1

cAMP Targeting of p38 MAP Kinase Inhibits Thrombin-induced NF-κB Activation

and ICAM-1 Expression in Endothelial Cells

Arshad Rahman1, Khandaker N. Anwar2, Mohd. Minhajuddin1, Kaiser M. Bijli1, Kamran Javaid2, Andrea L. True2, and Asrar B. Malik2

1Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

2Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612

Running head: cAMP inhibits ICAM-1 expression by targeting p38 MAPK

Address correspondence to: Arshad Rahman Department of Pediatrics, Box 850

University of Rochester School of Medicine 601 Elmwood Avenue

Rochester, New York 14642 Phone: (585) 275-5948 Fax: (585) 756-7780 E-mail: Arshad_Rahman@urmc.rochester.edu

Articles in PresS. Am J Physiol Lung Cell Mol Physiol (July 9, 2004). 10.1152/ajplung.00072.2004

Copyright © 2004 by the American Physiological Society.

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ABSTRACT

We investigated the mechanisms by which elevated intracellular cAMP concentration

inhibits the thrombin-induced ICAM-1 expression in endothelial cells. Exposure of human

umbilical vein endothelial cells (HUVEC) to forskolin or dibutyryl cAMP (dbcAMP), which

increase intracellular cAMP by separate mechanisms, inhibited the thrombin-induced ICAM-1

expression. This effect of cAMP was secondary to inhibition of NF-κB activity, the key

regulator of thrombin-induced ICAM-1 expression in endothelial cells. The action of cAMP

occurred downstream of IκBα degradation and was independent of NF-κB binding to the ICAM-

1 promoter. We observed that cAMP interfered with thrombin-induced phosphorylation of NF-

κBp65 (RelA) subunit, a crucial event promoting the activation of the DNA-bound NF-κB. As

p38 MAP kinase can induce transcriptional activity of RelA/p65 without altering the DNA

binding function of NF-κB, we addressed the possibility that cAMP antagonizes thrombin-

induced NF-κB activity and ICAM-1 expression by preventing the activation of p38 MAP

kinase. We observed that treating cells with forskolin blocked the activation of p38 MAP kinase

and inhibition of p38 MAP kinase interfered with phosphorylation of RelA/p65 induced by

thrombin. Our data demonstrate that increased intracellular cAMP concentration in endothelial

cells prevents thrombin-induced ICAM-1 expression by inhibiting p38 MAP kinase activation,

which in turn prevents phosphorylation of RelA/p65 and transcriptional activity of the bound NF-

κB.

Keywords: Endothelial adhesivity, polymorphonuclear leukocytes, IκΒα degradation,

RelA/p65 phosphorylation,

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INTRODUCTION

The procoagulant serine protease thrombin, released during intravascular coagulation

initiated by tissue injury or sepsis (11, 14), promotes the adhesion of polymorphonuclear

leukocytes (PMN) to the endothelium by a mechanism involving the endothelial cell surface

expression of intercellular adhesion molecule-1 (ICAM-1) (27, 29, 40). ICAM-1, a member of

the immunoglobulin supergene family (38), serves as a counter-receptor for leukocyte β2

integrins, CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1), and mediates the arrest of PMNs

and other leukocytes and their migration across the vessel wall barrier (36). We showed that the

transcription factor NF-κB p65 (RelA) is an essential regulator of thrombin-induced ICAM-1

gene transcription following thrombin-induced activation of the heterotrimeric G protein coupled

receptor, Protease-Activated Receptor-1 (PAR- 1) in endothelial cells (27, 30).

NF-κB, typically a heterodimer of 50 kDa (p50) and 65 kDa (RelA) subunits, is

sequestered in the cytoplasm bound to IκB proteins that mask the nuclear localization sequence

of NF-κB (3). NF-κB activity is in part regulated at the level of IκB degradation, which is

accomplished through serine phosphorylation (Ser32 and Ser36) of IκBα (41) by IκBβ kinase

(IKKβ) (16, 45). Phosphorylation targets IκBα for ubiquitination and proteasome-mediated

degradation (31). The liberated NF-κB is translocated to the nucleus and binds to NF-κB

responsive elements in genes. Studies have shown an additional regulatory pathway involving

phosphorylation of RelA/p65 that stimulates the activation of NF-κB (2, 23). In contrast to

IκBα phosphorylation, RelA/p65 phosphorylation can be mediated by several kinases depending

upon the stimulus and cell type (12, 33, 43, 44).

