This Article Abstract Full Text (PDF) All Versions of this Article: 96/2/164    most recent 01.RES.0000153669.24827.DFv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Huang, A. Articles by Keaney, J. F. Search for Related Content PubMed PubMed Citation Articles by Huang, A. Articles by Keaney, J. F., Jr Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Compound via MeSH Substance via MeSH Genetics Home Reference Hazardous Substances DB NITRIC OXIDE Related Collections Endothelium/vascular type/nitric oxide Cell signalling/signal transduction Gene regulation Related Article

(Circulation Research. 2005;96:164.)
© 2005 American Heart Association, Inc.

Molecular Medicine

Cytokine-Stimulated GTP Cyclohydrolase I Expression in Endothelial Cells Requires Coordinated Activation of Nuclear Factor-B and Stat1/Stat3

Annong Huang, Ying-Yi Zhang, Kai Chen, Kazuyuki Hatakeyama, John F. Keaney, Jr

From the Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute (A.H., Y.Z., K.C., J.F.K.Jr.), Boston University School of Medicine, Boston, Mass; and Department of Surgery (K.H.), University of Pittsburgh, Pa.

Correspondence to John F. Keaney Jr, Boston University School of Medicine, Whitaker Cardiovascular Institute, 715 Albany St, Rm W507, Boston, MA 02118. E-mail jkeaney{at}bu.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endothelial production of nitric oxide (NO) is dependent on adequate cellular levels of tetrahydrobiopterin (BH₄), an important cofactor for the nitric oxide synthases. Vascular diseases are often characterized by vessel wall inflammation and cytokine treatment of endothelial cells increases BH₄ levels, in part through the induction of GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme for BH₄ biosynthesis. However, the molecular mechanisms of cytokine-mediated GTPCH I induction in the endothelium are not entirely clear. We sought to investigate the signaling pathways whereby cytokines induce GTPCH I expression in human umbilical vein endothelial cells (HUVECs). Interferon- (IFN-) induced endothelial cell GTPCH I protein and BH₄ modestly, whereas high-level induction required combinations of IFN- and tumor necrosis factor- (TNF-). In the presence of IFN-, TNF- increased GTPCH I mRNA in a manner dependent on nuclear factor-B (NF-B), as this effect was abrogated by overexpression of a dominant-negative IB construct. HUVEC IFN- treatment resulted in signal transducer and activator of transcription 1 (Stat1) activation and DNA binding in a Jak2-dependent manner, as this was inhibited by AG490. Conversely, overexpression of Jak2 effectively substituted for IFN- in supporting TNF-–mediated GTPCH I induction. The role of IFN- was also Stat1-dependent as Stat1-null cells exhibited no GTPCH I induction in response to cytokines. However, Stat1 activation with oncostatin M failed to support TNF-–mediated GTPCH I induction because of concomitant Stat3 activation. Consistent with this notion, siRNA-mediated Stat3 gene silencing allowed oncostatin M to substitute for IFN- in this system. These data implicate both NF-B and Stat1 in endothelial cell cytokine-stimulated GTPCH I induction and highlight the role of Stat3 in modulating Stat1-supported gene transcription. Thus, IFN- and TNF- exert distinct but cooperative roles for BH₄ biosynthesis in endothelium that may have important implications for vascular function during vascular inflammation.

Key Words: biopterin • endothelium • inflammation • cytokines • signal transducers and activators of transcription • nuclear factor-B

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Nitric oxide (NO) is produced from L-arginine in the vascular endothelium by the endothelial isoform of nitric oxide synthase (NOS). Endothelial production of NO is critical for the control of vascular tone,¹ arterial pressure,² platelet adhesion to the endothelial surface,³ and smooth muscle cell proliferation.^4,5 Impaired endothelium-derived NO bioactivity is a common feature of many vascular diseases⁶ that predicts the development of clinical vascular complications.^7,8 Thus, clinical vascular disease involves a lapse in NO-mediated vascular homeostasis.

The production of NO by the endothelial isoform of nitric oxide synthase (eNOS) is subject to regulation through a variety of mechanisms including substrate availability, subcellular localization, protein-protein interactions, phosphorylation, and the availability of cofactors (see review⁹). With respect to the latter, considerable evidence indicates that tetrahydrobiopterin (BH₄) has an important role in regulating NOS activity. Recent studies with eNOS^10,11 and nNOS¹² indicate that the enzyme must be fully saturated with BH₄ to completely couple NADPH oxidation to NO production. In the setting of limited BH₄ levels, the enzyme functions in an "uncoupled" state in which NADPH-derived electrons are added to molecular O₂ rather than L-arginine, with superoxide and H₂O₂ as the reported products.^10,11 Uncoupling of eNOS has been implicated in a number of vascular diseases such as atherosclerosis¹³ and diabetes,¹⁴ consistent with findings that tissue levels of BH₄ are reduced in diabetes.¹⁵

