LXRβ/estrogen receptor-α signaling in lipid rafts preserves endothelial integrity

Liver X receptors (LXR) are stimulated by cholesterol-derived oxysterols and serve as transcription factors to regulate gene expression in response to alterations in cholesterol. In the present study, we investigated the role of LXRs in vascular endothelial cells (ECs) and discovered that LXRβ has nonnuclear function and stimulates EC migration by activating endothelial NOS (eNOS). This process is mediated by estrogen receptor-α (ERα). LXR activation promoted the direct binding of LXRβ to the ligand-binding domain of ERα and initiated an extranuclear signaling cascade that requires ERα Ser118 phosphorylation by PI3K/AKT. Further studies revealed that LXRβ and ERα are colocalized and functionally coupled in EC plasma membrane caveolae/lipid rafts. In isolated aortic rings, LXR activation of NOS caused relaxation, while in mice, LXR activation stimulated carotid artery reendothelialization via LXRβ- and ERα-dependent processes. These studies demonstrate that LXRβ has nonnuclear function in EC caveolae/lipid rafts that entails crosstalk with ERα, which promotes NO production and maintains endothelial monolayer integrity in vivo.

Liver X receptors (LXR) are members of the nuclear receptor superfamily that function as ligand-dependent transcription factors and play important roles in lipid metabolism (1–3). The 2 LXR isoforms, LXRα and LXRβ, share great similarity in protein structure and target genes, but they differ in tissue distribution. LXRα is primarily expressed in liver, kidney, intestine, adipose tissue, and macrophages, whereas LXRβ is ubiquitously expressed (4). Cholesterol-derived oxysterols such as 22(R)-hydroxycholesterol (22RHC) are endogenous ligands of LXR (5), and there are also synthetic LXR ligands that attenuate atherosclerosis development when administered to hypercholesterolemic mice (2, 6–8). The marked reductions in atherosclerosis in response to LXR agonists occur despite modest changes in plasma lipoprotein levels, suggesting that the underlying mechanism or mechanisms may involve direct effects on hematologic or vascular cell types of importance to atherogenesis. In macrophages, LXR upregulates a number of genes that promote reverse cholesterol transport to the liver (1, 9), and they inhibit macrophage inflammatory responses (3, 10). In vascular smooth muscle cells (VSMC), which also participate in atherogenesis and express LXR, LXR agonists attenuate mitogen-induced cell proliferation by inhibiting the expression of cell-cycle–regulating proteins (11). Whereas there are recognized functions of LXR in macrophages and VSMC, the potential roles of LXR in vascular endothelial cells (ECs), which are another cell type critically involved in atherogenesis (12), are unknown.

Similar to LXR, estrogen receptor-α and estrogen receptor-β (ERα and ERβ) are nuclear receptors that influence cardiovascular health and disease (13, 14). Estrogen treatment blunts atherosclerosis in mice, and the protection afforded by estrogen is not explained by changes in plasma lipoprotein levels (15, 16). Estrogen actions primarily via ERα promote reendothelialization following vascular injury, prevent neointima formation, and activate antiinflammatory responses (13). Along with their classical roles as transcriptional factors, both ERα and ERβ have nonnuclear function. These include the activation of kinases such as Akt in ECs, resulting in the stimulation of endothelial NOS (eNOS), increased production of the atheroprotective-signaling molecule NO, and the promotion of EC growth and migration (17–19). Nonnuclear actions of ERα and ERβ in ECs are mediated by receptor subpopulations that are associated with plasma membrane (PM) caveolae/lipid rafts (20, 21). There is recognized physiologic interplay between the LXR and ER ligand/receptor systems. LXR activation influences the synthesis and metabolism of estrogen (22, 23), estrogen causes attenuated expression of LXRα and its target genes in white adipose tissue (24), and estrogen also antagonizes LXR transcriptional activity in breast cancer cells (25). However, direct functional partnership between LXR and ER has not been previously observed in any biological context. In the current study, designed to elucidate the role of LXR in ECs, we discovered novel nonnuclear function of LXRβ that entails unique direct protein-protein interaction between LXRβ and ERα, and we show that these processes have important consequences on vascular NO production and the maintenance of endothelial monolayer integrity in vivo.

