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Originally published online as doi:10.1189/jlb.0603254 on August 11, 2003

Published online before print August 11, 2003
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(Journal of Leukocyte Biology. 2003;74:932-941.)
© 2003 by Society for Leukocyte Biology

ANP inhibits TNF-{alpha}-induced endothelial MCP-1 expression—involvement of p38 MAPK and MKP-1

Nina C. Weber*, Signe B. Blumenthal*, Thomas Hartung{dagger}, Angelika M. Vollmar* and Alexandra K. Kiemer*,1

* Department of Pharmacy, Center of Drug Research, University of Munich, Germany; and
{dagger} Institute of Biochemical Pharmacology, University of Konstanz, Germany

1Correspondence: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Atrial natriuretic peptide (ANP) has been shown to reduce tumor necrosis factor-{alpha} (TNF-{alpha})-induced activation of endothelial cells via inhibition of p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-{kappa}B pathways. The aim of this study was to determine whether ANP is able to inhibit TNF-{alpha}-induced expression of monocyte chemoattractant protein-1 (MCP-1) in endothelial cells and to elucidate the mechanisms involved. Pretreatment of human umbilical vein endothelial cells (HUVEC) with ANP significantly reduced TNF-{alpha}-induced expression of MCP-1 protein and mRNA. The effects of ANP were shown to be mediated via the guanylyl-cyclase (GC)-coupled A receptor. Activation of the other GC-coupled receptor (natriuretic peptide receptor-B) by the C-type natriuretic peptide as well as activation of soluble GC with S-nitroso-L-glutathione (GSNO) exerted similar effects as ANP, supporting a role for cyclic guanosine monophosphate (cGMP) in the signal transduction. Antisense experiments showed a requirement of MAPK phosphatase-1 (MKP-1) induction and therefore, inhibition of p38 MAPK in the ANP-mediated inhibition of TNF-{alpha}-induced expression of MCP-1. To investigate a potential interplay between TNF-{alpha}-induced activation of p38 MAPK and NF-{kappa}B, the p38 MAPK inhibitor SB203580 and a dominant-negative p38 MAPK mutant were used. The results indicated that the blockade of p38 MAPK activity leads to an increased activation of NF-{kappa}B and therefore, suggest a counter-regulatory action of p38 MAPK and NF-{kappa}B. As antisense experiments revealed a pivotal role for MKP-1 induction and therefore, p38 MAPK inhibition in ANP-mediated attenuation of MCP-1 expression, this action seems to be rather independent of NF-{kappa}B inhibition.

Key Words: natriuretic peptides • endothelial cells • chemokines • cytokines • inflammation • atherosclerosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The atrial natriuretic peptide (ANP) was the first-described member of the natriuretic peptide family, a family of cardiovascular cyclic peptide hormones. As a result of its natriuretic and diuretic properties, ANP exhibits important cardiovascular effects, such as regulation of blood pressure and plasma volume expansion [1 ]. ANP was shown to mediate most of its cardiovascular and renal effects through interaction with the guanylyl-cyclase (GC)-coupled natriuretic peptide receptor-A (NPR-A) via cyclic guanosine monophosphate (cGMP) as second messenger [1 ]. ANP also binds to the non-GC-linked "clearance" NPR (NPR-C) [2 ]. Besides the clearance function exerted by NPR-C, an NPR-C-mediated inhibition of adenylyl- cyclase was shown to be responsible for several in vitro effects of ANP, such as inhibition of astrocyte proliferation [3 ], reduction of endothelin production in endothelial cells [4 ], and inhibition of cyclooxygenase-2 (COX-2) induction in macrophages [5 ].

In the last years, natriuretic peptides and their receptors were found to be expressed in diverse tissues besides the cardiovascular and renal system. In this context, ANP and its receptors were shown to be expressed in macrophages [6 7 8 9 ]. Moreover, ANP was shown to attenuate the induction of the inducible nitric oxide synthase (iNOS), a central proinflammatory enzyme, in an autocrine manner [8 9 10 11 12 ]. Of further importance is that ANP was found to exert an inhibitory action on the production of tumor necrosis factor-{alpha} (TNF-{alpha}) in activated rodent macrophages and in whole human blood [13 14 15 ]. Taken together, ANP is suggested to be a regulator of macrophage/leukocyte activation and therefore, of inflammation.

One of the most important steps in inflammatory processes is leukocyte recruitment into inflamed tissue. Chemokines, such as monocyte chemoattractant protein-1 (MCP-1), have been shown to be critically involved in the TNF-{alpha}-induced recruitment of leukocytes [16 ]. These mediators exhibit chemoattractive activity and have been shown to be critically involved in a number of inflammatory diseases, such as sepsis and asthma [17 , 18 ]. Excessive expression of chemokines, such as MCP-1, has been recognized to mediate the initial steps in leukocyte recruitment in several pathophysiological conditions, such as atherosclerosis [19 , 20 ].

