Activation of NF-κB via the IκB kinase complex is both essential and

sufficient for proinflammatory gene expression in primary endothelial

cells

Andrea Denk1*, Matthias Goebeler2*, Sybille Schmid2, Ingolf Berberich4,

Olga Ritz1, Dirk Lindemann4, Stephan Ludwig3, and Thomas Wirth1,5

1 Dept. of Physiological Chemistry, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm,

Germany, 2 Dept. of Dermatology, Wuerzburg University, Josef-Schneider-Str. 2, 97078

Wuerzburg, Germany, 3 Institut fuer Medizinische Strahlenkunde und Zellforschung, MSZ,

Wuerzburg University, Versbacher-Str. 5, 97078 Wuerzburg, Germany, 4 Institut fuer

Virologie und Immunologie, Wuerzburg University, Versbacher Str. 7, 97078 Wuerzburg

Running title: The role of IKKs and IκBs in endothelial activation

This work was supported by grants GO 811/1-3 to M. G. as well as Wi789/2 and 2/3 from the

Deutsche Forschungsgemeinschaft and by Fonds der Chemischen Industrie to T. W.

* These two authors contributed equally to this work.

5) corresponding author

Tel.: +49 731 502 3270

FAX: +49 731 502 2892,

e-mail: thomas.wirth@medizin.uni-ulm.de

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 3, 2001 as Manuscript M102698200 by guest on February 19, 2018

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SUMMARY

Activation of transcription factor NF-κB is necessary for full expression of tumor necrosis

factor-α (TNF-α)-inducible endothelial chemokines and adhesion molecules. However, a

detailed analysis regarding contribution of the different NF-κB upstream components to

endothelial activation has not been performed yet. We employed a retroviral infection

approach to stably express transdominant (TD) mutants of IκBα, IκBβ or IκBε and dominant

negative (dn) versions of IKK1 or IKK2 as well as a constitutively active version of IKK2 in

human endothelial cells. TD IκBα, IκBβ and IκBε were not degraded upon TNF-α exposure

and each prevented NF-κB activation. These TD IκB mutants almost completely inhibited

induction of monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8),

intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1)

and E-selectin expression by TNF-α whereas interferon-γ-mediated upregulation of ICAM-

1 and HLA-DR was not affected. Expression of dn IKK2 completely blocked TNF-α-

induced upregulation whereas dn IKK1 showed a partial inhibition of expression of these

molecules. Importantly, expression of constitutively active IKK2 was sufficient to drive full

expression of all chemokines and adhesion molecules in the absence of cytokine. We

conclude that the IKK/IκB/NF-κB pathway is crucial and sufficient for proinflammatory

activation of endothelium.

ABBREVIATIONS

EMSA, electrophoretic mobility shift assay; FACS, fluorescence activated cell sorting; GFP,

green fluorescent protein; HUVEC, human umbilical vein endothelial cells; ICAM-1,

intercellular adhesion molecule-1; IFNγ, interferon-γ; IKK, IκB kinase; IL-8, interleukin-8;

MCP-1, monocyte chemoattractant protein-1; NF-κB, nuclear factor-κB; TNFα, tumor

necrosis factor-α; VCAM-1, vascular cell adhesion molecule-1

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INTRODUCTION

Activation of endothelial cells is the key process promoting initiation of inflammatory

reactions. It is associated with a multi-step cascade of events that results in the local

recruitment of leukocytes to sites of inflammatory challenge. Selectins and members of the

immunoglobulin superfamily of adhesion molecules are up-regulated by proinflammatory

factors and sequentially mediate rolling, adhesion and transmigration of leukocytes from the

blood stream to underlying tissues ((1,2), for review). In addition, chemoattractant cytokines

(chemokines) produced by and presented on endothelial cells deliver signals that further

trigger firm adhesion of rolling blood cells and transmigration, e. g. by modulation of

leukocyte integrin activity (3).

The process of endothelial activation underlies tight regulatory control mechanisms.

Proinflammatory stimuli targeting endothelium such as tumor necrosis factor-α (TNF- α),

interleukin-1 (IL-1) or bacterial lipopolysaccharides (LPS) elicit activation of intracellular

signal transduction cascades. One of the most important pathways results in activation of

members of the nuclear factor-κB (NF-κB)/Rel family of transcription factors. NF-κB was

suggested to be the major transcriptional regulator of endothelial adhesion molecules such as

E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1

(VCAM-1) as well as of chemokines such as monocyte-chemoattractant protein-1 (MCP-1)

or interleukin-8 (IL-8) (4).

