Published online before print May 7, 2009, doi: 10.1161/ATVBAHA.109.185280
© 2009 American Heart Association, Inc.
Cell Biology/Signaling |
Sphingosine-1-Phosphate
A Novel Nonhypoxic Activator of Hypoxia-Inducible Factor-1 in Vascular Cells
From the Centre de recherche du CHUQ (M.D.M., G.A.R., D.E.R.), L'Hôtel-Dieu de Québec and the Department of Medicine, Université Laval, Québec, QC, Canada; and the Laboratory of Endothelial Cell Biology (J.-P.G.), Institut de recherches cliniques de Montréal (IRCM), Université de Montréal, QC, Canada.
Correspondence to Darren E. Richard, Centre de Recherche du CHUQ, L'Hôtel-Dieu de Québec, 10 Rue McMahon, Québec, QC G1R 2J6, Canada. E-mail darren.richard{at}crhdq.ulaval.ca
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Abstract |
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Objective— Sphingosine-1-phosphate (S1P) is a potent bioactive phospholipid responsible for a variety of vascular cell responses. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of genes essential for adaptation to low oxygen. S1P and HIF-1 are both important mediators of vascular cell responses such as migation, proliferation, and survival. Studies have shown that nonhypoxic stimuli can activate HIF-1 in oxygenated conditions. Here, we attempt to determine whether S1P can modulate the vascular activation of HIF-1.
Methods and Results— We show that in vascular endothelial and smooth muscle cells, activation of the S1P type-2 receptor by S1P strongly increases HIF-1
protein levels, the active subunit of HIF-1. This is achieved through pVHL-independent stabilization of HIF-1. We demonstrate that the HIF-1 nuclear complex, formed on S1P stimulation, is transcriptionally active and specifically binds to a hypoxia-responsive elements. Moreover, S1P activates the expression of genes known to be closely regulated by HIF-1.
Conclusion— Our results identify S1P as a novel and potent nonhypoxic activator of HIF-1. We believe that understanding the role played by HIF-1 in S1P gene regulation will have a strong impact on different aspects of vascular biology.
This study demonstrates that sphingosine-1-phosphate (S1P) is a potent activator of hypoxia-inducible factor-1 (HIF-1) in vascular cells. Increases in cellular levels of HIF-1 is involved in S1P-mediated gene induction under normal oxygen conditions. We conclude that S1P is a novel nonhypoxic activator of HIF-1.
Key Words: sphingosine 1-phosphate • hypoxia-inducible factor-1 • gene expression • endothelial cells • vascular smooth muscle cells
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Introduction |
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The main cellular components of the vasculature are endothelial (ECs) and smooth muscle cells (VSMCs). Normally quiescent, these cells proliferate and migrate in response to vessel wall injury, atherosclerosis, and during angiogenesis associated with a variety of different conditions and diseases.1–3 Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor, has been shown to be involved in various functional responses in ECs and VSMCs.4,5 HIF-1 controls the expression of genes involved in adaptation responses to hypoxic stress.6 HIF-1 is formed after the interaction of a constitutive HIF-1β subunit with a tightly regulated HIF-1
subunit. In normal oxygen conditions, HIF-1 is hydroxylated through the action of HIF prolyl-hydroxylases. Hydroxylation of HIF-1 allows the binding of the product of the von Hippel–Lindau tumor suppressor gene (pVHL). As the recognition component of a E3 ubiquitin ligase complex, pVHL allows for HIF-1 polyubiquitination and subsequent proteasomal degradation.7 Although hypoxia is the ubiquitous inducer of HIF-1, accumulating evidence has revealed that under normal oxygen tension many other extracellular stimuli can also strongly induce the HIF-1 nuclear complex in a cell-specific manner.8 A clear understanding of the mechanisms regulating HIF-1 in vascular cells is fundamental and especially important in the endothelium where the loss of HIF-1 inhibits many aspects of EC behavior.4 Mainly released into circulation by activated platelets and erythrocytes, sphingosine-1-phosphate (S1P) is a potent and multifunctional phospholipid exerting a broad range of vascular cell responses including migration, proliferation and survival S1P has important impacts on physiological and pathological events such as wound healing, hemostasis, thrombosis, tumor progression, inflammation, and atherosclerosis.9–12
Given the importance of S1P and HIF-1 in a number of biological events, especially their central role in angiogenesis and concomitant implications in pathologies like atherosclerosis and tumor progression, the goal of the current study is to evaluate the potential role of S1P on HIF-1 activation. Here, we demonstrate that the treatment of vascular cells with S1P stabilizes HIF-1 protein levels in a time- and dose-dependent manner. The HIF-1 complex, formed on S1P stimulation, is transcriptionally active, specifically binds to hypoxia-response elements (HRE), and drives HIF-1–dependent gene expression. Taken together, these results identify S1P as a novel and potent normoxic activator of the HIF-1 transcriptional complex and HIF-1 as a mediator of S1P cellular responses.
