Retinoic acid receptor-related orphan receptor α reduces lipid droplets by upregulating neutral cholesterol ester hydrolase 1 in macrophages

doi:10.21203/rs.2.12171/v1

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Retinoic acid receptor-related orphan receptor α reduces lipid droplets by upregulating neutral cholesterol ester hydrolase 1 in macrophages

https://doi.org/10.21203/rs.2.12171/v1

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published 22 Apr, 2020

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Background: Neutral cholesterol ester hydrolase 1 (NCEH1) catalyzes the hydrolysis of cholesterol ester (CE) in macrophages. Genetic ablation of NCEH1 promotes CE-laden macrophages and the development of atherosclerosis in mice. Dysregulation of NCEH1 levels is involved in the pathogenesis of multiple disorders, including metabolic diseases and atherosclerosis; however, relatively little is known regarding the mechanisms regulating NCEH1. Retinoic acid receptor-related orphan receptor α (RORα)-deficient mice exhibit several phenotypes indicative of aberrant lipid metabolism, including dyslipidemia and increased susceptibility to atherosclerosis. Results: In this study, inhibition of lipid droplet formation by RORα positively regulated NCEH1 expression in monocyte-derived macrophages. In mammals, the NCEH1 promoter region had putative RORα response elements (ROREs). Electrophoretic mobility shift, chromatin immunoprecipitation, and luciferase reporter assays showed that RORα bound and responded to the ROREs in human NCEH1. Moreover, NCEH1 was upregulated through RORα in a phorbol myristate acetate-dependent mechanism during macrophage differentiation from THP1 cells. siRNA-mediated knockdown of RORα significantly downregulated NCEH1 and inhibited lipid droplet formation in human hepatoma cells. In contrast, NCEH1 was induced by treatment with RORα agonists. Conclusion: These data strongly suggested that NCEH1 was a direct RORα target, defining potential new roles for RORα in inhibition of lipid droplet formation through NCEH1.

General Cell Biology & Physiology

Molecular Biology

Lipid droplet

Macrophages

Transcriptional regulation

Nuclear receptor

RORα

NCEH1

Neutral cholesterol ester hydrolase 1 (NCEH1), also known as KIAA1363 or arylacetamide deacetylase-like 1, is a key enzyme that suppresses lipid droplet formation by removing cholesterol from macrophage foam cells [1, 2]. In contrast, ablation of NCEH1 accelerates atherosclerosis by promoting the formation of macrophage foam cells [3, 4]. In addition to cholesterol uptake, the balance of free cholesterol (FC) and cholesterol ester (CE) is also critical for regulation of intracellular cholesterol content in macrophage foam cells. After internalization, lipoproteins are localized to late endosomes/lysosomes, where CE is hydrolyzed into FC by lysosomal acid lipase. To prevent FC release, FC is re-esterified on the endoplasmic reticulum by acetyl-CoA acetyltransferase 1 and stored in cytoplasmic lipid droplets. If this pathway is persistently activated, excessive CE will accumulate in macrophages, thereby resulting in the formation of foam cells. The resulting CE is hydrolyzed by NCEH1 to release FC for transporter-mediated efflux, which is increasingly recognized as the rate-limiting step in FC outflow [5, 6].

Macrophage-specific overexpression of NCEH1 leads to a significant reduction in atherosclerotic lesions in low-density lipoprotein receptor (LDLR)^-/- mice because of enhanced FC efflux and reverse cholesterol transport [3]. NCEH1 is robustly expressed in macrophages and atherosclerotic lesions, and NCEH1 expression has been shown to be regulated by polyunsaturated fatty acids [7], pioglitazone [8], insulin [9], and interleukin (IL)–33 [10]. Moreover, NCEH1 transcripts are downregulated in cortical homogenates from peroxisome proliferator-activated receptor γ coactivator 1-α (PGC–1α)-knockout mice and increased by PGC–1 overexpression [11]. However, the specific mechanisms through which transcriptional regulators modulate NCEH1 expression are still unclear. Notably, two putative response elements for retinoic acid receptor-related orphan receptor α (RORα) are found at –1451/–1440 and 132/–121 regions upstream of the transcription start site (TSS) in the NCEH1 gene.

