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Originally published online as doi:10.1189/jlb.0907631 on February 8, 2008

Published online before print February 8, 2008
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(Journal of Leukocyte Biology. 2008;83:1258-1266.)
© 2008 by Society for Leukocyte Biology

Activation of cyclic-AMP response element binding protein contributes to adiponectin-stimulated interleukin-10 expression in raw 264.7 macrophages

Pil-hoon Park*, Honglian Huang*, Megan R. McMullen*, Kathryn Bryan* and Laura E. Nagy*,{dagger},1

* Departments of Pathobiology and
{dagger} Gastroenterology, Cleveland Clinic, Cleveland Ohio

1Correspondence: Cleveland Clinic Foundation, LRI/NE40, 9500 Euclid Ave., Cleveland, OH 44195, USA. E-mail: laura.nagy{at}case.edu


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ABSTRACT
 
Adiponectin, an adipokine predominantly secreted from adipose tissue, has potent anti-inflammatory properties. Although the mechanisms for the anti-inflammatory properties of adiponectin are not well understood, recent evidence suggests that increased production of interleukin-10 (IL-10), a potent immunomodulatory cytokine, is involved in the anti-inflammatory actions of adiponectin. Globular adiponectin (gAcrp) increased IL-10 promoter activity and IL-10 mRNA accumulation in RAW 264.7 macrophages. Deletion of the sequences from –416 and –369 in the IL-10 promoter, containing a cyclic AMP-response element (CRE), decreased gAcrp-induced IL-10 promoter activation. Treatment of RAW 264.7 macrophages with gAcrp increased the phosphorylation of cyclic AMP response element binding protein (CREB) at Ser133, as well as enhanced the DNA binding activity of CREB. Further, overexpression of a dominant negative form of CREB suppressed gAcrp-induced transcriptional activation of IL-10. gAcrp-stimulated CREB phosphorylation was mediated by the activation of both ERK1/2- and cAMP-dependent protein kinase (PKA)-dependent pathways. Inhibition of either ERK1/2 or PKA activity prevented gAcrp-stimulated CREB phosphorylation, as well as gAcrp-stimulated IL-10 promoter activation. Taken together, these data identify gAcrp-stimulated phospho-CREB as a key transcription factor responsible for gAcrp-induced IL-10 promoter activation.

Key Words: mitogen-activated protein kinase • tolerance • protein kinase A • transcription • inflammation


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INTRODUCTION
 
Adiponectin, also known as adipocyte complement-related protein or Acrp, is an adipokine primarily secreted by adipose tissue [1 ]. Decreased circulating adiponectin concentrations are related to a number of disease conditions, including obesity [2 ], type II diabetes [3 ], atherosclerosis [4 ], as well as alcoholic and nonalcoholic liver injury [5 , 6 ]. Adiponectin administration reduces hepatic triglyceride accumulation in obese ob/ob mice [5 ], stimulates glucose utilization in muscle and fatty acid oxidation in liver [7 ], and restores insulin sensitivity in diabetic animal models [8 ]. In addition to these well-characterized effects of adiponectin on the regulation of glucose and lipid metabolism, adiponectin also has potent anti-inflammatory activity. Adiponectin dampens the early phases of macrophage inflammatory responses, acting to inhibit the growth of myelomonocytic progenitor cells and decrease the ability of mature macrophages to respond to activation [9 ]. Adiponectin also suppresses the up-regulation of cellular adhesion molecules on endothelium in response to inflammatory signals, thus decreasing leukocyte adhesion to the endothelium [10 ]. Adiponectin suppresses phagocytic activity, as well as lipopolysaccharide (LPS)-stimulated cytokine production in macrophages [9 , 11 , 12 ]. In particular, LPS-stimulated nuclear factor-{kappa}B (NF-{kappa}B) and ERK1/2 activation are sensitive to the inhibitory effects of adiponectin [11 12 13 ]. Suppression of these Toll-like receptor 4 (TLR4)-mediated signaling pathways contributes to decreased activation of key transcription factors (NF-{kappa}B and early growth response protein 1 (Egr-1)) that mediate LPS-stimulated cytokine expression in macrophages [12 , 13 ].

Although the mechanisms for the long-term anti-inflammatory effects of adiponectin are not well understood, recent data suggest that adiponectin acts, at least in part, to increase the expression of anti-inflammatory mediators, such as IL-10 [14 , 15 ]. IL-10 is an important immunomodulatory cytokine with potent anti-inflammatory and immunosuppressive properties. IL-10 expression is induced by various inflammatory mediators, such as LPS and TNF-{alpha}. While the initial response of macrophages to inflammatory stimuli is an increased production of proinflammatory cytokines, there is a subsequent induction of IL-10 [16 ]. IL-10 then acts to limit excessive production of proinflammatory cytokines, including TNF-{alpha} and IL-1β [17 , 18 ] by decreasing cytokine gene transcription, as well as regulating the stability and/or translation of target mRNAs [19 20 21 ]. IL-10 can also act by deactivating macrophages and T cells to reduce inflammation [22 , 23 ]. Treatment of macrophages with adiponectin increases the expression of IL-10 mRNA and peptide [14 ]. Importantly, immunoneutralization of IL-10 prevents globular adiponectin (gAcrp)-mediated desensitization of the RAW 264.7 macrophages to subsequent activation by LPS [15 ].

Although recent investigations identify a role for IL-10 expression in mediating the anti-inflammatory effects of adiponectin in macrophages, the mechanisms by which adiponectin increases IL-10 expression are not well understood. In RAW 264.7 macrophages, we recently reported that gAcrp rapidly increases the production of TNF-{alpha}; this increase in TNF-{alpha} then contributes, at least in part, to increased IL-10 expression [15 ]. Here, we have further investigated the molecular mechanisms by which gAcrp increases IL-10 expression and identified a critical role for ERK1/2- and cAMP-dependent protein kinase (PKA)-mediated cAMP-response element binding protein (CREB) phosphorylation, interacting with the cAMP-response element (CRE) in the IL-10 promoter, in adiponectin stimulated IL-10 expression in RAW 264.7 macrophages.