Despite the fact that endothelial BH₄ levels are an important determinant of eNOS activity, relatively little is known about the control of endothelial BH₄ levels. In mammalian cells, the de novo biosynthesis of BH₄ begins with GTP cyclohydrolase I (GTPCH I), a homodecamer protein consisting of 25-kDa subunits. This enzyme catalyzes the rearrangement of GTP to dihydroneopterin triphosphate, a species subsequently converted to BH₄ by the sequential action of 6-pyruvoyltetrahydrobiopterin synthase and sepiapterin reductase (see review¹⁶). In contrast to the latter two enzymes, GTPCH I activity is limiting in most tissues,¹⁷ making it the major determinant of intracellular BH₄ content. In endothelial cells, GTPCH I expression is induced by cytokines^18–20 in much the same way that iNOS and GTPCH I are coordinately induced in other cell types.²¹ However, in contrast to other cell types,^22,23 the signaling mechanisms responsible for GTPCH I regulation in the endothelium are largely unknown. The purpose of this study, therefore, was to examine GTPCH I regulation in the endothelium as a function of cytokine exposure.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Human tumor necrosis factor- (TNF-), interferon-, interleukin (IL)-1ß, and oncostatin M were purchased from R&D Systems. The inhibitor of B (IB)-, signal transducer and activator of transcription 1 (Stat1) p84/91 antibodies, and Stat1 p82/p91 oligonucleotides were from Santa Cruz Biotechnology. The Jak2 inhibitor, AG490, was obtained from Calbiochem. Primary antibodies directed against total Stat1, Stat3, and phosphorylated forms of Stat1 (Tyr-701) and Stat3 (Tyr-705) as well as secondary peroxidase-labeled antibodies were from Cell Signaling Technology. Antibodies for total and phosphorylated Jak2 (Tyr-1007/Tyr-1008) were from Biosource International. The antibody for serine phosphorylated Stat1 (Ser-727). The GTPCH I antibody has been described previously.²⁴ Reverse transcriptase (AMV) was from Invitrogen. The TaKaRa Taq DNA polymerase was from Fisher Scientific, and gel shift assay kits and DNA labeling systems were from Promega. We obtained [-³²P]ATP from NEN Life Science. The siRNA construct for Jak2 was from Dharmacon and Stat3 siRNA was from Upstate. The dominant-negative IB construct was kindly provided by Dr Edward Schwarz, University of Rochester, Rochester, NY.²⁵ The human Jak2 cDNA construct was provided by Dr Sumiko Watanabe, University of Tokyo, Japan.²⁶ All other reagents were obtained from Sigma.

Cell Culture
Human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics, grown in endothelial cell growth medium (Clonetics, Inc), and used between passages 2 and 8. Confluent cells were stimulated in medium containing 1% serum without extra growth factors and hydrocortisone for the indicated period of time. Mouse fibroblast lines CD⁺ and CD^– derived from wide-type and the Stat1 knockout mice, respectively were kindly provided by Dr. David E. Levy, New York University Medical Center,^27,28 and were cultured in DMEM with 10% FBS.

Western Blotting and Tetrahydrobiopterin Determination
After treatments, cells were washed with ice-cold PBS twice, and lysed in loading buffer containing 50 mmol/L Tris-HCl (pH 6.8), 2% SDS, 200 mmol/L dithiothreitol, 20% glycerol, and 0.2% bromophenol blue. Proteins were fractionated by SDS-PAGE and Western blot analysis was performed as described previously.²⁹ Proteins were detected using an enhanced chemiluminescence detection kit from Amersham Pharmacia Biotech, Inc. Determination of tetrahydrobiopterin in HUVECs was performed as described.³⁰

Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared as described previously.^31,32 Double-stranded oligonucleotides for nuclear factor-B (NF-B) (5'-AGTTGAGGGGACTTTCCCAGGC-3') and Stat 1 (5'-CATGTTATGGATATTCCTGTAAGTG-3') were labeled with [-³²P]ATP using a T4 polynucleotide kinase kit (Promega). The binding reactions and electrophoresis of DNA-protein complexes were performed as described previously.³¹

Transfections
HUVECs were transfected with dominant-negative IB virus or a control (LacZ) as described previously.^25,33 Briefly, subconfluent cells in 6-well plates were transfected with a range of MOI for 16 hours before treatment with cytokines. Transfection was verified by examining the expression of mutated IB protein and NF-B transactivation by electrophoretic mobility shift assay (EMSA). Transfection of Stat3 or Jak2 siRNA was performed using RNAifect reagents from Qiagen and typically used siRNA concentrations of 20 to 40 nmol/L as outlined in the manufacturer’s instructions. Transfections with Jak2 plasmid were performed using Lipofectamine Plus reagents from Invitrogen according to the manufacturer’s instructions.

Reverse Transcription–Polymerase Chain Reaction
Total cellular RNA was isolated from endothelial cells using TRIzol from Life Technologies. The procedures for semiquantitative reverse transcription–polymerase chain reaction (RT-PCR) were as described³⁴ using forward (5'-GCCATGCAGTTCTTCACCAA-3') and reverse (5'-AGGCTTCCGTGATTGCTACA-3') primers corresponding to human GTPCH I mRNA. Reactions were run for 30 cycles at conditions as follows: denaturation for 30 seconds at 94°C, annealing for 30 seconds at 57°C, and extension for 30 seconds at 72°C. Constitutively expressed GADPH mRNA was amplified with forward (5'-ACCACAGTCCATGCCATCACTGCC-3') and reverse (5'-ACCAGGAAATGAGCTTGACAAAGT-3') primers in a similar manner for 26 cycles.

Statistical Analysis
Blots are representatives of three to four experiments. Comparisons among treatment groups were performed with one-way analysis of variance and a post hoc Tukey comparison. Statistical significance was accepted if the null hypothesis was rejected with P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cytokines Induce GTPCH I Expression and Tetrahydrobiopterin Biosynthesis in HUVECs
The effects of TNF-, interferon- (IFN-), and IL-1ß alone or in combinations were determined on the expression of GTPCH in HUVECs. As shown in Figure 1A, we observed constitutive low-level GTPCH I expression in resting cells that was unaltered by TNF- or IL-1ß alone. In contrast, IFN- alone increased GTPCH I protein by 2- to 3-fold and combinations of IFN- with either TNF- or IL-1ß increased protein levels >20-fold. We also observed time- and concentration-dependent induction of GTPCH I protein in HUVECs as a function of TNF- exposure in the presence of IFN- (Figure 1B and C). Consistent with this observation, intracellular BH₄ levels increased over control levels by 3- to 4-fold with IFN-, and 40- to 50-fold with the combination of IFN- and TNF- (Figure 1D). Although lipopolysaccharide (LPS) has been effective in GTPCH I induction in many cell types, we found no effect with this compound either alone or in combination with TNF-, IL-1ß, or IFN- (data not shown). Consistent with a prior report,²⁰ cytokine treatment of HUVECs with a combination of TNF- and IFN- increased GTPCH I mRNA levels by RT-PCR (Figure 1E).