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The p38 mitogen-activated protein (MAP) kinases are widely expressed serine threonine

kinases activated by a number of stimuli, including thrombin (21, 29, 34). Activation of p38

MAP kinase has been implicated in the mechanism of NF-κB activation (1, 29, 42). We have

recently shown that inhibition of p38 MAP kinase prevented thrombin-induced transcriptional

activity of NF-κB without altering its DNA binding function (29), indicating the involvement of

p38 MAP kinase in promoting the transactivation of NF-κB (42).

Cyclic adenosine monophospahte (cAMP) is an ubiquitous second messenger in cells.

An important function of cAMP is to activate protein kinase A (PKA) (39), which has been

implicated in the regulation of numerous genes through phosphorylation and activation of the

cAMP response element binding (CREB) protein (9). Studies showed that cAMP regulates the

activation of NF-κB in a cell-specific manner. In the pro-myelocytic cell line, HL 60, elevated

cAMP levels induced NF-κB activation (35), whereas exposure of endothelial cells to cAMP

prevented NF-κB activation (24). As cAMP modulation of NF-κB activity in endothelial cells

may be important in controlling ICAM-1 expression during inflammation, in the present study

we addressed the mechanism of this modulatory cAMP action. We provide evidence herein that

cAMP inhibits thrombin-induced NF-κB activity and ICAM-1 expression in endothelial cells by

blocking p38 MAP kinase activation, which in turns prevents the phosphorylation of RelA/p65,

and activation of the DNA-bound NF-κB.

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MATERIALS AND METHODS

Materials: Human thrombin (activity of 3170 NIH U/mg) was purchased from Enzyme

Research Laboratories (South Bend, IN). Polyclonal antibodies against p38 MAP kinase, IκBα,

and NF-κB p65 and a monoclonal antibody against ICAM-1 were obtained from Santa Cruz

Biotechnology (Santa Cruz, CA). Antibodies that detect p38 MAP kinase when activated by

dual phosphorylation at Thr180 and Tyr182, RelA/p65 when activated by phosphorylation at

Ser536 or IκBα when phosphorylated at Ser32 and Ser36 were obtained from Cell Signaling

Technology (Beverly, MA). In addition, polyvinylidene difluoride (PVDF) membrane was from

Millipore Corp. (Bradford, MA); Forskolin and dibutyryl cAMP (dbcAMP) from Sigma

Chemical Company (St. Louis, MO); SB203580 from Calbiochem-Novabiochem Corp. (La

Jolla, CA); protein assay kit and nitrocellulose membrane were from Bio-Rad Laboratories

(Hercules, CA); plasmid maxi kit from QIAGEN Inc. (Valencia, CA). All other materials were

from Fisher Scientific Company (Pittsburg, PA).

Cell Culture: Human umbilical vein endothelial cells (HUVEC; Clonetics, La Jolla, CA) were

cultured as described (29) in gelatin-coated flasks using endothelial basal medium 2 (EBM2)

with bullet kittm additives (Clonetics). Confluent cells were incubated for 2-12 h in heat

inactivated 0.5-1% FBS containing EBM2 prior to thrombin challenge. All experiments, except

where indicated, were made in cells under the 8th passage.

Flow Cytometry Analysis: Flow cytometry analysis was performed as described (27). Briefly,

HUVEC monolayers in six-well tissue culture dishes were pretreated with FSK (20 µM) or

dibutyryl cAMP (0.5 mM) for 30 min prior to stimulation with thrombin for 12-15 h. After

completion of incubation period, cells were washed twice with cold PBS, removed by careful

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trypsinization, and washed again with Ca2+/Mg2+-free PBS before incubating with 20% horse

serum for 30 min. Following two washes, cells were incubated with a mouse mAb directed

against human ICAM-1, BIRR0001 (kindly provided by Dr. Robert Rothlein, Boeringer

Ingleheim, Ridgefield, CT) (32), in Ca2+/Mg2+-free PBS containing 3% horse serum for 30 min at

4°C. Cells were then washed twice with PBS/horse serum and incubated for 30 min at 4°C with

a goat anti-mouse IgG FITC-conjugated secondary Ab. Cells were then fixed with 2%

paraformaldehyde, and analyzed by flow cytometry in a FACScan cytofluorometer (Becton

Dickinson, Mountain View, CA), and the results were gated for mean fluorescence intensity

above the fluorescence produced by the secondary Ab alone.

