Regulation of A-FABP expression by JNK _____________________________________________________________________________________ 1 Adipocyte Fatty Acid Binding Protein Modulates Inflammatory Responses in Macrophages through a Positive Feedback Loop Involving C-Jun N-terminal kinases and Activator Protein-1 Xiaoyan Hui 1,3# , Huiying Li 2,3# , Zhiguang Zhou 4 , Karen S L Lam 1,3 , Yang Xiao 4 , Donghai Wu 5 , Ke Ding 5 , Yu Wang 2,3 , Paul M. Vanhoutte 2,3 , Aimin Xu 1,2,3 * 1 Department of Medicine, 2 Department of Pharmacology and Pharmacy, 3 Research Center of Heart, Brain, Hormone, and Healthy Aging, the University of Hong Kong, Hong Kong; 4 Diabetes Center, Metabolic Syndrome Research Center, Institute of Metabolism and Endocrinology, Second Xiangya Hospital, Central South University, Changsha, China; 5 Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences. Guangzhou, China #. The authors contribute equally to the work. Running head: Regulation of A-FABP expression by JNK Address correspondence to: Aimin Xu, Ph D, L8-40, New Laboratory Block, 21 Sassoon Road, Hong Kong. Tel: 852-28199754; Fax: 852-28162095; E-mail: [email protected]SUMMARY Adipocyte fatty acid binding protein (A-FABP) has emerged as an important mediator of inflammation in macrophages. Macrophage-selective ablation of A-FABP alone is sufficient to prevent the development of high cholesterol diet-induced atherosclerosis in apoE-deficient mice. However, the precise mechanisms whereby A-FABP modulates inflammation remain elusive. Here we report that A-FABP forms a finely-tuned positive loop between c-Jun NH2-terminal kinases (JNK) and activator protein-1 (AP-1) to exacerbate LPS-induced inflammatory responses in macrophages. Real-time PCR and luciferase reporter analysis showed that lipopolysaccharide (LPS) induced A-FABP expression through transcriptional activation. This effect was mediated by (JNK), which promoted the recruitment of c-Jun to a highly conserved AP-1 consensus binding motif located within the proximal region of the A-FABP promoter. LPS-induced transactivation of the A-FABP gene was diminished by either pharmacological inhibition of JNK or knocking down c-Jun, or by mutating the AP-1 recognition site within the proximal region (-122 to -116 bp) of the A-FABP promoter.. Vice versa, The LPS-evoked phosphorylation of JNK, activation of AP-1 and production of pro-inflammatory cytokines were markedly attenuated by pharmacological or genetic suppression of A-FABP in macrophages. Furthermore, the LPS-induced elevation in A-FABP expression could also be prevented by the selective A-FABP inhibitor BMS309403. These findings support the notion that pharmacological inhibition of A-FABP represents a valid strategy for treating inflammation-related disorders such as atherosclerosis. http://www.jbc.org/cgi/doi/10.1074/jbc.M109.097907 The latest version is at JBC Papers in Press. Published on February 9, 2010 as Manuscript M109.097907 Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on April 8, 2019 http://www.jbc.org/ Downloaded from
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Regulation of A-FABP expression by JNK _____________________________________________________________________________________
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Adipocyte Fatty Acid Binding Protein Modulates Inflammatory Responses in Macrophages through a Positive Feedback Loop Involving C-Jun N-terminal
kinases and Activator Protein-1
Xiaoyan Hui1,3#, Huiying Li 2,3#, Zhiguang Zhou4, Karen S L Lam1,3, Yang Xiao4, Donghai Wu5, Ke Ding5, Yu Wang2,3, Paul M. Vanhoutte2,3, Aimin Xu1,2,3*
1 Department of Medicine, 2 Department of Pharmacology and Pharmacy, 3 Research Center of Heart, Brain, Hormone, and Healthy Aging, the University of Hong Kong, Hong Kong; 4 Diabetes Center, Metabolic Syndrome Research Center, Institute of Metabolism and Endocrinology, Second Xiangya Hospital, Central South University, Changsha, China; 5 Key Laboratory of Regenerative Biology,
Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences. Guangzhou, China
#. The authors contribute equally to the work.