Due to the role of ANP as a regulator of macrophage and endothelial [21 , 22 ] activation, we hypothesized that ANP might affect the TNF-{alpha}-induced expression of MCP-1 in endothelial cells. Furthermore, we determined the molecular signaling pathways involved in MCP-1 regulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Rat ANP 99-126 ("ANP"), C-type natriuretic peptide (CNP), and SB203580 were purchased from Calbiochem (Schwalbach, Germany) and cyclic atrial natriuretic factor (cANF) from Saxon Biochemicals (Hannover, Germany). S-nitroso-L-glutathione (GSNO) and NG-nitro-L-arginine (L-NNA) were from Alexis Biochemicals (Grünberg, Germany). Antiserum against the "von Willebrand" factor was from Serotec (Wiesbaden, Germany). Cell-culture medium (M199) and penicillin/streptomycine were from PAN (Aidenbach, Germany). Fetal calf serum (FCS) was from Biochrom (Berlin, Germany). All other materials were purchased from Sigma (Taufkirchen, Germany) or Merck-Eurolab (Munich, Germany).

Cell culture
Human umbilical vein endothelial cells (HUVEC) were prepared by digestion of umbilical veins with 0.1 g/L collagenase A (Roche, Mannheim, Germany). Cells were grown in M199 (PAN) supplemented with 20% heat-inactivated FCS, 1x endothelial medium supplement (Sigma), and penicillin (100 U/ml)/streptomycin (100 µg/ml). To compensate for interindividual differences, cells of at least two umbilical cords were combined in each cell preparation. For experiments, cells of passage number three or four were grown until confluence (plastic ware was from Peske, Aindling-Pichl, Germany). HUVEC were found >95% pure, as judged by fluorescence-activated cell sorter analysis (FACScan, Becton Dickinson, Heidelberg, Germany), using an antiserum against the von Willebrand factor.

Detection of MCP-1 secretion
HUVEC were grown until confluence in 24-well plates. Cells were left untreated or treated with TNF-{alpha} (10 ng/ml). The effect of the following substances on the expression of TNF-{alpha}-induced MCP-1 was determined: ANP (10-11–10-6 mol/L), cANF (10-8–10-6 mol/L), 8-Br-cGMP (10-10–10-3 mol/L), CNP (10-11–10-6 mol/L), GSNO (125 and 500 µmol/L), and L-NNA (125 and 500 µmol/L). Substances were added to the cells 30 min before TNF-{alpha}. After 6 h, the supernatants (200 µl) were transferred into 96-well microtiter plates, and the measurement of human MCP-1 release was determined by an enzyme-linked immunosorbent assay (ELISA) based on commercial antibody pairs (R&D Systems, Wiesbaden, Germany). Binding of biotinylated antibody was quantified using streptavidin-peroxidase (Jackson ImmunoResearch, West Grove, PA) and the substrate 3,3',5,5'-tetramethylbenzidine (Sigma). Recombinant MCP-1 (R&D Systems) served as standard.

Antisense experiment
HUVEC were cultured in 12-well plates until confluence. Phosphorothioate oligonucleotides (modifications are shown by small letters in the oligonucleotide sequence) were used in a final concentration of 0.03 µg/well for each transfection reaction. The used oligonucleotides for mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) were: antisense, 5'-cc-CACTTCCATGACCA-tgg-3'; sense, 5'-ccATGGTCATGGAAGT-ggg-3'. The cells were transfected using an EffecteneTM transfection kit (Qiagen, Hilden, Germany). The respective amount of antisense or sense DNA was dissolved in water, and after addition of an appropriate amount of enhancer, Effectene reagent was added, and the mixture was incubated for 10 min at room temperature to allow Effectene reagent-DNA complex formation. During this incubation time, HUVEC medium was removed from the monolayers, and fresh medium was added. The transfection complex was added to the cells and incubated for 3 h. Subsequently, transfection medium was removed, and fresh medium was added. Cells were left untreated or stimulated with TNF-{alpha} (10 ng/ml) in the presence or absence of ANP (10-6 mol/L), which was added to the cells alone or 30 min before TNF-{alpha} incubation. The stimulation time was 6 h for detection of human MCP-1 protein by ELISA.