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Materials and Methods |
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A detailed description of all methods is available in the supplemental materials (available online at http://atvb.ahajournals.org).
Cell Culture
The murine lung endothelial 1G11 cell line was originally obtained from Alberto Mantovani and Annunciata Vecchi (Instituto Ricerche Farmacologiche Mario Negri, Milan, Italy).13 VSMCs were isolated from thoracic aortas of young Wistar rats (Charles River). Bovine aortic endothelial cells (BAECs) were isolated from calf thoracic aortas (obtained from a local slaughterhouse). For all experiments, cells were rendered quiescent by overnight (16-hour) serum starvation. Hypoxic conditions were obtained by placing cells in a sealed hypoxic workstation (Ruskinn Technology Ltd).
RNA Interference
All siRNAs oligonucleotides were obtained from Applied Biosystems. To downregulate HIF-1 protein expression in VSMCs, a siRNA duplex targeting rat HIF-1 (accession no. NM_024359) was transfected by CaPO4 precipitation: sense: 5'-AGGACAAGUCACCACAGGAUU-3'. As a control oligonucleotide, the same siRNA duplex containing 2 mismatches was used: sense: 5'-AGGACAAGGCAUCACAGGAUU-3'. To downregulate S1P2 protein expression in BAECs, a siRNA duplex targeting bovine S1P2 receptor (accession no NM_001081541) was transfected by CaPO4 precipitation: sense: 5'-GCUCUACGGCAGCGACAAGtt-3'. As a control oligonucleotide, the Silencer Negative Control #2 siRNA was used.
Western Blot Analysis
Cell extracts were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore). Proteins were visualized with an enhanced chemiluminescence system (ECL; GE Healthcare Life Sciences). HIF-1 and anti–HIF-2 antiserum were previously described.14 Experiments are representative of at least 3 independent experiments.
Northern Blot Analysis
RNA was isolated with TRIzol reagent (Invitrogen), resolved on 1% agarose/6% formaldehyde gels, and transferred to Hybond N+ nylon membrane (GE Healthcare Life Sciences) before hybridization with a radioactive cDNA probes. Experiments are representative of at least 3 independent experiments.
pVHL Capture Assay
1G11 cytoplasmic extracts (250 µg) were incubated with sepharose-bound GST–HIF-1. GST pull-down assays for pVHL were performed as previously described.15 Experiments are representative of at least 3 independent experiments.
Transcription Factor Enzyme-Linked Immunoassay
Preparation of nuclear extracts and transcription factor enzyme-linked immunoassay (TFEIA) were performed as previously described.16 Experiments are an average ±SEM of at least 3 independent experiments performed in triplicate.
Luciferase Assays
Transient transfections were performed using 2 µg per well pGL3 (R2.2) 3HRE-tk-LUC or CMV-luc-HIF-1-ODDD luciferase reporter vectors. Renilla reniformis luciferase expression vector (250 ng/well) was used as a control for transfection efficiency. Luciferase assays were performed as previously described.15 Experiments are an average ±SEM of at least 3 independent experiments performed in triplicate.