The RORα gene encodes a ligand-dependent orphan nuclear receptor that acts as a transcriptional regulator and has been identified as a novel anti-atherosclerosis target gene. RORα regulates target gene expression mainly by binding as monomers to promoter response elements, which typically consist of a consensus AGGTCA half-site preceded by an A/T-rich sequence (ROR response elements [ROREs]) [12]. RORα is constitutively active, meaning that the protein remains in an active conformation in the absence of ligand and that ligand binding can actually suppress receptor activity. Although endogenous ligands of RORα have not yet been fully elucidated, recent evidence suggests that oxygenated sterols may function as high-affinity ligands. Indeed, 7-oxygenated sterols (e.g., 7α-OHC, 7β-OHC, and 7-ketocholesterol), 24-hydroxycholesterol (24-OHC), and 25-hydroxycholesterol (25-OHC) function as inverse agonists for RORα [13, 14]. RORα-deficient mice harboring a natural deletion in the ligand-binding domain exhibit cerebellar ataxia, a phenotype also observed in Staggerer (sg/sg) mutant mice [15], which express mutated RORα and present with vascular dysfunction, dyslipidemia, excessive inflammation, immune abnormalities, and diet-induced atherosclerosis [16–18]. Recent studies have demonstrated decreases in serum and liver　triglycerides and total and high-density lipoprotein serum cholesterol in sg/sg mice. These mice also exhibit decreased hepatic expression of sterol regulatory element-binding transcription factor 1 (SREBP–1c) and the reverse cholesterol transporters ABCA1 and ABCG1 [19]. Moreover, RORα positively regulates apolipoprotein A (APOA)-I and APOC-III, suggesting a role in lipid metabolism [20, 21]. The transcriptional activator steroid receptor coactivator–2 (SRC–2) functions as a coactivator with RORα to modulate the expression of glucose 6-phosphatase (G6Pase) [22] and phosphoenolpyruvate carboxykinase (PEPCK) [23] as essential gluconeogenesis genes, rate-limiting enzyme that controls glucose release into the plasma. Moreover, RORα deficiency and treatment with RORα inverse agonists inhibit PEPCK expression and glucose production in mice [24, 25]. Additionally, overexpression of Rev-Erbα, the physiological inhibitor of RORα, also suppresses the expression of gluconeogenesis genes in human liver cancer cell lines. Conversely, silencing of Rev-Erbα significantly induces the expression of gluconeogenesis-related genes [26–28].

In brain endothelial cells, claudin domain containing 1 (CLDND1), which is involved in tight junction formation, is regulated at the transcriptional level by RORα and at the post-transcriptional level by miR–124 [29, 30]. Moreover, decreased CLDND1 expression in the adult murine cerebellum results in cerebellar hemorrhage [31].

In macrophages, using the CRISPR-Cas9 system, RORA was deleted in human THP1 monocytic cells, and a dramatic increase was observed in basal expression of a subset of nuclear factor (NF)-κB-regulated anti-inflammatory genes, including tumor necrosis factor, IL–1β, and IL–6, both at the transcriptional and translational levels [32]. RORα is a negative regulator of the inflammatory response, functioning by NF-κB inhibition through IκB activation [33]. Moreover, NF-κB activation requires the removal of IκB from NF-κB by inducible proteolysis, which liberates this transcription factor for migration to the nucleus, where it binds to IκB-regulatory elements and induces transcription [34].

As described above, RORα is involved in many physiological processes, including regulation of metabolism, development, immunity, and the circadian rhythm. The recent characterization of endogenous ligands for these former orphan nuclear receptors has stimulated the development of synthetic ligands and provided insights into targeting these receptors to treat several diseases, including atherosclerosis, diabetes, autoimmunity, and cancer [14, 35, 36]. Nevertheless, the role of RORα in modulating NCEH1 promoter activity is not clear.