Detection of mRNA
HUVEC were treated with ANP (10-8–10-6 mol/L) or TNF-{alpha} (10 ng/ml), alone or in combination for 3 h. ANP was added to the cells 30 min before TNF-{alpha}. RNA was prepared using RNeasy® RNA isolation kit (Qiagen). Reverse transcription was performed using a reverse transcription system kit (Promega, Mannheim, Germany). Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments were performed with primers for MCP-1 (sense, 5'-GATGCAATCAATGCCCCAGT-3'; antisense, 5'-TTGCTTGTCCAGGTGGTCCAT-3'; MWG Biotech AG, Ebersberg, Germany) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sense, 5'-TCACTCAAGATTGTCAGCAA-3'; antisense, 5'-AGATCCACGACGGAC-ACATT-3'; MWG Biotech AG). PCR conditions were as follows: for MCP-1, 28 cycles of 94°C 50 s; 55°C 1 min; 72°C 1 min; and for GAPDH, 30 cycles of 93°C 24 s; 55°C 30 s; 72°C 1 min. PCR was followed by gel electrophoresis, ethidium bromide staining, and densitometric analysis (Kodak Image station, Kodak Digital Science, Stuttgart, Germany).

Transfection of human embryo kidney HEK293 cells
HEK293 cells (1.5x106) were seeded into 100 mm dishes the night before transfection. Transfections were performed by the calcium phosphate coprecipitation method using 0.1–1 µg DNA of the different plasmids. For luciferase reporter assay, the cells were transfected with pNF-{kappa}Bluc (Stratagene, Amsterdam, Netherlands) and pRL-TK (Promega) or pß-Gal (kindly provided by Gene Center, University of Munich, Germany) as an internal control to normalize variability in transfection efficiency. Salmon sperm DNA (kindly provided by Gene Center, University of Munich) was added to the transfection mixture as carrier. For the experiments with the dominant-negative (dn) version of p38 MAPK, cells were additionally transfected with kRSPA-Flag-p38 (AF) mutant or the empty expression vector [23 ]. dn p38 MAPK and vector plasmid were a kind gift from Professor Dr. Stephan Ludwig, University of Würzburg, Germany.

Luciferase reporter gene assay
Luciferase reporter gene assay was performed as described previously [24 ]. Briefly, transfected HEK293 cells were left untreated or were treated with 1 ng/ml TNF-{alpha} for 6 h with or without preincubation with ANP (10-9–10-6 mol/L, 30 min). Cells were washed twice and lysed with passive lysis buffer. Nuclear factor (NF)-{kappa}B activity was measured by the dual luciferase reporter assay or the luciferase assay system (both Promega), according to the manufacturer’s description, with an AutoLumat plus (Berthold, Pforzheim, Germany).

Electrophoretic mobility shift assay (EMSA)
HUVEC or HEK293 were grown in six-well plates and treated with TNF-{alpha} (10 ng/ml) for 60 min with or without pretreatment with SB203580 (5 µmol/L, 60 min). Nuclear extracts and EMSA experiments were performed as described previously [14 , 22 ]. A 22-mer double-stranded oligonucleotide probe containing a consensus-binding sequence for NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG C-3', Promega) was 5' end-labeled with [{gamma}32P]-adenosine 5'-triphosphate (10 µCi) using T4 polynucleotide kinase. Equal amounts of nuclear protein (10–20 µg) were incubated (20 min, room temperature) in a 15 µl reaction volume containing 10 mM Tris-HCl, pH 7.5, 5 x 104 cpm radiolabeled-oligonucleotide probe, 2 µg poly(dIdC), 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 50 mM NaCl, and 0.5 mM dithiothreitol. Nucleoprotein-oligonucleotide complexes were resolved by electrophoresis (4.5% nondenaturing polyacrylamide gel, 100 V), and bands were visualized by phosphorimaging (Packard, Meriden, CT).

cGMP measurement
HUVEC were cultured in 24-well plates until confluence. Cells were left untreated or treated with ANP (10-6 mol/L). Determination of cGMP content was performed with a commercially available kit (Amersham Pharmacia, Freiburg, Germany), based on a competitive enzyme immunoassay system. Cell lysis and assay performance were done as indicated by the manufacturer.

Statistical analysis
Unless stated otherwise, all experiments were done from cells of at least three different cell preparations. Each experiment was performed at least in triplicates. Data are expressed as mean ± SEM. Values with P < 0.05 were considered statistically different compared with 100% or onefold (TNF-{alpha}-treated cells; one sample t-test). Statistical analysis was performed with Graph Pad Prism (version 3.02)


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ANP inhibits TNF-{alpha}-induced secretion of MCP-1
TNF-{alpha} treatment (10 ng/ml) of cells significantly induced secretion of MCP-1 into the culture medium of HUVEC (Fig. 1 ). Pretreatment of cells with ANP dose-dependently (10-10–10-6 mol/L) reduced TNF-{alpha}-induced MCP-1 production (Fig. 1) . ANP (10-6 mol/L) alone elevated basal MCP-1 release. Values of ELISA experiments were expressed as percent of values for TNF-{alpha}-treated cells, whereby the range of MCP-1 concentration in the cell-culture medium after TNF-{alpha} treatment was between 1.2 and 4.2 µg/ml in the different experiments from different cell preparations.



