Visfatin contributes to the differentiation of monocytes into macrophages through the differential regulation of inflammatory cytokines in THP-1 cells

Cellular Signalling 26 (2014) 705–715

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Visfatin contributes to the differentiation of monocytes into macrophages through the differential regulation of inflammatory cytokines in THP-1 cells Mi Ran Yun a,b, Jeong Mi Seo a, Hyun Young Park a,⁎ a b

Division of Cardiovascular and Rare Diseases, Center for Biomedical Sciences, Korea National Institute of Health, Republic of Korea JE-UK Laboratory of Molecular Cancer Therapeutics, Yonsei Cancer Research Institute, College of Medicine, Yonsei University, Seoul, Republic of Korea

a r t i c l e

i n f o

Article history: Received 29 August 2013 Received in revised form 11 December 2013 Accepted 22 December 2013 Available online 27 December 2013 Keywords: CD36 IL-1β IL-6 Monocytes/macrophages differentiation NF-κB Visfatin

a b s t r a c t Visfatin is a novel multifunctional adipocytokine with inflammatory properties. Although a link between visfatin and atherosclerosis has recently been suggested, its actions in the development of atherosclerosis remain unknown. Therefore, we investigated a potential role and underlying mechanism(s) of visfatin in monocytes/macrophages differentiation, a critical early step in atherogenesis, using phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 cell models. The co-incubation of PMA with visfatin-induced CD36 expression with a concomitant increase in the phagocytosis of latex beads compared with PMA alone treatment. Moreover, visfatin markedly increased interleukin (IL)-1β secretion by enhancing IL-1β mRNA stability in a short-term incubation. Visfatin also significantly elevated the secretion of IL-6 as well as IL-1β in a longer incubation period, which was partially suppressed by nuclear factorκB (NF-κB) inhibitor, BAY11-7082, and c-Jun-N-terminal kinase (JNK) inhibitor, SP600125. Furthermore, silencing IL-1β successfully blocked IL-6 secretion, CD36 expression, and NF-κB activation in response to visfatin. Collectively, these results suggest that visfatin enhances the IL-1β-dependent induction of IL-6 and CD36 via distinct signaling pathways mediated by JNK and NF-κB, respectively, and consequently, leading to the acceleration of monocytes/ macrophages differentiation. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Atherosclerosis is an inflammatory disease characterized by the complex interactions among various cellular and molecular elements [1]. Of the cellular components within the atherosclerotic lesion, macrophages are the most dominant cell type and contribute to all stages of the process of atherosclerosis [2]. Macrophages are derived from monocytes differentiation in response to various stimuli in the arterial intima and play pivotal roles in inflammation by modulating the inflammatory response [3]. Monocyte-to-macrophage differentiation is associated with increased expression of several genes for the functionality of Abbreviations: DeM, differentiated macrophages; DiM, differentiating macrophages; ERK, extracellular signal-regulated kinase; FACS, fluorescence-activated cell sorter; FBS, fetal bovine serum; HRP, horseradish peroxidase; iNOS, inducible nitric oxide synthase; IL-1Ra, IL-1 receptor antagonist; IL, interleukin (IL); IκB-α, inhibitors of NF-κB-α; MMP9, matrix metalloproteinases-9; MO, monocytes; NAMPT, nicotinamide phosphoribosyltransferase; NF-κB, nuclear factor-κB; OD, optical density; PMA, phorbol12-myristate-13-acetate; PBEF, pre-B-cell colony-enhancing factor-1; RT-PCR, reverse transcriptase-PCR; SAPK/JNK, stress-activated protein kinase/c-Jun-N-terminal kinase; TNF-α, tumor necrosis factor-α; VSMCs, vascular smooth muscle cells. ⁎ Corresponding author at: Division of Cardiovascular and Rare Diseases, Center for Biomedical Sciences, Korea National Institute of Health, 187 Osongsaengmyeng2(i)-ro, Osong-eub, Cheongwon-gun, Chungbuk 363-951, Republic of Korea. Tel.: + 82 437198650; fax: +82 437198689. E-mail address: [email protected] (H.Y. Park). 0898-6568/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellsig.2013.12.010