LXRβ activation promotes EC migration via ERα and eNOS. To directly examine the role of LXR in EC, we evaluated cell migration in a scratch assay using the human EC line EA.hy926. A defined region of confluent ECs was removed with a cell scraper, cells were incubated under varying conditions for 20 hours, and the number of cells migrating past the wound edge was quantified. 17β-Estradiol (E₂, 10 nM) increased EC migration as previously reported (Figure 1A and refs. 19, 26), and the synthetic LXR agonists T0901317 (T1317) and GW3965 (GW) also promoted EC migration to degrees similar to those observed with 5% FBS. Evaluations of transcript abundance revealed that among the 2 known isoforms of LXR, LXRβ is the more abundant isoform in ECs (Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI66533DS1), and the abundance of ERα and LXRβ proteins was not changed under the experimental conditions tested (Supplemental Figure 1A). Studies performed in the presence versus absence of hydroxyurea, which blocks cell division (27), demonstrated that the responses to E₂ and LXR agonists are indicative of cell migration occurring independently of the stimulation of cell growth (Supplemental Figure 1B). Recognizing the important role of eNOS in EC migration induced by stimuli such as VEGF and E₂ (28), the involvement of NOS in the actions of LXR agonists was assessed by NOS inhibition. The responses to E₂, T1317, and GW were all fully prevented by the nonselective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) (Figure 1A). In contrast, neither iNOS- nor nNOS-specific inhibitors, 1400W and ARR17477, respectively, altered T1317-stimulated migration (Supplemental Figure 1C), indicating that the LXR-mediated processes are eNOS dependent. Primary ECs isolated from wild-type and LXR-null mice were then studied to determine whether the effect of the LXR agonist T1317 was LXR specific and to identify the operative receptor subtype(s). E₂ activated the migration of both Lxr^+/+ and Lxra/b^–/– ECs (Figure 1B). In contrast, whereas T1317 promoted the migration of EC from Lxr^+/+ mice, there was no migration stimulated by T1317 in ECs from Lxra/b^–/– mice. Furthermore, whereas the effect of T1317 on cell migration persisted in ECs from Lxra^–/– mice, it was lost in ECs from Lxrb^–/– mice, indicating that LXRβ is responsible for the EC migration stimulated by the LXR agonist. T1317 stimulated EC migration in a dose-dependent manner in the absence of E₂, and T1317 in the presence of a submaximal concentration of E₂ (10 nM) had neither an additive nor a synergistic effect (Figure 1C). Since the latter finding suggests that common mechanisms may mediate the responses to LXR and ER activation, potential functional linkage between the receptors was evaluated using the ER antagonist ICI 182,780 (ICI). ICI inhibited the EC migration stimulated by either E₂ or the LXR agonists T1317 and 22RHC (Figure 1D).

Figure 1

LXR activation promotes EC migration in a NOS-, LXRβ- and ER-dependent manner. EA.hy926 cells (A, C, and D) or isolated primary mouse EC (B) were grown to near-confluence, and a defined region was removed by a cell scraper. The cells were treated with vehicle control (Veh.), 10 nM E₂, 1 μM T1317, 1 μM GW, 10 μM 22RHC, or 5% FBS (as a positive control) in the absence or presence of 100 nM L-NAME (LN) (A) or 100 nM ICI (D) for 20 hours and stained with hematoxylin, and cells migrating past the wound edge (from left to right in A) were quantified. Original magnification, ×40. All graphs depict summary data (mean ± SEM, n = 3–4, *P < 0.05 vs. vehicle control; ^†P < 0.05 vs. –E₂). NT, not tested.

Next, we determined which ER isoform is responsible for the induction of migration by T1317 using the ERα- and ERβ-specific antagonists 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP) and 4-[2-phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), respectively. T1317-stimulated migration was prevented by the ERα-specific antagonist MPP, but not by the ERβ-specific antagonist PHTPP (Supplemental Figure 2A). Furthermore, whereas E₂ and T1317 stimulated migration in control ECs previously transfected with scrambled siRNA sequence (scRNA) (Supplemental Figure 2B), the response was lost in ECs in which ERα protein abundance was reduced by siRNA (Supplemental Figure 2B). These cumulative results indicate that LXRβ activation promotes EC migration via ERα and eNOS.

