Kinetic analyses of LPS-induced gene expression in WT or EP4^–/– BMDM with or without PGE₂ pretreatment. A, WT (EP4^+/+, as described under “Experimental Procedures”) BMDM were pretreated with 50 nm PGE₂ (PGE₂ (+)) or vehicle (PGE₂ (–)) for 1 h, followed by LPS stimulation (5 ng/ml) for various times. Total RNA was isolated and subjected to qPCR for indicated messages. mRNA levels of β-actin served as an internal control for normalization between samples. B, in EP4^–/– macrophages, PGE₂ does not inhibit rapid mRNA induction at the onset of LPS stimulation, especially for MIP-1β or TNFα. LPS-induced gene expression was measured by qPCR using EP4^–/– BMDM with or without PGE₂ pretreatment as described in A. In both studies (A and B), data represent at least three independent experiments on cells from different donors. Results are expressed as the mean -fold induction ± S.E. compared with unstimulated, vehicle-treated cells. *, p < 0.05; #, p < 0.01 for the indicated comparison (PGE₂ (–) versus PGE₂ (+) at each time point).

Forced EPRAP expression inhibits stimulus-induced p105 phosphorylation, degradation, and subsequent MEK1/2 activation. A, RAW264.7 cells were transiently transfected with mouse EPRAP expression construct (mEPRAP) or mock plasmid and then stimulated with LPS (10 ng/ml) for the indicated durations, followed by immunoblotting (representative of three different experiments). B, enforced EPRAP expression impairs inducible release of Tpl2 from p105. RAW264.7 cells transiently expressing either mouse EPRAP or mock plasmid were stimulated with LPS (50 ng/ml) or vehicle for 15 min. The same amounts of extracts were immunoprecipitated (IP) with anti-p105/p50 antibody, followed by immunoblotting with anti-Tpl2 or anti-p105/p50 antibody. To assess the protein levels of Tpl2, immunoblot analyses were performed using whole lysates (representative of three different experiments). C, densitometric quantification of M1-Tpl2/p105 ratio. Results are expressed relative to the values of samples without LPS treatment (defined as 1.0, from three different experiments) in mean ± S.E. *, p < 0.05 for the indicated comparison.

EPRAP directly interacts with NF-κB1 p105/p50 and forms a complex with EP4. A, EPRAP binds to NF-κB1 p105/p50. HEK293 cells were co-transfected with expression plasmids for V5-tagged EPRAP (EPRAP-V5) and GFP-tagged NF-κB proteins (p65-, p105-, and p50-GFP). Cell lysates were immunoprecipitated (IP) with rabbit anti-GFP antibody, followed by immunoblotting (IB) with anti-V5 or anti-GFP mAb (top). Cell extracts isolated from HEK293 cells with or without transient expression of EPRAP-V5 were immunoprecipitated with rabbit anti-p105/p50 antibody or control rabbit IgG, followed by immunoblotting with anti-V5 or anti-p105/p50 mAb (bottom). B, schematic representations for human EPRAP and deletion mutants. The positions of ankyrin repeat motif are indicated (top). HEK293 cells were co-transfected with expression plasmids for p50-GFP and V5-tagged EPRAP mutants. Cell lysates were immunoprecipitated with anti-V5 mAb, followed by immunoblotting with rabbit anti-GFP antibody (bottom). C, schematic representations for human NF-κB1 and deletion mutant. RHD, Rel homology domain; AR, ankyrin repeat; PEST, Pro-Glu-Ser-Thr-rich domain (top). HEK293 cells were co-transfected with expression plasmids for EPRAP-V5 and GFP-tagged p105 mutants (fulllength, p105-ΔN-GFP) (middle) or those for p105-ΔN-GFP and V5-tagged EPRAP mutants (N-AR, ΔAR, or fulllength) (bottom). Cell lysates were immunoprecipitated with anti-V5 mAb, followed by immunoblotting with anti-GFP antibody. D, EP4 forms a complex with EPRAP and NF-κB1 p105/p50. HEK293 cells were co-transfected with expression plasmids for FLAG-tagged EP4 (EP4-FLAG), EPRAP-V5, and p105-GFP (top) or p50-GFP (bottom). Cell lysates were immunoprecipitated with rabbit anti-FLAG antibody, followed by immunoblotting with anti-V5 or anti-GFP mAb. In these studies (B–D), expression of each recombinant protein was verified by immunoblotting using whole cell extracts (IP (–)).

PGE₂ augments IL-10 expression in LPS-stimulated macrophages but not through EP4-dependent mechanisms. A and B, LPS-induced gene expression was measured by qPCR using WT (A) or IL-10^–/– (B) BMDM with or without PGE₂ pretreatment as described in the legend to Fig. 1. Note that PGE₂ augments the expression of IL-10, which participates in the anti-inflammatory function of PGE₂, especially for IP-10 and RANTES. Data represent at least three independent experiments on cells from different donors. Results are expressed as the mean -fold induction ± S.E. compared with unstimulated, vehicle-treated cells. *, p < 0.05; #, p < 0.01 for the indicated comparison (PGE₂ (–) versus PGE₂ (+) at each time point). C, increased IL-10 production by PGE₂ is not mediated through EP4-specific signaling. WT (EP4^+/+) or EP4^–/– BMDM were pretreated with either vehicle Butaprost (10 μm) or PGE₂ (50 nm) for 1 h, followed by LPS (24 h). IL-10 protein levels in cell culture supernatants were measured by ELISA. Data are expressed relative to the values of samples from LPS-stimulated, vehicle-pretreated cells (defined as 1.0, from three different experiments) in mean ± S.E. #, p < 0.01 for the indicated comparison.

