Mol. Cells, Vol. 25, No. 3, pp. 347-351
Molecules
and
Cells
Minireview
©KSMCB 2008
A Lipid-derived Endogenous Inducer of COX-2: a Bridge
Between Inflammation and Oxidative Stress
Koji Uchida*
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
(Received April 30, 2008; Accepted May 2, 2008)
Several lines of evidence indicate that the oxidative modification of protein and the subsequent accumulation of
the modified proteins have been found in cells during
aging, oxidative stress, and in various pathological states
including premature diseases, muscular dystrophy,
rheumatoid arthritis, and atherosclerosis. The important
agents that give rise to the modification of a protein may
be represented by reactive aldehydic intermediates, such
as ketoaldehydes, 2-alkenals and 4-hydroxy-2-alkenals.
These reactive aldehydes are considered important mediators of cell damage due to their ability to covalently
modify biomolecules, which can disrupt important cellular functions and can cause mutations. Furthermore, the
adduction of aldehydes to apolipoprotein B in lowdensity lipoproteins (LDL) has been strongly implicated
in the mechanism by which LDL is converted to an
atherogenic form that is taken up by macrophages, leading to the formation of foam cells. During the search for
an endogenous inducer of cyclooxygenase-2 (COX-2), an
inducible isoform responsible for high levels of prostaglandin production during inflammation and immune
responses, 4-hydroxy-2-noennal (HNE), one of the most
representative lipid peroxidation product, has been identified as the potential inducer of COX-2. In addition, the
following study on the molecular mechanism of the COX2 induction by HNE has unequivocally established that a
serum component, which is eventually identified to be
denatured LDL, is essential for COX-2 induction. Here I
review current understanding of the mechanisms by
which HNE in cooperation with the serum component
activates gene expression of COX-2.
Keywords: 4-Hydroxy-2-Noennal; Atherosclerosis; Cyclooxygenase-2; Inflammation; Lipid Peroxidation; Low-Density
Lipoproteins; Oxidative Stress.
* To whom correspondence should be addressed.
Tel: 81-52-789-4127; Fax: 81-52-789-4127
E-mail: [email protected]
COX-2 and atherosclerosis
Atherosclerosis is a disorder of lipid metabolism as well as
a chronic inflammatory disease. Monocyte-derived macrophages play a prominent role in the formation and progression of atherosclerotic plaque, particularly after their transformation into foam cells. When activated by inflammatory
stimuli, the macrophages synthesize and secrete various
mediators, including cytokines, prothrombotic substances,
and eicosanoids, which cause the clinical manifestations
and acute clinical complications of atherosclerosis. The
eicosanoids derived from the metabolism of arachidonate
have been extensively investigated because several studies
have focused on their close relation to atherogenesis (FitzGerald et al., 2000; Gimbrone et al., 2000). In macrophages,
as well as in other cell types, arachidonate metabolites are
synthesized by the cyclooxygenase enzyme. Presently, two
isoforms of cyclooxygenase have been identified; cyclooxygenase-1 (COX-1), which is the constitutive form, and
cyclooxygenase-2 (COX-2), which is the inducible form.
COX-1 is present under normal conditions in most tissues
and is responsible for housekeeping functions. On the other
hand, COX-2 is not normally present under basal conditions or is present in very low amounts. COX-2 is rapidly
induced by various stimuli, including proinflammatory
cytokines, such as interleukin-1β and tumor necrosis facorα, growth factors and tumor promoters, to result in prostaglandin synthesis associated with inflammation and carcinogenesis (FitzGerald, 2003). Substantial evidence indicates that unregulated COX-2 expression and prostaglandin
synthesis influence chronic inflammatory conditions, including atherosclerosis and its complications (FitzGerald,
2003; Koki et al., 2002; Smith and Langenbach, 2001).
Abbreviations: COX-2, cyclooxygenase-2; EGFR, epidermal
growth factor receptor; HNE, 4-hydroxy-2-nonenal; LDL, lowdensity lipoproteins; oxLDL, oxidized low-density lipoproteins;
p38 MAPK, p38 mitogen-activated protein kinase.
348
An Endogenous Inducer of COX-2
Functional lipids originated from oxidized
LDL
Various lines of evidence indicate that an important part
of the pathogenesis of atherosclerosis is the modification
of plasma low-density lipoproteins (LDL) (Steinberg,
1995; Steinberg et al., 1989). A large number of pro-inflammatory and pro-atherogenic properties have been
ascribed to oxidatively modified LDL (oxLDL) and their
components (Glass and Witztum, 2001). In particular,
there is considerable evidence to support the role of oxidized fatty acids originating from the oxLDL as important
signaling molecules in the context of the athersclerotic
lesion. Podrez et al. (2002) have shown that oxLDL components, such as oxidized phosphatidylcholines, serve as
endogenous ligands for the scavenger receptor, CD36,
facilitating macrophage cholesterol accumulation and
foam cell formation. The work by Nagy et al. shows that
the oxidized fatty acids, such as 9- and 13-hydroxyoctadecadienoic acids and 15-hydroxyeicosatetraenoic acid,
regulate the macrophage gene expression through the
ligand activation of the peroxisome proliferators-activated
receptor γ (Nagy et al., 1998). In addition, the lipid peroxidation-derived short-chain aldehydes, such as acrolein
and 4-hydroxy-2-nonenal (HNE) (Fig. 1), have been
shown to modulate the NF-κB-dependent signaling pathways, which play an important role in gene regulation
during inflammatory and immune responses (Uchida,
2003). The oxidized lipids generated during oxidative
modification of LDL are therefore likely to be involved in
the process of macrophage transformation into the foam
cells during atherogenesis.
