5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability

Abstract

Background: The peroxisome proliferator-activated receptor (PPAR)-α activator, 5,8,11,14-eicosatetraynoic acid (ETYA), is an arachidonic acid analog. It is reported to inhibit up-regulation of pro-inflammatory genes; however, its underlying mechanism of action is largely unknown. In the present study, we focused on the inhibitory action of ETYA on the expression of the chemokine, CCL2/MCP-1, which plays a key role in the initiation and progression of inflammation.

Methods: To determine the effect of ETYA, primary cultured rat astrocytes and microglia were stimulated with IFN-γ in the presence of ETYA and then, expression of CCL2/MCP-1 and MAPK phosphatase (MKP-1) were determined using RT-PCR and ELISA. MKP-1 mRNA stability was evaluated by treating actinomycin D. The effect of MKP-1 and human antigen R (HuR) was analyzed by using specific siRNA transfection system. The localization of HuR was analyzed by immunocytochemistry and subcellular fractionation experiment.

Results: We found that ETYA suppressed CCL2/MCP-1 transcription and secretion of CCL2/MCP-1 protein through up-regulation of MKP-1mRNA levels, resulting in suppression of c-Jun N-terminal kinase (JNK) phosphorylation and activator protein 1 (AP1) activity in IFN-γ-stimulated brain glial cells. Moreover, these effects of ETYA were independent of PPAR-α. Experiments using actinomycin D revealed that the ETYA-induced increase in MKP-1 mRNA levels reflected an increase in transcript stability. Knockdown experiments using small interfering RNA demonstrated that this increase in MKP-1 mRNA stability depended on HuR, an RNA-binding protein known to promote enhanced mRNA stability. Furthermore, ETYA-induced, HuR-mediated mRNA stabilization resulted from HuR-MKP-1 nucleocytoplasmic translocation, which served to protect MKP-1 mRNA from the mRNA degradation machinery.

Conclusion: ETYA induces MKP-1 through HuR at the post-transcriptional level in a receptor-independent manner. The mechanism revealed here suggests eicosanoids as potential therapeutic modulators of inflammation that act through a novel target.

Figure 1

ETYA, but not fibrates, reduces CCL2/MCP-1 transcript levels and protein release in IFN-γ-stimulated astrocytes. (A and B) Primary astrocytes were stimulated with IFN-γ for 3 h (A) or 12 h (B) in the presence of the indicated levels of individual PPAR-α activators. Then, CCL2/MCP-1 and TNF-α mRNA levels and protein secretion into media were determined using qRT-PCR and ELISA, respectively. Data are presented as means ± SDs or SEMs of three independent experiments (*p < 0.01 versus IFN-γ group).

Figure 2

ETYA-induced CCL2/MCP-1 suppression depends on the inhibition of JNK-AP1 signals. (A) Astrocytes were transiently transfected with the indicated 5'-deleted CCL2/MCP-1 promoter constructs and incubated for 48 h. After stimulation with IFN-γ in the presence of the indicated activators for 6 h, cells were harvested and luciferase activity was measured and plotted as fold-induction over untreated controls. Data are presented as means ± SEMs for three independent experiments (*p < 0.05 versus IFN-γ group). (B) Astrocytes were stimulated with IFN-γ in the presence of the indicated PPAR-α activators, and nuclear factor binding activities of AP1 were measured by EMSA using oligonucleotide probes specific for the AP1 site of the rat CCL2/MCP-1 promoter (-129 to -111). The arrows represent specific DNA-protein complexes. (C) Astrocytes were treated with ETYA or WY-14643 and prepared for ChIP assays (see Materials and Methods for details). "Input" indicates control PCR and shows the amount of CCL2/MCP-1 promoter DNA present in each sample before ChIP. (D) After stimulating astrocytes with IFN-γ for 2 h in the presence of ETYA or WY-14643, JNK activity was measured by Western blot analysis using an anti-phospho-JNK antibody. The bar graph represents the intensities of pJNK bands normalized against those of α-tubulin (bottom panel). The data are presented as means ± SDs of three independent experiments. *p < 0.05, NS; non significant.

