Multiple control of interleukin-8 gene expression
Elke Hoffmann, Oliver Dittrich-Breiholz, Helmut Holtmann, and Michael Kracht
Institute of Pharmacology, Medical School Hannover, Germany
Abstract: Interleukin (IL)-8, a prototypic human
chemokine, was detected more than a decade ago
as the founding member of the chemokine superfamily. One of the most remarkable properties of
IL-8 is the variation of its expression levels. In
healthy tissues, IL-8 is barely detectable, but it is
rapidly induced by ten- to 100-fold in response to
proinflammatory cytokines such as tumor necrosis
factor or IL-1, bacterial or viral products, and
cellular stress. Recently, significant advances in the
understanding of signaling pathways, which coordinately regulate IL-8 transcription as well as
mRNA stabilization in response to external stimuli,
have been made. Maximal IL-8 amounts are generated by a combination of three different mechanisms: first, derepression of the gene promoter;
second, transcriptional activation of the gene by
nuclear factor-␬B and JUN-N-terminal protein kinase pathways; and third, stabilization of the
mRNA by the p38 mitogen-activated protein kinase pathway. In that way, cells are able to rapidly
increase and at the same time, to fine-tune the
amount of IL-8 secreted and thereby control the
extent of leukocytes attracted to sites of tissue
injury. J. Leukoc. Biol. 72: 847– 855; 2002.
Key Words: MAPK 䡠 NF-␬B 䡠 interleukin-8 䡠 transcription 䡠 mRNA
stability
INTRODUCTION
Infection or injury of the body results in inflammation. A
hallmark of this response is the recruitment of neutrophils from
the blood to the injured tissue. This process is directed by
chemotactic polypeptides of 8 –14 kD so-called chemokines.
About 40 human chemokines are known today [1]. Interleukin
(IL)-8 was detected more than a decade ago as the founding
member of this superfamily [2]. Many of its properties, including its three-dimensional structure, receptors, signaling mechanisms, and additional functions in angiogenesis, tumor progression, mitosis, and tissue remodeling, are well-known [2].
One of the most remarkable properties of IL-8 is the variation
of its expression levels. Normally, IL-8 protein is barely secreted from noninduced cells, but its production is rapidly
induced by a very wide range of stimuli encompassing proinflammatory cytokines such as tumor necrosis factor (TNF) or
IL-1 [3, 4], bacterial [5, 6] or viral products [7, 8], and cellular
stress [9 –12]. Remarkably, some stimuli, such as IL-1 or TNF,
up-regulate IL-8 by more than 100-fold [3, 4, 8, 12], whereas
others, such as certain bacteria or epidermal growth factor
(EGF), cause a more moderate five- to tenfold increase in IL-8
secretion [5, 6, 13]. Furthermore, IL-8 can be synthesized by
many different cell types (Table 1).
This raises the question of by which intracellular pathways
cells regulate the extent as well as the duration of IL-8 gene
expression. The aim of this review is to highlight recent advances in understanding of the signaling mechanisms involved
in IL-8 expression.
TRANSCRIPTIONAL REGULATION OF IL-8
Stimulus-dependent activation of IL-8 gene transcription has
been demonstrated in nuclear run-on experiments [3, 4]. In a
number of studies, it was found that a sequence spanning
nucleotides ⫺1 to ⫺133 within the 5⬘ flanking region of the
IL-8 gene is essential and sufficient for transcriptional regulation of the gene (refs. [55, 56] and reviewed in ref. [57]). As
demonstrated by mutational and deletional analyses, this promotor element contains a nuclear factor (NF)-␬B element that
is required for activation in all cell types studied. NF-␬B is a
dimeric transcription factor composed of a family of five subunits, namely NF-␬B1 (p50 and its precursor p105), NF-␬B2
(p52 and its precursor p100), and c-REL, REL A (p65), and
REL B [58]. NF-␬B is retained in the cytoplasm by its binding
to inhibitory (I␬B) proteins. I␬B phosphorylation results in
ubiquitination and rapid degradation of I␬Bs by the proteasome, allowing NF-␬B to translocate to the nucleus and bind to
DNA. This process is critical for NF-␬B activation, but enhanced NF-␬B-induced transcriptional activity might additionally require phosphorylation of the subunits as well as binding
of coactivators [58, 59]. By using chromatin immunoprecipitation, binding of p65 NF-␬B to the endogenous IL-8 promoter
and subsequent recruitment of RNA polymerase II are found
rapidly, within one-half hour of IL-1 stimulation, underscoring
the important role of NF-␬B in IL-8 transcription (Fig. 1).