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MATERIALS AND METHODS
 
Materials
Recombinant human gAcrp expressed in Escherichia coli was purchased from Peprotech Inc. (Rockey Hill, NJ, USA). LPS from Escherichia coli serotype 026:B6 (tissue culture tested) was purchased from Sigma (St. Louis, MO, USA). Endotoxin-free plasmid preparation kit was from Qiagen (Valencia, CA, USA). Inhibitors were purchased from the following sources: U0126: Cell Signaling Technology (Beverly, MA, USA), SB203580: Calbiochem (San Diego, CA, USA; SP600125, H89 and Rp-cAMPs: Biomol (Plymouth Meeting, PA, USA). Cell culture reagents were from Gibco/Invitrogen (Grand Island, NY, USA). Antibodies were from the following sources: total ERK1/2 (Upstate Biotechnology, Lake Placid, NY, USA), β-actin and phospho-CREB (Cell Signaling Technology), phospho-specific ERK1/2, phospho- and total p38, phospho-and total JNK and CREB [for electrophoretic mobility shift assay (EMSA) supershifts] (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse IgG2b control antibody (for EMSA supershifts) (Roche, Indianapolis, IN, USA). Anti-rabbit and anti-mouse IgG-peroxidase were purchased from Boehringer Mannheim (Indianapolis, IN, USA) and anti-rabbit Alexa Fluor 488 from Molecular Probes (Invitrogen, Indianapolis, IN, USA). A 1.6-kb fragment of the IL-10 promoter (–1538/+64)-luciferase construct, and the 5' successive deletion series were a kind gift from Dr. Stephen T. Smale at University of California at Los Angeles and have been described before [24 ]. Constructs containing dominant negative CREB133 or pCMV control plasmids were from Clontech (Mountain View, CA, USA). Path-detect CRE-LUC cis-reporter plasmid was from Stratagene (La Jolla, CA, USA). Endotoxin contamination was routinely monitored in the laboratory using a kinetic chromogenic test based on the Limulus amebocyte lysate assay (Kinetic-QCL, BioWhittaker, Walkersville, MD, USA).

Cell culture
The RAW 264.7 macrophage-like cell line was cultured routinely in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin at 37°C and 5% CO2. Primary cultures of hepatic macrophages were isolated from male Wistar rats and placed into primary culture, as described previously [12 ].

Transient transfection and luciferase assay
For luciferase reporter assays, RAW 264.7 macrophages were grown in 6-well plates to 70–80% confluency and then transiently transfected with expression vectors using Superfect transfection reagent (Qiagen), according to the manufacturer’s instructions. Cells were cotransfected with pTK-RL (Promega), an expression vector for Renilla luciferase under the control of the thymidine kinase promoter, as a control for transfection efficiency. gAcrp treatment had no effect on the expression of luciferase under the control of the TK promoter (data not shown). Transfected cells were subcultured and seeded at 10.2 x 104/cm2 in 96-well plates. After 24 h, media were removed and cells were stimulated with gAcrp in DMEM/FBS. We have previously observed that gAcrp is equally, or more, potent than full-length adiponectin in decreasing LPS-stimulated responses in RAW 264.7 macrophages [15 ]; therefore, we have made use of gAcrp in these experiments. After treatment, cells were extracted in lysis buffer and luciferase activities were measured using the DUAL luciferase assay system (Promega). Statistical analysis for luciferase expression was carried out on the ratios of relative luciferase activity/R. luciferase.

RNA isolation, reverse transcription and quantitative PCR
For the measurement of IL-10 mRNA, total RNA was isolated using RNeasy Micro Kit (Qiagen), with on-column DNA digestion using the RNase-free DNase set (Qiagen) according to the manufacturer’s instructions. Total RNA were reverse transcribed using the RETROscript kit (Ambion, Austin, TX, USA) with random decamers as primers. Real-time PCR amplification was performed in an Mx3000P-Stratagene machine using SYBR Green PCR Core Reagents (Applied Biosystems; Warrington, UK). The relative amount of target mRNA was determined using the comparative threshold (Ct) method by normalizing target mRNA Ct values to those for β-actin ({Delta}Ct). The primer sequences for mouse IL-10 was 5'-CCA AGC CTT ATC GGA AAT GA-3' (forward) and 5'-TTT TCA CAG GGG AGA AAT CG-3' (reverse). For mouse β-actin was 5'-CTT TGC AGC TCC TTC GTT GC-3' (forward) and 5'-ACG ATG GAG GGG AAT ACA GC-3' (reverse). All primers used for qPCR analysis were synthesized by Integrated DNA Technologies (Coralville, IA, USA). Statistical analysis of qPCR data was performed using {Delta}Ct values.

ELISA for IL-10 detection
RAW 264.7 macrophages were treated with gAcrp or LPS in 96-well plates. The cell culture media were collected and stored at –20°C. The amount of secreted IL-10 in the media was measured using IL-10 ELISA kits (BioSource, Camarillo, CA, USA), according to the manufacturer’s instructions.

Western blot analysis
Phosphorylation of ERK1/2, p38 MAPK and JNK were measured by Western blot analysis. After treatment of RAW 264.7 macrophages with gAcrp or LPS for 0-60 min, cells were washed by cold PBS and lysed in RIPA lysis buffer containing 100 µM of sodium orthovanadate. Total cellular extracts (20 µg) were separated in 12% SDS polyacrylamide gel and transferred onto PVDF membranes. Membranes were first probed with phospho-specific ERK 1/2, p38 MAPK, or JNK antibody, followed by secondary antibody conjugated with HRP and then visualized by ECL detection reagents (Amersham Biosciences). The membranes were then stripped and reprobed with total ERK 1/2, p38 MAPK, or β-actin antibody for loading control.

Isolation of nuclear extracts and EMSA
RAW 264.7 macrophages were treated with or without 1 µg/ml of gAcrp for 0-4 h. Nuclei were isolated, and nuclear proteins were extracted using the Nuclei EZ Prep kit (Sigma). Nuclear extracts (5 µg of protein) were then used to assess DNA binding activity by EMSA, as described previously [25 ], using an oligonucleotide probe containing the CRE promoter element in the murine IL-10 promoter: 5'-CAA TTT GTC CAC GTC ACT GTG ACC 3' [26 ] (Integrated DNA Technologies) or a mutant CRE oligo: 5'-AGA GAT TGC CTG TGG TCA GAG AGC TAG-3' (Santa Cruz Biotechnology).

Confocal microscopy
RAW 264.7 macrophages were cultured on LabTek chamber slides and then treated with or without 1 µg/ml of gAcrp for 1-2 h. In some experiments, cells were pretreated with inhibitor of MAPKs and/or cAMP-dependent protein kinase for 30 min. Cells were then fixed, permeabilized with 0.1% Triton X-100, and blocked with 2% bovine serum albumin and 1% fish gelatin in PBS. Primary antibody specific for phospho-CREB at Ser133 was added followed by an Alexa Fluor 488-conjugated sheep anti-rabbit secondary antibody. Slides were then mounted in Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). The percentage of pCREB positive nuclei was determined using Image-Pro software.

