Mechanism of glutamine inhibition of cytosolic phospholipase a₂ (cPLA₂): Evidence of physical interaction between glutamine-Induced mitogen-activated protein kinase phosphatase-1 and cPLA₂

Abstract

Non-essential amino acid L-glutamine (Gln) possesses anti-inflammatory activity via deactivating cytosolic phospholipase A₂ (cPLA₂). We showed previously that Gln deactivated cPLA₂ indirectly via dephosphorylating p38 mitogen-activated protein kinase (MAPK), the major kinase for cPLA₂ phosphorylation, through inducing MAPK phosphatase-1 (MKP-1). In this study, we investigated the precise mechanism underlying Gln deactivation of cPLA₂. In lipopolysaccharide (LPS)-treated mice, Gln injection resulted in dephosphorylation of phosphorylated cPLA₂ (p-cPLA₂), which coincided with rapid Gln induction of MKP-1. MKP-1 small interfering RNA (siRNA) abrogated the ability of Gln to induce MKP-1 as well as the dephosphorylation of cPLA₂. Co-immunoprecipitation and in-situ proximity ligation assay revealed a physical interaction between MKP-1 and p-cPLA₂. In a murine model of allergic asthma, we also demonstrated the physical interaction between MKP-1 and p-cPLA₂. Furthermore, Gln suppressed various allergic asthma phenotypes, such as neutrophil and eosinophil recruitments into the airway, airway levels of T helper type 2 (Th2) cytokines [interleukin (IL)-4, IL-5 and IL-13], airway hyperresponsiveness, mucin production and metabolites (leukotriene B₄ and platelet-activating factor) through inhibiting cPLA₂ in a MKP-1-dependent manner. These data suggest that MKP-1 uses cPLA₂, in addition to p38, as a substrate, which further potentiates the anti-inflammatory action of Gln.

Introduction

Phospholipase A₂ (PLA₂) is a family of proteins that consists of five subtypes: cytosolic PLA₂ (cPLA₂), secreted PLA₂, calcium-independent PLA₂, platelet-activating factor (PAF) acetylhydrolase and lysosomal PLAs 1,2. Among PLA₂s, many research studies have focused on cPLA₂ due to its preference for hydrolyzing membrane glycerophospholipids at position sn-2 to form arachidonic acid (AA) 3. AA is converted to potent inflammatory lipid mediator eicosanoids, including leukotrienes (LTs), lipoxins, thromboxanes and prostaglandins, through catalytic action of lipoxygenase or cyclooxygenase 4,5. The involvement of cPLA₂ in lipid mediator production makes it a potentially important pharmacological target to control inflammation and cancer. Despite its importance, the mechanisms for cPLA₂ down-regulation have not yet been determined. Understanding the mechanisms of down-regulation of cPLA₂ will provide a novel approach to improve the management of inflammatory diseases.

Non-essential amino acid L-glutamine (Gln), the most abundant amino acid in plasma 6, is an energy substrate for most cells 7,8. Consequently, Gln is important in multiple ways in the nitrogen- and carbon-skeleton exchange of different tissues 9. Human clinical trials with several patient populations have demonstrated that Gln treatment can decrease infectious complications, shorten hospitalization and decrease hospital costs 10. Additionally, extensive research studies using animal models of endotoxin shock, including severe injury models, have demonstrated that Gln supplementation can improve survival, enhance immune and gut barrier function, decrease bacteraemia and inhibit gut mucosal atrophy 11,12.

To understand the molecular mechanism of Gln anti-inflammatory activity, we have shown that Gln can protect animals effectively from endotoxin shock as well as inhibit bronchial allergic asthma by inhibiting cPLA₂ phosphorylation 13,14. To decipher the mechanism of Gln inhibition of cPLA₂, we demonstrated recently that Gln deactivated p38 and Janus kinase (JNK) mitogen-activated protein kinases (MAPKs) by rapidly inducing MAPK phosphatase (MKP)-1 protein 15, which preferentially dephosphorylated p38 and JNK 16,17, suggesting that Gln inhibited cPLA₂ phosphorylation indirectly by blocking the kinase activity of p38, a major upstream pathway for cPLA₂ phosphorylation 18.

In this study, we further investigated the precise mechanism of how Gln inhibits cPLA₂ phosphorylation. We found a physical interaction between Gln-induced MKP-1 and phosphorylated cPLA₂ (p-cPLA₂). Our data suggest that MKP-1 can directly dephosphorylate cPLA₂, thus further delineating the mechanism underlying Gln deactivation of cPLA₂.

