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. 2018 Apr 6;293(14):5295-5306.
doi: 10.1074/jbc.RA118.001593. Epub 2018 Feb 13.

Fatty Acid-Binding Protein 5 Controls Microsomal Prostaglandin E Synthase 1 (mPGES-1) Induction During Inflammation

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Free PMC article

Fatty Acid-Binding Protein 5 Controls Microsomal Prostaglandin E Synthase 1 (mPGES-1) Induction During Inflammation

Diane Bogdan et al. J Biol Chem. .
Free PMC article

Abstract

Fatty acid-binding proteins (FABPs) are intracellular lipid carriers that regulate inflammation, and pharmacological inhibition of FABP5 reduces inflammation and pain. The mechanism(s) underlying the anti-inflammatory effects associated with FABP5 inhibition is poorly understood. Herein, we identify a novel mechanism through which FABP5 modulates inflammation. In mice, intraplantar injection of carrageenan induces acute inflammation that is accompanied by edema, enhanced pain sensitivity, and elevations in proinflammatory cytokines and prostaglandin E2 (PGE2). Inhibition of FABP5 reduced pain, edema, cytokine, and PGE2 levels. PGE2 is a major eicosanoid that enhances pain in the setting of inflammation, and we focused on the mechanism(s) through which FABP5 modulates PGE2 production. Cyclooxygenase 2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1) are enzymes up-regulated at the site of inflammation and account for the bulk of PGE2 biosynthesis. Pharmacological or genetic FABP5 inhibition suppressed the induction of mPGES-1 but not COX-2 in carrageenan-injected paws, which occurred predominantly in macrophages. The cytokine interleukin 1β (IL-1β) is a major inducer of mPGES-1 during inflammation. Using A549 cells that express FABP5, IL-1β stimulation up-regulated mPGES-1 expression, and mPGES-1 induction was attenuated in A549 cells bearing a knockdown of FABP5. IL-1β up-regulates mPGES-1 via NF-κB, which activates the mPGES-1 promoter. Knockdown of FABP5 reduced the activation and nuclear translocation of NF-κB and attenuated mPGES-1 promoter activity. Deletion of NF-κB-binding sites within the mPGES-1 promoter abrogated the ability of FABP5 to inhibit mPGES-1 promoter activation. Collectively, these results position FABP5 as a novel regulator of mPGES-1 induction and PGE2 biosynthesis during inflammation.