View larger version (23K): [in this window] [in a new window]   Figure 1. Cytokines induce GTPCH I expression and tetrahydrobiopterin production in endothelial cells. A, HUVECs were incubated with TNF- (20 ng/mL), IFN- (50 ng/mL), or IL-1ß (20 ng/mL) as indicated for 18 hours. Cells were lysed, proteins resolved by SDS-PAGE, and the lysates probed with antibody against GTPCH I as indicated in Materials and Methods. B, HUVECs were incubated with IFN- (50 ng/mL) and TNF- (20 ng/mL) for the indicated time and GTPCH I determined as in A. C, HUVECs were incubated with IFN- (50 ng/mL) and the indicated concentration of TNF- for 20 hours and GTPCH I determined as in A. D, HUVECs were incubated with TNF- and/or IFN- as in A and tetrahydrobiopterin (BH4) levels were determined as described.29 E, HUVECs treated for 8 hours as in A were lysed, total RNA extracted, and mRNA levels for GTPCH I and GAPDH determined as in Materials and Methods. All blots or gels are representative of 3 to 4 independent experiments and composite data represent mean±SD from 4 independent experiments; *P<0.05 vs CTL by ANOVA and post hoc Tukey comparison.

NF-B Activation by TNF- Is Required for Optimal GTPCH I Induction
To determine the mechanisms involved in TNF- stimulation of HUVEC GTPCH I levels, we examined nuclear NF-B DNA binding activity and cytosolic IB content in response to cytokine stimulation. We found that TNF-, but not IFN-, stimulated degradation of IB and nuclear translocation of NF-B (Figure 2). Because TNF- markedly potentiated GTPCH I induction, the requirement for NF-B activation was evaluated by transfecting HUVECs with dominant-negative IB (dn.IB) resistant to ubiquitination²⁵ before stimulation with TNF- and IFN-. Transfected HUVECs expressed high levels of the dominant-negative IB protein that resulted in substantial inhibition of IB degradation, NF-B nuclear translocation, and induction of GTPCH I mRNA and protein (Figure 3). Transfection of vector or dn.IB alone did not alter the phosphorylation of Stat1 in these cells, and we observed similar results with the proteasome inhibitor, MG132 (data for both not shown). Thus, the TNF- component of cytokine-induced GTPCH I upregulation requires NF-B nuclear translocation.

View larger version (78K): [in this window] [in a new window]   Figure 2. Cytokine-mediated activation of NF-B in HUVECs. HUVECs were treated with TNF- (20 ng/mL), IFN- (50 ng/mL), both, or neither for 45 minutes. Nuclear proteins were extracted and NF-B DNA binding activity was determined by electrophoretic mobility shift assay (EMSA) as described in Materials and Methods. Cytosolic proteins were subjected to immunoblotting (IB) with antibody directed against IB. Data are representative of three independent experiments.

View larger version (69K): [in this window] [in a new window]   Figure 3. Cytokine-mediated GTPCH I induction requires NF-B. HUVECs were transfected with adenovirus expressing either LacZ (CTL; 50 MOI) or dominant-negative IB (dn.I-B) for 24 hours before incubation with IFN- (50 ng/mL) and TNF- (20 ng/mL) as indicated. After 15 minutes, cells were harvested for electrophoretic mobility shift assay (EMSA) of NF-B DNA binding as described in Materials and Methods. Time points for analysis of mRNA by RT-PCR (GTPCH I, GAPDH) and protein expression by immunoblot (IB; for GTPCH I and IB) were 8 and 18 hours, respectively. Data are representative of three independent experiments.

Activation of Jak2 by IFN- Is Required for GTPCH I Induction
Because responses to IFN- may involve activation of the Jak/Stat pathway,³⁵ we treated HUVECs with TNF- and/or IFN- and examined Jak2/Stat1 activation. We found that IFN-, but not TNF-, stimulated Stat1 tyrosine phosphorylation and nuclear translocation—two downstream events of Jak2 activation (Figure 4). The involvement of Jak2 in this process was confirmed using both pharmacological and molecular strategies. With regard to the former, the Jak2 inhibitor, AG490, dose-dependently attenuated Stat1 tyrosine phosphorylation and nuclear translocation as well as GTPCH I protein and mRNA upregulation in response to TNF- and IFN- (Figure 5A). As expected, we did not observe any effect of AG490 on IB degradation and NF-B nuclear translocation (data not shown). Consistent with this observation, Jak2 gene silencing via siRNA abrogated GTPCH I induction in response to TNF- and IFN- (Figure 5B). Conversely, overexpression of Jak2 enhanced phosphorylation of both Jak2 and Stat1 proteins and qualitatively recapitulated the effect of IFN- with regard to GTPCH I induction (Figure 5C). Thus, Jak2 activation is sufficient for GTPCH I upregulation in response to IFN- and its synergy with TNF-.

View larger version (81K): [in this window] [in a new window]   Figure 4. Cytokine-mediated activation of Stat1 in HUVECs. Cells were treated with TNF- (20 ng/mL), IFN- (50 ng/mL), both, or neither for 45 minutes. Nuclear proteins were extracted and Stat1 DNA binding activity was determined by EMSA as described in Materials and Methods. Total cell lysates were subjected to immunoblotting (IB) with antibodies against native or tyrosine phosphorylated (Tyr-701) Stat1 as indicated. Data are representative of three independent experiments.