Materials and methods

Animals

Specific pathogen-free female BALB/c mice were purchased from Samtako Bio Korea (Osan, Korea) and kept in our animal facility for at least 1 week before use. Mice used at the start of each experiment were at 7–8 weeks of age. All experimental animals used in this study protocol were approved by the Institutional Animal Care and Use Committee of Chonbuk National University Medical School.

Cell culture

MH-S murine alveolar macrophage cells were grown in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 1% antibiotics (Invitrogen) at 37°C in a 5% CO₂ atmosphere.

Reagents

Lipopolysaccharide (LPS) from Escherichia coli (O127:B8) and L-Gln (biotechnology performance certified, G-8540) were purchased from Sigma-Aldrich (St Louis, MO, USA). LPS (2·5 mg/mouse) was injected intravenously (i.v.) via the lateral tail vein. Gln was dissolved in sterilized distilled water to 3%. A volume of 0·5 ml (750 mg/kg) 12–15 was administered intraperitoneally (i.p.) 10 min before or 10 min after LPS injection.

Specific cPLA₂ inhibitor pyrrophenone (Cayman Chemical Company, Ann Arbor, MI, USA) was administered i.p. at 20 mg/kg 30 min before LPS injection. Triptolide (1 μM) (Calbiochem, Darmstadt, Germany), a MKP-1 inhibitor, was added to the culture 30 min before LPS stimulation. Antibodies against cPLA₂ and p-cPLA₂ were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-MKP-1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Immunoblotting analysis

Mice were killed by cervical dislocation. Lungs were collected immediately thereafter, washed briefly with cold phosphate-buffered saline (PBS) and dried with blotting paper. Isolated lung tissues were frozen in liquid nitrogen and stored at −70°C until analysis. Small lung specimens were homogenized in PhosphoSafe Extraction Reagent (Novagen, Madison, WI, USA) and subjected to immunoblotting analysis, as described previously 15.

Immunoprecipitation

Lungs and cells were lysed in non-denaturing lysis buffer consisting of 20 mM Tris HCl pH 8, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM ethylendiamine tetraacetic acid (EDTA), protease inhibitor cocktail and phosphatase inhibitor. Equal amounts (100 μg) of cell or tissue extracts were incubated with primary antibodies for 4 h at 4°C in the same total volume of lysis buffer. Protein A/G-conjugated agarose beads (Santa Cruz Biotechnology) were added and incubated overnight. The beads were washed four times with lysis buffer and boiled to separate proteins. The beads were removed by centrifugation. The supernatant was used for Western blot.

RNA interference

Small interfering RNA (siRNA) targeting mouse MKP-1 and non-targeting siRNA controls were purchased from Santa Cruz Biotechnology. In-vivo siRNA transfection was performed using polyethyleneimine (PEI) purchased from Polyplus-tansfection (BP 90018 ; F-67401 Illkirch Cedex, France) according to the manufacturer's instruction. siRNA and PEI dissolved in 5% glucose was mixed in a volume of 200 μl for i.v. or 30 μl for intratracheal (i.t.) injection. The mixture was incubated for 15 min at room temperature and injected 24 h before LPS treatment 12.

In-situ proximity ligation assay (PLA)

Interaction of p-cPLA₂ and MKP-1 was detected in situ by PLA technology. Duolink II fluorescence compound was purchased from Olink Bioscience (Uppsala, Sweden). The assay was performed according to the manufacturer's protocol. Briefly, 1 × 10⁴ of MH-S cells were cultured in a covered glass-bottomed dish overnight and treated with LPS (0·1 μg/ml). Cells were washed with PBS for 50 min, fixed with 4% paraformaldehyde for 15 min and treated with cold 100% methanol for 10 min for membrane permeabilization. Samples were blocked with blocking solution for 30 min and incubated with primary antibodies (anti-p-cPLA₂ at 1 : 500 or anti-MKP-1 at 1 : 500) at 4°C for 2 h. After washing twice for 5 min, samples were incubated with PLA probes at 37°C in a humidity chamber for 30 min and washed twice for 5 min. Oligonucleotides conjugated with PLA probes were ligated for 30 min and washed twice for 2 min. Ligated oligonucleotides were amplified for 60 min in a preheated humidity chamber and washed. Covered glass-bottomed dishes were mounted with mounting medium and covered with a coverslip. Samples were analysed by confocal microscopy.

