Cytokine-Stimulated GTP Cyclohydrolase I Expression in Endothelial Cells Requires Coordinated Activation of Nuclear Factor-B and Stat1/Stat3
http://www.100md.com
循环研究杂志 2005年第2期
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
Department of Surgery (K.H.), University of Pittsburgh, Pa.
Abstract
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-eCmediated 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-eCmediated 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
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 eNOS10,11 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 cytokines18eC20 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
Materials
Human tumor necrosis factor- (TNF-), interferon-, interleukin (IL)-1, 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 CDeC 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 TranscriptioneCPolymerase Chain Reaction
Total cellular RNA was isolated from endothelial cells using TRIzol from Life Technologies. The procedures for semiquantitative reverse transcriptioneCpolymerase 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
Cytokines Induce GTPCH I Expression and Tetrahydrobiopterin Biosynthesis in HUVECs
The effects of TNF-, interferon- (IFN-), and IL-1 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-1 alone. In contrast, IFN- alone increased GTPCH I protein by 2- to 3-fold and combinations of IFN- with either TNF- or IL-1 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-1, 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).
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.
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-.
Activation of Stat1 Is Essential for IFN-eCInduced 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-eCtreated 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-eCmediated Jak2 activation contributes to GTPCH I upregulation via mechanisms that go beyond simple Stat-1 phosphorylation at Tyr-701.
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 MeCmediated 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.
Discussion
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 mRNA19,20; 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-eCstimulated 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-eCdependent 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-eCdependent 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-eCdependent genes in endothelial cells. The data presented here indicate that oncostatin MeCmediated Stat3 activation explains the inability of this compound to substitute for IFN-. When we suppressed oncostatin MeCmediated Stat3 activation, we were able to recapitulate the effect of IFN- with regards to the induction of IFN-eCdependent 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-eCstimulated 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-1eCdriven 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.
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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: 88eC95.
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: 725eC735.
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: 331eC340.(Annong Huang, Ying-Yi Zha)
Department of Surgery (K.H.), University of Pittsburgh, Pa.
Abstract
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-eCmediated 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-eCmediated 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
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 eNOS10,11 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 cytokines18eC20 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
Materials
Human tumor necrosis factor- (TNF-), interferon-, interleukin (IL)-1, 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 CDeC 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 TranscriptioneCPolymerase Chain Reaction
Total cellular RNA was isolated from endothelial cells using TRIzol from Life Technologies. The procedures for semiquantitative reverse transcriptioneCpolymerase 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
Cytokines Induce GTPCH I Expression and Tetrahydrobiopterin Biosynthesis in HUVECs
The effects of TNF-, interferon- (IFN-), and IL-1 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-1 alone. In contrast, IFN- alone increased GTPCH I protein by 2- to 3-fold and combinations of IFN- with either TNF- or IL-1 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-1, 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).
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.
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-.
Activation of Stat1 Is Essential for IFN-eCInduced 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-eCtreated 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-eCmediated Jak2 activation contributes to GTPCH I upregulation via mechanisms that go beyond simple Stat-1 phosphorylation at Tyr-701.
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 MeCmediated 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.
Discussion
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 mRNA19,20; 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-eCstimulated 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-eCdependent 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-eCdependent 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-eCdependent genes in endothelial cells. The data presented here indicate that oncostatin MeCmediated Stat3 activation explains the inability of this compound to substitute for IFN-. When we suppressed oncostatin MeCmediated Stat3 activation, we were able to recapitulate the effect of IFN- with regards to the induction of IFN-eCdependent 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-eCstimulated 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-1eCdriven 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.
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