Cell Lysis and Immunoblotting: After treatment, the cells were lysed in radioimmune

precipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25 mM EDTA, 1% Triton-X100,

1% sodium deoxycholate, 5 mM sodium fluoride, 1 mM PMSF, and 1 µg/ml each of leupeptin,

pepstatin A, and aprotinin). Cell lysates were analyzed by SDS-PAGE and transferred onto

nitrocellulose (Bio-Rad Laboratories) or polyvinylidene difluoride (PVDF; Millipore Corp.,

Bradford, MA) membranes, and the residual binding sites on the filters were blocked by

incubating with 5% (w/v) nonfat dry milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05%

Tween 20) for 1 h at room temperature or overnight at 4 °C. The membranes were subsequently

incubated with indicated antibodies and developed using an enhanced chemiluminesence (ECL)

method as described (29).

p38 MAP Kinase Assay: Cells were serum-starved by overnight incubation in EBM2-1% FBS.

The cells were subsequently challenged with thrombin (5 U/ml) for 5 min in the absence and

presence of forskolin (20 µM) or dbcAMP (0.5 mM), which was added 30-60 min prior to

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thrombin treatment. The cells were then lysed with a phosphorylation lysis buffer (50 mM

HEPES, 150 mM NaCl, 200 µM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM

sodium fluoride, 1 mM EDTA, 1.5 mM magnesium chloride, 10% glycerol, 0.5-1% Triton X-

100, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin). Cell lysates were

immunoprecipitated with an antibody against p38 MAP kinase using protein G-Sepharose

(Amersham Pharmacia Biotech) as described (29). The immunocomplexes were washed three

times with phosphorylation lysis buffer and two times with kinase buffer (25 mM HEPES, pH

7.4, 25 mM MgCl2, 25 mM β-glycerophosphate, 2 mM DTT, 0.1 mM sodium orthovanadate, 20

µM ATP), and resuspended in 30 µl of kinase buffer containing 5 µg of ATF-2 and 20-30 µCi of

[γ32P]ATP was added. The reaction was incubated for 15-30 min at room temperature and was

terminated by the addition of SDS-sample buffer. Proteins were analyzed by SDS-PAGE, and

the phosphorylated form of ATF-2 was detected by autoradiography.

Northern Analysis: Total RNA was isolated from HUVEC with RNeasy kit (QIAGEN Inc.)

according to manufacturer’s recommendations. Quantification and purity of RNA were assessed

by A260/A280 absorption and an aliquot of RNA (20 µg) from samples with ratio above 1.6 was

fractionated using a 1% agarose formaldehyde gel. The RNA was transferred to Duralose-UVTM

nitrocellulose membrane (Stratagene, La Jolla, CA) and covalently linked by ultraviolet

irradiation using a Stratalinker UV crosslinker (Stratagene). Human ICAM-1 (0.96 kb SalI to

PstI fragment) (38) and rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.1 kb PstI

fragment) were labeled with [α 32P]dCTP using the random primer kit (Stratagene) and

hybridization was carried out as described (27). Briefly, the blots were prehybridized for 30 min

at 68 oC in QuikHybTM solution (Stratagene) and hybridized for 2 h at 68 oC with random primed

32P-labeled probes. After hybridization, the blots were washed 2x for 30 min at room

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temperature in 2x SSC with 0.1% SDS followed by 2 washes for 15 min each at 60 oC in 0.1x

SSC with 0.1% SDS. Autoradiography was performed with an intensifying screen at -70 oC for

12-24 h. The nitrocellulose membrane was soaked for stripping the probe with boiled water or

0.1x SSC with 0.1% SDS.

Reporter Gene Constructs, Endothelial Cell Transfection, and Luciferase Assay: The

plasmid pNF-κB-LUC containing 5 copies of consensus NF-κB sequences linked to a minimal

E1B promoter-luciferase gene was purchased from Stratagene. Transfections were performed

using DEAE-dextran method (22) with slight modifications (29). Briefly, 5 µg DNA was mixed

with 50 µg/ml DEAE-dextran in serum-free EBM2 and the mixture was added onto cells which

were 70-80% confluent. We used 0.125 µg pTKRLUC plasmid (Promega Corp., Madison, WI)

containing Renilla luciferase gene driven by the constitutively active thymidine kinase promoter

to normalize the transfection efficiencies. After 1 h, cells were incubated for 4 min with 10%

dimethyl sulfoxide (DMSO) in serum-free EBM2. The cells were then washed 2x with EBM2-

10% FBS and grown to confluency. Cell extracts were prepared and assayed for Firefly and

Renilla luciferase activities using Promega Biotech Dual Luciferase Reporter Assay System

(Promega, Madison, WI). The data were expressed as a ratio of Firefly and Renilla luciferase

activity.

