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Cellular cholesterol levels reflect a balance between uptake, efflux and endogenous synthesis. Here we show that the sterol-responsive nuclear receptor LXR helps maintain cholesterol homeostasis not only through promotion of cholesterol efflux, but also through suppression of low-density lipoprotein (LDL) uptake. LXR inhibits the LDL receptor (LDLR) pathway through transcriptional induction of Idol (Inducible Degrader of the LDLR), an E3 ubiquitin ligase that triggers ubiquitination of the LDLR on its cytoplasmic domain, thereby targeting it for degradation. LXR ligand reduces, whereas LXR knockout increases, LDLR protein levels in vivo in a tissue-selective manner. Idol knockdown in hepatocytes increases LDLR protein levels and promotes LDL uptake. Conversely, adenovirus-mediated expression of Idol in mouse liver promotes LDLR degradation and elevates plasma LDL levels. The LXR-Idol-LDLR axis defines a complementary pathway to sterol response element binding proteins for sterol regulation of cholesterol uptake.
The LDL receptor (LDLR) is central to the maintenance of plasma cholesterol levels (1). Mutations in this receptor are the leading cause of autosomal dominant hypercholesterolemia (ADH), characterized by elevated plasma cholesterol levels, and increased risk of cardiovascular disease (2, 3). In line with its pivotal role in cholesterol homeostasis, expression of the LDLR is tightly regulated. Transcription of the LDLR gene is coupled to cellular cholesterol levels through the action of the sterol response element binding protein (SREBP) transcription factors (4, 5). Enhanced processing of SREBPs to their mature forms when cellular sterol levels decline leads to increased LDLR transcription (6). Posttranscriptional regulation of LDLR expression is also a major determinant of lipoprotein metabolism. Genetic studies have identified mutations in the genes encoding the LDLR adaptor protein 1 (LDLRAP1/ARH) (7, 8) and the SREBP target gene proprotein convertase subtilisin/kexin 9 (PCSK9) that result in altered stability, endocytosis, or trafficking of the LDLR (9-13).
The Liver X Receptors (LXRs) are also important transcriptional regulators of cholesterol metabolism. LXRα (NR1H3) and LXRβ (NR1H2) are sterol-dependent nuclear receptors activated in response to cellular cholesterol excess (14). LXR target genes such as ABCA1 and ABCG1 promote the efflux of cellular cholesterol and help to maintain whole-body sterol homeostasis (15, 16). Mice lacking LXRs accumulate sterols in their tissues and manifest accelerated atherosclerosis, whereas synthetic LXR agonists promote reverse cholesterol transport and protect mice against atherosclerosis (17-19). The coordinated regulation of intracellular sterol levels by the LXR and SREBP signaling pathways led us to investigate whether LXRs control the uptake as well as efflux of cholesterol.
We initially tested the ability of LXRs to modulate LDL uptake in HepG2 human liver cells and primary mouse macrophages. Treatment with synthetic LXR ligand (GW3695 or T1317) decreased binding and uptake of BODIPY-labeled LDL (Fig. 1A). The LXR ligands did not induce changes in LDLR mRNA expression (Fig. S1A); however, they decreased LDLR protein levels rapidly and in a dose-dependent manner and this effect was independent of cellular sterol levels (Fig. 1B-D). Levels of ABCA1 protein, an established target of LXR, were reciprocally increased by LXR ligands (Fig. 1B-D). LXR ligands had no effect on LDLR levels in macrophages or mouse embryonic fibroblasts (MEFs) lacking LXRα and LXRβ (Fig. 1E and S1B). LXR activation also decreased LDLR protein but not mRNA levels in human SV589 fibroblasts (Fig. S1C,D) (20).
To investigate the link between endogenous LXR ligands and LDLR expression, we used an adenovirus vector encoding oxysterol sulfotransferase (Sult2b1) (21, 22). Depletion of oxysterol agonists by Sult2b1 in SV589 cells led to increased LDLR protein, and this effect was reversed by synthetic ligand (Fig. S1E). We further tested the effect of LXR agonists on LDLR produced from a transfected vector (i.e. not subject to endogenous SREBP regulation). In HepG2 cells stably expressing an LDLR-GFP fusion protein, LDLR-GFP expression was localized primarily on the plasma membrane (Fig. 1F). Ligand activation of LXR decreased LDLR-GFP expression and redistributed the protein from the plasma membrane to intracellular compartments.
