase I mRNA supports our conclusion that increased transcription of mRNA is responsible for the effect of cytokines. Treatment with actinomycin D also abolished the stimulatory effect of cytokines on production of tetrahydrobiopterin, reinforcing the concept that increased transcription of GTP cyclohydrolase I is an important regulatory mechanism responsible for increased biosynthesis of tetrahydrobiopterin.

Increased expression of GTP cyclohydrolase I mRNA in response to long-term treatment with cytokines appears to be independent of protein synthesis. Unlike results in cultured smooth muscle cells,¹³ the protein synthesis inhibitor cycloheximide did not enhance expression of the GTP cyclohydrolase I gene in endothelial cells. These findings suggest that different control mechanisms may be responsible for regulation of GTP cyclohydrolase I gene expression in vascular endothelium and smooth muscle. In contrast, cycloheximide inhibited production of tetrahydrobiopterin in endothelial cells, confirming that increased biosynthesis of tetrahydrobiopterin is dependent on protein synthesis. This finding is in agreement with a previous report on inhibition of tetrahydrobiopterin synthesis by cycloheximide in cultured dopamine neurons³ and most likely reflects reduction in de novo synthesis of GTP cyclohydrolase I protein.

Several previous studies indicated that cellular levels of cAMP may play an important role in regulation of tetrahydrobiopterin biosynthesis.^{14 15 16} In the pineal gland, receptor-mediated increase in cAMP levels inhibits synthesis of tetrahydrobiopterin.¹⁵ In contrast, in adrenal medullary cells and dopamine neurons, as well as in cultured rat aortic smooth muscle cells, GTP cyclohydrolase I enzymatic activity and tetrahydrobiopterin content are elevated by agents that increase intracellular levels of cAMP.^{14 16} The precise cAMP-dependent mechanisms responsible for these effects have not been identified. It is important to note that stimulation of vascular smooth muscle cells with forskolin induced expression of GTP cyclohydrolase I mRNA.¹⁶ In the present study, a 10^-3 mol/L concentration of 8-bromo-cAMP did not affect expression of the GTP cyclohydrolase I gene. We did not measure GTP cyclohydrolase I enzymatic activity or production of tetrahydrobiopterin in cells treated with cAMP analogue; therefore, we cannot rule out that cAMP may affect biosynthesis of tetrahydrobiopterin. However, our results suggest that in vascular endothelium, cAMP alone does not induce expression of GTP cyclohydrolase I mRNA.

In rats treated with lipopolysaccharide, increased expression of GTP cyclohydrolase I mRNA has been detected in lung, heart, and liver.^{9 18} These studies demonstrated that upregulation of GTP cyclohydrolase I gene expression does occur in vivo. More importantly, it appears that availability of tetrahydrobiopterin is a limiting factor in nitric oxide production after induction of nitric oxide synthase with lipopolysaccharide. Interestingly, glucocorticoids have an inhibitory effect on GTP cyclohydrolase I mRNA expression.⁹ This effect significantly contributes to reduction of nitric oxide formation in animals treated with lipopolysaccharide.⁹ The promoter region of the rat GTP cyclohydrolase I gene has been cloned, and it contains sites potentially responsive to nuclear factor-B, IFN-, nuclear factor IL6, and Activator Protein–1 (AP-1).¹⁹ The structure of the gene promoter is certainly consistent with results obtained with cytokines in the present study and may explain the reported ability of dexamethasone to inhibit induction of GTP cyclohydrolase I.

The major function of tetrahydrobiopterin synthesized in arterial wall is to support the activity of nitric oxide synthase and production of nitric oxide. Previous studies on cultured HUVECs demonstrated that availability of tetrahydrobiopterin may regulate activity of the constitutive (endothelial) isoform of nitric oxide synthase.^{2 3} Furthermore, in isolated cerebral arteries, increased intracellular concentrations of tetrahydrobiopterin may augment endothelium-dependent relaxations mediated by nitric oxide.²⁰ These findings and the results of the present study suggest that in endothelial cells, regulation of GTP cyclohydrolase I gene expression and tetrahydrobiopterin biosynthesis may have an important role in control of nitric oxide production.

	Selected Abbreviations and Acronyms

 

8-bromo-cAMP = 8-bromoadenosine 3':5'-cyclic monophosphate HUVEC = human umbilical vein endothelial cell IL-1ß = interleukin-1ß INF- = interferon- RT-PCR = reverse transcription polymerase chain reaction TNF- = tumor necrosis factor-

	Acknowledgments

 
This work was supported in part by National Heart, Lung, and Blood Institute grant HL-53542 and the Mayo Foundation. The authors would like to thank Dr T. Nagatsu, Fujita Health University School of Medicine, Japan, for the generous gift of human GTP cyclohydrolase I cDNA; Dr Emanuel Strehler, Mayo Clinic, for the gift of GAPDH; and Janet Beckman for typing the manuscript.

Received May 3, 1996; accepted September 8, 1997.

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