Cytoplasmic and Nuclear Extract Preparation: After treatment, cells were washed 2x with

ice-cold Tris-buffered saline (TBS) and resuspended in 400 µl of buffer A (10 mM HEPES, [pH

7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol [DTT], 0.5 mM

phenylmethylsulfonyl fluoride [PMSF]). After 15 min, Nonidet P-40 (NP-40) was added to a

final concentration of 0.6%. Samples were centrifuged to collect the supernatants containing

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cytosolic proteins for determining IκBα degradation by Western blot analysis. The pelleted

nuclei were resuspended in 50 µl of buffer B (20 mM HEPES [pH 7.9], 0.4 M NaCl, 1 mM

EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF). After 30 min at 4 0C, lysates were centrifuged

and supernatants containing the nuclear proteins were transferred to new vials. Protein

concentration of the extract was measured using a Bio-Rad protein determination kit (Bio-Rad

Laboratories).

Electrophoretic Mobility Shift Assay: Electrophoretic mobility shift assays (EMSA) were

performed as described (29). Briefly, 10 µg of nuclear extract was incubated with 1 µg of poly

(dI-dC) in a binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 0.5 mM DTT, 10%

glycerol [20 µl final volume] for 15 min at room temperature. Then end-labeled double-stranded

oligonucleotides containing an NF-κB site (30,000 cpm each) were added and the reaction

mixtures were incubated for 15 min at room temperature. The DNA-protein complexes were

resolved in 5% native polyacrylamide gel electrophoresis in low ionic strength buffer (0.25x

Tris-borate-EDTA). The oligonucleotide used for the gel shift analysis was

Ig-κB 5’-AGTTGAGGGGACTTTCCCAGGC-3’. The Ig-κB oligonucleotide contains the

consensus NF-κB binding site sequence. The sequence motifs within the oligonucleotides are

underlined.

PMN Adhesion Assay: PMN adhesion assay was performed as described (4). Briefly, HUVEC

grown on 12 mm circular cover slips were stimulated with thrombin, washed extensively to

remove residual thrombin prior to labeling with 3 µM fluorescent (red) Cell Tracker dye for 30

min. Freshly isolated human neutrophils (PMN) were stained with 5 µM fluorescent (green) Cell

Tracker dye, coincubated with endothelial cells for 20 min, washed with PBS, and visualized

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using a fluorescent microscope. The adherent PMN were counted and expressed as PMN/0.8

mm2 of endothelial cell.

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RESULTS

cAMP inhibits thrombin-induced ICAM-1 mRNA expression in endothelial cells: We

determined the effects of increasing intracellular cAMP on ICAM-1 mRNA expression in

response to thrombin challenge of endothelial cells. We used forskolin, an adenylate cyclase

activator, and dbcAMP, a synthetic cell-permeable cAMP analogue, to raise the intracellular

cAMP by two independent means. Northern blot analysis showed that pretreatment of HUVEC

monolayers with forskolin or dbcAMP, reduced thrombin-induced ICAM-1 transcript in a dose-

dependent manner (Fig. 1 A&B). In another experiment, we determined if cAMP can also

inhibit ICAM-1 mRNA expression in response to TNFα challenge of endothelial cells. These

results showed that forskolin, in contrast to its effect on thrombin response, failed to prevent

TNFα-induced ICAM-1 mRNA expression (Fig. 1C).

cAMP inhibits thrombin-induced ICAM-1 cell surface expression and endothelial

adhesivity towards PMN: We next evaluated the effects of cAMP on thrombin-induced

ICAM-1 cell surface expression and endothelial adhesivity towards naïve PMNs. FACS analysis

showed that thrombin challenge of HUVEC resulted in increased ICAM-1 cell surface

expression (Fig. 2A, panel b). Pretreatment of cells with forskolin or dbcAMP inhibited

thrombin-induced ICAM-1 cell surface expression (Fig. 2A, panels c&e). In control

experiments, forskolin or dbcAMP alone showed no effects on ICAM-1 cell surface expression

(Fig. 2A, panels a&d). As ICAM-1 expression induces PMN adhesion to endothelial cells (27,

29), we determined whether prevention of ICAM-1 cell surface expression interfered with

endothelial adhesivity. We found that increase in intracellular cAMP following pretreatment of

cells with forskolin prevented the thrombin-induced endothelial adhesivity towards naïve PMN

(Fig. 2B).

