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Cell Biology/Signaling

Transcriptional Activation of HIF-1 by RORα and its Role in Hypoxia Signaling

Eun-Jin Kim, Young-Gun Yoo, Woo-Kyeom Yang, Young-Soun Lim, Tae-Young Na, In-Kyu Lee, Mi-Ock Lee
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https://doi.org/10.1161/ATVBAHA.108.171546
Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:1796-1802
Originally published September 17, 2008
Eun-Jin Kim
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Young-Gun Yoo
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Woo-Kyeom Yang
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Young-Soun Lim
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Tae-Young Na
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In-Kyu Lee
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Mi-Ock Lee
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Abstract

Objective— Hypoxia-inducible factor 1α (HIF-1α) 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 α (RORα) have been implicated in cardiovascular diseases such as atherosclerosis. In this study, we have investigated the potential roles of RORα in the hypoxia signaling pathway in connection with activation of HIF-1α.

Methods and Results— Under hypoxic conditions, expression of RORα was induced. When RORα was introduced exogenously, protein level as well as transcriptional activity of HIF-1α was enhanced. Putative ligands of RORα, such as melatonin and cholesterol sulfate, induced transcriptional activity for HIF-1α, which was abolished by RNA interference against RORα. RORα was physically associated with HIF-1α through DNA binding domain, which was required to the RORα-induced stabilization and transcriptional activation of HIF-1α. Finally, either infection with adenovirus encoding RORα 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 RORα in amplification of hypoxia signaling and suggest a potential application of RORα ligands for the therapy of hypoxia-associated vascular diseases.

  • RORα
  • hypoxia
  • HIF-1α
  • melatonin
  • vascular endothelial growth factor

Retinoic acid receptor-related orphan receptor α (RORα; 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 RORα exists in 4 isoforms, RORα1, RORα2, RORα3, and RORα4 (also known as RZRα), 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 RORα, although these observations need to be clarified.4,5 Recently, analysis of the crystal structure of the ligand-binding domain of RORα 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 RORα.6,7 RORα 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, PPARγ, and Rev-Erbα, 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.

RORα 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 RORα 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, RORα mRNAs have been detected in human smooth muscle cells (SMCs), endothelial cells (ECs), as well as mammary arteries.16,18 RORα expression level is significantly decreased in human atherosclerotic plaques, whereas increased expression is observed after treatment with interleukin (IL) 1β, tumor necrosis factor (TNF) α, and lipopolysaccharide (LPS) in both ECs and human aortic SMCs.16,18 It thus appears that RORα 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 α 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-1α 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-1α at Pro564 of HIF-1α in the presence of oxygen, leading to the proteolysis of HIF-1α.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-1α.25–27 In addition to hypoxia, the stability and function of HIF-1α 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-1α.28,29

Recently, it was reported that RORα 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 TNFα.18,30,31 The potential implication of RORα in the hypoxia signaling pathway prompted us to study its role in transcriptional activation of HIF-1α. Here we report that RORα and its ligands activate HIF-1α, which leads to activation of a positive circuit for hypoxia signaling.

Methods

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 RORα, HIF-1α, VEGF, MDM2, VHL, FIH-1, CBP (Santa Cruz Biotechnology), FLAG (Sigma), or α-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-RORα1and pSG5-RORα4, 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-1α (GFP-HIF-1α), p3XFLAG™7.1-FLAG-HIF-1α, VP16-HIF-1α, and glutathione S-transferase (GST)-fused HIF-1α (pEBG-HIF-1α) and truncated constructs were described previously.27,29 The Gal4 DBD-fused RORα1, FLAG-tagged full-length and truncated RORα1 constructs, HA-tagged RORα4, and antisense (AS)-RORα1 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 RORα (si-RORα(1), 5′-CGCUGCCAACACUGUCGAUU ATT-3′ and 5′-UAAUCGACAGUGUUGGCAGUGTT-3′; si-RORα(2), 5′-GCACAGAAUAUAUCUAAAUTT-3′ and 5′-AUUUAGAUAUAUUCUGUGCTT-3′), HIF-1α (si-HIF-1α(1)27; si-HIF-1α(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-RORα(2) and si-HIF-1α(2) showed the same effects of si-RORα(1) and si-HIF-1α(1).

Adenovirus Production and Viral Infection

The RORα4 recombinant adenovirus vector was constructed by inserting full-length RORα4 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 (1×106 cells per dish) and incubated overnight. Cells were infected with adenovirus expressing either GFP or RORα at a density of 1.3×106 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).

