25-Hydroxycholesterol activates the expression of cholesterol 25-hydroxylase in an LXR-dependent mechanism

Cholesterol 25-hydroxylase (CH25H) catalyzes the production of 25-hydroxycholesterol (25-HC), an oxysterol which can play an important role in different biological processes. However, the mechanisms regulating CH25H expression have not been fully elucidated. In this study, we determined that CH25H is highly expressed in mouse liver and peritoneal macrophages. We identified several liver X receptor (LXR) response elements (LXREs) in the human CH25H promoter. In HepG2 cells, activation of LXR by 25-HC or other oxysterols and synthetic ligands [T0901317 (T317) and GW3965] induced CH25H protein expression which was associated with increased CH25H mRNA expression. 25-HC or T317 activated CH25H transcription in an LXRE-dependent manner. Thus, high expressing LXR  or LXR  activated CH25H expression, and the activation was further enhanced by LXR ligands. In contrast, inhibition of LXR  /  expression attenuated 25-HC or T317-induced CH25H expression. Deficiency of interferon  expression reduced, but did not block, LXR ligand-induced hepatic CH25H expression. Activation of LXR also substantially induced macrophage CH25H expression. In vivo , administration of GW3965 to mice increased CH25H expression in both liver and peritoneal macrophages. Taken together, our study demonstrates that 25-HC can activate CH25H expression in an LXR-dependent manner, which may be an important mechanism to exert the biological actions of 25-HC.

. For instance, LXR activation by synthetic ligands increases ATP-binding cassette transporter A1 (ABCA1) or ABCG1 to enhance excess cellular free cholesterol efflux to the extracellular cholesterol acceptor, apolipoprotein AI (apoA-I) or high-density lipoprotein (HDL), thereby inhibiting formation of lipid-laden macrophage/foam cells and the development of atherosclerosis (6). In addition to synthetic ligands, such as T0901317 (T317) and GW3965, several oxysterols including 25-HC, can function as endogenous LXR ligands.
Thus, 25-HC can also induce macrophage ABCA1/ABCG1 expression in an LXR-dependent manner (7). However, the effect of 25-HC on foam cell formation appears controversial. In by guest, on  www.jlr.org Downloaded from vitro, treatment of macrophages with 25-HC increases cellular cholesterol accumulation and foam cell formation. In vivo, a high 25-HC level is found in the atherosclerotic lesion areas of ApoE deficient (ApoE -/-) mice (8). Meanwhile, activation of LXR induces expression of SREBP-1 and its target genes in hepatocytes, such as fatty acid synthase (FASN) and acetyl-CoA carboxylase 1 (ACC1), which can result in severe lipid accumulation in the liver (9).
The effects of 25-HC on inflammation may need more investigation since the controversial results have also been reported. Activation of macrophages lacking CH25H expression leads to overproduction of inflammatory interleukin-1 (IL-1) family cytokines (3), which indicates the anti-inflammatory effects of 25-HC. 25-HC also reduces LPS-induced macrophage TNF- expression and secretion (10). In contrast, 25-HC enhances poly I:C-induced macrophage IL-6 production (11). In endothelial cells, 25-HC induces expression of adhesion molecules including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin, in an LXR-independent manner (12).  is also involved in other immunological processes. For instance, 25-HC secreted from activated macrophages can suppress TLR-induced immunoglobulin A production (13).

