Korean J Physiol Pharmacol 2021; 25(2): 111-118
Published online March 1, 2021 https://doi.org/10.4196/kjpp.2021.25.2.111
Copyright © Korean J Physiol Pharmacol.
Bo-Young Kim1, Yonghae Son1, Hyok-rae Cho2,*, Dongjun Lee3, Seong-Kug Eo4, and Koanhoi Kim1,*
1Department of Pharmacology, School of Medicine, Pusan National University, Yangsan 50612, 2Department of Neurosurgery, Kosin University College of Medicine, Busan 49267, 3Department of Convergence Medicine, School of Medicine, Pusan National University, Yangsan 50612, 4College of Veterinary Medicine and Bio-Safety Research Institute, Jeonbuk National University, Iksan 54596, Korea
Correspondence to:Hyok-rae Cho
E-mail: drchr@kosin.ac.kr
Koanhoi Kim
E-mail: koanhoi@pusan.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
27-Hydroxycholesterol (27OHChol) exhibits agonistic activity for liver X receptors (LXRs). To determine roles of the LXR agonistic activity in macrophage gene expression, we investigated the effects of LXR inhibition on the 27OHChol-induced genes. Treatment of human THP-1 cells with GSK 2033, a potent cell-active LXR antagonist, results in complete inhibition in the transcription of LXR target genes (such as LXRα and ABCA1) induced by 27OHChol or a synthetic LXR ligand TO 901317. Whereas expression of CCL2 and CCL4 remains unaffected by GSK 2033, TNF-α expression is further induced and 27OHChol-induced CCL3 and CXCL8 genes are suppressed at both the transcriptional and protein translation levels in the presence of GSK 2033. This LXR antagonist downregulates transcript levels and surface expression of CD163 and CD206 and suppresses the transcription of CD14, CD80, and CD86 genes without downregulating their surface levels. GSK 2033 alone had no effect on the basal expression levels of the aforementioned genes. Collectively, these results indicate that LXR inhibition leads to differential regulation of 27-hydroxycholesterolinduced genes in macrophages. We propose that 27OHChol induces gene expression and modulates macrophage functions via LXR-dependent and -independent mechanisms.
Keywords: Gene expression, Liver X receptors, Macrophage, 27-Hydroxycholesterol
Oxysterols are oxygenated derivatives of cholesterol. As compared to cholesterol, they contain an additional hydroxy, epoxide or ketone group in the sterol nucleus, and/or a hydroxyl group in the side chain [1]. 27-Hydroxycholesterol (27OHChol) is a side-chain oxysterol oxygenated at the 27th carbon atom of cholesterol. This oxysterol is produced via oxidation by sterol 27-hydroxylase (CYP27A1), and metabolized
27OHChol is a functional liver X receptors (LXR) agonist [12]. LXRα (NR1H3) and LXRβ (NR1H2) are two isoforms of LXRs expressed with overlapping, but a distinctive pattern. LXRα is dominant in the liver and expressed primarily in the intestine, adipose tissue, and macrophages, whereas LXRβ is widely expressed [13-15]. Since LXRs are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors, they exert their biological effects by controlling the expression of target genes [15]. Activation of LXRs with 27OHChol increases the transcription of LXR-responsive genes involved in cholesterol efflux, such as ABCA1 and ABCG1 in macrophages [12,14]. These findings indicate that 27OHChol induces gene expression
This study was therefore undertaken to determine the effects of GSK 2033, a potent cell-active LXR antagonist, on the transcription of 27OHChol-induced genes, including inflammatory and cell surface molecules as well as LXR target genes. Our results demonstrate that GSK 2033 differentially regulates the 27OHChol-induced genes in macrophages.
The human THP-1 monocyte/macrophage cell line (ATCC, #TIB-202) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO2. Penicillin (50 U/ml) and streptomycin (50 μg/ml) were added to prevent bacterial contamination. 27OHChol and antibodies against LXRα/β, CD14, CD80, and CD86 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GSK 2033 and TO 901317 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-ABCA1 antibody was purchased from Invitrogen (Eugene, OR, USA). Anti-CD163 antibody conjugated with FITC and anti-CD206 antibody conjugated with PE were purchased from BioLegend (San Diego, CA, USA).
