From the Medical Clinic B Research Unit (P.L., A.S., G.S.), Department of
Medicine, University Hospital of Zurich, and Department of Pediatrics (N.B.),
Division of Clinical Chemistry and Biochemistry, University Childrens Hospital
of Zurich, Zurich, Switzerland.
Correspondence to PD Dr Gabriele Schoedon, Department of Medicine, University Hospital of Zurich, Rämistrasse 100, CH-8091 Zurich, Switzerland. E-mail klinsog{at}usz.unizh.ch
Abstract
Background—The nitric oxide
synthase cofactor tetrahydrobiopterin (BH4) is involved in the
regulation of endothelium-dependent vascular functions
mediated by nitric oxide. Vascular endothelial cells
synthesize and secrete large amounts of BH4 on cytokine
activation. There is scant knowledge about molecular mechanisms of
cytokine-triggered BH4 production in endothelial
cells.
Methods and Results—Pteridine production, mRNA expression
of GTP cyclohydrolase (GTPCH) and 6-pyruvoyltetrahydropterin synthase
(PTPS) (both key enzymes of BH4 biosynthesis), and PTPS activity were
studied in human umbilical vein endothelial cells
(HUVECs) exposed to inflammatory cytokines. BH4 levels were
Conclusions—The present study demonstrates for the first time
the cytokine-dependent regulation of PTPS, the second enzyme in
BH4 synthesis. Although GTPCH is believed to be the rate-limiting step,
control of endothelial PTPS expression by
cytokines may play an important role in regulating
BH4-dependent nitric oxide production in the vascular system.
Nitric oxide (NO) has emerged as an important factor
controlling vascular tone under normal and pathological conditions.
There is increasing evidence that tetrahydrobiopterin (BH4), the
natural cofactor of NO synthases, plays a crucial role in the
regulation of NO synthesis in vascular cells.1
Intracellular BH4 availability modulates NO-mediated cGMP
production in human endothelial cells,
whereas BH4 synthesis is regulated by inflammatory and
anti-inflammatory cytokines.2 3
Interestingly, inflammatory cytokines enhance BH4 synthesis and
constitutive NO synthase (cNOS) specific activity, with a concomitant
decrease of cNOS enzyme in human vascular endothelial
cells.4 In vascular smooth muscle cells (SMCs),
inducible NO synthase (iNOS) is upregulated by inflammatory
cytokines, and therefore BH4 becomes limiting for maximal iNOS
activity.5 6 GTP cyclohydrolase I (GTPCH), the
first enzyme in the biosynthesis of BH4, is regulated by
cytokines7 8 and is commonly thought to
be the rate-limiting enzyme.9 However,
6-pyruvoyltetrahydropterin synthase (PTPS), the second enzyme in the
biosynthesis of BH4, can become rate limiting on induction of GTPCH,
especially in human cells, as reflected by neopterin
accumulation.7 8 Human
endothelial cells express GTPCH activity and synthesize
small amounts of BH4 constitutively.2 3 Under
inflammatory conditions, endothelial cells synthesize
and secrete large amounts of BH4 and little or no
neopterin,2 3 10 indicating that PTPS is not rate
limiting in these cells. Recently, GTPCH mRNA induction by
cytokines has been reported in human
endothelial cells,11 but nothing
is known about regulation of PTPS in those cells. Because vascular
endothelial cells are the main producers of BH4 in
physiological and pathological
conditions,3 4 10 12 we investigated the
expression of GTPCH and PTPS, the key enzymes of BH4 biosynthesis, at
the molecular level in human umbilical vein endothelial
cells (HUVECs) under normal and inflammatory conditions.
Methods
Cell Culture
Reverse Transcriptase–Polymerase Chain Reaction
Comparative Measurement of mRNA Levels
Determination of PTPS Activity
Measurement of Pterins
Statistical Analysis
Results
The effects of inflammatory cytokines on pteridine
synthesis and specific PTPS activity in HUVECs are shown in the
Table
PTPS activity was constitutively present in untreated HUVECs. After
incubation of HUVECs with IFN-
To investigate to what extent these metabolic
parameters reflect regulation at the molecular level, we
examined the effects of inflammatory cytokines on mRNA
expression of GTPCH and PTPS, the key enzymes of BH4 de novo synthesis.
