Post-transcriptional regulation of pro-inflammatory gene expression.

Abstract

The cytokine tumour necrosis factor (TNF)alpha is a vital mediator of the innate immune response, and a pleiotropic regulator of cellular function. Its involvement in rheumatoid arthritis is illustrated by the clinical benefits of TNF alpha blockade. Post-transcriptional regulation (the control of mRNA stability and translation) appears to play a critical role in the regulation of TNF alpha expression by mitogen activated protein kinase signal transduction pathways and by anti-inflammatory agents. The aim of this article is to review some recent advances in our understanding of these processes, and to speculate on mechanisms of regulation of TNF alpha and other pro-inflammatory genes.

Full text

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Commentary
Post-transcriptional regulation of pro-inflammatory gene
expression
Andy Clark
Kennedy Institute of Rheumatology, Imperial College School of Medicine, London, UK
Abstract
The cytokine tumour necrosis factor (TNF)α is a vital mediator of the innate immune
response, and a pleiotropic regulator of cellular function. Its involvement in rheumatoid
arthritis is illustrated by the clinical benefits of TNFα blockade. Post-transcriptional regulation
(the control of mRNA stability and translation) appears to play a critical role in the regulation
of TNFα expression by mitogen activated protein kinase signal transduction pathways and
by anti-inflammatory agents. The aim of this article is to review some recent advances in our
understanding of these processes, and to speculate on mechanisms of regulation of TNFα
and other pro-inflammatory genes.
Keywords: adenosine/uridine-rich element, dexamethasone, inflammation, mRNA stability, translation, tumour
necrosis factor α
Received: 16 February 2000
Accepted: 9 March 2000
Published: 31 March 2000
Arthritis Res 2000, 2:172–174
© Current Science Ltd
ARE = adenosine/uridine-rich elements; Cox-2 = cyclooxygenase-2; GM-CSF = granulocyte macrophage colony stimulating factor; IL = interleukin;
IFNγ = interferon γ; LPS = lipopolysaccharide; MAPK = mitogen activated protein kinase; MAPKAPK2 = mitogen activated protein kinase-activated
protein kinase 2; RA = rheumatoid arthritis; TNFα = tumour necrosis factor alpha; TTP = tristetraprolin; UTR = untranslated region.
http://arthritis-research.com/content/2/3/172
Post-transcriptional regulation of gene expression is gener-
ally mediated by the 5′or 3′untranslated regions (UTRs) of
the mature mRNA, which flank the protein coding sequence.
It is assumed that regulation is mediated by proteins that
specifically interact with these regions. Much recent atten-
tion has focused on adenosine/uridine-rich elements (AREs),
particularly repeats of the sequence AUUUA, which are
present in the 3′UTRs of many transiently expressed
cytokine and growth factor mRNAs, including TNFα, IL1α,
1β, 6 and 8, IFNγ and granulocyte macrophage colony stim-
ulating factor (GM-CSF) [1,2]. Several AREs have been
shown to destabilise heterologous transcripts into which
they are inserted [3]. Mammalian TNFα mRNAs possess a
3′UTR of almost 800 nucleotides, within which is a well-con-
served ARE containing seven or eight copies of the AUUUA
sequence. Among factors known to interact with the TNFα
ARE [4–6] is tristetraprolin (TTP), an unusual zinc finger
protein whose expression is induced by both lipopolysaccha-
ride (LPS) and TNFα itself. In mice deficient in TTP, TNFα
protein is overexpressed, and the stability of TNFα mRNA is
increased [6]. When coexpressed with a mimic TNFα
mRNA, TTP is able to promote shortening of the poly(A) tail,
normally an early step in mRNA degradation [7]. This prop-
erty of TTP is dependent upon a (more or less) intact TNFα
ARE. Therefore, TTP may participate in a negative feedback
loop whose effect is to limit the magnitude or duration of a
burst of TNFα expression by destabilising its mRNA in an
ARE-dependent manner.
Mimic transcripts and other ‘post-transcriptional reporters’
for the study of TNFα gene regulation do not faithfully repro-
duce all features of endogenous gene expression. A more
subtle approach has recently been used by Kollias et al, who
precisely deleted the ARE region from the mouse TNFα
genomic locus [8]. In the resulting (∆ARE) strain, the dynam-
ics of LPS-stimulated TNFα mRNA accumulation were