Identification of HNE as a putative inducer of
COX-2
It has been shown that COX-2 colocalizes with the lipid
peroxidation-specific epitopes in foamy macrophages
within human atheromatous lesions (Kumagai et al.,
2004). This finding suggests that the COX-2 expression
may be associated with the accumulation of lipid peroxidation products within the macrophages. Hence, Kumagai
et al. conducted a screen of oxidized fatty acids on COX2 induction in rat liver epithelial RL34 and mouse macrophage RAW264.7 cell lines and demonstrated that HNE
specifically stimulates the COX-2 expression (Kumagai et
al., 2000; 2004). Interestingly, they also observed that the
α,β-unsaturated aldehydes such as acrolein, crotonal, and
2-nonenal, possessing an analogous functionality to HNE,
were all inactive on the COX-2 induction. These studies
represent a first demonstration of a link between COX-2
and HNE.
It is known that the NF-κB signal transduction cascade
is a major stress response signaling pathway for the COX-
Fig. 1. Chemical structure of HNE.
2 gene expression. In mice and humans, the COX-2 promoter has many transcription factors including NF-κB in
the 5′ region of the cox-2 gene (Reddy et al., 2000), and
the requirement of the activation of NF-κB to induce the
expression of COX-2 in the lipopolysaccharide-stimulated
macrophages has been described (Huang et al., 1997). The
NF-κB pathway has also been implicated in the expression of COX-2 stimulated by tumor necrosis factor-α,
hypoxia, endothelin, and interleukin-1β in the endothelial
cells and hepatocytes. Thus, it is naturally suggested that
the NF-κB-dependent signaling pathway mediates the
HNE-stimulated COX-2 induction. However, in contrast
to the COX-2 expression by these endogenous and exogenous stimuli, no significant change in the IκB and NF-κB
levels is observed. Moreover, in agreement with this finding, the HNE-stimulated COX-2 induction has not been
associated with the induction of the NF-κB-dependent
gene products, such as iNOS. Meanwhile, Kumagai et al.
(2002) have shown that the p38 mitogen-activated protein
kinase (p38 MAPK) pathway, another stress response signaling pathway for the COX-2 gene expression, is involved in the HNE-stimulated COX-2 induction. The authors have observed that HNE stimuli elicit a rapid and
significant phosphorylation of p38 MAPK and activate
MMK3/MKK6, a specific MAPKK of p38 MAPK. In
addition, the observations that (i) the p38 MAPK specific
inhibitor, SB203580, inhibited COX-2 expression, (ii) the
overexpression of wild-type p38 MAPK enhanced COX-2
expression, and (iii) dominant negative p38 MAPK conversely decreased the HNE-induced COX-2 levels also
support the idea that the p38 MAPK signaling pathway
participates in the HNE-induced COX-2 expression. Furthermore, they have also examined the relationship between COX-2 mRNA stability and HNE-activated p38
MAPK pathway and found that the p38 MAPK specific
inhibitor accelerated COX-2 mRNA degradation. Thus, it
appears that the p38 MAPK pathway controls the HNEinduced COX-2 expression at posttranscriptional levels.
HNE is not a direct inducer of COX-2:
Identification of a bona fide inducer
Due to the potent cellular function of HNE, no one could
have doubts as to its COX-2 inducibility. However, several years after the first paper upon identification of HNE
as an inducer of COX-2 was published (Kumagai et al.,
Koji Uchida
2000), Kanayama et al. revealed that HNE was not a bona
fide inducer of COX-2. During the course of their study
on COX-2 induction by HNE, they accidentally found
that the serum was essential for the COX-2 expression
induced by HNE (Kanayama et al., 2007). In addition,
they separated the human serum by gel filtration tentatively and identified lipoproteins, such as VLDL and LDL,
as the co-inducers of COX-2 expression. At this stage,
they thought that the lipoproteins represent the bona fide
inducer of COX-2. However, they observed that LDL derived from pooled serum, but not LDL freshly prepared
from serum of healthy subjects, showed potent inducibility of COX-2 in the presence of HNE. In addition, several
modified forms of electronegative lipoprotein species,
including freeze-thawed, oxidized, and acetylated LDL,
significantly induced COX-2 expression in the presence
of HNE. Thus, the denatured LDL was eventually identified as a bona fide active component essential for the induction of COX-2 by HNE.