Rel family members (p65/RelA, RelB, c-Rel, p50, p52) form homo- or heterodimeric

complexes with each other which constitute the NF-κB complex (5,6). In resting cells, NF-

κB is inactive due to association with inhibitor κB (IκB) proteins which mask the nuclear

localization sequence of NF-κB thereby retaining it in the cytoplasm and preventing DNA-

binding. Several IκB proteins are involved in the control of NF-κB activity, three of these, i.

e. IκBα, IκBβ, and IκBε, act in a stimulus-dependent manner. Upon inflammatory activation,

IκB is phosphorylated in its N-terminal domain; subsequently it becomes ubiquitinylated and

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finally degraded by the proteasome. This allows nuclear translocation of NF-κB and binding

to cognate DNA motifs in the promoter region of target genes, which subsequently initiates

transcription. The critical step in NF-κB activation is the phosphorylation of IκB by a large

multisubunit kinase complex consisting of IκB kinases (IKK) 1/α and 2/β as well as an

additional essential protein, NEMO/IKKγ (reviewed in (6)). NEMO represents the regulatory

component of the IKK complex whereas IKK1 and IKK2 act as catalytic subunits. Both IKKs

can phosphorylate all three IκB proteins (α, β and ε) to a similar extent. In endothelial cells

all three IκBs are degraded upon stimulation with TNF-α but with slightly different kinetics

(7-9). There have been some investigations into the role of IκBα for the function of

endothelial cells (10-12). However, the relative contribution of the different IκB isoforms to

cytokine-induced endothelial gene expression has not been studied in detail. Furthermore,

while several studies reported a role for IKK2 in TNF-α-induced endothelial NF-κB

activation (8,13,14), the relevance of IKK1 for this process has not been elucidated yet.

We therefore initiated the present study to analyze the functional role of the IKKs and IκBs

during activation of primary human endothelium. Human umbilical vein endothelial cells

(HUVEC) were infected with retroviruses that express transdominant mutants of IκBα, β, and

ε, or kinase-inactive versions of IKK1 and IKK2. We then studied TNF-α-induced

expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin) and chemokines (MCP-

1, IL-8) in the infected cells. Whereas dominant negative IKK2 and transdominant IκBα, β,

and ε mutants completely inhibited expression of these molecules, dominant negative IKK1

blocked induction to a variable extent. Importantly, a constitutively active version of IKK2

was sufficient to yield maximal induction of the various target genes demonstrating that NF-

κB is not only essential but also sufficient for expression of activation markers on endothelial

cells. Our data thus show that the IKK/IκB/NF-κB signal transduction cascade is the

dominant control mechanism for inflammatory activation of endothelial cells.

EXPERIMENTAL PROCEDURES

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Cells and cell culture. Primary human endothelial cells derived from umbilical veins

(HUVEC) were obtained from Clonetics (via Cell Systems, St. Katharinen, Germany). They

were cultured in EBM medium (Clonetics) supplemented with 2 % fetal bovine serum, 1.0

µg/ml hydrocortisone, 10 ng/ml human epidermal growth factor, 12 µg/ml bovine brain

extract, 50 µg/ml gentamicin and 50 ng/ml amphotericin B which together constituted EGM

medium. Experiments were performed with cells of passage 3. The φNX amphotropic

retrovirus producer cells (gift from G. Nolan, Stanford USA; (15)) were cultured in DMEM

(Gibco BRL, Paisley, Scotland) containing 10 % fetal calf serum (PAN Systems, Aidenbach,

Germany), 100 U/ml penicillin (Gibco BRL) and 100 µg/ml streptomycin (Gibco BRL). Cells

were stimulated with human recombinant TNF-α (R&D Systems, Wiesbaden, Germany) or

human recombinant IFN-γ (Peprotech, London, UK) as indicated.

Retroviral vectors and stable producer cell lines. The pCFG5 IEGZ retroviral vector used for

infection has been described earlier (16). All cDNAs were inserted into blunted EcoRI/BamHI

sites. Mutant IκBα was provided by Patrick Baeuerle (Micromed, Munich, Germany), mutant

IκBβ and IκBε as well as the mutant IKK1 and IKK2 cDNAs by Alain Israel (Institut Pasteur,

Paris, France). Sequences of retroviral vectors were confirmed by DNA sequencing.

φNX producer cells plated at a density of 1x106 per 10 cm plate were transfected using the

calcium phosphate precipitation method with 10 µg of plasmid DNA as described (17).

