The Nuclear Receptor FXR Uncouples the Actions of miR-33 From SREBP-2Significance
Objective—To determine whether activation of farnesoid X receptor (FXR) alters cellular and plasma cholesterol homeostasis as a result of regulation of Srebp-2 and miR-33.
Approach and Results—Chromatin immunoprecipitation sequencing data identified an FXR response element within intron 10 of the Srebp-2 gene. Consistent with this observation, treatment of mice with FXR-specific agonists (GSK2324 or GW4064) rapidly increased hepatic levels of Srebp-2 mRNA, precursor sterol response element binding protein 2 (pSREBP-2) protein, and miR-33. Furthermore, miR-33 targets, that include ABCA1 (ATP binding cassette transporter A1), NSF (N-ethylmaleimide-sensitive factor), and CPT1 (carnitine palmitoyltransferase 1), were all reduced in GSK2324-treated mice. In contrast, neither nuclear SREBP-2 protein (nSREBP-2) nor SREBP-2 target genes were induced after FXR activation. The inability to process pSREBP-2 to nSREBP-2 is likely a consequence of the induction of insulin INSIG-2A (induced gene 2A) by FXR agonists. Finally, we show that FXR-dependent induction of both Srebp-2 and miR-33 is ablated in Scap–/– mice that lack nuclear SREBP-2.
Conclusions—We demonstrate that the activation of FXR uncouples the expression of nuclear SREBP-2 and miR-33, and the regulation of their respective target genes. Further, we conclude that the FXR agonist-dependent increase in miR-33 requires transcription of the Srebp-2 gene.
Sterol response element binding protein 2 (SREBP-2) is a master regulator of genes involved in both cholesterol synthesis and low-density lipoprotein endocytosis.1,2 The transcriptional regulation of the Srebp-2 gene and subsequent processing of precursor SREBP-2 (pSREBP-2) to generate mature nuclear, transcriptionally active SREBP-2 (nSREBP-2) is controlled at multiple levels by cellular sterols.1 Briefly, the precursor form of SREBP-2 contains 2 transmembrane domains that lead to its localization in the endoplasmic reticulum (ER) where it forms a complex with SREBP cleavage activated protein (SCAP) and insulin induced gene 2 (INSIG-2), a family of resident ER proteins. Decreased sterol content of the ER results in dissociation of INSIG from the pSREBP-2:SCAP complex and translocation of the latter to the Golgi. Two proteases cleave pSREBP-2 in the Golgi to release the soluble amino-terminal fragment of the protein (nSREBP-2). nSREBP-2 translocates to the nucleus, where it binds to sterol response elements (SREs) in target genes, resulting in transcriptional activation of these SRE-containing genes. Target genes include those encoding enzymes of cholesterol biosynthesis, the low-density lipoprotein receptor that facilitates endocytosis of low-density lipoprotein, and Srebp-2 itself.2 Therefore, SREBP-2 protein functions in a feed-forward pathway to maintain sterol homeostasis.1,2 SREBP-2 maturation is repressed when cells accumulate either lanosterol or oxysterols or both. These sterols interact with INSIG and SCAP and prevent the vesicular transport of SCAP:pSREBP-2 to the Golgi, thus attenuating proteolytic cleavage of pSREBP-2.3–5
See accompanying editorial on page 748
MicroRNAs are small (22 nucleotides) RNA molecules that fine-tune gene expression by binding to mRNAs containing complementary sequences to the miRNA seed region resulting in either degradation of the target mRNA or translational arrest.6,7 The net result is a decrease in functional protein.8 Recent studies identified a microRNA, miR-33a, that is localized to intron 16 of the Srebp-2 gene.9–11 Importantly, changes in cellular sterol levels result in similar fold changes in both Srebp-2 and miR-33.9–11 Further, miR-33 was shown to target Abca1 mRNA levels leading to decreases in ATP binding cassette transporter A1 (ABCA1) protein concentrations.9–11 This decrease in ABCA1 protein resulted in decreased efflux of cellular cholesterol to lipid-poor apoproteins and to a corresponding decrease in plasma high-density lipoprotein (HDL) levels. The combined effects of the co-ordinate induction of Srebp-2 mRNA, and subsequent synthesis of pSREBP-2 and nSREBP-2, and miR-33 results in the maintenance of cellular cholesterol homeostasis as a consequence of enhancing cholesterol synthesis and uptake while limiting ABCA1-dependent sterol efflux.
