Editorial
Regulation of TLR4 Expression Is a Tale About Tail
Zhong-qun Yan
T
oll-like receptor (TLR) 4 is the first identified mama-
lian homolog of the Drosophila Toll protein.1 TLR4
recognizes bacterial lipopolysaccharide (LPS), and
also senses endogenous ligands including hyaluronic acid,
oxidized low-density lipoprotein, and heat-shock proteins.
Engagement of TLR4 with its ligand triggers a cascade of
cellular signals through the intracellular signal transduction
domain, known as Toll/interleukin (IL)-1 receptor (TIR),
leading to the activation of nuclear factor-␬B (NF-␬B) and
mitogen-activated protein kinase (MAPK) signal transduction
pathways, consequently inducing expression of inflammatory
genes.2 Although the TLR family of molecules normally
contributes to host defense, excess TLR signaling has been
implicated in many inflammatory diseases. That TLR4 is
implicated in cardiovascular diseases was first suggested by
observations of increased expression of TLR4 in failing
hearts3 and in atherosclerotic lesions.4,5 The functional im-
portance of TLR4 has subsequently been demonstrated by the
attenuated vascular inflammation and reduced lesion size in
TLR4-deficient atherosclerosis prone mice,6 and by the asso-
ciation of hyporesponsive TLR4 variants with disease devel-
opment.7,8 Furthermore, excessive activation of TLR4 by LPS
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induces endotoxin shock, a serious systemic disorder with a
high mortality rate. Therefore TLR signaling must be tightly
controlled to avoid improper activation or suppression. Yet
regulation of TLR4 expression in cardiovascular disease
remains elusive. In the current issue of Arteriosclerosis,
Thrombosis, and Vascular Biology, two sister papers by
Feng-Yen Lin and colleagues9,10 lend new mechanistic in-
sights that oxidative stress induces posttranscriptional stabi-
lization of TLR4 mRNA in vascular cells.
See pages 2622 and 2630
Posttranscriptional regulation of mRNA stability is a critical
determinant in gene expression, in particular for genes
encoding cytokines, transcription factors, growth factors and
oncoproteins that have short half-lives. Although it remains
poorly understood, recent studies suggest that posttranscrip-
tional control is accomplished by a group of AU-rich element
(ARE)-binding proteins that interact in a specific manner
with AREs in 3⬘ untranslated regions (3⬘-UTR) of mRNA. In
most cases, binding of these proteins to AREs causes rapid
decay of mRNA. Among ARE-binding proteins, Human
From the Center for Molecular Medicine, Karolinska Institute, Swe-
den.
Correspondence to Zhong-qun Yan, Center for Molecular Medicine
L8:03, Karolinska Institute, 171 76 Stockholm, Sweden. E-mail Zhong-
qun.yan@ki.se
(Arterioscler Thromb Vasc Biol. 2006;26:2582-2584.)
© 2006 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
DOI: 10.1161/01.ATV.0000250933.92917.dd
2582
antigen R (HuR), also known as ELAV (embryonic lethal
abnormal vision)-like protein 1, is unique in that overexpres-
sion of HuR has been shown to stabilize and/or to activate
translation of a certain subset of genes. It has been postulated
that binding of HuR to ARE either competes with or displaces
other ARE-binding proteins, thus blocking ARE-containing
mRNA from the exosome-mediated degradation (for review
see Barreau et al11).
Being motivated by their previous observation that LPS
enhances TLR4 expression in human aortic smooth muscle
cells (HASMC) by stabilization of mRNA, Lin and col-
leagues in new studies explored the molecular mechanism for
posttranscriptional control of the TLR4 gene. The new study
shows that on a low dose of LPS stimulation, HuR rapidly
built up in the cytoplasm of HASMCs, as a result of
translocation from nucleus to cytoplasm. In contrast, two
other ARE-binding proteins, AUF1 and TTP1, did not re-
spond to the stimulation. These data suggest that LPS
specifically induces HuR mobilization in HASMCs. To
assess the functional relevance to TLR4 expression, HuR was
then knocked down by siRNA. This results in significant
reduction in t1/2 of TLR4 mRNA in the presence of actino-
mycin D, indicating that HuR functions as a stabilizing factor
in the posttranscriptional control of TLR4 mRNA. Using two
independent methods, namely cross-linking immunoprecipi-
tation assay and transfection of the cells with a plasmid
containing the luciferase reporter linked to the 3⬘-UTR of
TLR4 gene, Lin and colleagues demonstrate that LPS pro-
motes binding of HuR to the 3⬘-UTR of TLR4 mRNA, and by
interacting with cis-acting elements in TLR4 3⬘-UTR HuR
enhances gene expression, although the motifs that interact
with HuR in the 3⬘ UTR of TLR4 mRNA are yet to be
defined.