The core IL-8 promoter also contains activating protein
(AP)-1- and CAAT/enhancer-binding protein (C/EBP)-binding
sites (Fig. 2). The latter two sites are dispensable for transcriptional activation in some cells, but contribute to activation
in others. Thus, unlike the NF-␬B site, the AP-1 and C/EBP
sites are not essential for induction but are required for maximal gene expression [3– 8, 10, 57, 60, 65– 67].
Correspondence: Dr. Michael Kracht, Institute of Pharmacology, Medical
School Hannover, Carl-Neuberg Strasse 1, D-30625 Hannover, Germany.
E-mail: [email protected]
Received March 25, 2002; accepted April 23, 2002.
Journal of Leukocyte Biology Volume 72, November 2002 847
TABLE 1.
Evidence by Inhibition or Activation for the Involvement of Signalling Pathways in Regulation of IL-8 Expression
Abbreviations: a.s., Antisense; d.n., dominant negative; T, transcriptional; P, posttranscriptional.
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http://www.jleukbio.org
Fig. 1. IL-1 rapidly stimulates recruitment
of p65 NF-␬B and RNA polymerase II to
the endogenous IL-8 promoter. HeLa cells
were stimulated for 30 min with 10 ng/ml
IL-1␣ or left untreated. Protein-DNA complexes were cross-linked in intact cells by
formaldehyde treatment. Cells were lysed,
and p65 NF-␬B (A) or RNA polymerase II
(Pol-II; B) was immunoprecipitated with
specific antibodies. IL-8 promoter DNA bound to immune complexes was recovered after reversal of cross-links and was detected by polymerase chain reaction
(PCR). Shown are PCR products amplified with specific primers, which cover the IL-8 promoter region containing the NF-␬B-binding site and the tata box (as shown
in Fig. 2) or as a control, a region more than 800 bp 5⬘ in the IL-8 gene. As additional controls, PCR products were amplified with the same primer pairs from
increasing amounts of genomic DNA (Input).
The transcriptional regulator AP-1 is a homo- or heterodimer
composed of c-JUN, JUN D, JUN B, ATF-2, c-FOS, FRA-1,
FRA-2, and other family members [68, 69]. In contrast to
NF-␬B, AP-1 proteins are usually constitutively bound to their
cognate DNA element. Transcriptional activity of AP-1 proteins is regulated by their abundance, phosphorylation of transactivation domains, and by their binding to protein kinases
[68, 69].
It is important to note that in contrast to NF-␬B, whose p65
subunit binding to the IL-8 promoter has been analyzed at the
atomic level [62], the composition of the endogenous AP-1
dimer as a function of time and stimulus that modulates IL-8
transcription has not been determined.
Fig. 2. Regulation of basal and inducible IL-8 transcription. A part of the
IL-8 promoter, proximal, regulatory region is illustrated. Identified binding
sites for transcription factors are shaded [45, 57, 60]. In unstimulated cells, the
IL-8 promoter is repressed by three mechanisms: first, by binding of the
NF-␬B-repressing factor (NRF) to the negative regulatory element (NRE) that
overlaps the NF-␬B site (solid line) [45]; second, by binding of octamer-1
(OCT-1) to an octamer-binding site located on the complementary strand in the
opposite direction of the C/EBP site (dotted line) [67]; and third, by deacetylation of histone proteins by histone deacetylase 1 (HDAC-1) [61]. Upon
induction by IL-1␣ or TNF-␣, the p65 subunit of NF-␬B translocates to the
nucleus and binds to its site in close proximity to NRF [45, 62, 63]. OCT-1 is
replaced by C/EBP ␤ [63, 67], whereas NRF switches its function to act as a
coactivator [45]. Recruitment of CREB-binding protein (CBP)/p300 results in
hyperacetylation of histones and chromatin remodeling [64]. As a result,
repression of the promoter is relieved. AP-1 and NF-␬B proteins become
phosphorylated in a signal-dependent manner (see Fig. 3). Collectively, these
events facilitate initiation of transcription.