Results

RORα Increases the Transcriptional Activity of HIF-1

To study the role of RORα in regulation of hypoxic response, first, we examined expression pattern of RORα under hypoxia. As previously reported, expression levels of RORα in HepG2 were increased under hypoxic conditions.31,32 RORα1 and RORα4 proteins were induced dramatically; however, neither expression nor induction of RORα2 and RORα3 was detected (supplemental Figure IA, available online at http://atvb.ahajournals.org). Transcripts of RORα and VEGF were increased in a similar pattern, whereas the amount of HIF-1α mRNA was unchanged (supplemental Figure IB). A reporter encoding two copies of the RORE in the promoter of Iκ -Bα, was activated under hypoxia or in the presence of CoCl2 or desferroxamin, which are hypoxia-mimicking chemicals (supplemental Figure IC). These results demonstrate that RORα is induced under hypoxia at the level of transcription.

Therefore we asked whether this RORα induction had a functional importance in hypoxia signaling pathway. We tested the effect of RORα 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 RORα1 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-1α and VEGF proteins increased when RORα1 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 RORα by transfection with an antisense (AS)-RORα construct strongly suppressed hypoxia-induced HRE reporter activity (Figure 1C). Similarly, knockdown of RORα using silencing (si) RNA largely decreased the hypoxia-induced expression levels of HIF-1α and VEGF (Figure 1D). Both RORα1 and RORα4 showed the similar potency in activating HRE reporter activity and in increasing expression of HIF-1α and VEGF (supplemental Figure II). Together, these results indicate that RORα enhances the transcriptional activity of HIF-1 in the absence of hypoxic stress, and it may mediate hypoxia-induced HIF-1α activation.

Figure1
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Figure 1. RORα enhances transcriptional activity as well as protein level of HIF-1α. A, HepG2 cells were transfected with HRE-tk-Luc reporter and the expression vector for RORα1 or treated with CoCl2. B, HeLa cells were transfected with p3XFLAG 7.1-RORα1, or empty vector (EV). After transfection, cells were treated with or without CoCl2. Arrowhead indicates nonspecific bands. C, HepG2 cells were transfected with HRE-tk-Luc reporter and pcDNA3.0-AS-RORα. Transfected cells were treated with CoCl2. D, HepG2 cells were transfected with nonspecific siRNA (NS) or si-RORα,1 and then exposed to hypoxia or normoxia.

Melatonin Increases Transcriptional Activities of HIF-1 Through Activation of RORα

Because melatonin increases transcriptional activity of RORα,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 RORα may contribute to the induction of HIF-1α and VEGF (Figure 2A). Melatonin increased the expression levels of RORα, HIF-1α, and VEGF proteins as early as 10 minutes and continued up to 24 hours (Figure 2B and supplemental Figure III). When expression of either RORα or HIF-1α 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 RORα, which leads to transcriptional activation of HIF-1α and consequent induction of VEGF.

Figure2
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Figure 2. Melatonin increases transcriptional activity of HIF-1 through activation of RORα. A, HepG2 cells were transfected with RORE-tk-Luc, HRE-tk-luc, or VEGF promoter-Luc. After transfection, cells were treated with increasing concentrations of melatonin (Mel); 1, 5, 10, 25, 50, 75, and 100 μmol/L (left) or 10 and 50 μmol/L (center and right). CoCl2 or DFO was treated as positive controls. B, HepG2 cells were treated with 50 μmol/L Mel for the indicated time period. C, HepG2 cells were transfected with nonspecific siRNA (NS), si-RORα(1), or si-HIF-1α(1). After transfection, cells were treated with 100 μmol/L Mel.

RORα Interacts With HIF-1α and Increases the Stability of HIF-1α

To further characterize the cross-talk between RORα and HIF-1α, we examined whether these proteins were physically associated. RORα and HIF-1α were localized in the nucleus under hypoxic conditions (Figure 3A). Physical association of RORα with HIF-1α 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 RORα1 and the GST-fused HIF-1α constructs. The coimmunoprecipitation results showed that DBD of RORα served for binding site (Figure 3C). Inhibitory domain (ID), neither N terminus (N), ODD, nor CTAD of HIF-1α bound to RORα1 (supplemental Figure IVB and data not shown). The importance of this binding was demonstrated in that DBD of RORα was sufficiently active for the increases of protein-level as well as transcriptional activity of HIF-1α (supplemental Figure IVC and IVD). These results may suggest that protein–protein interaction of RORα with HIF-1α is required to the cross-talk of these molecules in hypoxia signaling.