macrophage. Among recognized differentiation markers, CD36 is upregulated during the late stages of monocyte differentiation [4] and also serves as scavenger receptor (SR) mediating the foam cell and fatty streak formation, one of the earliest stages in atherogenesis [5]. Martín-Fuentes et al. [6] addressed the individual variation in the expression of SRs according to different inflammatory responses. Therefore, elucidating the action mechanisms between CD36 expression and inflammatory cytokine during monocyte-to-macrophage differentiation is critical to early atherosclerosis prevention. Visfatin, known as pre-B-cell colony-enhancing factor-1 (PBEF) or nicotinamide phosphoribosyltransferase (NAMPT), was subsequently rediscovered as an adipocytokine released predominantly by visceral adipose tissue [7]. Visfatin can also be synthesized and released to the extracellular milieu by other cell types, where it affects the pathogenesis of inflammation-related diseases [8,9]. Previous studies have uncovered a link between visfatin and atherosclerosis [10–12]. A recent study by Spiroglou et al. [13] showed that visfatin was detected in vascular smooth muscle cells (VSMCs) within the atherosclerotic plaque. Extracellular visfatin enhanced the expression of inducible nitric oxide synthase (iNOS) by extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) activation in human VSMCs [14]. In the vascular endothelium, visfatin appears to promote endothelial dysfunction by increasing the release of inflammatory cytokines [15,16]. Interestingly, Curat et al. [17] reported that visfatin was released predominantly

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from macrophages rather than from adipocytes in visceral adipose tissue. Moreover, visfatin was detected in macrophages within unstable atherosclerotic lesions, and extracellular visfatin promoted the synthesis and release of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-8, and matrix metalloproteinases-9 (MMP9) activity by monocytes [10,18]. In addition, other studies demonstrated that serum visfatin levels positively correlated with circulating inflammatory markers [19,20], highlighting that visfatin may contribute to the development of atherosclerotic lesions by regulating the inflammatory responses in vascular cells. However, the pathophysiological role of visfatin on monocyte differentiation, an important step in early atherosclerosis, has not been explored. In the current study, therefore, we investigated whether visfatin affects the differentiation of monocytes into macrophages, focusing on inflammatory profile changes. 2. Materials and methods 2.1. Chemicals and antibodies Recombinant human visfatin was purchased from Peprotech (Rocky Hill, NJ). Visfatin protein used in this study was N 98% pure by SDS– PAGE gel and HPLC analysis and endotoxin levels were below the limit of detection (b 0.1 ng/μg of visfatin) as determined by the Limulus amebocyte lysate similar to the previous study [18]. FK866 was from Enzo Life Sciences (Plymouth Meeting, PA). Phorbol-12-myristate-13-acetate (PMA) was from Sigma Chemical Co. (St. Louis, MO). Pharmacologic inhibitors, including PD98059, SB203580, SP600125, LY294002, and BAY11-7082, were purchased from Calbiochem (La Jolla, CA). Antibodies for phospho- and total stress-activated protein kinase/c-Jun-N-terminal kinase (SAPK/JNK), and inhibitors of NF-κB-α (IκB-α) were purchased from Cell Signaling Technology, Inc. (Boston, MA). Anti-NF-κB p65, antiIL-1β, anti-CD36, CD11b, and anti-lamin A antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-visfatin antibody was from Enzo Life Sciences. Anti-β-actin antibody was from Sigma Chemical Co. 2.2. Cell culture and drug treatment THP-1 monocytic cells (ATCC, Rockville, MD) were grown in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics. Monocyte-to-macrophage differentiation was induced using the method of Zhou et al. [21], with some modifications. Briefly, after THP-1 cells were suspended in serum-free RPMI-1640 with 10 ng/ml PMA, they were seeded onto the appropriate culture plates and then immediately stimulated with varying visfatin concentrations, followed by the addition of visfatin every 24 h (DiM; differentiating macrophages). The vehicle controls were 0.001% DMSO and sterile PBS containing 0.1% BSA for PMA and visfatin, respectively (data not shown). In another series of experiments, THP-1 cells were directly stimulated with visfatin (MO: monocytes), or THP-1 cells were pre-incubated with 10 ng/ml PMA for 2 days, after which they were stimulated with visfatin (DeM; differentiated macrophages). In the present study, we used visfatin dose ranging from 30 to 300 ng/ml similar to the previous studies [10,18]. Although plasma visfatin concentrations in patients with diseases such as atherosclerosis and inflammation are usually b 30 ng/ml [22,23], visfatin concentrations in tissues may be much higher. Indeed, there are several reports demonstrating the enhanced tissue-specific visfatin expression in pathological conditions including coronary artery disease [24], atherosclerosis [17], and acute lung injury [25]. 2.3. Immunoblot analysis Cell lysates containing equal amounts of protein were separated by SDS–PAGE and transferred to a nitrocellulose membrane (Hybond, Amersham Biosciences, Piscataway, NJ) and then incubated with specific antibodies. Horseradish peroxidase (HRP)-conjugated IgG (Santa

