Down-regulation of lipoxin A4 receptor by thromboxane A2 signaling in RAW246.7 cells in vitro and bleomycin-induced lung fibrosis in vivo

doi:10.1016/j.biopha.2004.05.006

Biomedicine & Pharmacotherapy

Volume 58, Issues 6–7, July–August 2004, Pages 381-387

https://doi.org/10.1016/j.biopha.2004.05.006 Get rights and content

Abstract

Lipoxins (LXs) are members of eicosanoid family that can be endogenously produced during cell-to-cell interactions such as platelet–leukocyte interactions. Anti-inflammatory function of lipoxin A₄ (LXA₄) as “braking signals” is mediated by the receptor. On the other hand, thromboxane A₂ (TXA₂) produced by catalysis of cyclooxygenase and thromboxane synthetase is released during platelet aggregation as a vasoconstrictor and a pro-inflammatory factor. To investigate interaction of TXA₂ receptor (TP) and LXA₄ receptor, effects of a TP agonist and a thromboxane synthetase inhibitor on expression of LXA₄ receptor were examined in vitro and in vivo. A TP agonist, U46619 showed a down-regulation of LXA₄ receptor induced by interleukin-1β (IL-1β) in RAW246.7 cells. In bleomycin-induced lung fibrosis in mice, administration of a thromboxane synthetase inhibitor DP-1904 increased LXA₄ receptor mRNA and decreased type I collagen mRNA. In vitro experiments indicate that LXA₄ significantly prevented enhanced proliferation of NIH3T3 fibroblasts and the collagen expression by transforming growth factor-β (TGF-β). These results suggest that TXA₂-TP signaling could cause negative regulation of lipoxin A₄ receptor under the transcriptional level during inflammatory process mediated by IL-1β and TGF-β induce the expression of LXA₄ receptor. Furthermore, the down-regulation of LXA₄ receptor by TXA₂ implies a possibility that a cellular signaling by TXA₂ may have a novel and potential function as a pro-inflammatory factor to inhibit anti-inflammatory effect of LXA₄. Concomitantly, selective blockade of TXA₂-TP signaling could be suggested to lead to anti-inflammation through active role of LXA₄.

Introduction

Lipoxins (LXs) are members of the eicosanoid family that can be endogenously produced within the vascular lumen and at mucosal surface either from single cells or during cell-to-cell interactions such as platelet–leukocyte interactions and leukocyte–epithelial cell interactions [1]. Lipoxin A₄ (LXA₄) and lipoxin B₄ (LXB₄) are formed by the metabolism of arachidonic acid by lipoxygenases and are rapidly metabolized in vivo.

Platelet–leukocyte interaction promotes the formation of LX by transcellular conversion of the leukocyte 5-lipoxygenase epoxide product LXA₄. When platelets are adherent, platelet 12-lipoxygenase converts to LXA₄ and LXB₄ [2]. In addition, 15-lipoxygenase, which is abundant in eosinophils, alveolar macrophages, monocytes and epithelial cells, promotes oxygenation of arachidonic acid to generate 15-hydroperoxyeicosatetraenoic acid. The eicosanoid serves as a substrate for 5-lipoxygenase in leukocytes to generate LXs [3]. Aspirin can also trigger the transcellular biosynthesis if a series of 15-epimer (aspirin-triggered 15-epi-LXA₄, ATL), which share many anti-inflammatory activities with the native LXA₄ [4].

LX biosynthesis has also been shown to occur in many human diseases. Therefore, LXs could be involved in acute inflammation [5], aspirin-induced rhinosinusitis-asthma [6], [7], renal ischemia/reperfusion injury [8], intestinal inflammation [9], infection [10], [11], colitis [12], glomerulonephritis [13], thrombosis and atherosclerosis [14]. Thus, the LXs were the first to be identified and recognized as endogenous anti-inflammatory lipid mediators of resolution in that they can function as “braking signals” in inflammatory processes [15].

FPRL1/LXA₄R was originally identified as a low-affinity receptor for N-formyl-Met-Leu-Phe that shares 69% sequence identity with the high-affinity formyl peptide receptor FPR [16], [17], [18], [19]. It was subsequently reported that LXA₄ and aspirin-triggered 15-epi-LXA₄ are endogenous ligands for FPRL1/LXA₄R and exert their anti-inflammatory functions through this receptor [20]. LXA₄ receptor regulates neutrophil recruitment [21] activated phagocyte [22], stimulates a cytosolic Ca²⁺ increase in human bronchial epithelium [23], decreased enhanced secretion of interleukin-8 (IL-8) by tumor necrosis factor-α from enterocytes [24], induction of IL-8 secretion by serum amyloid A [25], and inhibit ear inflammation [5].