Running head: Regulation of A-FABP expression by JNK
Address correspondence to: Aimin Xu, Ph D, L8-40, New Laboratory Block, 21 Sassoon Road, Hong Kong. Tel: 852-28199754; Fax: 852-28162095; E-mail: [email protected]
SUMMARY
Adipocyte fatty acid binding protein (A-FABP) has emerged as an important mediator of inflammation in macrophages. Macrophage-selective ablation of A-FABP alone is sufficient to prevent the development of high cholesterol diet-induced atherosclerosis in apoE-deficient mice. However, the precise mechanisms whereby A-FABP modulates inflammation remain elusive. Here we report that A-FABP forms a finely-tuned positive loop between c-Jun NH2-terminal kinases (JNK) and activator protein-1 (AP-1) to exacerbate LPS-induced inflammatory responses in macrophages. Real-time PCR and luciferase reporter analysis showed that lipopolysaccharide (LPS) induced A-FABP expression through transcriptional activation. This effect was mediated by (JNK), which promoted the recruitment of c-Jun to a highly conserved AP-1 consensus binding motif located within
the proximal region of the A-FABP promoter. LPS-induced transactivation of the A-FABP gene was diminished by either pharmacological inhibition of JNK or knocking down c-Jun, or by mutating the AP-1 recognition site within the proximal region (-122 to -116 bp) of the A-FABP promoter.. Vice versa, The LPS-evoked phosphorylation of JNK, activation of AP-1 and production of pro-inflammatory cytokines were markedly attenuated by pharmacological or genetic suppression of A-FABP in macrophages. Furthermore, the LPS-induced elevation in A-FABP expression could also be prevented by the selective A-FABP inhibitor BMS309403. These findings support the notion that pharmacological inhibition of A-FABP represents a valid strategy for treating inflammation-related disorders such as atherosclerosis.
http://www.jbc.org/cgi/doi/10.1074/jbc.M109.097907The latest version is at JBC Papers in Press. Published on February 9, 2010 as Manuscript M109.097907
Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
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Fatty acid binding proteins (FABPs) are a family of small intracellular lipid chaperons that reversibly bind hydrophobic ligands, such as long chain fatty acids and eicosanoids, facilitating the intracellular diffusion of fatty acids between cellular compartments and enzymes (1,2). Adipocyte fatty acid binding protein (A-FABP, also known as aP2 and FABP4) is a member of the FABP family which is abundantly expressed in adipose tissue (1,2). In adipocytes, A-FABP regulates fatty acid storage and lipolysis. The latter effect is mediated by a direct interaction with hormone-sensitive lipase (HSL) (3,4). Mice deficient in A-FABP are partially protected against insulin resistance, dyslipidemia and fatty liver disease in both genetic and dietary obesity (5-7).
In addition to its role in regulating lipid metabolism and insulin sensitivity, mounting evidence suggests that A-FABP is also a key player in inflammation. A-FABP is highly expressed in macrophages, where it regulates cholesterol ester accumulation and inflammatory responses (8). Macrophage-specific A-FABP deficiency protects against both early and advanced atherosclerosis in apolipoprotein E-deficient (ApoE-/-) mice (8,9). This protection is comparable to that observed in ApoE-/- mice with total A-FABP deficiency, suggesting that the macrophage-specific actions of A-FABP are a predominant contributor to atherosclerotic lesion formation. At the molecular level, A-FABP deficiency in macrophages results in decreased production of a cluster of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)α, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1 and IL-1β. These findings are corroborated by the observation that pharmacological inhibition of A-FABP by its selective inhibitors protects mice against atherosclerosis and compromises inflammatory responses in macrophage cells
(10,11). In addition, the pivotal role of A-FABP in inflammation is highlighted by the findings that A-FABP deficient mice are resistant to develop several other inflammatory disorders, including allergic airway inflammation (12) and experimental autoimmune encephalomyelitis /multiple sclerosis (13).
Although initially thought to be an intracellular protein, recent studies showed that A-FABP is also secreted into the bloodstream in rodents and human (14,15). The circulating concentration of A-FABP significantly correlates with several features of the metabolic syndrome, including central adiposity, dyslipidemia, fasting glucose, the indices of insulin resistance and markers of inflammation (16,17). Furthermore, an elevated level of serum A-FABP has been observed in a number of obesity-related inflammatory diseases, including type 2 diabetes, atherosclerosis, coronary heart disease and nonalcoholic fatty liver disease (NAFLD) (15,18). A-FABP released from adipocytes suppresses cardiomyocyte contractile activity, implicating that it also functions as an endocrine factor (19). Taken in conjunction, these clinical and experimental data suggests that A-FABP might serve as an important mediator linking obesity with metabolic and cardiovascular diseases, partly through its pro-inflammatory actions in macrophages.
In macrophages, several pro-inflammatory stimuli have been shown to induce A-FABP expression, including phorbol 12-myristate 13-acetate (PMA), oxidized low-density lipoproteins and toll-like receptor (TLR) agonists (8,20-22). In particular, lipopolysaccharide (LPS), a major agonist of TLR4, is a potent inducer of A-FABP production in macrophages (21). TLR4, a key player in the innate immune system, is also actively involved in the etiology of obesity
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related disorders such as insulin resistance and atherosclerosis (23). Interestingly, an earlier report demonstrated that LPS-induced pro-inflammatory responses are decreased in macrophages derived from A-FABP knockout mice (8). However, the cellular mechanisms whereby LPS increases A-FABP expression and the precise role of A-FABP in LPS-induced pro-inflammatory pathways remain to be established.