In parallel, cNOS and iNOS mRNAs were also investigated. The results of
RT-PCR are shown in panel A of the
Figure
The extent of regulation of GTPCH, PTPS, and cNOS mRNAs in HUVECs
was evaluated by comparative limiting-dilution PCR analysis of
the respective cDNAs (panel B of the Figure
Discussion
In the present report, the regulation of mRNA abundance and
specific activity of PTPS, the second enzyme in the synthesis of BH4,
by inflammatory cytokines is described for the first time.
GTPCH, the first enzyme in the BH4 synthesis pathway, has been widely
described as the rate-limiting step in mammals.9
Up to a 40-fold regulation of GTPCH activity was found in cells treated
with inflammatory stimuli,2 8 which is
consistent with the strongly elevated GTPCH mRNA level reported
in the present article. In humans, PTPS was generally believed to
be constitutively present and to become rate limiting on induction
of GTPCH, reflected by production of higher neopterin than
biopterin concentrations.8 However, the
persistent low levels of neopterin in HUVECs even after inflammatory
activation (Table
The mechanism behind the upregulation of PTPS mRNA by cytokines
in HUVECs remains to be elucidated. However, our observation
demonstrates on the molecular level the relationship between
endothelial BH4 production and iNOS expression
in SMCs (Figure
Acknowledgments
This study is supported by grants of the Swiss National Science
Foundation, NF 32-42536.94 (Dr Schoedon) and NF 31-43380.95 (Dr
Blau).
Received July 17, 1998;
revision received August 18, 1998;
accepted August 27, 1998.
References
© 1998 American Heart Association, Inc.
Brief Rapid Communication
Regulation of 6-Pyruvoyltetrahydropterin Synthase Activity and Messenger RNA Abundance in Human Vascular Endothelial Cells
140-fold enhanced on treatment of HUVECs with combined
interferon-
/tumor necrosis factor-
/interleukin-1 (IFN/TNF/IL-1).
Specific PTPS activity was 
3-fold higher in cytokine-treated
HUVECs than in untreated cells. Reverse-transcription/limiting-dilution
polymerase chain reaction analysis showed that in response to
IFN/TNF/IL-1, mRNA abundance of GTPCH and PTPS was increased 64-fold
and 10-fold, respectively.
Key Words: cells • endothelium-derived factors • enzymes • vasculature
HUVECs and human aortic SMCs (ATCC) were maintained in
humidified air, 5% CO2 at 37°C. Cells from
passages 2 to 4 were seeded on 100-mm culture dishes in 10 mL of
medium. At confluence, cells were incubated with interferon-
(IFN-; 100 U/mL), tumor necrosis factor- (TNF-; 100 U/mL), and
interleukin-1 (IL-1; 20 U/mL) as indicated.
The relative expression of GTPCH, PTPS, cNOS, and iNOS
mRNAs compared with the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was evaluated in control and
cytokine-treated cells. Aliquots containing 5 µg of total
cellular RNA were reverse transcribed, and first-strand cDNA was used
as a template in polymerase chain reaction (PCR). cDNA aliquots were
amplified with the following specific primers: PTPS,
5'-TCGCTGCCAGGCACAAGTGT-3' (sense) and 5'-CCACAATATTATTGTCAGTT-3'
(antisense), yielding a 397-bp product located at base pairs 24 to
421 of the PTPS cDNA sequence (Genebank M97655); GTPCH,
5'-TTGGTTATCTTCCTAACAA-3' (sense) and 5'-GTGCTGGTCACAGTTTTGCT-3'
(antisense), yielding a 226-bp product located at base pairs 460 to
686 of the GTPCH sequence (Genebank U19523); cNOS,
5'-CACCGCTACAACATCCTGGAG-3' (sense) and 5'-CTGTGTTACTGGACTCCTTCC-3'
(antisense), yielding a 799-bp product located at base pairs 1130
to 1929 of the cNOS sequence (Genebank M95296); and iNOS,
5'-GGACATCAACAACAACGTG-3' (sense) and 5'-GACCTGATGTTGCCATTGTTG-3',
yielding a 649-bp product located at base pairs 282 to 931 of the
iNOS sequence (Genebank L09210). Primers specific for human
GAPDH were from Stratagene. The origin of amplified PCR
products from cDNAs of GTPCH, PTPS, cNOS, and iNOS, respectively,
was confirmed by sequencing. PCR products in agarose gels (Visigel
matrix, Stratagene) were analyzed by use of the Bio-Rad Gel Doc
1000 System.
For comparative determination of mRNA levels, limiting-dilution
PCRs of cDNA were performed.13 14 After reverse
transcription (RT) as described above, equal amounts of cDNA were
serially diluted 2-fold up to 256-fold and cycled as described
above.
