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The Journal of Immunology, 2007, 179, 6263 -6272
Copyright © 2007 by The American Association of Immunologists, Inc.
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Vasoactive Hormone Adrenomedullin and Its Binding Protein: Anti-Inflammatory Effects by Up-Regulating Peroxisome Proliferator-Activated Receptor-{gamma}1

Michael Miksa*,{dagger}, Rongqian Wu*,{dagger}, Xiaoxuan Cui*,{dagger}, Weifeng Dong*,{dagger}, Padmalaya Das*,{dagger}, H. Hank Simms{ddagger}, Thanjavur S. Ravikumar*,{dagger} and Ping Wang2,*,{dagger}

* Center for Immunology and Inflammation, The Feinstein Institute for Medical Research, and {dagger} Department of Surgery, North Shore University Hospital and Long Island Jewish Medical Center, Manhasset, NY 11030; and {ddagger} Department of Surgery, Albert Einstein Medical Center, Philadelphia, PA 19141


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sepsis is a critical inflammatory condition from which numerous patients die due to multiple organ failure and septic shock. The vasoactive hormone adrenomedullin (AM) and its binding protein (AMBP-1) are beneficial in sepsis by abrogating the progression to irreversible shock and decreasing proinflammatory cytokine release. To investigate the anti-inflammatory mechanism, we studied to determine the effect of the AM/AMBP-1 complex on peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) expression and activation by using RAW264.7 cells and a rat endotoxemia model. LPS treatment significantly decreased PPAR-{gamma} expression in vivo and in vitro and was associated with increased TNF-{alpha} production. Treatment with AM/AMBP-1 for 4 h completely restored PPAR-{gamma} levels in both models, resulting in TNF-{alpha} suppression. In a knockdown model using small interfering RNA in RAW264.7 macrophages, AM/AMBP-1 failed to suppress TNF-{alpha} production in the absence of PPAR-{gamma}. LPS caused the suppression of intracellular cyclic AMP (cAMP), which was prevented by simultaneous AM/AMBP-1 treatment. Although incubation with dibutyryl cAMP significantly decreased LPS-induced TNF-{alpha} release, it did not alter PPAR-{gamma} expression. Through inhibition studies using genistein and PD98059 we found that the Pyk-2 tyrosine kinase-ERK1/2 pathway is in part responsible for the AM/AMBP-1-mediated induction of PPAR-{gamma} and the anti-inflammatory effect. We conclude that AM/AMBP-1 is protective in sepsis due to its vasoactive properties and direct anti-inflammatory effects mediated through both the cAMP-dependent pathway and Pyk-2-ERK1/2-dependent induction of PPAR-{gamma}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sepsis is a systemic inflammatory response syndrome especially affecting patients with severe infections or trauma or already compromised patients with deficient immune systems or underlying chronic diseases such as diabetes mellitus, hypertension, or congestive heart failure (1). Despite advances in the intensive care of septic patients with improvements of in hospital deaths from 28 to 18% in the past 20 years, the total incidence and lethality are on a rise, leading to exploding costs in health care (1) and making effective targeted treatments more necessary than ever. Bacterial toxins (e.g., LPS) lead to an overstimulation of the innate immune system mediated through TLRs and trigger the extensive release of proinflammatory cytokines (e.g., TNF-{alpha}) followed by endocrine (e.g., relative adrenal insufficiency and hyperinsulinemia), metabolic (e.g., hyperglycemia and insulin resistance), coagulative, and cardiovascular dysregulation (2). Altogether, this may lead to septic shock, multiple organ failure, and death.

Adrenomedullin (AM)3 is a potent vasodilatory peptide that was first discovered in human pheochromocytoma and later found to be expressed in various organs and tissues including blood and endothelial and immune cells (3, 4). It acts as a circulating or paracrine hormone and mediates its function by binding to a G{alpha}S protein-coupled calcitonin receptor-like receptor (CRLR), thus activating membrane-bound adenylate cyclases and increasing intracellular cyclic AMP (cAMP) levels (5). Vascular smooth muscle cells consequently react by relaxation, which is both endothelial cell-dependent through the AM-induced release of NO (6) and endothelial cell-independent through a protein kinase A-dependent regulation of calcium dependent K+ channels (7). Recent studies have shown that up-regulation of AM plays a major role in initiating the hyperdynamic response during the early stage of sepsis and that a reduced vascular sensitivity to AM appears to be responsible for the transition from the hyperdynamic to the hypodynamic phase of shock during the progression of experimental polymicrobial sepsis that usually precedes death (8).

The decrease of a plasma protein with AM binding properties, adrenomedullin binding protein-1 (AMBP-1), was recently discovered to be responsible for the lack of AM response in sepsis (9, 10). AMBP-1 prevents AM degradation and possibly promotes AM receptor activation by the aggregation of the AM/AMBP-1 complex to adhesion molecules on an activated endothelium (11). This brings AM/AMBP-1 into close proximity with the CRLR, increasing the chance of activation of the cognate receptor for AM. The hepatic production and plasma concentration of AMBP-1 is dramatically reduced by over 50% during the late phase of cecal ligation and puncture (CLP)-induced sepsis in rats (12) and represents the pathophysiologic mechanism by which AM loses the ability to maintain its vasoactive function (9, 10). The administration of AM and AMBP-1 in combination protects against cardiovascular instability and mortality in experimental sepsis by preventing the increase in peripheral vascular resistance and maintaining oxygen delivery to terminal organs. Most interestingly, these agents in combination have also been shown to suppress proinflammatory cytokines in the circulation of septic animals as well as LPS-induced TNF-{alpha} secretion from peritoneal macrophages and RAW 264.7 cells in vitro (13). A general approach reducing the proinflammatory cytokine response has been shown to be beneficial in sepsis (14). Together with the vasoactive function, the anti-inflammatory effect of AM/AMBP-1 synergistically protects animals from dying of sepsis. However, the mechanism of the anti-inflammatory properties of AM/AMBP-1 remains elusive and cannot be satisfactorily explained by its effects on vascular function alone. To better understand the beneficial effect of AM/AMBP-1, we investigated the responsible mechanisms.

Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is a ligand-activated transcription factor, and the first described ligands were the recently launched antidiabetic thiazolidinediones. PPAR-{gamma} is mainly expressed in adipose tissues where it regulates fatty acid and glucose metabolism, cell growth, and differentiation of adipocytes (15, 16). Endogenous ligands of PPAR-{gamma} have been recently identified such as the prostaglandins of the D2 and J2 family, of which 15-deoxy {Delta}12,14 prostaglandin J2 (15dPGJ2) has been found to be the most potent activator (17, 18). After binding to ligands, PPAR-{gamma} dimerizes with the retinoid X receptor and induces gene transcription such as the up-regulation of glucose transporter 4, sensitizing cells to insulin by increasing glucose uptake (19). This is insofar relevant here because sepsis is associated with insulin resistance and PPAR-{gamma}-dependent liver dysfunction (20, 21, 22).

Most recently, PPAR-{gamma} was also found in endothelial cells and macrophages and seems to be involved in anti-inflammatory pathways (23) by down-regulating the proinflammatory cytokines IL-6, IL-10, IL-12, and TNF-{alpha} and the inducible NO synthase (24). PPAR-{gamma} has been reported to be up-regulated in activated macrophages as well as in neurons and astrocytes by activation of beta-adrenergic receptors (25), implying an influential role for cAMP as a second messenger in this pathway. Indeed, Michael et al. (26) demonstrated an up-regulation of PPAR-{gamma} in the cAMP-induced differentiation of type II pneumocytes. Therefore, we hypothesized that the anti-inflammatory properties of AM/AMBP-1 are mediated through the up-regulation of PPAR-{gamma} in macrophages. To test this hypothesis, we investigated whether AM/AMBP-1 is anti-inflammatory in endotoxemia, whether AM/AMBP-1 can up-regulate PPAR-{gamma} expression in vivo and in vitro, and what mechanism lies behind this action in an in vitro approach using murine RAW 264.7 and primary rat peritoneal macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Endotoxemia model and macrophage isolation

Male Sprague–Dawley rats (275–325 g) were fasted overnight but allowed water ad libitum. Rats were assigned to three different groups (sham, endotoxemia plus vehicle, and endotoxemia plus AM/AMBP-1) with six animals per group. To induce endotoxemia, rats were i.v. injected with 15 mg of LPS (Escherichia coli O55:B5, Difco Laboratories) per kilogram of body weight (BW) and received i.v. either normal saline (vehicle group) or AM/AMBP-1 (24 µg of AM from Phoenix Pharmaceuticals and 80 µg of AMBP-1 from Cortex) in 1 ml of normal saline 30 min before LPS injection. Sham animals received 1 ml of normal saline only. AMBP-1 is a native 120- to 140-kDa protein that was ultrapurified from human blood plasma with a purity of >98% by SDS-PAGE. Only trace amounts of IgG, IgA, IgM, factor I, factor B, factor P, C3, C4, and albumin can be detected. Rat AM is a highly purified (>99% by HPLC) synthetic peptide containing of 50 amino acids (5.73 kDa). AM and AMBP-1 were preincubated for 15 min before experiments in 1.5–3% of the final volume and then adjusted for the experiments to the required concentration with PBS (in vivo) or complete culture medium (in vitro). According to our previous studies using a rat model of sepsis produced by cecal ligation and puncture (9, 10), we initially used a dose of 12 µg of AM and 40 µg of AMBP-1 in vivo. This dose, however, proved to be ineffective in the present endotoxemia model. We therefore doubled the dose of AM and AMBP-1 in these endotoxemic rats, which was effective.