LXR have nonnuclear action and activate eNOS and Akt via ER. In previous studies, we demonstrated that nonnuclear ERα signaling underlies estrogen-induced EC migration (19). Having found that LXRβ-mediated EC migration occurs via ERα, the possibility was raised that LXR have nonnuclear action in ECs that influences cell motility. We tested this by assessing rapid changes in the actin cytoskeleton in response to LXR agonist in the absence or presence of actinomycin D using phalloidin staining. Under control conditions, ECs exhibited diffuse distribution of actin and few if any stress fibers (Figure 2A). T1317 treatment (10 minutes) caused stress fiber formation, and the response was not altered by actinomycin D. Since the EC migration promoted by LXR activation is eNOS-dependent (Figure 1A), we next determined whether LXR acutely stimulate eNOS activity using bovine aortic EC (BAEC), in which rapid eNOS activation is readily quantified by measuring ¹⁴C-l-arginine conversion to ¹⁴C-l-citrulline in intact cells during 15-minute incubations (19). Both E₂ and T1317 activated eNOS, and both responses were fully abolished by ICI (Figure 2B). eNOS activation by ERα is mediated by the phosphorylation of the serine residue at amino acid 1177 (Ser1177) of eNOS by Akt (18). E₂ and the LXR agonists T1317 and 22RHC caused increases in eNOS Ser1177 phosphorylation, and the promotion of phosphorylation by all agents was inhibited by ER antagonism with ICI (Figure 2C). E₂ and the LXR agonists also induced comparable phosphorylation of Akt at Ser473 (Figure 2D), the increase was inhibited by ICI, and in studies of T1317 activation of Akt, it was observed as early as 5 minutes after agonist exposure (Supplemental Figure 3).

Figure 2

LXRβ has nonnuclear action in EC and activates eNOS and Akt via ER. (A) EA.hy926 cells were treated with vehicle or 1 μM T1317 for 10 minutes, with or without prior and concurrent treatment with 5 μg/ml actinomycin D, and changes in the actin cytoskeleton were visualized using Alexa Fluor 488 phalloidin. Representative images from 3 separate experiments. Original magnification, ×200. (B) eNOS activation in BAEC was tested during 15-minute incubations of intact cells with either 10 nM E₂ or 1 μM T1317, with or without 100 nM ICI added. Values are mean ± SEM, n = 3–4; *P < 0.05 vs. vehicle control. (C and D) ECs were treated with 10 nM E₂, 1 μM T1317, or 10 μM 22RHC with or without 100 nM ICI for 20 minutes. Phosphorylated eNOS (Ser1177) and total eNOS (C), or phosphorylated Akt (Ser473) and total Akt (D) were then detected by immunoblot analysis, and the ratios of phospho-eNOS to total eNOS (C) and phospho-Akt to total Akt (D) were calculated (mean ± SEM, n = 3, *P < 0.05 vs. vehicle control). (E and F) scRNA (E) or siRNA against LXRβ (siLXRβ) (F) were introduced into BAEC, and 48 hours later, the cells were treated with 10 nM E₂, 1 μM T1317, or 1 μM GW for 20 minutes. The abundance of phospho-Akt relative to total Akt was determined by immunoblotting (mean ± SEM, n = 3, *P < 0.05 vs. vehicle control). In C–F, representative blots from 3 separate experiments are shown.

To then determine whether LXRβ is required for the effect of LXR agonists on Akt, siRNA against LXRβ was introduced into ECs. The siRNA targeting LXRβ markedly decreased LXRβ protein abundance, but not ERα expression in ECs (Supplemental Figure 4). Whereas E₂, T1317, and GW induced equal Akt phosphorylation in control cells (Figure 2E), the knockdown of LXRβ fully prevented Akt phosphorylation in response to T1317 and GW, but not in response to E₂ (Figure 2F). Taken together, these results indicate that LXRβ has nonnuclear action in EC, that LXRβ activates eNOS by stimulating its phosphorylation by Akt, and that these processes are entirely ER dependent. It is notable that, in contrast, nonnuclear ER signaling is not dependent on LXRβ.

