Published online before print June 2, 2008, doi: 10.1161/HYPERTENSIONAHA.108.114041
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(Hypertension. 2008;52:93.)
© 2008 American Heart Association, Inc.
Original Articles |
Erythropoietin Increases Endothelial Biosynthesis of Tetrahydrobiopterin by Activation of Protein Kinase B
/Akt1
From the Departments of Anesthesiology and Molecular, Pharmacology, and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, Minn.
Correspondence to Zvonimir S. Katusic or Livius V. d'Uscio, Departments of Anesthesiology and Molecular, Pharmacology, and Experimental Therapeutics, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail Katusic.Zvonimir{at}mayo.edu or dUscio.Livius@mayo.edu
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Abstract |
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Tetrahydrobiopterin (BH4) is an essential cofactor required for enzymatic activity of endothelial NO synthase. Recently, it has been shown that vascular protective effects of erythropoietin (EPO) are dependent on activation of endothelial NO synthase. Therefore, our objective was to characterize the effect of EPO on the biosynthesis of BH4 in the vascular wall. Incubation of isolated C57BL/6J mouse aortas for 18 hours with recombinant human EPO (1 to 50 U/mL) caused a concentration-dependent increase in intracellular BH4 levels and activity of GTP-cyclohydrolase I. Maximal biosynthesis of BH4 was detected at therapeutic concentrations of 5 U/mL. Removal of the endothelium abolished EPO-induced biosynthesis of BH4 demonstrating that the vascular endothelium is a major source of BH4. Treatment with a selective phosphatidylinositol 3-kinase inhibitor wortmannin significantly reduced BH4 biosynthesis stimulated by EPO. The stimulatory effect of EPO on vascular GTP-cyclohydrolase I activity, BH4 production, and phosphorylation of endothelial NO synthase was also detected in vivo in mice treated with recombinant human EPO. These effects of EPO were abolished in protein kinase B
/Akt1-deficient mice. In addition, EPO significantly increased systolic blood pressure and the number of circulating platelets in Akt1-deficient mice. Our results demonstrate that EPO stimulates biosynthesis of BH4 in vascular endothelium and that the increase in BH4 levels is caused by de novo biosynthesis of BH4 via the phosphatidylinositol 3-kinase/Akt1 pathway. This effect is most likely designed to provide optimal intracellular concentration of the cofactor necessary for EPO-induced elevation of endothelial NO synthase activity.
Key Words: tetrahydrobiopterin • endothelium • erythropoietin • GTP-cyclohydrolase I • protein kinase B • vasculature • mice
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Introduction |
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Nitric oxide generated by endothelial NO synthase (eNOS) in the vascular endothelium is a potent vasodilator and regulator of systemic blood pressure, vascular remodeling, and angiogenesis.1 Tetrahydrobiopterin (BH4) is an essential cofactor required for enzymatic activity of eNOS.1,2 The biosynthesis of BH4 depends on activity of the rate-limiting enzyme GTP-cyclohydrolase I (GTPCH I).3 During activation of the eNOS isoform, BH4 is needed for allosteric and redox activation of its enzymatic activity.4 Indeed, inhibition of GTPCH I with 2,4-diamino-6-hydroxy-pyrimidine results in almost complete depletion of intracellular BH4 and abolishes formation of NO in response to activation of eNOS.5
Erythropoietin (EPO) is a hypoxia-induced hormone that is essential for erythropoiesis in bone marrow mediated via activation of EPO receptors (EPORs).6 Recently, it has been demonstrated that classic homodimeric EPOR is also expressed in nonhematopoietic tissues, including endothelial cells,7 indicating that EPO has direct vascular effects, including NO production, inhibition of apoptosis, and stimulation of angiogenesis.8–11 In endothelial and other cells, EPOR is coupled to activation of the phosphatidylinositol-3 kinase (PI3-kinase)/protein kinase B (Akt) signaling pathway.12,13 PI3-kinase/Akt is a critical pathway modulating cell survival but is also a well-documented activator of eNOS.14 There are 3 different Akt isoforms. Akt1 or protein kinase B is the predominant isoform in endothelial cells.15 Indeed, genetic inactivation of Akt1 (but not Akt2) impairs revascularization after ischemia.15,16 Akt2/protein kinase Bβ–deficient mice display insulin resistance and exhibit a diabetic phenotype.17 Expression of Akt3/protein kinase B
is undetectable in vascular tissue.16 The exact molecular mechanisms underlying beneficial vascular effects of EPO in vivo are not well defined. Therefore, in the present study, we hypothesized that EPO stimulates vascular biosynthesis of BH4 by activation of the PI3-kinase/Akt1 signal transduction pathway.