Accordingly, in this study, the role of RORα in modulating NCEH1 expression was evaluated. The results suggested that control of NCEH1 expression by synthetic ligands of RORα may facilitate the development of novel anti-arteriosclerosis drugs.

Identification of ROREs in the NCEH1 promoter

Two putative response elements for RORα were found at –1451/–1440 (RORE1) and –132/–121 (RORE2) regions upstream of the TSS in NCEH1 (Fig. 1A). The ROREs contained a strongly conserved consensus sequence for RORα-binding sites based on analysis using the JASPER database (http://jaspar.genereg.net/) [37] (Fig. 1B). In particular, the consensus RGGTCA (R: A or G) half-site in NCEH1-ROREs was highly conserved in various mammals (Fig. 1C).

The ability of RORα to bind its putative response element in the NCEH1 promoter was tested by electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR). To further identify the RORα-binding site in the NCEH1 gene, H3K27Ac ChIP-sequencing (ChIP-seq) and DNaseI hypersensitivity assays were used as mapping data with the UCSC Genome Browser (http://genome.ucsc.edu/) [38] (Fig. 1A). The location and sequence of this response element are shown in Figure 1.

In addition to conventional EMSA, a 20-bp fragment spanning positions –132 to –121 and –1451 to –1440 of the NCEH1 promoter was generated as a cold probe and competitor for hot probes, end-labeled similar to IκB, which contains a known RORE, and incubated with RORα obtained by in vitro translation (Fig. 2A). RORα-induced sequence-specific mobility shifts were inhibited by the addition of excess unlabeled probe, such as IκB and wild-type NCEH1-RORE (RORE1 as a weak binding site and RORE2 as a strong binding site), but not by mutant NCEH1-RORE. Additionally, anti-RORα antibodies suppressed the formation of the DNA-protein complex, suggesting that RORα was present in the DNA-protein complex. Subsequent addition of anti-RORα antibodies destabilized the DNA complex by EMSA, and anti-early growth response protein I (EgrI) antibodies as a negative control resulted in further inhibition of the DNA-protein complex (Fig. 2B).

To further validate the transactivation ability of RORα on the NCEH1 promoter, ChIP assays were performed. The chromatin fragments of the NCEH1 promoter region from –1508 to –1257 and from –260 to –14, containing the RORE1 and RORE2 sites, could be immunoprecipitated by anti-RORα antibodies (Fig. 2C). As a control, the addition of IgG alone did not result in immunoprecipitation of the chromatin fragment of the NCEH1 promoter (Fig. 2C).

Characterization of ROREs in the NCEH1 promoter

In HEK293 cells, luciferase reporter assays were utilized to assess the activity of the putative RORE1 and RORE2 in the NCEH1 promoter. We used the UCSC Genome Browser to identify distal, conserved, putative ROREs in the 5′ region of the NCEH1 gene, which contained a promoter located within a DNase I hypersensitivity site. ChIP-seq assays indicated that acetylation of lysine 27 in histone H3 may be involved in enhancing transcription (Fig. 1A and B). Luciferase expression from cotransfection of the RORα expression plasmid with luciferase response plasmids containing three copies of the ROREs (pRORE1x3-wt, pRORE1x3-mt, pRORE2x3-wt, and pRORE2x3-mt) resulted in 0.93-, 1.00-, 1.80-, and 0.98-fold activation, respectively, compared with the empty control vector (PGVP2) containing the SV40 promoter (Fig. 3A). Furthermore, the region from –1689 to +128 of the NCEH1 gene was cloned into the luciferase reporter vector PGVB2 and transiently transfected into the human embryonic kidney cell line HEK293. Luciferase expression from pNCEH1(–1689/+128), which contained two ROREs (RORE1 and RORE2), and pNCEH1(–140/–128)-wt was increased along with transient overexpression of RORα (Fig. 3B). In contrast, pRORE1x3-mt, pRORE2x3-mt, and pNCEH1(–140/+128)-mt were not activated. These results suggested that RORα activated NCEH1 expression at the ROREs because a binding site was required in the NCEH1 promoter region.