Figure3
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Figure 3. RORα interacts with HIF-1α A, After NIH3T3 cells were exposed to hypoxia, cytoplasmic and nuclear extracts were obtained. The indicated proteins were analyzed by Western blot analysis. B, Whole cell lysates obtained form the NIH3T3 cells exposed to hypoxia were immunoprecipitated (IP) and probed with the indicated antibodies. C, Schematic representation of full-length and truncated RORα 1 constructs (upper). NIH3T3 cells were transfected with the indicated combinations of FLAG-tagged RORα 1 and GST-HIF-1α. Whole lysates were immunoprecipitated (IP) with anti-FLAG antibodies and probed with anti-GST antibodies. Arrow head indicates nonspecific band (lower).

To study the molecular mechanisms of RORα-induced activation of HIF-1, we tested whether RORα could enhance stability of HIF-1α. When HIF-1α protein stability was measured in the presence of cycloheximide, a blocker of de novo protein synthesis, overexpression of RORα1 or treatment of melatonin blocked degradation of HIF-1α, and this was as efficient as CoCl2 treatment (supplemental Figure VA). Also the protein stability of HIF-1α was examined by immunofluorescence study using green fluorescent protein (GFP) fused with HIF-1α. GFP–HIF-1α 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-1α protein through inhibiting ubiquitin-mediated proteasomal degradation pathways, because the binding of HIF-1α 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 RORα1 (NDhin) abolished the binding of HIF-1α to VHL (supplemental Figure VIB). In addition, Gal4–HIF-1α enhanced Gal4–tk-Luc activity in the presence of CoCl2 or melatonin, indicating that melatonin could directly enhance the transactivation function of HIF-1α (supplemental Figure VIC). Association of HIF-1α with coactivator CBP was increased, whereas that with FIH-1—shown to repress transcriptional activity of HIF-1α33—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 RORα.6,7 These putative ligands of RORα also induced the expression of RORα, HIF-1α, and VEGF (Figure 4A) and transcriptional activity of HRE in HepG2 (Figure 4B). Finally, we tested whether RORα 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 RORα4 was infected in HUVECs, expression of HIF-1α and VEGF was increased (Figure 5A). After the RORα4 virus infection, an extensive network was formed (Figure 5B and 5C). When HUVECs were treated with melatonin, induction of protein-level of RORα4, HIF-1α, and VEGF was observed. This induction was largely decreased by knockdown of RORα 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 RORα expression was repressed by transfection of si-RORα (Figure 5E). These results demonstrate that RORα and its ligands enhanced transcriptional activity of HIF-1α, leading to the production of VEGF and to tube formation by endothelial cells.

Figure4
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Figure 4. Potential ligands of RORα enhance expression as well as transcriptional activity of HIF-1. A, HepG2 cells were treated with 100 μmol/L melatonin (Mel), 10 μmol/L 22(R)-hydroxy-cholesterol (22(R)HCh), 10 μmol/L cholesterol sulfate (Ch-Sulfate), or 10 μmol/L 7-dehydrocholesterol (7-DHC). The expression of genes was analyzed by Western blot (upper) or RT-PCR analysis (lower). B, HepG2 cells were transfected with HRE-tk-luc, and the transfected cells were incubated in the presence of the indicated concentrations of Mel, Ch-Sulfate, or 7-DHC.

Figure5
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Figure 5. Melatonin increases protein level of HIF-1α as well as tube formation in human vascular endothelial cells. A, HUVECs were infected by the control adenovirus (Ad-GFP) or the adenovirus encoding RORα4 (Ad-RORα4). The expression of protein was analyzed by Western blot analysis. B, HUVECs were infected by Ad-GFP or Ad-RORα4, and then capillary-like tube formation was observed under fluorescent microscope. Data shown are the mean±SD of 3 independent experiments. C, Representative figures of capillary-like tube formation by HUVECs after adenovirus infection. D, HUVECs were transfected with nonspecific siRNA (NS) or si-RORα(1), and then cells were treated with melatonin (Mel). The expression of protein was analyzed by Western blot analysis. E, HUVECs were transfected with nonspecific siRNA (NS) or si-RORα(1), and then cells were treated with Mel. Capillary-like tube formation was observed under fluorescent microscope. Data shown are the mean±SD of 3 independent experiments.