Primary bile acids are synthesized in the liver and represent the major products of cholesterol catabolism.12 They function as detergents that facilitate the absorption of dietary lipid and as agonists for TGR5, a G-protein-coupled receptor, and several nuclear receptors including FXR.12 Activation of FXR affects many metabolic pathways including all aspects of the enterohepatic circulation and glucose homeostasis.12 We recently reported that FXR activation also led to induction of a bicystronic miRNA cluster that is processed into 2 distinct microRNAs, miR-144 and miR-451.13 We showed that miR-144, but not miR-451, targets ABCA1 resulting in decreased ABCA1 protein, cholesterol efflux, and plasma HDL. Activated hepatic FXR also induces hepatic expression of Scarb1 mRNA and protein.14 SCARB1/SR-B1 functions to remove HDL from the circulation providing a second mechanism by which FXR agonists lower plasma HDL cholesterol levels. Conversely, reduction of hepatic miR-144 levels resulted in increased levels of ABCA1 protein and plasma HDL, in the absence of changes in Scarb1.13
In this study, we report that activation of FXR also results in increased hepatic miR-33 levels and changes in miR-33 target genes. These changes in miR-33 levels and function occur in the absence of changes in the nuclear form of SREBP-2, and SREBP-2 target genes. Therefore, FXR activation identifies a novel pathway, in which increased miR-33 levels are dissociated from changes in nSREBP-2.
Materials and Methods
Materials and methods are available in the online-only Data Supplement.
FXR Activation Induces miR-33 and Srebp-2 Levels
Cholesterol homeostasis in the liver is tightly controlled by pathways that regulate sterol synthesis and uptake from the plasma, mediated by SREBPs, as well as cholesterol catabolism. Cholesterol catabolism occurs via conversion to bile acids, a pathway that is regulated by the nuclear receptor FXR. A link between these 2 pathways, which work together to maintain cholesterol and bile acid homeostasis, has never been shown at a physiological level. To investigate potential cross talk between FXR and Srebp-2, we investigated whether an FXR response element (FXRE) in the Srebp-2 locus15,16 might represent a link between FXR and SREBP-2. Analysis of 2 independent chromatin immunoprecipitation sequencing studies that used antibodies to FXR and mice treated with either vehicle or the FXR agonist GW4064, identified an FXRE in intron 10 of the Srebp-2 gene (Figure 1A). The Srebp-2 locus also contains a microRNA, miR-33, located within intron 16 of the Srebp-2 gene.9–11 Importantly, changes in the levels of cellular sterols were shown to result in co-ordinate regulation of Srebp-2, SREBP-2 target genes and miR-33.9–11 To determine whether the FXRE in the Srebp-2 locus is functional and regulates both Srebp-2 and miR-33 expression, we treated wild-type (C57BL/6) mice with a single dose of GSK2324, a potent and specific FXR agonist.13,17,18 Srebp-2 mRNA levels were increased significantly after 2 and 4 hours of GSK2324 treatment (Figure 1B), consistent with direct regulation by FXR. To demonstrate the specificity of the response to FXR agonists, we treated wild-type, Fxr–/– mice or mice that lack hepatic FXR (FxrL-KO) or their controls (floxed Fxr), for 3 days with either GSK2324 or GW4064. Induction of Srebp-2 mRNA levels was similar (1.6-fold) in wild-type treated with GSK2324 or GW4064 and floxed mice treated with GSK2324. In contrast, Srebp-2 induction was not observed in agonist treated whole body, or liver-specific Fxr–/– mice (Figure 1C through 1E).
