Published online before print July 24, 2008, doi: 10.1161/ATVBAHA.108.171546
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© 2008 American Heart Association, Inc.
Cell Biology/Signaling |
Transcriptional Activation of HIF-1 by ROR
and its Role in Hypoxia Signaling
From the College of Pharmacy and Bio-MAX institute (E.-J.K., Y.-G.Y., W.-K.Y., Y.-S.L., T.-Y.N., M.-O.L.), Seoul National University, and the Department of Internal Medicine (I.-K.L.), School of Medicine, Kyungpook National University, Daegu, Korea.
Correspondence to Mi-Ock Lee, College of Pharmacy and Bio-MAX institute, Seoul National University, Seoul 151-742, Korea. E-mail molee{at}snu.ac.kr
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Abstract |
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Objective— Hypoxia-inducible factor 1
(HIF-1) is primarily involved in the adapting of cells to changes in oxygen levels, which is essential for normal vascular function. Recently, physiological roles for retinoic acid–related orphan receptor (ROR) have been implicated in cardiovascular diseases such as atherosclerosis. In this study, we have investigated the potential roles of ROR in the hypoxia signaling pathway in connection with activation of HIF-1.
Methods and Results— Under hypoxic conditions, expression of ROR was induced. When ROR was introduced exogenously, protein level as well as transcriptional activity of HIF-1 was enhanced. Putative ligands of ROR, such as melatonin and cholesterol sulfate, induced transcriptional activity for HIF-1, which was abolished by RNA interference against ROR. ROR was physically associated with HIF-1 through DNA binding domain, which was required to the ROR-induced stabilization and transcriptional activation of HIF-1. Finally, either infection with adenovirus encoding ROR or treatment with ROR ligands enhanced the formation of capillary tubes by human umbilical vascular endothelial cells.
Conclusions— Our results provide a new insight for the function of ROR in amplification of hypoxia signaling and suggest a potential application of ROR ligands for the therapy of hypoxia-associated vascular diseases.
Key Words: ROR • hypoxia • HIF-1 • melatonin • vascular endothelial growth factor
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Introduction |
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Retinoic acid receptor-related orphan receptor
(ROR; NR1F1) is a member of the steroid/thyroid hormone receptor superfamily of transcriptional factors and is closely related to the retinoic acid receptors.1,2 ROR exists in 4 isoforms, ROR1, ROR2, ROR3, and ROR4 (also known as RZR), which are generated by a combination of alternative promoter use and exon splicing of the RORA gene.2 These isoforms comprise a common DNA-binding domain (DBD) and a putative ligand-binding domain (LBD), but differ by their N-terminal sequences.3 Melatonin and synthetic thiazolidine diones have been shown to transactivate ROR, although these observations need to be clarified.4,5 Recently, analysis of the crystal structure of the ligand-binding domain of ROR revealed that a ligand is present in the binding pocket. This was identified as cholesterol, suggesting that plasma and intracellular levels of cholesterol may be important in the regulation of transcriptional activity for ROR.6,7 ROR usually binds as a monomer to a ROR response element (RORE) consisting of a half site core AGGTCA motif or as a homodimer to a direct repeat of the core motif separated by 2 base pairs.1 Putative ROREs have been identified in the promoters, such as human fibrinogen β, Apo A-V, Apo A-I, Apo C-III, PPAR
, and Rev-Erb, which may suggest a role of this receptor in lipid metabolism and cardiovascular physiology.8–12 However, little is known regarding the internal and external stimuli that regulate the RORA gene expression.
ROR functions have been studied with the help of the staggerer (sg/sg) mutant mouse. A spontaneous mutation in the ligand-binding domain induces a frameshift that results in a protein truncated in its C terminus and generates the staggerer phenotype.13 These animals experience severe cerebellar ataxia caused by massive neurodegeneration of Purkinje cells.14 Moreover, the phenotype of these mice revealed that ROR is crucially involved in regulating the inflammatory and immune responses and lipid metabolism, which are closely related to vascular disorders such as atherosclerosis.15,16 In the staggerer mice, angiogenesis is enhanced markedly after ischemia induced by the ligation of the femoral artery.17 In the vascular system, ROR mRNAs have been detected in human smooth muscle cells (SMCs), endothelial cells (ECs), as well as mammary arteries.16,18 ROR expression level is significantly decreased in human atherosclerotic plaques, whereas increased expression is observed after treatment with interleukin (IL) 1β, tumor necrosis factor (TNF) , and lipopolysaccharide (LPS) in both ECs and human aortic SMCs.16,18 It thus appears that ROR has direct links to a number of age-related pathologies of great medical interest.