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Results |
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S1P Induces HIF-1
Protein LevelsNonhypoxic stimulations have been shown to strongly increase HIF-1
protein levels.8 Because S1P and HIF-1 share common downstream activities in the vasculature, we attempted to determine whether vascular cell stimulation with S1P could induce HIF-1 leading to activation and gene regulation. ECs were incubated in the presence of different concentrations of S1P followed by the evaluation of HIF-1 protein levels by Western blot (Figure 1A). Interestingly, the treatment of cells with S1P for 4 hours strongly increased HIF-1 protein levels. Treatment with 0.1 µmol/L S1P led to a detectable increase in HIF-1 protein levels, whereas maximal induction was attained at 2 µmol/L S1P. Time-course studies were then performed on these same cell lines treated with 2 µmol/L S1P (Figure 1B). An increase in HIF-1 protein levels was detected after 1 hour of S1P treatment, and a maximal induction was attained after 4 hours in the presence of S1P. After 6 hours, HIF-1 levels subsequently decreased. Phosphorylation of p42/p44 MAP kinase, a pathway known to be activated through S1P in vascular cells, was used as a control for S1P receptor activation. Similar results for HIF-1 induction by S1P treatment were also observed in VSMCs (supplemental Figure I, available at http://atvb/ahajournals.org) and in bovine aortic endothelial cells (BAECs) (results not shown). Taken together, these results demonstrate that S1P increases the expression of the inducible subunit of HIF-1 in vascular cells.
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Because hypoxia is the ubiquitous activator of HIF-1, we compared the hypoxic induction of HIF-1 protein with the normoxic induction elicited after S1P treatment. As shown in Figure 1C, the level of HIF-1 protein during S1P treatment of 1G11 cells is similar to the level induced by a 4-hour incubation in hypoxic conditions. This result demonstrates the potency of S1P for inducing HIF-1 in these cells. Similar results were observed in BAECs and VSMCs (results not shown). Interestingly, S1P was unable to induce HIF-2, a close homolog of HIF-1 also induced under hypoxic conditions in 1G11 cells (Figure 1C). This result indicates that S1P specifically targets the induction of the HIF-1 complex. Finally, the treatment of cells with S1P in the presence of hypoxia had an additive effect on HIF-1 induction (results not shown). This result suggests that different mechanisms are responsible for induction/activation of HIF-1 by S1P and hypoxia.
In vascular cells, S1P mediates its intracellular effects through S1P1, S1P2, and S1P3 receptors.17 We attempted to determine which S1P receptor led to HIF-1 induction. The treatment of cells with pertussis toxin, an inhibitor of Gi protein–coupled S1P receptors, did not block S1P-mediated HIF-1 induction, precluding the implication of S1P1, which couples exclusively through Gi (supplemental Figure IIIB).17 Pertussis toxin treatment did indeed block the phosphorylation of p42/p44 MAP kinase, a pathway known to be activated through Gi-coupled S1P receptors (supplemental Figure IIIB). However, the use of JTE-013, a potent S1P2-selective antagonist, strongly decreased HIF-1 levels after S1P treatment (Figure 2A; supplemental Figure IIA). Additionally, BAECs transfected with specific S1P2 siRNA oligonucleotide blocked HIF-1 levels after S1P treatment as compared to a control siRNA oligonucleotide (Figure 2B; supplemental Figure IIB). Effective silencing of S1P2 in BAECs is shown in the lower panel of Figure 2B. It is important to note that JTE-013 and S1P2 siRNA had no effect on hypoxic HIF-1 induction (Figure 2A and 2B). Finally, antagonists for S1P1 and S1P3, such as W146 (a S1P1 antagonist), VPC23019 (a S1P1/S1P3 antagonist), and suramin (a S1P3 antagonist), had no effect on HIF-1 protein levels (supplemental Figure IIIA). These results indicate that S1P acts through S1P2 to increase HIF-1 protein levels.
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S1P Regulates HIF-1 Protein Stabilization
We next investigated possible pathways involved in HIF-1 protein induction by S1P. In contrast to other nonhypoxic HIF-1 inducers,16,18 S1P did not modify HIF-1 transcript levels (supplemental Figure IVA). In hypoxia, the main mechanism involved in HIF-1 induction is protein stabilization by inhibiting the rapid proteasomal degradation of HIF-1.19 Given the elevated levels of HIF-1 protein during S1P treatment and the high instability of HIF-1 under normal oxygenation, we undertook studies to determine whether S1P treatment stabilized HIF-1 protein levels. The half-life of HIF-1 protein was evaluated using cycloheximide, a general protein synthesis inhibitor. 1G11 cells, stimulated with S1P or hypoxia, were then treated with cycloheximide for different periods of time to block all novel HIF-1 protein synthesis. Half-life of HIF-1 was evaluated by Western blotting. As seen in Figure 3A, the half-life of HIF-1 under S1P treatment was not significantly different from the half-life of HIF-1 under hypoxic treatment (16.3±1.2 versus 18.8±1.3 minutes, P>0.5). Because hypoxia stabilizes HIF-1, these results indicate that S1P treatment causes the stabilization of HIF-1 protein.