Blood was collected from animals by cardiac puncture 4 h after LPS injection. Macrophages were isolated by collecting peritoneal fluid through transabdominal puncture, centrifugation and resuspension in DMEM (Invitrogen Life Technologies) containing 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine (complete medium) and adherence for 2 h at 37°C. All experiments were performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. This project was approved by the Institutional Animal Care and Use Committee of the Feinstein Institute for Medical Research (Manhasset, NY).

ELISA

TNF-{alpha}, IL-6, and IL-10 levels were quantified in plasma and/or cell supernatant using ELISA kits specific for mouse TNF-{alpha}, IL-6, or IL-10 (BD Pharmingen). The assay was conducted according to the instructions provided by the manufacturer. Each sample was run in duplicate and the average was used for the calculation of the overall average in each group.

Lactate assay

Lactate was measured in the plasma of endotoxemic and sham animals 4 h after LPS or normal saline injection using a plasma lactate reagent set (Pointe Scientific) according to manufacturer’s instructions. Each sample was run in duplicate and the average was used for the calculation of the overall average in each group.

Cell culture

The murine macrophage cell line RAW 264.7 (American Type Culture Collection) was cultured in complete medium at 37°C in a humidified atmosphere containing 5% CO2. For all experiments, cells were grown to 80–90% confluence. After being washed twice with HBSS (Invitrogen Life Technologies), cells were cultured in medium containing LPS with or without AM/AMBP-1. For mechanistic studies, cells were preincubated with the PPAR-{gamma} activator 15dPGJ2 (Cayman), the cAMP analog dibutyryl cAMP (DBcAMP; Sigma-Aldrich), the nonspecific phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich), the cAMP-specific PDE IV inhibitor rolipram (ROL; Sigma-Aldrich), the tyrosine kinase inhibitor genistein (Sigma-Aldrich), the ERK1/2 inhibitor PD98059 (Calbiochem), the mitogen p38 inhibitor SB 203580 (Tocris Bioscience), or the JNK inhibitor SP600125 (Tocris Bioscience).

TNF-{alpha} and PPAR-{gamma} gene expression

Gene expression of PPAR-{gamma} was assessed by RT-PCR. The primers used were as follows: mouse PPAR-{gamma} (606 bp; GenBank accession no. U01664), 5'-ACCACTCGCATTCCTTTGAC-3' (forward) and 5'-TCAGCGGGA –AGGACTTTATG-3' (reverse); mouse TNF-{alpha} (356 bp; GenBank accession no. NM_013693), 5'-TTCTGTCCCTTTCACTCACTGG-3' (forward) and 5'-TTGGTGGTTTGCTACGACGTGG-3' (reverse); mouse beta-actin (540 bp; GenBank accession no. NM_007393), 5'-GTGGGCCGCTCTAGGCACCAA-3' (forward) and 5'-CTCTTTGATGTCACGCACGATTTC-3' (reverse). PCR was performed in a 25-µl reaction volume and amplified 28 times (94°C for 45 s, 60°C for 45 s, and 72°C for 120 s) followed by a final extension for 7 min at 72°C. Amplicons were electrophoresed in 1.5% agarose gel containing 0.22 µg/ml ethidium bromide. The gel was then analyzed on a Bio-Rad Image System and optical densities of gene bands were determined by ChemiImager 5500 software. For the silencing experiments, PPAR-{gamma} mRNA expression was assessed by quantitative PCR using the SYBR Green PCR master mix (Applied Biosystems). One microgram of RNA equivalent of cDNA was mixed with 80 nM each PPAR-{gamma} primer (forward, 5'-CAAGGCGAGGGCGATCTT-3' and reverse, 5'-CATGTCGTAGATGACAAATGGT-3') and run on a 7300 real-time PCR system (Applied Biosystems) in a final volume of 24 µl. Relative expression of mRNA was calculated by the {Delta}{Delta}Ct threshold cycle method and the results were expressed as fold change with respect to the corresponding experimental control.

PPAR-{gamma} Western blotting

Protein was extracted from RAW264.7 macrophages using TRI Reagent (Molecular Research Center) according to manufacturer’s protocol, dissolved in 1% SDS, and quantified using the DC Protein Assay (Bio-Rad). Sixteen micrograms of protein was fractionated on 4–12% Bis-Tris gel and transferred to a 0.2-µm nitrocellulose membrane. Nitrocellulose blots were blocked by incubation in TBST (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20) containing 5% milk for 1 h. Blots were incubated with rabbit anti-mouse PPAR-{gamma} IgG (1/1500; Cayman) overnight at 4°C and then washed for 10 min five times in TBST. Blots were incubated with HRP-labeled goat anti-rabbit IgG for 1 h at room temperature and chemiluminescent peroxidase substrate (ECL; Amersham Biosciences) was applied according to the manufacturer’s instructions. Membranes were briefly exposed to radiograph film and densities of bands were analyzed using the Bio-Rad Imaging system.

Immunohistochemistry

RAW264.7 macrophages were plated at a density of 2.5 x 104/well on a 16-well chamber slide (Lab-Tek; Nalge Nunc) and cultured in complete culture medium containing 100 ng/ml LPS with or without AM/AMBP-1 (200 nM AM/100 nM AMBP-1) or 15dPGJ2 (20 µM) for 4 h. Following the experiment, cells were fixed with 4% paraformaldehyde, washed in 0.1 M TBS (pH 7.4) containing 0.1% Triton X-100, and incubated in 2% hydrogen peroxide in 60% methanol for 15 min. Nonspecific binding sites were blocked with 3% goat serum followed by a 2-h incubation with 1/50 rabbit anti-mouse PPAR-{gamma} polyclonal Abs (Cayman). After washing with TBS, slides were incubated with 1/200 biotinylated anti-rabbit Ig G (Vector Laboratories) for 1 h at room temperature followed by 1 h of incubation in Vectastain ABC reagent (Vector Laboratories) and a final reaction in diaminobenzidine (DAB) solution (Vector Laboratories). Slides were viewed at 20°C with magnification at x10 (0.22 numerical aperture) and x40 (0.75 numerical aperture) using a Nikon Eclipse E600 (Japan) with Spot 3.5.2 acquisition software.

MTT assay

The MTT (Sigma-Aldrich) assay is based on the reduction of MTT by mitochondrial enzymes to an insoluble formazan dye that is dependent on metabolic activity. This method can be used to assess the viability of cultured cells. Briefly, 2 x 105 cells were cultured in 96-well-plates containing 200 µl of medium supplemented with 10 µl of 10 mg/ml MTT solution for 2 h at 37°C. The medium was removed and 100 µl of DMSO was added to dissolve the developed formazan. The absorbance was measured at 570 nm and correlated to control values to express the percentage of viability.

PPAR-{gamma} gene silencing by small interfering RNA (siRNA)

Dharmacon provided four sequences of double-stranded RNA engineered for >80% silencing effect in the SmartPool mixture. RAW 264.7 cells were incubated in 12-well plates (37°C and 5% CO2 at a concentration of 5 x 105 cells/ml in FBS and antibiotic-free DMEM. PPAR-{gamma} was silenced using SmartPool siRNA (Dharmacon) and the DharmaFECT4 transfection reagent as per the manufacturer’s protocol. The sequences of the four duplets used are as follows (mouse PPAR-{gamma} mRNA, GenBank accession no. NM_011146): 5'-CAACAGGCCTCATGAAGAA-3', 5'-GACATGAATTCCTTAATGA-3', 5'-GAAGAACCATCCGATTGAA-3', and 5'-CTGCATCTTTCCACCTTATTA-3'. Briefly, 0.5 µM siRNA was incubated for 20 min in 200 µl of DharmaFECT4 siRNA transfection reagent (diluted 1/100 with FBS and antibiotic-free DMEM). The reaction mix was then filled to 1 ml of DMEM supplemented with FBS with a final concentration of 10%. Transfection was performed by incubating RAW264.7 macrophages for 24 or 48 h at 37°C and 5% CO2. Nontargeting siRNA (Dharmacon) served as negative control. Successful transfection and knockdown of PPAR-{gamma} was verified by quantitative PCR and Western blotting. RAW 264.7 cells were then subjected to LPS stimulation with or without treatment with AM/AMBP-1 as described above.

cAMP enzyme immunoassay

Intracellular cAMP levels of RAW 264.7 macrophages were assessed after stimulation with LPS and treatment with AM/AMBP-1 in 96-well plates at 105 cells/well using the cAMP Biotrak enzyme immunoassay (Amersham Biosciences) according to the manufacturer’s instructions.

Signaling pathways

Activation of proline-rich tyrosine kinase-2 (Pyk-2) and p44/42 (ERK1/2) was assessed by measuring phosphorylated kinases via Western blotting. Briefly, cell lysates were resolved on a 4–12% Bis-Tris gel and transferred to a 0.2-µm nitrocellulose membrane. Nitrocellulose blots were blocked with TBST containing 5% BSA for 1 h, incubated with rabbit anti-mouse phospho-Pyk-2 IgG (1/1000; Cell Signaling Technology) overnight at 4°C, and washed five times in TBST. Blots were then incubated with HRP-labeled goat anti-rabbit IgG for 1 h at room temperature and washed as before. ECL was used to visualize bands on a radiograph.