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  		Figure 1. Inhibition of TNF-{alpha}-induced MCP-1 by ANP. HUVEC were cultured in medium alone (Co), in medium containing ANP (10-6 mol/L, 6h), or in medium containing TNF-{alpha} (10 ng/ml, 6 h) with or without pretreatment (30 min) of the cells with various concentrations of ANP (10-11–10-6 mol/L). ELISA detected MCP-1 secretion in the supernatants, as described in Materials and Methods. Data show means ± SEM of three independent experiments from different cell preparations performed in triplicates. **, P < 0.01, and ***, P < 0.001, represent significant differences compared with the values seen in TNF-{alpha}-activated cells, whereby TNF-{alpha} was set as 100%. ++, P < 0.01, represents significant differences compared with the values seen in control cells.

 
Receptor specificity of the ANP effects on MCP-1
To clarify the receptor specificity of the inhibitory effect of ANP on TNF-{alpha}-induced expression of MCP-1, the following substances were tested for their influence on TNF-{alpha}-induced MCP-1 release. The cell-permeable analog of the NPR-A second messenger cGMP, 8-Br-cGMP, significantly reduced TNF-{alpha}-induced MCP-1 production in a dose-dependent manner (10-6–10-3 mol/L, Fig. 2A ). 8-Br-cGMP alone did not affect basal MCP-1 release. The specific NPR-C ligand cANF did not affect the TNF-{alpha}-induced MCP-1 secretion. However, cANF markedly increased basal MCP-1 release (Fig. 2B) . The specific ligand for the other GC-coupled receptor (NPR-B), CNP, exerted similar effects as ANP. CNP pretreatment (10-6 mol/L) of the cells reduced TNF-{alpha}-induced MCP-1 secretion (Fig. 2C) , whereas basal MCP-1 production was slightly but not significantly increased (Fig. 2C) . These data suggest a role for cGMP and therefore, for the particulate GC-coupled NPR in the signal transduction of the inhibitory ANP effect on activated MCP-1 expression. To confirm the presence of functional NPR-A in HUVEC under our experimental conditions, we measured the cGMP response after ANP (10-6 mol/L) treatment for 20 min: ANP led to a 19.02 ± 1.38-fold increase of cellular cGMP content. To examine if the soluble GC (sGC) is also able to mediate inhibition of TNF-{alpha}-induced MCP-1 production, we tested whether NO, an activator of sGC, shows this effect.



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  		Figure 2. Characterization of the NPR responsible for inhibition of MCP-1 production. ELISA determined MCP-1 secretion of control cells (Co), of cells treated with TNF-{alpha} (10 ng/ml), or of the respective test substance for 6 h. (A) The effect of the NPR-A second-messenger analog 8-Br-cGMP (10-10–10-3 mol/L) on TNF-{alpha}-induced MCP-1 expression was determined after preincubation with 8-Br-cGMP for 30 min. (B) The effect of cANF (10-8–10-6 mol/L) on the expression of MCP-1 was determined for cANF added to the cells 30 min before TNF-{alpha}. (C) The NPR-B-specific ligand CNP (10-11–10-6 mol/L) was added to the cells in combination with TNF-{alpha} (30 min pretreatment). Data show means ± SEM of three independent experiments from different cell preparations, each performed in triplicates. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, represent significant differences compared with the values seen in TNF-{alpha}-activated cells, whereby TNF-{alpha} was set as 100%. ++, P < 0.01, represents significant differences compared with the values seen in control cells.

 
Modulation of MCP-1 secretion by NO
GSNO-pretreated cells showed an attenuated TNF-{alpha}-induced MCP-1 release (Fig. 3A ). This points to an inhibitory action of NO on TNF-{alpha}-induced MCP-1 secretion and further supports our finding of cGMP as second messenger in the signal-transduction pathway regulating MCP-1 induction. GSNO alone had no significant effect on MCP-1 production (data not shown). We were furthermore interested in the potential role of endogenous NO production in the regulation of MCP-1 secretion. Inhibition of NO production by preincubation (30 min) with the NOS inhibitor L-NNA (125 and 500 µmol/L), however, did not affect TNF-{alpha}-induced MCP-1 (Fig. 3B) , suggesting no role of endogenously produced NO.



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  		Figure 3. Modulation of MCP-1 expression by NO. HUVEC were cultured in medium alone (Co) or in medium containing TNF-{alpha} (10 ng/ml) with or without pretreatment (30 min) of the cells with various concentrations of GSNO (125 and 500 µmol/L, A) or L-NNA (125 and 500 µmol/L, B). MCP-1 production was assessed by ELISA measurement as described in Materials and Methods. Data show means ± SEM of three independent experiments from different cell preparations, each performed in triplicates. *, P < 0.05, represents significant differences compared with the values seen in TNF-{alpha}-activated cells, whereby TNF-{alpha} was set as 100%.