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altered, with a larger, slower and more sustained induction.
Altered TNFα mRNA stability was strongly implicated in this
change, but not directly demonstrated. Other workers have
shown that TNFα mRNA stability is regulated by LPS, and is
sensitive to protein kinase inhibitors in myeloid cell lines [9].
These studies should be interpreted with caution because of
their dependence upon toxic transcriptional inhibitors like
actinomycin D, which may give rise to artefacts.
Previous studies established that the mitogen activated
protein kinase (MAPK) p38 regulates TNFα production at
the translational level [10–12]. Specific inhibitors of p38
blocked TNFα protein synthesis, while downregulating
TNFα mRNA by 50% at most. The deletion of the TNFα
ARE resulted in the loss of sensitivity to a p38 inhibitor in
the mouse strain of Kollias et al [8]. A recent mouse
knockout study suggests that the effects of MAPK p38
may be mediated by mitogen activated protein kinase-acti-
vated protein kinase 2 (MAPKAPK2), a kinase that is
phosphorylated and activated by p38. In the absence of
MAPKAPK2 activity, reduced quantities of TNFα protein
were synthesised following an LPS challenge, while the
induction of TNFα mRNA was unaffected [13]. Together,
these findings suggest that p38 regulates TNFα mRNA
translation via MAPKAPK2, and by a mechanism involving
the TNFα ARE. In contrast, p38 and MAPKAPK2 regulate
the stability rather than the translation of IL-6, IL-8,
GM-CSF and cyclooxygenase-2 (Cox-2) mRNAs in the
human HeLa cell line [14] (Lasa et al, submitted). Regula-
tion of IL-8 and Cox-2 mRNA stability is mediated by
3′ AU-rich elements that closely resemble the TNFα ARE.
It is significant that these studies do not rely upon pharma-
cological inhibitors of transcription or of p38, and provide
independent confirmation of earlier reports that p38 is
able to regulate mRNA stability.
The parallels and differences between TNFα and IL-8 or
Cox-2 raise several questions. Do AREs such as those in
the TNFα and Cox-2 transcripts regulate translation, sta-
bility, or both? What of the p38 signalling pathway? Is it
conceivable that distinct mechanisms have evolved for the
regulation of both translation and stability by mitogen acti-
vated protein kinases, through extremely similar RNA
sequence motifs? Perhaps the apparently different conse-
quences of p38 inhibition reflect an underlying common
mechanism. It is suggested that both HeLa cells and
monocytes/macrophages can label mRNAs as ‘Not For
Use’, most probably by means of specific protein–mRNA
interactions that involve AREs and are sensitive to p38
(Fig. 1). The immediate consequence of this might be a
cessation of translation, with indirect consequences such
as mRNA degradation modulated in a species, cell type or
transcript specific manner.
Studies employing specific inhibitors have implicated
MAPK p38 in the post-transcriptional regulation of a
number of genes that could be broadly characterised as
pro-inflammatory. These include TNFα, IL-6, -8 and -1β,
GM-CSF, Cox-2 and the vascular cell adhesion molecule
VCAM-1 [12,15–17]. In the MAPKAPK2 knockout mouse
strain, LPS-induced expression of IL-6 was blocked at
both protein and mRNA levels, while expression of both
TNFα and IFNγ was blocked at the protein but not the
mRNA level [13]. The defective expression of TNFα and
IFNγ suggests translational regulation by MAPKAPK2
within both myeloid and T cell lineages.
Dexamethasone, a synthetic glucocorticoid and a potent
anti-inflammatory reagent, regulates the expression of
TNFα and Cox-2 partly at the post-transcriptional level.
Post-transcriptional reporter studies have implicated the
TNFα 3′ UTR in dexamethasone sensitivity [18], and the
deletion of the ARE diminishes the inhibitory effect of
dexamethasone upon TNFα gene expression in vivo [8]. In
a human pulmonary epithelial cell line, dexamethasone
destabilises Cox-2 mRNA by a mechanism that involves
shortening of the poly(A) tail, although it has not yet been
possible to map the RNA sequences that mediate this
effect [19]. Both TNFα and Cox-2 are subject to negative
regulation by anti-inflammatory cytokines such as IL-10,
which has been shown to regulate the stability of
ARE-containing transcripts [20,21], and preliminary evi-
dence indicates that its effects upon TNFα expression
may be partly mediated by the TNFα 3′ UTR (B. Foxwell,
personal communication). The responses of the mouse
∆ARE strain to anti-inflammatory cytokines would be inter-
esting to observe.
It is an intriguing possibility that these disparate observa-
tions reflect a common mechanism for the post-transcrip-
tional control of several pro-inflammatory genes. In other
words, the ARE represents a point of convergence of pro-
inflammatory signals (such as MAPK p38 activation) and
Figure 1
Convergence of regulatory signals at the 3′ untranslated region of a
pro-inflammatory mRNA. ARE, Adenosine/uridine-rich elements; UTR,
untranslated region; MAPKAPK2, mitogen activated protein kinase-
activated protein kinase 2.