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Methods |
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An expanded Methods section is available in the data supplement online at http://hyper.ahajournals.org.
Experimental Animals
Male C57BL/6J (wild-type) mice, heterozygous Akt1 (Akt1+/–) mice, homozygous Akt1 (Akt1–/–) mice (C57BL/6J-Akt1tm1Mbb), and homozygous eNOS (eNOS–/–) mice (C57BL/6J-Nos3tm1Unc) were obtained from Jackson Laboratory (Bar Harbor, Maine). EPO-transgenic mice were provided by Dr Max Gassmann (Vetsuisse Faculty, Zurich, Switzerland). Mice were maintained on standard chow with free access to drinking water. Housing facilities and all of the experimental protocols were approved by the institutional animal care and use committee of the Mayo Clinic and complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice were randomly distributed to a control group (PBS) and an EPO group (recombinant human EPO, 1000 U/kg body weight, biweekly, SC, Amgen).11 After 14 days of treatment, systolic blood pressure was recorded,11 the animals were euthanized (pentobarbital, 60 mg/kg of body weight IP), and aortas and lungs were harvested.
In Vitro Studies
Isolated aortic rings of wild-type mice were incubated with EPO at various concentrations in minimal essential medium for 18 hours at 37°C as described.5
Measurements of BH4, 7,8-Dihydrobiopterin, and GTPCH I Enzyme Activity
Biopterin levels and GTPCH I activity were determined in fresh aortas using the reverse-phase high-performance liquid chromatography method, as described previously.18
Western Blot Analysis
Primary antibodies against eNOS, Ser1177-phosphorylated eNOS (Transduction Laboratories), Akt1, Akt2, Ser473-phosphorylated Akt1 (Upstate), GTPCH I18, EPO-R (Santa Cruz), JAK2, Tyr1007/1008-phosphorylated JAK2 (Cell Signaling), and anti–β-actin (Sigma) were used.19
Calculations and Statistical Analysis
All of the results are expressed as means±SEMs, and "n" indicates the number of animals from which tissues were harvested. Single values were compared by 1-way ANOVA with Bonferroni’s correction for multiple comparisons. For simple comparisons between 2 groups, an unpaired Student t test was used where appropriate. P<0.05 was considered significant.
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Results |
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Stimulatory Effects of EPO on Endothelial BH4 Synthesis
Incubation of isolated wild-type mouse aorta for 18 hours in minimal essential medium supplemented with EPO (1 to 50 U/mL) causes a concentration-dependent increase in intracellular levels of BH4 levels, with maximum biosynthesis occurring at concentrations of 5 U/mL (P<0.05; Figure 1A). Oxidative products of BH4, 7,8-dihydrobiopterin levels, were unaffected by EPO, indicating that EPO does not increase oxidation of BH4 (data not shown). In addition, GTPCH I activity was augmented by EPO (P<0.05; Figure 1B). Inhibition of GTPCH I with 2,4-diamino-6-hydroxypyrimidine (10 mmol/L) abolished the increase in BH4 levels induced by EPO (data not shown; n=3). Furthermore, removal of the endothelium prevented the stimulatory effect of EPO on BH4 biosynthesis (P<0.05; Figure 1C).