Expression of CH25H protein in bone marrow-derived dendritic cells and macrophages
can be up-regulated by interferons (IFNs) (14). This finding has led to identification of CH25H as one of the IFN-stimulated genes, and 25-HC as a potent inhibitor of viral infection (15,16). Cellular  family cytokines and immunoglobulin A production by 25-HC is substantially impaired in mice lacking of CH25H expression (3,13). Macrophage CH25H expression can be activated by TLR4 ligands (e.g, LPS) and IFNs via an LXR independent manner (16). However, it remains unknown if the CH25H expression can be regulated by other mechanisms.
Interestingly, injection of mice with TLR ligand induces CH25H expression in multiple tissues, with the greatest effect in the liver. LPS treatment also elevates serum 25-HC levels which solely depends on CH25H expression (13). Collectively, these findings indicate that both macrophages and hepatocytes may be an important source for 25-HC production. Indeed, we determined high CH25H expression in mouse liver and peritoneal macrophages in this study. By completing a sequence alignment analysis, we identified several LXR response elements (LXREs) in the human CH25H promoter. Combining the facts that 25-HC is an endogenous LXR ligand, both LXR expression and oxysterol production can be detected in the cells with high activity of cholesterol metabolism, such as hepatocytes and macrophages, we hypothesized that 25-HC can activate CH25H expression in an LXR-dependent manner, which may be an important mechanism to exert the biological actions of 25-HC. Total RNA was extracted from cells followed by cDNA synthesis as described (19 onto a nylon-enhanced nitrocellulose membrane. The membrane was blocked with a solution of 0.1% Tween 20/PBS (PBS-T) containing 5% dry fat-free milk for 1 h followed by incubation with primary antibody overnight at 4°C. After re-blocking with PBS-T containing 5% milk, the blot was incubated with goat horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody or rabbit HRP-conjugated anti-mouse secondary antibody for 1 h at room temperature (RT). After washing 3 times for 10 min of each time with PBS-T, the membrane was incubated for 1 min in a mixture of equal volume of Western blot chemiluminescence reagent 1 and 2. The membrane was then exposed to X-ray film or subjected to C-DiGit Blot Scanner (Li-cor, Lincoln, NE).

Immunofluorescent staining
After treatment, expression of CH25H, HSPA5 or ATP1A1 protein in HepG2 cells was determined by immunofluorescent staining as described (21 and determination of promoter activity The cDNAs encoding LXR and LXR cloned into pEGFP-C2 (C2) vector were constructed, and expression of exogenous LXR and LXR protein was confirmed by Western blot as described (20).
The DNA for the human CH25H promoter (from -962 to +64) was generated by PCR with genomic DNA isolated from HepG2 cells and the following primers: forward, 5'-GGTACCTTGACGAACAACGCAGGTGG-3'; backward, 5'-GATATCGAGCAGTTGTGGCAGCTCAT-3'. The DNA was then ligated into pGL4.10 luciferase reporter vector, and the constructed normal CH25H promoter was named as pCH25H. The promoters with LXRE mutation(s) as indicated in the inserted box in Figure 3E was constructed with pCH25H and the primers containing mutated sequences using the Phusion Site-Directed Mutagenesis kit (New England Biolabs), respectively.
To determine normal or mutated pCH25H promoter activity, HepG2 cells were transfected with DNA for normal or mutated pCH25H promoter plus DNA for Renilla (for normalization of transfection efficiency) using Lipofectamine TM 2000 (Invitrogen). After 20 h of transfection plus treatment, cells were lysed with the lyse buffer supplied in the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). The cellular lysate was then used to determine firefly and Renilla luciferase activity, respectively, as described (22).
The promoter activity in each sample was initially obtained by dividing firefly luciferase activity with Renilla luciferase activity in the same sample. It was further normalized to the promoter activity of the control group (normal pCH25H promoter or normal pCH25H plus the C2 empty vector), and the promoter activity in the control group was defined as 1.

Chromatin immunoprecipitation (ChIP)-qPCR analysis
The binding of LXREs in the human CH25H promoter with LXR or LXR protein was determined by ChIP-qPCR methods. Briefly, 5x10 7 HepG2 cells were treated with 25-HC or T317 overnight followed by isolation of chromatin (22). The input PCR was conducted with DNA extracted from the sonicated chromatin after reversal of the cross-linking.

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Immunoprecipitation was conducted with the same amount of chromatin from each sample based on the input and anti-LXRα, anti-LXRβ or anti-STAT1 (as a positive control antibody) polyclonal antibody, or normal IgG (as a negative control antibody). The qPCR was conducted with the detailed protocol as described (24) and the corresponding primers were listed in the legend of Figure 3.

Determination of cellular total cholesterol and triglyceride levels
HepG2 cells in serum-free medium were treated with T317 or 25-HC for 16 h. After treatment, total cellular proteins and total lipids were extracted, respectively, followed by determination of total cholesterol and triglyceride levels using assay kits, and normalized by cellular protein levels (25).