 
ANP reduces TNF-{alpha}-induced MCP-1 mRNA expression
Our data showed a GC-mediated inhibition of TNF-{alpha}-induced MCP-1 secretion by ANP. The following experiments aimed to elucidate the molecular mechanisms leading to this inhibitory action of ANP. To get insight into whether the inhibition of TNF-{alpha}-induced MCP-1 secretion is regulated on the level of mRNA expression, we performed semiquantitative RT-PCR experiments. TNF-{alpha} (10 ng/ml) significantly induced MCP-1 mRNA in HUVEC (Fig. 4 ). In fact, ANP pretreatment (10-8–10-6 mol/L) reduced TNF-{alpha}-induced expression of MCP-1 mRNA. ANP alone, slightly but significantly, induced basal MCP-1 mRNA expression (Fig. 4) .



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  		Figure 4. Analysis of MCP-1 mRNA by RT-PCR. HUVEC were cultured in medium alone (Co), in medium containing ANP (10-6 mol/L), or in medium containing TNF-{alpha} (10 ng/ml) with or without pretreatment (30 min) of the cells with various concentrations of ANP (10-8–10-6 mol/L). After 3 h, mRNA expression of MCP-1 and GAPDH was determined by RT-PCR followed by gel electrophoresis and ethidium bromide staining. Data show one representative gel out of three independent experiments each. Graphs show densitometric evaluation of signal intensities normalized on GAPDH and are expressed as percentage of values for TNF-{alpha} treatment only, whereby values for TNF-{alpha} were set as 100%. Data show means ± SEM of three independent experiments from different cell preparations. **, P < 0.01, and *, P < 0.05, represent significant differences compared with the values seen in TNF-{alpha}-activated cells. ++, P < 0.01, represents significant differences compared with the values seen in control cells.

 
Involvement of MKP-1 induction in MCP-1 inhibition
The RT-PCR data suggested a transcriptional regulation of the ANP effects on TNF-{alpha}-induced MCP-1 expression. Activation of p38 MAPK has been suggested to play a pivotal role in the transcriptional regulation of MCP-1 expression [25 26 27 ]. As we published recently, ANP is able to inhibit TNF-{alpha}-induced activation of p38 MAPK in HUVEC [21 ]. This inhibitory action of ANP was shown to be mediated via induction of MKP-1 [21 ]. We here aimed to clarify a role for MKP-1 induction and therefore, p38 inhibition in the observed inhibition of MCP-1 expression by ANP. HUVEC were transfected with MKP-1 antisense or sense phosphorothioate-modified oligonucleotides. The effectiveness of this antisense approach has been demonstrated previously [21 ]. As demonstrated in Figure 5 , transfection with MKP-1 antisense but not sense oligonucleotides abrogated the inhibitory effect of ANP on MCP-1 expression. These data therefore provide a causal relationship between ANP-mediated induction of MKP-1 and inhibition of TNF-{alpha}-induced expression of MCP-1 by ANP, suggesting a central role for p38 MAPK in this signal-transduction pathway.



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  		Figure 5. Role for MKP-1 in MCP-1 inhibition. HUVEC were transfected with MKP-1 antisense or sense oligonucleotides as described in Materials and Methods. After addition of fresh medium, cells were left untreated or stimulated with TNF-{alpha} (10 ng/ml, 6 h) in the presence or absence of ANP (10-6 mol/L), which was added to the cells 30 min before TNF-{alpha}. ELISA for MCP-1 was performed as described in Materials and Methods. The histogram shows results of three independent experiments performed in triplicates. Values are expressed as percent of TNF-{alpha}-induced MCP-1 secretion and show means ± SEM, and values for TNF-{alpha} minus values for Co of the respective group were set as 100%. **, P < 0.01, and ***, P < 0.001, represent significant differences from values in TNF-{alpha}-activated cells. n.s., No significant difference.

 
Relationship between TNF-{alpha}-induced p38 MAPK and NF-{kappa}B activation
As ANP has previously been shown as an inhibitor of NF-{kappa}B activity in HUVEC [22 ], and p38 MAPK as well as NF-{kappa}B have been suggested as a regulator of MCP-1 induction [28 ], we aimed to clarify a potential interplay between these two inflammatory signaling pathways.

As we could recently describe, ANP down-regulates TNF-{alpha}-induced NF-{kappa}B DNA-binding activity in HUVEC [22 ]. We here confirmed these data by the use of a luciferase reporter gene assay. As a result of the difficulty of efficient HUVEC transfection [29 ], we used HEK293 for reporter gene assays: ANP pretreatment of the cells in fact significantly reduced TNF-{alpha}-induced NF-{kappa}B transcriptional activity.