Little is known about signaling pathways regulating C/EBPs,
including the family member C/EBP-␤ (also called NF-IL-6),
which was found to bind to the IL-8 promoter [60, 65– 67].
Therefore, in this review, we will focus on the role of pathways
targeting NF-␬B and AP-1 in IL-8 gene expression.
THE ROLE OF PROTEIN KINASE PATHWAYS
REGULATING NF-␬B AND AP-1 IN IL-8
TRANSCRIPTION
Virtually all stressful and proinflammatory stimuli known to
induce IL-8 production activate a number of protein kinases,
which in principal have the capacity to modulate NF-␬B or
AP-1 activity.
The rate-limiting step in NF-␬B activation—I␬B proteolysis—is regulated by recently identified I␬B kinases (IKK␣/␤
and IKK␥/Nemo), which specifically phosphorylate two adjacent serines in I␬B proteins [70]. In contrast, the pathway(s)
regulating NF-␬B transactivation, rather than translocation, are
less well-defined [59].
AP-1 is activated by mitogen-activated protein kinases
(MAPK). Three MAPK pathways contribute to IL-8 gene expression, the extracellular-regulated protein kinase (ERK),
JUN-N-terminal protein kinase (JNK), and p38 MAPK cascades. There are two closely related ERK (1 and 2), three JNK
whose differential splicing gives rise to 10 isoforms, and four
p38 MAPK isoforms [68, 69, 71, 72]. All of them are activated
by phosphorylation through dual-specificity MAPK kinases
(MKK). ERK are activated by MKK1 or 2, and JNK are
activated by MKK7, whereas p38 MAPK are activated by
MKK3 and 6. Additionally MKK4 activates JNK and p38
MAPK. MKK require phosphorylation in the conserved kinase
domain VIII for activation by upstream kinases (see below).
Several laboratories have used inactive kinase mutants, antisense RNA, or specific inhibitors to block these pathways at
various levels in intact human cells to study their contribution
to IL-8 gene expression (see Table 1). No mouse homologue of
IL-8 is known today [1], preventing the analysis of IL-8 expression in mice deficient for any of these signaling molecules.
However, information for regulation of IL-6, a cytokine whose
expression in many respects is regulated very similar to IL-8
[44, 59, 64], is available from mice deficient in individual
components of the pathways activating NF-␬B and AP-1.
As expected from the essential requirement of the NF-␬B
cis-element for IL-8 transcription, inhibition of NF-␬B by
Hoffmann et al. IL-8 gene expression
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dominant-negative mutants of IKK␤ [42] or I␬B [40, 41] or by
antisense to NF-␬B [37–39] strongly impairs IL-1- and TNFinduced transcriptional expression of IL-8 (see also Table 1).
In line with the importance of NF-␬B for inflammatory gene
expression, inducible IL-6 expression is lost in IKK␤- and
IKK␥-deficient mice [73, 74].
Compared with NF-␬B, the role of JNK in IL-8 regulation is
less widely explored. However, JNK, like NF-␬B, are strongly
activated under most conditions that induce IL-8. The observation that JNK are in fact essential for IL-8 (and IL-6)
expression was made initially in human epithelial cells by
blocking JNK activation by stable expression of antisense RNA
or by dominant-negative mutants [44]. The JNK inhibition was
specific and did not affect NF-␬B or p38 MAPK activation
[44]. The absolute requirement of JNK for IL-6 expression was
later confirmed in JNK2⫺/⫺ fibroblasts [73]. Recently, a
reversible ATP-competitive inhibitor, the anthrapyrazolone
SP600125, was developed, which preferentially inhibits JNK
in vitro (Ki 0.19␮M) and at concentrations above 5 ␮M in vivo.