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We next attempted to determine whether S1P could act on steps leading to HIF-1 protein degradation. HIF-1 ubiquitination and degradation is controlled through its oxygen-dependent degradation domain (ODDD). To evaluate the possibility that S1P could modulate the stability of this specific domain, we used a fusion protein construct comprised of amino acids 401 to 602 of the ODDD and the C-terminal end of the firefly luciferase protein. This construct generates an unstable form of luciferase when transfected into cells. The half-life of this luciferase construct is increased by oxygen deprivation and can be quantified by luciferase assays.20 As seen in Figure 3B, when BAECs were transiently transfected with the CMV-luc-HIF-1-ODDD vector and treated with MG132, an inhibitor of proteasomal degradation, increased luciferase activity was observed (2.2-fold over basal levels). More interestingly, the stimulation of BAECs with S1P also increased luciferase activity (1.5-fold over basal levels). This result indicates that S1P targets the HIF-1 ODDD to increase HIF-1 protein stabilization.
A crucial event leading to HIF-1 protein degradation is its binding to the product of the von Hippel–Lindau tumor suppressor gene (pVHL), which is a direct consequence of HIF-1 hydroxylation. To determine the effect of S1P treatment on pVHL binding, a pVHL capture assay was used. A GST–HIF-1 fusion protein, comprising amino acids 344 to 582 from human HIF-1, was subjected to modification by S1P-treated 1G11 cell extracts followed by interaction with in vitro translated pVHL protein. The treatment of cells with S1P did not decrease pVHL binding to HIF-1 (Figure 3C). As expected, the treatment of cells with CoCl2, a hypoxia mimetic that strongly inhibits HIF-1 hydroxylation, completely abolished pVHL binding to HIF-1. Taken together, our results demonstrate that S1P increases HIF-1 protein stability through a pathway independent of HIF-1 hydroxylation and pVHL binding.
S1P Activates the HIF-1 Nuclear Complex
We next attempted to determine whether increases in HIF-1 during S1P treatment led to the formation of an active HIF-1 complex with the constitutive nuclear subunit, HIF-1β. To perform these studies, we used a HIF-1 TFEIA. TFEIA uses a specific dsDNA oligonucleotide sequence (W26) fixed on a 96-well plate which corresponds to the sequence of a known hypoxic response element (HRE).16 Nuclear extracts from 1G11 cells maintained in hypoxic conditions or in the presence of S1P both demonstrated increased DNA-binding activity for HIF-1 and HIF-1β (Figure 4A). To control the specificity, we substituted the W26 dsDNA oligonucleotide sequence with a sequence mutated on 2 essential residues of the HIF-1-binding sequence (M26). In this case, very little HIF-1 binding could be observed (results not shown). These results demonstrate that endothelial HIF-1 protein induced by S1P can form the HIF-1 complex and bind to a HIF-1-specific promoter sequence. Similar results were obtained in VSMCs (results not shown).
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We then determined whether the HIF-1 complex induced by S1P is transcriptionally active. We measured HIF-1–dependent transcription in BAECs transiently transfected with a luciferase reporter gene driven by 3 HRE sequences (pGL3 [R2.2] 3HRE-tk-LUC). As shown in Figure 4B, S1P treatment increased reporter activity in BAECs by 2.5-fold over basal levels. In the same cells, hypoxia increased reporter activity by 3.6-fold over basal levels. Additionally, significant activation of HIF-1 transcriptional activity was observed at S1P concentrations as low as 0.5 µmol/L (results not shown). Similar results were also obtained in VSMCs (results not shown). These results indicate that S1P treatment leads to the formation of a transcriptionally active HIF-1 complex and could ultimately lead to the activation of different HIF-1 target genes.