Thus, biosynthesis of LX and the receptor is becoming to be well characterized. Moreover, observations that treatment with LXA₄, 15-epi-LXA₄, and the synthetic analogue protect against inflammation and other pathogenesis become to be accumulated as described above. However, information regarding to regulation of LX receptor is a quite few. The only report has shown to induce up-regulation of LXA₄ receptor by interleukin-1β (IL-1β) in normal human synovial fibroblasts, indicating post-transcriptional control for LX receptor [26]. In the present experiments, we studied possible interaction between thromboxane receptor (TP) and LXA₄ receptor. Thromboxane A₂ (TXA₂) is one of cyclooxygenase product, which is predominantly released from platelet during platelet aggregation, leading to thrombosis [27]. Therefore, production of LX and TX may be relevant to orchestrate during platelet–leukocyte interaction under pathophysiological condition. However, less is known about the regulation of LX receptor under transcription level. We report for the first time that a G-protein-coupled receptor agonist TXA₂ and the receptor TP could play a role in down-regulation of LXA₄ receptor, suggestion a possible regulation of LXA₄ mediated by TXA₂ signaling during interaction of platelet and leukocytes.

Section snippets

Cell cultures

Murine macrophage cell line RAW264.7 and murine fibroblast NIH3T3 were cultured in Dulbecco’s-modified Eagle’s medium (DMEM) (Sigma-Aldrich Chemical Inc, St. Louis, MO) supplemented with 10% heated-inactivated fetal calf serum (FCS) (Hyclone, Logan, UT), penicillin/streptomycin (100 U/ml) (Gibco BRL, Grand Island, NY) under standard 5% CO₂ humidified atmosphere at 37 °C. These cells were seeded in six-well plate and were cultured for 24 h. Then, the cells were placed into the quiescent state by

Suppressive effect of a TP agonist, U46619 on expression of LXA₄ receptor in RAW264.7 cells

Effect of a stable TXA₂ mimetic, U46619 on expression of LXA₄ receptor in RAW264.7 cells was examined. Expression of LXA₄ receptor in the cells was determined by RT-PCR amplification of RAW264.7 cells-derived total RNA. Amplified cDNA for the receptor corresponding to 130-bp of a fragment was detected by the PCR under resting condition of RAW264.7 cells (Fig. 1a). When the cells were stimulated with IL-1β (10 ng/ml) for 2 h, the expression was markedly increased to 10-fold compared to that in

Discussion and conclusion

The present results demonstrate that TX and the receptor could mediate negatively regulated expression of LXA₄ receptor. LXs are formed by the metabolism of arachidonic acid by lipoxygenases during platelet–leukocyte interactions and by peripheral blood monocytes during inflammation, thrombosis, and atherosclerosis [7]. During these pathological conditions, platelet aggregation is an important event of the haemostatic mechanism that prevents undesired bleeding and invasion of noxious

Acknowledgements

Part of this work was supported by a research grant from The Japanese Ministry of Education, Culture, Sports, Science and Technology (to I.H.), and a Research Project grant from the Graduate School of Medical Sciences, Kitasato University (to I.H. and H.K.). The authors thank Ms. Terumi Mizuno and Sachiko Kurihara for their expert technical assistance. The experimental Animal Center for animal care, and The Radioisotope Research Center of Kitasato University School of Medicine for experiment

References (48)

C.N Serhan
Lipoxins and novel aspirin-triggered 15-epi-lipoxins (ATL): a jungle of cell–cell interactions or a therapeutic opportunity?
Prostaglandins
(1997)
C.N Serhan
Lipoxins and aspirin-triggered 15-epi-lipoxin biosynthesis: an update and role in anti-inflammation and pro-resolution
Prostaglandins Other Lipid Mediat
(2002)
N.E Kieran et al.
Modification of the transcriptomic response to renal ischemia/reperfusion injury by lipoxin analog
Kidney Int
(2003)
J Goh et al.
Lipoxins: pro-resolution lipid mediators in intestinal inflammation
Gastroenterology
(2003)
R.D Ye et al.
Isolation of a cDNA that encodes a novel granulocyte N-formyl peptide receptor
Biochem Biophys Res Commun
(1992)
P.M Murphy et al.
A structural homologue of the N-formyl peptide receptor: characterization and chromosome mapping of a peptide chemoattractant receptor family
J Biol Chem
(1992)
L Bao et al.
Mapping genes for the human C5a receptor (C5AR), human FMLP receptor (FPR), and two FMLP receptor homologue orphan receptors (FPRH1, FPRH2) to chromosome 19
Genomics
(1992)
H.D Perez et al.
Cloning of a cDNA encoding a receptor related to the formyl peptide receptor of human neutrophils
Gene
(1992)
C Bonnans et al.
Lipoxin A₄ stimulates a cytosolic Ca²⁺ increase in human bronchial epithelium
J Biol Chem
(2003)
R He et al.
Serum amyloid A induces IL-8 secretion through a G protein-coupled receptor, FPRL1/LXA₄R
Blood
(2003)

M Takami et al.