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We next investigated whether EPO increases vascular BH4 production under in vivo conditions. Treatment with EPO for 3 days increased GTPCH I enzymatic activity and biosynthesis of BH4 in the aorta of wild-type mice (P<0.05; Figure 2A and 2B). Levels of 7,8-dihydrobiopterin were low in the aorta of wild-type mice (0.3±0.2 pmol/mg; n=5), and they were unaffected by EPO. Similar stimulatory effects of EPO on BH4 biosynthesis were observed in wild-type mice treated with EPO for 14 days (P<0.05; Figure 2C) or in EPO-transgenic mice (P<0.05; Figure 2D).
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Mechanisms of EPO-Induced BH4 Synthesis
We investigated signal transduction pathways coupling endothelial EPOR to BH4 metabolism. Here we provide the first evidence showing that EPOR is expressed in mouse aorta and that expression of EPOR was not affected by EPO treatment for 2 weeks (Figure 3A). Further analysis demonstrated that EPO increased phosphorylation of Janus kinase 2 (JAK2) at Tyr1007/1008, the key kinase in the signal transduction pathways activated by EPOR20 (P<0.05; Figure 3B). Indeed, in vitro treatment of intact isolated mouse aortas with a JAK2 inhibitor AG490 (5 µmol/L) inhibited the stimulatory effect of EPO on BH4 (P<0.05; Figure 3C), indicating that, in vascular endothelium, coupling between EPOR and JAK2 is an important step in the control of BH4 metabolism. We next analyzed downstream signaling pathways, such as PI3-kinase/Akt and protein kinase C, as well as signal transducer and activator of transcription factors. In isolated mouse aortas, inhibition of PI3-kinase by wortmannin (1 µmol/L) significantly diminished EPO-induced BH4 biosynthesis (P<0.05; Figure 3D). Wortmannin alone had no effect on BH4 metabolism (Figure 3D), suggesting that the stimulatory effect of EPO depends on activation of PI3-kinase. Furthermore, GTPCH I protein expression was unchanged in mouse aorta after in vitro incubation with EPO and in the presence of wortmannin or AG490 (Figure 3E). On the other hand, incubation of aortas with parthenolide (5 µmol/L), a specific inhibitor of signal transducer and activator of transcription factors, with general protein kinase inhibitor chelerythrine (3 µmol/L), or with casein kinase 2 (CK2) inhibitor 4,5,6,7-tetrabromobenzotriazole (5 µmol/L), did not abolish EPO-induced BH4 biosynthesis (data not shown; n=5 to 9).
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P<0.05 vs EPO-treated mouse aortas (ANOVA with Bonferroni’s).
Characteristics of Akt1-Deficient Mice
The number of red blood cells along with hematocrit and hemoglobin were increased after the administration of EPO for 14 days in wild-type, Akt1+/– and Akt1–/– mice (P<0.05; Table), but there was no difference among EPO-treated wild-type, Akt1+/–, and Akt1–/– mice. EPO treatment for 14 days did not increase the number of circulating white blood cells (Table). The number of platelets was significantly increased in EPO-treated Akt1–/– mice (P<0.05; Table). Furthermore, EPO significantly increases systolic blood pressure in Akt1–/– mice compared with wild-type and Akt1+/– mice after 14 days of treatment (P<0.05; Table).
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Effects of EPO on BH4 Synthesis in Akt1-Deficient Mice
To establish whether the stimulatory effect of EPO depends on activation of PI3-kinase–dependent Akt phosphorylation, we performed studies on Akt1-deficient mice. Western blot analysis confirmed that Akt1 protein expression was reduced in Akt1+/– mice and was absent in Akt1–/– mice (Figure 4A), consistent with previous findings obtained on other tissues of Akt1-deficient mice.16,21 Akt2 protein expression was unchanged in Akt1–/– mice and those treated with EPO (Figure 4A). The stimulatory effect of EPO on BH4 synthesis was abolished in Akt1–/– mice but not in Akt1+/– or eNOS–/– mice aortas (P<0.05; Figure 4B). In addition, increased enzymatic activity of GTPCH I by EPO treatment for 14 days was also abolished in aortas of Akt1–/– mice compared with wild-type and Akt1+/– mice (P<0.05; Figure 4C). GTPCH I protein expression was unaltered in EPO-treated wild-type and Akt1–/– mice as compared with untreated wild-type mice (Figure 4D).