Cruz Biotechnology) was used as the secondary antibody. Visualization of blot was performed using ECL system (Amersham Biosciences). The membrane was re-blotted with an anti-β-actin antibody as an internal control. Band intensities were quantified using an Alpha View SA (Cell Biosciences, Santa Clara, CA). 2.4. mRNA expression analysis Total RNA was extracted from cells using an RNeasy Mini kit (Qiagen GmbH, Qiagen Str.1, Hilden) according to the manufacturer's instructions, 1 μg of which was then converted to cDNA using the M-MLV reverse transcription system (Promega, Madison, WI). Reverse transcriptase-PCR (RT-PCR) was performed by accupower RT Premix, the cDNA, and forward and reverse primers for individual genes (Table S1) using the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). Bands were visualized using the AlphaImagerR HP (Cell Biosciences). Real-time PCR was also performed using the ABI 7900 HT (Applied Biosystems). Each reaction mixture consisted of the SYBR Green Master Mix and primers for genes (Table S1). Quantitative data were analyzed using the Sequence Detection System software (SDS version 2.0, Applied Biosystems). 2.5. Flow cytometric analysis Visfatin-stimulated THP-1 cells were resuspended in fluorescenceactivated cell sorter (FACS) buffer and stained with FITC-conjugated anti-CD36 antibodies. Fluorescence was analyzed using the FACScan system (Becton Dickinson, Franklin Lakes, NJ), quantifying 1 × 104 events using gates to exclude non-viable cells. 2.6. Phagocytosis assay Cells were incubated with visfatin (300 ng/ml) in the presence of PMA (10 ng/ml) for 48 h at 37 °C, and the phagocytosis of THP-1 cells was determined using phagocytosis assay kit (Cayman Chemical Co, Ann Arbor, MI), according to the manufacturer's instructions. Positive fluorescent phagocytic cells were visualized by fluorescence microscopy to ensure that the detected fluorescence was localized within the phagocytes and were quantified using a SpectraMax M4 microplate reader (Molecular Devices, Sunnyvale, CA). 2.7. Measurement of cytokine production Cell culture media were centrifuged at 10,000×g for 1 min at 4 °C and the supernatants were analyzed using commercially available milliplex™ map kits (Millipore, Billerica, MA), according to the manufacturer's instructions. The fluorescence intensity of each well was measured using a Luminex 200™ (Luminex Corporation, Austin, Texas). 2.8. IL-1β mRNA stability assay THP-1 cells were treated with PMA (10 ng/ml) or PMA plus visfatin (300 ng/ml) for 3 h (minimum time needed for IL-1β induction; see Fig. 3B), and then exposed to actinomycin D (1 μg/ml) to block RNA synthesis. The cells were harvested at 0, 1, 2, 4, or 6 h after the addition of actinomycin D. Total RNA was extracted, and then the rates of IL-1β mRNA decay were monitored using RT-PCR. 2.9. Caspase-1 fluorometric assay Caspase-1 activity was determined using a caspase-1 fluorometric assay kit (R&D Systems, Minneapolis, MN). In brief, after the various treatments, the cells were lysed. The supernatant obtained after centrifugation at 10,000×g was mixed with an equal volume of reaction buffer and caspase-1 fluorogenic substrate in a microplate and then incubated at 37 °C for 2 h. Fluorescence was analyzed using the FLX

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800 microplate fluorescence reader (Bio-Tek Instrument Inc., Winooski, VT) with an excitation of 400 nm and emission of 505 nm.

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2.11. siRNA transfection Cells were grown to 40–60% confluence in Opti-MEM. IL-1β siRNA (Santa Cruz Biotechnology) was delivered into THP-1 cells using Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. RT-PCR and western blot were conducted at 24 h after the transfection to evaluate the silencing effect of the siRNA on IL-1β expression. Control siRNA (Santa Cruz Biotechnology) was used as the negative control.

2.10. Cell fractionation The cells were centrifuged at 300×g for 5 min, and the pellets were resuspended in hypotonic buffer for 15 min on ice. Lysates were centrifuged at 14,000×g for 30 s at 4 °C, and the resultant supernatants (cytosolic fraction) were transferred to new tubes. The resultant pellets were resuspended in extraction buffer and shaken at 4 °C for 30 min on a shaking platform at 150 rpm. The nuclear extracts were centrifuged at 14,000×g for 10 min at 4 °C, and the resultant supernatants (nuclear fractions) were frozen (−70 °C).