Effects of DP-1904, a thromboxane synthetase inhibitor, on the antigen-induced airway hyperresponsiveness and infiltration of inflammatory cells in guinea-pigs

Prostaglandins Leukot Essent Fatty Acids

(1998)

U Diczfalusy et al.

A structural requirement for the conversion of prostaglandin endoperoxides to thromboxanes

FEBS Lett

(1979)

T Namba et al.

Mouse thromboxane A₂ receptor: cDNA cloning, expression and northern blot analysis

Biochem Biophys Res Commun

(1992)

D.B Chandler et al.

The in vitro synthesis and degradation of prostaglandins during the development of bleomycin-induced pulmonary fibrosis in hamsters

Prostaglandins Leukotrienes Med

(1983)

G Jenkins et al.

COX-2 overexpression, using an integrin-targeted gene delivery system (the LID vector), inhibits fibroblast proliferation in vitro and leads to increased prostaglandin E₂ in the lung

Chest

(2002)

C.B Keerthisingam et al.

Cyclooxygenase-2 deficiency results in a loss of the anti-proliferative response to transforming growth factor-β in human fibritic lung fibroblasts and promotes bleomycin-induced pulmonary fibrosis in mice

Am J Pathol

(2001)

C Risk et al.

Thromboxane and prostacyclin (epoprostenol) during exercise in diffuse pulmonary fibrosis

Lancet

(1982)

D.B Chandler et al.

The in vitro synthesis and degradation of prostaglandins during the development of bleomycin-induced pulmonary fibrosis in hamsters

Prostaglandins Leukot Med

(1983)

M Romano et al.

Lipoxin generation by permeabilized human platelets

Biochemistry

(1992)

B.D Levy et al.

Human alveolar macrophages have 15-lipoxygenase and generate 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid and lipoxins

J Clin Invest

(1993)

J Clària et al.

Aspirin-triggered lipoxins (15-epi-LX) are generated by the human lung adenocarcinoma cell line (A549)–neutrophil interactions and are potent inhibitors of cell proliferation

Mol Med

(1996)

T Takano et al.

Aspirin-triggered 15-epi-lipoxin A₄ (LXA₄) and LXA4₄ stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors

J Exp Med

(1997)

B.D Levy et al.

Multi-pronged inhibition of airway hyper-responsiveness and inflammation by lipoxin A₄

Nat Med

(2002)

J Aliberti et al.

Parasite-induced lipoxin A₄ is an endogenous regulator of IL-12 production and immunopathology in Toxoplasma gondii infection

J Exp Med

(2002)

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Original articleDown-regulation of lipoxin A4 receptor by thromboxane A2 signaling in RAW246.7 cells in vitro and bleomycin-induced lung fibrosis in vivo

Abstract

Introduction

Section snippets

Cell cultures

Suppressive effect of a TP agonist, U46619 on expression of LXA4 receptor in RAW264.7 cells

Discussion and conclusion

Acknowledgements

Prostaglandins

Prostaglandins Other Lipid Mediat

Kidney Int

Gastroenterology

Biochem Biophys Res Commun

J Biol Chem

Genomics

Gene

J Biol Chem

Blood

Prostaglandins Leukot Essent Fatty Acids

FEBS Lett

Biochem Biophys Res Commun

Prostaglandins Leukotrienes Med

Chest

Am J Pathol

Lancet

Prostaglandins Leukot Med

Lipoxin generation by permeabilized human platelets

Biochemistry

Human alveolar macrophages have 15-lipoxygenase and generate 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid and lipoxins

J Clin Invest

Aspirin-triggered lipoxins (15-epi-LX) are generated by the human lung adenocarcinoma cell line (A549)–neutrophil interactions and are potent inhibitors of cell proliferation

Mol Med

Aspirin-triggered 15-epi-lipoxin A4 (LXA4) and LXA44 stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors

J Exp Med

Multi-pronged inhibition of airway hyper-responsiveness and inflammation by lipoxin A4

Nat Med

Parasite-induced lipoxin A4 is an endogenous regulator of IL-12 production and immunopathology in Toxoplasma gondii infection

J Exp Med

Original article
Down-regulation of lipoxin A₄ receptor by thromboxane A₂ signaling in RAW246.7 cells in vitro and bleomycin-induced lung fibrosis in vivo

Suppressive effect of a TP agonist, U46619 on expression of LXA₄ receptor in RAW264.7 cells

Aspirin-triggered 15-epi-lipoxin A₄ (LXA₄) and LXA4₄ stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors

Multi-pronged inhibition of airway hyper-responsiveness and inflammation by lipoxin A₄

Parasite-induced lipoxin A₄ is an endogenous regulator of IL-12 production and immunopathology in Toxoplasma gondii infection