Keywords: FABP; NF-κB; fatty acid binding protein; inflammation; mPGES-1; pain; prostaglandin.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Biosynthetic pathway for PGE2. Arachidonic acid is converted into prostaglandin H2 by COX-1 and COX-2 enzymes. Prostaglandin H2 is subsequently converted into PGE2 by mPGES-1, mPGES-2, and cytosolic prostaglandin E synthase (cPGES) enzymes. COX-2 and mPGES-1 are the enzymes that contribute to the bulk of PGE2 biosynthesis during inflammation.
Figure 2.
Figure 2.
FABP5 inhibition produces anti-inflammatory and antinociceptive effects. A, WT, FABP4 KO, and FABP5 KO mice received intraplantar injections of carrageenan, and paw edema was measured 4 h later. The change in paw edema from baseline is indicated. *, p < 0.05 versus WT mice (n = 6). B, thermal withdrawal latencies in WT, FABP4 KO, and FABP5 KO mice before and 4 h after intraplantar injection of carrageenan. *, p < 0.05 versus WT mice after carrageenan injection (n = 9). C, CGRP release in sections of lumbar spinal cords obtained from carrageenan- and saline-injected WT mice. Mice received an intraplantar injection of carrageenan or saline, and CGRP release was measured 4 h later in spinal sections obtained from the ipsilateral (carrageenan-injected) or contralateral (saline-injected) side. **, p < 0.01 (n = 6). D, capsaicin evoked CGRP release in lumbar spinal slices from WT and FABP5 KO. Spinal sections were obtained as in C. CGRP release was quantified in ipsilateral (carrageenan-injected) slices before and after treatment with capsaicin (1 μm, 30 min). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (n = 6). E, TNFα, IL-1β, and IL-6 levels in paws at baseline and 4 h after carrageenan injection. Mice were injected with vehicle or SBFI26 (20 mg/kg, i.p.) 30 min before carrageenan injection. Cytokine levels were normalized to baseline levels in WT mice. *, p < 0.05 versus carrageenan-injected WT mice (n = 6). F, PGE2 levels in paws at baseline and 4 h after carrageenan injection in WT mice treated with vehicle or 20 mg/kg SBFI26 and in FABP5 KO mice. **, p < 0.01 versus carrageenan injected WT mice (n = 6). Error bars represent S.E.
Figure 3.
Figure 3.
FABP5 expression in paws after inflammation. A, immunolocalization of FABP5 in sections of paw tissue before and after intraplantar injection of carrageenan. Left panels, FABP5 was predominantly expressed in the epidermis of control paws with some expression in F4/80+ macrophages. Right panels, carrageenan injection resulted in the recruitment of FABP5-expressing F4/80+ macrophages to the site of inflammation. B, higher magnification merged images of those shown in A demonstrating robust expression of FABP5 in F4/80+ macrophages in carrageenan-injected paws.
Figure 4.
Figure 4.
Effect of FABP5 inhibition upon PGE2 levels in activated THP-1 cells. A, Western blot of COX-2 induction in THP-1 cells after incubation with 5 μg/ml LPS for 2 or 24 h. B, PGE2 levels in THP-1 cells before and after activation with LPS. The cells were incubated with vehicle (PBS; control) or 5 μg/ml LPS for 24 h to induce PGE2 biosynthesis. The cells were subsequently washed and incubated with SBFI26, diclofenac, or vehicle (0.1% DMSO) for 2 h, and PGE2 levels were quantified in the media. *, p < 0.05; **, p < 0.01 versus LPS-activated vehicle-treated cells (n = 6). C, effect of SBFI26 upon purified COX-1 and COX-2 activity (n = 4). D, PGE2 biosynthesis in membrane fractions of control and LPS-activated THP-1 cells. Membranes were isolated 24 h after LPS stimulation and incubated with 1 μm arachidonic acid in the presence or absence of SBFI26 or diclofenac. **, p < 0.01 versus LPS-activated vehicle-treated membranes (n = 6). Error bars represent S.E.
Figure 5.
Figure 5.
Effect of FABP5 inhibition upon COX-2 and mPGES-1 induction in carrageenan-injected paws. A, left, COX-2 expression in hind paws of WT and FABP5 KO mice injected with saline or carrageenan. The paws were harvested 24 h after carrageenan administration. Right, quantification of COX-2 and GAPDH intensity ratios. COX-2 levels are reported as the ratio of COX-2/GAPDH signal intensity (n = 6). B, left, mPGES-1 induction in hind paws of WT, FABP4 KO, and FABP5 KO mice injected with carrageenan. Right, quantification of mPGES-1 signal intensities. *, p < 0.05 versus carrageenan-injected WT mice (n = 6). C, left, induction of mPGES-1 in carrageenan-injected hind paws of WT mice receiving an intraperitoneal injection of vehicle or 20 mg/kg SBFI26. Right, quantification of mPGES-1 and GAPDH signal intensities. **, p < 0.01 versus carrageenan-injected mice (n = 6). D, immunofluorescence of mPGES-1 and F4/80+ macrophage expression in the paws of WT and FABP5 KO mice. Note the low mPGES-1 expression at baseline and its increase primarily in F4/80+ macrophages after carrageenan injection in WT mice. In FABP5 KO mice, carrageenan injection did not lead to an increase in mPGES-1 expression. E, higher magnification merged images of those shown in D. Error bars represent S.E.