Statistical analysis

All data are expressed as means ± SEM and compared by one-way ANOVA and Tukey’s test. Differences in values were considered significant if p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Beneficial effects of AM/AMBP-1 in endotoxemia

To test whether AM/AMBP-1 is beneficial in an endotoxemia model, we administered LPS (15 mg/kg BW) to rats that either received 1 ml of either vehicle or AM/AMBP-1 (24 and 80 µg/kg BW, respectively). This dose of LPS is known to induce septic shock and ~50% lethality within 24 h (27). Compared with sham-operated animals, plasma levels of TNF-{alpha} increased by 54-fold (from 15.4 ± 8.6 to 841.2 ± 183.9 pg/ml) (Fig. 1A) and lactate levels more than doubled (from 1.25 ± 0.16 to 2.73 ± 0.53 mmol/L) (Fig. 1B). Pretreatment with AM/AMBP-1, however, markedly attenuated TNF-{alpha} levels by 54% (Fig. 1A) and resulted in a complete abrogation of LPS-induced lactacidosis (Fig. 1B). One of the major sources of TNF-{alpha} seems to be peritoneal macrophages, because the TNF-{alpha} concentration in the peritoneal fluid of endotoxemic rats was 5.7 times higher compared with plasma levels (Fig. 1C). In peritoneal fluid, the AM/AMBP-1-mediated decrease of TNF-{alpha} release was even more dramatic than that in plasma (by >83%; Fig. 1C). This anti-inflammatory effect of AM/AMBP-1 was associated with a significant increase in PPAR-{gamma} gene expression in peritoneal macrophages by 77% (Fig. 1D). Plasma levels of the anti-inflammatory cytokine IL-10 were further increased in AM/AMBP-1-treated animals by 52.3% (Fig. 1E), whereas IL-6 levels did not show any significant difference (data not shown).


Figure 1
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  		FIGURE 1. Beneficial effects of AM/AMBP-1 in endotoxemia. A and B, Endotoxemia was induced in rats by i.v. injection of 15 mg/kg BW LPS. Six rats per group received PBS only (Sham), LPS plus PBS (Vehicle), or LPS plus AM/AMBP-1 (24 or 80 µg/kg BW) at the time of LPS-injection. Blood and peritoneal fluid were collected 4 h later. Plasma TNF-{alpha} (A) and lactate levels (B) were measured by ELISA. *, p < 0.05 vs sham; #, p < 0.05 vs vehicle; one-way ANOVA and Tukey’s test, n = 6. C and D, Peritoneal exudates were collected in endotoxemic rats 4 h after LPS injection with or without AM/AMBP-1 treatment and TNF-{alpha} concentration was measured by ELISA in undiluted samples (C). Peritoneal macrophages (pM{phi}) were recovered from the exudates and by peritoneal lavage from 6 individual rats in each group by centrifugation and plastic adherence for 2 h. PPAR-{gamma} gene expression was assessed by RT-PCR (D). #, p < 0.05 vs vehicle; Student’s t test, n = 6. Typical bands are shown above the graph. E, IL-10 was measured by ELISA in plasma samples of rats 4 h after challenging them with 15 mg/kg LPS and treated with either vehicle or AM/AMBP-1. *, p < 0.05 vs sham; #, p < 0.05 vs vehicle; ANOVA and Tukey’s test, n = 6.

 
LPS-mediated inhibition of PPAR-{gamma} expression is reconstituted by AM/AMBP-1

To evaluate the AM/AMBP-1-induced anti-inflammatory effects and its association with reconstituted PPAR-{gamma} levels, we used an in vitro model using the murine RAW 264.7 macrophage cell line. LPS (100 ng/ml) induced an increase in TNF-{alpha} mRNA expression by 63% (Fig. 2A) and TNF-{alpha} release by >280-fold (from 0.25 to 70.74 ng/ml; Fig. 2B). Administration of AM/AMBP-1 (100 nM AM/50 nM AMBP-1) to LPS-stimulated RAW264.7 macrophages decreased TNF-{alpha} mRNA expression (Fig. 2A) and its protein release (Fig. 2B) by 16.0 and 45.7%, respectively, and further reduced TNF-{alpha} mRNA expression at a dose of 200 nM AM/100 nM AMBP-1 (Fig. 2A). Incubation of RAW264.7 cells for 4 h with LPS at concentrations ranging from 0.1 to 1000 ng/ml resulted in a decrease in PPAR-{gamma} expression in a dose-dependent fashion (Fig. 2C). LPS at 10 ng/ml resulted in a significant (p = 0.028) decrease in PPAR-{gamma} expression compared with macrophages cultured in medium alone, decreasing even further (by 31%) at a concentration of 100 ng/ml LPS (Fig. 2C).


Figure 2
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  		FIGURE 2. LPS-mediated inhibition of PPAR-{gamma} expression is reconstituted by AM/AMBP-1. A and B, Suppression of LPS-stimulated TNF-{alpha} production by AM/AMBP-1 in vitro. RAW264.7 macrophages (2 x 106) were stimulated with LPS for 4 h with or without coincubation with AM/AMBP-1. TNF-{alpha} gene expression was measured by RT-PCR (six wells per group) (A) and TNF-{alpha} protein release by ELISA (eight wells per group) (B). C, Down-regulation of PPAR-{gamma} in RAW264.7 macrophages by LPS. PPAR-{gamma} expression was assessed 4 h after stimulating 2 x 106 RAW 264.7 macrophages with LPS (0.1–1,000 ng/ml; 6 wells per group). D and E, Prevention of LPS-mediated suppression of PPAR-{gamma} by AM/AMBP-1. RAW264.7 macrophages (2 x 106) were cultured in six individual wells with LPS with or without AM/AMBP-1 for 4 h, and PPAR-{gamma} gene expression (D) and protein levels (E) were assessed by RT-PCR and Western blotting, respectively. All data expressed as means ± SEM. *, p < 0.05 vs medium alone; #, p < 0.05 vs LPS 100 ng/ml alone; one-way ANOVA and Tukey’s test; n = 6 in A and C–E; n = 8 in B.

 
To determine whether AM/AMBP-1 reconstitutes LPS-induced reduction in PPAR-{gamma} expression, RAW264.7 macrophages were coincubated with AM/AMBP-1 at different concentrations (100–200 nM AM and 50–100 nM AMBP-1) based on previous reports (28, 29). Four hours of incubation induced a significant increase in PPAR-{gamma} mRNA expression (Fig. 2D) as well as protein levels (Fig. 2E) by 54 and 30%, respectively, at a dose of 100 nM AM/50 nM AMBP-1. Complete reconstitution of PPAR-{gamma} to unstimulated values was achieved at the incubation with 200 nM AM/100 nM AMBP-1 at both transcriptional and translational levels (Fig. 2, D and E). Inhibition of protein translation using cycloheximide (25 µM) in RAW264.7 cells resulted in the abrogation of PPAR-{gamma} up-regulation, indicating the importance of de novo protein synthesis in the up-regulation of PPAR-{gamma} (data not shown).

PPAR-{gamma} remains localized to the nucleus by AM/AMBP-1 after LPS exposure and is necessary for the inhibition of TNF-{alpha} expression

To further assess the role of PPAR-{gamma} role in the anti-inflammatory pathway induced by AM/AMBP-1, RAW 264.7 cells were stained with peroxidase-labeled anti-PPAR-{gamma} Abs. Microscopic evaluation revealed that nonstimulated macrophages were spherical and displayed strong nuclear staining, indicating a high expression and nuclear localization of PPAR-{gamma} (Fig. 3A). Four hours after LPS-stimulation, in contrast, the cells showed morphological signs of activation (spindle shape) with cytoplasmic and nuclear paucity of PPAR-{gamma} expression, indicating a down-regulation of this nuclear receptor (Fig. 3B). Coincubation with AM/AMBP-1 resulted in an almost complete inhibition of LPS-induced nuclear loss of PPAR-{gamma} with fewer morphological signs of activation (Fig. 3C). RAW 264.7 cells were incubated for 1 h with the PPAR-{gamma} agonist 15dPGJ2 before stimulation with 100 ng/ml LPS for 4 h. 15dPGJ2 at 10 and 20 nM significantly decreased TNF-{alpha}-release by 28 and 84%, respectively (Fig. 3D), without affecting the viability or proliferation of the macrophages at these concentrations as measured by MTT assay (98 ± 1.7% viability after preincubation with 20 µM 15dPGJ2; mean ± SEM compared with controls, n = 5). This suggests that PPAR-{gamma} plays a major role in the down-regulation of LPS-induced TNF-{alpha} release from macrophages. Furthermore, after PPAR-{gamma} siRNA transfection, RAW264.7 macrophages still responded to a 15dPGJ2-mediated suppression of LPS-stimulated TNF-{alpha} release, but to a significantly lower extent compared with control siRNA-transfected cells (57.3% vs 71.4% suppression, p < 0.05, n = 6). The reason for this effect may be due to the fact that as a prostaglandin, 15dPGJ2 may also suppress a proinflammatory response through PPAR-{gamma}-independent pathways (data not shown). To investigate the importance of PPAR-{gamma} in TNF-{alpha} suppression induced by AM/AMBP-1, RAW264.7 macrophages were transfected with either nontargeting RNA or PPAR-{gamma}-specific siRNA for 24–48 h. PPAR-{gamma} mRNA expression decreased at 24 h after transfection by 61.5 ± 8.2% (Fig. 3E) and protein levels were suppressed by 91.4 ± 0.7% within 48 h after transfection (Fig. 3F). Control-transfected RAW264.7 macrophages showed, as nontransfected macrophages, a significant suppression of LPS-stimulated TNF-{alpha} expression after preincubation with AM/AMBP-1 (Fig. 3G). Cells transfected with the PPAR-{gamma}-specific siRNA, however, showed that after almost complete suppression of PPAR-{gamma} the cells were unresponsive to AM/AMBP-1 (Fig. 3H). The same is true for TNF-{alpha} protein levels, which showed a significant suppression by AM/AMBP-1 in control-transfected cells (Fig. 3I) but failed to do so in PPAR-{gamma}-silenced cells (Fig. 3J). This indicates that PPAR-{gamma} is crucial for the AM/AMBP-1 mediated TNF-{alpha} suppression.