Based on the previous studies showing co-ordinate regulation of Srebp-2 and miR-33, we hypothesized that treatment of mice with FXR agonists would also increase the hepatic levels of miR-33 in parallel with the changes in Srebp-2 mRNA. Consistent with this hypothesis, we show that hepatic miR-33 levels were induced within 2 hours of treatment of wild-type mice with GSK2324 (Figure 1F). Hepatic miR-33 levels were further increased after 4 hours (Figure 1F), or after 3 days of treatment of wild-type mice with either GSK2324 or GW4064 (Figure 1G and 1H). In contrast, hepatic miR-33 levels were unchanged in Fxr–/– mice or after treatment of either Fxr–/– mice or mice that lack hepatic FXR (FxrL-KO) with either GSK2324 or GW4064 (Figure 1F through 1I). These data are consistent with a specific requirement for hepatic FXR. Taken together, the data in Figure 1 demonstrate that FXR activation with ≥2 different agonists specifically increases both Srebp-2 and miR-33 expression.
In a recent study, we used microRNA expression profiling followed by high stringency analysis to identify hepatic microRNAs that were regulated in response to FXR activation.13 This approach identified 5 microRNAs, including a microRNA cluster that encoded both miR-144 and miR-451, which we showed were regulated in response to FXR agonists in vivo.13 This initial and strict approach failed to identify miR-33. Based on the data of Figure 1, we reanalyzed our original microRNA expression data using less stringent analysis parameters (removing a fold-change cut off). Under the latter conditions, we identified additional microRNAs, including miR-33, whose levels were altered after FXR activation (Figure I in the online-only Data Supplement).
To further evaluate the function of the FXRE within the Srebp-2 locus, we generated a luciferase reporter gene, in which the FXRE was inserted 3′ of the luciferase coding sequence. In addition, we inserted the Srebp-2 proximal promoter, which contains ≥1 SRE, upstream of luciferase (Figure 1J). Plasmids containing this reporter gene and an expression plasmid for FXR were transfected into Hep3B cells, and the cells were then treated for 24 hours with either the FXR agonist GSK2324 or vehicle. Figure 1J shows that GSK2324 treatment resulted in a significant increase in luciferase activity. Taken together, these results suggest that the FXRE in intron 10 of the Srebp-2 gene is functional and that activation of hepatic FXR leads to induction of both Srebp-2 mRNA and miR-33. However, it remains unclear from these studies whether the increase in miR-33 is dependent on increased transcription of the Srebp-2 gene or whether FXR can independently activate the expression of miR-33 and Srebp-2.
Activation of Hepatic FXR Is Sufficient to Repress miR-33 Targets
Previous studies have shown that hepatic miR-33 levels change 1.5- to 2.5-fold under physiological conditions that alter Srebp-2 expression.9–11 In contrast, overexpression of miR-33, either from adenovirus-mediated overexpression or from mimetics, can result in supraphysiological changes of hepatic miR-33 that affect many targets.9–11 Consequently we were interested in determining whether the 1.6-fold increase in hepatic miR-33 levels that occurred after treatment of mice with GSK2324 (Figure 1) affected the mRNA and protein levels of known miR-33 targets.
To test this hypothesis, we treated wild-type and Fxr–/– mice with GSK2324 for 3 days before determining changes in specific hepatic mRNA and protein levels. The data of Figure 2A demonstrate that GSK2324 treatment of wild-type, but not of Fxr–/– mice, caused modest but significant decreases in Abca1, Cpt1a, and Hadhb mRNAs, 3 known miR-33 targets.9–11,19 However, mRNA levels of Nsf, a newly identified miR-33 target,19 were unchanged after GSK2324 treatment of either genotype (Figure 2A). Importantly, microRNAs often cause more robust decreases in protein levels than in the mRNA levels. Figure 2B and 2C shows that hepatic protein levels of ABCA1, carnitine palmitoyltransferase 1 (CPT1), carnitine O-octanoyltransferase 1 (CROT1), HADHB, and N-ethylmaleimide-sensitive factor (NSF) were all decreased after treatment of wild-type mice with GSK2324. No repression of the 5 proteins was observed after treatment of Fxr–/– mice with the FXR agonist consistent with the absolute requirement for FXR in mediating the effects of GSK2324 (Figure 2B and 2C).