To investigate the relationship between p38 MAPK and TNF-{alpha}-induced NF-{kappa}B activity, we performed EMSA using the chemical p38 MAPK inhibitor SB203580. These experiments were performed in HUVEC as well as in HEK293. As Figure 6B and 6C , clearly shows, TNF-{alpha}-stimulated HUVEC and HEK293 showed a significantly increased NF-{kappa}B DNA-binding activity after pretreatment with SB203580. As a result of potential, unspecific effects by the use of chemical kinase inhibitors, we additionally performed luciferase reporter gene assays using cells transfected with a dn p38 MAPK mutant [23 ]. Our observations from EMSA experiments were supported by the results of a luciferase reporter gene assay. As Figure 6D clearly demonstrates, the dn p38 mutant led to a significant increase in NF-{kappa}B transcriptional activity, compared with TNF-{alpha}-treated cells.



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  		Figure 6. Involvement of p38 in TNF-{alpha}-induced NF-{kappa}B activation. (A) pNF-{kappa}Bluc- and pRT-KL-transfected HEK293 cells were left untreated or pretreated with different concentrations of ANP (10-9–10-6 mol/L) for 30 min. Cells were then activated with 1 ng/ml TNF-{alpha} for an additional 6 h. Luciferase activity was measured by the dual luciferase reporter gene assay. Co represents luciferase activity of cells transfected with pNF-{kappa}Bluc. Bars represent means ± SEM of three independent experiments performed in triplicates. Values of TNF-{alpha} treatment were set as 100%. ***, P < 0.001, and *, P < 0.05, represent significant differences from values seen in TNF-{alpha}-activated cells. (B and C) HUVEC (B) or HEK293 (C) were cultured in medium alone (Co) or in medium containing TNF-{alpha} (HUVEC, 10 ng/ml; HEK293, 1 ng/ml) with or without pretreatment (60 min) of the cells with 5 µmol/L SB203580 (SB). NF-{kappa}B-binding activity was assessed by EMSA as described in Materials and Methods. Data show one representative out of two (HEK293) to three (HUVEC) independent experiments from different cell preparations with similar results performed in duplicates. The histograms show phosphorimaging analysis of EMSA experiments and represent means ± SEM, whereby values of untreated cells were set as 1. ***, P < 0.001, and **, P < 0.01, represent significant differences from values in control cells. ++, P < 0.01, and +, P < 0.05, represent differences compared with the values seen in TNF-{alpha}-activated cells. (D) HEK293 cells were transfected with pNF-{kappa}B and pß-Gal plus dn p38 MAPK or empty vector. Plasmid-transfected cells were left untreated (Co) or treated with 1 ng/ml TNF-{alpha} for 6 h. Luciferase activity was measured by the luciferase assay system. Bars represent means ± SEM of three independent experiments performed in triplicates, and values of TNF-{alpha}-treated cells were transfected with the empty vector plasmid set as 100%. ***, P < 0.001, represents significant differences from values seen in TNF-{alpha}-activated cells transfected with the empty vector plasmid.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
MCP-1 has been shown to play a key role in inflammation. In this context, it has been demonstrated to be critically involved in the pathogenesis of atherosclerosis [19 , 20 , 30 ], where its local overexpression induces macrophage infiltration and formation of atherosclerotic lesions [31 , 32 ].

In the present study, we demonstrate that the cardiovascular hormone ANP is able to suppress the TNF-{alpha}-induced expression of MCP-1 via induction of MKP-1 in endothelial cells. This inhibitory action of ANP on MCP-1 secretion in human endothelial cells might contribute to an anti-inflammatory and antiatherogenic potential of this cardiovascular hormone. Little is known about the potential of other endogenous compounds regulating MCP-1 expression. For instance, the mechanisms responsible for the inhibitory action of estrogen on MCP-1 expression are as yet completely unknown [33 ]. The attenuation of TNF-{alpha}-induced MCP-1 expression by transforming growth factor-ß1 was suggested to be mediated via a down-regulation of TNF receptors [34 ]. Thioredoxin was also shown to inhibit lipopolysaccharide (LPS)-induced MCP-1 expression [35 ]. It is interesting that this inhibitory action of thioredoxin was linked to its potency to attenuate p38 MAPK activation. Our data also suggest p38 MAPK as an important factor of endogenous MCP-1 regulation.

cGMP-mediated inhibition of TNF-{alpha}-induced MCP-1 release
The results presented here indicate that the inhibitory action of ANP on TNF-{alpha}-induced MCP-1 expression is mediated via cGMP. 8-Br-cGMP mimicked the effect of ANP on MCP-1 release, and CNP, the ligand for the other GC-coupled receptor NPR-B, also shows inhibitory action on MCP-1 secretion. Furthermore, the specific NPR-C agonist cANF did not affect TNF-{alpha}-induced MCP-1 secretion. To our knowledge, the regulatory action of cGMP on TNF-{alpha}-induced MCP-1 release has as yet been completely unknown. Therefore, our data are the first to report the inhibitory action of cGMP on TNF-{alpha}-induced MCP-1 expression.