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Two weeks of treatment with EPO also increased protein expressions of phosphorylated Akt1 at Ser473 and eNOS at Ser1177 in wild-type mice (P<0.05; Figure 5A and 5B, respectively). Maintained serine phosphorylation and activation of eNOS were prevented in Akt1–/– mice (P<0.05; Figure 5B). In contrast, EPO did not affect protein expressions of ' and subunits of protein kinase CK2 in wild-type and Akt1–/– mice (Figure 5C).
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Discussion |
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In the present study, we report several novel findings. First, therapeutic concentrations of EPO activate vascular GTPCH I (rate-limiting enzyme in biosynthesis of BH4), thereby increasing the intracellular concentration of BH4. Second, EPO stimulates production of BH4 exclusively in endothelial cells. Third, the effect of EPO is independent of shear stress imposed on the vascular endothelium by an elevated number of circulating red blood cells. Fourth, pharmacological and molecular genetic analysis established that activation of the EPOR/PI3-kinase/Akt1 signal transduction pathway is responsible for the stimulatory effect of EPO on the production of BH4. Fifth, genetic inactivation of Akt1 signaling favors the prohypertensive effect of EPO. In aggregate, these findings provide new insights into the molecular mechanisms underlying nonhematopoietic vascular effects of EPO.
Previous studies from several groups, including ours, demonstrated that the vascular protective effect of EPO is mediated by phosphorylation of Akt, eNOS, and a subsequent increase in the production of NO.9,11,13 Because enzymatic activity of eNOS is critically dependent on the availability of BH4, it was of major interest to determine the effect of EPO on the metabolism of BH4. Our results demonstrate that EPO causes a concentration-dependent increase in intracellular levels of BH4, with the maximal BH4 biosynthesis detected at therapeutic concentration of 5 U/mL. Pharmacological inhibition of GTPCH I abolished the stimulatory effect of EPO on BH4, thereby reinforcing the conclusion regarding the importance of GTPCH I activation as a major molecular mechanism responsible for the EPO effect. We and others have demonstrated that endothelium is a major source of BH4 in intact mouse aorta.18,22 Consistent with these observations, mechanical removal of endothelial cells abolished the stimulatory effect of EPO on BH4 biosynthesis, thus demonstrating that activation of endothelial cells by EPO results in increased availability of BH4. Detected increase in BH4 levels in arteries treated with EPO is quantitatively similar to the levels reported in genetically modified mice overexpressing GTPCH I specifically in endothelial cells.23,24 Indeed, a number of previous studies demonstrated that elevation of BH4 concentration in vascular endothelium detected in our experiments is vascular protective and can prevent endothelial dysfunction induced by hypercholesterolemia, diabetes, and hypertension.22–24
Recently, our group reported that in vivo increases in blood flow cause upregulation of GTPCH I activity in the arterial wall.25 This finding was corroborated by in vitro studies demonstrating a stimulatory effect of shear stress on BH4 production in cultured endothelial cells.26 Because EPO increases the number of circulating red blood cells, thereby causing elevation of shear stress imposed by the circulating blood on the endothelial layer of blood vessel,27 it was important to rule out the possibility that the effect of EPO is mediated indirectly by changes in shear stress rather than direct activation of EPOR. Therefore, we performed experiments on mice treated with EPO only for 3 days. Short-term exposure to EPO does not affect the production of red blood cells,11 thus excluding increased shear stress as an explanation for the observed effects of EPO. Indeed, elevation of BH4 observed 3 days after treatment with EPO was not significantly different from the elevation detected in the aorta of mice treated with EPO for 2 weeks. Based on these observations, we concluded that EPO has a direct stimulatory effect on GTPCH I activity and BH4 biosynthesis in vascular endothelium and that this effect is independent of hematopoietic effects of EPO. This conclusion is further reinforced by the fact that the promoter region of the mouse GTPCH I28 does not have shear stress response elements.29 In addition, in our previous study we demonstrated that EPO treatment for 3 or 14 days stimulated phosphorylation of eNOS in the arterial wall to a similar degree,11 thus demonstrating that a high number of red blood cells and subsequent elevation of shear stress are not responsible for the effect of EPO.