LXRβ interacts directly with the ligand-binding domain of ERα. To investigate potential functional interaction between LXRβ and ERα of consequence to nonnuclear LXRβ signaling in ECs, we first performed coimmunoprecipitation experiments using EC whole-cell lysates and an anti-ERα antibody. LXRβ was coimmunoprecipitated with ERα, the amount of LXRβ coimmunoprecipitated with ERα was increased by EC treatment with T1317 or E₂, and the increase in interaction caused by T1317 was inhibited by ICI (Figure 3A). To determine whether the LXRβ-ERα interaction is a direct protein-protein interaction, coimmunoprecipitation assays were performed using purified, recombinant Flag-tagged ERα, recombinant LXRβ, and anti-LXRβ antibody. Full-length, wild-type Flag-ERα was pulled down with anti-LXRβ antibody (Figure 3B, construct 1), and in separate experiments the interaction between LXRβ and ERα was increased by the addition of either T1317 or E₂ (Supplemental Figure 5A). In addition, it was found that LXRβ is capable of interaction with ERβ, but modulation by ligand was not observed (Supplemental Figure 5B).

Figure 3

LXRβ and ERα interact directly via the ligand-binding domain of ERα. (A) EA.hy926 cells were treated with vehicle, 10 nM E₂, 1 μM T1317, or T1317 plus 100 nM ICI for 20 minutes, and cell lysates were prepared for immunoprecipitation with control IgG (cont. IgG) or anti-ERα antibody. Immunoprecipitated proteins were detected by immunoblotting with anti-LXRβ or anti-ERα antibodies, and the ratio of immunoprecipitated LXRβ to ERα proteins against vehicle control was calculated (mean ± SEM, n = 3, *P < 0.05 vs. vehicle control, ^†P < 0.05 vs. T1317 alone). For the ERα immunoblot, the lanes were run on the same gel but were noncontiguous. (B) In vitro coimmunoprecipitation assays were performed using recombinant wild-type Flag-ERα or deletion mutant proteins and LXRβ protein. Linear depiction of the ERα constructs tested (numbered 1–7) is provided in the upper panel. The proteins were incubated with 1 μM T1317 for 2 hours at 4°C, and immunoprecipitation was performed with control IgG or anti-LXRβ antibody. Immunoprecipitated proteins were separated by SDS-PAGE and detected by immunoblot analysis with anti-Flag or anti-LXRβ antibodies. Results shown in A and B were confirmed in at least 3 independent experiments.

To determine which domain of ERα is involved in the direct protein-protein interaction between LXRβ and ERα, recombinant Flag-tagged deletion mutants of ERα were used in the coimmunoprecipitation assay (Figure 3B). Deletion mutants lacking the N-terminal 175 residues of ERα (construct 2) or portions of the DNA-binding domain or C domain of ERα (construct 3 and 4) displayed direct interaction with LXRβ similar to wild-type, full-length receptor. Mutants lacking the N-terminal portion of the ligand-binding domain or E domain (construct 5 and 7) did not display interaction, whereas a mutant lacking amino acids 271–300 (construct 6) showed interaction. These cumulative observations indicate that LXRβ and ERα interact in ECs, that there is direct protein-protein interaction between the receptors that involves amino acids 300–330 within the ligand-binding domain of ERα, and that the interaction is dynamically regulated by ligand binding to either receptor.

LXRβ is functionally coupled to ERα in EC caveolae/lipid rafts. Because nonnuclear ERα functions in ECs are mediated by receptors localized to PM caveolae/lipid rafts (28), we next determined by subfractionation whether a subpopulation of LXRβ is colocalized with ERα in EC PM caveolae/lipid rafts. We employed our established caveolae membrane isolation method, which yields a fraction that is free of nuclear proteins (20, 29). Both ERα and LXRβ proteins were associated with the PM fraction and also with the caveolae membranes (Figure 4A), and as expected, both receptor proteins were abundant in the nuclear fraction. Mirroring the LXRβ-ERα interaction observed in EC whole-cell lysates (Figure 3A), their interaction was also demonstrated in nuclear and PM fractions by coimmunoprecipitation using anti-ERα antibody (Figure 4B). More importantly, in isolated caveolae membranes, T1317 activated eNOS, and the activation was fully prevented by ER antagonism with ICI (Figure 4C). In addition, Akt activation stimulated by T1317 in intact ECs was prevented by cell treatment with methyl-β-cyclodextrin, which destroys caveolae structure (30), providing further evidence that the localization of LXR in caveolae/lipid rafts is required for nonnuclear signaling by the receptor (Supplemental Figure 6). Thus, there is a subpopulation of LXRβ colocalized with ERα in EC caveolae/lipid rafts, and in the microdomain, there is functional coupling of LXRβ with ERα that enables LXRβ to modulate kinase-dependent processes.