Disruption of oxygen homeostasis represents a major aspect of the pathophysiology of inflammatory vascular diseases such as atherosclerosis. When cellular oxygen availability decreases, the transcription factor hypoxia-inducible factor 1 (HIF-1) plays a central role in cellular adaptation by stimulating the transcription of diverse genes, which encode proteins that function to increase oxygen delivery, to allow metabolic adaptation, and to promote cell survival.19,20 HIF-1 consists of and β subunits, both of which belong to the basic helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) protein family. Whereas HIF-1β is quite stable under normoxic conditions, HIF-1 is extremely unstable and is quickly degraded by the ubiquitin-proteasome system.21,22 The tumor-suppressor von Hippel-Lindau (VHL) protein interacts with hydroxylated HIF-1 at Pro564 of HIF-1 in the presence of oxygen, leading to the proteolysis of HIF-1.23,24 Posttranscriptional modifications of the oxygen-dependent degradation (ODD) domain such as hydroxylation, acetylation, and deacetylation are important in the regulation of protein stability and transcriptional activity of HIF-1.25–27 In addition to hypoxia, the stability and function of HIF-1 is modulated by a variety of intracellular proteins. We have previously shown that the Nur77 family of orphan nuclear receptors and their activators stabilize and transactivate HIF-1.28,29
Recently, it was reported that ROR expression is upregulated in several types of cells, including human SMCs and ECs, under hypoxia or in the presence of inflammatory cytokines such as IL-1β and TNF.18,30,31 The potential implication of ROR in the hypoxia signaling pathway prompted us to study its role in transcriptional activation of HIF-1. Here we report that ROR and its ligands activate HIF-1, which leads to activation of a positive circuit for hypoxia signaling.
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Methods |
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Cell Culture and Hypoxic Treatment
HepG2, HeLa, HEK293, and NIH3T3 were obtained from American type culture collection. Human umbilical vein endothelial cells (HUVECs) were obtained from Cambrex. Cells were exposed to hypoxia, 0.1% O2, by incubating cells at 37°C in 5% CO2/10% H2/85% N2 anaerobic incubator (Forma Scientific). Hypoxia was also induced chemically by treating cells with CoCl2 or desferrioxamine (DFO). For ligand treatment, cells were plated in medium supplemented with charcoal-stripped fetal bovine serum.
Western Blotting, Immunoprecipitation, and Subcellular Fractionation
Western blotting was basically performed as previously described using specific antibodies against ROR, HIF-1, VEGF, MDM2, VHL, FIH-1, CBP (Santa Cruz Biotechnology), FLAG (Sigma), or -tubulin (Oncogene).27,29 Immunoprecipitation and subcellular fractionation was carried out basically as described previously.27,28
Plasmids and Transient Transfection
The RORE-tk-Luc, and the eukaryotic expression vectors, pSG5-ROR1and pSG5-ROR4, were kindly provided by Drs Bart Staels (Université de Lille 2) and Hendrik Stunnenberg (Radboud University Nijmegen Medical Center), respectively. The HRE-tk-Luc, VEGF promoter (–2068 to +50)-Luc, and Gal4-tk-Luc reporter constructs have been described previously.8,27,29 The eukaryotic expression vectors such as pEGFP-C3-HIF-1 (GFP-HIF-1), p3XFLAGTM7.1-FLAG-HIF-1, VP16-HIF-1, and glutathione S-transferase (GST)-fused HIF-1 (pEBG-HIF-1) and truncated constructs were described previously.27,29 The Gal4 DBD-fused ROR1, FLAG-tagged full-length and truncated ROR1 constructs, HA-tagged ROR4, and antisense (AS)-ROR1 plasmids were constructed by conventional rDNA technology. Transient expression of proteins and reporter gene analysis were as previously described.29
RT-PCR and Real-Time PCR
RT-PCR reaction was performed as described previously.29 Real-time PCR amplifications were performed using Fast Start DNA Master SYBR Green I Mixture Kits (Roche Diagnostics) in a Light Cycler system (Roche Diagnostics) following manufacturer’s protocol using specific primers.