Figure 3
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  		FIGURE 3. PPAR-{gamma} remains localized to the nucleus by AM/AMBP-1 after LPS exposure and is necessary for the inhibition of TNF-{alpha} expression. For immunohistochemistry RAW264.7 macrophages were cultured in medium without stimulation (A), stimulation with LPS (100 ng/ml) (B), or LPS plus AM/AMBP-1 (200 nM AM/100 nM AMBP-1) (C) for 4 h, labeled with PPAR-{gamma}-specific Abs, and made visible by peroxidase reaction. Unstimulated macrophages are of round shape and display a small seam of cytoplasm and a strong nuclear staining, indicating a high expression and nuclear location of PPAR-{gamma} (A). LPS-stimulated macrophages show morphological signs of activation (pseudopodia and firm adhesion to the surface) with nuclear and cytoplasmic paucity of PPAR-{gamma} expression (B). AM/AMBP-1 treatment almost completely maintained nuclear localization of PPAR-{gamma} (C). Size bar, 20 µm. Activation of PPAR-{gamma} by its ligand 15dPGJ2 leads to a dose-dependent decrease of TNF-{alpha} release from cultured RAW264.7 macrophages (D). *, p < 0.05 vs LPS alone; one-way ANOVA and Tukey’s test, n = 6. PPAR-{gamma} silencing abrogates AM/AMBP-1-mediated suppression of TNF-{alpha} expression. RAW264.7 macrophages were transfected with either nontargeting siRNA (Control) or PPAR-{gamma}-specific siRNA (siRNA) for 24 (E) or 48 h (F). PPAR-{gamma} mRNA expression was evaluated after 24 h by quantitative PCR (E), and protein levels were assessed by Western blotting after 48 h (F). *, p < 0.05 vs control; t test, n = 3. RAW264.7 macrophages that were either control-transfected (G and I) or transfected with PPAR-{gamma}-specific siRNA (H and J) for 48 h were stimulated with 10 ng/ml LPS for 4 h with or without a preincubation with 200 nM AM and 100 nM AMBP-1 for 1 h prior to stimulation. TNF-{alpha} gene expression was assessed by RT-PCR (G and H; representative gels are shown above graphs) and protein levels were assessed by ELISA (I and J). *, p < 0.05 vs control; #, p < 0.05 vs LPS alon; ANOVA and Tukey’s test, n = 3. All experiments were performed at least three times (three separate experiments) with three replicates in each experiment.

 
AM/AMBP-1-mediated increase in intracellular cAMP is partially responsible for the suppression of TNF-{alpha} release

Because AM is known to produce its vasoactive effects through the activation of G{alpha}S-mediated increases in cAMP, we measured intracellular cAMP concentrations in RAW264.7 macrophages by enzyme immunoassay using the same conditions as described above. To our surprise, we found a similar pattern of changes as those found in PPAR-{gamma} expression. cAMP levels were dramatically reduced in LPS-treated macrophages by 84% (from 490 ± 116 to 78.7 ± 29.2 fmol/105 cells) and almost completely reconstituted after coincubation with 100 nM AM/50 nM AMBP-1 (Fig. 4A). To determine whether cAMP is able to reduce inflammatory cytokine release from macrophages, RAW264.7 cells were incubated with LPS (100 ng/ml) for 30 min after preincubation with different concentrations of DBcAMP. DBcAMP considerably reduced TNF-{alpha} release from LPS-stimulated macrophages in a dose-dependent fashion and was highly effective at concentrations of 1 and 10 mM, where it reduced TNF-{alpha} release by >73% (Fig. 4B). Note, however, that the extracellular concentration of DBcAMP necessary to induce an inhibitory effect on TNF-{alpha} release was at least 250 times higher than the intracellular cAMP levels we observed. Similar results were found after preincubation with the nonspecific and cAMP-specific (type IV) PDE inhibitors IBMX and ROL, respectively, and stimulation with 10 ng/ml LPS. These agents were also able to significantly reduce TNF-{alpha} release from RAW264.7 macrophages (–43% by IBMX and –55% by ROL) even if their efficacy was not as pronounced as that of DBcAMP (Fig. 4C). To investigate whether cAMP plays a role in PPAR-{gamma} regulation in activated macrophages, cells were incubated with DBcAMP (10 µM) or ROL (200 µM) before stimulation with LPS. However, these agents were not able to reconstitute LPS-induced PPAR-{gamma} suppression (Fig. 4D) at the biologically active concentrations that significantly reduced TNF-{alpha} release (Fig. 4C).


Figure 4
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  		FIGURE 4. A, AM/AMBP-1-mediated increase in intracellular cAMP is partially responsible for the down-regulation of proinflammatory cytokines. LPS-stimulation of RAW264.7 macrophages decreases intracellular cAMP levels and AM/AMBP-1 reconstituted these levels to 84% of control. *, p < 0.05 vs medium; #, p < 0.05 vs LPS; one-way ANOVA and Tukey’s test, n = 3. B, Preincubation with DBcAMP for 30 min dose-dependently decreased TNF-{alpha} release from LPS-stimulated (100 ng/ml for 4 h) RAW264.7 macrophages. *, p < 0.05 vs control; #, p < 0.05 vs LPS alone; one-way ANOVA and Tukey’s test, n = 6. C, The PDE inhibitors IBMX (10 nM) and ROL (200 µM) were used to preincubate (30 min) RAW264.7 cells before stimulation with 10 ng/ml LPS for 4 h. *, p < 0.05 vs medium; #, p < 0.05 vs LPS alone (10 ng/ml); one-way ANOVA and Tukey’s test, n = 5. D, LPS-mediated suppression of PPAR-{gamma} cannot be recovered by cAMP. RAW264.7 cells were preincubated with DBcAMP or ROL for 30 min before LPS-stimulation and PPAR-{gamma} expression was assessed by RT-PCR 4 h later. *, p < 0.05 vs control; one-way ANOVA and Tukey’s test, n = 8 for control and LPS; n = 6 for LPS plus DBcAMP or LPS plus ROL.

 
AM/AMBP-1-induced up-regulation of PPAR-{gamma} is mediated by the activation of the Pyk-2-ERK1/2 pathway

According to previous reports, AM was able to induce the proliferation of several cell types via the activation of the Pyk-2 tyrosine kinase and ERK1/2 pathway (28). We therefore investigated whether Pyk-2 could also be activated in macrophages by AM/AMBP-1. Within 10 min of AM/AMBP-1 stimulation, RAW264.7 macrophages displayed a massive activation of Pyk-2 as assessed by phosphorylation of the tyrosine kinase (Fig. 5A). Pyk-2 activation peaked at 60 min and then decreased to constitutive levels after 2 h. AM/AMBP-1 also induced the phosphorylation of ERK1/2 in RAW264.7 macrophages that started within 10 min and remained high after 2 h (Fig. 5B), when LPS-induced phospho-ERK1/2 expression had already declined (Fig. 5C). In RAW264.7 cells that were stimulated with both AM/AMBP-1 and LPS there was a dose-dependent increase in phospho-ERK1/2 expression even after 4 h as shown in Fig. 5D. To investigate the role of MAPKs in the suppression of PPAR-{gamma}, we used inhibitors for the three kinases ERK1/2 (PD98059), JNK (SP600125), and p38 (SB203580). However, none of those was able to prevent LPS-mediated down-regulation of PPAR-{gamma} (Figs. 5, E and F). To investigate whether the activation of the Pyk-2 tyrosine kinase and ERK1/2 is causative for the up-regulation of PPAR-{gamma} by AM/AMBP-1, we inhibited these two kinases by preincubation with genistein (Fig. 5G) and PD98059 (Fig. 5H), respectively. Both genistein and PD98059 were able to dose-dependently abrogate AM/AMBP-1-mediated reconstitution of PPAR-{gamma} in LPS-stimulated RAW264.7 macrophages, strongly suggesting the influence of the Pyk-2-ERK1/2 pathway in the up-regulation of PPAR-{gamma} (Fig. 5, G and H). Up-regulation of PPAR-{gamma} protein levels by AM/AMBP-1 was also abrogated by the preincubation with the ERK1/2-inhibitor PD98059 (Fig. 5I), indicating that the ERK1/2 pathway plays a crucial role in the AM/AMBP-1-mediated regulation of PPAR-{gamma}. Preincubation of RAW264.7 macrophages with the MEK1/2 inhibitor PD89059, genistein, or tyrphostin-23 blocked the phosphorylation of ERK1/2 in these cells (Fig. 5I). At the same time, PD98059 preincubation also partially blocked the AM/AMBP-1-mediated inhibition of TNF-{alpha} gene expression (Fig. 5K) and protein levels (Fig. 5L), indicating an influential role of the ERK1/2-mediated up-regulation of PPAR-{gamma} in the anti-inflammatory effect of AM/AMBP-1.