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cAMP inhibits thrombin-induced NF-κB activity without affecting its DNA binding

function: As NF-κB activation is essential for the thrombin-induced ICAM-1 gene transcription

(27), we addressed the possibility that cAMP exerts its effect on ICAM-1 expression by

inhibiting the NF-κB activity. HUVEC were co-transfected with pNF-κBLUC containing 5

copies of consensus NF-κB sequence linked to a minimal adenovirus E1B promoter-luciferase

reporter gene. As shown in Fig. 3, thrombin-induced NF-κB activity was markedly reduced in

cells pretreated with forskolin.

We also determined the effect of cAMP on thrombin-induced IκBα degradation, a

requirement for NF-κB activation (6, 41). Western blot analysis showed that thrombin exposure

of endothelial cells resulted in IκBα degradation (Fig. 4A). In contrast to forskolin’s effect on

NF-κB activity (Fig. 3), forskolin failed to prevent the thrombin-induced IκBα degradation (Fig.

4A). As IκBα degradation results in nuclear translocation and DNA binding of NF-κB, we also

evaluated the effects of cAMP on thrombin-induced nuclear uptake and DNA binding function

of NF-κB. Pretreatment of cells with forskolin failed to prevent the nuclear translocation of

RelA/p65 (Fig. 4B). Electrophoretic mobility shift assay showed that forskolin failed to inhibit

the DNA binding activity of NF-κB (Fig. 4C) consistent with its lack of effect on nuclear uptake

of RelA/p65 (Fig. 4B).

cAMP inhibits thrombin-induced phosphorylation of RelA/p65: Because activation of

RelA/p65 requires phosphorylation of serine 536 in the transactivation domain (20, 44), we

explored the possibility that cAMP inhibits thrombin-induced transcriptional activity of

RelA/p65 by preventing its phosphorylation. We determined the effect of cAMP on the

phosphorylation status of RelA/p65 using an antibody that detects RelA/p65 phosphorylated at

serine 536. Co-immunoprecipitation studies demonstrated that thrombin challenge resulted in

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increased phosphorylation of RelA/p65 at serine 536 and this response was inhibited in cells

treated with forskolin prior to the thrombin challenge (Fig. 5A).

cAMP inhibits thrombin-induced activation of p38 MAP kinase: We have previously shown

that p38 MAP kinase is a critical signaling intermediate required for thrombin-induced ICAM-1

transcription in endothelial cells (29). Our results showed that pretreatment of cells with

SB203580, a p38 MAP kinase inhibitor, prevented the thrombin-induced phosphorylation of

serine 536 of RelA/p65 (Fig. 5A). These findings led us to investigate if cAMP functions by

interfering with the activation of p38 MAP kinase in response to thrombin challenge, and thereby

inhibits phosphorylation of serine 536 of RelA/p65 and its activation. Thus, we determined the

effects of cAMP on thrombin-induced p38 MAP kinase phosphorylation, and found that thrombin

activated p38 MAP kinase (Thr180/Tyr182) phosphorylation in a manner dependent on cAMP

(Fig. 5B). Preincubation of cells with dbcAMP also prevented the thrombin-induced

phosphorylation of p38 MAP kinase (data not shown). We next determined if the effect of cAMP

on p38 MAP kinase phosphorylation could be ascribed to its inhibition of p38 MAP kinase

activation. In an in vitro kinase assay using ATF-2 as a substrate, we observed that the p38 MAP

kinase immunoprecipitated from thrombin-challenged cells resulted in increased phosphorylation

of ATF-2 (Fig. 5C) indicative of activation of p38 MAP kinase by thrombin. Pretreatment of cells

with dbcAMP prevented the thrombin-induced p38 MAP kinase activation (Fig. 5C). Taken

together, these data indicate that cAMP inhibits thrombin response by interfering with the

activation of p38 MAP kinase.

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DISCUSSION

The present study demonstrates that p38 MAP kinase is a critical target mediating the

cAMP-dependent inhibition of thrombin-induced NF-κB activation and ICAM-1 expression in

endothelial cells. We showed that inhibition of p38 MAP kinase by cAMP prevented the

thrombin-activated phosphorylation of RelA/p65, and thus interfered with transcriptional

competency of this DNA-bound NF-κB subunit.

The inhibitory effect of cAMP was only evident when thrombin was used as the agonist

to elicit ICAM-1 expression. cAMP had no significant effect on the TNFα-induced ICAM-1

expression. This latter finding agrees with studies showing that increased intracellular levels of

cAMP failed to inhibit ICAM-1 expression in response to TNFα challenge of endothelial cells

(24, 26). However, we showed clearly that increased cAMP level following incubation of cells

with forskolin significantly reduced the thrombin-induced NF-κB-dependent reporter gene

activity, indicating that cAMP prevented the activation of NF-κB, and thus ICAM-1 expression.

