Novel insights into the regulation of chemerin expression: role of acute-phase cytokines and DNA methylation

Chemerin is a chemoattractant protein with adipokine properties encoded by the retinoic acid receptor responder 2 (RARRES2) gene. It has gained more attention over the past few years due to its multilevel impact on metabolism and immune responses. The pleiotropic actions of chemerin include chemotaxis of dendritic cells, macrophages and natural killers (NK) subsets, bactericidal activity as well as regulation of adipogenesis and glucose metabolism. Therefore, reflecting the pleiotropic actions of chemerin, expression of RARRES2 is regulated by a variety of inflammatory and metabolic mediators. However, for most cell types, the molecular mechanisms controlling constitutive and regulated chemerin expression are poorly characterized. Here we show that RARRES2 mRNA levels in murine adipocytes are upregulated in vitro and in vivo by acute-phase cytokines, IL-1β and OSM. In contrast to adipocytes, these cytokines exerted a weak, if any, response in mouse hepatocytes, suggesting that the effect of IL-1β and OSM on chemerin expression is specific to fat tissue. Moreover, we show that DNA methylation controls the constitutive expression of chemerin. Bisulfite sequencing analysis showed low methylation levels within −735 to +258 bp of the murine RARRES2 gene promoter in unstimulated adipocytes and hepatocytes. In contrast to these cells, the RARRES2 promoter is highly methylated in B lymphocytes, cells that do not produce chemerin. Together, our findings reveal previously uncharacterized mediators and mechanisms controlling chemerin expression in various cells.

The gene encoding chemerin is known as retinoic acid receptor responder 2 (RARRES2) (12), or tazarotene induced gene 2 (TIG2) since it was first discovered in tazarotene-treated psoriatic skin lesions (13,14). Liver and adipose tissue are reported as the major sites of chemerin production; nonetheless, RARRES2 mRNA is detected in many other tissues, including the adrenal gland, ovary, pancreas, lung, kidney, and skin (2,15). Thus, reflecting the pleiotropic actions of chemerin, its association with inflammatory, metabolic and cell differentiation processes, and RARRES2 tissue mRNA expression pattern, chemerin production is regulated by a variety of inflammatory and metabolic mediators that can be broadly classified as A) agonists of nuclear receptors (retinoids, vitamin D, glucocorticoids), B) factors mainly associated with metabolic processes (e.g. fatty acids, insulin, glucose) and C) immunomodulatory mediators (e.g. cytokines of acute or chronic inflammation and lipopolysaccharide (LPS) (1).
Although more than 20 years have passed since the RARRES2 was discovered, still little is known about the molecular mechanisms regulating the transcription, translation, and secretion of chemerin in different cells. Chemerin expression may be constitutive or regulated (1). It is likely that these pathways are controlled differentially. For instance, adipocytes and hepatocytes show high constitutive RARRES2 mRNA levels (15). However, pro-inflammatory cytokines, like TNF, upregulate RARRES2 mRNA mainly in adipocytes but not in hepatocytes (16). Eukaryotic gene expression can be regulated by a variety of mechanisms. This includes modifications of DNA (e.g. DNA methylation), binding of transcription factors to a gene promoter, alternative splicing, miRNAs and many others. Comprehensive functional analysis of the chemerin promoter has not yet been performed. Only two functional, in silico predicted, transcription factor binding sites have been validated in vitro. These studies identified functional response elements for the peroxisome proliferator-activated receptor  (PPARγ), farnesoid X receptor (FXR), and sterol regulatory elementbinding protein 2 (SREBP2) in the mouse chemerin promoter (17)(18)(19). These factors are regulated by lipids (PPAR), bile acids (FXR) or free fatty acids (SREBP2). So far, the DNA methylation status of the RARRES2 promoter and the role of methylation in the regulation of chemerin transcription has not yet been established. Therefore, in this work we use adipocytes and hepatocytes to investigate constitutive and regulated expression of RARRES2 after stimulation with interleukin 1 (IL-1) and oncostatin M (OSM) pro-inflammatory cytokines. IL-1 and OSM are important acute-phase mediators that act on distant tissues to induce systemic effects of the acute-phase response to inflammatory stimuli like injury or infection. In vitro, IL-1 and OSM were shown to induce RARRES2 mRNA and chemerin protein expression in cultured human skin equivalents (2) or 3T3-L1 adipocytes (IL-1 solely) (20).
However, the effects of these cytokines on chemerin expression in primary hepatocytes and adipocytes have not been studied yet.
Here we show that acute-phase cytokines, IL1 and OSM, regulate chemerin expression in visceral white adipose tissue (vWAT) derived mouse primary adipocytes but not in primary hepatocytes, both in vitro and in vivo. Moreover, for the first time, we provide mechanistic information on the role of methylation in controlling constitutive chemerin expression.