Discussion

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 RORα and its ligands increase the transcriptional activity of HIF-1α and the expression of VEGF. This finding may provide new insight into the clinical applications of the RORα ligands in targeting various human vascular diseases.

The novel function of RORα could be an important contribution to a strong positive circuit for the activation of HIF-1α under hypoxic conditions. Expression of RORα was dramatically enhanced under hypoxia (supplemental Figure I). This induction of RORα may be attributable to presence of a putative HRE and a cluster of GC-rich sequences in the RORα promoter that was identified earlier by Chauvet et al.31 RORα in turn enhanced transcriptional activity of HIF-1α by enhancing protein stability as well as transactivation function of HIF-1α (Figure 1 and supplemental Figure VI). These data suggest that RORα may induce multiple posttranslation modifications on HIF-1α that affect stability and transactivation function. Molecular details such as the effects of RORα on prolyl hydroxylation or acetylation on the ODD and C-terminal activation domains of HIF-1α, as well as mechanisms of RORα-induced regulation of MDM2 and FIH-1, may help us to understand the RORα-mediated positive regulatory circuit of hypoxia signaling.

RORα 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 RORα, although there were controversies.5 Recently, Kallen et al (2002) determined the crystal structure of the ligand-binding domain of RORα and revealed the presence of cholesterol in the ligand-binding pocket. Further experiments on purified RORα 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 RORα as well as HIF-1α (Figures 2 and 4⇑). RNA interference study demonstrated that these effects were largely dependent on the presence of RORα (Figure 2), suggesting that melatonin is a real natural activator of RORα. Further studies on how melatonin works as RORα 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 RORα in vascular remodeling has been implicated, in that it was detected in human SMCs, ECs, and mammary arteries.16,18 Further RORα expression is significantly decreased in human atherosclerotic plaques.18 Here we showed that RORα and its ligands enhanced HIF-1 activity and capillary tube formation capability of HUVECs (Figure 5), suggesting a potential involvement of RORα 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 RORα 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 RORα in new vessel formation. We observed here that DBD of RORα 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 RORα protein in its C-terminal part, which is similar to NDhin in our investigation (Figure 3C).13,35 Therefore, the RORα in the staggerer mice may not be defective in the RORα-mediated hypoxia signaling. Further approaches employing genetically controlled RORα-deficient mice or transgenic mice that overexpress specific functional domains of RORα, may be useful to better understand the molecular actions of RORα in vascular biology. In conclusion, our findings that small compounds such as melatonin and cholesterol activate HIF-1 through RORα suggest that RORα could thus constitute the missing molecular link and be an important novel drug target.

Acknowledgments

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.

Footnotes

  • Original received February 25, 2008; final version accepted July 2, 2008.