RORα induced NCEH1 expression at both the mRNA and protein levels in macrophages

RORα positively regulates the expression and activity of endogenous NCEH1. To confirm this finding under our experimental conditions, THP1 cells were treated with 50 or 100 nM phorbol 12-myristate 13-acetate (PMA) to induce differentiation of macrophages. Real-time PCR analysis showed that endogenous NCEH1 expression was induced by RORα in THP1 cells at 12 h (Fig. 2A and B). Similarly, western blot analysis showed that RORα expression induced NCEH1 protein expression in THP1 cells at 24 h (Fig. 2E and F). Moreover, macrophage differentiation was monitored by measuring cluster of differentiation (CD) 11b and matrix metalloproteinase (MMP) 9 expression as markers of macrophages (Fig. 2C, D). The results demonstrated that RORα upregulated endogenous NCEH1 expression at both the mRNA and protein levels in THP1 macrophages.

siRNA targeting RORα reduced NCEH1 expression and lipid droplet formation

To further investigate the function of RORα knockdown, we determined whether RORα knockdown blocked accumulation of lipid droplets through downregulation of NCEH1 expression in HepG2 cells. Indeed, siRNA targeting the sequences around 258 and 1388 bp downstream of the RORα start codon suppressed NCEH1 transcription by 82.0% (Fig. 5A). Negative control siRNA (siGFP, targeting green fluorescent protein) did not affect NCEH1 transcription. The effects of siRNA transfection on cell viability were estimated by measurement of lactate dehydrogenase (LDH) activity at 48 h after transfected with siRORα. The results showed that LDH activities in siGFP- and siRORα-transfected cells (95.6% and 89.3%, respectively) were similar to that in untreated cells. Subsequently, cells were treated with oleic acid for 24 h, and Oil Red O staining was performed. Notably, lipid droplet accumulation in cells transfected with siRORα was markedly increased compared with that in negative controls (fold-changes in cells transfected with siGFP at 100 and 400 nM: 1.75 and 1.69, respectively; Fig. 5C and D).

Effects of agonist-induced RORα activation on NCEH1 expression

Analyses were performed in HepG2 cells treated for 48 or 72 h with 5 µM SR1078 (RORα agonist) or vehicle (DMSO). The mRNA levels of RORα were not altered in HepG2 cells treated with 5 µM SR1078 (Fig. 6A). In contrast, NCEH1 expression levels were increased following treatment with an RORα agonist (Fig. 6B). Additionally, the expression levels of BMAL1, as a positive control RORα-target gene, were increased (Fig. 6C).

NCEH1 is a key enzyme that suppresses lipid droplet formation by removing cholesterol in macrophage foam cells [1, 2]. Downregulation of NCEH1 promotes atherosclerosis by increasing the generation of macrophage foam cells [3, 4]. Macrophage-specific overexpression of NCEH1 leads to a significant reduction in the atherosclerotic lesion area in LDLR^-/- mice because of enhanced FC efflux and reverse cholesterol transport [3]. RORα-deficient mice harboring a natural deletion in the ligand-binding domain exhibit cerebellar ataxia, a phenotype also observed in sg/sg mice [15], which express mutated RORα and present with dyslipidemia, excessive inflammation, immune abnormalities, vascular dysfunction, and diet-induced atherosclerosis [16–18].