Figure 4

LXRβ is colocalized and functionally coupled to ERα in EC caveolae/lipid rafts. (A) ERα and LXRβ abundance in BAEC nuclear (Nuc), cytoplasmic (Cyto), PM and caveolae/lipid raft (Cav) fractions were evaluated by immunoblot analysis. Lamin B and caveolin-1 (Cav1) proteins served as markers of nuclear and caveolae/lipid raft fractions, respectively. (B) BAEC nuclear and PM proteins isolated in A were subjected to immunoprecipitation using control IgG or anti-ERα antibody in the presence of 1 μM T1317 for 1 hour, followed by immunoblot analysis with anti-LXRβ or anti-ERα antibodies. In A and B, protein distributions were assessed in 2 parallel samples. (C) eNOS activity in the caveolae/lipid raft fraction of BAEC was quantified during 60-minute incubations with vehicle, 1 μM T1317, or T1317 plus 100 nM ICI. Values are mean ± SEM, n = 3, *P < 0.05 vs. vehicle control; ^†P < 0.05 vs. T1317 alone. Findings were confirmed in 2 independent experiments.

Nonnuclear LXRβ signaling requires PI3K-Akt–dependent ERα Ser118 phosphorylation. In the absence of estrogen, peptide growth factors such as EGF and IGF-1 cause the phosphorylation of ERα at Ser118. This process is mediated by MAP kinase, and it enhances the binding of the receptor to p160 coactivators (31, 32). Since we have discovered that LXR has nonnuclear function in ECs mediated by ERα in the absence of estrogen, we determined whether LXR activation causes the phosphorylation of ERα. Either E₂ or T1317 treatment for 20 minutes resulted in an increase in ERα Ser118 phosphorylation, and ICI inhibited T1317-induced phosphorylation (Figure 5A). To identify the signaling mechanisms that underlie ERα Ser118 phosphorylation by LXR, pharmacologic inhibitors of kinases known to be required for ERα phosphorylation were used (33). ERα phosphorylation induced by the LXR agonist GW was attenuated by the PI3K inhibitor LY294002 and by the Akt inhibitor Akti-1/2, but not by the MEK1/2 inhibitor PD0325901 (Figure 5B). Thus, PI3K and Akt are required for ERα Ser118 phosphorylation in response to LXR activation. LY294002 and Akti-1/2 also inhibited Akt Ser473 phosphorylation activated by LXR agonist (Figure 5C). In parallel studies, PI3K inhibition by LY294002 blunted both E₂- and T1317-induced activation of Akt Ser473 phosphorylation and EC migration (Supplemental Figure 7).

Figure 5

LXR activation causes EC migration via PI3K–Akt-dependent ERα S118 phosphorylation. (A) EC were treated with vehicle (Veh.), 10 nM E₂, 1 μM T1317, or T1317 plus 100 nM ICI, and protein abundance of phosphorylated ERα (Ser118) and total ERα were detected by immunoblot analysis (mean ± SEM, n = 3, *P < 0.05 vs. vehicle control; ^†P < 0.05 vs. GW alone). (B and C) ECs were treated with vehicle, 10 μM LY294002 (LY), 10 μM Akti-1/2, or 25 nM PD 0325901 (PD) for 1 hour, followed by vehicle or 1 μM GW treatment for 20 minutes. Cells were harvested and phosphorylated ERα (Ser118), total ERα, phosphorylated Akt (Ser473), and total Akt were detected (mean ± SEM, n = 3, *P < 0.05 vs. vehicle control; ^†P < 0.05 vs. GW alone). (D–F) Parental HEK293 cells (D), or HEK293 cells stably transfected with wild-type ERα (E) or ERα S118A (F) were treated with vehicle, 10 nM E₂, 1 μM T1317, or 1 μM GW for 20 minutes, and phosphorylated Akt (Ser473) and total Akt were detected by immunoblot analysis (mean ± SEM, n = 3, *P < 0.05 vs. vehicle control). In A–F, representative immunoblots from 3 separate experiments are shown; summary data are shown for phosphorylated ERα and Akt protein abundance normalized to total ERα and Akt abundance, respectively. (G) BAEC were transfected with ERα or ERα S118A expression plasmids, and cell migration in response to vehicle, 10 nM E₂, or 1 μM T1317 treatment for 20 hours was evaluated (mean ± SEM, n = 3–4, *P < 0.05 vs. vehicle control).

