doi: 10.1161/CIRCRESAHA.107.162503
© 2007 American Heart Association, Inc.
Editorials |
Endothelial Cell Tetrahydrobiopterin
Going With the Flow
From the Department of Cardiovascular Medicine, University of Oxford, UK.
Correspondence to Prof Keith M. Channon, Department of Cardiovascular Medicine, John Radcliffe Hospital, Oxford, OX3 9DU, UK. E-mail keith.channon{at}cardiov.ox.ac.uk
See related article, pages 830–838
Key Words: blood flow • nitric oxide • shear stress
The predilection of atherosclerosis for specific locations in the vasculature strongly suggests that the response of the arterial wall to alterations in blood flow is an important factor in vascular disease pathogenesis. Turbulent rather than laminar blood flow reduces shear stress and leads to endothelial cell dysfunction.1 Better understanding of the mechanisms linking laminar shear stress to endothelial cell homeostasis would improve our knowledge of the pathogenesis of atherosclerosis and may provide new therapeutic targets to combat vascular disease.
One major effect of laminar shear stress on endothelial cells is the increased production of nitric oxide (NO), through increased expression of endothelial nitric oxide synthase (eNOS) and by eNOS activation through phosphorylation.2 However, upregulation of eNOS protein alone can have paradoxical deleterious effects on vascular disease pathogenesis.3 In particular, eNOS activity is critically dependent on the cofactor tetrahydrobiopterin (BH4); in the absence of BH4 the eNOS enzyme becomes uncoupled, producing superoxide rather than NO.4,5 Indeed, recent studies in models of atherosclerosis, diabetes, or hypertension indicate that loss of BH4 is an important contributor to endothelial dysfunction.6 Restoration or augmentation of BH4, either pharmacologically or by increasing endothelial cell BH4 biosynthesis, improves NO-mediated endothelial function, with salutary effects on vascular disease pathogenesis.3,7,8 However, the mechanisms that regulate endogenous BH4 levels in the endothelium remain unclear.
In this issue of Circulation Research, Widder and colleagues describe important new mechanisms that link laminar shear stress with increased BH4 levels in human endothelial cells.9 They elegantly reveal how endothelial cell BH4 biosynthesis is regulated by shear stress through novel signaling pathways that modulate the activity of the BH4 biosynthetic enzyme, GTP cyclohydrolase I, by phosphorylation.
GTP cyclohydrolase (GTPCH) catalyzes the first and rate limiting step in the de novo synthesis of BH4 from GTP.10 The regulation of GTPCH at the transcriptional level has been the focus of several previous studies that demonstrated increased BH4 synthesis in inflammatory cells and hepatocytes after cytokine stimulation. The GCH1 gene contains a CRE- response element and transcription is upregulated by cytokines, lipopolysaccharide, or insulin, whereas in the endothelium hydrogen peroxide also stimulates GTPCH expression. However, the relevance of cytokine-mediated GCH1 induction in regulating endothelial cell BH4 levels in vivo is uncertain, because vascular disease states, where BH4 levels are reduced, are associated with increased inflammatory activation.