However, at higher concentrations (50 ␮M), it also inhibited
p38 MAPK [75]. In contrast to the findings above, the inhibitor
had no effect on IL-8 expression in lipopolysaccharide (LPS)stimulated human monocytes [75]. Therefore, it remains to be
seen if it affects IL-8 expression in other systems.
JNK are the only c-JUN kinases identified to date. Therefore, they are generally thought to activate inflammatory genes
via c-JUN and the AP-1 cis-element [76, 77]. However, adenoviral expression of a dominant-negative mutant of c-JUN, in
which the transactivation domain was deleted, failed to inhibit
IL-8 induction in one report [40], but this mutant did affect
IL-8 expression in another study [46]. These findings are
consistent with the known variable contribution of the AP-1
site to IL-8 transcription and may point to a less significant role
for c-JUN in IL-8 regulation in some conditions [57].
Taken together, available data suggest that the NF-␬B and
JNK pathways are indispensable for inducible IL-8 regulation.
Blockade of one pathway in the presence of normal activation
of the other drastically reduces IL-8 secretion. To further
elucidate the individual contribution of NF-␬B and JNK (and
also p38; see below), they were activated in the absence of
extracellular stimulation. This was achieved by transient expression of constitutively active protein kinase mutants. Although nonphysiological, this approach allows selective activation of individual pathways alone or in combination. Constitutively active MKK7, a specific activator of the JNK pathway,
or NF-␬B-inducing kinase (NIK), a specific upstream activator
of NF-␬B, induced low IL-8 synthesis and transcription from a
minimal IL-8 promotor [13]. However, MKK7 synergized in
both effects with NIK. Activation of the IL-8 promotor by either
of the kinases required functional NF-␬B and AP-1 sites [13].
What might be the significance of this pathway cooperativity? The cis-elements for NF-␬B, AP-1, and other transcription
factors are located in close proximity within less than 200 base
pairs of the IL-8 promoter (refs. [45, 57] and Fig. 2). This
suggests the formation of higher-order nucleoprotein complexes. Such a complex, termed transcriptional enhanceosome,
has been analyzed in detail for the virus-inducible cytokine
interferon (IFN)-␤ [78, 79]. The purpose of the enhanceosome
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is to provide a multiprotein surface that makes optimal contact
with the proteins of the basal transcriptional machinery and
thus facilitates maximal gene transcription [80]. Contacts between the proteins are enabled or improved by post-translational modifications, chromatin-remodeling via histone acetylation or histone phosphorylation [81], or large coactivator
proteins such as p300/CBP [82]. The latter provide multidocking platforms and possess intrinsic acetyltransferase activities [83].
Indeed, recent observations indicate that enhanceosomelike structures modulate IL-8 expression. CBP/p300 potentiates p65-mediated IL-8 transcription in an acetylation-dependent manner [64], whereas the HDAC-1 negatively affects IL-8
promoter activity [61]. Using the chromatin immunoprecipitation technique, binding of p65 NF-␬B and the formation of a
TNF-stimulated, preinitiation complex at the IL-8 promoter
containing inducibly phosphorylated RNA polymerase II were
recently demonstrated [84].
THE NUCLEAR PROTEIN NRF IS REQUIRED
FOR BASAL AND INDUCIBLE IL-8
TRANSCRIPTION
The conclusion that IL-8 transcription is regulated by a multiprotein complex is also supported by the recent identification
of a signal-responsive cis-element that overlaps with the NF␬B-binding site.
IL-8 expression is low in unstimulated cells. This is partly a
result of transcriptional repression of the promoter. By sequence comparison with the IFN-␤ promoter, a negative regulatory element (NRE) was found in the IL-8 promoter overlapping partially with the NF-␬B response element [85].