Figure 5
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  		FIGURE 5. AM/AMBP-1 up-regulates PPAR-{gamma} through the Pyk-2-ERK1/2 pathway. A, RAW264.7 macrophages were incubated with 200 nM AM/100 nM AMBP-1 for 4 h and Pyk-2 tyrosine kinase phosphorylation was assessed by Western blotting. *, p < 0.05 vs 0 min; one-way ANOVA and Tukey’s test, n = 4. B, Subsequent ERK1/2 activation by 200 nM AM/100 nM AMBP-1 (phospho-p44/42 Western blot). C, Transient ERK1/2 activation by LPS. RAW 264.7 cells were stimulated with LPS (100 ng/ml) and phospho-ERK1/2 was assessed by Western blotting. D, AM/AMBP-1 maintains ERK1/2 phosphorylation at 4 h in a dose-dependent manner in LPS-stimulated macrophages. RAW264.7 macrophages were stimulated with 100 ng/ml LPS and coincubated with AM/AMBP-1 for 4 h. *, p < 0.05 vs medium; one-way ANOVA and Tukey’s test, n = 4. E and F, LPS-induced suppression of PPAR-{gamma} is mediated by a MAPK-independent pathway. RAW264.7 macrophages were stimulated with 100 ng/ml LPS for 4 h after a 1-h preincubation with inhibitors of different mitogen-activated protein kinases. ERK, JNK, and p38 were inhibited with PD98059 (30 µM), SP600125 (25 µM), and SB203580 (20 µM), respectively. Cells were lysed and analyzed for PPAR-{gamma} mRNA expression by quantitative PCR (E) or protein expression by Western blotting (F). *, p < 0.05 vs control; one-way ANOVA and Tukey’s test. The experiment was repeated three times. Protein levels are shown in a representative blot (F). G–I, Inhibition of Pyk-2 by genistein (G) or MEK1/2 by PD98059 (H and I) 30 min before the experiment dose-dependently abrogated AM/AMBP-1 induced reconstitution of PPAR-{gamma} gene (RT-PCR, G and H) and protein (Western blotting, I) expression in RAW264.7 cells. *, p < 0.05 vs control; #, p < 0.05 vs LPS alone; {dagger}, p < 0.05 vs LPS plus AM/AMBP-1; one-way ANOVA and Tukey’s test, n = 4. J, Suppression of AM/AMBP-1-mediated ERK1/2 activation by PD98059 (30 µM), genistein (50 µM), and tyrphostin-23 (Tyr23) (100 µM). RAW264.7 cell were stimulated with LPS with or without AM/AMBP-1 for 10 min with or without prior incubation with the inhibitors and analyzed by Western blotting of phospho-ERK1/2. K–L, Inhibition of MEK1/2 abrogates the anti-inflammatory effect of AM/AMBP-1. RAW264.7 cells were preincubated with PD98059 for 30 min before stimulation with LPS for 4 h. TNF-{alpha} mRNA (K) and protein (L) levels were assessed by RT-PCR and Western blotting, respectively. *, p < 0.05 vs control; #, p < 0.05 vs LPS; {dagger}, p < 0.05 vs LPS plus AM/AMBP-1; one-way ANOVA and Tukey’s test; n = 4 for control, LPS, and LPS plus AM/AMBP-1 plus PD98059 and n = 6 for LPS plus AM/AMBP-1. n represents the number of experiments performed with two to three replicates in each experiment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Sepsis, trauma, injury, and severe infection lead to a systemic inflammatory response and are associated with neurohumoral activation and increased cytokines, catecholamines, and glucocorticoids. The extensive inflammatory and endocrine response is associated with physiological dysfunctions. The initially increased metabolic and cardiovascular response (hyperglycemia and hyperdynamic circulation, i.e., increased cardiac output) is rapidly followed by an abrupt deterioration (hypoglycemia and hypodynamic circulation, i.e., reduced cardiac output and increased peripheral vascular resistance) and impaired immune function (9, 10). However, it is unclear what triggers the transition from the hyperdynamic phase of septic shock to the preterminal hypodynamic phase. Wang and coworkers (9, 10) have shown that part of this transition may be triggered by the loss of AMBP-1 in late sepsis, which, in turn, leads to vascular hyporesponsiveness to AM. They have shown that the administration of AM in conjunction with AMBP-1 could prevent this transition and salvage animals from otherwise lethal sepsis (13, 27). This was associated with significantly reduced proinflammatory mediators, which raised the question how inflammation is suppressed by AM/AMBP-1. Because the beneficial mechanism has never been investigated before, the present work used endotoxemia as an acute systemic inflammatory model. This model is different from the previously described CLP-induced sepsis. Contrary to CLP, endotoxemia is not associated with primary bacteremia and the inflammatory reaction seen is more rapid and more severe than after CLP. Aside from providing further evidence of the effectiveness of AM/AMBP-1 in multiple disease models, this strong proinflammatory response made endotoxemia a specifically reliable model to investigate the anti-inflammatory properties of AM/AMBP-1.

A previous study has shown that AM/AMBP-1 effectively suppresses LPS-induced TNF-{alpha} release from macrophages and that this effect is markedly higher in the combination of both substances than in either alone (13, 30). The reason for this is still not completely clear, yet studies have shown that the binding of AM to AMBP-1 prevents its degradation and augments its receptor stimulatory effect (5). The more pronounced reduction of TNF-{alpha} gene expression compared with the inhibition of the actual release of cytokines by AM/AMBP-1 indicates that AM/AMBP-1 modulates the proinflammatory response on a transcriptional rather than a secretory level.

AM binds to its receptor and activates adenylate cyclases, thus increasing intracellular cAMP levels (5, 31). This second messenger has been reported to inhibit certain proinflammatory pathways such as the LPS-induced p38 and JNK pathways (32, 33, 34). However, in this study we show that AM/AMBP-1 also induces PPAR-{gamma} expression in LPS stimulated macrophages in vitro and in vivo. There has been ample research on the role of PPAR-{gamma} in improving insulin resistance. This effect is now widely used for the treatment of type 2 diabetic patients (35). Insulin resistance is also an issue occurring in septic patients, which emphasizes the role of PPAR-{gamma} in maintaining blood sugar levels in sepsis. However, down-regulation of PPAR-{gamma} has also been shown to be associated with liver dysfunction in sepsis (21), and there are numerous reports of its function in the suppression of inflammation (20, 36, 37, 38). We have shown in Fig. 1D that peritoneal macrophage PPAR-{gamma} is up-regulated in vivo by AM/AMBP-1 in endotoxemic rats. Together with our previous report showing a suppressive effect of AM/AMBP-1 on TNF-{alpha} gene expression and protein release in isolated and cultured Kupffer cells (13), we have sufficient evidence that the anti-inflammatory effect of AM/AMBP-1 through the up-regulation of PPAR-{gamma} holds true under in vivo conditions.

The implication of the three key molecules investigated here can be highlighted by previous research performed on mice lacking AM, AMBP-1, or PPAR-{gamma}. AM–/– embryos die at midgestation with extreme hydrops fetalis and cardiovascular abnormalities (39). Heterozygous AM+/– mice have reduced fertility (40) and express a severe perivascular inflammation after angiotensin II administration in conjunction with a high salt diet (41). Homozygous mutations of AMBP-1 results in markedly reduced serum C3, abnormal renal histology, spontaneous membranoproliferative glomerulonephritis, hematuria, proteinuria, and increased mortality at 8 mo of age. Pickering et al. (42) also showed that AMBP-1–/– mice are hypersensitive to developing renal injury caused by immune complexes. The lack of the PPAR-{gamma} gene in mice leads to interference with the terminal differentiation of the trophoblast and placental vascularization, causing severe myocardial thinning and early embryonic death. Other lethal pathologies include lipodystrophy and multiple hemorrhages (43). Targeted PPAR-{gamma} deficiency in macrophages increases atherosclerosis in hypercholesterolemia (40). The endogenous ligand of PPAR-{gamma}, 15dPGJ2, is synthesized by the hemopoietic prostaglandin D synthase (PGDS), which is the key enzyme for production of the D and J series of prostanoids in the immune system. PGDS–/– mice display severe inflammation in response to a delayed-type hypersensitivity reaction (44). Hence, inflammation seems to play a crucial role in the animals lacking either of the molecules mentioned above. In this study our further supportive data suggest a causative link between these molecules that leads to protection from an overwhelming inflammatory response. The majority of the knockout mice described above are not viable and most of these animals also display an overwhelming inflammatory response. This constitutes a strong limitation for the use of these models in our study and can only be bypassed by the use of specific targeted knockout models in the future. Another useful approach is the use of gene silencing in specific tissues in the future that can generate results of a similar value.

Our present results indicate that LPS down-regulates both intracellular cAMP levels and PPAR-{gamma} expression. As expected, administration of AM/AMBP-1 reconstitutes LPS-suppressed cAMP levels and PPAR-{gamma} expression at the transcriptional and translational levels. Increased concentrations of intracellular cAMP have been shown to down-regulate cytokine release in dendritic cells (36), microglial cells (32), alveolar cells (20, 45), and other macrophages (13, 46). In our present study, the cAMP analog DBcAMP and PDE inhibitors were able to decrease LPS-induced TNF-{alpha} release from RAW 264.7 macrophages. DBcAMP is an analog of the naturally occurring cAMP and has been shown to activate the downstream signaling pathways of the natural second messenger cAMP (33, 47). There is a variety of PDEs that rapidly terminate the effect of the second messenger cAMP. Nonspecific PDE inhibitors such as IBMX and the cAMP-specific inhibitors of type IV PDEs such as ROL maintain the second messenger signaling by preventing the degradation of cAMP (45). We inquired whether cAMP could be anti-inflammatory by up-regulating PPAR-{gamma}. However, none of the agents used proved to alter PPAR-{gamma} expression in LPS-stimulated macrophages. This might be due to the fact that analogues (e.g., DBcAMP) do activate certain pathways downstream of cAMP, such as protein kinase A, but fail to activate other pathways that are cAMP dependent. Another reason could be that this event is cAMP independent. Our data further showed that inhibitors for the three MAPK kinases ERK1/2 (PD98059), JNK (SP600125), and p38 (SB203580) were unable to prevent LPS-mediated suppression of PPAR-{gamma} (Fig. 5, E and F). We believe that in RAW264.7 macrophages the suppression of PPAR-{gamma} is mediated through a MAPK-independent pathway. In this regard, we recently found that TNF-{alpha}, released by the macrophages themselves, may play a role for the LPS-mediated suppression of PPAR-{gamma} via an autocrine mechanism (48). Of note, genistein also did not alter PPAR-{gamma} expression after LPS-stimulation alone, indicating that receptor tyrosine kinases may not play a role in LPS-mediated suppression of PPAR-{gamma}.