References

  1. ↵
    Jetten AM, Kurebayashi S, Ueda E. The ROR nuclear orphan receptor subfamily: critical regulators of multiple biological processes. Prog Nucleic Acid Res Mol Biol. 2001; 69: 205–247.
    OpenUrlCrossRefPubMed
  2. ↵
    Jetten AM. Recent advances in the mechanisms of action and physiological functions of the retinoid-related orphan receptors (RORs). Curr Drug Targets Inflamm Allergy. 2004; 3: 395–412.
    OpenUrlCrossRefPubMed
  3. ↵
    Giguère V, Tini M, Flock G, Ong E, Evans RM, Otulakowski G. Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR alpha, a novel family of orphan hormone nuclear receptors. Genes Dev. 1994; 8: 538–553.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Missbach M, Jagher B, Sigg I, Nayeri S, Carlberg C, Wiesenberg I. Thiazolidine diones, specific ligands of the nuclear receptor retinoid Z receptor/retinoid acid receptor-related orphan receptor alpha with potent antiarthritic activity. J Biol Chem. 1996; 271: 13515–13522.
    OpenUrlCrossRefPubMed
  5. ↵
    Wiesenberg I, Missbach M, Kahlen JP, Schrader M, Carlberg C. Transcriptional activation of the nuclear receptor RZR alpha by the pineal gland hormone melatonin and identification of CGP 52608 as a synthetic ligand. Nucleic Acids Res. 1995; 23: 327–333.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Kallen JA, Schlaeppi JM, Bitsch F, Geisse S, Geiser M, Delhon I, Fournier B. X-ray structure of the hRORalpha LBD at 1.63 A: structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of RORalpha. Structure. 2002; 10: 1697–1707.
    OpenUrlCrossRefPubMed
  7. ↵
    Kallen JA, Schlaeppi JM, Bitsch F, Delhon I, Fournier B. Crystal structure of the human RORalpha Ligand binding domain in complex with cholesterol sulfate at 2.2 A. J Biol Chem. 2004; 279: 14033–14038.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Vu-Dac N, Gervois P, Grotzinger T, De Vos P, Schoonjans K, Fruchart JC, Auwerx J, Mariani J, Tedgui A, Staels B. Transcriptional regulation of apolipoprotein A-I gene expression by the nuclear receptor RORalpha. J Biol Chem. 1997; 272: 22401–22404.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Sundvold H, Lien S. Identification of a novel peroxisome proliferator-activated receptor (PPAR) gamma promoter in man and transactivation by the nuclear receptor RORalpha1. Biochem Biophys Res Commun. 2001; 287: 383–390.
    OpenUrlCrossRefPubMed
  10. ↵
    Raspè E, Mautino G, Duval C, Fontaine C, Duez H, Barbier O, Monte D, Fruchart J, Fruchart JC, Staels B. Transcriptional regulation of human Rev-erbalpha gene expression by the orphan nuclear receptor retinoic acid-related orphan receptor alpha. J Biol Chem. 2002; 277: 49275–49281.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Chauvet C, Bois-Joyeux B, Fontaine C, Gervois P, Bernard MA, Staels B, Danan JL. The gene encoding fibrinogen-beta is a target for retinoic acid receptor-related orphan receptor alpha. Mol Endocrinol. 2005; 19: 2517–2526.
    OpenUrlCrossRefPubMed
  12. ↵
    Lind U, Nilsson T, McPheat J, Stromstedt PE, Bamberg K, Balendran C, Kang D. Identification of the human ApoAV gene as a novel RORalpha target gene. Biochem Biophys Res Commun. 2005; 330: 233–241.
    OpenUrlCrossRefPubMed
  13. ↵
    Hamilton BA, Frankel WN, Kerrebrock AW, Hawkins TL, FitzHugh W, Kusumi K, Russell LB, Mueller KL, van Berkel V, Birren BW, Kruglyak L, Lander ES. Disruption of the nuclear hormone receptor RORalpha in staggerer mice. Nature. 1996; 379: 736–739.
    OpenUrlCrossRefPubMed
  14. ↵
    Herrup K, Mullen RJ. Regional variation and absence of large neurons in the cerebellum of the staggerer mouse. Brain Res. 1979; 172: 1–12.
    OpenUrlCrossRefPubMed
  15. ↵
    Mamontova A, Seguret-Mace S, Esposito B, Chaniale C, Bouly M, Delhaye-Bouchaud N, Luc G, Staels B, Duverger N, Mariani J, Tedgui A. Severe atherosclerosis and hypoalphalipoproteinemia in the staggerer mouse, a mutant of the nuclear receptor RORalpha. Circulation. 1998; 98: 2738–2743.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Delerive P, Monte D, Dubois G, Trottein F, Fruchart-Najib J, Mariani J, Fruchart JC, Staels B. The orphan nuclear receptor ROR alpha is a negative regulator of the inflammatory response. EMBO Rep. 2001; 2: 42–48.
    OpenUrlAbstract
  17. ↵
    Besnard S, Silvestre JS, Duriez M, Bakouche J, Lemaigre-Dubreuil Y, Mariani J, Levy BI, Tedgui A. Increased ischemia-induced angiogenesis in the staggerer mouse, a mutant of the nuclear receptor Roralpha. Circ Res. 2001; 89: 1209–1215.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Besnard S, Heymes C, Merval R, Rodriguez M, Galizzi JP, Boutin JA, Mariani J, Tedgui A. Expression and regulation of the nuclear receptor RORalpha in human vascular cells. FEBS Lett. 2002; 511: 36–40.