The reasons for the differential effects of cAMP in thrombin- versus TNFα-induced ICAM-1

expression are not clear; but raise important question whether raising the cAMP concentration

would be protective in inflammatory diseases associated with the generation of cytokines such as

TNFα. While NF-κB is essential and sufficient for thrombin-induced ICAM-1 transcription

(27), the TNFα-induced ICAM-1 expression in endothelial cells required the cooperation of

multiple transcription factors including NF-κB and CCAAT/enhancer binding protein (C/EBP)

(19). A likely explanation for the failure of cAMP to prevent the TNFα-induced ICAM-1

expression may be that increased intracellular cAMP concentration can independently result in

activation of C/EBP (5, 13). Further support for this notion comes from studies (24, 26) in which

elevated cAMP levels in endothelial cells inhibited the TNFα-induced expression of genes

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encoding E-selectin and VCAM-1, which are both NF-κB-dependent but do not require the

cooperation of C/EBP (8) as is the case with ICAM-1. Studies have shown that transactivation

of genes by NF-κB requires DNA binding secondary to phosphorylation and degradation of

IκBα and translocation of RelA/p65 to the nucleus (3, 16, 41). We determined if the increase in

cAMP level could regulate NF-κB activation by influencing these events. Preincubation of cells

with forskolin failed to prevent the NF-κB DNA binding activity induced by thrombin. Also

increased cAMP concentration did not inhibit the thrombin-induced IκBα degradation and

translocation of RelA/p65 to the nucleus. Thus, cAMP appears to exert its inhibitory effect on

ICAM-1 expression without interfering with activation of NF-κB in the cytosol and its

translocation to the nucleus.

Another important mechanism regulating NF-κB activity involves the phosphorylation of

the DNA-bound RelA/p65 NF-κB subunit (2, 23,). Studies showed that phosphorylation of

RelA/p65 at serine 536 increases the transcriptional competency of NF-κB bound to the

promoter (20, 44). We recently showed that inhibition of p38 MAP kinase by pharmacological

and genetic approaches significantly reduced the thrombin-induced ICAM-1 expression

secondary to inhibition of transactivation activity of the nuclear NF-κB, but did not interfere

with its cytosolic activation (29). Thus, we addressed the possibility that elevation in cAMP

levels could inhibit thrombin-induced transcriptional activity of NF-κB by preventing the

phosphorylation of RelA/p65 at serine 536. Further, we determined whether this effect of cAMP

could be ascribed to inhibition of p38 MAP kinase activation. We observed, consistent with our

hypothesis, that increase in intracellular cAMP concentration (elicited by pretreating cells with

forskolin) prevented serine 536 phosphorylation of RelA/p65 in response to thrombin. We also

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found, interestingly, that cAMP prevented the activation of p38 MAP kinase, and that this was in

fact responsible for inhibiting the serine 536 phosphorylation of RelA/p65 induced by thrombin.

In contrast, elevated cAMP levels failed to prevent the TNFα-induced RelA/p65 phosphorylation

in endothelial cells (24). Recently, Duran et al., (12) have reported that the TNFα-induced

RelA/p65 phosphorylation is mediated by PKC-ζ, suggesting that other kinases are also capable

of transactivating the DNA-bound RelA/p65. As PKC-ζ is required for TNFα-induced NF-κB

activation in endothelial cells (2, 28), it is possible that cAMP has no effect on PKC-ζ activation,

which may thus explain why cAMP had no effect on TNFα-induced RelA/p65 phosphorylation.

Thus, our findings demonstrate that the mechanism of action of cAMP in preventing thrombin-

induced NF-κB activation involves inhibition of p38 MAP kinase activation, which in turn

prevents phosphorylation of RelA/p65 bound to the ICAM-1 promoter.

In addition to the effect of p38 MAP kinase in inducing phosphorylation of RelA/p65A as

discussed above, another possible mechanism by which p38 MAP kinase can mediate NF-κB

activation is through phosphorylation of TATA binding protein (TBP), a subunit of transcription

factor IID (TFIID). Phosphorylation of TBP by p38 MAP kinase is necessary for TBP binding

to the TATA box (7). Inhibition of phosphorylation of TBP was shown to reduce its binding to

the TATA box and interaction with the NF-κB p65 subunit (7). Thus, it is possible that

inhibition of p38 MAP kinase by cAMP can abrogate ICAM-1 expression by preventing the

phosphorylation of TBP. This idea has support from the findings of Delgado and Ganea (10)

showing that vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating

polypeptide (PACAP) prevented the lipopolysaccharide-induced NF-κB activation through the

cAMP-dependent inhibition of p38 MAP kinase phosphorylation of TBP.