Statistical analysis
Values are reported as mean ± SEM. Data were analyzed by general linear models procedure followed by least square means analysis of differences between groups (SAS, Carey, IN, USA).


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RESULTS
 
Globular adiponectin increases IL-10 mRNA expression and induces transcriptional activation of IL-10 in RAW 264.7 macrophages
Previous studies have shown that adiponectin stimulates the secretion of IL-10 in porcine macrophages, RAW 264.7 macrophages and human leukocytes [11 , 14 , 15 ]. Here, we find that treatment of primary cultures of hepatic macrophages (Kupffer cells) with gAcrp rapidly increases the accumulation of IL-10 mRNA (Fig. 1A ). Because increased IL-10 is critical in mediating the anti-inflammatory effects of adiponectin in RAW 264.7 macrophages [15 ], here, we have investigated the mechanisms by which adiponectin increases IL-10 expression. gAcrp treatment increased IL-10 mRNA accumulation by more than 40-fold in RAW 264.7 macrophages (Fig. 1B) [15 ]. This increase in IL-10 mRNA was associated with an increase in IL-10 promoter activity; gAcrp increased luciferase activity driven by the full-length IL-10 promoter (from nucleotide –1538 to nucleotide +64, relative to the +1 transcription start site) by 3.5-fold after 6 h and further increasing to 200-fold after 18 h treatment with gAcrp (Fig. 1C) . To exclude the possibility that endotoxin contamination in the gAcrp preparation contributed to increased IL-10 promoter activity, cells were pretreated with polymyxin B, an antibiotic that binds to and inactivates LPS, prior to stimulation with either 100 ng/ml LPS or 1 µg/ml gAcrp. While polymyxin B decreased IL-10 promoter activation stimulated by LPS, it had no effect on the gAcrp-induced IL-10 promoter activation (Fig. 1D) .


Figure 1
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  		Figure 1. Globular adiponectin (gAcrp) increased interleukin-10 (IL-10) mRNA accumulation and promoter activity. (A) Primary cultures of hepatic macrophages were treated with 1 µg/ml of globular adiponectin (gAcrp) for up to 5 h and expression of IL-10 mRNA measured by real-time PCR. Values represent IL-10 mRNA normalized to β-actin mRNA, means ± SEM, n = 9. *, P < 0.05. (B) RAW 264.7 macrophages were treated with 1 µg/ml of globular adiponectin (gAcrp) for 5 h and expression of IL-10 mRNA was measured by real-time PCR. Values represent IL-10 mRNA normalized to β-actin mRNA, means ± SEM, n = 9. *, P < 0.05. (C) The effect of gAcrp on IL-10 promoter activity was measured in RAW 264.7 macrophages. Cells were transiently transfected with the IL-10 promoter-luciferase reporter construct, along with a Renilla luciferase to control for transcription efficiency. After 24 h of transfection, cells were treated with 1 µg/ml of gAcrp for up to 18 h. Luciferase expression level driven by IL-10 promoter was measured and normalized to R. luciferase activity. Values represent means ± SEM, n = 5. *P < 0.05 compared with untreated cells. (D) Pretreatment with polymyxin B did not affect gAcrp-induced IL-10 promoter activation. RAW 264.7 macrophages were cotransfected with the IL-10 promoter luciferase reporter and a R. luciferase reporter and then, after 24 h, pretreated for 30 min with polymyxin B. Cells were then treated with 100 ng/ml of LPS or 1 µg/ml of gAcrp for 8 h. Fold activation by LPS was 47 ± 14 and gAcrp was 66 ± 19. Values represent relative suppression of luciferase activity (corrected for R. luciferase activity) compared with cells not treated with polymyxin B, means ± SEM, n = 6. *, P < 0.05 compared with cells not treated with polymyxin B.

Identification of the promoter region responsible for gAcrp-induced IL-10 promoter activation
To identify the promoter region and transcription factors involved in gAcrp-induced IL-10 promoter activation, the effect of gAcrp on luciferase reporter activity in constructs driven by the full-length IL-10 promoter construct was compared with constructs containing successive deletions from 5' end of the IL-10 promoter [24 ]. While deletions in the sequences between –1538 and –416 had no effect on gAcrp-induced luciferase activity (Fig. 2 ), deletion of the sequences between –416 and –369 decreased gAcrp-induced luciferase activity by ~50% compared with the full-length IL-10 promoter (–1548/+64) (Fig. 2) . These data suggest that the region between –416 and –369, which contains a putative CREB binding site [26 , 27 ], is a critical region for mediating gAcrp-induced IL-10 promoter activation. A further decrease in luceriferase activity was observed in the –78 truncation, which contains a predicted SP-1 binding site (Fig. 2) .


Figure 2
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  		Figure 2. Identification of IL-10 promoter region responsible for gAcrp-induced transcriptional activation. RAW 264.7 macrophages were transiently transfected with the full-length IL-10 promoter-luciferase construct (–1538/+64) or a series of 5' deletion mutants of the IL-10 promoter-luciferase constructs, along with a R. luciferase construct to control for transfection efficiency. After 24 h, transfected cells were then treated with gAcrp for 18 h. Data are expressed relative to the activity of the full-length IL-10 promoter (–1538/+64). gAcrp increased the activity of the full-length promoter 133 ± 27-fold over basal (n=7). The baseline ratio of luciferase over R. luciferase was 0.24 ± 0.07 (n=7). Values represent means ± SEM (n=4–7).

The transcription factor CREB mediates gAcrp-induced IL-10 promoter activation
Because the CRE in the IL-10 promoter was required for gAcrp-induced promoter activation, we next investigated the role of CREB in gAcrp-induced IL-10 expression. CREB is activated via phosphorylation at Ser133 [28 ]. Therefore, we assessed the effect of gAcrp on CREB phosphorylation by confocal microscopy by using an antibody specific for phospho-Ser133 CREB. CREB phosphorylation was minimal at baseline, but gAcrp increased phosphorylation of CREB in the nucleus of RAW 264.7 macrophages after 1-2 h of exposure to gAcrp (Fig. 3A ). The percentage of pCREB positive nuclei was 10 ± 2 in nontreated cells and 89 ± 3 (n=4) after treatment with gAcrp for 1 h. pCREB immunoreactivity completely colocalized with DAPI staining of the nuclei (data not shown). Treatment of RAW 264.7 macrophages with gAcrp increased the binding of nuclear proteins to an oligonucleotide corresponding to the CRE site in IL-10 promoter (Fig. 3B) . The binding of CREB to the CRE site was confirmed in supershift assays using specific antibodies against CREB (Fig. 3B) . gAcrp also stimulated the expression of a luciferase reporter construct containing a consensus CRE (Fig. 3C) , demonstrating that the gAcrp-stimulated phosphorylation of CREB resulted in the generation of transcriptionally active CREB in RAW 264.7 macrophages.