Activation of PPAR-{gamma} by 15dPGJ2 has been shown to down-regulate TNF-{alpha} release in vitro and in vivo (49). Jiang et al (50) and Ricote et al. (25) have both reported that 15dPGJ2 suppresses the inflammatory response of murine macrophages and human monocytes in a PPAR-{gamma}-dependent manner. However, because 15dPGJ2 is a prostaglandin, the anti-inflammatory effects may in part be caused by yet unknown PPAR-{gamma}-independent pathways, thereby explaining the incomplete reversal of TNF-{alpha} release after blocking PPAR-{gamma} either with a specific inhibitor or with siRNA. Because PPAR-{gamma} gene and protein expressions are up-regulated and its protein localization to the nucleus is promoted by AM/AMBP-1, we propose that this is a major pathway of the anti-inflammatory effect of AM/AMBP-1. We have previously shown that PPAR-{gamma} activation is in fact responsible for the down-regulation of LPS-induced TNF-{alpha} release in inhibition studies using GW9662, a PPAR-{gamma} selective antagonist (51). In this model, the AM/AMBP-1-mediated up-regulation of PPAR-{gamma} seems to play a greater role than the LPS-mediated suppression of PPAR-{gamma} in the regulation of TNF-{alpha} response.

Endogenous ligands for the PPARs are free fatty acids and eicosanoids. PPAR-{gamma} can be activated by the anti-inflammatory PGJ2. 15dPGJ2, a specific PPAR-{gamma} ligand, is detectable in low amounts in vivo, which makes its tracking very difficult. It has been reported that LPS-induced inflammation does not change 15dPGJ2 production in vivo. Similarly, in the synovial fluid of arthritis patients, for example, 15dPGJ2 levels were not altered (52). There may be other endogenous PPAR-{gamma} ligands that change in concentration during inflammation. We have shown here that the levels of PPAR-{gamma} expression decrease in macrophages, leading to an increased priming of those cells and an enhanced proinflammatory response. However, the mechanism responsible for the up-regulation of PPAR-{gamma} by AM/AMBP-1 remains unknown. Using inhibitors, we have shown that AM/AMBP-1 is able to increase cellular PPAR-{gamma} mRNA as well as protein levels by inducing gene transcription and de novo protein synthesis. Furthermore, we have shown that by knocking down PPAR-{gamma} using siRNA gene silencing, the suppressive effect of AM/AMBP-1 on TNF-{alpha} production was abrogated (Fig. 3).

Various inducers of PPAR-{gamma} expression have been reported, including growth factors, oxidized low-density lipoprotein (oxLDL), and even PPAR-{gamma} ligands themselves activating different pathways (53). Fortunately, our knowledge concerning the signal transduction of the AM receptor is constantly increasing. The AM receptor is a protein complex of the CRLR and the receptor activity-modifying protein (RAMP). CRLR is a seven-transmembrane domain protein and RAMP is a single transmembrane protein. There are three subtypes of RAMP, and AM-specific receptors are formed by CRLR and RAMP-2 or RAMP-3 (31, 54). Recently it has been shown that lack of the receptor component protein (RCP) leads to abrogation of the receptor-mediated signal transduction of AM (54, 55). The activation of protein kinases that are independent of cAMP assigns RCP a role in this signal transduction. We have convincingly shown that AM/AMBP-1-mediated signal transduction involves the Pyk-2-ERK1/2 pathway that is responsible for the up-regulation of PPAR-{gamma} under inflammatory conditions (i.e., LPS-stimulation). RCP-associated tyrosine kinase activation, which is proposed to be responsible for AM-mediated proliferation, also plays a role in the induction of the anti-inflammatory PPAR-{gamma} in macrophages. In fact, in this study we have shown that AM/AMBP-1 induces the phosphorylation of Pyk-2 tyrosine kinase and sustains ERK1/2 phosphorylation over 4 h (Fig. 5D). The inhibition of either kinase leads to an abrogation of AM/AMBP-1 mediated up-regulation of PPAR-{gamma}, indicating the importance of the Pyk-2-ERK1/2 pathway for this regulation. PPAR-{gamma} transactivation then leads to a reduction of proinflammatory cytokine production and release (Fig. 6). This novel finding can be further confirmed by knocking down the Pyk-2 and/or ERK1/2 kinases in macrophages. Although such an approach would support our data, gene silencing of the ERK1/2 kinase pathway has limitations because it is a crucial cell cycle protein that may lead to premature cell death (56, 57). The coinciding cAMP-mediated suppression of inflammation seems to act independently of PPAR-{gamma} activation. Multiple PKA-dependent and -independent pathways have been shown to be involved in the anti-inflammatory effects of cAMP. These include the direct inhibition of the IFN-{gamma}-activated STAT-signaling pathway, the decrease of LPS-induced phosphorylation of p38 and JNK MAPK, and also of NF-{kappa}B-mediated transcription (58), as graphically presented in Fig. 6.


Figure 6
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  		FIGURE 6. Signaling pathways of AM/AMBP-1-mediated anti-inflammatory effect. AMBP-1-bound AM binds its cognate receptor on the cell surface consisting of a CRLR and one of two receptor activity modifying proteins (RAMP2 or RAMP3). This G{alpha}S protein-coupled receptor is a multiprotein complex associated with another RCP thought to have protein kinase-activating properties. LPS activates several proinflammatory pathways, including the JNK and NF-{kappa}B signaling cascades after binding to the TLR4/CD14/MD-2 receptor complex. AM/AMBP-1 mediates an increase in intracellular cAMP through the activation of the adenylate cyclase (AC), which is known to inhibit proinflammatory JNK pathways. AM-receptor complex activation leads to phosphorylation of the Pyk-2 tyrosine kinase and ERK1/2 (which can be blocked by the inhibitor tyrphostin-23 (Tyr-23) and the MEK1/2 inhibitor PD98059, respectively. ERK1/2 increases PPAR-{gamma} expression (which can be suppressed by siRNA) that, upon activation with endogenous ligands (e.g., J2 prostaglandins), dimerizes with the retinoid X receptor (RXR) and translocates into the nucleus (suppressed by the PPAR-{gamma} inhibitor GW9662). On a transcriptional level PPAR-{gamma} suppresses the expression of proinflammatory cytokines.

 
Previous publications strongly suggest that the activation of ERK1/2 has a role in the proinflammatory response of macrophages, especially because ERK1/2 is transiently activated by LPS (59, 60). However, unlike the roles played by JNK and p38 MAPK, the role of ERK1/2 is not quite as clear. In several cases, it has been shown that ERK1/2 activation is associated with beneficial rather than detrimental effects by conferring a cytoprotective effect in different cell types (58). Although there are publications stating that LPS induces TNF-{alpha} release through the ERK1/2-Egr1 pathway (57), other data shows that this causative link is not so obvious. Conditions such as timing, preconditioning, and the combination of different pathway activations seem to influence the outcome (61, 62, 63). Although the role of p38 and JNK have been consistently shown to be responsible for a proinflammatory response of macrophages, the role of ERK1/2 still remains elusive. In that respect it may depend on the coexisting environmental and intracellular factors that determine the role of ERK as being either proinflammatory or anti-inflammatory, similar to what has been observed in NF-{kappa}B signaling. The specific role of the diverging effects of ERK1/2 activation is another very important issue that needs to be investigated in the future. Our data strongly suggest that in the case of AM/AMBP-1, ERK1/2 activation is maintained over a long period (4 h) and leads to an anti-inflammatory effect by overriding other eventually proinflammatory signaling pathways within the stimulated macrophage.

In summary, AM/AMBP-1-induced suppression of TNF-{alpha} release is mediated by mechanisms involving intracellular cAMP increase through the activation of AM-specific G-protein coupled receptors and by up-regulating the nuclear receptor PPAR-{gamma} through a Pyk-2 tyrosine kinase-ERK1/2 dependent pathway. This in turn seems to be responsible for the suppressive effect on inflammation and consequently contributes to the overall beneficial effects of AM/AMBP-1 in sepsis.