We have previously shown that FXR agonists also increase the hepatic expression of miR-144 and that miR-144 targets ABCA1 independent of miR-33.13 Therefore, we conclude that activation of hepatic FXR in vivo induces 2 microRNAs (miR-33 and miR-144) that independently target ABCA1 leading to decreased ABCA1 protein and function. Previous cell-based studies demonstrated that both miR-33 and miR-144 could act synergistically to target ABCA1.20
FXR Activation Causes Accumulation of pSREBP-2, but No Change in nSREBP-2
SREBP-2 regulates gene expression by binding to sterol response elements in target genes.1,2 Among the most well-characterized SREBP-2 target genes are enzymes of the sterol biosynthesis pathway.2 We first wanted to determine whether SREBP-2 target genes were increased after FXR activation, because we demonstrated that Srebp-2 expression is increased under the same conditions. Despite the increase in Srebp-2 mRNA, the expression of SREBP-2 target genes, that included cytochrome P450 51 (Cyp51), farnesyl diphosphate synthase (Fdps), HMG-CoA reductase (Hmgcr), and squalene synthase (Sqle), was unchanged (Figure 3A). Western blots confirmed that GSK2324 treatment did not result in changes in farnesyl diphosphate synthase or HMG-CoA reductase protein (Figure 3B and 3C).
SREBP-2 protein is synthesized as an ER-membrane bound precursor that undergoes sterol-dependent proteolytic cleavage to generate nSREBP-2 to activate target genes. Having failed to observe a change in SREBP-2 activity, despite changes in Srebp-2 mRNA, we determined whether SREBP-2 protein was altered after GSK2324 treatment. We observed a 3-fold increase in pSREBP-2, but no change in nSREBP-2 (Figure 3B and 3C). Hepatic pSREBP-2 protein levels were unchanged after treatment of Fxr–/– mice with GSK2324, consistent with the absence of changes in Srebp-2 mRNA (Figures 1B and 3B and 3C). Taken together, these results demonstrate that FXR activation increases SREBP-2 expression and precursor protein, in the absence of proteolytic processing of SREBP-2 to the nucleus and subsequent activation of SREBP-2 target genes.
Two proteins, SCAP and INSIG,1 have been identified that regulate nSREBP-2 levels by controlling SREBP-2 retention in the ER. In the absence of SCAP, the translocation of pSREBP-2 to the Golgi for processing before nuclear localization is greatly attenuated.21 In contrast, increased levels of INSIG in the ER impair translocation of pSREBP-2 to the Golgi.22 A previous report demonstrated that treatment of mice for 1 to 10 days with the FXR agonist GW4064 resulted in increased levels Insig-2a mRNA, but no change in SREBP-2 target genes.23 Our data in Figure 3A through 3C provide direct proof to support the hypothesis proposed by Hubbert et al,23 as GSK2324 treatment of wild-type mice led to a 3-fold increase in pSREBP-2 protein levels but to no change in nSREBP-2 protein. We also measured Insig-2a expression after treatment with the novel FXR agonist GSK2324. Insig-2a expression was rapidly induced within 2 hours and reached a 6-fold increase 4 hours after a single dose of GSK2324 (Figure 3D). Further, the induction of Insig-2a mRNA by GSK2324 was abolished in mice lacking FXR (Figure 3E). Consistent with previous findings,23 Insig-2a was induced in wild-type, but not in Fxr–/– mice treated with GW4064 (Figure 3F). Finally, analysis of chromatin immunoprecipitation sequencing data identified several FXREs at the Insig-2a locus, consistent with Insig-2a being a direct FXR target (Figure II in the online-only Data Supplement). These results provide additional support for the hypothesis that the inability to process pSREBP-2 to nSREBP-2 correctly after the treatment of mice with FXR agonists is likely a result of the induction of INSIG-2a protein. The effects of FXR activation on Srebp-2 mRNA, as well as pSREBP-2 and nSREBP-2 protein, are illustrated in Figure 3G.