To further delineate whether ERα Ser118 phosphorylation is necessary for LXR nonnuclear signaling, we tested LXR agonist activation of Akt phosphorylation in HEK293 cells that have endogenous LXRβ but not ERα (Supplemental Figure 8A). E₂, T1317, and GW failed to induce Akt phosphorylation in the parental ERα-negative HEK293 cells (Figure 5D). In contrast, in HEK293 cells expressing ERα, Akt phosphorylation was activated by E₂ or the LXR agonists, and the activation by all 3 compounds was inhibited by ICI (Figure 5E). However, E₂ and the LXR agonists failed to activate Akt phosphorylation in cells expressing a mutant form of ERα in which Ser118 was mutated to alanine (ERα S118A; Figure 5F and Supplemental Figure 8A). Using immunoblotting of the PM-associated receptors, we further determined that the failure of Akt phosphorylation in response to E₂ and LXR agonists in ERα Ser118A-expressing cells was not due to altered PM localization of either endogenous LXRβ or the mutant ERα protein (Supplemental Figure 8B). Furthermore, the overexpression of ERα Ser118A in ECs had a dominant negative effect, completely preventing the stimulation of EC migration by either E₂ or T1317 (Figure 5G). Taken together, these results demonstrate that ERα Ser118 phosphorylation by PI3K-Akt is required for nonnuclear signaling and resulting cell migration provoked by LXRβ.

LXR activation relaxes precontracted aortic rings via NOS stimulation. To translate the observations regarding LXR coupling to eNOS in cultured ECs to the function of the receptor in intact endothelium, tensiometry was performed using isolated rings of rat aorta, in which NOS activation by ER and resulting effects on vascular tension can be quantified (26, 34). Aortas were incubated with E₂ or T1317 for 24 hours, and contraction induced by 100 nM phenylephrine (PE) was measured. The stimulation of contraction by PE was blunted by treatment with E₂ or T1317 (Figure 6, A and B, and Supplemental Figure 9). We confirmed that the endothelium was intact in all samples by evaluating relaxation in response to acetylcholine at the end of the assay. The inhibition of PE-induced contraction by T1317 was dose dependent (Supplemental Figure 9), and the vasodilating effects of both E₂ and T1317 were not observed in the presence of the NOS antagonist NG-methyl-l-arginine (L-NNA), indicating that they are NOS dependent. Importantly, the effects of E₂ and T1317 on aortic contraction were not related to changes in eNOS protein abundance (Figure 6C). These findings indicate that LXR activation causes vascular relaxation via the stimulation of NOS activity in intact endothelium.

Figure 6

LXR activation stimulates NOS in intact aortic endothelium. (A) PE-stimulated contraction was measured in rat aortic rings previously incubated with vehicle, 10 nM E₂, or 1 μM T1317 for 24 hours. L-NNA (100 μM) was added to selected rings 20 minutes before PE stimulation. After tension reached maximum level, 10 μM acetylcholine was added to evaluate endothelium-dependent, NOS-dependent relaxation. (B) Summary data for aortic tension are expressed as gram tension per tissue wet weight (mean ± SEM; n = 8–12 per group; *P < 0.05 vs. vehicle control). (C) Aorta samples incubated with compound for 24 hours were homogenized, and protein abundance of eNOS and β-actin was determined by immunoblotting. Each lane represents individual aorta samples.