Twenty-four h later, transfection efficiencies were determined by monitoring GFP expression

by fluorescence microscopy (Improvision, Heidelberg, Germany) or flow cytometry

(FACScalibur; Becton Dickinson, Heidelberg, Germany). Transfection efficiencies usually

ranged between 70 to 80 %. Twenty-four h after transfection, 1 mg/ml zeocin (Invitrogen,

Groningen, The Netherlands) was added to the cells which were then grown in the presence of

this agent for another two weeks until all the cells were positive for GFP.

Retroviral infection of endothelial cells with supernatant from φNX producer cells. Four days

before infection, endothelial cells were seeded in six-well-plates at a density of 5x104 cells

per well in EGM medium. Twenty-four h before infection, the culture medium of the

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producer cells was changed from DMEM to EGM. At the day of infection, φNX cell

supernatant was obtained, filtered through a 0.45 µm-filter and 5 µg/ml polybrene (Sigma,

Munich, Germany) was added to the filtrate. Thereafter, EGM medium was removed from

endothelial cell cultures and replaced by φNX cell supernatant containing the retrovirus.

Culture plates were centrifuged at 1000 x g for 3 h, supernatants then removed and replaced

by conventional EGM. The infection procedure was repeated one day later to increase the

efficiency of infection, which was finally monitored by fluorescence microscopy or flow

cytometry as described above. Infection efficiencies of endothelial cells ranged between 10

and 70 % depending on the retrovirus used.

Western blot analysis and electrophoretic mobility shift assay (EMSA). Preparation of whole

cell extracts was performed as described earlier (18). For Western blot analysis, 50 µg of

protein extracts per lane were separated on 12.5 % polyacrylamide gels and transferred onto

polyvinyldifluoride membranes (Millipore, Bedford, USA). Membranes were blocked with

7.5 % dry milk in PBS containing 0.2 % Tween-20. For subsequent washes, 0.2 % Tween-20

in PBS was used. Membranes were labeled with affinity-purified rabbit antisera against IκBα

or JNK (Santa Cruz Biotechnology, Santa Cruz, USA) or phospho-JNK (New England

Biolabs, Beverly, USA). Thereafter, membranes were stained with horseradish peroxidase-

coupled secondary donkey-anti-rabbit IgG antibody (Dianova, Hamburg, Germany) which

was visualized by enhanced chemiluminescence (ECL, Pharmacia-Amersham, Freiburg,

Germany). EMSAs were performed essentially as described before (18).

Flow cytometry analysis. Chemokines MCP-1 and IL-8 were detected by an intracellular

flow cytometry staining procedure as described before (13). Endothelial cells were stimulated

with cytokine (or medium as control) for the time intervals indicated in the presence of 2 µM

monensin in order to avoid secretion via the Golgi pathway. Cells were subsequently

harvested, washed and fixed with 4 % paraformaldehyde at 4°C for 20 min. Endothelial cells

were subsequently incubated with mouse monoclonal antibodies against MCP-1, IL-8 or

isotype control antibodies (all obtained from Becton Dickinson, Heidelberg, Germany) which

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had been diluted in permeabilisation buffer (0.1 % saponin/1 % FCS/PBS). Thereafter, cells

were successively stained with biotin-SP-conjugated goat-anti-mouse IgG (Dianova,

Hamburg, Germany) and streptavidin-Cy-chrome (Becton Dickinson) and fluorescence of

induced proteins determined in the FL3 channel using a FACScalibur (Becton Dickinson).

For analysis of adhesion molecule expression on the cell surface, endothelial cells were

stimulated with cytokines as indicated, incubated with 1 % BSA in PBS to block non-specific

binding and immunostained with mouse monoclonal antibodies against E-selectin, VCAM-1

(both obtained from Dianova) or ICAM-1 (Immunotech, Marseille, France) diluted in 1 %

BSA/2 % normal goat serum/PBS. Thereafter, cells were successively stained with biotin-

SP-conjugated goat-anti-mouse IgG (Dianova) and streptavidin-Cy-chrome (Becton

Dickinson). Adhesion molecule expression of the GFP+ population as detected in the FL3

channel was determined with a FACScalibur. Nonviable cells were excluded from analysis by

means of FSC and SSC parameters. Quantification of expression levels determined in the

FACS was done by determining the geo mean in the CellQuest Software, which represents the

mean value of the area covered by the curve.

All flow cytometry experiments were repeated at least twice and revealed essentially similar results.