   Acknowledgments
 
We thank Dhruv Amin and Kavin Shah for the help with the Western blots and PCR, Dr. Mian Zhou for the help with immunohistochemical staining, and Dr. Asha Varghese for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants R01 HL076179 and R01 GM057468 (to P.W.). Back

2 Address correspondence and reprint requests to Dr. Ping Wang, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, NY 11030. E-mail address: PWang{at}nshs.edu Back

3 Abbreviations used in this paper: AM, adrenomedullin; AMBP-1, adrenomedullin binding protein; BW, body weight; cAMP, cyclic AMP; CLP, cell ligation and puncture; CRLR, calcitonin receptor-like receptor; DBcAMP, dibutyryl cAMP; 15dPGJ2, 15-deoxy {Delta}12,14 prostaglandin J2; IBMX, 1-methyl-3-isobutylxanthine; PDE, phosphodiesterase; PPAR-{gamma}, peroxisome proliferator-activated receptor-{gamma}; Pyk-2, proline-rich tyrosine kinase-2; RAMP, receptor activity-modifying protein; siRNA, small interfering RNA; RCP, receptor component protein; ROL, rolipram. Back

Received for publication November 16, 2006. Accepted for publication August 24, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Martin, G. S., D. M. Mannino, S. Eaton, M. Moss. 2003. The epidemiology of sepsis in the United States from 1979 through 2000. N. Engl. J. Med. 348: 1546-1554. [Abstract/Free Full Text]
  2. Caille, V., P. Bossi, D. Grimaldi, A. Vieillard-Baro. 2004. Physiopathology of severe sepsis (In French). Presse Med. 33: 256-261. discussion 269. [Medline]
  3. Kitamura, K., J. Sakata, K. Kangawa, M. Kojima, H. Matsuo, T. Eto. 1993. Cloning and characterization of cDNA encoding a precursor for human adrenomedullin. Biochem. Biophys. Res. Commun. 194: 720-725. [Medline]
  4. Kato, H., M. Shichiri, F. Marumo, Y. Hirata. 1997. Adrenomedullin as an autocrine/paracrine apoptosis survival factor for rat endothelial cells. Endocrinology 138: 2615-2620. [Abstract/Free Full Text]
  5. Coppock, H. A., A. A. Owji, C. Austin, P. D. Upton, M. L. Jackson, J. V. Gardiner, M. A. Ghatei, S. R. Bloom, D. M. Smith. 1999. Rat-2 fibroblasts express specific adrenomedullin receptors, but not calcitonin-gene-related-peptide receptors, which mediate increased intracellular cAMP and inhibit mitogen-activated protein kinase activity. Biochem. J. 338: 15-22. [Medline]
  6. Vallet, B.. 2002. Endothelial cell dysfunction and abnormal tissue perfusion. Crit. Care Med. 30: S229-S234. [Medline]
  7. Ross, G. R., C. Yallampalli. 2006. Endothelium-independent relaxation by adrenomedullin in pregnant rat mesenteric artery: role of cAMP-dependent protein kinase A and calcium-activated potassium channels. J. Pharmacol. Exp. Ther. 317: 1269-1275. [Abstract/Free Full Text]
  8. Wang, P., Z. F. Ba, W. G. Cioffi, K. I. Bland, I. H. Chaudry. 1998. The pivotal role of adrenomedullin in producing hyperdynamic circulation during the early stage of sepsis. Arch. Surg. 133: 1298-1304. [Abstract/Free Full Text]
  9. Yang, S., M. Zhou, I. H. Chaudry, P. Wang. 2002. Novel approach to prevent the transition from the hyperdynamic phase to the hypodynamic phase of sepsis: role of adrenomedullin and adrenomedullin binding protein-1. Ann. Surg. 236: 625-633. [Medline]
  10. Zhou, M., Z. F. Ba, I. H. Chaudry, P. Wang. 2002. Adrenomedullin binding protein-1 modulates vascular responsiveness to adrenomedullin in late sepsis. Am. J. Physiol. 283: R553-R560.
  11. Malhotra, R., M. Ward, R. B. Sim, M. I. Bird. 1999. Identification of human complement Factor H as a ligand for L-selectin. Biochem. J. 341: 61-69. [Medline]
  12. Cui, Y., Y. Ji, R. Wu, M. Zhou, P. Wang. 2006. Adrenomedullin binding protein-1 is downregulated during polymicrobial sepsis in the rat. Int. J. Mol. Med. 17: 925-929. [Medline]
  13. Wu, R., M. Zhou, P. Wang. 2003. Adrenomedullin and adrenomedullin binding protein-1 downregulate TNF-{alpha} in macrophage cell line and rat Kupffer cells. Regul. Pept. 112: 19-26. [Medline]
  14. Hotchkiss, R. S., I. E. Karl. 2003. The pathophysiology and treatment of sepsis. N. Engl. J. Med. 348: 138-150. [Free Full Text]
  15. Tontonoz, P., E. Hu, B. M. Spiegelman. 1994. Stimulation of adipogenesis in fibroblasts by PPAR {gamma} 2, a lipid-activated transcription factor. Cell 79: 1147-1156. [Medline]
  16. Elbrecht, A., Y. Chen, C. A. Cullinan, N. Hayes, M. Leibowitz, D. E. Moller, J. Berger. 1996. Molecular cloning, expression and characterization of human peroxisome proliferator activated receptors {gamma} 1 and {gamma} 2. Biochem. Biophys. Res. Commun. 224: 431-437. [Medline]
  17. Dello Russo, C., V. Gavrilyuk, G. Weinberg, A. Almeida, J. P. Bolanos, J. Palmer, D. Pelligrino, E. Galea, D. L. Feinstein. 2003. Peroxisome proliferator-activated receptor {gamma} thiazolidinedione agonists increase glucose metabolism in astrocytes. J. Biol. Chem. 278: 5828-5836. [Abstract/Free Full Text]
  18. MacDougald, O. A., M. D. Lane. 1995. Transcriptional regulation of gene expression during adipocyte differentiation. Annu. Rev. Biochem. 64: 345-373. [Medline]
  19. Carlson, G. L.. 2003. Insulin resistance in sepsis. Br. J. Surg. 90: 259-260. [Medline]
  20. Zingarelli, B., M. Sheehan, P. W. Hake, M. O’Connor, A. Denenberg, J. A. Cook. 2003. Peroxisome proliferator activator receptor-{gamma} ligands, 15-deoxy-{delta}(12,14)-prostaglandin J2 and ciglitazone, reduce systemic inflammation in polymicrobial sepsis by modulation of signal transduction pathways. J. Immunol. 171: 6827-6837. [Abstract/Free Full Text]
  21. Collin, M., M. Abdelrahman, C. Thiemermann. 2004. Endogenous ligands of PPAR-{gamma} reduce the liver injury in haemorrhagic shock. Eur. J. Pharmacol. 486: 233-235. [Medline]
  22. Enomoto, N., Y. Takei, M. Hirose, A. Konno, T. Shibuya, S. Matsuyama, S. Suzuki, K. I. Kitamura, N. Sato. 2003. Prevention of ethanol-induced liver injury in rats by an agonist of peroxisome proliferator-activated receptor-{gamma}, pioglitazone. J. Pharmacol. Exp. Ther. 306: 846-854. [Abstract/Free Full Text]
  23. Alleva, D. G., E. B. Johnson, F. M. Lio, S. A. Boehme, P. J. Conlon, P. D. Crowe. 2002. Regulation of murine macrophage proinflammatory and anti-inflammatory cytokines by ligands for peroxisome proliferator-activated receptor-{gamma}: counter-regulatory activity by IFN-{gamma}. J. Leukocyte Biol. 71: 677-685. [Abstract/Free Full Text]
  24. Wang, P., P. Yoo, M. Zhou, W. G. Cioffi, Z. F. Ba, I. H. Chaudry. 1999. Reduction in vascular responsiveness to adrenomedullin during sepsis. J. Surg. Res. 85: 59-65. [Medline]
  25. Ricote, M., A. C. Li, T. M. Willson, C. J. Kelly, C. K. Glass. 1998. The peroxisome proliferator-activated receptor-{gamma} is a negative regulator of macrophage activation. Nature 391: 79-82. [Medline]
  26. Michael, L. F., M. A. Lazar, C. R. Mendelson. 1997. Peroxisome proliferator-activated receptor {gamma}1 expression is induced during cyclic adenosine monophosphate-stimulated differentiation of alveolar type II pneumonocytes. Endocrinology 138: 3695-3703. [Abstract/Free Full Text]
  27. Hotchkiss, R. S., K. W. Tinsley, P. E. Swanson, I. E. Karl. 2002. Endothelial cell apoptosis in sepsis. Crit. Care Med. 30: S225-S228. [Medline]
  28. Iwasaki, H., M. Shichiri, F. Marumo, Y. Hirata. 2001. Adrenomedullin stimulates proline-rich tyrosine kinase 2 in vascular smooth muscle cells. Endocrinology 142: 564-572. [Abstract/Free Full Text]
  29. Osajima, A., H. Kato, Y. Uezono, T. Suda, M. Okazaki, Y. Oishi, M. Tamura, H. Tanaka, F. Izumi, Y. Nakashima. 1999. Adrenomedullin inhibits transmural pressure induced mesangial cell proliferation through activation of protein kinase A. Nephron. 83: 352-357. [Medline]
  30. Pio, R., A. Martinez, E. J. Unsworth, J. A. Kowalak, J. A. Bengoechea, P. F. Zipfel, T. H. Elsasser, F. Cuttitta. 2001. Complement factor H is a serum-binding protein for adrenomedullin, and the resulting complex modulates the bioactivities of both partners. J. Biol. Chem. 276: 12292-12300. [Abstract/Free Full Text]
  31. Kamitani, S., M. Asakawa, Y. Shimekake, K. Kuwasako, K. Nakahara, T. Sakata. 1999. The RAMP2/CRLR complex is a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells. FEBS Lett. 448: 111-114. [Medline]