LXRβ activation promotes reendothelialization via ERα. To determine how LXR regulate vascular endothelium in vivo, we studied carotid artery reendothelialization after perivascular electric injury in mice, which is an established model in which nonnuclear ERα activation promotes endothelial repair (19). Male mice were treated with vehicle or T1317 for 3 days, and carotid artery endothelium was denuded by perivascular electric injury. The mice subsequently received treatment for 4 additional days, and the amount of remaining denudation 4 days after injury was visualized using Evans blue dye. Figure 7A shows representative images of the carotid artery intimal surface, and summary findings are given in Figure 7B. In Lxra/b^+/+ and Lxra^–/– mice, markedly more reendothelialization occurred with T1317 versus vehicle treatment, as indicated by the smaller area of remaining denudation. In contrast, the promotion of reendothelialization by T1317 did not occur in Lxra/b^–/– or in Lxrb^–/– mice. We next determined whether LXRβ-mediated reendothelialization requires ERα. The effect of the LXR agonist T1317 on reendothelialization was compared in Era^+/+ male mice treated with vehicle versus the ER antagonist ICI for 3 days prior to injury and for 4 days after injury. In Era^+/+ mice, T1317 promoted reendothelialization, and this was fully prevented by concomitant treatment with ICI (Figure 7, C and D). Furthermore, T1317-induced reendothelialization was absent in Era^–/– mice. These findings mirror the observation regarding ERα-dependent, LXRβ-induced activation of migration by cultured ECs (Figure 1). Thus, LXRβ activation promotes reendothelialization in vivo, and the process requires ERα.

Figure 7

LXRβ activation promotes reendothelialization in mice in an ERα–dependent manner. (A and B) Reendothelialization of the carotid artery was evaluated in male Lxra/b^+/+, Lxra/b^–/–, Lxra^–/–, and Lxrb^–/– mice fed a diet containing either vehicle or T1317. (C and D) Reendothelialization experiments were performed in Era^+/+ mice, in Era^+/+ mice treated with ICI, and in Era^–/– mice fed a diet containing either vehicle or T1317. Four days after perivascular electric injury, mice were injected with Evans blue dye and the area of intimal surface dye incorporation was quantified. (A and C) Intimal surfaces of representative carotid arteries are shown. (B and D) Remaining area of denudation (arbitrary pixel units of blue stain) was quantified, and summary data are expressed as percentage of vehicle-treated Lxra/b^+/+ or Era^+/+ controls (mean ± SEM; n = 6–7 per group; *P < 0.05 vs. vehicle treatment).

In the current study, we investigated the role of LXR in the regulation of EC function and discovered a pathway by which nonnuclear actions of LXRβ promote EC migration. Using complementary EC and reconstitution cell-culture models as well as in vitro protein-protein interaction assays, we found that LXRβ interacts directly with ERα via the ligand-binding domain of ERα, that upon activation by its ligands, LXRβ has increased interaction with ERα and causes ERα Ser118 phosphorylation through PI3K-Akt, and that the phosphorylation of ERα Ser118 is required for resulting eNOS-dependent stimulation of EC migration.

We and others have previously demonstrated that a PM-associated subpopulation of ER causes potent activation of EC growth and migration and the inhibition of apoptosis in EC (17, 35). These processes are mediated by ER in caveolae/lipid rafts that are coupled to multiple signaling molecules including Src, PI3K, and Akt, and thereby also to eNOS (20, 28). In the present study, we provide what we believe is the first evidence that a subpopulation of LXRβ is associated with caveolae/lipid rafts on the EC PM and that through partnership with ERα in caveolae, LXRβ is operative in nonnuclear signaling of consequence to EC behavior. The biochemical approach used for the caveolae/lipid raft fractionation is a well-established method, and we and others have confirmed that the caveolae membranes obtained are not contaminated by proteins from the nucleus (20, 29), where LXR is abundant. Importantly, we demonstrate functional coupling of LXR with ER that results in eNOS activation in isolated caveolae/lipid rafts. Further evidence of LXR function on the PM was obtained by the observation that LXR agonist activation of Akt in intact ECs was prevented by the disruption of caveolae architecture with cyclodextrin. Interestingly, in VSMCs, LXR activation alters angiotensin II–induced phosphorylation of Erk and also the phosphorylation of the transcription factor SP1 (36). Thus, nonnuclear signaling by LXR may serve important functions in additional cell types besides ECs.

Our work also indicates that LXRβ interacts directly with ERα and that the interaction is increased by LXR agonist or by E₂. It has been reported that LXR directly binds to AP1 (37), which is a well-known molecular and functional partner of ERα. However, we believe the current findings are the first to demonstrate direct interaction between LXR and a steroid hormone receptor. We further delineated that the interaction involves the N terminus of the ERα ligand–binding domain and that the ER antagonist ICI fully inhibits the promotion of LXRβ-ERα interaction by LXR agonist. Since we previously demonstrated that structurally different ER ligands induce distinct conformational changes in ERα (38), the latter finding suggests that the dynamic interaction between LXRβ and ERα is strongly influenced by the tertiary structures of the 2 receptor proteins.