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Arthritis Research Vol 2 No 3Clark
anti-inflammatory signals (such as activation of IL-10 or
glucocorticoid signalling pathways; Fig. 1). In theory, this
might allow a cell to modulate its sensitivity to pro- or anti-
inflammatory stimuli through changes in the expression of
ARE binding factors, and to rapidly and coordinately shut
down the expression of several inflammatory mediators by
means of phosphorylation-regulated changes in protein–
RNA interactions. In the simplest model, all of these regu-
latory processes could be mediated by a single ARE
binding factor; however, in reality, the situation is likely to
be rather more complex. One can imagine post-transcrip-
tional regulation involving competition between positive
and negative regulatory RNA binding proteins, and other
subtleties such as cell-specific differences in the levels of
expression of these factors. The assessment of this
hypothesis requires the characterisation of putative post-
transcriptional regulators, and their functional and physical
interactions. It may also be instructive to look for additional
targets of suspected regulatory factors such as TTP.
Both the TTP knockout and the ∆ARE mouse strains
display striking inflammatory pathologies, including an
erosive arthritis with features of rheumatoid arthritis (RA)
in man [8,22]. Thus, the disruption of either cis-acting
(ARE) or trans-acting (TTP) components of negative regu-
latory control of TNFα synthesis can cause joint disease.
One wonders whether ‘off phase’ or negative regulatory
mechanisms for the restraint of TNFα biosynthesis are
perturbed in RA. Even if not, post-transcriptional control
may represent a target for novel anti-inflammatory thera-
pies. For example, a recent paper reports disease modify-
ing activity of a p38 inhibitor in an animal model of RA,
with downregulation of both TNFα and IL-6 expression
[23]. In the event of unwanted side effects of intervention
with MAPK or glucocorticoid signal transduction path-
ways, it might be possible to target downstream effectors,
the putative RNA binding coordinators of pro- and anti-
inflammatory post-transcriptional regulation. It is conceiv-
able that reactivation or reinforcement of ‘off phase’
regulatory mechanisms may have quite broad anti-inflam-
matory effects. A recent paper [24] reports that TTP regu-
lates the stability of GM-CSF mRNA.
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Author affiliation: Kennedy Institute of Rheumatology, Imperial
College School of Medicine, London, UK
Correspondence: Andy Clark, Kennedy Institute of Rheumatology,
Imperial College School of Medicine, London W6 8LH, UK.
Fax: +44 (0)20 8383 4499; e-mail: a.clark@cxwms.ac.uk