View larger version (39K): [in this window] [in a new window]   Figure 5. Cytokine-stimulated GTPCH I induction requires Jak2. A, HUVECs were incubated with AG490 as indicated for 45 minutes before stimulation with IFN- (50 ng/mL) and TNF- (20 ng/mL) for 8 hours and cells were analyzed for Stat1 DNA binding activity and GTPCH I mRNA expression as described in Materials and Methods. After 18 hours of incubation, cells were assayed for GTPCH I protein and Stat1 tyrosine phosphorylation by immunoblotting (IB). B, HUVECs were treated with Jak2 (+) or control (–) siRNA for 48 hours before incubation with IFN- and TNF- as in A. Cells were harvested after 18 hours for assay of Jak2 protein, Stat1 tyrosine phosphorylation, GTPCH I, IFN- response factor (IRF-1), and ß-actin by immunoblotting. C, HUVECs were transfected with human Jak2 and cultured for 24 hours before incubation with IFN- (50 ng/mL) and/or TNF- (20 ng/mL) as indicated. After 18 hours, cell lysates were subjected to immunoblotting with antibodies specific for GTPCH I, ß-actin, or the phosphorylated forms of Stat1 (Tyr-701) and Jak2 (Tyr-1007/Tyr-1008). Data are representative of 3 to 4 independent experiments.

Activation of Stat1 Is Essential for IFN-–Induced GTPCH I
Although many Jak2-mediated responses to IFN- involve Stat1, there is a growing appreciation that some IFN- responses are Stat1-independent.³⁵ To investigate the role of Stat1 in cytokine-induced GTPCH expression, we examined the extent of GTPCH I upregulation in response to TNF- and IFN- as a function of Stat1. As shown in Figure 6A, CD+ wild-type fibroblasts exhibited synergistic induction of GTPCH I in response to TNF- and IFN-, whereas this response was absent in Stat1-null CD-fibroblasts, suggesting a dependence on Stat1. To determine whether Stat1 phosphorylation is sufficient for GTPCH I induction, we treated HUVECs with oncostatin M, a cytokine that also activates Stat1 independent of IFN- receptor activation.³⁶ We found that oncostatin M could not substitute for IFN- to support GTPCH I induction in TNF-–treated HUVECs despite inducing phosphorylation of Stat1 on Tyr-701 and Ser-727 (Figure 6B). Moreover, oncostatin M was unable to enhance interferon- response factor-1 (IRF-1) activation to any greater extent than TNF- alone (Figure 6B). These data indicate that IFN-–mediated Jak2 activation contributes to GTPCH I upregulation via mechanisms that go beyond simple Stat-1 phosphorylation at Tyr-701.

View larger version (55K): [in this window] [in a new window]   Figure 6. Stat1 phosphorylation is required but not sufficient for GTPCH I induction. A, Wild-type and Stat1-null (Stat1–/–) murine fibroblasts were incubated with TNF- (20 ng/mL) and/or IFN- (50 ng/mL) for 18 hours. Cells were then lysed, proteins resolved, and subjected to immunoblotting with antibodies against GTPCH I, tyrosine phosphorylated Stat1 (Tyr-701), interferon- response factor-1 (IRF-1), or inducible nitric oxide synthase (iNOS). B, HUVECs were incubated with TNF- (20 ng/mL), oncostatin M (OnM; 50 ng/mL), and/or IFN- (50 ng/mL) as indicated. Cells were then lysed and immunoblots probed with antibodies against GTPCH I, IRF-1, and the indicated phosphorylated forms of Stat1 and Stat3. Data are representative of four independent experiments, and Stat blots were obtained after a 15-minute incubation, whereas all others represent a 20-hour duration of treatment.

Coordination Between Stat1 and Stat3 Activation Determine GTPCH I Induction
An important difference between oncostatin M and IFN- is the former produces robust activation of Stat3 (Figure 6B). Indeed, after several hours of oncostatin M treatment, HUVECs exhibit only Stat3 activation, whereas similar treatment with IFN- results in Stat1 activation (Figure 7A). These data suggest a negative influence of Stat3 on GTPCH I induction. To evaluate this possibility, we suppressed Stat3 protein expression using siRNA (Figure 7B). Suppression of Stat3 expression also abrogated oncostatin M–mediated Stat3 phosphorylation and this effect afforded robust IRF1 and GTPCH I induction in combination with TNF-. Indeed, suppression of Stat3 effectively "converts" the oncostatin M response to one resembling that of IFN- (Figure 7B). These data indicate that coordinated Stat1 and NF-B activation, along with the lack of Stat3 activation, accounts for cytokine-stimulated GTPCH I induction in endothelial cells.

View larger version (67K): [in this window] [in a new window]   Figure 7. Stat3 activation inhibits Stat1-mediated GTPCH I induction. A, HUVECs were incubated with TNF-, IFN-, and oncostatin M (OnM) as in Figure 6B except for 20 hours. Cells were then lysed and probed for Stat1 and Stat3 phosphorylation as in Figure 6B. B, HUVECs were transfected with Stat3 (+) or control (–) siRNA for two days before stimulation with TNF- (20 ng/mL) and IFN- (50 ng/mL). Cells were then lysed after 20 hours and subjected to immunoblotting with antibodies against Stat3, GTPCH I, and IRF-1. Parallel experiments using only a 15-minute incubation were immunoblotted for the indicated phosphorylated forms of Stat1 and Stat3. Data are representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major finding of this study is that cytokine-induced GTPCH I upregulation in endothelial cells in response to TNF- and IFN- involves cooperative activation of both the IKK/NF-B and Jak2/Stat pathways. The activation of NF-B is downstream of TNF-, whereas the contribution of IFN- involves coordination of Stat1 activation and Stat3 inhibition. We found these two pathways to be independent, as TNF- had no effect on Jak2/Stat, and IFN- played no role in modulation of NF-B activation by TNF-. We also found that Stat1 was required for the effect of IFN-, but coordinated inactivation of Stat3 was necessary to support GTPCH I induction in endothelial cells.