Animal studies
Male 8 -12 week old C57BL/6 mice were used in these studies. Mice were housed under pathogenfree conditions in the animal facility at the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University. IL-1 and OSM were injected intraperitoneally at a dose of 10 g/kg BW and 160 g/kg BW, respectively. After 48 h, the liver and epididymal white adipose tissue (eWAT) were isolated and subjected to RT-QPCR analysis. All experimental procedures were approved by the First Local Ethical Committee on Animal Testing at the Jagiellonian University in Krakow, Poland (permit no: 41/2014), in accordance with the Guidelines for Animal Care and Treatment of the European Community. Mice were sacrificed by overdose of anaesthesia (a mixture of ketamine and xylasine) followed by cervical dislocation.

Primary hepatocytes isolation and culture
Primary hepatocytes were isolated from C57BL6 mice with a modified two-step perfusion method according to the protocol described by Seglen (21). Briefly, the mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) i.p., and the abdomen was opened under sterile conditions. After cannulation of the portal vein, the liver was perfused with the solution I (100 µM EGTA in Krebs-Ringer (K-R) buffer), followed by the solution II (1 mg/ml collagenase D (Roche) in K-R buffer supplemented with 150 µM CaCl2). Then the liver was dissected, passed through a 100 µm cell strainer and centrifuged (60 g, 5min, 10°C). The isolated hepatocytes were suspended in DMEM:F12 medium supplemented with 10% FBS, 50 µg/ml gentamycin, 6 ng/ml insulin, and 400 ng/ml dexamethasone. Viable cells were counted using trypan blue staining and seeded on a collagencoated plate at a density of 1 x 10 5 cells/cm 2 . Cells were cultured at 37°C in presence of 5% CO2 for 4 hours. Afterwards, plated cells were washed with DMEM:F12 medium and stimulated with IL-1β (10 ng/ml) and OSM (50 ng/ml) for 48 hours. At the time of harvest, cell culture media supernatants were collected and RNA lysis buffer (Fenozol Plus, A&A Biotechnology, Poland) or RIPA buffer containing protease inhibitors (Roche) were added to the specified wells. Collected samples were subjected to RT-QPCR or ELISA analysis.

Isolation and culture of adipose tissue-derived stromal vascular fraction (SVF)
Epididymal white adipose tissue depots were isolated from 8 -10 week old male C57BL6 mice, minced and digested with collagenase D (3,5 mg/ml) in K-R buffer supplemented with 2% BSA and 150 M CaCl2 for 1 hour at 37°C water bath with shaking every 5 min. Digested tissue were then centrifuged (280 g, 10 min, 15°C), washed, filtered through 100 μm cell strainer and centrifuged. Then the pellet was suspended in red blood cell lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0,1 mM EDTA) for 3min at room temperature (RT), centrifuged and suspended in DMEM:F12 medium (20 % FBS, gentamycin 50 g/ml). Isolated SVF cells were then seeded at a density of 9 x 10 4 cells/cm 2 in culture plate. Attached cells were washed and replenished with fresh medium after 24 hours to discard unattached dead cells or immune cells, and by day 3 more than 90% cells displayed typical fibroblastic morphology. Then, the SVF culture were subjected to adipocyte differentiation.

Isolation and culture of adipogenic progenitors
Adipogenic progenitors (APs) were isolated from C57BL6 mice according to the protocol described by Lee at al. (22). Epididymal WAT was digested, filtered, and washed as described above.
Adipocyte progenitors were then isolated using first negative selection of CD45 and CD31 followed by positive selection for SCA-1. Briefly, SVF cells were suspended in MACS buffer (0,5% BSA, 2mM EDTA, 50µg/ml gentamycin) and preincubated with Fc block (rat-anti-mouse CD16/32 mAbs, 45 µg/ml) followed by incubation with biotin-conjugated antibodies against CD45 (rat-anti-mouse CD45 mAb) and CD31 (rat-anti-mouse CD31 mAb), both 7,5 g/ml. Then SVF cells were incubated with streptavidin-conjugated magnetic beads, washed and passed over a lineage depletion (LD) column to exclude endothelial cells and leukocytes. The flow-through was collected, washed, and incubated with PE-conjugated anti-SCA-1 (rat-anti-mouse Ly-6A/E mAb, 4,8 g/ml ) followed by anti-PE-conjugated magnetic beads. Cells were then washed and passed over a lineage selection (LS) column. Labeled cells were collected, suspended in DMEM:F12 medium (20 % FBS, gentamycin 50 g/ml), counted, seeded on a cell culture flask and expanded. Then the cells were replated at a density of 2,5 x 10 4 cells/cm 2 and subjected to adipocyte differentiation. in adipocyte maintenance medium
The cells were stimulated with cytokines as described above.