NF-␬B repressing factor (NRF) binds to the IL-8-promoter
NF-␬B-NRE element [45, 86]. Reduction of cellular NRF by
expressing NRF-antisense RNA results in spontaneous IL-8
gene expression. In contrast, IL-1-induced IL-8 secretion is
strongly impaired by expressing NRF-antisense RNA. Mutation of the NRE site results in loss of NRF binding and
increased basal IL-8 transcription. Conversely, IL-1-induced
IL-8 transcription is decreased by mutating the NRE element.
These data provide evidence for a dual role of the NRF in IL-8
transcription. Although in the absence of stimulation, it is
involved in transcriptional silencing, in IL-1-induced cells, it
is required for full induction of the IL-8 promoter [45].
The mechanism by which NRF contributes to IL-1-induced
IL-8-promoter activation remains elusive. Overexpressed NRF
alone did not enhance NF-␬B-mediated IL-8 transcription.
However, when p65 NF-␬B was expressed together with active
nuclear JNK, cotransfected NRF enhanced IL-8 transcription
further. These results suggest that NRF is modified itself by a
JNK-dependent mechanism or that it is interacting on the IL-8
promoter with kinase-activated transcription factor(s). NRF
coactivation required not only an intact NRE site but also
binding of NF-␬B and AP-1 proteins to their cis-elements [45].
This suggests that the binding of all three transcription factors
to their DNA binding sites and their physical interaction is
needed for maximal induction of the IL-8 promoter. The NRE
overlaps with the NF-␬B site, and indeed, it was shown prehttp://www.jleukbio.org
viously that NRF is able to interact directly with members of
the rel family [86]. NF-␬B is modified by protein kinases
[87– 89], interacts with various proteins that enhance its transcriptional activity [63, 90 –92], and contacts through its Cterminus components of the basal transcriptional machinery
[93]. Thus, it is possible that in IL-1-stimulated cells, NRF
enhances NF-␬B activity in concert with AP-1 by acting on one
or more of these mechanisms. The combination of NF-␬B and
NRE sites is conserved in a variety of genes relevant to
inflammation [85, 86, 94]. The NRE element does not overlap
the NF-␬B site in all cases. However, it was shown previously
that the inhibitory effect of NRE on various NF-␬B elements is
exhibited over distances up to 2.5 kbp [86]. Thus, it is tempting
to speculate that NRF constitutes a major transcription factor,
which prevents uncontrolled expression of proinflammatory
proteins and contributes to their effective synthesis during
disease.
Another mechanism for repression of the IL-8 gene has been
described earlier [67]. In this model, transcription of the IL-8
promoter is induced by replacing the repressor OCT-1 with
NF-␬B and C/EBP as a consequence of IL-1 stimulation [67].
In contrast to this competitive mechanism, NRF represses the
IL-8 promoter, most likely by a noncompetitive mechanism, as
it is not replaced by NF-␬B after stimulation by IL-1 (Fig. 2),
and as outlined above, it alters its function and is required for
maximal promoter activity during stimulation.
As summarized in Figure 2, these findings are best reconciled with a model, whereby IL-8 transcription is effectively
repressed in unstimulated cells by a combination of three
mechanisms involving deacetylation of histones [61, 95],
OCT-1 binding [67], and active repression by NRF [45].
Induction of strong IL-8 transcription by the cytokines IL-1
and TNF requires the actively promoted formation of an enhanceosome-like structure at the promoter by at least two
signals, one provided by nuclear translocation of NF-␬B and
the other by activation of the JNK pathway. Presumably,
through these pathways, the enhanceosome integrates various
external signals by a combinatorial usage of a limited set of
activators and thereby is able to regulate the magnitude of IL-8
gene transcription (see Fig. 4). Further insight into this mechanism requires the identification of all proteins involved, including the target(s) of JNK.
