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. 2011 Sep 1;187(5):2696-701.
doi: 10.4049/jimmunol.1101149. Epub 2011 Jul 22.

Zinc finger protein tristetraprolin interacts with CCL3 mRNA and regulates tissue inflammation

Ju-Gyeong Kang  1 , Marcelo J Amar, Alan T Remaley, Jaeyul Kwon, Perry J Blackshear, Ping-yuan Wang, Paul M Hwang
Affiliations

Zinc finger protein tristetraprolin interacts with CCL3 mRNA and regulates tissue inflammation

Ju-Gyeong Kang et al. J Immunol. .

Abstract

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, TTP(-/-) macrophages produced higher levels of LPS-induced CCL3. In addition, the plasma level of CCL3 in TTP(-/-) mice was markedly higher than that in wild-type mice. To determine the in vivo significance of TTP-regulated CCL3, we generated CCL3(-/-)TTP(-/-) double-knockout mice. Along with decreased proinflammatory cytokines in their paw joints, there were significant functional and histologic improvements in the inflammatory arthritis of TTP(-/-) mice 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 APOE(-/-) mouse 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.

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Figures

FIGURE 1
FIGURE 1
TTP interacts with CCL3 mRNA and its knockdown increases CCL3 mRNA level in an activated human monocytic cell line. A, Scheme to identify all mRNAs interacting with TTP by immunoprecipitation and subsequent expression profiling using Serial Analysis of Gene Expression (SAGE). B, Confirmation of CCL3 mRNA binding to TTP protein by RT-PCR in the anti-TTP antibody versus nonspecific control immunoglobulin (IgG) bound RNA fractions of PMA-activated THP-1 cells. C, TTP depletion using siRNA, confirmed by western blotting, increases the levels of its target mRNAs. THP1 cells were transfected with either nonspecific (NS) or TTP-specific (TTP) siRNA, activated with PMA, and the target transcripts measured at 48 h by RT-PCR. Data presented as mean ± SEM, n = 3. *P < 0.05 compared to respective nonspecific controls.
FIGURE 2
FIGURE 2
CCL3 expression is increased in TTP−/− mouse bone marrow-derived macrophages (BMDM). A, CCL3 mRNA levels after LPS (10 ng/ml) stimulation are higher in TTP−/− compared to TTP+/+ BMDM. The time course of TTP protein induction by LPS treatment is shown by western blotting. B, CCL3 protein in the medium was quantified by ELISA. C, CCL3 and TNF mRNA half-lives are increased in TTP−/− versus TTP+/+ BMDM. After stimulating with LPS for 90 min, actinomycin D (Act. D) was added to the cells. At the indicated times (Time after Act. D), mRNA was isolated from the cells and quantified by RT-PCR. Dashed line indicates 50% level. Data presented as mean ± SEM, n = 3 to 6.
FIGURE 3
FIGURE 3
TTP destablizes CCL3 mRNA through conserved AU-rich elements in HEK293 cells. A, TTP cDNA expression specifically decreases the level of cotransfected wild-type full length CCL3 mRNA while control GAPDH mRNA levels are unchanged. RT-PCR was used to measure mRNA levels 48 h after cotransfection into HEK293. B, Alignment of the CCL3 mRNA 3′ untranslated regions from the indicated species identifies conserved AREs (shaded and numbered). A-to-C point mutations were introduced into the CCL3 AREs as shown (Mutant). C, The conserved AREs either individually or in combination contribute to the destabilization of CCL3 mRNA by TTP. TTP cDNA or empty vector plasmid was cotransfected with either wild-type (WT) or mutant CCL3 into HEK293 cells. Data presented as mean ± SEM, n = 3. *P < 0.05.
FIGURE 4
FIGURE 4
Deletion of CCL3 reduces the inflammatory arthritis of TTP−/− mice. A, Paw grip strength is improved in the absence of CCL3. Four-paw grip strength of 9 wk-old female mice was measured by using a digital grip strength meter. TTP+/+ (square), TTP−/− (open circle), and CCL3−/− TTP−/− (filled circle). B, Bone erosion and tissue inflammation of TTP−/− mouse paws are reduced in the absence of CCL3 (TTP−/− CCL3−/− genotype). Representative H&E stained sections of front (upper panel) and rear (lower panel) paw joints of female mice in the 22 to 35 wk range. Synovial space (arrowhead); bone (asterisk). Scale bar: 200 μm.
FIGURE 5
FIGURE 5
Deletion of CCL3 dissociates localized from systemic inflammation in TTP−/− mice. A, Growth curves of female TTP+/+, TTP−/−, and CCL3−/− TTP−/− mice are shown. Arrowheads indicate the closely apposed growth curves of TTP−/− mice and CCL3−/− TTP−/− mice (n= 4 to 7). B, Body mass composition determined by NMR (n = 3 to 6). C, The splenomegaly of TTP−/− mice is unaltered in the absence of CCL3 (n = 4). D, The plasma protein levels of TTP targets TNF and IL-1β continue to be elevated in the absence of CCL3 in TTP−/− mice (n = 3). E, The joint tissue mRNA levels of TTP targets TNF and IL-1β are reduced in the absence of CCL3 in TTP−/− mice (n = 3). Except as indicated in (A) the mice used were in the age range of 17 to 22 wk. Data are presented as mean ± SEM. *P < 0.05 compared to TTP+/+ mice.
FIGURE 6
FIGURE 6
Deletion of CCL3 prevents the increase in aortic atherosclerosis of TTP−/− APOE−/− mice. A, The increased aortic atherosclerotic plaque area (Sudan IV stained) of female TTP−/− APOE−/− mice (23 to 25 wk-old) is prevented by deleting CCL3. Scale bar: 2 mm. B, Plasma lipids levels are reduced both in TTP−/− APOE−/− and in CCL3−/− TTP−/− APOE−/− mice compared to APOE−/− mice (n = 6 to 8). Data are presented as mean ± SEM. *P < 0.05 compared to APOE−/− mice. C, Body weights of 23 to 25 wk-old female mice of the indicated genotypes. Data shown as mean ± SEM, n = 4 to 8. *P < 0.05 compared to APOE−/− mice.
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