Multiple steroid and nonsteroid nuclear receptors such as ER, progesterone receptor, androgen receptor, retinoic acid receptor, retinoid X receptor, and vitamin D receptor are activated in a ligand-independent manner by phosphorylation (33, 39, 40). ERα has a protein-protein interaction with the IGF-1 receptor and is phosphorylated at Ser118 by IGF-1 through MAPK in the absence of E₂ (31), and this process influences the transcriptional activity of ERα and contributes to the development of ER-positive cancer cell resistance to endocrine therapy (33). Our discovery that ERα Ser118 phosphorylation is required for either E₂ or LXR agonist to invoke Akt activation is the first demonstration, to our knowledge, that Ser118 phosphorylation promotes the nonnuclear function of ERα. LXR modulation of ERα Ser118 phosphorylation differs from the process by which IGF-1 does so because MAPK is not required. In addition, it is notable that Akt participates both upstream and downstream of ERα in this newly identified LXRβ nonnuclear signaling cascade. Recognizing that multiple isoforms of Akt are expressed in EC and that the Akt isoforms serve distinct functions (41–43), one possibility worthy of future study is that different Akt isoforms participate proximal versus distal to ERα.

Our observations regarding mechanistic linkage between LXRβ and ERα and the resulting promotion of both NO production by the endothelium of intact arteries and endothelial monolayer integrity in vivo have potentially important implications for cardiovascular health and disease. NO is a potent atheroprotective molecule via numerous mechanisms, and NO deficiency contributes to the earliest process in atherogenesis (28, 44). The disruption of the endothelial monolayer plays an integral role in the initiation of both restenosis and atherosclerosis (12). It perturbs the ability of the endothelium to modulate local hemostasis and thrombolysis, and in the absence of the tempering influence of the endothelium, VSMC proliferate and produce extracellular matrix proteins, resulting in the formation of neointima or atherosclerotic lesions (12). Consistent with our discovery that LXRβ partnership with ERα avidly promote NO production and endothelial monolayer integrity through processes that are independent of LXRα, previous studies have shown that LXRβ activation attenuates atherosclerosis in the absence of LXRα (45, 46). Interestingly, it has been observed that the atheroprotection afforded by LXR agonists is greater in male versus female mice. Since female mice (6) are less prone to atherosclerosis than males due to estrogen- and ERα-mediated processes (16), the decreased atheroprotection provided by LXR agonists in females versus males may be consistent with our finding that LXR and ER modify EC behavior through common mechanisms. Thus, our discovery of the capacity of LXRβ to beneficially affect both NO production and endothelial repair via coupling with ERα may explain certain prior observations regarding the role of LXRβ in the modulation of vascular health and disease. A limit of the current work is that the discrete role of EC LXRβ in protection from atherosclerosis and other vascular disorders has not been directly interrogated by cell-specific gene silencing in vivo. However, based on the new functional pathway that we report, future studies of this nature are warranted.

Using complementary cell culture and in vivo models and both genetic and pharmacologic manipulation, we have identified an unanticipated nonnuclear function of LXRβ in ECs and show that this entails unique crosstalk between LXRβ and ERα, with important consequences on both NO production and endothelial monolayer integrity. Further work targeting endothelial LXRβ-mediated processes may lead to new prophylactic or therapeutic approaches to restenosis, atherosclerosis, and other vascular diseases.

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The authors thank K. Chambliss, C. Mineo, K. Kamm, K. Luby-Phelps, and K. Rothberg for help on experiments; C.-J. Edgell for providing EA.hy926 cells; D. Mangelsdorf and S. Kliewer for critical review of the manuscript; and members of the Shaul and Mango/Kliewer labs for helpful suggestions. This work was supported by grants from the American Heart Association (0865158F to M. Umetani), the NIH (HL087564 to P.W. Shaul), and the Lowe Foundation (to P.W. Shaul).

Address correspondence to: Michihisa Umetani, Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390-9063, USA. Phone: 214.648.9180; Fax: 214.648.2096; E-mail: Michihisa.Umetani@utsouthwestern.edu.

Conflict of interest: Philip W. Shaul receives additional research support provided by Pfizer.

Reference information: J Clin Invest. 2013;123(8):3488–3497. doi:10.1172/JCI66533.

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