2.12. NF-kB (p65) transcription factor assay The binding activity of NF-κB in nuclear extracts was measured using an NF-kB (p65) transcription factor assay kit (Rockland

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Fig. 1. Effect of visfatin on CD36 expression. (A) CD36 protein expression was determined by immunoblotting of various cell extracts as described in the Materials and Methods section. (Top) Blots are representative of four independent experiments. (Bottom) Densitometric data of blots after normalization relative to β-actin expressed as the fold change versus control situation 1-fold. Mo, monocytes; DiM, differentiating macrophages; DeM, differentiated macrophages.*p b 0.05, **p b 0.01 vs. the value (1-fold) at concentration 0 in each groups (onesample t-test). (B) Surface expression of CD36 was quantified in DiM at 48 h by flow cytometry using an FITC-conjugated CD36 antibody. Numbers indicate percentages of cells expressing CD36. **p b 0.01 vs. without visfatin (Student's t-test). (C) CD36 mRNA levels in DiM were quantified by real-time PCR, normalized to GAPDH, and expressed as the fold change versus control situation 1-fold. *p b 0.05, **p b 0.01 vs. the value (1-fold) at concentration 0 (one-sample t-test). All data are presented as means ± SEM of four independent experiments.

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Immunochemicals Inc, Gilbertsville, PA), according to the manufacturer's instructions. The optical density (OD) at 450 nm was measured using a SpectraMax 250 microplate reader (Molecular Devices, Downingtown, PA).

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All data were expressed as means ± SEM of three or more individual experiments. Statistical significance was estimated by Student's t-test for unpaired observations between two groups or by ANOVA with Dunnett's post-test for comparisons of multiple groups. A one-sample t-test was used for comparison of group when control was calculated by 1-fold. P b 0.05 was regarded as significant.

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0 3. Result 3.1. Visfatin increases CD36 expression in differentiating THP-1 cells We investigated the effect of visfatin on the expression of CD36, the cell-surface marker associated with macrophage differentiation [4], in various cell types, including monocytic (Mo), differentiating (DiM), or differentiated (DeM) THP-1 cells. As shown in Fig. 1A, visfatin dosedependently caused a significant increase in CD36 protein expression in DiM cells at 48 and 72 h, but not in Mo and DeM cells. Consistent with these results, CD36 surface expression was enhanced in DiM cells with visfatin (77.9 ± 15.6%) compared to without (46.7 ± 3.3%) at 48 h (Fig. 1B). Real-time PCR analysis revealed that visfatin transiently up-regulated CD36 mRNA expression in DiM cells at 24 h (Fig. 1C). Moreover, the protein expression of CD11b, another differentiation marker [26], was increased by visfatin in differentiating THP-1 cells (Fig. S1). These results suggest that visfatin influences monocytes/macrophages differentiation. 3.2. Visfatin increases phagocytosis in differentiating THP-1 cells To further confirm the synergistic effect of visfatin on differentiation, we next determined whether visfatin-treated THP-1 cells exhibit the enhanced phagocytic capacity, a functional characteristic of macrophages [27]. As shown in Fig. 2, visfatin treatment resulted in an increased phagocytosis of beads by THP-1 cells at 48 h compared with control, indicating that THP-1 cells were co-incubated with visfatin actively displaying the function of an effective phagocyte than that treated with PMA alone. 3.3. Visfatin enhances cytokine production in differentiating THP-1 cells We next examined whether visfatin affects the production of inflammatory cytokines, including IL-1β, IL-6, and TNF-α by DiM cells. Fig. 3A shows that IL-1β secretion significantly was increased after 6 h incubation in the presence of 30 and 300 ng/ml visfatin, which remained elevated for up to 48 h. In contrast, both proteins of IL-6 and TNF-α were significantly released at 18 h after addition of 300 ng/ml visfatin, reaching a maximal level at 48 h. We also assessed the mRNA expression of these three cytokines using real-time PCR. Interestingly, the level of IL-1β mRNA started to increase at 18 h after incubation with visfatin, suggesting that the visfatin-induced initial increase of IL-1β

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Fig. 2. Effect of visfatin on phagocytosis. THP-1 cells were treated with or without visfatin (300 ng/ml) in the presence of PMA (10 ng/ml) for 48 h, and then challenged with latex beads for 2 h as described in the Materials and Methods section. (Top) Representative images of positive fluorescent phagocytes were visualized by fluorescence microscopy. (Bottom) Positive fluorescent phagocytes that had ingested fluorescence-labeled latex beads were quantified using microplate reader. Data are presented as means ± SEM from three independent experiments. **p b 0.01 vs. without visfatin (Student's t-test).

secretion was caused by a transcriptional regulation-independent mechanism. On the other hand, increased levels of IL-6 mRNA were observed at 3 and 6 h after visfatin treatment, but levels progressively decreased after 12 h, irrespective of the presence of visfatin. Later on, IL-6 mRNA levels increased again at 36 h in cells incubated with visfatin, suggesting that the visfatin-regulated late production of IL-6 might be a secondary response to some other mediator. The levels of TNF-α mRNA were significantly enhanced at 1 h in cells treated with visfatin and were sustained until 48 h. Similar to the IL-6 mRNA expression, TNF-α mRNA levels were reduced between 6 and 48 h but remained higher in cells with visfatin than in those without visfatin (Fig. 3B). These results suggest that the production of these three cytokines may be regulated by fundamentally different mechanisms.