In this study, the results showed that the NCEH1 gene was a direct target of RORα in macrophages and was related to lipid droplet regulation. First, a global search for ROREs as RORα-binding sites involved in transcriptional regulation within –1500 to +500 from the TSS in human genes in the DBTSS database [39] and by TFBIND [40] analysis as software for searching transcription factor binding sites on DNA sequences identified the promoter region of NCEH1 as a putative target of RORα. Therefore, in this study, the promoter region of NCEH1 was isolated and characterized. Using ENCODE data, two distal, conserved, putative ROREs were identified in the 5′ region of the NCEH1 gene containing the promoter and a highly conserved RGGTCA (R: A or G) half-site motif. One of the ROREs, RORE2, was located within a DNase I hypersensitivity site, and H3K27Ac ChIP-seq indicated transcriptional enhancement at RORE2, but not RORE1. A comparison of the human RORE with those of rhesus monkeys, rats, and mice revealed that the element was highly conserved between humans and rhesus monkeys. However, low sequence conservation was observed with rats and mice, suggesting that this element may not have a major role in the transcriptional regulation of RORα in these animals. The results also showed that RORα bound to this response element and that the receptor modestly stimulated expression. In addition, PMA-induced stimulation of NCEH1 expression depended on RORα induction in macrophages, and suppression of RORα expression by siRNA significantly decreased NCEH1 transcription and lipid droplet accumulation. In contrast, NCEH1 was induced in RORα agonist-treated cells. Taken together, these data strongly indicated that NCEH1 was a direct target of RORα.

Macrophages from RORα-deficient sg/sg mice show the ability to accumulate lipids and therefore harbor large lipid droplets. Bone marrow-derived macrophages from sg/sg mice exhibited significantly reduced mRNA and protein levels of cholesterol 25-hydroxylase (Ch25h) and deficiencies in phagocytosis. Ch25h produces 25-OHC from cholesterol; 25-OHC functions as an agonist for liver X receptor α (LXRα) and is an inverse agonist for RORα, functioning through transcriptional regulation of target genes [41, 42]. Recent work has also defined a role for LXRs in the regulation of gene expression in response to cellular lipid loading. ABCA1 and ABCG1, two members of the ATP-binding cassette (ABC) family of transporter proteins, are highly induced in lipid-loaded macrophages [43, 44]. LXRs activate target genes by binding to DNA sequences associated with target genes. LXRs then bind to consensus elements (LXREs) as heterodimers with isoforms of retinoid X receptors. LXRE consists of two direct repeats of the consensus sequence AGGTCA separated by four nucleotides [45, 46]. In contrast, RORα regulates target gene expression mainly by binding as monomers to promoter response elements, which typically consist of a consensus AGGTCA half-site preceded by an A/T-rich sequence [12]. The response elements on NCEH1 are not two direct repeats, but are instead consensus ROREs involved in direct transcriptional regulation by RORα. RORα has been shown to directly regulate hepatic expression of SREBP–1c and the reverse cholesterol transporters ABCA1 and ABCG1 [19]. Moreover, RORα positively regulates APOA-I and APOC-III, suggesting a role for the receptor in lipid metabolism [20, 21] and lipid homeostasis [47, 48]. Additionally, the transcriptional activator SRC–2 functions as a coactivator with RORα to modulate G6Pase [22] and PEPCK expression [23]. Thus, RORα and LXR share many common target genes. In macrophages, RORα and 25-OHC crosstalk regulates lipid droplet homeostasis, whereas the absence of NCEH1 augments 25-OHC-induced endoplasmic reticulum stress and apoptosis [49, 50]. 25-OHC functions as an oxysterol to regulate RORα activation and NCEH1 expression. Moreover, treatment of liver macrophages with two synthetic RORα ligands was found to modulate nonalcoholic steatohepatitis (NASH); activation of RORα by SR1078 [51] as an RORα agonist results in protection against NASH and loss of RORα function, whereas inhibition of RORα function by SR3335 [52], an RORα-selective inverse agonist, results in progression of NASH [53]. Further pharmacological and pathophysiological studies are currently investigating the development of candidate RORα ligands for various applications [54, 55].