Transfection of siRNA Duplexs
The siRNA duplexs targeting all isoforms of ROR (si-ROR(1), 5'-CGCUGCCAACACUGUCGAUU ATT-3' and 5'-UAAUCGACAGUGUUGGCAGUGTT-3'; si-ROR(2), 5'-GCACAGAAUAUAUCUAAAUTT-3' and 5'-AUUUAGAUAUAUUCUGUGCTT-3'), HIF-1 (si-HIF-1(1)27; si-HIF-1(2), 5'-GCACAGAAUAUAUCUAAAUTT-3' and 5'-AUUUAGAUAUAUUCUGUGCTT-3') and control nonspecific siRNA, siGL3, were synthesized and purified by Shamchully Pharm Co.27 HepG2 cells were transfected with siRNAs using Lipofectamine 2000 (Invitrogen) reagent according to the manufacturer’s protocol. si-ROR(2) and si-HIF-1(2) showed the same effects of si-ROR(1) and si-HIF-1(1).
Adenovirus Production and Viral Infection
The ROR4 recombinant adenovirus vector was constructed by inserting full-length ROR4 cDNA into the pShuttle-IRES-hrGFP-2 vector (Stratagene). The resulting shuttle vector was transfected into HEK 293 cells. Recombinants were identified, amplified, and isolated using CsCl. The adenovirus preparations were desalted and titers were determined by plaque counts. For adenovirus infection, HUVECs were seeded in 60-cm2 dishes (1x106 cells per dish) and incubated overnight. Cells were infected with adenovirus expressing either GFP or ROR at a density of 1.3x106 GTU/mL and incubated for 3 hours with agitation. At the end of infection, media containing virus was replaced with fresh media and the incubation was continued for 24 hours.
Capillary Tube Formation Assay
The 96-well plates were coated with Matrigel (BD Biosciences) by incubating at 37°C for 1 hour. HUVECs were suspended in media supplemented with 2% FBS and endothelial cell growth supplement, and then plated in the coated plates. The morphological changes in the cells and the closed networks of vessel-like tubes were observed and photographed using a fluorescent microscope (TH4-200, Olympus).
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Results |
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ROR
Increases the Transcriptional Activity of HIF-1To study the role of ROR
in regulation of hypoxic response, first, we examined expression pattern of ROR under hypoxia. As previously reported, expression levels of ROR in HepG2 were increased under hypoxic conditions.31,32 ROR1 and ROR4 proteins were induced dramatically; however, neither expression nor induction of ROR2 and ROR3 was detected (supplemental Figure IA, available online at http://atvb.ahajournals.org). Transcripts of ROR and VEGF were increased in a similar pattern, whereas the amount of HIF-1 mRNA was unchanged (supplemental Figure IB). A reporter encoding two copies of the RORE in the promoter of I
-B, was activated under hypoxia or in the presence of CoCl2 or desferroxamin, which are hypoxia-mimicking chemicals (supplemental Figure IC). These results demonstrate that ROR is induced under hypoxia at the level of transcription.
Therefore we asked whether this ROR induction had a functional importance in hypoxia signaling pathway. We tested the effect of ROR expression on the transcriptional activity of HIF-1, using the HRE-tk-Luc reporter construct containing hypoxia response element (HRE) sequences of the 3' enhancer of erythropoietin gene. Cotransfection of the ROR1 expression vector into HepG2 cells activated the reporter activity in a dose-dependent manner (Figure 1A). Consistent with the results from reporter gene analysis, the levels of both HIF-1 and VEGF proteins increased when ROR1 was exogenously introduced. The induction level was similar to that caused by CoCl2, and it was not further increased by CoCl2 (Figure 1B). Importantly, repression of ROR by transfection with an antisense (AS)-ROR construct strongly suppressed hypoxia-induced HRE reporter activity (Figure 1C). Similarly, knockdown of ROR using silencing (si) RNA largely decreased the hypoxia-induced expression levels of HIF-1 and VEGF (Figure 1D). Both ROR1 and ROR4 showed the similar potency in activating HRE reporter activity and in increasing expression of HIF-1 and VEGF (supplemental Figure II). Together, these results indicate that ROR enhances the transcriptional activity of HIF-1 in the absence of hypoxic stress, and it may mediate hypoxia-induced HIF-1 activation.
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Melatonin Increases Transcriptional Activities of HIF-1 Through Activation of ROR
Because melatonin increases transcriptional activity of ROR,5 we examined whether it enhances HIF-1 activity. Consistent with previous reports, melatonin induced the RORE-tk-Luc reporter in a dose-dependent manner. Melatonin also increased activities of HRE and VEGF promoter, suggesting that the melatonin-induced ROR may contribute to the induction of HIF-1 and VEGF (Figure 2A). Melatonin increased the expression levels of ROR, HIF-1, and VEGF proteins as early as 10 minutes and continued up to 24 hours (Figure 2B and supplemental Figure III). When expression of either ROR or HIF-1 was repressed by transfection with the corresponding si-RNAs, the melatonin-induced increase of VEGF was diminished dramatically (Figure 2C). Together these results clearly showed that melatonin activates ROR, which leads to transcriptional activation of HIF-1 and consequent induction of VEGF.