    OpenUrlCrossRefPubMed
  19. ↵
    Hirota K, Semenza GL. Regulation of angiogenesis by hypoxia-inducible factor 1. Crit Rev Oncol Hemato. 2006; 59: 15–26.
    OpenUrlCrossRef
  20. ↵
    Ke Q, Costa M. Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol. 2006; 70: 1469–1480.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Huang LE, Gu J, Schau M, Bunn HF. Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci U S A. 1998; 95: 7987–7992.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Kallio PJ, Wilson WJ, O'Brien S, Makino Y, Poellinger L. Regulation of the hypoxia-inducible transcription factor 1alpha by the ubiquitin-proteasome pathway. J Biol Chem. 1999; 274: 6519–6525.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Kamura T, Sato S, Iwai K, Czyzyk-Krzeska M, Conaway RC, Conaway JW. Activation of HIF1alpha ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex. Proc Natl Acad Sci U S A. 2000; 97: 10430–10435.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Tanimoto K, Makino Y, Pereira T, Poellinger L. Mechanism of regulation of the hypoxia-inducible factor-1 alpha by the von Hippel-Lindau tumor suppressor protein. EMBO J. 2000; 19: 4298–4309.
    OpenUrlAbstract
  25. ↵
    Jeong JW, Bae MK, Ahn MY, Kim SH, Sohn TK, Bae MH, Yoo MA, Song EJ, Lee KJ, Kim KW. Regulation and destabilization of HIF-1alpha by ARD1-mediated acetylation. Cell. 2002; 111: 709–720.
    OpenUrlCrossRefPubMed
  26. ↵
    Brahimi-Horn C, Mazure N, Pouysségur J. Signalling via the hypoxia-inducible factor-1alpha requires multiple posttranslational modifications. Cell Signal. 2005; 17: 1–9.
    OpenUrlCrossRefPubMed
  27. ↵
    Yoo YG, Kong G, Lee MO. Metastasis-associated protein 1 enhances stability of hypoxia-inducible factor-1alpha protein by recruiting histone deacetylase 1. EMBO J. 2006; 25: 1231–1241.
    OpenUrlAbstract
  28. ↵
    Yoo YG, Yeo MG, Kim DK, Park H, Lee MO. Novel function of orphan nuclear receptor Nur77 in stabilizing hypoxia-inducible factor-1alpha. J Biol Chem. 2004; 279: 53365–53373.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Yoo YG, Na TY, Yang WK, Kim HJ, Lee IK, Kong G, Chung JH, Lee MO. 6-Mercaptopurine, an activator of Nur77, enhances transcriptional activity of HIF-1alpha resulting in new vessel formation. Oncogene. 2007; 26: 3823–3834.
    OpenUrlCrossRefPubMed
  30. ↵
    Miki N, Ikuta M, Matsui T. Hypoxia-induced activation of the retinoic acid receptor-related orphan receptor alpha4 gene by an interaction between hypoxia-inducible factor-1 and Sp1. J Biol Chem. 2004; 279: 15025–15031.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Chauvet C, Bois-Joyeux B, Berra E, Pouyssegur J, Danan JL. The gene encoding human retinoic acid-receptor-related orphan receptor alpha is a target for hypoxia-inducible factor 1. Biochem J. 2004; 384: 79–85.
    OpenUrlCrossRefPubMed
  32. ↵
    Chauvet C, Bois-Joyeux B, Danan JL. Retinoic acid receptor-related orphan receptor (ROR) alpha4 is the predominant isoform of the nuclear receptor RORalpha in the liver and is up-regulated by hypoxia in HepG2 human hepatoma cells. Biochem J. 2002; 364: 449–456.
    OpenUrlCrossRefPubMed
  33. ↵
    Mahon PC, Hirota K, Semenza GL. FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev. 2001; 15: 2675–2686.
    OpenUrlAbstract/FREE Full Text
  34. Roe JS, Youn HD. The positive regulation of p53 by the tumor suppressor VHL. Cell Cycle. 2006; 5: 2054–2056.
    OpenUrlPubMed
  35. ↵
    Matysiak-Scholze U, Nehls M. The structural integrity of ROR alpha isoforms is mutated in staggerer mice: cerebellar coexpression of ROR alpha1 and ROR alpha4. Genomics. 1997; 43: 78–84.
    OpenUrlPubMed
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    Transcriptional Activation of HIF-1 by RORα and its Role in Hypoxia Signaling
    Eun-Jin Kim, Young-Gun Yoo, Woo-Kyeom Yang, Young-Soun Lim, Tae-Young Na, In-Kyu Lee and Mi-Ock Lee
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:1796-1802, originally published September 17, 2008
    https://doi.org/10.1161/ATVBAHA.108.171546

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    Eun-Jin Kim, Young-Gun Yoo, Woo-Kyeom Yang, Young-Soun Lim, Tae-Young Na, In-Kyu Lee and Mi-Ock Lee
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:1796-1802, originally published September 17, 2008
    https://doi.org/10.1161/ATVBAHA.108.171546
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