Role of HIF-1 in S1P-Mediated Gene Expression
We next investigated the role of HIF-1 induction in the expression of known HIF-1 target genes. As seen in the upper panels of Figure 5, VSMCs treated with S1P increased the expression of VEGF, GLUT-1, and PAI-1 mRNA (2.9±0.3-, 5.3±0.5-, and 5.5±0.6-fold over basal levels respectively); 3 transcripts shown to be upregulated during HIF-1 activation.21–23 Similar results were observed with 1G11 ECs (supplemental Figure IVA). Increased expression of HIF-1–responsive gene induction was also seen with S1P concentrations as low as 0.5 µmol/L (supplemental Figure IVB). To determine the implication of HIF-1 in these responses, VSMCs were transfected with specific HIF-1 siRNA oligonucleotides. During S1P treatment, HIF-1 siRNAs significantly decreased the expression of all 3 transcripts as compared to control siRNA oligonucleotides, which were mismatched by 2 base pairs (Figure 5, upper panel; supplemental Figure V). Effective silencing of HIF-1 in VSMCs is shown in the lower panels of Figure 5. These results indicate that HIF-1 is involved in activating downstream target genes during cell stimulation with S1P and suggest a functional importance of these pathways in the vascular activity of S1P.
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Discussion |
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The past decade has seen great advances in understanding the function and biological actions of lysophospholipids. Precisely because of its proliferative, invasive, and migrative effects on vascular cells, S1P has been implicated in the pathophysiology of cancer and certain cardiovascular diseases.24,25 Because the modulation of HIF-1 is also implicated in the progression of aforementionned diseases26–28 and recent studies have shown that a number of nonhypoxic stimuli can activate HIF-1 in a cell-specific manner,8 the present study set out to investigate the possible interactions between HIF-1 and S1P in vascular cells and subsequent activation of HIF-1 target genes. Here, we show that in vascular cells, HIF-1
protein levels are strikingly increased on S1P stimulation, which leads to HIF-1 complex formation and transcriptional activity.
This study identifies S1P as an activator of the HIF-1 complex in oxygenated conditions and demonstrates that HIF-1 can activate hypoxia-responsive genes after S1P stimulation. Indeed, we report that in VSMCs, HIF-1 is involved in the expression of VEGF, GLUT-1, and PAI-1 mRNA elicited by S1P, because all 3 transcripts are diminished when HIF-1 protein levels are reduced by RNA interference. As previously mentioned, ECs and VSMCs respond very strongly to the bioactive lipid S1P to promote a range of crucial intracellular events through multiple signaling pathways.9 Moreover, in these 2 cell types, a tight regulation in the expression of the HIF-1 transcription factor is very important for a variety of responses that can lead to cell survival or cell death.4,27 We believe that during S1P treatment, along with other pathways and transcription factors, HIF-1 will permit the maximal activation of certain genes, leading to key cellular responses.
When cells are subjected to hypoxia, HIF-1 protein is stabilized and rapidly accumulated to permit a rapid adaptative response. Our results demonstrate that S1P increases HIF-1 protein stability through a pathway which is independent of HIF-1 hydroxylation and pVHL binding. Recent studies have also revealed that HIF-1 protein stabilization can occur through pathways independent of pVHL under normal oxygen conditions. Two studies are of particular interest. The first involves HSP90, a molecular chaperone that protects client proteins from misfolding and degradation, and the receptor of activated protein kinase C (RACK1). By competing with HSP90 for binding to HIF-1, RACK1 mediates a proteasomal degradation pathway that is mechanistically similar to the pVHL pathway but independent of O2, hydroxylation, and pVHL binding.29 Using a specific inhibitor of HSP90, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), we were unable to block HIF-1 induction or stabilization in our cell models, indicating that HSP90 is not implicated in HIF-1 stabilization by S1P (results not shown). A second study involves glycogen synthase kinase 3 (GSK-3) which appears to exert its effect on HIF-1 in such a way that the activation of GSK-3 downregulates HIF-1 protein levels. The activation of signaling pathways, such as the phosphatidylinositol 3-kinase (PI3K) pathway, leads to an inhibition of GSK-3 activity during the treatment of cells with various growth factors and thus the stabilization of HIF-1 protein.30 In vascular cells, S1P is a known activator of the PI3K pathway leading to the phosphorylation and inactivation of GSK-3 (31and our unpublished results). We are now evaluating the possible role of the PI3K/GSK-3 pathway in the regulation of HIF-1 by S1P.