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ROR Interacts With HIF-1 and Increases the Stability of HIF-1
To further characterize the cross-talk between ROR and HIF-1, we examined whether these proteins were physically associated. ROR and HIF-1 were localized in the nucleus under hypoxic conditions (Figure 3A). Physical association of ROR with HIF-1 were demonstrated by reciprocal coimmunoprecipitation and mammalian two-hybrid assays (Figure 3B and Supplemental Figure IVA). Next, we delineated the interaction domains using the FLAG-tagged truncated ROR1 and the GST-fused HIF-1 constructs. The coimmunoprecipitation results showed that DBD of ROR served for binding site (Figure 3C). Inhibitory domain (ID), neither N terminus (N), ODD, nor CTAD of HIF-1 bound to ROR1 (supplemental Figure IVB and data not shown). The importance of this binding was demonstrated in that DBD of ROR was sufficiently active for the increases of protein-level as well as transcriptional activity of HIF-1 (supplemental Figure IVC and IVD). These results may suggest that protein–protein interaction of ROR with HIF-1 is required to the cross-talk of these molecules in hypoxia signaling.
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To study the molecular mechanisms of ROR-induced activation of HIF-1, we tested whether ROR could enhance stability of HIF-1. When HIF-1 protein stability was measured in the presence of cycloheximide, a blocker of de novo protein synthesis, overexpression of ROR1 or treatment of melatonin blocked degradation of HIF-1, and this was as efficient as CoCl2 treatment (supplemental Figure VA). Also the protein stability of HIF-1 was examined by immunofluorescence study using green fluorescent protein (GFP) fused with HIF-1. GFP–HIF-1 was barely detected under normoxia, whereas it enhanced and accumulated in the nucleus in the presence of either CoCl2 or melatonin (supplemental Figure VB). Melatonin may enhance stability of HIF-1 protein through inhibiting ubiquitin-mediated proteasomal degradation pathways, because the binding of HIF-1 to VHL or MDM2 was strong in the presence of the proteasome inhibitor MG132; however, these effects were largely diminished when cells were treated with melatonin (supplemental Figure IVA). Overexpression of the full-length as well as the LBD-truncated ROR1 (NDhin) abolished the binding of HIF-1 to VHL (supplemental Figure VIB). In addition, Gal4–HIF-1 enhanced Gal4–tk-Luc activity in the presence of CoCl2 or melatonin, indicating that melatonin could directly enhance the transactivation function of HIF-1 (supplemental Figure VIC). Association of HIF-1 with coactivator CBP was increased, whereas that with FIH-1—shown to repress transcriptional activity of HIF-133—was completely abolished in the presence of melatonin, further supporting this notion (supplemental Figure VID).
Putative ROR Ligands Induce Capillary Tube Formation by Human Umbilical Vein Endothelial Cells
In addition to melatonin, cholesterol sulfate, 22(R)-hydroxycholesterol, and 7-dehydrocholesterol are known to modulate the transcriptional activity of ROR.6,7 These putative ligands of ROR also induced the expression of ROR, HIF-1, and VEGF (Figure 4A) and transcriptional activity of HRE in HepG2 (Figure 4B). Finally, we tested whether ROR and melatonin affected the capability of human umbilical vein endothelial cells (HUVECs) to form capillary tubes, a key phenotype of angiogenesis induced by VEGF. When the adenovirus encoding ROR4 was infected in HUVECs, expression of HIF-1 and VEGF was increased (Figure 5A). After the ROR4 virus infection, an extensive network was formed (Figure 5B and 5C). When HUVECs were treated with melatonin, induction of protein-level of ROR4, HIF-1, and VEGF was observed. This induction was largely decreased by knockdown of ROR by RNA interference (Figure 5D). Similar results were obtained when cells were incubated under hypoxic conditions (supplemental Figure VII). Treatment with melatonin and cholesterol sulfate also enhanced formation of capillary tubes, which was comparable to the increase by hypoxia (Figure 5D). The number of tubes was dramatically decreased when ROR expression was repressed by transfection of si-ROR (Figure 5E). These results demonstrate that ROR and its ligands enhanced transcriptional activity of HIF-1, leading to the production of VEGF and to tube formation by endothelial cells.