Cytokine-stimulated induction of GTPCH I has been described in endothelial cells. Werner-Felmayer and colleagues¹⁸ reported that HUVECs exposed to TNF- and IFN- exhibited a 40-fold increase in GTPCH I enzymatic activity and an intracellular accumulation of BH₄. Subsequent studies demonstrated this effect was attributable to induction of GTPCH I mRNA^19,20; however, the molecular mechanisms responsible for these effects were not known. The data presented in this study extend this body of knowledge by demonstrating that full GTPCH I mRNA upregulation in endothelial cells requires activation of both NF-B and the Jak2/Stat1 pathway. The effect of TNF- on HUVEC GTPCH I is mediated by NF-B, whereas the IFN- response is dependent on Jak2/Stat1. Evidence to support the former includes our demonstration that inhibition of the ubiquitination pathway with either a degradation-resistant IB mutant or MG132 prevented cytokine-induced upregulation of GTPCH I mRNA and protein (Figure 3). The role of Jak2 and Stat1 in this process is supported by observations that full GTPCH I induction was abrogated by the Jak2 inhibitor, AG490, and in cells that lacked Stat1 (Figure 6A). The precise nature of the cooperativity between these two pathways in GTPCH I upregulation is not clear, but likely occurs at the transcriptional level, as we found no crossover between these two pathways. Specifically, TNF- had no effect on Stat1 nuclear translocation (Figure 4) and IFN- did not alter NF-B activation in response to TNF- (Figure 2).

Interferon- classically exerts its effects through its interaction with the heterodimeric IFN- receptor, resulting in receptor oligomerization and activation of Jak1 and Jak2 by transphosphorylation.³⁵ Subsequent Jak-mediated phosphorylation of the IFN- receptor intracellular domain results in Stat1 recruitment and phosphorylation on tyrosine 701, thereby facilitating Stat1 dimerization and nuclear translocation (see review³⁵). With the availability of Stat1 null mice, however, it has become clear that some IFN- responses are Stat1-independent. For example, IFN- induces c-myc and c-Jun in Stat1 null cells but not in wild-type cells.³⁷ Many genes induced in wild-type cells by IFN- are also induced in Stat1 null cells, including osteopontin and PDGF.³⁸ In the present study, we found that IFN-–stimulated GTPCH I induction was dependent on Stat1, but appeared to involve other aspects of IFN- signaling. Indeed, Stat1 activation by oncostatin M was not sufficient to substitute for IFN- in supporting GTPCH I induction despite promoting rapid Stat1 phosphorylation comparable to IFN- (Figure 6B). These findings are consistent with those of Mahboubi and Pober³⁶ demonstrating that endothelial cell Stat1 activation in response to oncostatin M was not sufficient to promote transcription of IFN-–dependent genes, including IRF-1.

There is precedent for the notion that Stat1 tyrosine phosphorylation is not sufficient for full activity of this transcription factor. For example, phosphorylation of Stat1 on Ser-727 also appears important for full activation of IFN-–dependent genes.^39,40 In this study, we did not observe any material difference in Stat1 Ser-727 phosphorylation as a function of TNF-, IFN-, or oncostatin M, suggesting this event did not explain the inability of oncostatin M to substitute for IFN-. There is also evidence for BRCA1 in modulating both IFN-⁴¹ and TNF- responses,⁴² yet we were unable to demonstrate any difference between oncostatin M or IFN- in promoting complex formation between BRCA1 and Stat1 (data not shown). We also noted that IFN- promoted more persistent Stat1 activation than did oncostatin M, suggesting the duration of Stat1 activation could be important. However, this concept has been refuted by Mahboubi and Pober,³⁶ who found that even prolonged Stat1 activation could not drive transcription of IFN-–dependent genes in endothelial cells. The data presented here indicate that oncostatin M–mediated Stat3 activation explains the inability of this compound to substitute for IFN-. When we suppressed oncostatin M–mediated Stat3 activation, we were able to recapitulate the effect of IFN- with regards to the induction of IFN-–dependent genes such as GTPCH I and IRF-1 (Figure 7). Thus, Stat3 activation exerts a modulatory influence on the Stat1-dependent gene transcription.

The finding that IFN- and TNF- have a synergistic impact on GTPCH I induction is not particularly surprising. For example, it has long been known that optimal NOS2 induction in macrophages requires the combination of these two cytokines.²¹ Another IFN-–stimulated gene, indolamine 2,3,-dioxygenase, is optimally upregulated in the presence of both TNF- and IFN-.⁴³ With regard to this latter report, transcription of Stat1- and IRF-1–driven promoters were enhanced by the combinations of IFN- and TNF- compared with IFN- alone.⁴³ The precise nature of this synergism is not yet evident. Previous studies have indicated that NF-B from TNF- stimulation could interact with IRF-1, but whether such an effect is operative in the present study is unclear at this point.