Induction of miR-33 by FXR Agonists Requires nSREBP-2
Some microRNAs are generated as a result of splicing from introns of mRNAs after transcription of the host gene. One example is that miR-33 is localized within exon 16 of the Srebp-2 gene.9–11 Other microRNAs, such as miR-144, have their own promoter that controls the synthesis of the pri-microRNA, independent of the expression of adjacent genes.20 The data presented in Figures 1 through 3 demonstrate that treatment of wild-type mice with FXR-specific agonists induces both Srebp-2 mRNA and miR-33 levels. However, it remained unclear whether the FXRE within intron 10 of the Srebp-2 gene activated miR-33 directly or activated Srebp-2 transcription before splicing of the intronic miR-33.
In addition, under the experimental conditions of FXR activation described in Figures 1 through 3, the nucleus contains detectable nSREBP-2 protein, despite impairment of the maturation of pSREBP-2 (Figure 3B and 3C). Further, nSREBP-2 is known to bind to the promoter of the Srebp-2 gene and to act as a feed-forward activator of Srebp-2 transcription.1,2 Therefore, whether the induction of miR-33 in response to FXR activation was dependent or independent of Srebp-2 transcription and nSREBP-2 protein remained unresolved. To distinguish between these possibilities, we generated and then used mice that lack hepatic SCAP (ScapL-KO).
Previous studies have shown that in the absence of SCAP, processing of pSREBP-2 in the Golgi is greatly attenuated and results in extremely low nuclear levels of SREBP-2.21 Our initial analysis showed that mRNA and protein levels of farnesyl diphosphate synthase (Fpds) and Srebp-2, both known targets of nSREBP-2, were reduced >90% in untreated ScapL-KO when compared with that in Scapflox/flox mice (Figure 4A and 4B). We next treated Scapflox/flox and ScapL-KO mice for 3 days with GSK2324 or vehicle. GSK2324 treatment of both Scapflox/flox and ScapL-KO mice led to a robust induction of Shp, Insig-2a, and miR-144 (Figure 4C and 4D). Therefore, we conclude that loss of SCAP does not interfere with the normal induction of hepatic FXR target genes by FXR agonists. In contrast, treatment of ScapL-KO mice with GSK2324 failed to induce either Srebp-2 (Figure 4E) or miR-33 (Figure 4F). Together, these data support the hypothesis that the FXR-dependent increase in hepatic miR-33 levels requires increased transcription of the Srebp-2 gene. The data also suggest that the FXRE within the Srebp-2 locus does not directly activate miR-33 expression but rather functions to increase transcription of the Srebp-2 gene with the subsequent excision and processing of miR-33 from intron 16.
Reduction in Plasma Lipids After FXR Activation Involves Multiple Mechanisms
Previous reports have shown that activation of hepatic FXR leads to decreased plasma HDL levels13 and that this was likely a result of increased hepatic expression of both the HDL receptor (Scarb1), that clears HDL from the plasma, and miR-144, that reduces hepatic ABCA1 protein thus limiting HDL generation.13,14 However, this study demonstrates that FXR agonists also induce hepatic expression of miR-33 that subsequently targets ABCA1 and lowers plasma HDL levels.9–11
In an attempt to determine the relative importance of miR-33 and miR-144 in regulating hepatic ABCA1 and plasma HDL after FXR activation, we performed the experiments described in Figure 5. Both plasma total and HDL cholesterol levels are reduced in untreated ScapL-KO when compared with that in Scapflox/flox mice (Figure 5A). GSK2324 treatment of both Scapflox/flox and ScapL-KO mice resulted in further decreases in both total plasma cholesterol and HDL cholesterol (Figure 5A). However, the decrease in plasma HDL was greatest in the GSK2324-treated Scapflox/flox mice (Figure 5A). These latter mice responded to GSK2324 by the combined induction of miR-33, miR-144 (Figure 4D and 4F), and Scarb1 (Figure 5B). In contrast, GSK2324 treatment of ScapL-KO mice induces miR-144 (Figure 4D) and Scarb1 (Figure 5B), but not miR-33 (Figure 4F). Consistent with this lack of induction, miR-33 target genes CPT1, CROT1, HADHB, and NSF were not reduced in ScapL-KO mice treated with GSK2324 (Figure 5D and 5E). The changes in plasma HDL observed in ScapL-KO mice (Figure 5A) were paralleled by similar changes in hepatic Abca1 mRNA and ABCA1 protein (Figure 5C through 5E). These results demonstrate cross talk and corequirement of FXR and SREBP-2 for the specific regulation of the SREBP-2 locus, but not at other genes.