Isolation of B lymphocytes
Popliteal, axillary lymph nodes and spleen were dissected and pressed through the 40 µm mesh.
Cells were washed with RPMI-1640, centrifuged (300 g, 6 min, 4°C), and pellet was suspended in red blood cell lysis buffer for 5 min at RT. Cells were centrifuged and suspended in MACS buffer (3 % FBS, 10 mM EDTA in PBS). B cell magnetic sorting was performed using MagniSort Negative Selection Protocol II (MagniSort Mouse B cell Enrichment Kit, Affymetrix). Negatively selected cells were suspended in PBS and DNA extraction was performed.

3T3-L1 cell line culture and cytokine stimulation
Primary mouse preadipocyte cell line 3T3-L1 was purchased from ATCC. 3T3-L1 cells were grown in DMEM medium supplemented with 10% FBS and gentamycin (50 µg/ml). Cells were seeded at a density of 8 x 10 3 cells/cm 2 in culture plate. After 24-hours medium was changed and cells were stimulated with IL-1β (10 ng/ml) and OSM (50 ng/ml) for 48 hours. At the time of harvest, cell culture media was removed and RNA lysis buffer was added. Collected samples were subjected to RT-QPCR analysis.
The plates were then washed with PBS containing 0.1% Tween 20, and nonspecific protein-binding sites were blocked with 3% BSA in PBS. Mouse recombinant chemerin was used as a standard.
The results were normalized to total protein content in the corresponding cell RIPA lysates (Quick Start Bradford Protein Assay, Bio-Rad). The ELISA detects both the 163S and 157S chemerin.

Bisulfite genomic DNA sequencing
Genomic DNA was extracted from B cells, hepatocytes or differentiated APs using the GeneJET After transformation and culturing of the competent bacteria (Top10 E.coli) overnight on an LB/agar/ampicillin plate, colonies (at least 8) were randomly selected, plasmids was recovered with GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific) and the DNA was sequenced with M13 common sequencing primers. The results were analyzed using QUMA online tool (RIKEN, Japan).

Plasmid construction and in vitro methylation
The liver was dissected from C57BL6 mice and genomic DNA was isolated using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific). The RARRES2 promoter was amplified using Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific) and the following overlap The efficiency of the methylation reaction was verified by resistance to cleavage by the methylationsensitive restriction enzyme HpaII (New England BioLabs). The methylated and mock-methylated chemerin promoter fragments were re-ligated into the parent vector, purified using Clean-Up Concentrator (A&A Biotechnology) and used for transfection.

Transient transfection and luciferase assay
3T3-L1 cells were seeded at a density of 1,5 x 10 4 cells in 24-well culture plate and 24 hours later cells were transfected using ViaFect transfection reagent (Promega   (Fig.1A), as well as a trend to higher chemerin protein levels ( Fig.   1B) after 48-hour stimulation. In contrast to adipocytes, downregulation of RARRES2 mRNA was detected in hepatocytes in response to OSM or OSM + IL-1β but not IL-1β alone. Cytokines did not affect chemerin protein levels in hepatocytes cell culture supernatants (Fig. 1B). Similar results were obtained after in vivo IL-1β + OSM administration. RARRES2 mRNA was upregulated only in vWAT ( Fig. 1D-E) but not in the liver. We confirmed that primary hepatocytes and mouse liver responded to stimulation since SAA3 mRNA, encoding an acute-phase protein, was markedly elevated (Fig. 1C and   1F). Together, these results suggest that the effect of the cytokines that results in upregulation of chemerin expression is specific to fat tissue.