Figure 3
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  		Figure 3. Activation of cAMP-response element binding protein (CREB) by gAcrp. (A) RAW 264.7 macrophages were cultured in LabTek chamber slides and treated with or without 1 µg/ml of gAcrp for 1-2 h. Phospho CREB was then visualized by confocal microscopy. Nuclei were stained with DAPI (data not shown). Images are representative of four independent experiments. (B) RAW 264.7 macrophages were treated with 1 µg/ml of gAcrp for up to 4 h. Nuclear extracts were then prepared and used to measure DNA binding activity to oligonucleotide probes specific for the CRE binding site in the murine IL-10 promoter. At 4 h, CREB binding was increased to 1.7 ± 0.2-fold over baseline (P<0.05, n=3). In some experiments, extracts were incubated with antibody to CREB or normal IgG2b for supershift assays or unlabeled competitor DNA included. Nuclear extracts were also used to assess DNA binding activity to a mutant form of the CRE binding site (mCRE). Images are representative for 3 separate experiments. (C) RAW 264.7 macrophages were cotransfected with a CRE-luciferase reporter construct and R. luciferase. After 24 h, cells were treated with gAcrp (1 µg/ml) for 18 h. Values represent relative luciferase activity (corrected for R. luciferase), means ± SEM (n=4). Values with different superscripts are significantly different from each other, P < 0.05. (D) RAW 264.7 macrophages were cotransfected with full-length IL-10 promoter-luciferase (–1538/+64) and R. luciferase, along with a plasmid encoding a mutated form of CREB (CREB133) or empty vector (pCMV). After 24 h, cells were treated with gAcrp (1 µg/ml) for 18 h. Values represent gAcrp-stimulated luciferase activity (corrected for R. luciferase) compared with basal, means ± SEM (n=5). *, P < 0.05.

If CREB activity is required for gAcrp-induced IL-10 promoter activation, then gAcrp-stimulated IL-10 promoter activity should be decreased in cells cotransfected with a plasmid containing the dominant negative form of CREB, termed CREB133. As shown in Fig. 3D , gAcrp increased IL-10 promoter activation in cells cotransfected with empty vector, consistent with previous experiments. However, overexpression of dominant negative CREB133 suppressed IL-10 promoter activation induced by gAcrp (Fig. 3D) , demonstrating a critical role of CREB in mediating the gAcrp-induced IL-10 promoter activation.

gAcrp-induced IL-10 production is dependent on protein kinase A signaling pathways
CREB can be phosphorylated at Ser133 by activated protein kinase A [29 ]. Since PKA signaling has been implicated in mediating the effects of adiponectin in other cell types [30 ], we hypothesized that the PKA signaling pathway would be involved in gAcrp-induced IL-10 production. When cells were pretreated with H89, a selective PKA inhibitor, the ability of gAcrp to increase pCREB immunoreactivity was suppressed (Fig. 4A ). The percentage of pCREB-positive nuclei was 89 ± 2 in cells treated with gAcrp for 1 h and 14 ± 3 (n=4) in cells pretreated with H89 prior to stimulation with gAcrp. A second selective PKA inhibitor, Rp-cAMPs, also effectively prevented gAcrp-stimulated increases in pCREB immunoreactivity (data not shown). Similarly, when cells were pretreated with H89, IL-10 promoter activation induced by gAcrp was inhibited by 50% (Fig. 4B) .


Figure 4
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  		Figure 4. Inhibition of cAMP-dependent protein kinase suppresses gAcrp-induced CREB phosphorylation and IL-10 promoter activation. (A) RAW 264.7 macrophages were cultured in LabTek chamber slides and then pretreated with 50 µM H89 for 60 min before stimulation with or without 1 µg/ml of gAcrp for 1 h. Phospho-CREB was then visualized by confocal microscopy. Images are representative of four independent experiments. (B) RAW 264.7 macrophages were cotransfected with IL-10 promoter-luciferase (–1538/+64) construct and R. luciferase. Transfected cells were pretreated with H89 (5 µM) for 60 min followed by treatment with gAcrp for 18 h. Luciferase expression driven by IL-10 promoter was measured and corrected for R. luciferase. Values represent means ± SEM (n=4). *, P < 0.05 compared with cells not treated with H89.

ERK 1/2 signaling pathways contributes to gAcrp-induced CREB phosphorylation and IL-10 expression
Multiple MAPK family members (ERK1/2, p38 MAPK, and JNK) can contribute to IL-10 production in various cell types [31 32 33 ]. Further, CREB phosphorylation at Ser133 can also be mediated via activation of ERK1/2 [34 ]. To investigate the potential contribution of MAPKs to gAcrp-stimulated IL-10 expression, we first assessed the ability of gAcrp to activate ERK1/2, p38, and JNK in RAW 264.7 macrophages. Globular adiponectin treatment rapidly increased phosphorylation of ERK1/2, p38 MAPK, and JNK (Fig. 5 ). The time course of phosphorylation of each MAPK by gAcrp was quite similar; initial increases in phosphorylation were observed after 10 min and maximal phosphorylation observed at 30 min exposure to gAcrp (Fig. 5) . While phosphorylation of p38 and JNK returned to baseline by 60 min, ERK1/2 phosphorylation remained elevated at 60 min. Pretreatment with polymyxin B did not affect gAcrp-induced phosphorylation of MAPKs but inhibited LPS-stimulated MAPK phosphorylation (Fig. 5) .


Figure 5
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  		Figure 5. gAcrp rapidly increased phosphorylation of ERK1/2, p38 MAPK and JNK. RAW 264.7 macrophages were treated with or without 1 µg/ml of gAcrp or 100 ng/ml of LPS for 0-60 min and phosphorylation of ERK1/2, p38 MAPK, and JNK assessed by Western blot analysis. In some samples, cells were pretreated with polymyxin B for 30 min before stimulation with LPS or gAcrp. Images are representative of 3 independent experiments for the response to gAcrp or LPS and two independent experiments, including polymyxin B.