POST-TRANSCRIPTIONAL REGULATION OF
IL-8 BY THE P38 MAPK PATHWAY
Inhibition of p38 MAPK by the pyridylimidazole analogues
SB203580 or 202190 suppresses induction of IL-8 in some
cells (Table 1) but not in others [96]. Moreover, inhibition of
IL-1- and TNF-induced IL-8 secretion by SB compounds is
only partial [27]. Care should be taken in interpreting many of
the data obtained with the SB inhibitors, as at concentrations
above 2 ␮M, they will also inhibit kinases other than p38 [97].
However, available data with low concentrations of the drugs
suggest that the p38 pathway significantly contributes to IL-8
expression but unlike NF-␬B, is not essential [27, 29]. This
interpretation is supported by observations made in MKK3- or
p38␣-deficient mice. In these animals, the expression of IL-6
is still inducible by IL-1 or TNF, although to a much lesser
extent than in wild-type mice [98, 99].
These findings may be explained by the observation that
the p38 MAPK pathway regulates a specific, post-transcriptional step in IL-8 gene expression. The very low amount of
IL-8 found in unstimulated cells is not only a result of
repressed transcription but also the result of a very unstable
mRNA. The rapid adenine and uracil degradation of the
IL-8 mRNA is mediated by absorbance unit (AU)-rich ciselements (ARE) contained in its 3⬘ untranslated region. It is
interesting that this part of the mRNA is also required for
signal-mediated stabilization of the IL-8 transcript. By measuring IL-8 mRNA stability with tetracycline-regulatable
reporter gene constructs [13, 31, 43], it was recently shown
that activation of the p38 MAPK pathway stabilizes the IL-8
mRNA. Specifically, these data showed that an active form
of MKK6, which selectively activates p38 MAPK, induced
marked stabilization of the IL-8 transcript. In addition, an
active form of the protein kinase MAPK-activated protein
kinase-2 (MK2), a downstream substrate of the MKK6-p38
MAPK pathway, also induced mRNA stabilization, whereas
kinase-negative mutants of p38 MAPK or of MK2 interfered
with MKK6-induced stabilization. Furthermore, a dominantnegative mutant of p38 MAPK interfered with mitogenactivated ERK kinase kinase 1 (MEKK1) as well as with
IL-1-induced stabilization [13, 31, 43]. NIK and MKK7,
which as outlined above, cooperatively induce IL-8 transcription, did not affect degradation of IL-8 mRNA. However, they synergized with MKK6 in induction of IL-8
protein [13].
Collectively, these findings indicate that the p38 MAPK
pathway contributes to cytokine/stress-induced IL-8 gene expression by stabilizing mRNAs through an MK2-dependent,
ARE-targeted mechanism (Figs. 3 and 4).
REGULATION OF IL-8 EXPRESSION BY THE
ERK PATHWAY
Although, as reported so far, the role of the NF-␬B, JNK, and
p38 pathways in IL-8 gene regulation has been analyzed in
detail, information about the role of other signaling molecules
(MKK1) is very limited (see Table 1). Based on use of the
MEK1 inhibitors PD9805 and U0126, there is some evidence
that the ERK pathway contributes to IL-8 expression (Table 1).
We found that EGF, a physiological activator of ERK, weakly
induces IL-8 in a JNK- and NF-␬B-independent manner [13].
Furthermore, expression of a constitutively active mutant of
MEK1 causes some IL-8 transcription but fails to induce
significant IL-8 protein (M. Kracht and E. Hoffmann, unpublished observations). These data suggest that the ERK pathway
on its own is not a very potent inducer of IL-8 but has the
potential to contribute to IL-8 induction stimulated by NF-␬B
and other pathways.