Adipocytes are key cells expressing chemerin after stimulation with acute-phase cytokines.
The stromal vascular fraction (SVF) of adipose tissue consists of a heterogeneous population of cells that includes adipocyte precursors, hematopoietic stem cells, endothelial cells, fibroblasts, and immune cells (27). Therefore, we next asked if adipocytes are the main cells that express chemerin in response to acute-phase cytokines. Indeed, we found a similar chemerin expression pattern and levels in both vWAT-derived adipocytes and in adipocytes derived from sorted SCA-1 + adipogenic progenitors ( Fig. 2A-D). Chemerin transcript levels continue to rise throughout a 5-day time course.
Stimulation with IL-1 and IL-1 + OSM showed the highest mRNA levels (approx. 12-13-fold increase over the 1-day control) in both types of adipocyte cell culture ( Fig. 2A and 2C). However, statistically significant upregulation of chemerin protein levels was observed only in IL-1β + OSM treated vWAT-derived adipocytes or in the cell culture of sorted adipogenic precursors after IL-1 and IL-1 + OSM stimulation. Accordingly, we concluded that IL-1 and OSM regulate chemerin expression primarily in adipocytes. Chemerin protein production was the highest in response to IL-1β + OSM, suggesting additive effects.
DNA methylation affects RARRES2 expression. The functional significance of DNA methylation in the regulation of RARRES2 transcription has not yet been elucidated. Therefore, using bisulfite sequencing we determined the methylation status of the murine RARRES2 gene in unstimulated and cytokine-treated adipocytes and hepatocytes. B cells were used as a reference since these cells do not produce chemerin. We found 17 CpG sites located within -735/+258 bp of RARRES2 (Fig. 3A). However, computational analysis of this sequence did not identify any CpG island (data not shown). The results showed low methylation levels within the murine chemerin promoter in unstimulated adipocytes and hepatocytes (Fig. 3B). In contrast to these cells, the chemerin  (Fig. 4A). We used 3T3-L1 adipocyte precursors for transient transfection since these cells produce chemerin and respond to cytokine stimulation like differentiated adipocytes (Fig. 4B). Chemerin_Proximal and Chemerin_Full had the highest promoter activity, as shown by luciferase assay (Fig. 4C). The Chemerin_Distal construct had similar promoter activity compared to the empty vector. We observed downregulation of promoter activity of Chemerin_Proximal and Chemerin_Full after 48-hour stimulation of 3T3-L1 cells with IL-1 + OSM.
To investigate the role of DNA methylation in the regulation of chemerin expression, we excised the promoter sequence from pNL1.1[Nluc] constructs with restriction enzymes, treated the eluted DNA with SssI methylase or left it untreated, and religated the methylated and unmethylated DNA inserts into the parent vector. Promoter activity of the methylated Chemerin_Proximal was five-times lower than that of the unmethylated construct (Fig. 4D). In line with the results of bisulfite sequencing (Fig.   3), promoter activity of the methylated Chemerin_Distal was almost four-times higher than the unmethylated construct. Together, these data suggest that the DNA sequence of the RARRES2 promoter located from -252 to +258 bp is critical for chemerin transcription and its methylation suppresses the transcription. Interestingly, the distal part of the chemerin promoter (position -735/-253 bp) shows minimal activity but seems to play a regulatory role since DNA methylation increases its activity.

Discussion
In this study, we demonstrate differential RARRES2 gene regulation in IL-1 and OSM treated murine adipocytes and hepatocytes, both in vitro and in vivo. Our data also reveal the key importance of methylation of specific regions of the chemerin promoter in the regulation of chemerin expression.
For the first time we show that cells expressing chemerin have a different methylation profile of RARRES2 compared to cells that do not produce this protein.
Chemerin is expressed at the highest levels in the liver and white adipose tissue (1), organs that contribute to fatty acid metabolism, respond to numerous cytokines, and are involved in the pathophysiology of obesity (25). Many studies confirmed a relationship between serum chemerin and fat mass in overweight/obese individuals and correlations with obesity-associated traits, like low-grade inflammation, hypertension, and insulin resistance in some but not all of the patient cohorts studied (36). Both cytokines, IL-1β and OSM, are linked with low-grade inflammation in obesity. IL-1β can influence the metabolic function of adipocytes and induce insulin resistance (37). OSM inhibits adipocyte development, modulates glucose and lipid metabolism (38), and contributes to hepatic insulin resistance and the development of non-alcoholic steatohepatitis (NASH) (39). IL-1β and OSM can induce production of C-reactive protein (CRP) in the liver, independently of IL-6 (25). In vitro, IL-1β increased chemerin protein concentrations in conditioned media from 3T3-L1 adipocytes and the expression was upregulated in these cells in a time-and dose-dependent manner (20). IL-1β and OSM upregulated chemerin expression in human skin cultures (2). Our studies revealed that a combination of both cytokines considerably increased RARRES2 mRNA and protein levels in cell cultures of murine vWAT-derived adipocytes and differentiated SCA1+ APs but not in primary hepatocytes. In vivo experiments confirmed the results shown in vitro. RARRES2 mRNA was elevated in vWAT but not in liver. vWAT plays a key metabolic and endocrine role in overweight individuals (40). This led us to the conclusion that adipocytes have an important role in sensing and responding to cytokines by supporting chemerin production. In contrast to adipocytes, IL-1β and OSM did not affect chemerin expression in hepatocytes. So far, only free fatty acids (FFA) and GW4064, a synthetic farnesoid X receptor (FXR) agonist, were shown to influence chemerin expression in hepatocytes (18).
FXR is known as a primary bile acid nuclear receptor, which plays a critical role in maintaining lipid and glucose homeostasis (18). A pro-inflammatory cytokine, TNF was reported to affect chemerin expression in various cells like adipocytes (16), human synoviocytes (35), or fetal human intestinal cells (41). However, it did not induce the expression of chemerin in the liver or primary hepatocytes, which implicated adipocytes as a potential source of elevated serum chemerin after TNF exposure in vivo (16). Taken