Isolation of primary hepatocytes and in vivo studies
Both C57BL/6 wild type mice and IFN- deficient (IFN- -/-, C57BL/6 background) mice were purchased from the Animal Center of Nanjing University, and the protocol for the animal study was approved by the Animal Ethics Committee of Nankai University.
Mouse primary hepatocytes were isolated from C57BL/6 or IFN- -/mice by a collagenase perfusion method. Briefly, after the animals were anesthetized, the midline laparotomy was performed, and the inferior vena cava was cannulated with an angiocatheter. 400 m screening size filter by flushing with the cold DMEM medium. The isolated hepatocytes were collected after centrifuge for 5 min at 50 g, re-suspended with DMEM medium and plated in 6-well plates (the cell density is ~1x10 6 cells/well). The viability of the isolated hepatocytes was ~90% which was determined by the trypan blue exclusion method.
To determine if LXR activation can induce CH25H expression in mouse liver and peritoneal macrophages, C57BL/6 mice (males, ~8-week old) were randomly divided into two groups (5/group), and they then received the following treatment for one week: Control group, mice were fed normal chow; GW3965 group, mice were fed normal chow containing GW3965, a synthetic LXR ligand, at a dose of 20 mg/day/kg bodyweight. At the end of treatment, mice were anesthetized and sacrificed. Mouse blood, peritoneal macrophage and liver samples were collected. Blood was used to prepare serum. A piece of liver was used to prepare frozen sections or extract total lipids. The hepatic lipid content was determined by Oil-Red O staining of liver frozen sections and triglyceride (TG) quantitative assay with the liver total lipid extract as described (26). Total cellular protein and RNA were extracted from a piece of liver or peritoneal macrophages followed by determination of CH25H protein and mRNA expression by Western blot and qRT-PCR, respectively.

Data analysis
All experiments were repeated at least three times, and the representative results are presented. Data were presented as mean ± standard errors, and analyzed by a Student's t-test using Prism 5 (GraphPad Software, Inc.

Activation of LXR induces CH25H protein expression in HepG2 cells
Cellular 25-HC is mainly produced from cholesterol in the reaction catalyzed by CH25H.
Therefore, CH25H protein levels in tissue/cell types may decide the rate of 25-HC production.
We initially extracted total cellular proteins from mouse tissues and peritoneal macrophages, and then determined CH25H protein expression by Western blot. As shown in Figure 1A, CH25H protein is expressed in multiple tissues, particularly in the liver. Meanwhile, a high expression of CH25H protein was found in mouse peritoneal macrophages. In addition, we determined that CH25H mRNA levels in mouse brain, intestine, heart, adipose tissue and muscle are much lower than that found in peritoneal macrophages, by using a qRT-PCR method ( Figure 1B). Therefore, in mice, liver or macrophages could be one of the main sources for CH25H expression as well as 25-HC production in vivo.
By completing a sequence alignment analysis, we identified several LXREs in the promoter of the human CH25H gene ( Figure 3D). Thus, we speculated that CH25H  Figure 1C demonstrate that hepatic CH25H protein expression was induced by T317, GW3965 and 25-HC, in a concentration-dependent manner.
The time course study ( Figure 1D)  of CH25H expression by these LXR ligands can last for 24 hours after treatment ( Figure 1D).
In addition to 25-HC, other oxysterols can also function as endogenous LXR ligands to activate expression of LXR target genes. For instance, 22(R)-hydroxycholesterol is a potent LXR ligand inducing macrophage ABCA1 expression, while its stereo isoform, 22(S)-hydroxycholesterol, can also induce ABCA1 expression but to a lesser extent (7). In this study, we determined that both 22(R)-hydroxycholesterol and 22(S)-hydroxycholesterol induced CH25H expression, with a greater effect by 22(R)-hydroxycholesterol ( Figure 1E).
Thus, induction of CH25H expression by oxysterols may depend on its ability to activate LXR.
To further confirm the induction of hepatic CH25H protein expression by LXR activation, we conducted an immunofluorescent staining study with intact HepG2 cells following treatment with 25-HC and T317. Similar to the results found from our Western blots, Figure   1F demonstrates that 25-HC or T317 substantially increased CH25H protein expression in HepG2 cells.
In addition, by completing a co-immunofluorescent staining study with anti-CH25H and anti-HSPA5 (heat shock protein 5, a marker of ER) or anti-ATP1A1 (Na + /K + -transporting ATPase subunit -1, a marker of plasma membrane) antibodies, we determined that CH25H co-localized with HSPA5 protein (up panel, Figure 1G), but not the ATPA1 protein (bottom panel, Figure 1G), indicating that CH25H likely is an ER protein.