In our studies, the strong induction of HIF-1 protein levels elicited by S1P was specific to vascular cells. We were unable to increase HIF-1 in other S1P-responsive cell models such as macrophages, fibroblasts, HEK293, and HeLa (results not shown). However, recent evidence also indicates that S1P may be involved in the expression of HIF1 isoforms in lymphocytes.32 Whereas hypoxia is the ubiquitous HIF-1 activator, most nonhypoxic stimuli seem to function in a more cell type–specific manner.8 It is possible that given the importance of HIF-1 gene induction in a variety of cellular functions and the lack of controlled stimuli, cells specifically develop mechanisms for HIF-1 induction to suit their needs. Because S1P is a key cardiovascular signaling molecule in the regulation of vascular function and homeostasis and HIF-1 is the master regulator of various genes in vascular cells that are particularly related to cell survival and angiogenesis, specific interactions between these 2 components in vascular cells are likely to play an important role in different physio- and pathological functions in the vessel wall.
It is also interesting to note that HIF-1 is induced by S1P concentrations that are in the physiological range. Because high levels of S1P are found in blood, this bioactive lipid is in an effective position to regulate the vascular endothelium, which is a major S1P-responsive cell type. S1P concentrations in serum, estimated to be around 0.4 µmol/L, can be twice as elevated in plasma, attaining concentrations similar as those used in the present study.12 Moreover, in conditions of platelet activation, such as in injured vessels during wound healing, the concentration of S1P could be increased considerably.10 This could also be the case in various atherosclerotic diseases where the levels of lipids and lipoproteins are altered and endothelial functions are disturbed.33
Our studies also demonstrate that the effects of S1P on HIFs are specific to HIF-1 and not to HIF-2. This finding concurs with studies demonstrating that effects of HIF on EC migration are critically dependent on HIF-1 and not HIF-2.4,34 This result also demonstrates that when HIF-1–silenced vascular cells are treated with S1P, HIF-2 will not act as an alternative and compensatory pathway. Recently, there has been considerable interest in determining the specific roles of different HIF family members.34,35 Our findings are interesting at this level for identifying model systems to study signals which pass exclusively through HIF-1 as compared to hypoxia, which will induce both HIF-1 and HIF-2. A recent study has shown that another bioactive lipid, lysophosphatidic acid (LPA), also activates HIF-1 in various cancer cell lines.36 Taken together, these results identify S1P and LPA as a new class of nonhypoxic activators of HIF-1.
S1P receptors (S1PR) are expressed in almost every cell type, but in the vascular system, S1P1, S1P2, and S1P3 are predominant.37 In our EC model, HIF-1 protein expression is abrogated by pretreatment of cells with JTE-013, a specific antagonist of S1P2, or a specific S1P2 siRNA. On the other hand, different inhibitors and antagonists for S1P1 and S1P3 (pertussis toxin, W146, VPC23019 and suramin) were ineffective. Therefore, our results indicate that S1P induces HIF-1 through S1P2. It is interesting to note that human umbilical vessel endothelial cells (HUVECs), which express very low levels of S1P2, do not increase HIF-1 levels after S1P treatment (results not shown).39 In line with our results, S1P increases PAI-1 expression after S1P2 activation in glioblastoma cells.38 S1P also increases vascular permeability through S1P2.39 Because VEGF has a central role in the regulation of vascular permeabitily and is highly regulated through HIF-1, the activation of HIF-1 (and subsequently VEGF) by S1P2 activation could collaborate with direct S1P2 signaling events in the regulation of vascular permeability by S1P. Finally, an interesting study has shown that in the developing limbs of S1P1 knockout animals, HIF-1 protein levels and VEGF expression is increased, suggesting a repressive role of S1P on HIF-1 expression.40 However, severe hypoxia exists in the limbs of these animals. It is likely that defective vascular development induced by lack of S1P1 caused this hypoxia, leading to enhanced HIF-1 and VEGF induction.
In conclusion, our work identifies S1P as a novel nonhypoxic activator of the HIF-1 transcription factor in vascular cells. The elevation of HIF-1 protein levels by S1P leads to the formation of an active HIF-1 complex and to the expression of downstream target genes. These events are likely to have important physiological implications given the wide spectrum of genes possibly activated through this pathway and their roles in various areas of vascular biology.
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Acknowledgments |
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Sources of Funding
This work was supported by grants to D.E.R. from the Canadian Institutes of Health Research (CIHR, MOP-49609) and the Heart and Stroke Foundation. M.D.M. is a recipient of a Canada Graduate Scholarship Doctoral Award from the CIHR. J.P.G. holds a Canada Research Chair. D.E.R. is a recipient of a CIHR New Investigator Award.
Disclosures
None.
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Footnotes |
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Received November 12, 2007; revision accepted March 31, 2009.
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