Immunization and challenge

Mice were immunized i.p. with 20 μg OVA (grade V; Sigma-Aldrich) plus 1·0 mg aluminium hydroxide adjuvant (Imject® Alum; Pierce, Rockford, IL, USA) on day 0 and ovalbumin (OVA) alone without alum on day 14. The immunized mice were exposed to aerosolized OVA on days 28 and 35. Aerosolization of OVA was performed using a chamber adapted for mice. Animals were exposed to 1% OVA using a model NE-U12 ultrasonic nebulizer at output of 0·8 ml/min (Omron, Tokyo, Japan) for 30 min at first challenge and 10 min at second challenge 19. Gln (750 mg/kg) was administered i.p. immediately after the cessation of the second challenge. Pyrrophenone (20 mg/kg) was administered i.p. 30 min before the second airway challenge.

Bronchoalveolar lavage (BAL)

Mice were anaesthetized and the trachea was cannulated while gently massaging the thorax. Lungs were lavaged with 800 μl of saline. An equal volume of BAL fluid sample was centrifuged. The supernatant was collected and stored at −70° until cytokine assay. The pellet was resuspended in saline and cytospin preparations (5 min, 202 g) of BAL cells were stained with Diff-Quik (International Reagents, Kobe, Japan). Different cell types were enumerated based on morphology and staining profile.

Determination of airway hyperresponsiveness (AHR)

AHR was determined as changes in airway function after challenge by aerosolized methacholine using published procedures 14,19. After methacholine challenge, airway resistance (RL) data were collected continuously. Maximum RL values representing percentage of change from baseline after aerosol saline challenge were used to express changes in airway function.

Histological analysis

Lungs were fixed with 10% formalin. Tissues were embedded in paraffin. To detect mucin, sections (4 μm) were stained with periodic acid-Schiff and counterstained with haematoxylin.

Enzyme-linked immunosorbent assay (ELISA)

Levels of T helper type 2 (Th2) cytokines interleukin (IL)-4, IL-5 and IL-13 in BAL fluids were measured using ELISA according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). The lower limits of detection for the cytokines were as follows: IL-4 (>2 pg/ml), IL-5 (>7 pg/ml) and IL-13 (>1·5 pg/ml). Levels of lipid mediators, leukotriene B₄ (LTB₄) and PAF in the blood and BAL fluids were also measured using ELISA according to the manufacturer's instructions. The lower limits of detection for lipid mediators were: LTB₄ (>13 pg/ml; Cayman Chemical Company) and PAF (>0·78 ng/ml; MyBiosource, San Diego, CA, USA).

Statistical analysis

Data were expressed as mean ± standard deviation (s.d.). Statistical comparisons were performed using one-way analysis of variance (anova) followed by Tukey's test. Statistically significant difference was considered when P-values were less than 0·05.

Results

Gln dephosphorylates p-cPLA₂ in a MKP-1-dependent manner

In the course of our studies on Gln deactivation of cPLA₂, we have found consistently that Gln could deactivate cPLA₂ by dephosphorylating p-cPLA₂ 13,14,20. In this study, we confirmed this finding in the lungs of LPS-injected mice. Time kinetics of cPLA₂ phosphorylation and MKP-1 induction are shown in Fig. 1a. When Gln was administered 10 min after LPS injection, Gln rapidly dephosphorylated the p-cPLA₂ within 5 min, concurrent with cPLA₂ phosphorylation (Fig. 1b). Gln administration also resulted in an early induction of MKP-1 protein (Fig. 1b), consistent with our previous report 15. MKP-1 siRNA potentiated cPLA₂ phosphorylation and suppressed MKP-1 protein induction (Fig. 1c). In mice treated with MKP-1 siRNA, Gln could no longer dephosphorylate cPLA₂ (Fig. 1d). To verify the MKP-1/cPLA₂ linkage, cPLA₂ phosphorylation and MKP-1 expression in response to LPS were evaluated in MH-S murine alveolar macrophage cells. The time kinetics of cPLA₂ phosphorylation and MKP-1 induction are shown in Fig. 1e. Addition of Gln resulted in decreased phosphorylation of cPLA₂ and early induction of MKP-1 (Fig. 1f). Inhibition of MKP-1 using MKP-1 inhibitor triptolide enhanced the magnitude of cPLA₂ phosphorylation but reduced MKP-1 expression (Fig. 1g). Gln-mediated inhibition of cPLA₂ phosphorylation was reversed by triptolide (Fig. 1h). Taken together, the observation that Gln deactivated cPLA₂ by dephosphorylating it only when cPLA₂ was phosphorylated in response to LPS in a MKP-1-dependent manner suggested that Gln could dephosphorylate cPLA₂ directly through the induction of MKP-1.