The findings reported here have important implications for vascular homeostasis in vascular diseases. Tetrahydrobiopterin is an important cofactor for nitric oxide synthases, including eNOS, and a relative deficiency of BH₄ may "uncouple" eNOS NADPH oxidation from NO production, leading to instability of the eNOS F=O⁺ species resulting in eNOS-mediated conversion of NADPH-derived electrons into superoxide. Diabetes is consistently associated with relative reductions in BH₄ and eNOS uncoupling,^14,15,44 whereas findings in atherosclerosis are controversial.^13,45,46 Overexpression of GTPCH I partially restores endothelial function in diabetic mice,⁴⁷ suggesting that endothelial availability of reduced biopterin is an important determinant of endothelial cell NO bioactivity. We have found that full upregulation of GTPCH I, the rate-limiting enzyme for BH₄ synthesis, requires coordinated action of two cytokine species. Thus, optimal endothelial cell BH₄ levels are not simply a consequence of any cytokine stimulation of endothelial cells. Although the precise cytokine milieu of the atherosclerotic plaque is not known, one might speculate that the maintenance of fully reduced biopterin in the endothelium is a fragile process. As a consequence, any inflammation and its associated increase of ROS flux could have profound implications for the intracellular availability of BH₄ and, as a result, eNOS uncoupling and NO bioactivity. In fact, recent observations that overexpression of eNOS accelerates atherosclerosis⁴⁸ would tend to support the idea that the eNOS/BH₄ balance is critical to the maintenance of vascular homeostasis.

In summary, the data presented in this study indicate that cytokine-mediated GTPCH I upregulation in endothelial cells requires coordinated activation of NF-B and the Jak2/Stat pathway. Moreover, activation of the Jak2/Stat pathway has a particular requirement for Stat1 activation in the absence of Stat3 stimulation. These data add to the increasing body of evidence that the relative activation of Stat1 and Stat3 dictate the phenotypic implications for cytokine stimulation.

	Acknowledgments

 
This work was supported by grants DK55656 and HL60886 from the NIH and was performed, in part, while J.F.K.Jr. was an Established Investigator of the American Heart Association.