While gAcrp activated all three MAPK pathways, only ERK1/2 activation contributed to gAcrp-stimulated increases in CREB phosphorylation. Pretreatment of RAW 264.7 macrophages with the ERK1/2 inhibitor, U0126, blunted gAcrp-induced CREB phosphorylation (Fig. 6A ). The percentage of pCREB-positive nuclei was 89 ± 4 in cells treated with gAcrp for 1 h. In cells pretreated with U0126 prior to stimulation with gAcrp, the percentage of pCREB-positive nuclei was 6 ± 2 (U0126). In contrast, pretreatment with p38 MAPK (SB203580) or JNK (SP60015) inhibitors had no effect on gAcrp-induced CREB phosphorylation. The percentage of pCREB-positive nuclei was 84 ± 3 in cells pretreated with SB203580 and 89 ± 6 in cells pretreated with SP600125 prior to their stimulation with gAcrp for 1 h (Fig. 6A) . The inhibition of CREB phosphorylation by pretreatment with U0126 was paralleled by a suppression in gAcrp-stimulated IL-10 promoter activity (Fig. 6B) and IL-10 mRNA accumulation (Fig. 6C) . These observations suggest that ERK1/2 activation contributes to gAcrp-induced IL-10 production via phosphorylation and activation of CREB.


Figure 6
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  		Figure 6. Inhibition of ERK1/2 decreased gAcrp-stimulated CREB phosphorylation and IL-10 expression. (A) RAW 264.7 macrophages were cultured in LabTek chamber slides and then pretreated with or without MAPK inhibitors (10 µM U0126, 20 nM SB203580, or 30 µM SP600125) for 60 min prior to stimulation with or without 1 µg/ml of gAcrp for 1 h. Phospho-CREB was then visualized by confocal microscopy. Inhibitors had no effect on baseline phospho-CREB (data not shown). Images are representative of 4 independent experiments. (B) RAW 264.7 macrophages were cotransfected with IL-10 promoter luciferase reporter construct along with R. luciferase. After 24 h, cells were preincubated with or without U0126 for 30 min prior to stimulation with or without gAcrp (1 µg/ml) for 8 h. Values represent luciferase activity driven by IL-10 promoter (corrected for R. luciferase), means ± SEM, n = 4. *, P < 0.05. (C) RAW 264.7 macrophages were preincubated with or without U0126 for 30 min prior to stimulation with or without gAcrp (1 µg/ml) for 5 h. The expression level of IL-10 mRNA was measured by real-time PCR. Values represent IL-10 mRNA normalized to β-actin mRNA, means ± SEM, n = 6. *, P < 0.05.

In some cell types, the cAMP/PKA pathway is located upstream of ERK1/2; ERK1/2 can be activated or inhibited by cAMP in a cell-specific manner [35 ]. Because the present data indicate that both PKA and ERK1/2 signaling pathways are involved in gAcrp-induced IL-10 expression through CREB phosphorylation, we next investigated the relationship between gAcrp-mediated PKA and ERK1/2 signaling. Pretreatment of RAW 264.7 macrophages with H89 had no effect on gAcrp-induced ERK phosphorylation (Fig. 7A ), suggesting that PKA does not act upstream of gAcrp-stimulated ERK1/2 phosphorylation. Pretreatment of RAW 264.7 macrophages with H89 and/or U0126 also had an additive effect in inhibiting gAcrp-stimulated CRE-luciferase activity (Fig. 7B) , providing further support to independent contributions of PKA- and ERK1/2-mediated activation of CREB in response to gAcrp.


Figure 7
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  		Figure 7. cAMP-dependent protein kinase and ERK1/2 contribute in parallel to activation of CREB by gAcrp (A). Raw 264.7 macrophages were pretreated with 0-10 µM H89 for 60 min and then stimulated with or without 1 µg/ml of adiponectin for 30 min. Phosphorylated ERK1/2 was assessed by Western blot analysis. Images are representative of 4 separate experiments. (B) RAW 264.7 macrophages were cotransfected with a CRE-luciferase reporter construct and R. luciferase. After 24 h, cells were pretreated with 10 µM H89 and/or 10 µM U0126 for 30 min and then stimulated with gAcrp (1 µg/ml) for 18 h. Values represent relative luciferase activity (corrected for R. luciferase), means ± SEM (n=6). Values with different superscripts are significantly different from each other, P < 0.05.


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DISCUSSION
 
Adiponectin is an adipokine with potent anti-inflammatory properties. A growing body of evidence indicates that adiponectin has potential therapeutic efficacy in a number of inflammatory diseases. For example, increasing adiponectin expression in mice using recombinant adenoviral constructs suppresses the development of atherosclerosis in apolipoprotein E-deficient mice, at least in part by decreasing the production of TNF-{alpha} [36 ]. Treatment with exogenous adiponectin protected mice from ethanol-induced liver injury by decreasing both hepatic steatosis and TNF-{alpha} expression [5 ]. Similarly, pretreatment of mice with adiponectin protects from LPS-induced liver injury in KK-Ay obese mice by suppressing TNF-{alpha} expression [37 ]. In contrast, adiponectin-deficient mice exhibit increased sensitivity to galactosamine/LPS-induced liver injury [38 ]. Taken together, these studies demonstrate an important protective effect of adiponectin in diseases characterized by increased inflammatory responses.

These protective effects of adiponectin are associated with a down-regulation of proinflammatory cytokine production, as well as an increased production of anti-inflammatory mediators, including IL-10 and IL-1 receptor antagonist [14 ]. In particular, increased production of IL-10 is important in mediating the anti-inflammatory effects of adiponectin. For example, neutralization of IL-10 diminishes anti-inflammatory effect of adiponectin in ConA-treated mice liver [39 ], inhibits adiponectin-induced tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in human macrophages [40 ] and ameliorates the inhibitory effects of adiponectin on LPS-stimulated TNF-{alpha} production in RAW 264.7 macrophages [15 ]. Although it is clear that increased IL-10 expression plays a crucial role in the anti-inflammatory effect of adiponectin, the molecular mechanisms by which adiponectin increases IL-10 expression are not well understood. We have recently reported that gAcrp rapidly increases the expression of TNF-{alpha} in RAW 264.7 macrophages; this increase in TNF-{alpha} contributes to gAcrp-stimulated IL-10 expression [15 ]. Here, we have further characterized the signaling pathways required for gAcrp-stimulated IL-10 expression, identifying a critical contribution of phospho-CREB and the CRE in the IL-10 promoter in the response of RAW 264.7 macrophages to gAcrp. Importantly, gAcrp-stimulation of CREB phosphorylation involves both ERK1/2- and PKA-dependent pathways, demonstrating that multiple signaling cascades activated by gAcrp work in combination to mediate the anti-inflammatory effects of gAcrp in RAW 264.7 macrophages.