Total RNA isolated were reverse-transcribed for 1 h at 42°C with 100 U Moloney murine leukemia virus reverse transcriptase in a 10 μl reaction volume, containing 50 mM Tris-HCl (pH 8.3 at 25°C), 55 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 μg oligo (dT) 15 primers, 0.125 mM each dNTP, and 40 U RNase inhibitor. Subsequent qPCR was performed in triplicate using a LightCycler 96 Real-Time PCR System (Roche, Mannheim, Germany), as previously described [8]. Each 20 μl reaction mixture consisted of 10 μl SYBR Green Master Mix, 2 μl forward and reverse primers (10 pM each) of the gene to be quantified, and cDNA template. The thermal cycling conditions consisted of 95°C for 10 min, followed by 45 cycles of 95°C for 10 sec, 50°C for 10 sec, and 72°C for 10 sec. The relative expression of each gene was calculated as the ratio to the GAPDH gene using the LightCycler 96 software (Version 1.1.0.1320; Roche). The primers used were as follows in Table 1.
Table 1 . List of primers used in this study.
Gene | Sequence | |
---|---|---|
Forward | 5’-AAG CCC TGC ATG CCT ACG T-3’ | |
Reverse | 5’-TGC AGA CGC AGT GCA AAC A-3’ | |
Forward | 5’-TGT CCA GTC CAG TAA TGG TTC TGT-3’ | |
Reverse | 5’-CGA GAT ATG GTC CGG ATT GC-3’ | |
Forward | 5’-CAG CCA GAT GCA ATC AAT GCC-3’ | |
Reverse | 5’-TGG AAT CCT GAA CCC ACT TCT-3’ | |
Forward | 5’-AGT TCT CTG CAT CAC TTG CTG-3’ | |
Reverse | 5’-CGG CTT CGC TTG GTT AGG AA-3’ | |
Forward | 5’-CTG GGT CCA GGA GTA CGT GT-3’ | |
Reverse | 5’-GCG GAG AGG AGT CCT GAG TA-3’ | |
Forward | 5’-TCT GCA GCT CTG TGT GAA GG-3’ | |
Reverse | 5’-AAT TTC TGT GTT GGC GCA GT-3’ | |
Forward | 5’-CCC AGG GAC CTC TCT CTA ATC-3’ | |
Reverse | 5’-ATG GGC TAC AGG CTT GTC ACT-3’ | |
Forward | 5’-ACG CCA GAA CCT TGT GAG C-3’ | |
Reverse | 5’-GCA TGG ATC TCC ACC TCT ACT G-3’ ; | |
Forward | 5’-GCA GGG AAC ATC ACC ATC CA-3’ | |
Reverse | 5’-TCA CGT GGA TAA CAC CTG AAC A-3’ | |
Forward | 5’-GGA CTA GCA CAG ACA CAC GGA-3’ | |
Reverse | 5’-CTT CAG AGG AGC AGC ACC AGA-3’ | |
Forward | 5’-AAA AAG CCA CAA CAG GTC GC-3’ | |
Reverse | 5’-CTT GAG GAA ACT GCA AGC CG-3’ | |
Forward | 5’-TGA ATT GTA CTG GTC TGT CCT-3’ | |
Reverse | 5’-CTG TGG TGC TGT GCA TTT ATC T-3’ | |
Forward | 5’-GAA GGT GAA GGT CGG AGT-3’ | |
Reverse | 5’-GAA GAT GGT GAT GGG ATT TC-3’ |
Cell lysates were separated by 10% SDS-PAGE, and resolved proteins were transferred to nitrocellulose membranes. After blocking for 1 h in 1% skim milk in TBS (pH 7.4) containing 0.05% Tween-20, membranes were incubated with antibodies against LXRα/β, ABCA1 or β-actin at 4°C overnight. Membranes were washed three times with 0.05% Tween 20/TBS for 10 min each and incubated for 1 h with HRP conjugated secondary Abs (1:5,000 dilution) at room temperature. After washing with 0.05% Tween 20/TBS, membranes were exposed to chemiluminescent detection reagents (Pierce ECL Western Blotting Substrate; Thermo Scientific, Rockford, IL, USA). Chemiluminescence images were captured by using an Amersham Imager 680 (GE Healthcare Life Scicences, Pittsburgh, PA, USA).
THP-1 cells were harvested by centrifugation at 200 × g for 5 min at room temperature and incubated for 2 h with antibodies against CD14, CD80 or CD86 (1:100 dilution) in the FACS buffer containing 2 mM EDTA and 0.2% BSA in PBS. After washing with cold-PBS, cells were incubated for 1 h with Alexa Fluor 488-conjugated secondary antibodies (1:200 dilution) at 4°C. Or the harvested cells were incubated with the fluorescent dye-conjugated antibodies (1:100 dilution) against CD163 or CD206 for 1 h at 4°C. Cells were washed with cold-PBS, resuspended in 1% paraformaldehyde, and analyzed by flow cytometry.