3.4. Visfatin enhances IL-1β mRNA stability IL-1β secretion can be controlled by the induction of pro-IL-1β expression via transcriptional/post-transcriptional regulation and by pro-IL-1β processing via caspase-1 activation [27,28]. On the basis of our real-time PCR studies, it seemed likely that the early increase in IL-1β secretion was not associated with enhanced mRNA expression. Hence, we examined the effect of visfatin on IL-1β mRNA stabilization by assessing the decay kinetics of IL-1β mRNA following actinomycin D treatment using RT-PCR. Visfatin treatment markedly improved the stability of the IL-1β transcript, with 81.7% ± 7.6% of the original IL1β mRNA remaining after 6 h actinomycin D treatment relative to in the absence of visfatin (55.5% ± 4.9%) (Fig. 4A). To assess the contribution of processing pathways to visfatin-induced early IL-1β secretion, we next performed a caspase-1 assay on lysates of THP-1 cells. However there was no significant difference due to visfatin (Fig. 4B). These results suggest that visfatin may induce early IL-1β secretion by enhancing the stability of pro-IL-1β.

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Fig. 3. Effect of visfatin on cytokine production. (A) At the indicated times, cytokine secretions were analyzed in the conditioned media (CM) using commercially available Milliplex™ map. *p b 0.05, **p b 0.01 vs. corresponding without visfatin value (ANOVA with Dunnett's post-test). (B) Total RNAs were extracted from CM-subjected cells, and cytokine mRNA levels were quantified by real-time PCR. *p b 0.05, **p b 0.01 vs. corresponding without visfatin value (Student's t-test). All data are presented as means ± SEM of four independent experiments.

3.5. Visfatin-enhanced production of IL-6 and CD36 is regulated by IL-1β Because inflammatory cytokines, such as IL-1β and TNF-α, autoregulate their own expression and trigger production of secondary inflammatory mediators via autocrine or paracrine pathways [29], we determined whether visfatin-induced the early secretion of IL-1β could affect the late elevation of other cytokines. When THP-1 monocytic cells were transfected with IL-1β siRNA before being stimulated with visfatin, we found that the knockdown of IL-1β resulted in a significant reduction in IL-6 and IL-1β secretion at 48 h, but not that of TNF-α (Fig. 5A). We next tested the potential effect of released IL-1β on cytokine secretion using an IL-1 receptor antagonist (IL-1Ra). Similar to the siRNA results, pre-treatment of THP-1 cells with IL-1Ra partially inhibited the increased secretion of IL-6 and IL-1β at 48 h (Fig. 5B). Furthermore, when early-secreted IL-1β was eliminated by changing to IL1β-free fresh medium at 24 h, lower levels of IL-6 and IL-1β were observed than when the medium was unchanged for 48 h (Fig. 5C). These results suggest that the visfatin-triggered IL-1β pathway may contribute to IL-6 secretion and also to the auto-amplification of IL-1β itself in the late phase. In addition, the suppression of IL-1β with IL-1β siRNA successfully diminished CD36 expression compared to when

control siRNA was transfected into THP-1 cells, indicating the involvement of the IL-1β pathway in the visfatin-mediated up-regulation of CD36 expression (Fig. 5D). 3.6. Visfatin-induced effects are mediated by NF-κB pathway Next, we determined the effect of visfatin on activation of NF-κB, a major transcriptional regulator of pro-inflammatory cytokines [30]. Immunoblot analysis demonstrated that visfatin increased the phosphorylation of IκB-α in the cytosolic fraction with a concomitant increase the nuclear translocation of the p65 subunit of NF-κB, appearing within 30 min after the second addition of visfatin (at 24 h after first addition of visfatin) and remaining for longer than 2 h (Fig. 6A). Immunofluorescence data confirmed an increased association of p65 with the nucleus at 1 h after the second visfatin treatment (Fig. 6B). In addition, the NF-κB (p65) transcription factor ELISA assay showed that the binding activity of NF-κB was significantly enhanced in the nuclear extracts from visfatin-stimulated THP-1 cells, which was attenuated by the knockdown of IL-1β with IL-1β siRNA (Fig. 6C and D). These results suggest that visfatin-triggered early IL-1β release is involved in the NF-κB activation. This pathway may regulate the IL-1β release-dependent

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120 Reportedly, exogenous visfatin has been shown to participate in inflammatory processes via its NAMPT enzymatic activity [14,31]. Thus, we asked if NAMPT enzymatic activity mediates the pro-inflammatory action of visfatin during the differentiation of THP-1. To address this question, we indirectly checked whether NAMPT activity is implicated in visfatin-induced effects using FK866, a pharmacological inhibitor of NAMPT activity. FK866 inhibited in part NF-κB activation and late secretion of IL-1β in response to visfatin (Fig. 8) but failed to prevent the initial secretion of IL-1β as well as IL-6 and CD36 (data not shown). These result indicated that NAMPT enzymatic activity may be involved in the visfatin-enhanced late auto-amplification of IL-1β via NF-κB pathways.