  32. Woo, M. S., P. G. Jang, J. S. Park, W. K. Kim, T. H. Joh, H. S. Kim. 2003. Selective modulation of lipopolysaccharide-stimulated cytokine expression and mitogen-activated protein kinase pathways by dibutyryl-cAMP in BV2 microglial cells. Brain Res. Mol. Brain Res. 113: 86-96. [Medline]
  33. Zhang, B., L. Yang, Y. Konishi, N. Maeda, M. Sakanaka, J. Tanaka. 2002. Suppressive effects of phosphodiesterase type IV inhibitors on rat cultured microglial cells: comparison with other types of cAMP-elevating agents. Neuropharmacology 42: 262-269. [Medline]
  34. Mizukami, J., T. Taniguchi. 1997. The antidiabetic agent thiazolidinedione stimulates the interaction between PPAR {gamma} and CBP. Biochem. Biophys. Res. Commun. 240: 61-64. [Medline]
  35. Kubota, N., Y. Terauchi, H. Miki, H. Tamemoto, T. Yamauchi, K. Komeda, S. Satoh, R. Nakano, C. Ishii, T. Sugiyama, et al 1999. PPAR {gamma} mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance. Mol. Cell 4: 597-609. [Medline]
  36. Hammad, H., H. J. de Heer, T. Soullie, V. Angeli, F. Trottein, H. C. Hoogsteden, B. N. Lambrecht. 2004. Activation of peroxisome proliferator-activated receptor-{gamma} in dendritic cells inhibits the development of eosinophilic airway inflammation in a mouse model of asthma. Am. J. Pathol. 164: 263-271. [Abstract/Free Full Text]
  37. Reddy, R. C., V. G. Keshamouni, S. H. Jaigirdar, X. Zeng, T. Leff, V. J. Thannickal, T. J. Standiford. 2004. Deactivation of murine alveolar macrophages by peroxisome proliferator-activated receptor-{gamma} ligands. Am. J. Physiol. 286: L613-L619.
  38. Yuan, Z., Y. Liu, J. Zhang, C. Kishimoto, Y. Wang, A. Ma, Z. Liu. 2003. Peroxisome proliferation-activated receptor-{gamma} ligands ameliorate experimental autoimmune myocarditis. Cardiovasc. Res. 59: 685-694. [Abstract/Free Full Text]
  39. Caron, K. M., O. Smithies. 2001. Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional adrenomedullin gene. Proc. Natl. Acad. Sci. USA 98: 615-619. [Abstract/Free Full Text]
  40. Cui, Y., K. Miyoshi, E. Claudio, U. K. Siebenlist, F. J. Gonzalez, J. Flaws, K. U. Wagner, L. Hennighausen. 2002. Loss of the peroxisome proliferation-activated receptor {gamma} (PPAR{gamma}) does not affect mammary development and propensity for tumor formation but leads to reduced fertility. J. Biol. Chem. 277: 17830-17835. [Abstract/Free Full Text]
  41. Shimosawa, T., Y. Shibagaki, K. Ishibashi, K. Kitamura, K. Kangawa, S. Kato, K. Ando, T. Fujita. 2002. Adrenomedullin, an endogenous peptide, counteracts cardiovascular damage. Circulation 105: 106-111. [Abstract/Free Full Text]
  42. Pickering, M. C., H. T. Cook, J. Warren, A. E. Bygrave, J. Moss, M. J. Walport, M. Botto. 2002. Uncontrolled C3 activation causes membranoproliferative glomerulonephritis in mice deficient in complement factor H. Nat. Genet. 31: 424-428. [Medline]
  43. Barak, Y., M. C. Nelson, E. S. Ong, Y. Z. Jones, P. Ruiz-Lozano, K. R. Chien, A. Koder, R. M. Evans. 1999. PPAR {gamma} is required for placental, cardiac, and adipose tissue development. Mol. Cell 4: 585-595. [Medline]
  44. Trivedi, S. G., J. Newson, R. Rajakariar, T. S. Jacques, R. Hannon, Y. Kanaoka, N. Eguchi, P. Colville-Nash, D. W. Gilroy. 2006. Essential role for hematopoietic prostaglandin D2 synthase in the control of delayed type hypersensitivity. Proc. Natl. Acad. Sci. USA 103: 5179-5184. [Abstract/Free Full Text]
  45. Beshay, E., F. Croze, G. J. Prud’homme. 2001. The phosphodiesterase inhibitors pentoxifylline and rolipram suppress macrophage activation and nitric oxide production in vitro and in vivo. Clin. Immunol. 98: 272-279. [Medline]
  46. Seternes, O. M., R. Sorensen, B. Johansen, U. Moens. 1999. Activation of protein kinase A by dibutyryl cAMP treatment of NIH 3T3 cells inhibits proliferation but fails to induce Ser-133 phosphorylation and transcriptional activation of CREB. Cell. Signal. 11: 211-219. [Medline]
  47. Meng, X., L. Ao, B. D. Shames, A. H. Harken. 2001. Inhibition of cyclic-3',5'-nucleotide phosphodiesterase abrogates the synergism of hypoxia with lipopolysaccharide in the induction of macrophage TNF-{alpha} production. J. Surg. Res. 101: 210-215. [Medline]
  48. Zhou, M., R. Wu, W. Dong, H. H. Simms, P. Wang. 2004. Hepatic peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is downregulated in sepsis. Shock 21: 39 (Abstr.).
  49. Siddiqui, A. M., X. Cui, R. Wu, W. Dong, M. Zhou, M. Hu, H. H. Simms, P. Wang. 2006. The anti-inflammatory effect of curcumin in an experimental model of sepsis is mediated by up-regulation of peroxisome proliferator-activated receptor-{gamma}. Crit. Care Med. 34: 1874-1882. [Medline]
  50. Jiang, C., A. T. Ting, B. Seed. 1998. PPAR-{gamma} agonists inhibit production of monocyte inflammatory cytokines. Nature 391: 82-86. [Medline]
  51. Harris, S. G., R. S. Smith, R. P. Phipps. 2002. 15-Deoxy-{delta}12,1412,14-PGJ2 induces IL-8 production in human T cells by a mitogen-activated protein kinase pathway. J. Immunol. 168: 1372-1379. [Abstract/Free Full Text]
  52. Bell-Parikh, L. C., T. Ide, J. A. Lawson, P. McNamara, M. Reilly, G. A. FitzGerald. 2003. Biosynthesis of 15-deoxy-{delta}12,14-PGJ2 and the ligation of PPAR{gamma}. J. Clin. Invest. 112: 945-955. [Medline]
  53. Chawla, A., W. A. Boisvert, C.-H. Lee, B. A. Laffitte, Y. Barak, S. B. Joseph, D. Liao, L. Nagy, P. A. Edwards, L. K. Curtiss. 2001. A PPAR{gamma}-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol. Cell 7: 161-171. [Medline]
  54. Evans, B. N., M. I. Rosenblatt, L. O. Mnayer, K. R. Oliver, I. M. Dickerson. 2000. CGRP-RCP, a novel protein required for signal transduction at calcitonin gene-related peptide and adrenomedullin receptors. J. Biol. Chem. 275: 31438-31443. [Abstract/Free Full Text]
  55. Prado, M. A., B. Evans-Bain, K. R. Oliver, I. M. Dickerson. 2001. The role of the CGRP-receptor component protein (RCP) in adrenomedullin receptor signal transduction. Peptides 22: 1773-1781. [Medline]
  56. Squires, M. S., P. M. Nixon, S. J. Cook. 2002. Cell-cycle arrest by PD184352 requires inhibition of extracellular signal-regulated kinases (ERK) 1/2 but not ERK5/BMK1. Biochem. J. 366: 673-680. [Medline]
  57. Troadec, J. D., M. Marien, S. Mourlevat, T. Debeir, M. Ruberg, F. Colpaert, P. P. Michel. 2002. Activation of the mitogen-activated protein kinase (ERK(1/2)) signaling pathway by cyclic AMP potentiates the neuroprotective effect of the neurotransmitter noradrenaline on dopaminergic neurons. Mol. Pharmacol. 62: 1043-1052. [Abstract/Free Full Text]
  58. Shi, L., R. Kishore, M. R. McMullen, L. E. Nagy. 2002. Lipopolysaccharide stimulation of ERK1/2 increases TNF-{alpha} production via Egr-1. Am. J. Physiol. 282: C1205-C1211.
  59. Guha, M., M. A. O’Connell, R. Pawlinski, A. Hollis, P. McGovern, S.-F. Yan, D. Stern, N. Mackman. 2001. Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor {alpha} expression by inducing Elk-1 phosphorylation and Egr-1 expression. Blood 98: 1429-1439. [Abstract/Free Full Text]
  60. Van der Bruggen, T., S. Nijenhuis, E. van Raaij, J. Verhoef, B. Sweder van Asbeck. 1999. Lipopolysaccharide-induced tumor necrosis factor {alpha} production by human monocytes involves the Raf-1/MEK1-MEK2/ERK1-ERK2 pathway. Infect. Immun. 67: 3824-3829. [Abstract/Free Full Text]
  61. Nelson, D. E., A. E. Ihekwaba, M. Elliott, J. R. Johnson, C. A. Gibney, B. E. Foreman, G. Nelson, V. See, C. A. Horton, D. G. Spiller, et al 2004. Oscillations in NF-{kappa}B signaling control the dynamics of gene expression. Science 306: 704-708. [Abstract/Free Full Text]
  62. Chung, Y. J., H. R. Zhou, J. J. Pestka. 2003. Transcriptional and posttranscriptional roles for p38 mitogen-activated protein kinase in upregulation of TNF-{alpha} expression by deoxynivalenol (vomitoxin). Toxicol. Appl. Pharmacol. 193: 188-201. [Medline]
  63. Rawadi, G., V. Ramez, B. Lemercier, S. Roman-Roman. 1998. Activation of mitogen-activated protein kinase pathways by Mycoplasma fermentans membrane lipoproteins in murine macrophages: involvement in cytokine synthesis. J. Immunol. 160: 1330-1339. [Abstract/Free Full Text]




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