Together, our previous studies13 and this study demonstrate that the reduction in ABCA1 protein after FXR activation in C57BL/6 mice involves both miR-144 and miR-33. Indeed, GSK2324 treatment of mice pretreated with an antisense oligonucleotide to miR-144 resulted in a small but significant decrease in ABCA1 protein and plasma total and HDL cholesterol levels (Figure IV in the online-only Data Supplement). Therefore, suppression of the FXR-dependent induction of miR-144 or miR-33 alone is not sufficient to abolish the regulation of ABCA1 by FXR.
Here, we show that the Srebp-2 gene is a direct FXR target gene that contains a functional FXRE within intron 10. Importantly, we demonstrate that FXR agonists lead to a rapid and sustained induction of both Srebp-2 and miR-33 and that the changes in miR-33 are sufficient to repress known targets that include ABCA1, CPT1a, CROT1, HADHB, and NSF. Whether other miR-33 targets are also regulated by FXR remains to be established. However, among the many newly identified miR-33 targets is Srebp1,24 which is known to be repressed by FXR.25 Therefore, our results suggest that the repression of Srebp1c by FXR may also involve miR-33. To understand the mechanism of induction of miR-33 by FXR agonists, we used mice that lack hepatic expression of SCAP. As a result, nSREBP-2 levels and the levels of most SREBP-2 target genes are significantly repressed when compared with wild-type mice. Treatment of ScapL-KO mice with FXR agonists failed to induce Srebp-2 mRNA or miR-33, although known FXR target genes that include Shp, Insig-2a, and miR-144 were induced normally. These data are consistent with a model in which agonist activation of FXR bound to the FXRE within the Srebp-2 gene, leads to increased transcription of the Srebp-2 locus and the subsequent increase in miR-33 is a result of splicing the RNA from intron 16 (Figure 5F). Further, these data do not support a model in which the FXRE functions independently to directly induce miR-33.
This report confirms and extends an earlier study in which Hubbert et al23 showed that activation of FXR induced Insig-2a mRNA. They proposed that the increased levels of INSIG-2a might be sufficient to attenuate the maturation of pSREBP-2 to nSREBP-2. Our new analysis of chromatin immunoprecipitation sequencing data now identifies multiple FXREs within the Insig-2a locus consistent with this gene being a direct target of FXR. Further, the demonstration that treatment of wild-type mice with FXR agonists results in significant increases in pSREBP-2 protein in the absence of changes in nSREBP-2 protein is entirely consistent with a model in which the increase in INSIG-2a protein prevents the translocation of the pSREBP-2:SCAP complex to the Golgi, thus preventing the normal maturation of pSREBP-2 to nSREBP-2. Therefore, FXR activation does not increase sterol synthesis, the major process regulated by nSREBP-2, as maturation of the accumulating pSREBP-2 is impaired (Figure 2). Increased sterol synthesis would seem undesirable in conditions, where FXR is activated. FXR is active when bile acids, the breakdown products of cholesterol, are elevated, and a further elevation in cholesterol under those conditions would seem physiologically counter productive.
So what would be the physiological reason for FXR agonists to induce Srebp-2 mRNA expression without increasing the functional level and activity of nuclear SREBP-2? We propose that the answer lies with the microRNA, miR-33, that is generated from intron 16 of Srebp-2. We show that expression and activity (ie, reduction of target genes) of miR-33 are both increased after FXR activation (Figure 3). The most responsive miR-33 target gene identified thus far is ABCA1, which contains ≥2 miR-33 binding sites.9–11 We have observed more subtle regulation of other miR-33 target genes, consistent with miR-33 fine-tuning the FXR response. In the case of the regulation of ABCA1, it seems that FXR uses 2 independent microRNAs to reduce ABCA1 levels. We previously showed that the reduction in ABCA1 is part of a larger FXR response to enhance cholesterol movement into the gall bladder.13 The relative contribution of miR-33 in the FXR-dependent regulation of ABCA1 remains to be established. The dual microRNA regulation of ABCA1 by miR-33 and miR-144 would likely ensure a more robust regulation of ABCA1, consistent with our observations.