17

Yet another explanation for the inhibitory effect of cAMP on NF-κB activation is the

finding of Parry and Mackman (25) showing that cAMP promotes the phosphorylation of the

CRE-binding protein (CREB), and subsequent binding to the CREB-binding protein (CBP),

which in turn can block NF-κB activation. CBP is a co-activator known to associate with

RelA/p65 (15, 25). The presence of CBP in the nucleus may lead to a decrease in RelA-CBP

complex formation, and impaired NF-κB activation (25). As the phosphorylation status of

RelA/p65 is critical for its association with CBP (12, 46), it may be that cAMP-dependent

inhibition of p38 MAP kinase activation and of phosphorylation of RelA/p65 can impair

transcriptional competency of the bound NF-κB.

Increase in cellular cAMP levels induced by pharmacologic agents, pentoxifylline,

rolipram and amrinone, suppress expression of proinflammatory genes and are beneficial in

inflammatory conditions such as autoimmune encephalomyelitis and acute cardiac allograft

rejection (17, 18, 37). In this study, we have shown that elevation of endothelial cell cAMP

levels inhibits NF-κB activation induced by thrombin and this has important functional

consequences in preventing ICAM-1 expression and endothelial adhesivity towards PMNs. We

have also shown that cAMP acts by targeting p38 MAP kinase activation and preventing the

phosphorylation of the RelA/p65 subunit of NF-κB bound to the ICAM-1 promoter. Thus, the

present results provide a basis for the mechanism of anti-inflammatory action of cAMP, which

may have implications in novel therapeutics targeting p38 MAP kinase.

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ACKNOWLEGDMENTS

This work was supported by NHLBI grants HL67424, HL46350, and HL64573.

19

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ABBREVIATIONS

ICAM-1, intercellular adhesion molecule-1; PMN, polymorphonuclear leukocytes; PKA, protein

kinase A; MAPK, mitogen-activated protein kinase; TNF-α, tumor necrosis factor α; HUVEC,

human umbilical vein endothelial cells; FSK, Forskolin; dbcAMP, dibutyryl cyclic AMP;

EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; LUC,

luciferase; NF-κB, nuclear factor-κB; bp, base pair(s); TBP, TATA-binding protein; PAR-1,

protease-activated receptor-1, CREB, cyclic AMP response element binding protein; CBP,

CREB-binding protein

27

FIGURE LEGENDS

Figure 1. Elevated cAMP concentration in endothelial cells inhibits thrombin-induced

ICAM-1 mRNA expression. Confluent HUVEC monolayers were pretreated with (A&C)

forskolin (FSK) or (B) dibutyryl cyclic AMP (dbcAMP) at the indicated concentrations. After

30 min, cells were challenged with (A, B&C) thrombin (2.5 U/ml) for 3 h or (C) TNFα (100

U/ml) for 2 h. Total RNA was isolated and analyzed by Northern hybridization with a human

ICAM-1 cDNA, which hybridizes to a 3.3-kb transcript. Blots were stripped and re-probed to

determine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression as a

measure of RNA loading. Results are representative of 3 separate experiments.

Figure 2A. Elevated cAMP concentration inhibits thrombin-induced ICAM-1 expression

on endothelial cell surface. Confluent HUVEC monolayers were pretreated with (a&c) FSK

(20 µM ) or (d&e) dbcAMP (0.5 mM) for 30 min prior to stimulation with thrombin (2.5 U/ml)

for 12-15 h. ICAM-1 expression was quantitated by flow cytometry using mAb against ICAM-1

(BIRR0001) or mAb against IgG, as described in Materials and Methods. Results are

representative of two separate experiments.

Figure 2B. Elevated cAMP concentration inhibits thrombin-induced endothelial adhesivity

towards PMN. Confluent HUVEC monolayers were pretreated with FSK (20 µM ) prior to

challenge with thrombin (5 U/ml). Expression of endothelial adhesiveness was determined by

PMN adhesion assays as described in Materials and Methods. Data are mean ± S.E. (n= 3 for

each condition).

Figure 3. Elevated cAMP concentration inhibits thrombin-induced NF-κB activity.

HUVEC were transfected with NF-κBLUC construct using DEAE-dextran method as described

(Lopata et al., 1984). Cells were pretreated with FSK (20 µM) prior to stimulation for 8 h with

28

thrombin (5 U/ml). Cell extracts were prepared and assayed for Firefly and Renilla luciferase

activities using Promega Biotech Dual luciferase Assay System (Promega, Madison, WI). The

data are expressed as Firefly/Renilla luciferase activity. Data are mean ± S.E. (n= 3 for each

condition).