Activation of LXR induces CH25H expression at the transcriptional level
The results above demonstrate that activation of LXR by T317 and 25-HC can induce To further define LXR activates CH25H transcription, we initially investigated the effect of LXR activation on CH25H mRNA expression. As shown in Figures 2B and 2C, both 25-HC and T317 increased CH25H mRNA levels in both concentration-and time-dependent manners. To directly determine the effect of LXR activation on CH25H transcription, we constructed a CH25H promoter, and found that activity of the normal CH25H promoter was increased by T317 or 25-HC treatment ( Figure 3A); and that high expressing LXR or LXR also induced CH25H promoter activity ( Figure 3B, 3C).
By completing a sequence alignment analysis, we found 5 putative LXREs in the proximal region (~1,000 bp) of the human CH25H promoter ( Figure 3D). LXRE is also named as a direct repeat 4 (DR4) since the 6 nucleotides on each side, which are separated by any four nucleotides, are repeated. We found that 5 of 6 nucleotides on each side are repeated in the LXRE1, which is more than any other LXREs, indicating that LXRE1 could be most  Figure 3E). We determined that the normal CH25H promoter was activated by 25-HC and the high expressing LXR/.
Meanwhile, we found that the mutation in each LXRE reduced CH25H promoter activity, with most occurring in the LXRE1 mutation, at the basal level. In addition, we determined that the promoter with the mutation in a single LXRE was still activated by LXR activation.
Consequently, the mutations of all the 5 LXREs totally abolished the promoter basal activity, and no induction occurred to this mutated CH25H promoter by LXR activation ( Figure 3E).
Compared with other LXRE, we determined that the mutation of LXRE1 reduced promoter basal activity most, and the LXRE1 mutated CH25H promoter is barely activated by LXR activation ( Figure 3E). These results further confirm that LXRE1 is important for CH25H transcription induced by LXR activation.
Next, we conducted a ChIP-qPCR to analyze the binding of LXRE1 with LXR/ protein. In addition to anti-LXR or anti-LXR antibody, we used normal IgG as a negative control antibody and anti-STAT1 as a positive control antibody because of an IFN-activated site (GAS) element in the CH25H promoter. At the basal level, compared with the substantial binding of the STAT1 protein with GAS, the binding of LXR or LXR protein with LXRE1 was relatively moderate in control sample, ( Figure 3F). However, treatment of cells with 25-HC or T317 clearly increased the binding of LXR or LXR protein to the LXRE1 (left and middle panels, Figure 3F).

LXR expression is critical for induction of CH25H expression by 25-HC and T317
To determine if the induction of CH25H expression by T317 or 25-HC is directly mediated by LXR, we initially transfected HepG2 cells with an LXR or LXR expression vector, and then treated the transfected cells with 25-HC. Figure 4A shows that high expressing LXR or LXR induced CH25H expression, and the induction was further enhanced by 25-HC treatment.
Next, we established an LXR or LXR knockout HepG2 cell line by the CRISPR-Cas9 method (23), and defined them as CRISPR-LXR or CRISPR-LXR cells. The deletion of LXR or LXR protein expression in these cells was confirmed by Western blot ( Figure 4B).
As shown in Figure 4C Activation of LXR has been demonstrated to play an important role in lipid metabolism.
For instance, LXR induces lipogenesis by activating the regulatory element binding protein 1 (SREBP1) which consequently enhances FASN expression (9). In contrast to activation of LXR  Figure 5A). Associated with inhibition of HMGCR expression, we found that cellular total cholesterol levels were reduced ( Figure 5C). Meanwhile, we observed that T317 and 25-HC influenced expression of SREBP-1 and FASN differently. T317 substantially induced SREBP-1 and FASN expression, while 25-HC had a slight effect on both ( Figure 5B).
As a result, cellular TG levels were increased by T317, but not by 25-HC ( Figure 5D).
Activation of LXR can induce ABCA1 and ABCG1 expression to enhance cellular cholesterol efflux. We also determined that both T317 and 25-HC increased ABCA1 and ABCG1 levels in HepG2 cells ( Figure 5E), which may be another mechanism contributing to the reduction of cellular cholesterol levels in response to T317 or 25-HC treatment ( Figure 5C). Expression of CH25H has been reported to be induced in LPS-or IFN-activated macrophages. We previously reported that IFN- is an LXR target gene (22). Thus, we speculated that IFN- might be involved in LXR-induced CH25H expression. To address this hypothesis, we isolated primary hepatocytes from wild type and IFN- knockout (IFN- -/-) mice, and treated these cells with 25-HC or T317. We found that deficiency of IFN- expression reduced CH25H expression at basal levels in control cells indicating the importance of IFN- in controlling CH25H expression. However, expression of CH25H was still activated by 25-HC or T317 in IFN- -/cells ( Figure 6A). In addition, we determined that 25-HC or T317 induced CH25H expression in a time-dependent manner, in hepatocytes isolated from wild type or IFN- -/mice ( Figure 6B). Taken together, the results in Figure 6 suggest that IFN- is important for CH25H expression; but the induction of CH25H expression by T317 or 25-HC occurs in an IFN- independent manner.