To address these questions, we investigated the detailed signaling events underlying LPS-induced A-FABP expression in murine macrophages. Our results demonstrated that LPS induces the transactivation of the A-FABP gene through c-Jun NH2-terminal kinases (JNK), which in turn promotes the recruitment of c-Jun to a highly conserved activator protein-1 (AP-1) recognition site within the proximal region (-122 to -116 bp) of the A-FABP promoter. Interestingly, we found that A-FABP potentiates LPS-induced activation of JNK/AP-1 signaling pathway, suggesting that it controls an auto-regulatory loop aggravating the pro-inflammatory responses in macrophages.
EXPERIMENTAL PROCEDURES
Reagents⎯ The antibody against c-Jun and siRNA against mouse c-Jun and scrambled siRNA were from Santa Cruz Biotechnology (Delaware, CA, USA) and antibodies against β-tubulin, phospho-JNK and total JNK1 and ChIP grade protein G beads were from Cell Signaling Technology (Beverly, MA,USA). Anti-mouse A-FABP was produced in house by immunizing female New Zealand rabbits with recombinant mouse A-FABP expressed in E. coli, and was affinity purified as described.(24) Trizol Reagent, SYBR Green and cell culture medium were purchased from Invitrogen (Carlsbad, CA, USA). Superscript first-strand
cDNA synthesis system was purchased from Promega (Madison, WI, USA). Streptavidin agarose beads and LPS (E. coli 026:B6) were from Sigma-Aldrich (St. Louis, MO,USA). The AP-1-luc reporter plasmid was obtained from Stratagene (La Jolla, CA, USA). The A-FABP inhibitor BMS309403 was synthesized as described (25) and dissolved in dimethyl sulfoxide (DMSO).
Construction of luciferase reporter vectors driven by the mouse A-FABP promoters⎯ The mouse A-FABP promoter/enhancer regions spanning from -5160 bp to +22 bp and -962 bp to +22 bp were subcloned into pGL3-Basic vector (Promega) to obtain the 5kb-luc reporter vector and 1kb-luc reporter vector respectively. The putative AP-1 binding site was mutated by mutagenesis PCR using their wild type vectors as the templates. The constructs were confirmed by DNA sequencing. The sequences of all the primers used for the vector construction are listed in Supplemental Table 1.
Transient transfection and luciferase assay⎯ The murine macrophage cell line RAW 264.7 was obtained from American Type Culture Collection (Manassas, VA,USA) and cultured in DMEM supplemented with 10% FBS. Cells were co-transfected with the luciferase reporters and siRNA (50 μM) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, followed by treatment with various concentrations of LPS for different periods as specified in figure legends. Afterwards, cells were lysed in Reporter Lysis Buffer and the luciferase activity was measured using Bright-GloTM Luciferase Assay System (Promega) as described (26). The siRNA sequences for A-FABP are listed in supplemental Table 2. A plasmid encoding a constitutively active JNK (CA JNK)(27) was introduced into cells by electroporation using Genepulser Xcell (Bio-Rad, Hercules, CA,USA).
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Quantitative real-time PCR⎯ Total RNA was extracted from RAW 264.7 cells using Trizol reagent. 1 μg of total RNA was reverse transcribed into cDNA using ImProm-II Reverse. Each cDNA sample was analyzed for gene expression by quantitative real-time PCR using the SYBR Green reagent on an Applied Biosystems Prism 7000 sequence detection system as described.(24) Analysis was performed with ABI Prism 7000 SDS Software. Sequences of the primers used for real time PCR are listed in Supplemental Table 1.
Preparation of nuclear extracts⎯ Nuclear extracts were prepared as previously described (28) with minor modifications. In brief, 80–90% confluent RAW 264.7 cells were harvested by scraping, washed in cold PBS, and incubated in three packed cell volumes of hypertonic buffer (10 mM HEPES, pH 8.0, 1.5 mM MgCl2, 200 mM sucrose, 0.5% Nonidet P-40, 10 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol plus protease and phosphatase inhibitors) for five minutes at 4 °C. The crude nuclei were collected by microcentrifugation, rinsed twice in hypertonic buffer, and resuspended in lysis buffer (PBS, pH 7.4, 1.0 mM EDTA, 2.5% glycerol, 1.0 mM dithiothreitol plus protease and phosphatase inhibitors). Nuclei were disrupted by sonication at 4 °C. The debris was removed by centrifugation at 4°C and 13,500 g for 10 minutes.