gAcrp increased the transcriptional activity of the full-length IL-10 promoter within the context of a heterologous luciferase reporter plasmid in RAW 264.7 macrophages (Fig. 1) . A number of transcription factors can contribute to the regulation of IL-10 transcription; the relative importance of each transcription factor is dependent on cell type and activating stimulus. Sp1 and CCAAT/enhancer binding protein (C/EBP) can increase IL-10 expression [24 ]. Further, NF-{kappa}B1 homodimers regulate IL-10 transcription through the formation of complexes with CREB binding protein (CBP) [41 ]. The promoter region of IL-10 contains several putative CREs [27 ] and cAMP-dependent signaling pathways are implicated in IL-10 production through activation of CREB [26 , 42 ]. Making use of a series of IL-10 promoter-deletion constructs, here we found that the CRE element in the IL-10 promoter made a critical contribution to gAcrp-induced IL-10 promoter activation in RAW 264.7 macrophages (Fig. 2) . When the CRE was deleted, gAcrp-stimulated IL-10 promoter activity was reduced by ~50%. A similar extent of inhibition was observed when RAW 264.7 macrophages were cotransfected with a dominant-negative form of CREB (Fig. 3C) . Together, these data point to an important contribution of the phospho-CREB/CRE pathway in gAcrp-stimulated IL-10 expression.

These data also indicate that gAcrp-stimulated IL-10 transcriptional control is distinct from LPS-stimulated responses, in which the SP-1 promoter elements play an important role [24 ], particularly at the SP-1 site at position -636 to -617 bp [43 ]. We find that deletion beyond –118 of the IL-10 promoter further decreases gAcrp-stimulated IL-10 promoter activity (Fig. 2) . There is a second, putative SP-1 site at position –80 of the IL-10 promoter, as well as an NF-{kappa}B 1 (p50) binding site at –55 [41 ]. Further, in some reports, there is predicted TATA box at –79 of the IL-10 promoter [26 ]. Investigations are under way to determine the possible role of Sp-1 or p50 binding to this region in the IL-10 promoter to gAcrp-stimulated expression [43 ].

The upstream signal transduction pathways regulating IL-10 transcription are also not completely understood. Mitogen-activated protein kinase (MAPK) signaling pathways, including ERK1/2, p38, and JNK, are thought to play an important role in the regulation of IL-10 transcription. While the relative involvement of individual MAPK family members depends on cell type and stimulus, all three MAPKs contribute to LPS-stimulated IL-10 induction in macrophages [33 ]. Further, cAMP-dependent signaling is linked to stress-induced IL-10 expression through CREB activation [42 ]. In RAW 264.7 macrophages, gAcrp increased the phosphorylation of ERK1/2, p38, and JNK with similar kinetics, except that phosphorylation of ERK1/2 was sustained, while phosphorylation of p38 and JNK were transient (Fig. 5) . Importantly, both PKA and ERK1/2 contributed to the stimulation of CREB phosphorylation by gAcrp in RAW 264.7 macrophages. Thus, ERK1/2 phosphorylation appears to be involved in the two major pathways mediating gAcrp-stimulated IL-10 expression, contributing both to gAcrp-induced TNF-{alpha} production via increasing Egr-1 expression [15 ] and activation of pCREB pathway (Fig. 6) .

Cyclic AMP-dependent signaling pathways have been linked to the anti-inflammatory effects of adiponectin in human aortic endothelial cells (HAECs) [44 , 45 ]. In HAECs, long-term treatment (18 h) with adiponectin suppresses TNF-{alpha}-dependent NF-{kappa}B activation and interleukin-8 synthesis [44 , 45 ]. These responses were prevented by pretreatment with an inhibitor of PKA and were associated with increased cAMP accumulation [44 , 45 ]. Similarly, inhibition of high glucose-induced reactive oxygen species production by adiponectin is mediated by cAMP in endothelial cells [30 ]. These responses in endothelial cells are consistent with the role of PKA/pCREB in mediating the anti-inflammatory effects of gAcrp in RAW 264.7 macrophages reported in the current study.

In most inflammatory conditions, both positive and negative regulators of the inflammatory response are released. Macrophages are very responsive to the balance of both proinflammatory and anti-inflammatory signals in their local environment. Accumulating evidence indicates that reduced expression of adiponectin contributes to an increased sensitivity of macrophages to activation [5 , 46 ]. Interestingly, adiponectin, while ultimately producing anti-inflammatory responses, initiates these anti-inflammatory responses in macrophages via stimulation of pathways typically characterized as proinflammatory, including activation of NF-{kappa}B and Egr-1, as well as increasing the production of TNF-{alpha} [15 ]. Here, we demonstrate that activation of pCREB/CRE-dependent transcription of the IL-10 promoter contributes to gAcrp-stimulated production of the potent anti-inflammatory cytokine IL-10. Taken together, these data demonstrate that adiponectin uses two "classic" anti-inflammatory signaling pathways, IL-10 and cAMP, with both pathways contributing to the potent ability of adiponectin to suppress macrophage activation.


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ACKNOWLEDGEMENTS
 
This work was supported by United States Public Health Service grants AA011975 and AA013868.

Received September 14, 2007; revised January 8, 2008; accepted January 18, 2008.