Specific PTPS activity was measured after 48 hours as described
previously.15
Pterins were determined after 24 hours by
high-performance liquid chromatography after
acidic oxidation as described previously.16
Statistical analysis was performed by ANOVA with
Dunnett's correction for comparison of multiple groups with 1 control
by use of a computer-based program (Instat 3.0, Graphpad). 
. Inflammatory cytokines
predominantly caused enhancement of BH4 and no or only moderate
increase of neopterin. A 140-fold increase of extracellular BH4 (20 to
2920 pmol/mg) was found on incubation with mixed cytokines.
Under these conditions, extracellular BH4 (2920 pmol/mg) was
116-fold higher than the corresponding intracellular portion (25
pmol/mg), indicating that the major part of the cofactor is
secreted.
View this table:
[in a new window]
Table 1. Regulation of PTPS Activity and Pteridine Synthesis in
HUVECs /TNF-/IL-1, specific PTPS activity
was enhanced 3-fold (Table
). . In untreated HUVECs, mRNAs of all
enzymes except iNOS were constitutively present, GTPCH and PTPS in
low amounts and cNOS in higher amounts. Treatment with IFN- and
TNF- enhanced GTPCH mRNA abundance, whereas IFN- and TNF-
alone had no effect on PTPS mRNA. In contrast, IL-1 alone did not
influence GTPCH mRNA but significantly enhanced PTPS mRNA.
Combination of the 3 inflammatory cytokines further enhanced
GTPCH mRNA and, most interestingly, PTPS mRNA abundance. Under
inflammatory conditions, cNOS mRNA was diminished and GAPDH
mRNA levels remained unchanged. iNOS mRNA was not detectable in HUVECs.
In human vascular SMCs, however, large amounts of iNOS mRNA were
expressed after treatment with IFN-/TNF-/IL-1 (Figure).
View larger version (56K):
[in a new window]
Figure 1. Effects of inflammatory cytokines on mRNA expression
of PTPS, GTPCH, cNOS, and iNOS in HUVECs. Human aortic SMCs served as
positive control for iNOS expression. Cells were incubated with the
indicated agents for 24 hours. A, RT-PCR analysis of GTPCH,
PTPS, cNOS, and iNOS. B, Comparative limiting-dilution PCR
analysis of PTPS, GTPCH, and cNOS cDNAs from untreated and
cytokine-treated (cyt/mix) HUVECs. Results are
representative of 
5 independent
experiments.). In cells treated with
mixed inflammatory cytokines, PTPS mRNA abundance was
10-fold higher than in untreated cells. mRNA of GTPCH was 64-fold
enhanced, whereas mRNA of cNOS declined to undetectable levels. ), suggest that PTPS is either present with high
constitutive activity or that its expression is upregulated by
cytokines. We demonstrated that PTPS is upregulated in HUVECs
by a combination of inflammatory cytokines not only by
measuring PTPS enzyme activity using an assay system generally approved
for diagnosis of PTPS deficiencies,15 16 but also
by RT-PCR analysis of PTPS mRNA abundance. IL-1 seems to be the
leading signal for PTPS regulation in HUVECs, because it is effective
as a single stimulus and strongly synergizes with IFN-/TNF-
(Figure), each of which was ineffective alone. This finding is in
accordance with a previous report on PTPS activity in HUVECs in which
IFN- and TNF- did not alter PTPS activity.2
Although cytokine regulation of PTPS is a novel finding in
cells of the vascular system, regulation of PTPS mRNA abundance has
been described in other organs: a 3- to 4-fold elevation of PTPS mRNA
has been reported in rat adrenal gland after treatment with
reserpine.17 Furthermore, both GTPCH and PTPS
enzyme activities have been described to be coordinately induced by
cytokines in T cells.18 ), substantiating previous findings concerning human
vascular cell functions: cNOS and iNOS are compartmentalized in
endothelium and SMCs, and there is a coordinated
switching of NO production from endothelial
cNOS to smooth muscle iNOS in the presence of inflammatory
cytokines.19 Because BH4 is limiting for
maximal NO production in SMCs,5 6 BH4
secreted by activated endothelial cells could
serve as an additional cofactor necessary for prolonged NO
production for iNOS expressed in adjacent
SMCs.14 20
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