Fig 1.

L-glutamine (Gln) dephosphorylates phosphorylated cPLA₂ in a mitogen-activated protein kinase phosphatase-1 (MKP-1)-dependent manner. (a) Lungs were removed at the indicated time-points after lipopolysaccharide (LPS) injection [50 μg/mouse, intravenously (i.v.)]; (b) Gln [750 mg/kg, intraperitoneally (i.p.)] was injected 10 min before or 10 min after LPS and mice were killed at the indicated time-points. (c) MKP-1 small interfering RNA (siRNA) or control siRNA (0·4 nM each) was injected i.v. 24 h before LPS; (d) siRNA and Gln were injected 24 h before and 10 min after LPS, respectively; (e) LPS (0·1 μg/ml) was added to the culture medium and cells were harvested at the indicated time-points; (f,h) Gln (20 mM) was added 10 min after LPS; (g,h) triptolide (1 μM) was added 3 h before LPS. (a–d) A representative of three to five independent experiments with three mice/time-point/experiment is shown. (e–g) A representative of two or three independent experiments with three dished/time-point/experiment is shown.

Physical interaction between MKP-1 and p-cPLA₂

How could Gln dephosphorylate p-cPLA₂ within 5 min? cPLA₂ phosphorylation is known to depend upon the kinase activity of p38. In fact, pretreatment of p38 inhibitor SB202190 inhibited cPLA₂ phosphorylation significantly 20. Injection of Gln resulted in a rapid dephosphorylation of p38 through MKP-1 induction 15. However, the extent to which Gln-mediated cPLA₂ dephosphorylation is p38-dependent remains to be elucidated. Phosphatase activity of MKP-1 could be another possible mechanism underlying Gln dephosphorylation of cPLA₂ based on the finding that the activity of Gln was exclusively MKP-1-dependent. To test this hypothesis, we investigated whether or not there was a physical interaction between MKP-1 and cPLA₂. Lung lysates from LPS-injected mice were co-immunoprecipitated using anti-MKP-1 and anti-p-cPLA₂ antibodies targeting the Ser⁵⁰⁵ residue. MKP-1 was detected in immunoprecipitates using anti-p-cPLA₂ (Fig. 2a). A reciprocal immunoprecipitation and Western blotting experiment using anti-MKP-1 antibody also detected p-cPLA₂ (Fig. 2b). A co-immunoprecipitation assay using LPS-treated MH-S cells showed similar results (Fig. 2c,d). However, immunoprecipitates were not detected using anti-MKP-1 or anti-cPLA₂ antibodies (Fig. 2e,f). These data support the theory that there was a physical interaction between MKP-1 and p-cPLA₂, but not cPLA₂ itself. In-situ PLA was performed to confirm further the physical interaction between MKP-1 and p-cPLA₂. After MH-S cells were treated with LPS for 50 min, cells were probed with anti-p-cPLA₂ and anti-MKP-1 antibodies and conjugated with oligonucleotide PLA probes. When the complementary PLA probes were in close proximity (<40 nm), oligonucleotide strands could be hybridized and amplified via the rolling circle method. As a result of this process, red dots were visualized. When viewed with a fluorescence microscope, the red fluorescence was observed mainly around nucleus (Fig. 2g). However, application of either anti-p-cPLA₂ or anti-MKP-1 failed to produce red fluorescence, confirming that there was physical interaction between MKP-1 and p-cPLA₂.

Fig 2.

Physical interaction between mitogen-activated protein kinase phosphatase-1 (MKP-1) and phosphorylated cytosolic phospholipase A2 (cPLA₂). (a–d) Lung and cell lysates were immunoprecipitated using anti-p-cPLA₂ and anti-MKP-1 antibodies. (e,f) Lung and cell lysates were immunoprecipitated using anti-cPLA₂ antibody. (g) Cells were probed with anti-phospho-cPLA₂ or anti-MKP-1 antibody and conjugated with oligonucleotide PLA probe: nucleus was counterstained with 4',6-diamidino-2-phenylindole (DAPI). A representative of two or three independent experiments with three mice/time-point/experiment is shown in (a–f). A representative of four independent experiments is shown in (g).