RESULTS

Retroviral transduction of endothelial cells with dominant-interfering mutants of the

IKK/NF-κB pathway. To study the contribution of the IKK/NF-κB signaling module to

endothelial gene expression after inflammatory activation we used retroviral gene transfer to

express dominant interfering mutants of this pathway. Negative modulation of NF-κB

signaling was achieved using transdominant mutants of different IκB proteins as well as

kinase-inactive mutants of IKK1 and IKK2. For selective activation of the IKK/NF-κB

module we used an active mutant of IKK2 to investigate whether constitutive NF-κB activity

is sufficient to drive expression of the examined target genes. Genes for the interfering

mutants were transduced by a retroviral infection approach (Fig. 1a) which results in DNA

integration and stable chromatin-embedded expression of the mutant proteins. The LTR-

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mediated expression of the inserted gene is coupled to the expression of a GFP-zeocin

resistance fusion gene through an internal ribosome entry site (IRES) (16). This allowed us to

distinguish between infected and non-infected cells by flow cytometric analysis of GFP

expression. The percentage of GFP-expressing cells was used as an indicator for transduction

efficiencies. Furthermore, this method permitted us to compare equally treated uninfected and

infected cells in the same dish regarding the effects of mutant proteins on expression of

endothelial adhesion molecules and chemokines.

Expression of transdominant mutants of the IκB proteins results in a blockade of

proinflammatory NF-κB activation in endothelial cells. To analyze whether the

transdominant IκB mutants are functional in inhibiting NF-κB activation, endothelial cells

(HUVEC) were infected with different retroviruses and stimulated with TNF-α for various

time intervals. Subsequently, expression of endogenous (wildtype) and mutant IκB proteins

was studied by Western blot analysis. Endogenous IκBα was degraded within five minutes of

TNF-α exposure and the protein completely disappeared after 30 minutes (Fig. 1b). In

contrast, the mutant protein remained stable over the whole observation period (Fig. 1c).

Endothelial cells transduced with the empty vector showed maximal NF-κB DNA-binding

activity after five minutes and remained at this level over the period investigated (Fig. 1b).

Supershift analysis revealed that these DNA-binding complexes predominantly consisted of

p50/RelA heterodimers (data not shown). Endothelial cells expressing transdominant IκBα

displayed almost no NF-κB DNA-binding activity in response to TNF-α (Fig. 1c). The

weak residual activity is most likely contributed by the uninfected cells. The same results

were obtained when transdominant IκBβ and IκBε mutants were studied (data not shown).

Thus, expression of transdominant IκB proteins results in an efficient inhibition of induced

NF-κB activation in human primary endothelial cells.

Expression of transdominant IκB mutants completely abolishes TNF-α-induced upregulation

of endothelial adhesion molecules and chemokines. To examine the effect of mutant IκB

protein expression on proinflammatory induction of endothelial proteins, endothelial cells

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were infected with retroviruses expressing transdominant IκBα or the parental vector as a

control. Cells were then exposed to TNF-α and expression of chemokines and adhesion

molecules analyzed by flow cytometry using specific antibodies.

In the representative experiment depicted in Fig. 2, about 70 % of the cells expressed GFP

indicating successful transduction of the transgenes. GFP+ and GFP- populations of the same

sample were analyzed for TNF-α-induced adhesion molecule (VCAM-1 in Fig. 2) and

chemokine expression (see below). Cells transduced with the empty vector showed a strong

upregulation of VCAM-1 in both the GFP+ and the GFP- population. A comparable

induction of VCAM-1 was detected in the GFP- population of endothelial cells which had

been treated with the transdominant IκBα virus (Fig. 2). However, there is also a population

that does not efficiently upregulate VCAM-1 in response to TNF-α. These are most likely

cells that had been infected with the virus, but which show only low GFP levels and therefore

fall in the lower gate. In contrast, VCAM-1 was not inducible in GFP+ cells, i. e. in the

successfully infected subpopulations expressing the transdominant IκBα protein. Isotype

controls have been performed for this and all the following experiments and showed that the

observed effects were specific for the mutants used. For the sake of clarity, these isotype

controls have been omitted from the figures. Similar experiments were performed regarding

TNF-α-induced upregulation of adhesion molecules ICAM-1 and E-selectin and

chemokines MCP-1 and IL-8. Expression of transdominant IκBα virtually completely

abolished inducibility of these molecules by TNF-α (Fig. 3a). Furthermore, transdominant

mutants of IκBβ and IκBε were capable of inhibiting induction of these adhesion molecules

and chemokines as well (Fig 3a). Our data indicate that activation of NF-κB is essential for

the expression of all these proteins investigated. Moreover, all three IκB mutant proteins act

as equally competent inhibitors of adhesion molecule and chemokine expression in

endothelial cells (Fig. 3a) indicating that each of the IκBs may critically contribute to the

regulation of NF-κB-mediated proinflammatory activation of endothelium.