To investigate the mechanism by which LXR affects the LDLR we examined LXR target genes by transcriptional profiling. We identified a potential mediator, denoted on our array as 9430057C20Rik (Table. S1), that corresponds to a protein originally identified on the basis of its interaction with myosin regulatory light chain (23). We propose that this protein, which has been variably referred to as Mir and Mylip, be renamed Idol (for Inducible Degrader of the LDLR) to reflect its biologic function (see below). Idol contains a band 4.1 and Ezrin/Radixin/Moesin homology (FERM) domain that mediates interactions with cytoplasmic domains of transmembrane proteins (24, 25). Unique among FERM domain-containing proteins, Idol also contains a C-terminal RING domain and has been proposed to act as an E3-ubiquitin ligase, although its biological substrate(s) have not been identified (23, 26). Idol mRNA is widely expressed in mice in vivo (Fig. S2A). Exposure of cells to increasing concentrations of LDL induced expression of both Idol and Abca1 mRNA, indicating that their expression is responsive to extracellular cholesterol levels (Fig. S2B). Furthermore, LXR agonists induced Idol expression in multiple cells in an LXR-dependent manner, including primary hepatocytes, primary macrophages, MEFs, and HepG2 cells (Fig. 2A and S2C-E). Treatment of mice with GW3965 induced expression of Idol in multiple tissues, including spleen, intestine and adrenals (Fig. 2B). Interestingly, only modest regulation of Idol by LXR agonist was observed in liver, consistent with the degree of Abca1 regulation (Fig S2F). Idol mRNA levels were also substantially decreased in spleen and liver of Lxrαβ-/- mice compared to wild-type controls (Fig. S2G).
LXR regulation of Idol was not sensitive to cycloheximide, suggesting that it was a direct transcriptional effect (Fig. S3A). It was not secondary to induction of SREBP-1c, because oxysterols that block SREBP processing still induced Idol expression (Fig. S3B). LXRs activate target genes by binding to consensus elements (LXREs) in their promoters. We identified an LXRE approximately 2.5 kb upstream of the mouse Idol translation start site (Fig. S3C) and generated a reporter construct encompassing this region. Activation by LXRα and GW3965 resulted in a ~4-fold increase in reporter activity that was largely abolished in the absence of a functional LXRE (Fig. S3D). Electromobility shift analysis showed that LXR/RXR bound to wild-type but not mutant versions of the Idol LXRE (Fig. S3E).
Given that Idol is a putative E3 ubiquitin ligase, we hypothesized that Idol induction might underlie the ability of LXRs to regulate LDLR abundance. Using a cotransfection system in human HEK293T kidney cells, we found that Idol expression redistributed LDLR-GFP from the plasma membrane to an intracellular compartment (Fig. 2C) and reduced the level of LDLR-GFP protein in a dose-dependent manner (Fig. 2D). Indeed, even levels of Idol protein too low to be detected by our antibody were effective. By contrast, Idol carrying a point mutation (C387A) in the catalytic RING domain (26) had no effect on the LDLR (Fig. 2C and S4A). Idol levels are very low, even when driven from an exogenous vector, suggesting that Idol is an unstable protein. In support of this idea, Idol levels were greatly enhanced when the RING domain was mutated, raising the possibility that Idol might catalyze its own degradation (Fig. S4A).
The effect of Idol on membrane proteins appears to be selective for the LDLR. Levels of transfected LRP1-GFP or APP-GFP proteins, both of which undergo regulated endocytosis similar to the LDLR, were unaffected by Idol expression (Fig. S4A). Similarly, Idol did not influence protein levels of ABCA1-GFP, endogenous transferrin receptor, endogenous myosin regulatory light chain, or the LDLR family members Lrp4 and SorLA (Fig. S4A,B). The very closely related family member ApoER2 was marginally affected by Idol. Adenovirus-mediated expression of Idol reduced LDLR protein levels in primary hepatocytes, HepG2 cells, and McR-H7777 rat hepatocytes (Fig. 2E) and also reduced LDL uptake in MEFs and McR-H7777 cells (Fig. 2F).
To explore the role of endogenous Idol in LDLR regulation, we developed shRNAs targeting Idol (Fig. S5). Idol-specific shRNAs increased LDLR protein levels in MEFs (Fig. 3A) and McR-H7777 cells (Fig. 3B), without affecting LDLR or LXR or SREBP2 target mRNAs (Figs. 3C and S5), suggesting that Idol activity is a physiological mechanism for regulating LDLR abundance. In support of this idea, Idol-specific shRNAs increased LDL uptake in both fibroblasts (Fig. 3D) and McR-H7777 cells (Fig. 3E). Finally, the ability of an LXR ligand to reduce LDLR protein levels was diminished by Idol shRNA, implicating Idol in LXR-dependent regulation of the LDLR (Fig. 3F).
Pulse-chase labeling studies showed that Idol did not block LDLR mRNA translation or appearance of the immature protein (p), but it completely prevented appearance of the mature (m) glycosylated form (Fig. 4A). Given that mutation of the RING domain inactivates Idol, the most parsimonious explanation for our data is that Idol acts an E3 ligase to trigger ubiquitination of the LDLR itself, thereby marking it for degradation. We found that ubiquitination of transfected LDLR in 293T cells was dramatically enhanced by expression of active but not mutant Idol (Fig. 4B). Analysis of a series of LDLR receptor mutants that become trapped at various stages of the maturation or recycling pathway revealed that Idol is capable of acting in the ER. LDLR G546D, which is unable to exit the ER (27), could still be degraded by Idol (Fig. 4C). Treatment of cells with brefeldin A, which blocks protein trafficking out of the ER, did not inhibit ubiquitination of the WT LDLR (Fig. S6A) or the G546D mutant (Fig. S6B). Treatment with ammonium chloride, a disruptor of lysosomal pH, inhibited LXR-induced endogenous LDLR degradation (Fig. S6C). These data suggest that Idol can ubiquitinate the precursor LDLR in the ER and that subsequent trafficking to the lysosome is required for degradation. Importantly, we also observed LXR ligand-dependent ubiquitination of endogenous LDLR in primary macrophages (Fig. 4D) and Idol-dependent ubiquitination of endogenous LDLR in primary hepatocytes (Fig. 4E). GW3965 triggered LDLR ubiquitination within 4 hours, consistent with the time course of LDLR degradation (Fig. 4D, cf. Fig. 1D).