The possible posttranscriptional mechanisms that might regulate GTPCH activity have until now focused on the potential roles of GTP cyclohydrolase 1 feedback regulatory protein (GFRP)11 and on putative phosphorylation sites, but in neither of these have physiologic roles been demonstrated in the endothelium. Interaction between GFRP and GTPCH can either inhibit or stimulate GTPCH activity through binding of either BH4 or phenylalanine, respectively, a mechanism that may be most important in hepatocytes. GTPCH phosphorylation has been proposed on the basis of several putative phosphorylation sites for protein kinase CK2 (previously known as casein kinase II [CK2]), and a single putative phosphorylation site for protein kinase C (PKC).12 Previous studies have demonstrated that GTPCH can be phosphorylated by both CK2 and PKC,13,14 and that GTPCH phosphorylation appeared to be associated with increased GTPCH activity.15
Widder et al have now provided, for the first time, definitive evidence showing how endothelial cell GTPCH activity is directly regulated by phosphorylation in response to laminar shear stress.9 They showed that shear stress–induced phosphorylation of GTPCH by the alpha prime (
') subunit of CK2 increases GTPCH activity without a change in GTPCH protein levels. In contrast to earlier studies, inhibition of PKC did not have any effect on GTPCH phosphorylation or activity. Using a panel of phosphospecific antibodies against each of the 5 putative phosphorylation sites, Widder et al went on to demonstrate that shear stress–induced GTPCH phosphorylation occurred exclusively at a single site, serine 81, and that the phosphorylation was specifically increased by laminar rather than oscillatory shear stress. Identification of this single phosphorylation site offers the potential for targeted mutation to further investigate the functional importance of CK2-dependent GTPCH phosphorylation. Inhibitor and siRNA knockdown studies showed that GTPCH and CK2a' specifically (as distinct from CK2a) were required for the increase in endothelial cell BH4 in response to laminar shear stress. Furthermore, this increase in BH4 was in turn a required step in the shear stress–induced increase in NO production: inhibition of GTPCH prevented the increase in endothelial cell BH4 in response to shear stress and led to an increase in eNOS-derived superoxide production and reduced eNOS dimer levels, features of eNOS uncoupling. Thus, CK2a'-mediated GTPCH phosphorylation at serine 81 is a novel and critical regulator of eNOS activity and coupling in response to laminar shear stress, mediated through endothelial cell BH4 availability.
CK2, found ubiquitously in eukaryotic cells, is a tetramer consisting of 2 identical regulatory subunits (CK2ß) and 2 catalytic subunits (either CK2 or CK2‘).16 It is the most highly pleiotropic of all the cellular protein kinases with many hundreds of protein substrates identified, the majority of which are signaling proteins or are involved in control of gene expression.17 However, a number of metabolic enzymes have also been identified as targets, including angiotensin-converting enzyme and the p47 phox subunit of NADPH oxidase, a major source of superoxide within the endothelium, which is deactivated by CK2-mediated phosphorylation.18,19 The regulation of CK2 is complex; activity is not affected by the typical small molecule second messengers such as cyclic nucleotides, lipids, or calcium, but depends on phosphorylation and direct interactions with other proteins.16 Although the mechanisms by which shear stress activates CK2 are not clear, Dunzendorfer have also shown that shear stress in endothelial cells induces CK2-mediated phosphorylation of SP-1, preventing SP-1–induced upregulation of Toll-like receptor 2 in response to inflammatory stimuli, suggesting that activation of CK2 may represent an important common pathway mediating the beneficial effects of laminar shear stress on the endothelium.20
A number of interesting further questions are highlighted by these new findings. It will be important to investigate the relative dependence of endothelial BH4 levels in vivo on shear stress–induced GTPCH activation versus GTPCH protein levels, and the importance of GTPCH regulation by laminar shear stress in relation to changes in BH4 in response to physiologic stimuli such as physical exercise, and in response to vascular disease states. For example, reduced endothelial BH4 levels in vascular disease states in vivo appear to result, at least in part, from BH4 oxidation rather than altered BH4 biosynthesis alone.21 However, transgenic overexpression of endothelial GTPCH in vivo is sufficient to augment vascular BH4 in vivo, even in the presence of increased BH4 oxidation, suggesting that changes in GTPCH protein levels remain an important determinant of BH4 levels.22 Indeed, the only previous study of the effect of shear stress on vascular biopterin levels reported a moderate increase in BH4 in a rat aorto-caval fistula model that was directly related to an increase in GTPCH protein and enzymatic activity, without implicating other regulatory mechanisms—although GTPCH phosphorylation was not investigated.23 There may be a biologic rationale for these differences; the rat GCH1 promoter contains a greater number of putative shear stress response elements than the human gene.23
The observations reported by Widder et al represent a major breakthrough in our understanding of the regulation of BH4 synthesis in the vascular endothelium. They not only extend our understanding of the beneficial consequences of shear stress on vascular homeostasis, but also suggest novel mechanisms underlying the association between alternations in blood flow, endothelial dysfunction, and vascular disease pathogenesis.
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Acknowledgments |
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Sources of Funding
The authors are funded by the British Heart Foundation and the Bristol Myers Squibb Research Fellowship.
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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Related Article:
Circ. Res. 2007 101: 830-838.
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