To prove specificity of transdominant IκB-mediated inhibition of endothelial gene expression

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for the NF-κB pathway, infected endothelial cells were also stimulated with IFN-γ, a known

inducer of ICAM-1 expression in endothelial cells. IFN-γ employs the JAK/STAT rather

than the NF-κB signalosome. None of the mutant IκBs was capable of inhibiting IFN-γ-

induced expression of ICAM-1 (Fig. 3b). In addition, IFN-γ-induced upregulation of

endothelial HLA-DR was not blocked by transdominant IκB mutants either (data not shown).

These observations clearly demonstrate that there is no general signaling defect in the stably

transduced cells. Retrovirally infected endothelial cells expressing mutant IκB proteins rather

show a selective blockade of the NF-κB signaling cascade.

A kinase-deficient mutant of SEK attenuates TNF-α-induced upregulation of specific

adhesion molecules. Promoter regions of many proinflammatory cytokine-inducible genes

carry binding motifs not only for NF-κB but also for transcription factors of the activator

protein–1 (AP-1) family. Activity of several AP-1 factors such as c-Jun, ATF-2 and JunD is

regulated via phosphorylation by Jun-N-terminal kinases (JNKs). Like NF-κB, JNK is

activated by TNF-α/TNFR- signaling which activates the upstream kinase SEK which then

in turn phosphorylates JNK. To investigate the extent of SEK/JNK/AP-1 involvement in the

regulation of endothelial adhesion molecules and chemokines, endothelial cells were

retrovirally transduced with a kinase-deficient mutant of SEK. To prove the functionality of

the mutant, we first monitored the phosphorylation of JNK in response to TNF-α treatment in

the stably transfected producer cell line. In cells stably transfected with the parental vector,

JNK was phosphorylated after 10 minutes of TNF-α treatment. In contrast, cells expressing

the SEK mutant did not show measurable JNK phosphorylation in response to TNF-α (Fig.

4a). endothelial cells expressing the kinase-deficient SEK mutant were exposed to TNF-α

and expression of MCP-1, IL-8, ICAM-1, VCAM-1 and E-selectin was analyzed. In all

cases we could still see significant upregulation of the activation markers (Fig. 4b). However,

we consistently saw a weak attenuation of the upregulation of E-selectin and VCAM-1. For

the other target molecules we could not reproducibly see such a downregulation. However, in

no instance could we observe a complete inhibition of chemokine/adhesion molecule

expression as we saw it for the NF-κB inhibitors. These data indicate that JNK and AP-1

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only play a minor role with respect to the control of endothelial chemokine and adhesion

molecule expression.

IKK1 and IKK2 differentially contribute to TNF-α-induced expression of endothelial

chemokines and adhesion molecules. IKK1 and IKK2, kinases present in the IκB kinase

complex, are both capable of phosphorylating IκB proteins in response to extracellular

stimulation. So far, the relative contribution of these to TNF-α-induced endothelial gene

expression remained elusive. To address this aspect, endothelial cells were retrovirally

transduced with kinase-inactive mutants of IKK1 or IKK2 and subsequently analyzed for

TNF-α-mediated chemokine and adhesion molecule expression as described above.

Kinase-inactive IKK2 virtually completely inhibited TNF-α-induced expression of all these

endothelial molecules (Fig. 5a). In contrast, kinase-inactive IKK1 showed a more selective

effect: while induction of IL-8 and VCAM-1 was blocked by both mutant kinases to an

almost similar extent, dominant negative IKK1 only partially inhibited the synthesis of MCP-

1, ICAM-1 and E-selectin (see discussion). Paralleling the results obtained with the

transdominant IκB proteins, kinase-deficient IKK1 and IKK2 mutants did not interfere with

IFN-γ-induced ICAM-1 expression (data not shown). These data indicate that the kinase

activity of IKK2 is absolutely required for induction of all the proteins investigated whereas

IKK1 appears to be essential for only a subset of endothelial NF-κB target genes.

A constitutively active IKK2 mutant is sufficient to drive the expression of chemokines and

adhesion molecules in primary endothelial cells. Since IKK2 obviously plays an important

role in regulating endothelial gene expression, we next addressed the question whether

constitutively active IKK2 by itself is sufficient to induce expression of target genes.