In vitro DNA-protein binding assay⎯ Binding of c-Jun to the A-FABP promoter DNA was assayed as described previously (29). Biotin-labeled oligonucleotide primers were synthesized by Invitrogen (CA, Carlsbad, USA) and used to amplify a 200-bp region in the mouse A-FABP promoter (-217 to -27) containing the putative AP-1 cis element. The forward and reverse primer pairs used are listed in supplemental table 1. The binding assay was
performed by mixing 500 μg of nuclear extract proteins purified as above, 5 μg of biotin-labeled DNA and 30 μl of streptavidin agarose beads for one hour at room temperature. Beads were then pelleted and washed four times with cold PBS. The binding proteins were eluted by boiling in 50 μl of SDS-PAGE loading sample buffer and separated on 10% SDS-PAGE, followed by Western blot analysis.
Chromatin immunoprecipitation (ChIP) ⎯The ChIP assay was performed as described (30) with minor modifications. 80–90% confluent RAW 264.7 cells were treated with or without LPS for various time periods, fixed with 1% formaldehyde for 15 min at room temperature. Cells were lysed and chromatin was sheared by sonication at 4°C. 25 μg of the lysates were incubated overnight at 4°C with 2 μg of anti-c-Jun antibody or rabbit non-immune IgG as negative control, followed by precipitation with ChIP grade protein G beads. The precipitates were washed extensively and eluted with the elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature for 15 min. Input chromatin and immunoprecipitated chromatin were incubated at 65°C overnight to reverse the crosslinks. After digestion with protease K, DNA was extracted with phenol-chloroform and precipitated with ethanol. Purified DNA was analyzed by quantitative real time PCR using specific A-FABP promoter primers as listed in supplemental table 1. All results were normalized to the respective input values.
Western blot analysis⎯60 μg of RAW 264.7 cell lysates were separated by SDS-PAGE, and probed with various primary antibodies as indicated. The proteins were visualized by chemiluminescence detection, and the relative band densities were quantified using the MultiAnalyst software package (Bio-Rad) as described(24).
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Statistical analysis⎯Data are expressed as mean ± standard deviation (SD). Statistical significance was determined by one-way ANOVA or student’s t test. In all statistical comparisons, a P value less than 0.05 was accepted to indicate a statistically significant difference.
RESULTS
LPS induces A-FABP expression through transcriptional activation in macrophages⎯ Quantitative real-time PCR analysis showed that treatment of RAW 264.7 murine macrophage cells with LPS significantly increased the mRNA abundance of the A-FABP gene in a time- and concentration-dependent manner (Figure 1A and 1B). The mRNA abundance of A-FABP was increased approximately three and seven fold respectively following stimulation with LPS at 1 ng/ml and 100 ng/ml for 24 hours. The stimulatory effect of LPS on A-FABP mRNA expression was slightly attenuated when its concentration was further increased to above 1000 ng/ml. Consistent with the changes in A-FABP mRNA abundance, the protein level of A-FABP in RAW 264.7 murine macrophages was also elevated following LPS stimulation (Figure 1C). Induction of A-FABP mRNA and protein expression by LPS was also observed in human THP1 macrophages (data not shown).
In the absence of LPS, treatment of RAW 264.7 macrophages with the transcription inhibitor actinomycine D for a period of 12 hours did not cause significant change in A-FABP mRNA abundance, suggesting that A-FABP mRNA is highly stable in this cell line (Online supplemental Figure 1). However, the LPS-induced elevation of A-FABP mRNA was abolished when cells were incubated with actinomycin D, suggesting that the stimulatory
effect of LPS on A-FABP mRNA expression in macrophages is due to the increased transcriptional activation, but not due to the enhanced mRNA stability.
LPS-evoked transactivation of the A-FABP gene is mediated by a putative AP-1 recognition site located between -122 to -116 bp of the promoter⎯To further elucidate the molecular events by which LPS induces the transactivation of the A-FABP gene, we constructed two luciferase reporter vectors driven by the 1 kb and 5 kb of the mouse A-FABP promoter, namely 1kb-luc and 5kb-luc. These reporter vectors were transfected into RAW 264.7 cells to test their response to LPS stimulation. This analysis revealed that LPS increased the luciferase activity driven by both 1 kb and 5 kb A-FABP gene promoter (online supplemental Figure 2). Noticeably, time- and concentration-dependent studies showed that the kinetic changes of the luciferase activity driven by both 1 kb and 5 kb A-FABP promoters mirrored the changes in A-FABP mRNA abundance in response to LPS stimulation (Figure 1). The maximal promoter activity was observed at 24 hours after stimulation with 100 ng/ml LPS. The magnitude of LPS-mediated increase of the luciferase activity was comparable between the 1 kb and 5 kb A-FABP promoter, suggesting that the LPS-responsive DNA element is located within the 1 kb promoter region of the A-FABP gene.