Figure 4. Elevated cAMP concentration fails to prevent thrombin-induced IκBα

degradation, nuclear translocation, and NF-κB DNA binding activity. Confluent HUVEC

monolayers were pretreated for 30 min with FSK (20 µM) prior to challenge with thrombin (2.5

U/ml) for indicated time periods. Cytoplasmic (A) and nuclear (B&C) extracts were prepared

and assayed for IκBα degradation (A) and NF-κB nuclear translocation (B) by Western blot

analysis and NF-κB DNA binding activity (C) by EMSA as described in Materials and Methods.

Results are representative of 2-3 separate experiments.

Figure 5A. Elevated cAMP concentration and p38 MAP kinase inhibition prevent

thrombin-induced serine 536 phosphorylation of RelA/p65. Confluent HUVEC monolayers

were pretreated with FSK (20 µM) or SB203580 (10 µM) for 30 min prior to challenge with

thrombin (5 U/ml) for 1 h. Cell lysates were immunoprecipitated with an antibody against p65,

immunoblotted with an antibody against the phosphorylated (Ser536) form of RelA/p65. The

blots were subsequently stripped and re-probed with an antibody against RelA/p65. Results are

representative of 3 separate experiments.

Figure 5B&C. Elevated cAMP concentration inhibits thrombin-induced p38 MAP kinase

activation. B. Confluent HUVEC monolayers were pretreated with FSK (20 µM) for 30 min

prior to challenge with thrombin (5 U/ml) for 5 min. Total cell lysates (10 µg/lane) were

separated by SDS-PAGE and immunoblotted with an antibody against phosphorylated

29

(Thr180/tyr182) form of p38 MAP kinase. The blots were subsequently stripped and re-probed

with an antibody against p38 MAP kinase. C. Confluent HUVEC monolayers were pretreated

with dbcAMP (0.5 mM) for 30 min prior to challenge with thrombin (5 U/ml) for 5 min. Cell

lysates were immunoprecipitated with an antibody against p38 MAP kinase, and in vitro kinase

assays were carried out on immunoprecipitates using ATF-2 as an exogenous substrate. Proteins

were analyzed by SDS-PAGE, transferred to nitrocellulose membrane and the phosphorylated

form of ATF-2 was detected by autoradiography. The blots were subsequently stripped and re-

probed with an antibody against p38 Map kinase. Results are representative of 3 separate

experiments.

Rahman et al.Figure 1

A

ICAM-1

GAPDH

FSK (µM) - 50 - 5 20 50Thrombin (2.5 U/ml) - - + + + +

B

ICAM-1

dbcAMP (mM) - 0.5 - 0.1 0.5Thrombin (2.5 U/ml) - - + + +

GAPDH

C

ICAM-1

GAPDHFSK (20 µM) - - + + - +

Thrombin (5 U/ml) - + - + - -TNFα (100 U/ml) - - - - + +

Figure 2ARahman et al.

a b c

d e

Rahman et al.

Figure 2B

0

5

10

15

20

25

FSK (20 µM) - + - + Thrombin (5 U/ml) - - + +

PMN

Adh

esio

n(N

umbe

r of P

MN

/0.8

8 m

m2

of E

C)

Figure 3Rahman et al.

0.0

0.5

1.0

1.5

2.0

______________________ NF-κBLUC

Thrombin - + - + __________ FSK

NF-

κB A

ctiv

ity(F

irefly

/ Ren

illa

Luci

fera

se A

ctiv

ity)

Rahman et al.Figure 4

A

IκBα

FSK - - + +Thrombin (h) - 1 - 1

Actin

B

p65FSK - + - + - +

Thrombin (h) - - 0.5 0.5 1 1

FSK - - + +Thrombin (h) - 1 - 1

NF-κΒ

Free probe

C

Rahman et al.

Figure 5A

A

p65Blot: anti-p65 S536-P

p65Blot: anti-p65IP: anti-p65 + + + + + +

FSK (20 µM) - - + + - -SB203580 (10 µM) - - - - + +Thrombin (5 U/ml) - + - + - +

Rahman et al.

Figure 5B&C

B

Blot: anti-phospho-p38 p38

p38Blot: anti-p38

FSK (20 µM) - + - + Thrombin (5 U/ml) - - + +

C

ATF-2

dbcAMP (0.5 mM) - - + Thrombin (5 U/ml) - + +

p38