We determined the structural requirements for Idol-dependent LDLR degradation using mutational analysis. Idol had no effect on an LDLR lacking the entire 50-amino acid intracellular domain (Fig. S6D). The LDLR intracellular domain contains 3 highly conserved lysines and one cysteine that could be potential sites for ubiquitination (Fig. 4F) (28). Single mutations of any of these residues, or combined mutation of all three lysine residues, did not prevent Idol from degrading the LDLR (Fig. 4G and S6E). However, superimposing a cysteine mutation on constructs containing two or three mutated lysines rendered the LDLR insensitive to degradation. Additional mutagenesis revealed that either an intact K20 or an intact C29 was required for Idol-mediated degradation (Figs. 4G and S6E). Finally, not only did combined mutation of the K20 and C29 residues block LDLR degradation by Idol, it also blocked ubiquitination (Fig. 4H). Interestingly, the proteasome blocker MG132, despite stabilizing Idol protein, did not stabilize the LDLR, consistent with previous reports that degradation of the LDLR occurs in the lysosome (Fig. 4H) (29, 30).
To investigate whether activation of the LXR-Idol pathway affects LDLR expression in vivo, we treated mice with GW3965. LXR agonist reduced LDLR protein levels in a tissue-selective manner, concordant with the degree of Idol induction (Fig. 5A, cf. Fig 2C). Whereas prominent effects were observed in intestine and peritoneal macrophages, LXR ligand had minimal effect on Idol mRNA and LDLR protein levels in liver. Remarkably, we observed a reciprocal effect on LDLR protein levels in resident macrophages and intestine when we analyzed mice in which the LXR-Idol pathway is inactive (Fig. 5B). LDLR protein levels were substantially higher in macrophages and intestine of LXRαβ-/- mice compared to wild-type controls. LDLR levels were also slightly higher in liver. Thus, gain or loss of LXR-Idol activity affects LDLR expression in vivo.
Since Idol was not subject to strong LXR regulation in liver, we employed an alternative strategy to test its function in this tissue. We infected mice with adenoviral vectors encoding β-galactosidase or mouse Idol. Idol expression increased plasma levels of total and unesterified cholesterol, whereas levels of triglycerides, free fatty acids and glucose were not significantly altered (Fig. 5C and S7A). Fractionation of plasma lipoproteins revealed that Idol expression caused a phenotype reminiscent of that exhibited by Ldlr(–/–) mice, with the appearance of a prominent LDL peak not present in the control mice (Fig. 5D and S7B). Plasma apoB protein levels were approximately 3-fold increased in in Idol-transduced mice and there was no difference in PCSK9 levels (Fig. S7C,D). Consistent with our in vitro results, hepatic expression of LXR and SREBP-2 target genes was not affected by Idol expression (Fig. S7E), but LDLR protein levels were markedly reduced (Fig. 5E). In contrast, transferrin receptor levels were not altered by Idol. Finally, Idol adenovirus had no effect on plasma cholesterol levels (Fig. S8A) or lipoprotein profiles (Fig. S8B) in Ldlr(–/–) mice, demonstrating that Idol requires LDLR expression for these effects.
In summary, we have shown that the sterol-sensitive nuclear receptor LXR regulates LDLR-dependent cholesterol uptake through a pathway independent of the SREBPs. LXR induces expression of Idol, which in turn catalyzes the ubiquitination of the LDLR, thereby targeting it for degradation. These results provide a potential explanation for an earlier observation that an ectopically-expressed LDLR could still be regulated by sterols (31). Identification of the Idol-LDLR pathway fills a gap in our understanding of how LXRs control cholesterol homeostasis. The ability of LXRs to respond to excess cellular cholesterol by promoting efflux through ABC transporters has been extensively documented (14). The LXR-Idol-LDLR pathway provides a mechanism to simultaneously limit LDL cholesterol uptake. Idol and ABCA1 are coordinately regulated by LXR in a cell-type selective manner, consistent with this functional link.
Interestingly, the LXR-Idol pathway appears to be most active in peripheral cells such as macrophages, adrenals and intestine. At the same time, however, Idol is constitutively expressed in liver, and gain or loss of Idol function in cultured heptocytes regulates LDLR protein levels and affects LDL uptake. Forced expression of Idol in liver in vivo profoundly reduces LDLR levels, further indicating that Idol is capable of degrading LDLR in this tissue.
Whether Idol expression is required for LDLR degradation in vivo is a critical question that remains to be addressed. If it is required, then conceivably the Idol pathway could be targeted pharmacologically so as to increase LDLR levels and enhance LDL clearance.