Deficiency of NF-κB1 in macrophages attenuates the anti-inflammatory effect of PGE₂. BMDM isolated from nfkb1^–/– or WT (nfkb1^+/+) mice were pretreated with or without PGE₂ (50 nm, 1 h), followed by LPS stimulation (5 ng/ml, 24 h). MIP-1β (A) or IL-10 (B) production was measured in cell culture supernatants by ELISA. Data are representative of three independent experiments. Results are presented as the mean -fold induction ± S.E. compared with the values of LPS treatment alone (defined as 1.0). #, p < 0.01 for the indicated comparison.

PGE₂ suppresses LPS-induced phosphorylation and degradation of NF-κB1 p105 through EP4-dependent mechanisms. Results of immunoblot analyses using LPS-stimulated WT (EP4^+/+)(A) or EP4^–/– (B) BMDM with or without PGE₂ pretreatment as described in the legend to Fig. 1 (left, representative blots; right, densitometric quantification of phosphorylated p105 in cell extracts). Data are expressed relative to the values at 15 min after LPS stimulation in the absence of PGE₂ pretreatment (defined as 1.0) in mean ± S.E. (from three different experiments). #, p < 0.01 for the indicated comparison (PGE₂ (–) versus PGE₂ (+) at each time point).

EPRAP overexpression attenuates NF-κB activation and nuclear translocation induced by proinflammatory stimuli. A, EPRAP overexpression attenuates NF-κB activation induced by various stimuli in a dose-dependent manner. NF-κB reporter gene analyses were performed using HEK293 cells cotransfected with the secreted alkaline phosphatase reporter plasmid, LacZ expression plasmid, and increasing amounts of human EPRAP expression construct (hEPRAP) as described under “Experimental Procedures.” Transfection efficiency was normalized by β-galactosidase (β-Gal) activity. Data are representative of three independent experiments. Results are expressed relative to the values of unstimulated cells in mean ± S.E. #, p < 0.01; †, p < 0.005 for the indicated comparison. B, nuclear translocation of NF-κB subunits. HEK293 cells transfected with either human EPRAP expression plasmid or mock plasmid were stimulated by TNFα (25 ng/ml) for increasing durations. Nuclear (nuc) and cytoplasmic (cyt) extracts were analyzed by immunoblotting using antibodies against p50, p65, or lamin B1 (loading control) (representative of three different experiments). C, DNA binding activity of p50 and p65 in the nuclear extracts of TNFα-stimulated HEK293 cells was determined by an ELISA-based DNA-binding assay. Data are representative of three independent experiments. Results were expressed as the mean OD value (450 nm) ± S.E. *, p < 0.05; #, p < 0.01 for the indicated comparison.

PGE₂ enhances the EPRAP-mediated antagonism of stimulus-induced p105 phosphorylation through EP4. HEK293 cells were co-transfected with expression plasmids for either human EP4, EPRAP, or mock control (EP4-/EPRAP-, EP4-/EPRAP+, or EP4+/EPRAP+). At 48 h after transfection, cells were incubated with PGE₂ (100 nm) or vehicle for 1 h, followed by TNFα (25 ng/ml) for 15 min. Cell extracts were immunoblotted with the indicated antibodies (representative of three independent experiments). Note that only in EP4/EPRAP-overexpressing cells (EP4+/EPRAP+) did PGE₂ demonstrate substantial inhibition of TNFα-induced p105 phosphorylation.

EPRAP knockdown in macrophages impairs the anti-inflammatory action of PGE₂. A and B, RAW264.7 cells were transfected with mouse EP4 expression construct (mEP4) or mock plasmid, together with either EPRAP-specific or control siRNA. At 48 h after transfection, cells were incubated with or without PGE₂ (100 nm, 1 h), followed by LPS (10 ng/ml, 24 h). MIP-1β (A) or IL-10 (B) production was measured in cell culture supernatants by ELISA. Data represent three independent experiments. Results are expressed relative to the values of samples without PGE₂ pretreatment (defined as 1.0) in mean ± S.E. *, p < 0.05 for the indicated comparison. Note that in EP4-overexpressing cells, PGE₂ suppressed MIP-1β production, but ERPAP knock-down impaired this effect. EPRAP silencing did not affect IL-10 production. C, EPRAP knockdown impairs the protective effect of PGE₂-EP4 signaling on LPS-induced p105 phosphorylation. EP4-transfected RAW264.7 cells with or without EPRAP silencing were incubated with PGE₂ (100 nm, 1 h), followed by LPS (50 ng/ml, 15 min). Protein extracts were subjected to immunoblotting (left) and quantified by densitometry (right). Data are expressed relative to the values of samples without PGE₂ pretreatment (defined as 1.0, from three different experiments) in mean ± S.E. *, p < 0.05 for the indicated comparison.

Schematic diagram depicting dual pathways for anti-inflammatory action of PGE₂ in macrophages. Based on the data presented here, we propose that PGE₂-EP4 signaling attenuates NF-κB as well as MEK activation under proinflammatory settings by inhibiting p105 phosphorylation and degradation through EPRAP, which directly interacts and forms a complex with NF-κB1 p105. PGE₂ also increases production of anti-inflammatory cytokine IL-10 but through EP4- or EPRAP-independent mechanisms.