Gln inhibits the production of cPLA₂ products

We assessed whether Gln inhibition of cPLA₂ phosphorylation could lead to the inhibition of cPLA₂ products LTB₄ and PAF. Serum levels of LTB₄ (Fig. 3a) and PAF (Fig. 3b) were increased progressively after LPS injection. Gln administration resulted in the inhibition of both LTB₄ (Fig. 3c) and PAF (Fig. 3d). The suppressive activity of Gln was comparable to those of pyrrophenone, the specific cPLA₂ inhibitor.

Fig 3.

L-glutamine (Gln) and pyrrophenone suppress serum levels of leukotriene B4 (LTB₄) and platelet activating factor (PAF). Serum levels of LTB₄ (a) and PAF (b) were determined at the indicated time-points. Gln (750 mg/kg, i.p.) and pyrrophenone [20 mg/kg, intraperitoneally (i.p.)] were administered 10 min after and 30 min before LPS injection, respectively. LTB₄ (c) and PAF (d) were determined at 1 and 4 h after LPS. Data representing the mean ± standard deviation (s.d.) of three independent experiments with three to five mice/group/experiment are shown. *P < 0·05 compared to saline control; **P < 0·01 compared to saline control; #P < 0·05 compared to the LPS group.

Physical interaction between MKP-1 and p-cPLA₂ in asthmatic lungs

We have reported previously a beneficial effect of Gln on allergic asthma in mice 14,20. In this study, we assessed whether cPLA₂/MKP-1 linkage was involved in the beneficial effect of Gln. Phosphorylation of cPLA₂ was prominent at 10–30 min after the second airway challenge. Expression of MKP-1 was at 30–60 min after the cessation of the second OVA airway challenge (Fig. 4a). MKP-1 siRNA abrogated MKP-1 induction and potentiated cPLA₂ phosphorylation in asthmatic lungs (Fig. 4b). Administration of Gln immediately after the cessation of the second airway challenge resulted in the dephosphorylation of cPLA₂ and induced early strong induction of MKP-1 protein (Fig. 4c). MKP-1 siRNA reversed Gln inhibition of cPLA₂ phosphorylation as well as MKP-1 induction (Fig. 4d). We assessed the interaction between MKP-1 and p-cPLA₂ in asthmatic lungs. MKP-1 was detected in immunoprecipitates using anti-p-cPLA₂ antibody (Fig. 4e). Immunoprecipitates using anti-MKP-1 antibody showed p-cPLA₂ (Fig. 4f). Taken together, these data indicated that MKP-1-deactivated cPLA₂, through their physical interaction, also occurred in asthmatic lungs.

Fig 4.

L-glutamine (Gln) deactivates phosphorylated (p)-cytosolic phospholipase A2 (cPLA₂) through mitogen-activated protein kinase phosphatase-1 (MKP-1) in asthmatic lungs. After a second ovalbumin (OVA) airway challenge, lungs were removed at the indicated time-points (a–c). Small interfering RNAs (siRNAs) (0·2 nM) were administered intratracheally (i.t.) 24 h before the second challenge (b,d). Gln (750 mg/kg) was administered intraperitoneally (i.p.) immediately after the cessation of the second challenge (c,d). Lungs were removed at 10 min post-challenge (d). Lungs were removed at the indicated time-points. Lung lysates were immunoprecipitated using anti-p-cPLA₂ or anti-MKP-1 antibodies (e,f). A representative of three to five independent experiments with three to five mice/time-point/experiment is shown.

Gln suppresses allergic pulmonary inflammation in a MKP-1-dependent manner

We evaluated the role of MKP-1 in the suppression of allergic asthmatic reactions by Gln using MKP-1 siRNA. Gln and pyrrophenone suppressed the recruitment of neutrophils (Fig. 5a) and eosinophils (Fig. 5b), but not macrophages (Fig. 5c), BAL fluid levels of Th2 cytokines (Fig. 5d), AHR (Fig. 5e) or mucus secretion (Fig. 5f). MKP-1 siRNA abrogated all the suppressive effects of Gln. We also assessed LTB₄ and PAF in the airways. BAL fluid levels of LTB₄ (Fig. 6a) and PAF (Fig. 6b) were increased progressively up to 3 h after challenge, but declined thereafter. Gln and pyrrophenone suppressed the BAL levels of both LTB₄ and PAF. The suppressive effect of Gln was reversed by MKP-1 siRNA (Fig. 6c). These data indicate that Gln ameliorated allergic asthma by inhibiting cPLA₂ after MKP-1 induction.