Hoffmann et al. IL-8 gene expression
851
Fig. 3. Essential signal transduction steps in cytokine-mediated IL-8 gene regulation. In unstimulated
cells, the NRF prevents IL-8 transcription [45]. IL-1 or
TNF-␣ binding to the cell surface results in formation
of multimeric receptor complexes that recruit the adaptor proteins TRAF6 or TRAF2, respectively [32, 100,
101]. TRAF oligomerization triggers activation of MAPKKK, such as TAK1, NIK, or MEKK1 by unknown
mechanisms [32, 100, 101]. TAK1 or MEKK1 activates
the MKK7-JNK and the IKK-␤/-␥ pathways [13, 43,
45, 73, 74]. The direct targets of JNK as well as the
proteins binding to the AP-1 site have not been identified, whereas IKK phosphorylates I␬B, allowing release of NF-␬B [70]. The p65 subunit of NF-␬B translocates to the nucleus and binds to the NF-␬B site of
the IL-8 promoter [62, 45]. There, it interacts with
constitutively bound NRF and AP-1 transcription factors [13, 45]. Post-translational modifications, such as
phosphorylation of the transactivation domains of AP-1
[69] and NF-␬B [59], coactivator (CBP/p300) recruitment [64], and histone phosphorylation or acetylation
[61, 64, 95], result in chromatin remodeling and strong
IL-8 transcription. The cis-elements for AP-1, NF-␬B,
or NRF cannot be altered without decreasing JNK or
NF-␬B-mediated IL-8 trancription [13, 45], favoring a
model where all the proteins involved in transcription
interact to form a multiprotein complex. This enhanceosome-like structure favors maximal contact with the
RNA polymerase II holoenzyme, which itself becomes
phosphorylated [84]. The newly synthesized transcript
is then rapidly stabilized by the p38 MAPK pathway,
which targets ARE in the IL-8 mRNA through an
unknown mechanism that may involve proteins binding
to the ARE [e.g., AU-binding factor 1 (AUF1) or embryonic lethal abnormal vision-like RNA-binding protein HuR] [31]. Essential steps in signal transmission
and gene expression are shaded in gray. eIF, eukaryotic
initiation factor; P, phosphorylation; PABP, polyAbinding protein.
COORDINATED ACTIVATION OF THE NF-␬B,
JNK, AND P38 MAPK PATHWAYS BY
MAPKK KINASES (MAPKKK) STRONGLY
INDUCES IL-8
The data discussed so far support a model where significant
IL-8 expression requires activation of NF-␬B and at least one
or two MAPK pathways. This raises the question of how cytokine receptors coordinate this activation.
MAPKKK comprise a remarkably large group of distinct
enzymes, namely Raf-1, A-Raf, B-Raf, Mos, NIK, MEKK1– 4,
MLK2, MLK3, DLK/MUK, ASK1, Tpl-2/Cot, and transforming
growth factor-␤-activated protein kinase 1 (TAK1). Although
structurally quite different, MAPKKK phosphorylate and activate MAPKK and thereby activate MAPK. The JNK and p38
MAPK signaling pathways can be activated by ectopically
expressed MEKK1– 4, MLK2, MLK3, DLK/MUK, ASK1, Tpl2/Cot, and TAK1, albeit to varying degrees [102].
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IKK are activated by phosphorylation of serine 176 and 180
in the T-loop of the kinase domain [103]. This motif resembles
the regulatory sequence in MAPKK, which is phosphorylated
by MAPKKK. In agreement with that observation, an increasing number of MAPKKK have been shown recently to phosphorylate IKK and activate NF-␬B. Of those, MEKK1, 2, and
3, Tpl-2/Cot, and TAK1 also activate JNK and p38 MAPK, and
NIK is the only NF-␬B-specific MAPKKK [13, 31, 43, 45,
100 –102, 104]. Thus, apparently some MAPKKK, like cellular
stress or the proinflammatory cytokines IL-1 or TNF, can
trigger two or three stress-kinase pathways simultaneously.
However, knowledge is still limited as to which of the many
MAPKKK link these pathways to the receptors for physiological inducers of IL-8, such as IL-1 or TNF [104].
It is interesting that the MAPKKK, MEKK1, and TAK1,
which can activate NF-␬B, JNK, and p38 MAPK, very potently
induce IL-8 formation [13, 43, 45]. Furthermore, both kinases
are activated by oligomerized adaptor proteins, TNF-receptorhttp://www.jleukbio.org
tantly, this three-pathway model of IL-8 induction is operative
in response to a physiological stimulus IL-1, and the MAPKKK
TAK1 plays a central and nonredundant role in coupling the
IL-1 receptor to transcriptional and RNA-targeted mechanisms
mediated by the three pathways [13, 31, 43, 45].