The next question is how LPS induces mobilization of
HuR. LPS has been known to activate NADPH oxidase and
the MAPK signaling pathway in vascular cells. They found
that pretreatment of HASMC with the NADPH oxidase
inhibitor diphenylene iodonium, or knockdown of Rac1, a
small GTPase which is essentially required for activation of
NADPH oxidase, significantly reduces the t1/2 of TLR4
mRNA. This was associated with reduced cytoplasmic levels
of HuR. Blockade of the MAPK signaling pathway also led to
the same effect on TLR4 mRNA stability and HuR mobili-
zation as inhibition of NADPH oxidase. These findings
propose that LPS-induced posttranscriptional stabilization of
TRL4 involves activation of NADPH oxidase and the MAPK
signaling pathway.
This hypothesis was further tested in the 2nd study by
means of pharmacological inhibition of the enzyme activity.
Their data depict an intriguing model for the LPS signal in the
process of posttranscription regulation. It shows that LPS
may initially activate NADPH oxidase through induction of
 Yan Regulation of TLR4 Expression Is a Tale About Tail
Dual model for TLR4 gene upregulation in vascular cells.
Expression of TLR4 gene is controlled by two distinct molecular
mechanisms. First, TLR4 gene is upregulated by transcriptional
activation. Under most circumstances, this involves activation of
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NF-␬B and MAPK signal transduction pathways, subsequently
the activated transcription factors, NF-␬B and AP-1, bind to the
promoter of TLR4 gene, leading to transcriptional upregulation
of TLR4 gene. Extent of the transcriptional upregulation is deter-
mined by the activity of transduction signaling. Second, upregu-
lation of TLR4 gene can also result from posttranscriptional sta-
bilization of mRNA. For instance, activation of TLR4 on vascular
cells by LPS triggers NADPH oxidase activation, in turn leading
to generation of reactive oxygen species (ROS) and further to
the activation of MAPK signaling. This promotes nuclear export
of ARE-binding protein HuR in parallel with an increase in a
binding of HuR to the AREs in 3⬘UTR of TLR4 mRNA, conse-
quently preventing TRL4 mRNA from exosome-mediated degra-
dation. The posttranscriptional regulation is particularly prevalent
among mRNAs encoding proteins related to the inflammatory
response.
p47phox translocation to the cell membrane and activation of
Rac1, leading to generation of reactive oxygen species (ROS)
and further to the activation of the MAPK signaling pathway.
Both NADPH oxidase and MAPK signaling are indispens-
able for the stabilization of TLR4 mRNA. Obviously, the
current model is vastly simplified, and does not enlighten
whether HuR is the downstream target and phosphorylated by
the activated MAPK, and whether NADPH oxidase generated
ROS also directly regulates the interaction between HuR and
cis-acting elements in 3⬘-UTR. Nevertheless the study of Lin
at all sheds light on the neglected role of NADPH oxidase
signaling in posttranscription regulation (Figure).
Finally, results from a study of balloon injured arteries lend
further support to the role of LPS signal in upregulation of
TLR4. And consistently with the recent experimental study
on TLR4 deficient mice,12 their work suggests that inappro-
2583
priate TLR4 signaling affects vascular repair in a remarkable
way.
The work of Lin and colleagues represents a major advance
in the mechanistic understanding of TLR4 expression under
oxidative stress and is apt to stimulate future studies of the
posttranscriptional regulation of genes implicated in the
pathogenesis of atherosclerosis. An incomplete survey of
published studies reveals that a number of genes implicated in
vascular inflammation bear AUR motifs in mRNA, thus are
very likely subjected to oxidative stress induced posttran-
scriptional regulation in the disease process. These include
genes encoding IL-1, IL-2, IL-3, IL-4, IL-6, IL-8, GM-colony
stimulating factor (CSF), IFN-␥, tumor necrosis factor
(TNF)-␣, COX2, and iNOS. Evidently, the list will expand in
the near future. More importantly, we also need to assess the
importance of enhanced mRNA stability under conditions of
chronic inflammation in atherosclerosis and other pathologi-
cal conditions.
The importance of cytokine mRNA stabilization has been
demonstrated in mice with genomic deletion of ARE motifs
in TNF-␣ mRNA or the ARE-binding protein TTP. Both
models display elevated levels of circulating TNF-␣ and
markedly prolonged t1/2 of TNF-␣ mRNA in macrophages.
More importantly, the mutant mice spontaneously develop
inflammatory and autoimmune pathologies in multiple or-
gans, which can be attributed to excess TNF-␣.13,14 Hence,
alteration in cytokine mRNA stability is a potential mecha-
nism in the pathogenesis of atherosclerosis. Answers to these
questions will open a new avenue for modulating inflamma-
tory reactions and thus progression of the disease.
Acknowledgments
I would like to thank Dr Göran K. Hansson for helpful discussion.
Sources of Funding
Zhong-qun Yan is supported by grants from the Swedish Research
Council and Swedish Heart and Lung Foundation.
Disclosures
None.
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