It has been shown that human plasma contains a denatured, electronegative LDL subfraction that possesses
atherogenic properties and is associated with increased
cardiovascular disease risk (Sevanian et al., 1997; 1999).
Most mechanisms that describe the formation of such
modified LDLs involve the oxidative modification of particles (Sevanian et al., 1996; Ziouzenkova et al., 1999).
Although the detailed mechanism for the oxidative modification of lipoproteins has not yet been established, it is
generally accepted that the primary generation of lipid
hydroperoxides initiates a reaction cascade leading to the
rapid propagation and amplification of the number of reactive oxygen species formed; this ultimately leads to
extensive fragmentation of the fatty acid chains and conversion of the LDL into a more atherogenic form (Quinn
et al., 1987). Thus, it was speculated that the oxidative
modification of LDL leads to simultaneous production of
HNE and denatured, electronegative LDL, both of which
contribute to the induction of COX-2 gene expression.
HNE and denatured LDL cooperatively
induce COX-2
The identification of denatured LDL as the inducer of
COX-2 suggested that scavenger receptor(s) might be
involved in the induction mechanisms. Hence, Kanayama
et al. (2007) examined the changes in the mRNA levels of
scavenger receptors in RAW264.7 macrophages exposed
to HNE in the presence and absence of oxidized LDL and
demonstrated that exposure of macrophages to HNE alone
resulted in the increased expression of CD36, a major
receptor responsible for the binding and uptake of modified LDL in macrophages (Kanayama et al., 2007). To
further obtain evidence for the involvement of HNE in the
CD36 expression in vivo, they examined the pathohis-
349
tologic location of the protein-bound HNE and CD36 in
human atherosclerotic lesions and observed the colocalization of protein-bound HNE with CD36 in the cytoplasm
of foamy macrophages. These data led to the assumption
that the HNE-induced CD36 expression might be associated with the induction of COX-2 expression. This hypothesis was supported, at least in part, by the observations that (i) the reduction of CD36 expression by treatment with CD36 siRNA resulted in the reduced expression of COX-2 induced by the combination of HNE and
oxLDL and (ii) overexpression of CD36 resulted in the
enhanced expression of COX-2 induced by oxLDL alone
(Kanayama et al., 2007). Thus, it was established that the
oxidized lipid component of oxLDL particles could function as an endogenous inducer of CD36 gene expression.
These findings suggest the existence of a mechanism
whereby the oxidized lipid promotes the uptake of denatured LDL by increasing the level of the scavenger receptor
expression, leading to the expression of COX-2 (Kanayama
et al., 2007). This association of HNE with CD36 may be
consistent with the previously defined role for oxLDL in
the regulation of CD36 expression (Nagy et al., 1998).
Cellular target of HNE
It is challenging to define a target molecule that triggers
signal transduction pathways leading to CD36 expression
by HNE. Based on the previous observations that the inhibitors of the EGFR tyrosine kinases significantly suppress the HNE-induced COX-2 expression (Kumagai et
al., 2004), it has been speculated that EGFR may represent one of the upstream targets of HNE in CD36 gene
expression. The EGFR, a transmembrane receptor tyrosine kinase shared by several growth factors, is implicated
in various biological processes such as cell proliferation
or differentiation and has been suggested to be involved in
the genesis or progression of atherosclerosis and a number
of human malignancies. EGFR activation is associated
with the stimulation of its intrinsic tyrosine kinase, with
autophosphorylation of its own tyrosine residues, and with
phosphorylation of intracellular substrate proteins. Indeed,
HNE up-regulates the catalytic actions of EGFR for autophosphorylation in RAW264.7 macrophages and that the
inhibitors of EGFR tyrosine kinase down-regulate the
HNE-induced CD36 gene expression (Kanayama, M., and
Uchida, K., unpublished data). These data are consistent
with the previous findings that HNE activates the EGFR
tyrosine kinase and subsequent signaling pathways in endothelial cells (Suc et al., 1998). While the mechanisms
that activate EGFR are not well defined, previous studies
have suggested that the receptor may be activated via covalent modification by HNE. Concomitantly to the activation of EGFR, the HNE-modified EGFR has been detected in cultured endothelial cells treated with oxLDL or
350
An Endogenous Inducer of COX-2
sensitive signaling mechanisms. They are mostly relevant
in atheromata, where close contact between the macrophages and the oxidized lipids might ultimately result in
the development of cellular responses, together with a cell
failure to repair tissue damage. These functions may represent an important contributing feature during an early
step in the process of macrophage transformation into the
foam cells composing the fatty streak, a primary histologic aspect of incipient atherosclerosis.
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Fig. 2. A proposed mechanism for induction of COX-2 by the
combined stimulus of HNE and denatured LDL. HNE, generated during oxidative stress and LDL oxidation, up-regulates
gene expression of the scavenger receptor CD36. The upregulation of CD36 is accompanied by the enhanced uptake of
denatured LDL through CD36 and promotes the CD36-mediated
COX-2 induction by denatured LDL.
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