To map precisely the minimal cis-DNA element that mediates LPS-induced A-FABP expression, we analyzed potential transcription factor binding sites within the 1 kb promoter region of the mouse A-FABP gene using Promoter 2.0 Prediction Server (http://www.cbs.dtu.dk/services/Promoter/). This analysis identified a 7-bp DNA motif located between -122 and -116 bp of the mouse A-FABP gene promoter (Figure 2A) that
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matches the consensus binding sequence for AP-1, a transcription factor that plays a key role in mediating the inflammatory responses and production of pro-inflammatory cytokines(31). Noticeably, this putative AP-1 binding site is highly conserved among eight mammalian species.
To investigate whether or not the putative AP-1 recognition site is indeed a critical cis-element mediating LPS-induced transactivation of the A-FABP gene, we generated two mutant luciferase reporter vectors, 1kbmu-luc and 5kbmu-luc, in which three nucleotides within the motif were mutated from 5’-TGACTCA-3’ to 5’-TGACCTG-3’ (Figure 2A). In RAW 264.7 macrophage cells, the luciferase activity driven by either 1 kb or 5 kb mutated promoter was decreased by approximately 90% compared to their wild type form under the basal condition (Figure 2B and 2C). Furthermore, the mutation of these three nucleotides within the putative AP-1 cis-element led to an almost complete abolition of LPS-induced increase in luciferase activity, suggesting that the putative AP-1 recognition motif between -122 and -116 bp of the A-FABP promoter is indispensible for both basal and LPS-induced A-FABP expression in macrophage cells.
LPS induces the recruitment of c-Jun to the AP-1 binding site within the A-FABP promoter⎯ The transcription factor AP-1 is a heterodimeric protein consisting of the Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra1, and Fra2) families (32). We next investigated whether c-Jun mediates LPS-induced A-FABP expression through its interaction with the AP-1 recognition motif using a streptavidin pull-down assay (29). This analysis showed that the double-stranded DNA fragment spanning the sequence from -217 to -27 in the A-FABP promoter was bound to
c-Jun derived from nuclear extracts of the macrophage cells, and that the interaction was further increased by more than six-fold upon stimulation with LPS (Figure 3A). By contrast, this interaction was abrogated by mutation of the three nucleotides within the AP-1 recognition motif, suggesting that c-Jun is indeed physically associated with the 7-bp cis-DNA element within the A-FABP promoter.
To further confirm the binding of c-Jun to the endogenous A-FABP gene promoter, RAW 264.7 cells were treated with LPS for various periods and subjected to ChIP assay using rabbit anti-c-Jun antibody or non-immune rabbit IgG as a negative control (Figure 3B). Quantitative real-time PCR analysis showed that the soluble chromatin samples obtained from each time point (input) had equal amounts of chromatin fragments containing the A-FABP promoter. The association between c-Jun and the endogenous A-FABP promoter was detectable in untreated cells, started to increase at 15 minutes and peaked at 30 minutes after LPS stimulation, after which the association was gradually diminished. This result suggests that LPS induces the recruitment of c-Jun to the endogenous A-FABP promoter in a temporal manner.
LPS-induced A-FABP expression is dependent on the JNK/AP-1 signaling cascade⎯ We next investigated whether c-Jun is directly involved in LPS-induced A-FABP expression by knocking down its expression in RAW 264.7 cells. Transfection of c-Jun siRNA for 24 hours led to an over 50% decrease in the protein level of c-Jun compared to that transfected with the scrambled control (Figure 4A). Knockdown of c-Jun expression by siRNA inhibited A-FABP mRNA expression by 36% in untreated cells and 48% in LPS-treated cells respectively (Figure 4B and 4C). Accordingly, both basal and LPS-stimulated promoter activity of the
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A-FABP gene was significantly reduced in cells transfected with c-Jun siRNA.
The c-Jun NH2-terminal kinase (JNK), also known as stress-activated MAP kinases (SAPK) enhance AP-1-dependent transcriptional activity of c-Jun by phosphorylating c-Jun at Ser-63 and Ser-73 (33,34). We next tested the role of JNK in regulating A-FABP expression. Inhibition of JNK by its chemical inhibitor SP600125 markedly suppressed both basal and LPS-stimulated A-FABP expression (Figure 5A). Furthermore, the ectopic expression of a constitutively active form of JNK alone was sufficient to increase A-FABP mRNA expression even in the absence of LPS stimulation (Figure 5B). Taken together, these data suggest JNK as a key mediator in LPS-induced A-FABP expression in macrophages.
A-FABP mediates LPS-induced activation of JNK/AP-1 signaling cascade and production of pro-inflammatory cytokines⎯ A number of recent studies demonstrate that A-FABP is an important player in inflammation, actively participating in the pro-inflammatory responses evoked by various stimuli (21,22,35). We therefore speculate that there might be a positive feedback loop between A-FABP and JNK/AP-1, which exacerbates LPS-induced inflammation in macrophages. We tested this hypothesis by knocking down A-FABP expression in RAW 264.7 cells. Transfection of A-FABP specific siRNA for 24 hours resulted in an approximately 50% reduction in A-FABP expression compared to scrambled control (Figure 6A). This decrease in A-FABP expression was accompanied by a significant reduction in LPS-induced phosphorylation of JNK, activation of the AP-1 transcriptional activity and secretion of a panel of pro-inflammatory cytokines, including TNFα, IL6 and MCP-1 (Figure 6B-D).