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REFERENCES
 
    1
  1. Berg, A. H., Combs, T. P., Scherer, P. E. (2002) ACRP30/adiponectin: an adipokine regulating glucose and lipid metabolism Trends Endocrinol. Metab. 13,84-89[CrossRef][Medline]
    2. 2
  2. Ouchi, N., Kihara, S., Funahashi, T., Matsuzawa, Y., Walsh, K. (2003) Obesity, adiponectin and vascular inflammatory disease Curr. Opin. Lipidol. 14,561-566[CrossRef][Medline]
    3. 3
  3. Berg, A. H., Combs, T. P., Du, X., Brownlee, M., Scherer, P. E. (2001) The adipocyte-secreted protein Acrp30 enhances hepatic insulin action Nat. Med. 7,947-953[CrossRef][Medline]
    4. 4
  4. Matsuzawa, Y. (2005) Adiponectin: Identification, physiology and clinical relevance in metabolic and vascular disease Atheroscler. Suppl. 6,7-14[Medline]
    5. 5
  5. Xu, A., Wang, Y., Keshaw, H., Xu, L. Y., Lam, K. S., Cooper, G. J. (2003) The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice J. Clin. Invest. 112,91-100[CrossRef][Medline]
    6. 6
  6. You, M., Considine, R. V., Leone, T. C., Kelly, D. P., Crabb, D. W. (2005) Role of adiponectin in the protective action of dietary saturated fat against alcoholic fatty liver in mice Hepatology 42,568-577[CrossRef][Medline]
    7. 7
  7. Yamauchi, T., Kamon, J., Minokoshi, Y., Ito, Y., Waki, H., Uchida, S., Yamashita, S., Noda, M., Kita, S., Ueki, K., et al (2002) Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase Nat. Med. 8,1288-1295[CrossRef][Medline]
    8. 8
  8. Maeda, N., Shimomura, I., Kishida, K., Nishizawa, H., Matsuda, M., Nagaretani, H., Furuyama, N., Kondo, H., Takahashi, M., Arita, Y., et al (2002) Diet-induced insulin resistance in mice lacking adiponectin/ACRP30 Nat. Med. 8,731-737[CrossRef][Medline]
    9. 9
  9. Yokota, T., Oritani, K., Takahashi, I., Ishikawa, J., Matsuyama, A., Ouchi, N., Kihara, S., Funahashi, T., Tenner, A. J., Tomiyama, Y., et al (2000) Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages Blood 96,1723-1732[Abstract/Free Full Text]
    10. 10
  10. Ouedraogo, R., Gong, Y., Berzins, B., Wu, X., Mahadev, K., Hough, K., Chan, L., Goldstein, B. J., Scalia, R. (2007) Adiponectin deficiency increases leukocyte-endothelium interactions via upregulation of endothelial cell adhesion molecules in vivo J. Clin. Invest. 117,1718-1726[CrossRef][Medline]
    11. 11
  11. Wulster-Radcliffe, M. C., Ajuwon, K. M., Wang, J., Christian, J. A., Spurlock, M. E. (2004) Adiponectin differentially regulates cytokines in porcine macrophages Biochem. Biophys. Res. Commun. 316,924-929[CrossRef][Medline]
    12. 12
  12. Thakur, V., Pritchard, M. T., McMullen, M. R., Nagy, L. E. (2006) Adiponectin normalizes LPS-stimulated TNF-{alpha} production by rat Kupffer cells after chronic ethanol feeding Am. J. Physiol. Gastrointest. Liver Physiol. 290,G998-G1007[Abstract/Free Full Text]
    13. 13
  13. Yamaguchi, N., Argueta, J. G., Masuhiro, Y., Kagishita, M., Nonaka, K., Saito, T., Hanazawa, S., Yamashita, Y. (2005) Adiponectin inhibits Toll-like receptor family-induced signaling FEBS Lett. 579,6821-6826[CrossRef][Medline]
    14. 14
  14. Wolf, A. M., Wolf, D., Rumpold, H., Enrich, B., Tilg, H. (2004) Adiponectin induces the anti-inflammatory cytokines IL-10 and IL-1RA in human leukocytes Biochem. Biophys. Res. Commun. 323,630-635[CrossRef][Medline]
    15. 15
  15. Park, P. H., McMullen, M. R., Huang, H., Thakur, V., Nagy, L. E. (2007) Short-term treatment of RAW264.7 macrophages with adiponectin increases tumor necrosis factor-{alpha} expression via ERK1/2 activation and Egr-1 expression: Role of TNF-{alpha} in adiponectin-stimulated IL-10 production J. Biol. Chem. 282,21695-21703[Abstract/Free Full Text]
    16. 16
  16. Sato, T. A., Keelan, J. A., Mitchell, M. D. (2003) Critical paracrine interactions between TNF-alpha and IL-10 regulate lipopolysaccharide-stimulated human choriodecidual cytokine and prostaglandin E2 production J. Immunol. 170,158-166[Abstract/Free Full Text]
    17. 17
  17. Fiorentino, D. F., Zlotnik, A., Mosmann, T. R., Howard, M., O’Garra, A. (1991) IL-10 inhibits cytokine production by activated macrophages J. Immunol. 147,3815-3822[Abstract]
    18. 18
  18. De Waal Malefyt, R., Abrams, J., Bennett, B., Figdor, C. G., de Vries, J. E. (1991) Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes J. Exp. Med. 174,1209-1220[Abstract/Free Full Text]
    19. 19
  19. Moore, K. W., O’Garra, A., de Waal Malefyt, R., Vieira, P., Mosmann, T. R. (1993) Interleukin-10 Annu. Rev. Immunol. 11,165-190[CrossRef][Medline]
    20. 20
  20. Kishore, R., Tebo, J. M., Kolosov, M., Hamilton, T. A. (1999) Cutting edge: clustered AU-rich elements are the target of IL-10-mediated mRNA destabilization in mouse macrophages J. Immunol. 162,2457-2461[Abstract/Free Full Text]
    21. 21
  21. Brown, C. Y., Lagnado, C. A., Vadas, M. A., Goodall, G. J. (1996) Differential regulation of the stability of cytokine mRNAs in lipopolysaccharide-activated blood monocytes in response to interleukin-10 J. Biol. Chem. 271,20108-20112[Abstract/Free Full Text]
    22. 22
  22. Bogdan, C., Vodovotz, Y., Nathan, C. (1991) Macrophage deactivation by interleukin 10 J. Exp. Med. 174,1549-1555[Abstract/Free Full Text]
    23. 23
  23. Akdis, C. A., Joss, A., Akdis, M., Faith, A., Blaser, K. (2000) A molecular basis for T cell suppression by IL-10: CD28-associated IL-10 receptor inhibits CD28 tyrosine phosphorylation and phosphatidylinositol 3-kinase binding FASEB J. 14,1666-1668[Free Full Text]
    24. 24
  24. Brightbill, H. D., Plevy, S. E., Modlin, R. L., Smale, S. T. (2000) A prominent role for Sp1 during lipopolysaccharide-mediated induction of the IL-10 promoter in macrophages J. Immunol. 164,1940-1951[Abstract/Free Full Text]
    25. 25
  25. Shi, L., Kishore, R., McMullen, M. R., Nagy, L. E. (2002) Chronic ethanol increases lipopolysaccharide-stimulated Egr-1 expression in RAW 264.7 macrophages: contribution to enhanced tumor necrosis factor alpha production J. Biol. Chem. 277,14777-14785[Abstract/Free Full Text]