In pharmacological therapies involving nuclear receptor ligands, increased activity of lipoprotein lipase (LPL) in response to various peroxisome proliferator-activated receptors (PPARs) may explain the hypotriglyceridemic effects of fibrates, thiazolindinediones, and fatty acids, which are known activators (and/or ligands) of the various PPARs. Treatment with compounds that preferentially activate PPARα, such as fenofibrate, induces LPL expression exclusively in the livers of rats. In addition, the antidiabetic thiazolidinedione, a high-affinity ligand for PPAR, has no effect on the liver, but induces LPL expression in rat adipose tissues [56]. Moreover, endocrine therapy targeting the estrogen receptor (ER) is a standard of care for the treatment of postmenopausal women with ER-positive breast cancer. Given the dependence of these tumors on active ER signaling, the predominant treatment strategy has been to inhibit various aspects of this pathway, including directly antagonizing the ER with the use of selective ER modulators. Selective ER modulators have been used for the treatment of advanced breast cancer and are currently being evaluated for all stages of ER-positive disease [57]. Selective receptor modulators (SRMs) are receptor ligands that exhibit agonistic or antagonistic biocharacteristics in a cell- and tissue context-dependent manner. SRM-induced alterations in the conformation of the ligand-binding domains of nuclear receptors influence their abilities to interact with other proteins, such as coactivators and corepressors [58]. In contrast, RORα ligands may be able to control some RORα target genes, although it is currently not possible to achieve pathological tissue-specific selectivity.

In summary, the data from this study suggested that human NCEH1 was a direct target of RORα, containing two functional response elements in the NCEH1 promoter region. Our results defined the potential new roles of RORα in macrophage cholesterol metabolism, possibly by regulating NCEH1 expression. Improving our understanding of the interactions between RORα and its ligands may facilitate the development of specific drugs to treat inflammation, metabolic diseases, obesity, and atherosclerosis.

Cell culture

Human THP1 monocytic cells were used as a model for monocytes and were maintained in RPMI–1640 supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. THP1 cells were differentiated into macrophages following treatment with 100 nM PMA for 12 or 24 h. HEK293 embryonic kidney cells and HepG2 hepatoma cells were maintained in Dulbecco’s modified Eagle’s medium (D-MEM) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA).

EMSA

EMSAs were performed as described previously [23]. The IκB probe was prepared by radiolabeling synthetic double-stranded DNA using [–³²P] ATP (Perkin-Elmer, Waltham, MA, USA). Unlabeled probe was used as a competitive inhibitor, and IκB, which contained a known RORE [33], was used as a positive competitive control. In this assay, 0.02 pmol ³²P-radiolabeled probe was incubated with RORα crude product from in vitro translation, along with 0.1 and 0.4 pmol unlabeled DNA as wild-type and mutant NCEH1-RORE1 and RORE2. Probe sequences are listed in Supplementary Table S1. For supershift assays, anti-RORα antibodies (cat. no. sc–28612; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-EgrI antibodies (cat. no. sc–110; Santa Cruz Biotechnology) as a negative control were pre-incubated with the RORα crude product prior to the addition of the oligonucleotide probe. Gel electrophoresis was conducted at 120 V using 4% native polyacrylamide gels and 0.5× TBE buffer. The autoradiograms were obtained and quantiﬁed using a Typhoon 9400　variable mode imager (GE Healthcare, Little Chalfont, UK).

ChIP assays

ChIP assays were performed using a OneDay ChIP Kit (Diagenode, Liege, Belgium). THP1 monocytes and differentiated macrophages were used as samples. Samples containing protein/chromatin complexes were incubated with antibodies specific for RORα (cat. no. sc–28612; Santa Cruz Biotechnology) or nonimmunized IgG as a negative control overnight at 4°C. Immunoprecipitated complexes were eluted with elution buffer (1% sodium dodecyl sulfate [SDS], 50 mM Tris-HCl [pH 7.5], and 10 mM ethylenediaminetetraacetic acid). Sample DNA was extracted using a supplied column and amplified by PCR using designed primers in the NCEH1 promoter region. Primer sequences are listed in Supplementary Table S1. The band intensities of PCR products were analyzed with a CS Analyzer (Atto, Tokyo, Japan).