The amounts of CCL2, CCL3, CCL4, CXCL8, and TNF-α secreted in culture media were quantified using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA), following the manufacturer's instructions.
Statistical analysis was performed via one-way analysis of variance, followed by Dunnett's multiple comparison test, using PRISM (version 5.0) (GraphPad Software Inc., San Diego, CA, USA). A p-value less than 0.05 (p < 0.05) is considered to indicate a statistically significant difference.
The human LXRα and ABCA1/ABCG1 are targets for regulation by LXR, and ligands for the receptor induce their expression in macrophages [12]. We investigated whether 27OHChol affects expression of the genes along with TO 901317, a synthetic LXR ligand. We observed levels of
27OHChol induces expression of C-C chemokines, including CCL2, CCL3, and CCL4 [11]. We undertook to determine effects of GSK 2033 on the transcription of chemokines to understand the function of LXR inhibition in the induction of C-C chemokines. 27OHChol exposure elevated the levels of
27OHChol as well as LXR agonists induces the
27OHChol affect levels of both M1 and M2 markers of macrophages [8]. We used GSK 2033 to estimate involvement of LXR agonistic activity in the induction of M1/M2 markers. 27OHChol increased levels of transcripts and surface expression of M2 markers of CD163 and CD206. Exposure to GSK 2033 resulted in suppressing the transcription of
The LXRs are members of the nuclear receptor superfamily that modulate metabolism, development, proliferation and inflammation through positive and negative regulation of gene expression [13]. LXRs have crucial functions in the regulation of immune responses. Activation of LXR with agonists downregulates the expression of inflammatory genes through a process known as transrepression, and several studies have demonstrated the anti-inflammatory activities of synthetic LXR agonists using different mouse models of inflammatory diseases [19-23]. Endogenous oxysterols, like 27OHChol, are known to activate LXRs [12]. It is evident that this oxysterol is a pro-inflammatory molecule activating macrophages. Activated cells increase cytokine production and upregulate cell surface molecules involved in the immune response [7,9,10]. The current study investigated whether LXR-agonistic activity of 27OHChol affects the cytokine production and upregulates surface molecules after exposure to cell-active GSK 2033 [16], and thereby explored the biological functions of LXRs in macrophage activation.
27OHChol induces expression of diverse genes involved in lipid metabolism, inflammation, and cell differentiation [9,10,12]. Consistent with previous reports [12,14], we found that 27OHChol induces the LXR target genes of
27OHChol elevates transcripts of the
Cell surface molecules such as CD14, CD80, CD86, CD163, and CD206 are specific markers whose levels are enhanced by 27OHChol on monocytic cells [8]. Among them, CD163 and CD206 belong to M2 polarization markers, and CD80, and CD86 to M1 markers [28]. The results of Figs. 4B and 5B imply that expression of CD163 and CD206 protein is positively regulated by the LXR pathway while CD14, CD80, and CD86 are negatively regulated at protein level in the presence of 27OHChol. Collectively, these results suggest that LXR activity of 27OHChol is also involved in M2 polarization, which agrees with a previous study that reported that 27OHChol drives M2 polarization of human macrophages [29].
In summary, LXR inhibition with GSK 2033 leads to inhibited expression of CCL3 and CXCL8, but not of CCL2 and CCL4, enhanced TNF-α expression, and differential expression of M1/M2 markers on cell surface. We believe that the diverse effects of GSK 2033 are likely to be specific. GSK 2033 alone did not affect cell viability (Supplementary Fig. 2) and had no effect on the basal expression of the aforementioned genes. These results indicate that 27OHChol activates macrophages and affects immune cells
Supplementary data including two figures can be found with this article online at https://doi.org/10.4196/kjpp.2021.25.2.111.
kjpp-25-2-111-supple.pdfThis research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1I1A3A01055344).
B-Y.K. and Y.S. performed all experiments. H-r.C. and K.K. designed the study. B-Y.K., D.L., and S-K.E. analyzed and interpreted data. B-Y. K., D.L., H-r.C., S-K.E., and K.K. drafted the manuscript. All authors review the manuscript.
The authors declare no conflicts of interest.
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