Expression of an active IKK2 mutant, which is characterized by the replacement of two

serines of the activation loop by glutamic acid (19,20), in the producer cell line φNX resulted

in a strong constitutive DNA-binding of NF-κB (Fig. 6a). Although infection efficiencies of

endothelial cells infected with a retrovirus containing active IKK2 only reached around 10 %,

the transduced subset within the pool of cells analyzed by EMSA still gave rise to a

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measurable increase in NF-κB DNA-binding activity in the absence of TNF-α (Fig. 6a).

Endothelial cells expressing constitutively active IKK2 showed a pronounced up-regulation

of ICAM-1, VCAM-1, E-selectin, MCP-1 and IL-8 already in the absence of a

proinflammatory stimulus (Fig. 6b). The protein levels induced were even higher than those

observed upon exposure to TNF-α. The active IKK mutant acts specifically on NF-κB since

endothelial HLA-DR expression which is not dependent on NF-κB activity was not affected

(data not shown). These observations indicate that the IKK mutant is not a general activator of

endothelial cells but selectively targets genes which are NF-κB-dependent. Interestingly,

when cells expressing the active IKK mutant were additionally exposed to TNF-α, protein

synthesis of the above mentioned read-out molecules was not further enhanced but down-

regulated to the level of TNF-α-stimulated empty vector-infected control cells. Taken

together, our data indicate that expression of a constitutively active IKK2 mutant widely

mimics proinflammatory activation of endothelial cells since it is sufficient to drive full

expression of endothelial chemokines and adhesion molecules.

DISCUSSION

Activation of endothelium is a critical step in the onset of inflammatory responses and is

associated with the up-regulation of a variety of chemokines and adhesion molecules. Here

we have analyzed the contribution of the IKK/IκB/NF-κB signaling pathway to endothelial

protein expression upon inflammatory stimulation. We used a retroviral transduction approach

which allowed expression of dominant interfering mutants of components of the NF-κB

signaling pathway. The consequences of this modulation of NF-κB activity on the expression

of endogenous genes in endothelial cells was analyzed. With this approach we were able to

demonstrate that inhibition of NF-κB signaling by expression of transdominant mutants of

IκBα, β, and ε or a kinase-deficient form of IKK2 results in a complete blockade of TNF-α-

induced expression of chemokines MCP-1 and IL-8 and adhesion molecules ICAM-1,

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VCAM-1 and E-selectin. In contrast, a constitutively active version of IKK2 by itself was

sufficient to induce maximal expression of these genes. A dominant-negative SEK mutant

that interfered with the activation of the AP-1 transcription factor had only marginal effects

on some of the investigated molecules.

Each isoform of the IκB proteins inhibited the expression of endothelial activation markers to

a similar extent. The main dimer induced upon TNF-α treatment of endothelial cells is the

p50/RelA heterodimer ((21-23) and data not shown) for which it was reported before that it

can be efficiently inhibited by IκBα and IκBβ (24,25). Interestingly, the publications

describing the inhibition profile of IκBε were somehow contradictory with respect to the

inhibition of p50/RelA- containing complexes and it had been suggested that IκBε may play

a special role in regulating c-Rel-containing complexes (9,26-28). On the basis of inhibition

of c-Rel-containing complexes, a selective role for IκBε in ICAM-1 regulation was

proposed (9). Here we could show that IκBε was able to inhibit TNF-α-induced gene

expression as effectively as IκBα and IκBβ and in other experiments we could demonstrate

that IκBε indeed inhibits p50/RelA activated transcription (data not shown). From our results

we conclude that the expression of ICAM-1 is not exclusively regulated by NF-κB

complexes bound to IκBε, but also by complexes controlled by IκBα and IκBβ.

Our results demonstrate that the activation of NF-κB is essential for the expression of various

target genes in endothelial cells. Furthermore they show that the signal transduction cascade

critically involves the IκB-kinase complex, most notably the IKK2 kinase. During

preparation of this manuscript, Oitzinger and colleagues described the effect of a dominant-

negative IKK2 mutant on inflammatory marker genes. Consistent with our results, they found

that adenoviral expression of dn IKK2 completely inhibits the activation of endothelial cells

in response to proinflammatory cytokines (14).

While IKK2 appears to play a very prominent role for NF-κB activation and gene expression

in endothelial cells, the contribution of IKK1 is less pronounced and differs for the target

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molecules analyzed. Nevertheless, this observation is intriguing since it had been suggested

earlier from gene disruption studies and transient transfections that IKK1 is not required for

cytokine-induced NF-κB activation at all (20,29,30). Our data show that at least in

endothelial cells IKK1 contributes to the TNF-α-induced expression of some NF-κB target

genes. This might be due to the fact that IKK1 is able to phosphorylate and thereby activate

IKK2 both in unstimulated and in TNF-α-stimulated cells (31,32). A strong overexpression

of kinase-deficient IKK1 might therefore titrate the endogenous wildtype IKK1 away from

the IκB- kinase complex and thereby inhibit this phosphorylation. The difference among the

various NF-κB target genes might be due to different dependencies of these molecules on

IKK1 kinase activity.