Treatment of RAW 264.7 cells with BMS309403, a selective inhibitor of A-FABP,(10) also attenuated phosphorylation of JNK (Figure 7A) and reduced the transactivation activity of AP-1 (Figure 7B) in response to LPS stimulation. Interestingly, the LPS-induced elevation of endogenous A-FABP expression and the A-FABP promoter activity were also significantly suppressed by the A-FABP selective inhibitor BMS309403 (Figure 7C and 7D), further highlighting the existence of a positive feedback loop that modulates A-FABP expression and inflammation. Real time PCR and Western blot analysis demonstrated that either pharmacological inhibition of A-FABP by BMS309403 or siRNA-mediated knockdown of A-FABP expression had no obvious effect on either mRNA or protein levels of c-Jun (data not shown) or JNK (Figure 6B and 7A) under basal or LPS-stimulated condition.
DISCUSSION
The present study provides evidence showing that A-FABP potentiates LPS-induced inflammation by forming a positive feedback loop with the JNK signaling cascade (Figure 8). In response to LPS stimulation, activated JNK increases A-FABP expression by inducing the phosphorylation of c-Jun, which in turn binds to a highly conserved AP-1 cis-element within the A-FABP gene promoter and enhances the gene transcription. Vice versa, elevated A-FABP potentiates LPS-elicited JNK phosphorylation and subsequent activation of the AP-1 complex. Interestingly, pharmacological inhibition of A-FABP not only reduces LPS-induced JNK phosphorylation and AP1 activity, but also decreases A-FABP expression by suppressing the transcriptional activation of the gene promoter, suggesting the existence of an auto-regulatory mechanism that tightly controls the feedback loop between
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A-FABP, JNK and its downstream target c-Jun.
Consistent with our findings, an intimate connection between A-FABP and JNK has been implicated in a number of previous animal studies demonstrating that JNK and A-FABP null mice exhibit similar protection against obesity-induced insulin resistance, metabolic syndrome and fatty liver disease (5,8,36-39). Furthermore, genetic ablation of A-FABP and JNK2 (one of the three JNK isoforms) renders apoE-deficient mice a comparable degree of resistance to high cholesterol diet-induced atherosclerosis (38). In apoE-deficient mice, JNK is selectively activated in the atherosclerotic lesion area (38), and this change is accompanied by elevated A-FABP expression (unpublished observations, P Vanhoutte and A Xu).
A growing body of evidence suggests JNK as a central mediator of obesity-related pathologies, including insulin resistance, type 2 diabetes and vascular dysfunction (37,39). In both dietary and genetic obesity, the activity of JNK is markedly elevated in several metabolically active organs, especially in adipose tissue (37). Activated JNK can directly induce insulin resistance by increasing serine phosphorylation of insulin receptor substrates 1 and 2 (IRS1 and IRS2) and thereby preventing them from being recruited to activated insulin receptors. In addition, JNK in macrophages can cause insulin resistance indirectly, by mediating stress-induced production of pro-inflammatory cytokines, which in turn trigger local and systemic inflammation (36). Selective ablation of JNK1 in either adipose tissue or macrophages is sufficient to alleviate high fat diet-induced systemic insulin resistance and glucose intolerance (36,39). Likewise, obesity-induced macrophage infiltration in adipose tissue and production of pro-inflammatory cytokines are markedly attenuated by selective ablation of JNK1 in
hematopoietic cells (mainly macrophages), suggesting a central role of JNK in obesity-related inflammation (36). Since both adipocytes and macrophages are the major sites of A-FABP expression, it is conceivable that A-FABP and JNK act in a synergistic manner to perpetuate macrophage infiltration and inflammation in adipose tissue, consequently leading to systemic insulin resistance and metabolic syndrome.
Another notable finding of the present study is that both siRNA-mediated knockdown of A-FABP and pharmacological inhibition of this protein attenuate LPS-induced activation of JNK and its downstream AP-1 complex in macrophages, suggesting that A-FABP is required for the maximal activation of the JNK signaling. In support of this notion, the oral administration of the A-FABP inhibitor BMS309403 in mice decreases obesity-induced JNK activation in adipose tissue (10). However, the mechanism by which A-FABP potentiates JNK activation is unknown at this stage. Two members of the MAPK kinase family, including MKK4 and MKK7, are directly involved in JNK activation by inducing dual phosphorylation on threonine and tyrosine residues.(40) In addition, JNK activity can be modulated by its interacting proteins (JIPs) (41). Beyond its role as a lipid chaperone, A-FABP also modulates signal transduction and gene expression by fatty acid-dependent interaction with a number of intracellular proteins, including HSL(3), Jak2 (42), and peroxisome proliferator activated receptor γ (PPARγ) (43). The PPAR agonists induce the expression of c-Jun in HT-29 colon cancer cells (44). However, the present study found that neither pharmacological nor genetic inhibition of A-FABP affects the expression of c-Jun or JNK. Noticeably, a recent study showed that A-FABP mediates lipids-induced endoplasmic reticulum (ER) stress in macrophages (45). The ER stress has been shown to be a potent
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activator of JNK (46). Whether or not A-FABP potentiates LPS-induced activation of the JNK/c-Jun signaling pathway through ER stress warrants further investigation.