The regulation of miR-33 by FXR is modest, ≈50%. This is in line with the physiological response of expression Srebp-2, which changes no >2-fold under conditions of cellular sterol deprivation (high SREBP-2 activity) or sterol excess (low SREBP-2 activity).1,2 Therefore, the changes observed in miR-33 target genes after FXR activation are reflective of the physiological role of miR-33, which is different from the supraphysiological conditions that would be expected when miR-33 is overexpressed using mimetics or adenovirus particles.
Finally, we also demonstrate that the FXR-dependent coregulation of Srebp-2 mRNA and miR-33 requires SCAP. SCAP is required for transportation of pSREBP-2 from the endoplasmic reticulum to the Golgi where pSREBP-2 is processed to nSREBP-2 protein, before nuclear localization and subsequent activation of target genes that include Srebp-2 itself. The most facile interpretation is that the increase in miR-33 after FXR activation requires Srebp-2 transcription and that the latter process is dependent on nuclear levels of SREBP-2 protein. Whether FXR and SREBP-2 physically interact to regulate the SREBP-2 locus remains to be determined, but there is a corequirement for both, at least in this specific context. The end result of this intricate and complex regulation is that FXR uncouples the actions of SREBP-2 from those of miR-33. Although both are coregulated by FXR, it seems that the requirement for the actions of miR-33 have outweighed the actions of Srebp-2.
We thank Dr Peter Edwards for the many useful discussions and careful reading of this article, as well as for FPDS and HMG-CoA reductase antibodies. We thank Dr John Parks for the ABCA1 antibody and Dr Tim Osborne and Peter Phelan for the SREBP-2 antibody. We thank Dr Christy Esau and Regulus Therapeutics for the control and anti miR-144 compounds.
Sources of Funding
This work was supported in part by National Institutes of Health grant to Dr Tarling (HL118161). Drs Tarling and Vallim were partially supported by National Institutes of Health grants DK102559 and HL30568. Dr Tarling was partially supported by an American Heart Association (Western States Affiliate) Beginning Grant-in-Aid (13BGIA17080038). Dr Vallim was partially supported by an American Heart Association (National Affiliate) Scientist Development Grant (14SDG18440015), a University of California Los Angeles (UCLA) Clinical and Translational Science Institute (CTSI) grant (UL1TR000124), a UCLA Center for Ulcer Research and Education (CURE): Digestive Diseases Research Center (DDRC) grant (DK41301) and a UCLA Diabetes Research Center (DRC) grant (DK063491).
Dr Vallim has patents and disclosures related to the use of antimiR-144 as a therapeutic agent, which are owned by the University of California Los Angeles. The other authors report no conflicts.
This manuscript was sent to Kathryn Moore, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.304179/-/DC1.
- Nonstandard Abbreviations and Acronyms
- ATP binding cassette transporter A1
- endoplasmic reticulum
- farnesoid X receptor
- FXR response element
- high-density lipoprotein
- insulin induced gene 2A
- nuclear SREBP
- precursor SREBP
- real-time quantitative polymerase chain reaction
- SREBP cleavage activated protein
- sterol response element binding protein
- sterol response element
- untranslated region
- Received June 17, 2014.
- Accepted December 30, 2014.
- © 2015 American Heart Association, Inc.
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MicroRNAs are known to play important roles as fine-tuning regulators of gene expression. Here, we show that the bile acid-regulated nuclear receptor farnesoid X receptor (FXR) controls the hepatic expression of both miR-33 and Srebp-2. We identify a functional FXR response element in intron 10 of the Srebp-2 gene allowing us to identify the molecular mechanism for the FXR-dependent regulation of Srebp-2/miR-33. Further we show that FXR activation uncouples the activities of the parent gene Srebp-2 from the microRNA (miR-33) via the induction of Insig-2a. Therefore, we have identified a physiological context where miR-33 and Srebp-2 are coregulated (ie, after FXR activation) but the net effect of this regulation is a specific induction of miR-33.