Fig 5.

Mitogen-activated protein kinase phosphatase-1 (MKP-1) Small interfering RNA (siRNA) abrogates the suppressive effect of L-glutamine (Gln) in allergic pulmonary inflammation. Small interfering RNAs (siRNA) and Gln were administered as described in Fig. 4. Pyrrophenone (Pyr; 20 mg/kg) was administered intraperitoneally (i.p.) 30 min before the second challenge. Bronchoalveolar lavage (BAL) fluids were collected 12 h (a), 48 h (b) and (c) and 18 h (d) after the second ovalbumin (OVA) challenge. Airway hyperresponsiveness (AHR) was performed at 48 h (e). Lungs were removed at 48 h post-challenge for mucin production (f). Data representing the mean ± standard deviation (s.d.) of three independent experiments with three to five mice/group/experiment are shown. *P < 0·05 compared to saline control; **P < 0·01 compared to saline control; #P < 0·05 compared to the OVA group; ##P < 0·05 compared to the Gln group.

Fig 6.

L-glutamine (Gln) and pyrrophenone suppress bronchoalveolar lavage (BAL) levels of leukotriene B4 (LTB₄) and platelet activating factor (PAF). BAL fluid levels of LTB₄ (a) and PAF (b) were determined at the indicated time-points. Gln [750 mg/kg, intraperitoneally (i.p.)] and pyrrophenone (Pyr; 20 mg/kg, i.p.) were administered immediately after and 30 min before the second airway challenge, respectively. (c) LTB₄ and PAF were measured at 3 h. Data representing the mean ± standard deviation (s.d.) of three independent experiments with three to five mice/group/experiment are shown. *P < 0·05 compared to saline control; **P < 0·01 compared to saline control; #P < 0·05 compared to ovalbumin (OVA) challenge group.

Discussion

Our data demonstrate a physical interaction between Gln-induced MKP-1 and p-cPLA₂, suggesting that Gln could deactivate cPLA₂ directly via early induction of MKP-1. Our findings not only provided the underlying mechanism of Gln's anti-inflammatory activity, but also delineated a possible mechanism underlying the down-regulation of cPLA₂. Gln deactivates p38 MAPK by rapidly inducing MKP-1 protein 15, which dephosphorylates p38 and JNK preferentially 16,17. Both MKP-1 induction and MKP-1 mediated p38 dephosphorylation within 5 min following Gln administration, suggesting that Gln dephosphorylated cPLA₂ indirectly through MKP-1-mediated p38 dephosphorylation. However, Gln dephosphorylated p-cPLA₂ rapidly within 5 min, as in the case of p38 in LPS-injected mice 13,14,20, suggesting that phosphatase is involved. Gln injection resulted in a rapid induction of MKP-1 within 5 min, which coincided with Gln-induced dephosphorylation of cPLA₂ (Fig. 1b). MKP-1 siRNA abrogated the ability of Gln to induce dephosphorylation of cPLA₂, suggesting that MKP-1 was involved in the dephosphorylation of cPLA₂ by Gln. Our time-kinetic study revealed that Gln failed to deactivate cPLA₂, consistent with our previous report 13. As shown in this and other studies 15,20, Gln dephosphorylation of cPLA₂ is an exclusively MKP-1-dependent phenomenon. MKP-1 has been reported to be a labile protein that is normally degraded via the ubiquitin/proteasome pathway. Its phosphorylation reduces its ubiquitination and degradation 21,22 and phosphorylation of MKP-1 is mediated by transient activation of extracellular-regulated kinase (ERK), which enhances MKP-1 stabilization 23. Therefore, in the absence of ERK activation by an exogenous stimulus, Gln cannot up-regulate MKP-1 induction. This is the probable reason why Gln failed to deactivate cPLA₂ when it was administered before LPS injection.