An important outcome of our experiments was the finding that activation of NF-κB alone (by

means of the constitutively active IKK2) was sufficient for full induction of endothelial

effector proteins in the absence of additional proinflammatory stimuli. Thus, activation of

NF-κB via IKK2 is both essential and sufficient for endothelial chemokine and adhesion

molecule expression. Interestingly, expression of the various target genes was even higher in

cells expressing the constitutively active IKK2 protein as compared to TNF-α-induced cells.

When cells expressing constitutively active IKK2 were further stimulated with TNF-α, the

expression of chemokines and adhesion molecules was attenuated. A potential explanation for

this observation is that TNF-α induces some modulators of the transcriptional activity of NF-

κB in addition to stimulating NF-κB activity. One candidate for such a modulator is A20, an

anti-apoptotic protein which is induced by TNF-α and whose expression depends on NF-κB

(33-35). Furthermore, it was shown that A20 can inhibit NF-κB and thereby also endothelial

activation (36,37). The exact mechanism of A20 function is not clear yet. Recently it was

shown that A20 interacts with the IKK complex upon TNF receptor stimulation (38). Thereby

it could act directly on the IκB kinases. In addition to A20, other anti-apoptotic proteins like

A1, Bcl-2 and Bcl-XL have been described as inhibitors of NF-κB and endothelial

activation (39,40). TNF-α by stimulating pathways in addition to NF-κB may increase the

expression level of these proteins and thereby attenuate the NF-κB response. Such a

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desensitizing signal may help to protect the cell from a signaling overflow and may be part of

a switch-off mechanism once the positive signal has fulfilled its task. Another possibility for

downregulation of target gene expression by TNF-α could be that TNF-α induces other

transcription factors in addition to NF-κB which titrate away cofactors which are necessary

for NF-κB transcriptional activation. Such a competitive interaction was recently shown for

NF-κB and Smad proteins activated by TGF-β. When endothelial cells were treated with

TGF-β or transfected with Smad expression vectors in addition to IL-1β stimulation, E-

selectin gene expression was inhibited because the coactivator CBP was titrated away from

the NF-κB dimers (41). A similar mechanism could also be initiated by TNF-α in a situation

where NF-κB activation is already maximal.

It was reported earlier that signal transduction cascades apart from the NF-κB pathway such

as the JNK/SAPK mitogen-activated protein kinase pathway initiated by TNF-α are

important for induction of endothelial genes, e. g. for expression of E-selectin (42,43). By

expressing a dominant negative mutant of SEK, the upstream activator of JNK, we observed

only marginal changes in induced expression of E-selectin and VCAM-1, and no

reproducible effects on ICAM-1, MCP-1 and IL-8 although the kinase-deficient mutant

efficiently blocked TNF-α-induced JNK activation. Thus, the JNK pathway appears to play a

modulating rather than an essential role in regulating endothelial gene expression upon

inflammatory activation

Acknowledgements:

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LEGENDS TO FIGURES

Figure 1

Effect of retrovirally expressed transdominant IκBα mutant on TNF-α-induced IκBα degradation

and NF-κB activation. (A) Schematic representation of the retrovirus used for the expression

of IκB, IKK and SEK mutants.

Endothelial cells were infected with parental vector (B) or a retrovirus expressing

transdominant IκBα (C) as described in Experimental Procedures. Twenty-four h after

infection, endothelial cells were stimulated with 2 ng/ml TNF-α for the time intervals

indicated. Whole cell lysates were obtained and IκBα degradation visualized by Western blot

analysis (upper panels). Protein bands of endogenous and transdominant (TD) IκBα are

indicated. TNF-α-induced NF-κB DNA-binding activity was studied by electrophoretic

mobility shift assay (EMSA; lower panels). Cells extracts were prepared and processed as

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described in Experimental Procedures. NF-κB complexes are indicated.

Figure 2

Expression of transdominant IκBα inhibits TNF-α-induced up-regulation of VCAM-1.