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Time (h) Fig. 4. Effect of visfatin on the stability of IL-1β mRNA. THP-1 cells were treated with visfatin as described in the Materials and Methods section. (A) At the indicated time points after addition of actinomycin D, IL-1β mRNA expression was quantified by RT-PCR and normalized to GAPDH. All values were normalized to 100% at the 0-h time point. (B) At the indicated times, the caspase-1 activity was measured by a fluorometric assay of whole lysates. Data are presented as means ± SEM of three independent experiments. *p b 0.05, **p b 0.01 vs. corresponding without visfatin value (Student's t-test).

late production of cytokines. Indeed, in the late phase, pre-treatment with Bay-117082, an NF-κB inhibitor, completely abrogated TNF-α secretion as well as the increased secretion of IL-6 and IL-1β (Fig. 6E). The inhibition of NF-κB also strongly blocked visfatin-increased CD36 expression, indicating the involvement of the IL-1β/NF-κB pathways in the visfatin-mediated up-regulation of CD36 expression (Fig. 6F).

3.7. JNK is partially involved in visfatin-mediated production of IL-1β, IL-6 and CD36 To identify the signal transduction pathway(s) involved in the production of the cytokines, several specific pharmacologic inhibitors, including MAPK inhibitors (PD98059, an ERK inhibitor; SB203580, a p38 inhibitor; and SP600125, a JNK inhibitor) and PI3-kinase inhibitor (LY294002), were tested. As shown in Fig. 7A, visfatin-elevated IL-1β secretion was significantly blocked by JNK inhibitor, but not other inhibitors. Interestingly, visfatin-elevated IL-6 secretion was partially diminished by inhibitors of JNK and ERK, respectively. For TNF-α production, however, none of the inhibitors tested had significant effects (Fig. 7A). We also observed that JNK inhibitor notably reduced visfatin-mediated CD36 up-regulation (Fig. 7B), but other inhibitors had no effect (data not shown). In line with these results, visfatin caused JNK phosphorylation, but not ERK (Fig. S2). Collectively, these results suggest that JNK might participate in the regulation of visfatininduced production of IL-1β, IL-6, and CD36.

We demonstrated for the first time that visfatin can induce the IL1β-dependent production of IL-6 and CD36 through distinct mechanisms mediated by JNK and NF-κB, which might accelerate monocytes/macrophages differentiation, causing the development of atherosclerosis. Monocyte-to-macrophage differentiation is an important step in early atherogenesis [2]. PMA-treated THP-1 cells are commonly used to study the differentiation of monocyte/macrophage, which is characterized by increased cell adherence, a distinct morphology, and specific surface markers [32]. Using PMA on THP-1 monocytic cell line, we designed three experimental cell groups that included monocytic, differentiating, or differentiated cell types and evaluated the role of visfatin on differentiation marker expression in these three groups. We found that visfatin up-regulated the expression of surface markers, including CD36 (Fig. 1) and CD11b (Fig. S1) in only differentiating THP-1 cells. It is widely recognized that CD36 is differentially regulated during the late stages of monocyte differentiation [4] and CD11b is also associated with both monocyte maturation and differentiation to macrophages [26]. We also observed that visfatin resulted in a dramatic increase of PMA-induced cell adhesion in a dose-dependent manner. To confirm this finding, we verified dynamic monitoring of cell adhesion using electrical impedance cell sensor technology. Under experimental conditions as described in the Materials and Methods section, the elevation of cell adhesion by visfatin exposure appeared after 15 min of stimulation, reaching a maximal level about 1 h and sustaining until 2 h (Fig. S3). Moreover, visfatin exposure to THP-1 cells exhibited the enhanced phagocytic capacity as macrophage functionality (Fig. 2). Similar to our results in THP-1 cells, both CD36 protein levels and phagocytic ability for latex beads were markedly enhanced in bone marrow-derived monocytes were co-treated with visfatin when compared with that incubated with M-CSF alone (Fig. S4), suggesting that visfatin may cause a synergistic effect on the differentiation of monocytes into macrophages. Visfatin, a multifunctional adipocytokine [33], modulates inflammatory cytokines in endothelial cells [15,16], smooth muscle cells [13,14], and monocytes [18], which play an important role in the pathogenesis of inflammation-related atherosclerosis [9]. Recent studies reported that visfatin was detected in macrophage within unstable atherosclerotic lesions, where it exerts a baneful effect in plaque destabilization [10,17]. IL-1β, IL-6, and TNF-α are elevated during monocytes/macrophages differentiation and are key inflammatory cytokines in the progression and destabilization of plaques [3]. Similar to previous reports, our present study showed that visfatin increased production of IL-1β, IL-6, and TNF-α in differentiating THP-1 cells (Fig. 3). Intriguingly, our results revealed two novel observations. First, visfatin increased IL-1β secretion at an earlier time point than IL-6 and TNF-α, which was associated with increased IL-1β mRNA stability. IL-1β secretion requires the induction of IL-1β production by transcriptional or post-transcriptional regulation and caspase-1-activation by inflammasomes [28]. However, in our study, visfatin failed to induce both the enhanced pro-IL-1β mRNA levels and caspase-1 activation at the same time or before