If MKP-1 could dephosphorylate cPLA₂, these two molecules should interact physically with each other. Co-immunoprecipitation and in-situ PLA experiments revealed a physical interaction between MKP-1 and p-cPLA₂, identifying cPLA₂ as a novel MKP-1-interacting protein. In-situ PLA revealed red fluorescence in nucleus and the immediate vicinity. MKP-1 is a nuclear protein 24. Activated cPLA₂ translocates from the cytosol to cell membranes, such as those of endoplasmic reticulum, Golgi apparatus and perinuclear region 5. Thus, MKP-1 may interact with cPLA₂ during its translocation to the perinuclear membrane. These data suggest that Gln could deactivate cPLA₂ directly by MKP-1-dependent cPLA₂ dephosphorylation. Taken together, these data suggest that two mechanisms are involved in the Gln deactivation of cPLA₂. The first mechanism is by indirect cPLA₂ dephosphorylation through MKP-1-mediated p38 inactivation; the other mechanism is by direct physical interaction between Gln-induced MKP-1 and p-cPLA₂.

In this study, we used anti-p-cPLA₂ antibody to recognize phosphorylated Ser⁵⁰⁵. When MKP-1 was induced by Gln, the antibody could no longer detect p-cPLA₂, indicating that MKP-1 could bind to at least the Ser⁵⁰⁵ residue of cPLA₂. Further studies are required to address the possible involvement of phosphorylation site besides Ser⁵⁰⁵. MKP-1, an archetypal member of the MKP family, plays a pivotal role in the negative control of p38 and JNK. p38 has a role in the production of inflammatory molecules through both transcription-dependent mechanism and post-transcriptional regulation 25–29. MKP-1 functions as a critical negative regulator of inflammation. In a septic shock model, MKP-1-deficient mice produced greater amounts of proinflammatory cytokines and chemokines, with increased incidence and severity of multi-organ failure as well as higher mortality 30–32. These studies identified MKP-1 as an important target for the treatment of inflammatory diseases. Thus, developing novel ligands to up-regulate MKP-1 would be a therapeutically attractive anti-inflammatory strategy.

It is important to discuss the mechanism underlying Gln suppression of neutrophilic infiltration into the airway and Th2-dependent asthmatic phenotypes. Many investigators have reported an association between cPLA₂ metabolites and Th2 response in asthmatic reactions 33–36. Importantly, LTs play a key role in dendritic cell influx into airways in asthma 37–39 which, in turn, results in inhibition of recruitment of T cells and eosinophils into asthmatic airways and late AHR. Regarding neutrophil infiltration, we reported previously that airway neutrophilia in the asthmatic lungs is mediated by LTB4 rather than CXC chemokines such as keratinocyte-derived chemokine and macrophage inflammatory protein (MIP)-2 20, which are principally chemotactic for neutrophils. Taken together, cPLA₂ metabolites play an important role in induction of airway neutrophilia and Th2-dependent asthmatic phenotypes. As a result, Gln can suppress these parameters. In contrast to neutrophils and eosinophils, macrophage infiltration was not suppressed by Gln. This may be due to the fact that macrophage migration is not influenced by cPLA₂ metabolites; therefore, the monocytes/macrophages chemokines, C-C chemokines, such as monocyte chemotactic protein-1 and MIP-1, are not inhibited by Gln. In fact, the C-C chemokines are not suppressed by Gln in asthmatic lungs (data not shown). cPLA₂ has high selectivity in liberating arachidonic acid that is metabolized subsequently by a panel of downstream enzymes for eicosanoid production. Absence of cPLA₂ in mice resulted in their resistance to a variety of inflammatory diseases 40–44, indicating the potential contribution of cPLA₂ to inflammation. Therefore, cPLA₂ has attracted considerable attention as a potential therapeutic target for the control of inflammation and cancer. Inhibition of cPLA₂ as a pharmacological strategy has been proposed for the treatment of asthma 45, inflammatory skin diseases 46,47 and arthritis 48,49. Despite promising preclinical data, the use of cPLA₂ inhibitors as a therapeutic approach in inflammatory diseases has not yet been confirmed in clinical trials, due probably to toxicity 50 or poor bioavailability 51. In this regard, our findings showing that Gln exerted a potent anti-inflammatory action by deactivating both p38 and cPLA₂, probably the most potent enzymes involved in inflammatory reactions, might make Gln a useful therapeutic agent for many inflammatory diseases that involve cPLA₂ in their pathogenesis.

Disclosures

The authors declare that there are no conflicts of interest.