Endothelial cells infected with parental vector as control (left panels) or a retrovirus carrying a

transdominant mutant of IκBα (right panels) were exposed to 2 ng/ml TNF-α 24 h after

infection for another 16 h. Cells were then harvested, immunostained for VCAM-1 surface

expression and processed for flow cytometry as described in Experimental Procedures.

Successfully infected cells, i. e. cells expressing the IκBα mutant, are labeled by co-

expressed green fluorescent protein (GFP; upper panel). The flow cytometric profiles in the

middle and lower panels show endothelial expression of VCAM-1 after exposure to TNF-α

(green line) or to medium as control (black line). The middle panel shows VCAM-1

expression in the uninfected, GFP- population , whereas the lower panel shows VCAM-1

expression in the infected, GFP+ population.

Figure 3

Transdominant IκB proteins block expression of TNF-α-, but not of IFN-γ-inducible target

genes in endothelium. Endothelial cells were infected with retroviruses carrying

transdominant mutants of IκBα, IκBβ or IκBε or empty control vector as indicated. Twenty-

four h after infection, cells were stimulated with (A) 2 ng/ml TNF-α for 6 h (E-selectin) or

16 h (MCP-1, IL-8, ICAM-1, VCAM-1), or, (B), 25 ng/ml IFN-γ for 24 h, respectively.

Endothelial cells were then processed for flow cytometry and intracellular expression of

chemokines and surface expression of adhesion molecules determined as described in

Experimental Procedures. For the sake of clearness, only profiles of the GFP+ populations are

depicted. Expression of chemokines or adhesion molecules is shown after stimulation with

(A) TNF-α or (B) IFN-γ (green lines) or medium as control (black lines) as indicated.

Figure 4

A kinase-deficient mutant of SEK inhibits agonist-induced phosphorylation of JNK but fails

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to block up-regulation of endothelial chemokines and adhesion molecules. (A) φNX cells

stably expressing a kinase-deficient SEK mutant were stimulated with 80 ng/ml TNF-α for

10 minutes. Whole cell lysates were obtained and studied for phosphorylation of JNK by

Western blot using a phosphospecific antibody. Loading controls were performed with

antibodies against non-phosphorylated JNK as described in Experimental Procedures. (B)

Endothelial cells stably expressing a kinase-deficient mutant of SEK or appropriate controls

were exposed to 2 ng/ml TNF-α (green profile lines) or medium (black profile lines) for 6 h

(E-selectin) or 16 h (MCP-1, IL-8, ICAM-1, VCAM-1) and expression of chemokines and

adhesion molecules was evaluated by flow cytometry as described in Experimental

Procedures. Only the GFP+ population is considered for analysis. Quantitative evaluation of

the expression levels was performed by analyzing the mean of the area covered by the curve

(i. e. geo mean in CellQuest software).

Figure 5

Dominant-negative mutants of IKK1 and IKK2 inhibit TNF-α-induced up-regulation of

chemokines and adhesion molecules in endothelial cells. Endothelial cells expressing

dominant-negative IKK1 (i. e., IKK1 KD), IKK2 (i. e., IKK2 KD) or empty vector were

stimulated with 2 ng/ml TNF-α for 6 h (E-selectin) or 16 h (MCP-1, IL-8, ICAM-1,

VCAM-1) and studied for expression of chemokines and adhesion molecules. Only the GFP+

population is evaluated with the green profile lines showing TNF-α-exposed cells and the

black profile lines representing cells exposed to medium as control.

Figure 6

Expression of a constitutively active mutant of IKK2 is sufficient to activate NF-κB and to

drive the expression of chemokines and adhesion molecules in endothelial cells. Endothelial

cells were infected with a retrovirus carrying a constitutively active IKK2 mutant the parental

vector. Subsequently, endothelial cells were exposed to 2 ng/ml TNF-α (φNX producer cells

to 80 ng/ml TNF-α) or medium as control for 15 min (A). Whole cell lysates were obtained

and studied for NF-κB DNA-binding activity by EMSA as described in Experimental

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Procedures. (B) Endothelial cells expressing constitutively active IKK2 or controls were

exposed to 2 ng/ml TNF-α (green profile lines) or medium as control (black profile lines) and

evaluated for expression of chemokines and adhesion molecules by flow cytometry as

described in Experimental procedures. Only the GFP+ population is considered for analysis.

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Lindemann, Stephan Ludwig and Thomas WirthAndrea Denk, Matthias Goebeler, Sybille Schmid, Ingolf Berberich, Olga Ritz, Dirk

proinflammatory gene expression in primary endothelial cellsActivation of NF-kB via the IkB kinase complex is both essential and sufficient for

published online May 3, 2001J. Biol. Chem. 

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