	Footnotes

 
Original received November 15, 2004; revision received December 3, 2004; accepted December 7, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Lefroy DC, Crake T, Uren NG, Davies GJ, Maseri A. Effect of inhibition of nitric oxide synthesis on epicardial coronary artery caliber and coronary blood flow in humans. Circulation. 1993; 88: 43–54.[Abstract/Free Full Text]
2. Rees DD, Palmer RM, Moncada S. Role of endothelium-derived nitric oxide in the regulation of blood pressure. Proc Natl Acad Sci U S A. 1989; 86: 3375–3378.[Abstract/Free Full Text]
3. Azuma H, Ishikawa M, Sekizaki S. Endothelium-dependent inhibition of platelet aggregation. Br J Pharmacol. 1986; 88: 411–415.[Medline] [Order article via Infotrieve]
4. Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cGMP inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989; 83: 1774–1777.[Medline] [Order article via Infotrieve]
5. Rudic RD, Shesely EG, Maeda N, Smithies O, Segal SS, Sessa WC. Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling. J Clin Invest. 1998; 101: 731–736.[Medline] [Order article via Infotrieve]
6. Widlansky ME, Gokce N, Keaney JF, Jr., Vita JA. The clinical implications of endothelial dysfunction. J Am Coll Cardiol. 2003; 42: 1149–1160.[Abstract/Free Full Text]
7. Schachinger V, Britten MB, Zeiher AM. Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease. Circulation. 2000; 101: 1899–1906.[Abstract/Free Full Text]
8. Gokce N, Keaney JF, Jr, Hunter LM, Watkins MT, Menzoian JO, Vita JA. Risk stratification for postoperative cardiovascular events via noninvasive assessment of endothelial function: a prospective study. Circulation. 2002; 105: 1567–1572.[Abstract/Free Full Text]
9. Govers R, Rabelink TJ. Cellular regulation of endothelial nitric oxide synthase. Am J Physiol Renal Physiol. 2001; 280: F193–F206.[Abstract/Free Full Text]
10. Xia Y, Tsai AL, Berka V, Zweier JL. Superoxide generation from endothelial nitric-oxide synthase. A Ca²⁺/calmodulin-dependent and tetrahydrobiopterin regulatory process. J Biol Chem. 1998; 273: 25804–25808.[Abstract/Free Full Text]
11. Vasquez-Vivar J, Kalyanaraman B, Martasek P, Hogg N, Masters BS, Karoui H, tordo P, Pritchard KA, Jr. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci U S A. 1998; 95: 9220–9225.[Abstract/Free Full Text]
12. Presta A, Siddhanta U, Wu C, Sennequier N, Huang L, Abu-Soud HM, Erzurum S, Stuehr DJ. Comparative functioning of dihydro- and tetrahydropterins in supporting electron transfer, catalysis, and subunit dimerization in inducible nitric oxide synthase. Biochemistry. 1998; 37: 298–310.[CrossRef][Medline] [Order article via Infotrieve]
13. Laursen JB, Somers M, Kurz S, McCann L, Warnholtz A, Freeman BA, Tarpey M, Fukai T, Harrison DG. Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation. 2001; 103: 1282–1288.[Abstract/Free Full Text]
14. Hink U, Li H, Mollnau H, Oelze M, Matheis E, Hartmann M, Skatchkov M, Thaiss F, Stahl RA, Warnholtz A, Meinertz T, Griendling K, Harrison DG, Forstermann U, Munzel T. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res. 2001; 88: e14–e22.[Medline] [Order article via Infotrieve]
15. Shinozaki K, Kashiwagi A, Nishio Y, Okamura T, Yoshida Y, Masada M, Toda N, Kikkawa R. Abnormal biopterin metabolism is a major cause of impaired endothelium-dependent relaxation through nitric oxide/O₂^– imbalance in insulin-resistant rat aorta. Diabetes. 1999; 48: 2437–2445.[Abstract]
16. Nichol CA, Smith GK, Duch DS. Biosynthesis and metabolism of tetrahydrobiopterin and molybdopterin. Annu Rev Biochem. 1985; 54: 729–764.[CrossRef][Medline] [Order article via Infotrieve]
17. Werner ER, Werner-Felmayer G, Fuchs D, Hausen A, Reibnegger G, Yim JJ, Pfleiderer W, Wachter H. Tetrahydrobiopterin biosynthetic activities in human macrophages, fibroblasts, THP-1, and T 24 cells. GTP-cyclohydrolase I is stimulated by interferon-, and 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are constitutively present. J Biol Chem. 1990; 265: 3189–3192.[Abstract/Free Full Text]
18. Werner-Felmayer G, Werner ER, Fuchs D, Hausen A, Reibnegger G, Schmidt K, Weiss G, Wachter H. Pteridine biosynthesis in human endothelial cells: impact on nitric oxide-mediated formation of cyclic GMP. J Biol Chem. 1993; 268: 1842–1846.[Abstract/Free Full Text]
19. Hattori Y, Nakanishi N, Kasai K, Shimoda SI. GTP cyclohydrolase I mRNA induction and tetrahydrobiopterin synthesis in human endothelial cells. Biochim Biophys Acta. 1997; 1358: 61–66.[Medline] [Order article via Infotrieve]
20. Katusic ZS, Stelter A, Milstien S. Cytokines stimulate GTP cyclohydrolase I gene expression in cultured human umbilical vein endothelial cells. Arterioscler Thromb Vasc Biol. 1998; 18: 27–32.[Abstract/Free Full Text]
21. Gross SS, Levi R. Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J Biol Chem. 1992; 267: 25722–25729.[Abstract/Free Full Text]
22. Ganster RW, Taylor BS, Shao L, Geller DA. Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-B. Proc Natl Acad Sci U S A. 2001; 98: 8638–8643.[Abstract/Free Full Text]
23. Feng X, Guo Z, Nourbakhsh M, Hauser H, Ganster R, Shao L, Geller DA. Identification of a negative response element in the human inducible nitric-oxide synthase (hiNOS) promoter: the role of NF-kappa B-repressing factor (NRF) in basal repression of the hiNOS gene. Proc Natl Acad Sci U S A. 2002; 99: 14212–14217.[Abstract/Free Full Text]
24. Yoneyama T, Hatakeyama K. Decameric GTP cyclohydrolase I forms complexes with two pentameric GTP cyclohydrolase I feedback regulatory proteins in the presence of phenylalanine or of a combination of tetrahydrobiopterin and GTP. J Biol Chem. 1998; 273: 20102–20108.[Abstract/Free Full Text]
25. Li P, Sanz I, O’Keefe RJ, Schwarz EM. NF-kappa B regulates VCAM-1 expression on fibroblast-like synoviocytes. J Immunol. 2000; 164: 5990–5997.[Abstract/Free Full Text]
26. Watanabe S, Itoh T, Arai K. JAK2 is essential for activation of c-fos and c-myc promoters and cell proliferation through the human granulocyte-macrophage colony-stimulating factor receptor in BA/F3 cells. J Biol Chem. 1996; 271: 12681–12686.[Abstract/Free Full Text]
27. Durbin JE, Hackenmiller R, Simon MC, Levy DE. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell. 1996; 84: 443–450.[CrossRef][Medline] [Order article via Infotrieve]
28. Lee CK, Smith E, Gimeno R, Gertner R, Levy DE. STAT1 affects lymphocyte survival and proliferation partially independent of its role downstream of IFN-gamma. J Immunol. 2000; 164: 1286–1292.[Abstract/Free Full Text]
29. Huang A, Vita JA, Venema RC, Keaney JF, Jr. Ascorbic acid enhances endothelial nitric oxide synthase activity by increasing intracellular tetrahydrobiopterin. J Biol Chem. 2000; 275: 17399–17406.[Abstract/Free Full Text]
30. Howells DW, Hyland K. Direct analysis of tetrahydrobiopterin in cerebrospinal fluid by high-performance liquid chromatography with redox electrochemistry: prevention of autoxidation during storage and analysis. Clin Chim Acta. 1987; 167: 23–30.[CrossRef][Medline] [Order article via Infotrieve]
31. Huang A, Li C, Kao RL, Stone WL. Lipid hydroperoxides inhibit nitric oxide production in RAW264.7 macrophages. Free Radic Biol Med. 1999; 26: 526–537.[CrossRef][Medline] [Order article via Infotrieve]
32. Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983; 11: 1475–1489.[Abstract/Free Full Text]
33. Okuda M, Takahashi M, Suero J, Murry CE, Traub O, Kawakatsu H, Berk BC. Shear stress stimulation of p130(cas) tyrosine phosphorylation requires calcium-dependent c-Src activation. J Biol Chem. 1999; 274: 26803–26809.[Abstract/Free Full Text]
34. Chen K, Albano A, Ho A, Keaney JF, Jr. Activation of p53 by Oxidative Stress Involves Platelet-derived Growth Factor-ß Receptor-mediated Ataxia Telangiectasia Mutated (ATM) Kinase Activation. J Biol Chem. 2003; 278: 39527–39533.[Abstract/Free Full Text]
35. Ramana CV, Gil MP, Schreiber RD, Stark GR. Stat1-dependent and -independent pathways in IFN-gamma-dependent signaling. Trends Immunol. 2002; 23: 96–101.[CrossRef][Medline] [Order article via Infotrieve]
36. Mahboubi K, Pober JS. Activation of signal transducer and activator of transcription 1 (STAT1) is not sufficient for the induction of STAT1-dependent genes in endothelial cells. Comparison of interferon-gamma and oncostatin M. J Biol Chem. 2002; 277: 8012–8021.[Abstract/Free Full Text]
37. Ramana CV, Grammatikakis N, Chernov M, Nguyen H, Goh KC, Williams BR, Stark GR. Regulation of c-myc expression by IFN-gamma through Stat1-dependent and -independent pathways. EMBO J. 2000; 19: 263–272.[CrossRef][Medline] [Order article via Infotrieve]
38. Ramana CV, Gil MP, Han Y, Ransohoff RM, Schreiber RD, Stark GR. Stat1-independent regulation of gene expression in response to IFN-gamma. Proc Natl Acad Sci U S A. 2001; 98: 6674–6679.[Abstract/Free Full Text]
39. Goh KC, Haque SJ, Williams BR. p38 MAP kinase is required for STAT1 serine phosphorylation and transcriptional activation induced by interferons. EMBO J. 1999; 18: 5601–5608.[CrossRef][Medline] [Order article via Infotrieve]
40. Xu B, Bhattacharjee A, Roy B, Xu HM, Anthony D, Frank DA, Feldman GM, Cathcart MK. Interleukin-13 induction of 15-lipoxygenase gene expression requires p38 mitogen-activated protein kinase-mediated serine 727 phosphorylation of Stat1 and Stat3. Mol Cell Biol. 2003; 23: 3918–3928.[Abstract/Free Full Text]
41. Ouchi T, Lee SW, Ouchi M, Aaronson SA, Horvath CM. Collaboration of signal transducer and activator of transcription 1 (STAT1) and BRCA1 in differential regulation of IFN- target genes. Proc Natl Acad Sci U S A. 2000; 97: 5208–5213.[Abstract/Free Full Text]
42. Zielinski CC, Budinsky AC, Wagner TM, Wolfram RM, Kostler WJ, Kubista M, Brodowicz T, Kubista E, Wiltschke C. Defect of tumour necrosis factor-alpha (TNF-) production and TNF-alpha-induced ICAM-1-expression in BRCA1 mutations carriers. Breast Cancer Res Treat. 2003; 81: 99–105.[Medline] [Order article via Infotrieve]
43. Robinson CM, Shirey KA, Carlin JM. Synergistic transcriptional activation of indoleamine dioxygenase by IFN-gamma and tumor necrosis factor-alpha. J Interferon Cytokine Res. 2003; 23: 413–421.[CrossRef][Medline] [Order article via Infotrieve]
44. Guzik TJ, Mussa S, Gastaldi D, Sadowski J, Ratnatunga C, Pillai R, Channon KM. Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase. Circulation. 2002; 105: 1656–1662.[Abstract/Free Full Text]
45. Vasquez-Vivar J, Duquaine D, Whitsett J, Kalyanaraman B, Rajagopalan S. Altered tetrahydrobiopterin metabolism in atherosclerosis: implications for use of oxidized tetrahydrobiopterin analogues and thiol antioxidants. Arterioscler Thromb Vasc Biol. 2002; 22: 1655–1661.[Abstract/Free Full Text]
46. d’Uscio LV, Milstien S, Richardson D, Smith L, Katusic ZS. Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity. Circ Res. 2003; 92: 88–95.[Abstract/Free Full Text]
47. Alp NJ, Mussa S, Khoo J, Cai S, Guzik T, Jefferson A, Goh N, Rockett KA, Channon KM. Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I overexpression. J Clin Invest. 2003; 112: 725–735.[CrossRef][Medline] [Order article via Infotrieve]
48. Ozaki M, Kawashima S, Yamashita T, Hirase T, Namiki M, Inoue N, Hirata K, Yasui H, Sakurai H, Yoshida Y, Masada M, Yokoyama M. Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoE-deficient mice. J Clin Invest. 2002; 110: 331–340.[CrossRef][Medline] [Order article via Infotrieve]