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Fig. 5. Effect of interfering with IL-1β on visfatin-induced cytokine secretion and CD36 expression. (A) IL-1β siRNA or a negative control (NC) siRNA were transfected into THP-1 cells and incubated with visfatin (300 ng/ml) for 48 h; cytokine secretion was then measured by Milliplex™ map. Inset in A shows representative immunoblots and RT-PCR of IL-1β expression in cells transfected with IL-1β siRNA for 24 h, respectively. Data are presented as means ± SEM of five independent experiments. **p b 0.01 vs. without visfatin in each group (Student's ttest), #p b 0.05, ##p b0.01 vs. with visfatin in did not transfected (NT) (ANOVA with Dunnett's post-test). (B) In IL-1Ra-pre-treated THP-1 cells, cytokine secretion was measured by Milliplex™ map. **p b 0.01 vs. without visfatin in each group (Student's t-test), #p b 0.05, ##p b 0.01 vs. with visfatin in each group (ANOVA with Dunnett's post-test). (C) After the stimulation of THP-1 cells with visfatin (300 ng/ml) for 24 h, visfatin was added to the cells with (black) or without (white) a fresh medium change. At 48 h of total incubation, cytokine secretion was measured by Milliplex™ map. **p b 0.01 vs. without medium change in each group (Student's t-test). (D) CD36 expression in THP-1 cells transfected with IL-1β siRNA was detected at 48 h after the incubation of visfatin by immunoblotting. (Left) Blots are representative of four independent experiments. (Right) Densitometric data of blots after normalization relative to β-actin expressed as the fold change versus without visfatin in NT situation 1-fold. **p b 0.01 vs. without visfatin in each group (one-sample t-test or Student's t-test), ##p b 0.01 vs. with visfatin in NT (ANOVA with Dunnett's post-test).

point started to increase IL-1β secretion. Thus, we focused on posttranscriptional regulation as a possible mechanism for the increase of early IL-1β secretion because post-transcriptional control mechanisms have been showed to affect cytokine production by regulating decay and translation of cytokine transcripts [34,35]. Fig. 4 showed that visfatin decreased the decay of pre-existing pro-IL-1β mRNA following actinomycin D addition, but not in terms of different amounts of available mRNA for pro-IL-1β. Moreover, it has recently been demonstrated that IL-1β secretion could be controlled, at least in part, by the synthesis of its precursor without activating of caspase-1 or distinct release pathway such as noncytolytic, noncaspase-1, and process for the release of pro-IL-1β [36]. Abdalla et al. [37] also reported that a pathogen-infected dendritic cells leads to partially caspase-1-independent, but

inflammasome components, NLRP3- and ASC-dependent IL-1β secretion. In light of these finding, visfatin may increase the early secretion of IL-1β by regulating the stability of IL-1β mRNA and caspase-1independent release pathway. Second, the early IL-1β release participated in visfatin-induced IL-6 secretion and CD36 expression as well as the late IL-1β secretion. These findings are supported by other reports in which IL-1β induces the production of IL-6, or auto-regulates its own expression via autocrine and paracrine signals [29]. Our results also agree with a report that CD36 expression correlates positively with IL1β [6]. Moreover, Kirii et al. [38] reported that IL-1β−/−/ApoE−/−mice exhibited a significant decrease in atherosclerosis compared with that seen in IL-1β-expressing ApoE deficient mice. Considering our results with other reports, it seems that visfatin-evoked initial IL-1β may be