Luciferase reporters

The luciferase reporter plasmids pRORE1x3-wt, pRORE2x3-wt, pRORE1x3-mt, and pRORE2x3-mt containing triplet repeats of NCEH1-ROREs were constructed. Synthetic oligonucleotides corresponding to sense and antisense ROREs, as used for EMSA (Supplementary Table S1), were phosphorylated with T4 DNA polynucleotide kinase (Takara Bio, Shiga, Japan), mixed, and annealed. Each resulting double-stranded oligonucleotide was cloned into the SmaI site of the reporter vector PGVP2 (Nippon Gene, Tokyo, Japan) containing the SV40 promoter. Moreover, the human NCEH1 promoter [pNCEH1(–1689/+128)], from –1689 to +128 relative to the TSS, was amplified by PCR and inserted as a KpnI/MluI-fragment into the promoterless luciferase expression vector PGVB2 (Nippon Gene). The wild-type and mutant promoter sequences for RORE2 [pNCEH1(–140/+128)-wt and -mt] were generated by PCR using the pNCEH1(–1689/+128) plasmid as a template. Briefly, the primer sets Promoter-RORE2-FW-KpnI, Promoter-RORE2mt-FW-KpnI, and Promoter-RV-MluI were synthesized to incorporate the desired mutation (Supplementary Table S1), and a continuous fragment was produced. This fragment was then cloned as a KpnI/MluI-fragment into PGVB2. All cloned plasmids were purified using a Qiagen Plasmid Mini Kit (Qiagen, Valencia, CA, USA). Inserts were confirmed by sequencing using PGVB2-FW and PGVB2-RV primers.

Transfection and luciferase activity assay

HEK293 cells were transfected using Lipofectamine 2000 (Life Technologies, Gaithersburg, MD, USA). Briefly, cells were seeded at 1 × 10⁵ cells/well in D-MEM containing 10% fetal calf serum. After 1 day at 37°C and 5% CO₂, cells in each well were transfected for 16 h with a mixture of 100 ng various luciferase reporter plasmid, 100 ng RORα expression plasmid (pRORα) or empty plasmid (pSG5), plus 100 ng -galactosidase reporter plasmid (pSV-gal), which was used to normalize luciferase activity. Cells were grown for an additional 24–32 h in fresh medium and finally lysed. The lysate was assayed for luciferase activities using a PicaGene Luminescence Kit (Toyo Inc., Tokyo, Japan) with a Luminescencer-PSN AB–2200 (Atto). Data were collected from at least four independent experiments.

Quantitative reverse transcription-PCR (qRT-PCR)

THP1 cells were seeded at 1 × 10⁵ cells/well in RPMI–1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. After 24 h at 37°C and 5% CO₂, THP1 cells were differentiated into macrophages by treatment with 100 nM PMA for 12 h. In contrast, HepG2 cells were seeded at 1 × 10⁵ cells/well in D-MEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. After 24 h at 37°C and 5% CO₂, HepG2 cells were induced by treatment with 5 μM SR1078 as an RORα agonist for 48 or 72 h. qRT-PCR using a SYBR green reaction were performed as described previously [23]. RORα and NCEH1 expression levels were measured, with CD11b and MMP9 as positive controls of macrophage-specific genes and BMAL1 as positive controls of RORα-target genes. These genes were quantified using specific primer sets with the following protocol: 40 cycles at 95°C for 10 s, 56°C for 10 s, and 72°C for 15 s, following initial denaturation at 95°C for 2 min. The 18S rRNA gene was used as an internal control. Primer sequences of all genes are listed in Supplementary Table S1. Data were collected from at least three independent experiments.