Figure 3

ETYA acts in a PPAR-α-independent manner to suppress CCL2/MCP-1 expression through induction of MKP-1 expression and phosphatase activity. (A and B) ETYA- or WY-14643-treated astrocytes were stimulated with IFN-γ for 2 h. MKP-1 protein levels and JNK phosphorylation were analyzed by Western blotting (A), and MKP-1 and CCL2/MCP-1 transcript levels were determined by qRT-PCR (B). (C and D) Cell lysates immunoprecipitated with an anti-MKP-1 antibody were incubated with p-NPP for 4 h and then analyzed spectrophotometrically at 405 nm (C), or incubated with lysates from IFN-γ-activated astrocytes followed by Western blot analysis of eluates (D). (E-I) Primary astrocytes were transfected with an MKP-1-specific (E and F) or PPAR-α-specific siRNA duplex (G-I) or a nonsilencing control siRNA. Forty-eight h after transfection, cells were stimulated with IFN-γ for 2 h or 12 h in the absence or presence of ETYA. MKP-1 protein levels and phospho-JNK, and transcript levels of MKP-1 and CCL2/MCP-1 were determined by Western blot analysis (E and G) and qRT-PCR (F and H). MCP-1 protein secretion was analyzed by ELISA (I). Efficiency of siRNA-mediated PPAR-α silencing was demonstrated by monitoring expression of acetyl CoA synthase (ACS), a PPAR-α-dependent gene (data not shown). Values are means ± SDs. of three independent experiments. *p < 0.05, ** p < 0.01, NS; non significant.

Figure 4

ETYA suppresses CCL2/MCP-1 expression by inducing MKP-1 expression in brain microglia. (A - C) Primary microglia were stimulated with 10 U/ml IFN-γ for 2 h or 12 h in the presence of ETYA or WY-14643. MKP-1 protein expression and JNK phosphorylation were analyzed by Western blotting (A), and MKP-1 and CCL2/MCP-1 transcript levels and protein release were examined by qRT-PCR (B) and ELISA (C). (D - G) Rat microglia were transfected with a siRNA duplex specific for MKP-1 (D and E) or PPAR-α (F and G). After 48 h, cells were stimulated with IFN-γ for 2 h in the presence or absence of ETYA. MKP-1 protein and JNK phosphorylation were analyzed by Western blotting (D and F), and MKP-1 and CCL2/MCP-1 transcript levels were examined by RT-PCR (E and G). Values are means ± SDs of three independent experiments. *p < 0.05, **p < 0.01, NS; non significant.

Figure 5

The ETYA-induced increase in MKP-1 expression involves a HuR-mediated increase in MKP-1 mRNA stability resulting from HuR cytoplasmic translocation. (A) Astrocytes were treated with Act D for the indicated periods in the presence of ETYA, after which MKP-1 mRNA levels were determined by conventional RT-PCR (left, upper panel) and qRT-PCR (right panel). The bar graph represents the intensities of MKP-1 bands normalized against those of GAPDH (left, bottom panel). All data are presented as means ± SDs of three independent experiments (*p < 0.05). (B and C) Astrocytes were transfected with a HuR-specific or nonsilencing control siRNA. Forty-eight h after transfection, cells were stimulated with IFN-γ in the absence or presence of ETYA. MKP-1 mRNA and protein levels were determined by RT-PCR and Western blot analysis (B), and MKP-1 mRNA stability was determined by qRT-PCR (C). The data are presented as means ± SDs of three independent experiments (*p < 0.05 versus IFN-γ + ETYA + con siRNA group). (D) Astrocytes were stimulated with IFN-γ in the presence or absence of ETYA, and nuclear extracts (NE) and cytosolic extracts (CE) were prepared for Western blotting. (E) Confocal microscopic images of astrocytes immunostained with antibodies against HuR, GFAP (astrocyte marker), and DAPI (nuclear marker), under the indicated conditions. Insets are magnified images of the corresponding boxed regions. Scale bars, 20 μm.

Figure 6

A model showing the anti-inflammatory mechanism of ETYA in IFN-γ-stimulated glial cell. IFN-γ induces JNK/Jun phosphorylation and increases CCL2/MCP-1 gene expression. ETYA, but not fibrates, increases MKP-1 mRNA stability by inducing HuR cytoplasmic shuttling, and thereby suppresses IFN-γ-induced JNK signaling and CCL2/MCP-1 expression.