The present study shows that the potentiating effect of A-FABP on JNK activation is dependent on its fatty acid binding capacity, since the LPS-induced phosphorylation of JNK and activation of the AP1 complex are largely abrogated by BMS309403, a selective inhibitor of A-FABP that competitively binds within the fatty acid-binding pocket and inhibits the binding of A-FABP to endogenous free fatty acids (25). These findings further support the notion that A-FABP is at the crossroads of lipid metabolism and inflammation, and also raise the possibility that long chain fatty acids-induced JNK activation in macrophages may also depend on A-FABP.
In summary, the present study shows that A-FABP, along with JNK and AP-1, forms a finely-tuned positive feedback loop to perpetuate inflammatory responses in macrophages. Disruption of this feedback loop by molecular or pharmacological inhibition of either A-FABP or JNK causes a similar effect in attenuating JNK activation and production of
pro-inflammatory cytokines. Indeed, both JNK and A-FABP have been proposed as promising therapeutic targets for obesity-related metabolic disorders and atherosclerosis (1,47). The small molecule inhibitors of both JNK and A-FABP have been shown to be efficacious in alleviating insulin resistance, hyperglycemia, fatty liver and atherosclerosis in rodent models (10,47). However, the use of small molecule JNK inhibitors for treating obesity-related metabolic disease and atherosclerosis faces several formidable challenges: Firstly, there exist three isoforms of JNK involved in a wide range of biological functions, such as cell growth, differentiation and apoptosis (48). Secondly, JNK1 and JNK2 are expressed in almost all the cells and tissues, and it appears difficult to develop an isoform-specific inhibitor that would inhibit JNK specifically in adipose tissue or macrophages. In this connection, A-FABP inhibitors may have a more favorable selectivity than JNK inhibitors, since the expression of A-FABP is restricted mainly to adipocytes and macrophages. Therefore, pharmacological inhibition of A-FABP may represent a promising strategy to selectively block the inflammation associated with obesity and atherosclerosis.
Acknowledgement
This work was supported by a grant from the NSFC/RGC Joint Research Scheme sponsored by the Research Grants Council of Hong Kong and the National Natural Science Foundation of China (Project No: N_HKU 735/08 and 30831160518.), and the collaborative research fund (HKU 2/07C) from the Research Grants Council of Hong Kong.
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FIGURE LEGENDS Figure 1: LPS potently induces A-FABP expression in RAW 264.7 macrophages. A: Cells were stimulated with 100 ng/ml LPS or vehicle control, and the mRNA level of A-FABP was determined by quantitative real-time PCR at the specified time points. B: RAW 264.7 cells were treated with various concentrations of LPS, and the mRNA level of A-FABP was determined at 24 hours after the treatment. C: The protein level of intracellular A-FABP was analyzed by Western blotting at 12 and 24 hr after stimulation with 100 ng/ml LPS. * p<0.05, ** p<0.01, versus vehicle control at each time point (n=5-6). Figure 2: Identification of a key promoter element responsible for LPS-induced transactivation of the A-FABP gene. A: Multiple sequence alignment analysis of -150 to -100 bp of the A-FABP promoter region adjacent to the transcription initiation site from eight species. The 7-bp DNA motif inside the box is evolutionarily conserved and is identical to the AP-1 consensus recognition motif (TGACTCA). B: Schematic presentation of luciferase reporter vectors driven by the 1 kb A-FABP gene promoter (1kb-luc), mutated 1 kb A-FABP gene promoter (1kbmu-luc), 5 kb A-FABP gene promoter (5kb-luc), and 5 kb mutated A-FABP gene promoter (5kb-luc). The three underlined nucleotides within the AP-1 recognition motif were mutated. C and D: Comparison of the luciferase activity driven by the wild type and mutant A-FABP promoters as in panel B. RAW 264.7 macrophage cells were transfected with the luciferase reporter vector described in panel B, stimulated with 100 ng/ml LPS for 24 hours, and then harvested for the luciferase assay. ** p<0.01 versus wild type promoters (n=4). Figure 3: LPS induces the recruitment of c-Jun to the A-FABP gene promoter through the AP-1 recognition site. A. Streptavidin pull-down assay to detect the complex formation between c-Jun and the A-FABP promoter in vitro. Nuclear extract proteins from RAW 264.7 cells treated without or