Western blotting analysis

Cells were washed in phosphate-buffered saline (PBS) and homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.0), 200 mM sucrose, 1 mM ethylenediaminetetraacetic acid, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 0.5 mM phenylmethylsulfonyl fluoride, and 1% SDS on ice. The samples were subjected to SDS-polyacrylamide gel electrophoresis on 12% gels. Proteins on the SDS-slab gel were transferred to Immobilon-P membranes (Merck Millipore, Billerica, MA, USA) by electrophoresis. Detection of proteins was performed using rabbit anti-RORα (cat. no. sc–28612; Santa Cruz Biotechnology), anti-NCEH1 (cat. no. PA5–50285; Thermo Fisher Scientific, Waltham, MA, USA), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (cat. no. sc–20357; Santa Cruz Biotechnology). Subsequently, the membranes were rinsed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG. Bound antibodies were detected with enhanced chemiluminescence western blotting detection regents (GE Healthcare) according to the manufacturer’s instructions. The band intensities were analyzed with a CS Analyzer (Atto).

siRNA experiments

siRNAs targeting different sequences in RORα (siRORa–258 and siRORa–1388) were generated using an in vitro transcription T7 kit (Takara Bio). siRNA against green fluorescent protein (siGFP) was used as a negative control. siRNA oligonucleotide sequences are listed in Supplementary Table S1. HepG2 cells were seeded in 24-well plates at 0.5 × 10⁵ cells/well and transfected using Lipofectamine 2000 (Life Technologies) with siRNA the following day. Cells were harvested 48 h after transfection, and total RNA was prepared using ISOGEN reagent (Wako Pure Chemical). RORα and NCEH1 levels were quantified by qRT-PCR as described above. The effects of siRNA transfection on cell viability were estimated by measuring LDH activity using a Cytotoxicity Detection Kit PLUS (Roche), following the manufacturer’s protocol. Data were collected from at least three independent experiments.

Lipid droplet assays

Oil Red O stock solution was prepared in isopropanol (0.3 g/100 mL). siRNA-transfected HepG2 cells were fixed with 4% paraformaldehyde for 10 min and washed with PBS. The cells were then soaked in 60% Oil Red O stock solution diluted with distilled water for 20 min, and stained cells were washed with PBS. Cells were observed using a Leica TCS-SPE DMI4000B microscope (Leica Microsystems, Wetzlar, Germany). Moreover, lipid droplet accumulation was measured by determining the absorbance at 510 nm in isopropanol-eluted samples from stained cells. Normalization was carried out according to the protein concentrations determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical analysis

All data are expressed as means ± standard errors unless otherwise stated. Comparisons between two groups were made with unpaired Student’s t-tests. In all cases, results with P values of less than 0.05 were considered significant.

Acknowledgments

We thank Ms. Arisa Uda and Mr. Sou Kobayashi from Fukuyama University for technical assistance. This work was supported by a Fukuyama University Grant for Academic Research Projects (grant no. GARP 2018–103). This work was supported by a grant from NIG-JOINT (grant no. 73A2018) to H. Matsuoka. We are grateful to Dr. Kazuho Ikeo (Center for Information Biology, National Institute of Genetics) for advice on selection of the putative RORα target genes and for helpful discussions and suggestions.

Supplementary information

Supplementary Table S1.

Conflict of interest

The authors have no conflicts of interest.

Author contributions

HM shared responsibility for the writing of the manuscript with AM. All authors were responsible for the study conception and design. RT, YH, MK, KM, KS, AO, JK, and AS carried out the experiments. All authors contributed to critical revision of the manuscript.

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Retinoic acid receptor-related orphan receptor α reduces lipid droplets by upregulating neutral cholesterol ester hydrolase 1 in macrophages

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Results

Identification of ROREs in the NCEH1 promoter

Characterization of ROREs in the NCEH1 promoter

RORα induced NCEH1 expression at both the mRNA and protein levels in macrophages

siRNA targeting RORα reduced NCEH1 expression and lipid droplet formation

Effects of agonist-induced RORα activation on NCEH1 expression

Discussion

Conclusion

Methods

Cell culture

EMSA

ChIP assays

Luciferase reporters

Transfection and luciferase activity assay

Quantitative reverse transcription-PCR (qRT-PCR)

Western blotting analysis

siRNA experiments

Lipid droplet assays

Statistical analysis

Declarations

Acknowledgments

Supplementary information

Conflict of interest

Author contributions

References

Supplementary Files

Status:

Journal Publication

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