More information exists on the regulatory mechanisms of endothelial MCP-1 expression induced by stimuli other than TNF-{alpha}. It is interesting that these reports indicate no relevance for cGMP-dependent pathways in regulating MCP-1. For instance Chien and coworkers [36 ] reported that cGMP-dependent kinases play no role in the regulation of mechanical strain stress-induced MCP-1 expression in HUVEC. Another report by Okada et al. [37 ] also showed no involvement of cGMP-dependent kinases on cyclic stretch-induced up-regulation of MCP-1 in HUVEC. In this context, one should take into account that it has been shown by the groups of van Hinsbergh [38 ] and Lohmann [39 ] that HUVEC do not seem to express cGMP kinases. It is well described that cGMP might not only exert its different biological actions via activation of cGMP-dependent kinases but also via phosphodiesterases [40 , 41 ] or cGMP-dependent ion channels [42 , 43 ]. Therefore, we would not support cGMP-dependent kinases as central cellular downstream targets in HUVEC.

Modulation of TNF-{alpha}-induced MCP-1 release by NO
The fact that cGMP seems to be the second messenger for ANP-mediated inhibition of TNF-{alpha}-induced MCP-1 led us to focus on an important activator of cGMP release, NO, which activates the sGC and thereby increases intracellular cGMP. Our data show for the first time that a NO donor attenuates TNF-{alpha}-induced MCP-1 release in HUVEC. Another group recently reported decreased shear stress-induced MCP-1 levels after pretreatment of HUVEC with a NO donor [44 ]. These results support our hypothesis of NO as a modulator of MCP-1 induction in HUVEC.

As endothelial cells themselves represent a source of NO production [45 ], we aimed to determine a potential role for endogenously produced NO in our cell system. As the NOS inhibitor L-NNA did not affect TNF-{alpha}-induced MCP-1 release, endogenous NO production appears not to be involved in regulating MCP-1 induction.

Regulation of basal MCP-1 production
Despite the lack of information on effects of NO on TNF-{alpha}-induced MCP-1 expression in the literature, NO has been recognized to regulate basal MCP-1 expression in HUVEC. Zeiher et al. [46 ] report a NO-dependent decrease in basal MCP-1 expression. However, in our experimental settings, the NO donor GSNO did not affect basal MCP-1 release. This discrepancy of our data to the literature might be explained by the use of different NO donors and stimulation times.

8-Br-cGMP did not affect basal MCP-1 expression in our experiments excluding a role for cGMP in basal regulation of MCP-1. These observations are supported by the group of Zeiher [46 ], who demonstrated that elevation of endothelial cGMP levels has no effect on basal MCP-1 expression.

It is interesting that ANP and the specific NPR-C agonist cANF increased basal MCP-1 secretion. Besides its role in inflammatory processes, MCP-1 has been described to play a crucial role in angiogenesis. MCP-1 has been shown to increase collateral and peripheral conductance after femoral artery occlusion [47 ]. By affecting basal MCP-1 production, ANP might therefore be associated with repair of the vasculature.

The activation of NPR-C by ANP is known to result in an inhibition of adenylyl-cyclase activity and thereby decreased cyclic adenosine monophosphate (cAMP) levels in endothelial cells [4 ]. Therefore, our data suggest that NPR-C and cAMP might play a role in the signal-transduction pathway inducing basal MCP-1 expression. Conversely, some groups describe an increased MCP-1 expression as a result of increased cAMP levels [46 , 48 , 49 ].

It has furthermore been described that there might be differences in the regulation of basal and stimulus-dependent production of chemokines. In this context, e.g., peroxisome proliferator-activated receptor agonists have been shown to induce basal MCP-1, whereas they inhibit C-reactive protein-induced MCP-1 expression in HUVEC [50 ]. These data support our findings that there is a clear difference between basal- and stimulus-dependent MCP-1 regulation. In this context, it seems quite interesting to know that we could recently describe that ANP induces the basal DNA-binding activity of the transcription factor activated protein-1 (AP-1) [24 ]. As the MCP-1 gene has been reported to be positively regulated by the AP-1-binding sites in its promotor [28 ], these findings point to an involvement of ANP-mediated, AP-1 induction in regulation of basal MCP-1 induction.

Molecular mechanisms of reduced MCP-1 induction by ANP
Characterizing the molecular mechanisms responsible for the inhibitory action of ANP on MCP-1 release, our data show that ANP reduces MCP-1 mRNA expression. A transcriptional regulation of MCP-1 mRNA expression has been increasingly recognized to be mediated via the p38 MAPK pathway [27 , 35 ]. For instance, Takaishi and coworkers [27 ] recently reported the involvement of p38 MAPK in high glucose-induced MCP-1 expression in vascular endothelial cells, and the group of Nakamura [35 ] showed an inhibitory action of thioredoxin on LPS-induced MCP-1 expression via suppressed p38 phosphorylation.