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closely and critically associated with acceleration of monocytes/macrophages differentiation. NF-κB is an important transcription factor that regulates the expression of a large number of genes involved in the inflammatory response [30] and is also linked to CD36 signaling [39]. Unexpectedly, we found that visfatin-triggered NF-κB activation was observed at later time points than those at which mRNA levels of the three cytokines increased. These results suggest that NF-κB may be a secondary response that is indirectly modulated through the downstream activity of the early-released cytokines. Indeed, the knockdown of IL-1β with IL-1β siRNA significantly attenuated the visfatin-induced nuclear binding

activity of NF-κB (Fig. 6D). Interestingly, in the late phase, NF-κB inhibitor completely abrogated the increase in IL-6 secretion and CD36 expression and also partially diminished IL-1β secretion (Fig. 6E and F), but not that in the early phase (Data not shown). It is generally agreed that inflammatory cytokines such as IL-1β and TNF-α can rapidly induce NF-κB activation and subsequently cause the up-regulation of NF-κBdependent genes [40]. These results suggest that the early IL-1β release-mediated NF-κB activation in part contribute to the visfatininduced production of IL-6 and CD36. However, the transcription factors involved in the early expression of IL-6 mRNA still remain unclear. According to previous reports, IL-6 is regulated by several transcription

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factors in a highly stimulus-specific or cell-specific manner [41]. In addition, although visfatin increased the TNF-α production, we did not identify the signaling pathways involved in TNF-α production other than NF-κB. Thus, further experiments are necessary to determine the possible signal transduction pathways that regulate IL-6 and TNF-α. Several lines of evidence indicate that mitogen-activated protein kinases (MAPKs) also contribute to the control of the expression of inflammatory cytokines during the inflammatory process [42]. A previous report noted that inhibitors of p38 and MEK1 prevented the visfatin-induced production of IL-1β, IL-6, and TNF-α in monocytes [18]. However, in the present study using differentiating THP-1 cells we clearly demonstrated that only the JNK inhibitor significantly suppressed the increased secretion of IL-6 and late IL-1β and CD36 expression during long-term exposure of THP-1 cells to visfatin (Fig. 7). Moreover, similar to NF-κB activation, the phosphorylation of JNK was increased in a time-dependent manner after the second visfatin treatment (Fig. S2). JNK is reported to be responsible for AP-1 transcriptional activity and PMA-induced IL-1β production in THP-1 cells is mediated by AP-1 [43]. However, in our study, visfatin failed to induce AP-1 activation (data not shown). These results suggest that JNK may play a role as a secondary mediator of some signaling pathways rather than transcriptional regulator in mediating visfatin-induced cytokine production. Recently, exogenous visfatin has been shown to participate in inflammatory processes via its NAMPT enzymatic activity [14]. In our study, pre-treatment of FK866, an NAMPT inhibitor, significantly inhibited the visfatin-elevated late secretion of IL-1β and NF-κB

activation, but not early IL-β secretion (Fig. 8). Moreover, FK866 failed to prevent the increased production of IL-6 and CD36 in response to visfatin (data not shown). These results suggest that endogenous NAMPT may be involved in the auto-amplification of IL-1β via the NFκB pathway in the late phase. It is still unknown whether the exogenous visfatin initiate the inflammatory action via cellular surface receptor or intracellular target. Although Fukuhara et al. [7] reported that visfatin exert pathophysiological actions through binding to the insulin receptor (IR), such a statement was later retracted and is still debatable [14,18,44,45]. Thus, we first measured both intracellular and extracellular visfatin levels after visfatin treatment in THP-1 cells in the presence of PMA by modified methods of Park et al. [46]. After visfatin treatment for 1 h, visfatin levels in medium, but not in cell lysates, were increased compared with that without visfatin (Fig. S5A and B). However, the responses such as TNF-α mRNA expression (Fig. 3B) and cell adhesion (Fig. S3) already occurred within 1 h after visfatin treatment. We also observed that visfatin failed to cause the phosphorylation of IRS-1/2 and Akt, IR signaling pathways (Fig. S5C), and LY294002, a PI3-kinase inhibitor, had also no significant in visfatin-elevated cytokine secretion (Fig. 7A). These results suggested that the biological effects by visfatin in the present study may occur via unidentified receptor. It is considered that further study is required to identify the hypothetical receptor of visfatin. In conclusion, visfatin regulates the IL-1β-elicited induction of CD36 and IL-6 via separate pathways of NF-κB and JNK, indicating a novel mechanism by which visfatin affects the differentiation of monocytes into

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macrophages (Fig. 9). Hence, these findings represent a potential novel therapeutic approach for the early stages of atherosclerosis. Acknowledgments This work was supported by the Korea National Institute of Health intramural research grant (2010-N63003-00). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cellsig.2013.12.010. References [1] [2] [3] [4] [5] [6] [7]

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