Related Article:

Transcribing the Cross-Talk of Cytokine-Induced Tetrahydrobiopterin Synthesis in Endothelial Cells

Timothy E. Peterson and Zvonimir S. Katusic
Circ. Res. 2005 96: 141-143. [Full Text] [PDF]

This article has been cited by other articles:

				C. Antoniades, C. Shirodaria, T. Van Assche, C. Cunnington, I. Tegeder, J. Lotsch, T. J. Guzik, P. Leeson, J. Diesch, D. Tousoulis, et al. GCH1 Haplotype Determines Vascular and Plasma Biopterin Availability in Coronary Artery Disease Effects on Vascular Superoxide Production and Endothelial Function. J. Am. Coll. Cardiol., July 8, 2008; 52(2): 158 - 165. [Abstract] [Full Text] [PDF]

				J. D. Widder, W. Chen, L. Li, S. Dikalov, B. Thony, K. Hatakeyama, and D. G. Harrison Regulation of Tetrahydrobiopterin Biosynthesis by Shear Stress Circ. Res., October 12, 2007; 101(8): 830 - 838. [Abstract] [Full Text] [PDF]

				E. Takimoto and D. A. Kass Role of Oxidative Stress in Cardiac Hypertrophy and Remodeling Hypertension, February 1, 2007; 49(2): 241 - 248. [Full Text] [PDF]

				D. E. Berkowitz Myocyte Nitroso-Redox Imbalance in Sepsis: NO Simple Answer Circ. Res., January 5, 2007; 100(1): 1 - 4. [Full Text] [PDF]

				A. L. Moens and D. A. Kass Tetrahydrobiopterin and Cardiovascular Disease Arterioscler. Thromb. Vasc. Biol., November 1, 2006; 26(11): 2439 - 2444. [Abstract] [Full Text] [PDF]

				L. Zhang, W.H. L. Kao, Y. Berthier-Schaad, Y. Liu, L. Plantinga, B. G. Jaar, N. Fink, N. Powe, M. J. Klag, M. W. Smith, et al. Haplotype of Signal Transducer and Activator of Transcription 3 Gene Predicts Cardiovascular Disease in Dialysis Patients J. Am. Soc. Nephrol., August 1, 2006; 17(8): 2285 - 2292. [Abstract] [Full Text] [PDF]

<