CONCLUSIONS
Fig. 4. Quantitative control of IL-8 synthesis by cooperation of at least three
signaling pathways. Virtually no IL-8 synthesis is detectable in most cells or
tissues as a result of transcriptional repression and destabilization of its
mRNA. Activation of NF-␬B alone is essential for transcription of the IL-8
gene but like activation of JNK, results in only low secretion of IL-8. Activation
of NF-␬B and JNK (and ERK or other pathways?) results in moderate IL-8
mRNA synthesis and secretion. When a third signal by the p38 MAPK
pathway is provided, the transcribed RNA is rapidly stabilized, and high
amounts of the protein are produced [13].
associated factors (TRAF)-2 or -6, respectively. TRAFs link
the IL-1 or TNF receptors to cytosolic signaling pathways by,
so far, poorly characterized mechanisms [32, 100].
Evidence for a central role of the TAK1 protein kinase in
IL-8 expression was found recently. Expression of a kinaseinactive mutant of TAK1 largely blocked IL-1- or TNF-induced
transcription and mRNA stabilization (ref. [43], and M. Kracht
and J. Enninga, unpublished observations).
Activation of TAK1 is crucially dependent on interaction
with the adaptor protein TAB1 [100]. A truncated version of
TAB1, lacking the TAK1-binding domain, or a TAK1-derived
peptide containing a TAK1-autoinhibitory domain was also
efficient in inhibition. TAB1-activated TAK1 induced IL-8
transcription, mRNA stabilization, and protein formation [43].
These data establish the TAK1-TAB1 complex as an intracellular effector that controls the main steps of IL-8 gene expression. Additional experiments showed that downstream of
TAK1, signaling diverges to regulate distinct steps in IL-8
expression. TAB1-TAK1-induced transcription was blocked by
a kinase-inactive mutant of JNK2, whereas a kinase-inactive
mutant of p38 MAPK had no effect. Conversely, TAB1-TAK1induced mRNA stabilization was blocked by a kinase-inactive
mutant of p38 MAPK but not by a kinase-inactive mutant of
JNK2. Thus, downstream of active TAK1, NF-␬B and JNK2 as
well as p38 MAPK are targeted to distinct gene-regulatory
functions by, so far, unknown mechanisms [43].
These results provide evidence that maximal IL-8 gene
expression requires the coordinate action of at least three
different signal transduction pathways that cooperate to induce
mRNA synthesis and suppress mRNA degradation. Impor-
From these data, a model for signal-dependent IL-8 gene
regulation can be derived. IL-8 production is actively kept low
in the absence of external stimulation (Fig. 2). During stimulation, conserved signaling pathways activate IL-8 expression
at the transcriptional and post-transcriptional levels (Figs. 3
and 4). Maximal IL-8 amounts can only be generated if the
gene promoter is derepressed, NF-␬B and JNK pathways are
activated to induce transcription, and the resulting mRNA is
rapidly stabilized by the p38 MAPK pathway (Figs. 3 and 4).
In that way, cells are able to rapidly increase and at the same
time, to fine-tune the amount of IL-8 secreted and thereby
control the extent of leukocytes attracted to sites of tissue
injury. The components of the involved pathways are not only
ubiquitously expressed but are also activated by numerous
stimuli, a fact that explains why so many cells are capable to
react with a uniform, biological response, i.e., IL-8 secretion to
external challenge. It is also important to note that the type of
signal-dependent gene expression described here is not restricted to IL-8 but may also be relevant for many other
proteins, e.g., IL-6 expressed during an inflammatory response.
Therefore, therapeutic targeting of the production of IL-8 (and
of other inflammatory proteins) may most effectively be
achieved by inhibiting key intracellular signaling molecules.
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