Expression and activation of EPOR on endothelium are well established.7,30 Furthermore, it has been shown that EPO induces homodimerization of cell surface EPOR with subsequent autophosphorylation and activation of the receptor-associated JAK2 tyrosine kinase leading to activation of several downstream cellular pathways, including PI3-kinase/Akt, signal transducer and activator of transcription factors, and protein kinase C.6,20 Of interest, we detected higher expression of phosphorylated JAK2 in EPO-treated mice, suggesting that increased phosphorylation and activation of JAK2 are the molecular mechanisms underlying the observed effect of EPO on BH4 synthesis. Indeed, pharmacological inhibition of JAK2 with AG490 abolished EPO-induced BH4 biosynthesis. Existing literature indicates that, in endothelial and other cells, EPOR/JAK2 is coupled to activation of the PI3-kinase/Akt signaling pathway.12,13,31 Our study showed that PI3-kinase activity was an upstream activator of Akt1, because pharmacological and genetic inactivation of PI3-kinase/Akt1 abolished the stimulatory effects of EPO on GTPCH I activity and biosynthesis of BH4 in mouse aorta. These observations provide new insights into the molecular mechanisms responsible for EPO-induced elevation of vascular BH4. Inspection of mouse GTPCH I protein sequence (National Center for Biotechnology Information accession No. NP-032128) revealed that, in contrast to eNOS, GTPCH I does not contain the consensus sequence for phosphorylation by Akt1 (RXRXXS/T).32 Most recently, it has been reported that GTPCH I possesses 6 consensus sequences for phosphorylation by CK2.26 Furthermore, it has been shown that shear stress-induced activation of GTPCH I is mediated through activation of catalytic subunits of CK2' in endothelial cells.26 However, in our study, we could not detect any effect of EPO on protein expressions of ' and subunits of CK2, whereas inhibition of CK2 failed to prevent EPO-induced BH4 synthesis. Thus, the exact molecular mechanisms responsible for the Akt1-mediated activation of GTPCH I remain to be determined.
We also wish to point out that treatment with EPO caused a significant increase in arterial blood pressure and in the number of circulating platelets in Akt1-deficient mice. This observation may help to explain adverse cardiovascular effects of EPO, including hypertension and thrombosis.11,33 Although the analysis of the exact mechanism of EPO-induced hypertension is beyond the scope of our study, we wish to point out that a number of previous studies demonstrated an important role for BH4 in the pathogenesis of hypertension and attendant dysfunction of the endothelium.22,24,34–36 The clinical importance of the impaired EPOR/Akt1 signal transduction pathway in the development of adverse cardiovascular effects of EPO requires further studies.
Perspectives
Akt1 is an important protein responsible for eNOS activation in response to stimulation of several signal transduction pathways. Akt1 phosphorylates and activates eNOS, whereas impaired Akt1 kinase activity results in endothelial dysfunction. Furthermore, it is well established that BH4 is a critical cofactor for enzymatic activity of eNOS, by which NO is generated in endothelium, and that the vascular protective effects of EPO are dependent on Akt1-induced phosphorylation and activation of eNOS. The results of the present study provide the first direct evidence that EPO stimulates BH4 synthesis through the de novo synthetic pathway involving activation of GTPCH I via the PI3-kinase/Akt1 signaling pathway. The ability of EPO to upregulate GTPCH I activity and phosphorylation of eNOS in a coordinated fashion is most likely designed to optimize the production of NO in vascular endothelium. Our results also suggest that impaired Akt1 signaling is an important mechanism underlying the prohypertensive effect of EPO.
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Acknowledgments |
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We thank Suzanne M. Greiner for assistance with blood cell counts.
Sources of Funding
This work was supported by National Institutes of Health grant HL-53524, by Roche Foundation for Anemia Research, and by the Mayo Foundation. L.V.D. is the recipient of a Scientist Development Grant from the American Heart Association (07-30133N).
Disclosures
None.
Received March 27, 2008; first decision April 21, 2008; accepted May 2, 2008.
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