The Journal of Immunology
Zinc Finger Protein Tristetraprolin Interacts with CCL3
mRNA and Regulates Tissue Inflammation
Ju-Gyeong Kang,* Marcelo J. Amar,†Alan T. Remaley,†Jaeyul Kwon,‡
Perry J. Blackshear,xPing-yuan Wang,* and Paul M. Hwang*
Zinc finger protein tristetraprolin (TTP) modulates macrophage inflammatory activity by destabilizing cytokine mRNAs. In this
study, through a screen of TTP-bound mRNAs in activated human macrophages, we have identified CCL3 mRNA as the most
abundantly bound TTP target mRNA and have characterized this interaction via conserved AU-rich elements. Compared to
the wild-type cells, TTP2/2macrophages produced higher levels of LPS-induced CCL3. In addition, the plasma level of CCL3
in TTP2/2mice was markedly higher than that in wild-type mice. To determine the in vivo significance of TTP-regulated CCL3,
we generated CCL32/2TTP2/2double-knockout mice. Along with decreased proinflammatory cytokines in their paw joints, there
were significant functional and histologic improvements in the inflammatory arthritis of TTP2/2mice when CCL3 was absent,
although cachexia, reflecting systemic inflammation, was notably unaffected. Furthermore, the marked exacerbation of aortic
plaque formation caused by TTP deficiency in the APOE2/2mouse model of atherosclerosis was also rescued by disrupting CCL3.
Taken together, our data indicate that the interaction between TTP and CCL3 mRNA plays an important role in modulating
localized inflammatory processes in tissues that are dissociated from the systemic manifestations of chronic inflammation.
Journal of Immunology, 2011, 187: 2696–2701.
signaling events that when deranged can result in diseases such as
autoimmune arthritis (1, 2). The identification of tristetraprolin
(TTP), encoded by the zinc finger protein 36 gene, as an essential
factor that binds to AU-rich elements (AREs) and destablizes TNF
mRNA provided a novel mechanism for modulating a central
inflammatory cytokine (3). Mice with genetic disruption of TTP
display an inflammatory syndrome comprised of cachexia, erosive
arthritis, myeloid hyperplasia, and serologies consistent with au-
toimmune disorders (4). Since this seminal report, the expanding
number of cytokines with ARE sequences that bind to TTP and
other similar proteins have underscored the importance of this
mechanism in modulating various immune responses (2, 5).
Identifying the molecular components involved in activating
monocytes into effector macrophages may provide new insights
into inflammatory diseases such as atherosclerosis (6, 7). Through
gene expression analysis, we previously identified TTP as one of
he temporal regulation of cytokine and chemokine mRNA
levels, through posttranscriptional mechanisms, plays a
critical role in regulating the cascades of inflammatory
the most highly expressed transcriptional regulatory genes in
macrophages purified from human atherosclerotic plaques com-
pared with circulating monocytes (8). To further elucidate how
TTP might regulate these inflammatory cells, we screened for
TTP-interacting mRNA species in activated human macrophages
by combining TTP immunoprecipitation with a sequencing-based
gene expression technique. One mRNA species bound to TTP at
high levels was CCL3 (MIP-1a), a cytokine belonging to the CC
motif subfamily of chemokines that is involved in both acute and
chronic inflammation (9).
CCL3 is secreted by activated macrophages and other in-
flammatory cells for diverse functions such as chemotaxis,
phagocytosis, and mediator release (9, 10). Thus, as a chemokine
present at high tissue concentrations, CCL3 promotes inflam-
mation and has been proposed to be involved in a spectrum of
diseases from asthma to multiple sclerosis (11–13). CCL3 is also
known to be highly expressed both in the synovial fluid of patients
with rheumatoid arthritis and in the plaque tissues of patients
with atherosclerosis (14–17). As there is a well-established associ-
ation between rheumatoid arthritis and atherosclerosis (18), CCL3
could serve as a common chemokine for the recruitment of in-
flammatory cells at disparate disease sites. Given our previous
observation of high TTP expression in atherosclerotic plaque
macrophages (8), we speculated that regulation of CCL3 mRNA
by TTP may play an important role in atherosclerosis pathogen-
esis. Although CCL3 mRNA has been reported to have ARE
sequences and can be inhibited by TTP (19, 20), the molecular
mechanism and functional consequences of the interaction be-
tween CCL3 mRNA and TTP have not been well studied. In this
study, we characterize the interaction between CCL3 mRNA and
TTP. We provide genetic evidence that the loss of this interaction
significantly contributes to the increase in inflammatory arthritis
and atherosclerosis of TTP-deficient mice, highlighting CCL3 as
a pathogenically significant target of TTP in these two common
*Center for Molecular Medicine, National Heart, Lung, and Blood Institute, National
Institutes of Health, Bethesda, MD 20892;†Cardio-Pulmonary Branch, National
Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892;
‡Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, MD 20892; andxLaboratory of Signal Trans-
duction, National Institute of Environmental Health Sciences, Research Triangle
Park, NC 27709
Received for publication April 21, 2011. Accepted for publication June 27, 2011.
This work was supported by funds from the Division of Intramural Research, Na-
tional Heart, Lung, and Blood Institute, National Institutes of Health.
Address correspondence and reprint requests to Dr. Paul M. Hwang, Center for
Molecular Medicine, National Heart, Lung, and Blood Institute, National Institutes
of Health, Building 10-CRC, Room 5-5330, 10 Center Drive, Bethesda, MD 20892-
1454. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
Abbreviations used in this article: ARE, AU-rich element; BMDM, bone marrow-
derived macrophages; SAGE, serial analysis of gene expression; siRNA, small in-
terfering RNA; TTP, tristetraprolin.
tion in a mouse model is applicable to human disease, as there is
abundant expression of both TTP and CCL3 in human atheroscle-
rotic lesions (8, 17). In addition, CCR5 is also present in arterial
smooth muscle cells, which are involved in plaque formation (17).
Although hyperlipidemia is thought to be a primary driver of ath-
erosclerosis, the unregulated expression of CCL3 appears to be piv-
otal in worsening atherosclerosis in TTP2/2APOE2/2mice despite
their significantly lower lipid levels compared with APOE2/2mice.
The CCL3 pathway may represent a potential target for site-
specific modulation of autoimmune arthritis and atherosclerotic
disease activity. Circulating cytokines have pleiotropic effects on
multiple systems, and their chronic blockade may lead to potential
complications such as the immune suppression seen with TNF an-
tagonist treatments. As chemokines spatially and selectively target
subsets of leukocytes, their blockade may reduce the likelihood of
systemic side effects (11). By disrupting CCL3 chemokine signal-
ing, the vicious cycle of recruiting more inflammatory cells to the
disease site may be prevented. Given our observations using two
different disease models, TTP regulation of CCL3 is likely to be
important for the pathogenesis of other common inflammatory
conditions and may merit therapeutic targeting.
We thank Milton Pryor, Rafael Molina, Deborah Stumpo, Cory Lago, Ho
Joong Sung, Wenzhe Ma, and Toren Finkel for helpful advice and assistance.
The authors have no financial conflicts of interest.
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The Journal of Immunology2701