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Discussion |
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Cellular adaptation to changes in oxygen tension is an important process for a wide range of events, including normal embryonic development and the pathophysiology of ischemic vascular disorders. When cellular oxygen availability decreases, HIF-1 plays a central role in cellular adaptation to hypoxic conditions.19,20 Therefore, great attention has been paid to the factors that control the activity of HIF-1 with the aim of developing new therapies for human ailments such as cardiovascular disease and cancer. Here, we report that ROR
and its ligands increase the transcriptional activity of HIF-1 and the expression of VEGF. This finding may provide new insight into the clinical applications of the ROR ligands in targeting various human vascular diseases.
The novel function of ROR could be an important contribution to a strong positive circuit for the activation of HIF-1 under hypoxic conditions. Expression of ROR was dramatically enhanced under hypoxia (supplemental Figure I). This induction of ROR may be attributable to presence of a putative HRE and a cluster of GC-rich sequences in the ROR promoter that was identified earlier by Chauvet et al.31 ROR in turn enhanced transcriptional activity of HIF-1 by enhancing protein stability as well as transactivation function of HIF-1 (Figure 1 and supplemental Figure VI). These data suggest that ROR may induce multiple posttranslation modifications on HIF-1 that affect stability and transactivation function. Molecular details such as the effects of ROR on prolyl hydroxylation or acetylation on the ODD and C-terminal activation domains of HIF-1, as well as mechanisms of ROR-induced regulation of MDM2 and FIH-1, may help us to understand the ROR-mediated positive regulatory circuit of hypoxia signaling.
ROR was initially described as an orphan nuclear receptor and has long been considered a constitutive activator of transcription in its exogenous ligands. Later, ligands such as melatonin and certain synthetic thiazolidinediones were shown to bind and transactivate ROR, although there were controversies.5 Recently, Kallen et al (2002) determined the crystal structure of the ligand-binding domain of ROR and revealed the presence of cholesterol in the ligand-binding pocket. Further experiments on purified ROR ligand-binding domain have shown that cholesterol sulfate and 7-hydroxycholesterol are the most active forms of cholesterol derivatives for ligand binding.7 Here we demonstrated that both cholesterol sulfate and melatonin induced transcriptional function of ROR as well as HIF-1 (Figures 2 and 4
). RNA interference study demonstrated that these effects were largely dependent on the presence of ROR (Figure 2), suggesting that melatonin is a real natural activator of ROR. Further studies on how melatonin works as ROR activator and the potential involvement of other factors in melatonin-induced hypoxia signaling, such as melatonin receptors in cytomembrane and antioxidative properties, are required in future.
Vascular remodeling represents alterations in growth of both vascular endothelial cells and SMCs, which are critical in the pathological processes of vascular diseases including atherosclerosis. A role for ROR in vascular remodeling has been implicated, in that it was detected in human SMCs, ECs, and mammary arteries.16,18 Further ROR expression is significantly decreased in human atherosclerotic plaques.18 Here we showed that ROR and its ligands enhanced HIF-1 activity and capillary tube formation capability of HUVECs (Figure 5), suggesting a potential involvement of ROR in the major symptoms of atherosclerosis and therapy against this diseases. However, increased postischemic angiogenesis was observed in the hindlimbs of homozygous staggerer mice, which may suggest ROR as a negative regulator of ischemia-induced angiogenesis.17 One of the potential explanations of this discrepancy could be differential roles of functional domains of ROR in new vessel formation. We observed here that DBD of ROR was sufficient to induce protein stability as well as transcriptional activation of HIF-1 (supplemental Figure IV). Staggerer mice have the sg/sg genotype, which results in truncated ROR protein in its C-terminal part, which is similar to NDhin in our investigation (Figure 3C).13,35 Therefore, the ROR in the staggerer mice may not be defective in the ROR-mediated hypoxia signaling. Further approaches employing genetically controlled ROR-deficient mice or transgenic mice that overexpress specific functional domains of ROR, may be useful to better understand the molecular actions of ROR in vascular biology. In conclusion, our findings that small compounds such as melatonin and cholesterol activate HIF-1 through ROR suggest that ROR could thus constitute the missing molecular link and be an important novel drug target.
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Acknowledgments |
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Sources of Funding
This study was supported by grants from the Korea Science and Engineering Foundation (2006-02634), the SRC/ERC program of MOST/KOSEF (R11-2007-107-01001-0), and the Ministry of Education as The Brain Korea 21 Project.
Disclosures
None.
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Footnotes |
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Original received February 25, 2008; final version accepted July 2, 2008.
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