    26. 26
  26. Gee, K., Angel, J. B., Ma, W., Mishra, S., Gajanayaka, N., Parato, K., Kumar, A. (2006) Intracellular HIV-Tat expression induces IL-10 synthesis by the CREB-1 transcription factor through Ser133 phosphorylation and its regulation by the ERK1/2 MAPK in human monocytic cells J. Biol. Chem. 281,31647-31658[Abstract/Free Full Text]
    27. 27
  27. Platzer, C., Fritsch, E., Elsner, T., Lehmann, M. H., Volk, H. D., Prosch, S. (1999) Cyclic adenosine monophosphate-responsive elements are involved in the transcriptional activation of the human IL-10 gene in monocytic cells Eur. J. Immunol. 29,3098-3104[CrossRef][Medline]
    28. 28
  28. Gonzalez, G. A., Montminy, M. R. (1989) Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133 Cell 59,675-680[CrossRef][Medline]
    29. 29
  29. Nichols, M., Weih, F., Schmid, W., DeVack, C., Kowenz-Leutz, E., Luckow, B., Boshart, M., Schutz, G. (1992) Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP-induced gene transcription EMBO J. 11,3337-3346[Medline]
    30. 30
  30. Ouedraogo, R., Wu, X., Xu, S. Q., Fuchsel, L., Motoshima, H., Mahadev, K., Hough, K., Scalia, R., Goldstein, B. J. (2006) Adiponectin suppression of high-glucose-induced reactive oxygen species in vascular endothelial cells: evidence for involvement of a cAMP signaling pathway Diabetes 55,1840-1846[Abstract/Free Full Text]
    31. 31
  31. Foey, A. D., Parry, S. L., Williams, L. M., Feldmann, M., Foxwell, B. M., Brennan, F. M. (1998) Regulation of monocyte IL-10 synthesis by endogenous IL-1 and TNF-alpha: role of the p38 and p42/44 mitogen-activated protein kinases J. Immunol. 160,920-928[Abstract/Free Full Text]
    32. 32
  32. Lucas, M., Zhang, X., Prasanna, V., Mosser, D. M. (2005) ERK activation following macrophage Fc{gamma}R ligation leads to chromatin modifications at the IL-10 locus J. Immunol. 175,469-477[Abstract/Free Full Text]
    33. 33
  33. Liu, Y. W., Chen, C. C., Tseng, H. P., Chang, W. C. (2006) Lipopolysaccharide-induced transcriptional activation of interleukin-10 is mediated by MAPK- and NF-{kappa}B-induced CCAAT/enhancer-binding protein delta in mouse macrophages Cell. Signal. 18,1492-1500[CrossRef][Medline]
    34. 34
  34. McCubrey, J. A., May, W. S., Duronio, V., Mufson, A. (2000) Serine/threonine phosphorylation in cytokine signal transduction Leukemia 14,9-21[CrossRef][Medline]
    35. 35
  35. Stork, P. J., Schmitt, J. M. (2002) Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation Trends Cell Biol. 12,258-266[CrossRef][Medline]
    36. 36
  36. Okamoto, Y., Kihara, S., Ouchi, N., Nishida, M., Arita, Y., Kumada, M., Ohashi, K., Sakai, N., Shimomura, I., Kobayashi, H., et al (2002) Adiponectin reduces atherosclerosis in apolipoprotein E-deficient mice Circulation 106,2767-2770[Abstract/Free Full Text]
    37. 37
  37. Masaki, T., Chiba, S., Tatsukawa, H., Yasuda, T., Noguchi, H., Seike, M., Yoshimatsu, H. (2004) Adiponectin protects LPS-induced liver injury through modulation of TNF-{alpha} in KK-Ay obese mice Hepatology 40,177-184[CrossRef][Medline]
    38. 38
  38. Matsumoto, H., Tamura, S., Kamada, Y., Kiso, S., Fukushima, J., Wada, A., Maeda, N., Kihara, S., Funahashi, T., Matsuzawa, Y., et al (2006) Adiponectin deficiency exacerbates lipopolysaccharide/D-galactosamine-induced liver injury in mice World J. Gastroenterol. 12,3352-3358[Medline]
    39. 39
  39. Wolf, A. M., Wolf, D., Avila, M. A., Moschen, A. R., Berasain, C., Enrich, B., Rumpold, H., Tilg, H. (2006) Up-regulation of the anti-inflammatory adipokine adiponectin in acute liver failure in mice J. Hepatol. 44,537-543[CrossRef][Medline]
    40. 40
  40. Kumada, M., Kihara, S., Ouchi, N., Kobayashi, H., Okamoto, Y., Ohashi, K., Maeda, K., Nagaretani, H., Kishida, K., Maeda, N., et al (2004) Adiponectin specifically increased tissue inhibitor of metalloproteinase-1 through interleukin-10 expression in human macrophages Circulation 109,2046-2049[Abstract/Free Full Text]
    41. 41
  41. Cao, S., Zhang, X., Edwards, J. P., Mosser, D. M. (2006) NF-{kappa}B1 (p50) homodimers differentially regulate pro- and anti-inflammatory cytokines in macrophages J. Biol. Chem. 281,26041-26050[Abstract/Free Full Text]
    42. 42
  42. Platzer, C., Docke, W., Volk, H., Prosch, S. (2000) Catecholamines trigger IL-10 release in acute systemic stress reaction by direct stimulation of its promoter/enhancer activity in monocytic cells J. Neuroimmunol. 105,31-38[CrossRef][Medline]
    43. 43
  43. Ma, W., Lim, W., Gee, K., Aucoin, S., Nandan, D., Kozlowski, M., Diaz-Mitoma, F., Kumar, A. (2001) The p38 mitogen-activated kinase pathway regulates the human interleukin-10 promoter via the activation of Sp1 transcription factor in lipopolysaccharide-stimulated human macrophages J. Biol. Chem. 276,13664-13674[Abstract/Free Full Text]
    44. 44
  44. Ouchi, N., Kihara, S., Arita, Y., Okamoto, Y., Maeda, K., Kuriyama, H., Hotta, K., Nishida, M., Takahashi, M., Muraguchi, M., et al (2000) Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-{kappa}B signaling through a cAMP-dependent pathway Circulation 102,1296-1301[Abstract/Free Full Text]
    45. 45
  45. Kobashi, C., Urakaze, M., Kishida, M., Kibayashi, E., Kobayashi, H., Kihara, S., Funahashi, T., Takata, M., Temaru, R., Sato, A., et al (2005) Adiponectin inhibits endothelial synthesis of interleukin-8 Circ. Res. 97,1245-1252[Abstract/Free Full Text]
    46. 46
  46. Matsumoto, H., Tamura, S., Kamada, Y., Kiso, S., Fukushima, J., Wada, A., Maeda, N., Kihara, S., Funahashi, T., Matsuzawa, Y., et al (2006) Adiponectin deficiency exacerbates lipopolysaccharide/D-galactosamine-induced liver injury in mice World J. Gastroenterol. 12,3352-3358[Medline]



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