Activation of LXR also induces macrophage CH25H expression
Previous reports have demonstrated that macrophage CH25H is an IFN-induced gene (15,16). Macrophage is a major cell type producing IFNs, such as IFN-. Our results in this study demonstrate that the macrophage is another cell type which highly expresses CH25H ( Figure 1A). Therefore, we completed the following experiment to determine if activation of LXR can also induce macrophage CH25H protein expression. Similar to hepatocytes, we determined that 25-HC, T317 or GW3965 induced CH25H protein expression in a dose-dependent manner ( Figure 7A), and the induction occurred quickly ( Figure 7B), in peritoneal macrophages collected from wild type mice. In RAW264.7 macrophage cell line, we also observed that CH25H protein expression was activated by T317, GW3965 or 25-HC in both concentration-and time-dependent manners ( Figure 7C, 7D). Combining the results we obtained from the studies with mouse primary hepatocytes ( Figure 6) and HepG2 cells (Figures 1-4), we can make a conclusion that induction of CH25H expression by LXR activation can be independent of cell type or species.

Activation of LXR induces CH25H expression in vivo
To confirm if activation of LXR can induce CH25H expression in vivo, particularly in hepatocytes and macrophages, we fed wild-type mice normal chow or normal chow containing GW3965 at a dose of 20 mg/day/kg bodyweight for 1 week. After treatment, we collected mouse blood, liver and peritoneal macrophage samples individually. Similar to our in vitro studies, we found that expression of CH25H mRNA in liver and macrophages was increased by GW3965 ( Figure 8A). Associated with increased CH25H mRNA expression, CH25H protein expression in liver or macrophages was also elevated by GW3965 treatment ( Figure 8B).
Activation of LXR can induce hepatic lipogenesis and hypertriglyceridemia. Similar to previous reports (6), in this study we also determined that after one week of GW3965 treatment, TG levels in both serum and liver were increased ( Figure 8C). Meanwhile, hepatic lipid accumulation induced by GW3965 was confirmed by Oil-Red O staining of liver frozen sections ( Figure 8D). In this study, we demonstrated that the activated CH25H protein is localized in the ER (up panel, Figure 1F), which may be linked to the physiological relevance of its product, Staphylococcus aureus and TLR2/6 by lipoteichoic acid) can also activate macrophage CH25H expression (11,13). Treatment of macrophages or dendritic cells with IFN-,  or induced CH25H expression in a STAT1-dependent manner (14,16). Taken together, these studies indicate that CH25H expression in the immune system can be regulated by multiple pathways. Interestingly, administration of KDO can activate CH25H expression in mouse tissues with the greatest effect on the liver (13), suggesting that CH25H can be expressed by different cell types, and CH25H expression can be regulated by different mechanisms.
Consistently, in this study, we observed that activation of LXR by 25-HC and synthetic LXR ligands increased CH25H expression in both hepatocytes and macrophages (Figure 1, 6, 7).
And in vivo, we determined that administration of GW3965 to mice increased CH25H expression in both liver and peritoneal macrophages (Figure 8). Expression of macrophage CH25H can be induced by IFNs. Previously, we identified that IFN- is an LXR target gene (22). Activation of LXR by ligands induces IFN- expression and secretion (22,32). Therefore, the LXR-induced CH25H expression may be involved by activation of IFN-. Indeed, we found that lack of IFN- expression reduced the basal levels of CH25H protein indicating the importance of IFN- in the activation of CH25H expression.