As we recently reported, ANP inhibits TNF-{alpha}-induced p38 MAPK activation in HUVEC [21 ]. We demonstrated that this inhibitory action of ANP was mediated via an early transcriptional induction of the p38 MKP-1 by ANP [21 ]. Considering these facts, we were interested in the causal relationship between MKP-1 induction by ANP and the observed inhibition of MCP-1 release. The data presented here show indeed a causal role for MKP-1 induction in ANP-mediated MCP-1 inhibition, as MKP-1 antisense but not sense oligonucleotides abrogated the inhibitory effect of ANP on TNF-{alpha}-induced MCP-1 expression. In contrast to our observations, induction of MKP-1 has been reported to be required for MCP-1 expression induced by an oxidized phospholipid in human aortic endothelial cells [51 ]. These different data may point to stimulus-dependent differences in MCP-1 regulation.

It is interesting that we could recently report that ANP can induce basal activity of p38 MAPK in the whole rat liver model [52 ]. Carini et al. [53 ] confirmed these results by showing p38 MAPK activation by ANP in rat hepatocytes. As previously published [21 ], there is no induction of basal p38 MAPK phosphorylation after ANP treatment in HUVEC. This observation points to represent species- and cell type-dependent differences in the effects of ANP on basal p38 MAPK activity.

In the present work, however, we investigated the TNF-{alpha}-induced p38 MAPK phosphorylation and not the basal status of p38 MAPK. Our observation that ANP inhibits p38 MAPK in activated cells was also reported for cytokine-activated macrophages [15 ] and for vascular endothelial growth factor-activated vascular endothelial cells [54 ].

Relationship between TNF-{alpha}-induced p38 MAPK and NF-{kappa}B activation
Based on our previously published data concerning inhibitory action of ANP on NF-{kappa}B DNA-binding activity [22 ], we were interested in a potential connection between the NF-{kappa}B and the p38 MAPK pathway. As our results demonstrate, the use of a chemical p38 MAPK inhibitor SB203580 as well as a dn p38 MAPK mutant revealed that the inhibition of p38 MAPK leads to significant activation of NF-{kappa}B transcriptional activity in TNF-{alpha}-pretreated HUVEC and HEK293. This inter-relationship between activation of the transcription factor NF-{kappa}B and p38 MAPK has been controversially discussed in the literature. Several groups, reporting that p38 MAPK activation can inhibit NF-{kappa}B transcriptional activity in monkey kidney, human colon adenocarcinoma, human erythroleukaemia, and HeLa cell lines [55 56 57 58] , support our observation that the blockade of p38 MAPK activity leads to a significant induction of NF-{kappa}B transcriptional activity. Conversely, Tsai and coworkers [59 ] showed that heregulin-ß1-induced NF-{kappa}B activation was effectively blocked by SB203580 [59 ]. These divergent data may point to stimulus- and cell type-dependent differences in NF-{kappa}B regulation. In our setting, the observed inhibition of TNF-{alpha}-induced p38 MAPK activation and the inhibition of NF-{kappa}B by ANP [22 ] seem to be two independent mechanisms.

Taken together, our data show that ANP is able to inhibit TNF-{alpha}-induced MCP-1 expression in endothelial cells. Moreover, our work provides insight into mechanisms by which ANP regulates MCP-1 expression, namely by an induction of MKP-1. These observations are of special importance, as there is clear evidence that an increased release of MCP-1 is a key step in the formation of atherosclerotic lesions.

The observed effects of ANP therefore point to an anti-inflammatory and antiatherogenic potential of this cardiovascular hormone. It is interesting that there exist several reports showing increased natriuretic peptide levels in inflammatory situations such as sepsis [60 61 62 63 ]. Furthermore, several preclinical and clinical trials show that a novel group of pharmacological agents, vasopeptidase inhibitors, which suppress degradation of natriuretic peptides, is highly effective in the treatment of endothelial dysfunction, hypertension, and heart failure [64 ]. Most importantly, a recent clinical trial shows that an ANP polymorphism plays a central role in coronary blood flow regulation and development of atherosclerosis [65 ]. These clinical data together with our present results therefore draw attention to ANP as an endogenous antiatherosclerotic and anti-inflammatory regulator.


   ACKNOWLEDGEMENTS
 
A. K. K. is a recipient of the "Bayerischer Habilitationsförderpreis." The excellent technical support of Brigitte Weiss and Cornelia Niemann is gratefully acknowledged. We thank Robert Fürst for his help in manuscript preparation and Nicole Bildner for cGMP measurement. We thank Prof. Dr. Stephan Ludwig (Würzburg, Germany) for providing the dn p38 plasmids and the Gene Center and University of Munich for providing the ß-Gal plasmid. The staff of the Department of Gynecology of the Klinikum Grosshadern, University of Munich, is gratefully acknowledged for providing umbilical cords.

Received June 3, 2003; revised June 3, 2003; accepted July 2, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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