with 100 ng/ml LPS were incubated with a biotinylated wild type (WT) mouse A-FABP promoter spanning from -217 to – 27 bp or a mutant A-FABP promoter (MUT) in which the three nucleotides within the AP-1 recognition motif were mutated as in Figure 2. Following precipitation using streptovidin-coupled agarose bead, the proteins in the complexes were separated by SDS-PAGE and probed with anti c-Jun. The bar chart in the lower panel shows the relative amount of c-Jun that binds to the A-FABP promoter probes. Open bars: WT probes; black bars: MUT probes. Data is expressed as fold of the wild type promoter DNA probe in untreated cells. ** p<0.01 versus untreated WT control group (n=3). B. ChIP assay to quantify the interaction between c-Jun and the endogenous A-FABP promoter. Chromatin isolated from RAW 264.7 cells treated with 100 ng/ml LPS for various periods was subjected to immunoprecipiatation using anti- c-Jun antibody as described in Methods. The relative abundance of the 200bp A-FABP promoter enriched by ChIP was quantified by real time PCR and expressed as fold of untreated control. * p<0.05, ** p<0.01 versus the untreated control group (n=4). Figure 4: Knockdown of c-Jun expression attenuates LPS-induced transcriptional activation of the A-FABP gene in murine macrophages. A: Western blot analysis for c-Jun protein levels from RAW 264.7 cells transfected with siRNA specific to c-Jun (sic-Jun) or scrambled control (sc) for 24 hours.
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The bar chart below is the densitometric quantification of the blot. B: Real time PCR analysis for A-FABP mRNA abundance in cells transfected with c-Jun siRNA or scrambled control in response to stimulation with 100 ng/ml LPS. C. Effect of knocking down c-Jun on the 1 kb A-FABP promoter activity in the absence or presence of 100 ng/ml LPS for 24 hours. * p<0.05, ** p<0.01 versus the scrambled control group (n=5-6). Figure 5: JNK is a key regulator of A-FABP expression in murine macrophages. A: Pharmacological inhibition of JNK suppresses LPS-induced A-FABP expression. RAW 264.7 cells were treated with the JNK inhibitor SP600125 (5 μM) for two hours prior to stimulation with 100 ng/ml LPS for 24 hours. The A-FABP mRNA level was determined by real time PCR. B: Ectopic expression of constitutively active (CA) JNK induces A-FABP expression. RAW 264.7 cells were transfected with an empty plasmid (pcDNA3.1) or a plasmid encoding HA-tagged CA JNK for 48 hours. The expression of constitutive active JNK (CA JNK) and phosphorylation of c-Jun were analyzed by Western blotting (upper panel). The relative A-FABP mRNA abundance was quantified by real time PCR (lower panel) ** p<0.01 versus control (n=5). Figure 6: LPS-induced activation of the JNK/AP-1 signaling cascade and production of pro-inflammatory cytokines are compromised by knocking down A-FABP expression in macrophages. RAW 264.7 cells were transfected with A-FABP specific siRNA (siA-FABP) or scrambled control (sc) for 48 hours. A:. A-FABP expression as determined by real time PCR. B: Cells were treated without or with 100 ng/ml LPS for 10 min, and phosphorylation of JNK was determined by semi-quantitative Western blot. C: The luciferase activity in cells transfected with an AP-1 reporter vector. The cells were co-transfected with the reporter vector and siRNA for 24 hours, followed by stimulation with 100 ng/ml LPS for another 24 hours. D: The levels of pro-inflammatory cytokines (TNFα, IL6 and MCP-1) in the conditioned medium. * p<0.05, ** p<0.01 versus scrambled control (n=5-7). Figure 7: The selective A-FABP inhibitor BMS309403 attenuates LPS-induced activation of the JNK/AP-1 signaling pathway and A-FABP expression in macrophages. RAW 264.7 cells were pre-treated with BMS309403 (50 μM) for two hours followed by stimulation with 100 ng/ml LPS. LPS. A: Western blot analysis to detect JNK phosphorylation at 10 minutes after stimulation with or without LPS. B: The luciferase activity in cells transfected with an AP-1 reporter vector for 24 hours in the absence or presence of LPS. C: Real time PCR analysis for A-FABP mRNA abundance in the absence or presence of LPS and/or BMS309403 as indicated for 24 hours. D: The luciferase activity driven by 1 kb A-FABP promoter at 24 hours after LPS stimulation. * p<0.05, ** p<0.01 versus the control group without BMS309403 (n=5-6). Figure 8: Schematic representation of the molecular pathway whereby A-FABP mediates the inflammatory responses by forming a positive feedback loop with JNK and AP-1 in macrophages. FFA: free fatty acids.