Figure 6.
Figure 6.
Involvement of cannabinoid and PPAR receptors in mPGES-1 induction. A, induction of mPGES-1 in WT and FABP5 KO mouse hind paws after injection with carrageenan. Mice were administered vehicle, the cannabinoid receptor 1 and 2 antagonists AM251 and AM630 (3 mg/kg, i.p.), or the PPARα antagonist GW6471 (4 mg/kg, i.p.). Paws were harvested 24 h after carrageenan injection. B, quantification of mPGES-1/GAPDH band intensities of Western blots described in A and C. *, p < 0.05 versus carrageenan-injected WT mice (n = 6–8). C, induction of mPGES-1 in paws of WT and FABP5 KO mice treated with vehicle or the PPARγ antagonist GW9662 (2 mg/kg, i.p.). D, PGE2 levels in hind paws of WT and FABP5 KO mice administered AM251/AM630, GW6471, or GW9662. PGE2 levels were quantified at baseline and 24 h after carrageenan injection. *, p < 0.05; **, p < 0.01 versus carrageenan-injected WT mice (n = 6). Error bars represent S.E.
Figure 7.
Figure 7.
Effect of FABP5 inhibition upon mPGES-1 induction and NF-κB activity in A549 cells. A, left, induction of mPGES-1 and COX-2 in control and FABP5 shRNA–expressing A549 cells treated with 1 ng/ml IL-1β or vehicle. Right, quantification of Western blots. *, p < 0.05 versus IL-1β–treated control cells at the same time point (n = 6). B, left, knockdown of FABP5 by shRNA. Right, quantification of Western blots. *, p < 0.05 (n = 3). C, left, phosphorylated and total NF-κB in control and FABP5 shRNA–expressing A549 cells treated with 1 ng/ml IL-1β. Right, quantification of phosphorylated/total NF-κB. *, p < 0.05 versus IL-1β–treated control cells at the same time point (n = 6). D, left, Western blot of nuclear and cytoplasmic NF-κB in control and FABP5 shRNA–expressing A549 cells incubated with 1 ng/ml IL-1β or vehicle for 30 min. The cells underwent fractionation, and nuclear and cytosolic fractions were collected. The purity of the fractions was confirmed using GAPDH and histone H3 as cytosolic and nuclear markers, respectively. C, cytosolic fraction; N, nuclear fraction. Right, quantification of nuclear NF-κB/histone H3 levels. *, p < 0.05 versus IL-1β–treated control cells (n = 4). E, NF-κB reporter activity in control and FABP5 shRNA A549 cells expressing the NF-κB luciferase reporter construct. The cells were treated with 1 ng/ml IL-1β or vehicle for 6 h, and luciferase and β-galactosidase signals were quantified. Results are reported as luciferase/β-galactosidase signal and normalized to control cells. **, p < 0.01 versus IL-1β–treated control cells (n = 6). Error bars represent S.E.
Figure 8.
Figure 8.
FABP5 modulates mPGES-1 promoter activity in A549 cells. A, schematic of the mPGES-1 promoter luciferase constructs, which contain the human mPGES-1 promoter upstream of luciferase. The ΔNF-κB construct lacks base pairs −631 to −177 that contain the NF-κB–binding sites. B, WT mPGES-1 promoter activity in control and FABP5 shRNA A549 cells treated with 1 ng/ml IL-1β or vehicle for 6 h. Promoter activity is represented as luciferase/β-galactosidase and is normalized to control A549 cells. *, p < 0.05; ***, p < 0.001. ##, p < 0.01 versus control cells (n = 6). C, baseline promoter activity in control and FABP5 shRNA–expressing A549 cells transfected with the WT or ΔNF-κB constructs. **, p < 0.01 versus control cells expressing the WT construct (n = 6). D, promoter activity in control and FABP5 shRNA A549 cells expressing the ΔNF-κB promoter construct. The cells were treated with 1 ng/ml IL-1β or vehicle for 6 h, and luciferase and β-galactosidase activities were quantified. Data are normalized to control cells. *, p < 0.05 (n = 6). Error bars represent S.E.
Figure 9.
Figure 9.
Schematic representation of the pathways through which FABP5 modulates PGE2 biosynthesis during inflammation. A, activation of the interleukin 1 receptor (IL-1R) by IL-1β triggers NF-κB phosphorylation and translocation to the nucleus wherein it binds to the mPGES-1 promoter and initiates mPGES-1 transcription. In parallel, FABP5 may transport arachidonic acid to COX-1/2 and mPGES-1, the main biosynthetic enzymes for PGE2, which reside on the endoplasmic reticulum. B, inhibition of FABP5 blunts NF-κB activation and nuclear translocation, resulting in attenuated induction of mPGES-1. FABP5 inhibition may additionally reduce arachidonic acid delivery to COX-1/2, resulting in suppression of PGE2 biosynthesis.

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